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Simple, Intuitive Calculations of Free Energy of Binding for Protein−Ligand Complexes. 3. The Free Energy Contribution of Structural Water Molecules in HIV-1 Protease Complexes
Abstract
Structural water molecules within protein active sites are relevant for ligand-protein recognition because they modify the active site geometry and contribute to binding affinity. In this work an analysis of the interactions between 23 ligands and dimeric HIV-1 protease is reported. The X-ray structures of these complexes show the presence of four types of structural water molecules: water 301 (on the symmetry axis), water 313, water 313bis, and peripheral waters. Except for water 301, these are generally complemented with a symmetry-related set. The GRID program was used both for checking water locations and for placing water molecules that appear to be missing from the complexes due to crystallographic uncertainty. Hydropathic analysis of the energetic contributions using HINT indicates a significant improvement of the correlation between HINT scores and the experimentally determined binding constants when the appropriate bridging water molecules are taken into account. In the absence of water r2 = 0.30 with a standard error of +/- 1.30 kcal mol(-1) and when the energetic contributions of the constrained waters are included r2 = 0.61 with a standard error of +/- 0.98 kcal mol(-1). HINT was shown to be able to map quantitatively the contribution of individual structural waters to binding energy. The order of relevance for the various types of water is water 301 > water 313 > water 313bis > peripheral waters. Thus, to obtain the most reliable free energy predictions, the contributions of structural water molecules should be included. However, care must be taken to include the effects of water molecules that add information value and not just noise.
... 27 Many inhibitory compounds were designed based on this structure, and an impressive number of cocrystal structures were solved and reported in just a few years. One particularly interesting series of compounds for this story was the cyclic urea class developed by the DuPont Merck Pharmaceutical Co. 28 −30 In addition to an open conformation, the unbound protease's (pdb 1g6l 31 ) active site ( Figure 1A) contains several water molecules that are nominally displaced by the incoming ligands, a few that adapt to support ligand binding 32,33 and another that is tightly bound and generally retained. Water "300", found between the two catalytic aspartyls, 34 is an easy target for displacement, as are a handful of other water (300), five structural waters (301, 313, 313′, 313bis, and 313bis′), and a number of (hot) waters appearing to simply occupy the active site (a−g) were located by X-ray crystallography. ...
... More than a decade ago, we explored the energetics of six such inhibitors as well as 17 of the acyclics by calculating their HINT binding scores with respect to HIV-1 protease. 33 The overall scaled 193 protein−ligand HINT scores for the acyclic inhibitors averaged to −6.6 ± 1.1 kcal mol −1 , and, indeed, the averaged HINT scores for the six members of the cyclic set averaged to −8.1 ± 1.5 kcal mol −1 , which seems to confirm the rationale for water displacement. However, it must be considered that the former compounds also have a significant interaction with water 301, which averaged to −2.1 ± 0.5 kcal mol −1 . ...
... We also have applied HINT scoring, wherein the water is treated as a ligand and all surrounding atoms as a pseudoreceptor to evaluate water placement and orientation ( Figure 9J). We have previously reviewed HINT, and its numerous applications in biomolecular structure and drug discovery/design scenarios, 33,190,225,234,262 so only the important operation details will be described here. The HINT score (H TOTAL ) provides an empirical, but quantitative, evaluation of a molecular interaction as a sum of all individual atom−atom interactions using ...
The value of thoroughly understanding the thermodynamics specific to a drug discovery/design study is well known. Over the past decade, the crucial roles of water molecules in protein structure, function, and dynamics have also become increasingly appreciated. This Perspective explores water in the biological environment by adopting its point of view in such phenomena. The prevailing thermodynamic models of the past, where water was seen largely in terms of an entropic gain after its displacement by a ligand, are now known to be much too simplistic. We adopt a set of terminology that describes water molecules as being "hot" and "cold", which we have defined as being easy and difficult to displace, respectively. The basis of these designations, which involve both enthalpic and entropic water contributions, are explored in several classes of biomolecules and structural motifs. The hallmarks for characterizing water molecules are examined, and computational tools for evaluating water-centric thermodynamics are reviewed. This Perspective's summary features guidelines for exploiting water molecules in drug discovery.
... The K m values indicate the enzymes affinity for substrates. Thus, the K m s have been chosen among the various biochemical parameters for the comparison with the computed scores since the HINT scoring may correlate the pocket-ligand affinity proportionally [6,[21][22][23][24][25]. ABTS showed a clearly defined rank of K m s among the various isoforms, with the following order: 88 µM in beta < 359 µM in gamma < 2262 µM in delta [26]. ...
... Software setting and rescoring procedures reported by Ehrlich were used [47]. In more detail, HINT score provides the evaluation of thermodynamic benefits of protein-ligand interaction, and relates with the ∆G • of complex formation [22,24,25]. Specifically, the empirical HINT scoring function implicitly considers enthalpic and entropic aspects of protein-ligand interaction using experimental Log Po/w measurements (partition coefficient for 1-octanol/water) as the basis of its force field. ...
... The HINT score is the sum of the all inter-atomic contributions from binding, thereby providing an empirical and quantitative estimate of the favors of the host-guest interaction from an atomic point of view. Thus, the higher the score, the more favored is the arrangement of ligands within the binding site [22,24,25,48]. The HINT equation is the following: ...
Mycotoxins are secondary metabolites of fungi that contaminate food and feed, and are involved in a series of foodborne illnesses and disorders in humans and animals. The mitigation of mycotoxin content via enzymatic degradation is a strategy to ensure safer food and feed, and to address the forthcoming issues in view of the global trade and sustainability. Nevertheless, the search for active enzymes is still challenging and time-consuming. The in silico analysis may strongly support the research by providing the evidence-based hierarchization of enzymes for a rational design of more effective experimental trials. The present work dealt with the degradation of aflatoxin B1 and M1 by laccase enzymes from Trametes versicolor. The enzymes–substrate interaction for various enzyme isoforms was investigated through 3D molecular modeling techniques. Structural differences among the isoforms have been pinpointed, which may cause different patterns of interaction between aflatoxin B1 and M1. The possible formation of different products of degradation can be argued accordingly. Moreover, the laccase gamma isoform was identified as the most suitable for protein engineering aimed at ameliorating the substrate specificity. Overall, 3D modeling proved to be an effective analytical tool to assess the enzyme–substrate interaction and provided a solid foothold for supporting the search of degrading enzyme at the early stage.
... Early stage inhibitors were designed to provide appropriate hydrogen bond acceptors for the two protons of the water – a task made easier by the inherent symmetry of the site. However, in one of the triumphs of structure-based drug discovery, ligands that were designed to displace this water [22] [23] were even more tight binders, where the additional free energy was hypothesized to arise from entropy [14], i.e., generated by its release to the bulk solvent, but Fornabaio et al. suggested that it was more of an enthalpic/desolvation effect [24]. ...
... There are a few other water molecules buried within the ligand binding site that are not always detected by crystallographic analysis: Wat313 (and its symmetrical Wat313') and Wat313bis (and its symmetrical Wat313bis') being the most conserved. The interaction energies of twelve waters, observed with varying degrees of conservation in the HIV-1 protease active site, were calculated by Fornabaio et al. [24] using the HINT program (vide infra). It turns out that, while the value of understanding and accounting for the contribution of " important " water molecules in biological computations is obvious, the identification of these waters is not always so obvious or easy: it is not simply a matter of proximity. ...
... Since it is impossible to obtain all (or usually any) hydrogen positions from X-ray analyses, and the pH at which crystals are grown are generally chosen for the quality of the resulting crystals for diffraction rather than their biological relevance, experiment does not reveal the ionization states of the acidic or basic sidechains (or ligand functional groups). In general, when hydrogens are considered, the assumption is made that all residues are protonated as at pH 7. In our work [24], the ionization states of the two catalytic aspartates (25 and 25) were examined and modeled based on the reports of Smith [26] and Wang [27], who had earlier shown with NMR that only one of these aspartates can be protonated in the pH range 2.5 – 6.5. In one complex, where the Glu-Asp-Leu peptide was liganded to HIV-1 protease in solution at pH values between 3.0 and 5.0, Louis et al. [28] demonstrated pHdependent binding as protonation states of the two protein aspartates and in the peptide were accessed. ...
... The binding energy calculated through MM-GBSA OPLS-2005 was much accurate than the XP Gscore [44]. The binding energies were calculated by the following equations [45,46]. ...
... G BSA continuum model was used to carry out the simulations by using Prime v4.1 [47,48]. For better representation, Gaussian surface area model was employed instead of vdW surface area model [46]. ...
An endless drug-resistant strains of Helicobacter pylori and multitudinous drug reactions are obstacles in the treatment of H. pylori infections, thereby ambitious novel proof-of-concept for inhibitor design was practiced in advancement of medication. Dihydropteroate synthase (DHPS) is an alluring target that plays a great role in folate synthesis pathway essential for amino acids biosynthesis was selected for designing novel drugs to prevent infections caused by pathogenic H. pylori. In the present study, a reliable tertiary structure of DHPS in complex with inhibitor 6MB was constructed by Modeler 9v19. DrugBank compounds of DHPS, published inhibitors, and co-crystal ligand (6MB) were docked against DHPS. The best docked compounds were screened against 28.5 million compounds resulted 1186 structural analogs. Virtual screening workflow and quantum polarized ligand dockings of these compounds against DHPS resulted three leads that showed better XP Gscores, ADME properties, and binding-free energies compared to 6MB, DrugBank compounds, and published inhibitors. The proposed leads were also validated by receiver operative characteristic (ROC) curve metrics in the presence of thousand decoys and the best docked existing compounds against DHPS. Long-range molecular dynamics (MD) simulations for 100 ns were executed after post-docking evaluations. Trajectory analysis showed the lead–DHPS docking complex’s inter-molecular interactions were stable throughout the entire runtime of MD simulations than 6MB–DHPS complex and Eliglustat–DHPS complex. The study outcomes showed good competitive binding propensity and active-tunneling of leads over the existing inhibitors, thereby these leads could be ideal inhibitors against DHPS to target H. pylori.
... With the aim to model a potential endocrine disrupting activity, the interaction between chemicals under investigation (i.e., phthalates, bisphenols, parabens) and some NRs (i.e., ER , ER , ARwt and ARmut) was evaluated by using the coupling of docking simulations and proper rescoring procedures. Specifically, the coupling of GOLD, as docking software, and HINT (Hydropathic INTeraction) [19], as rescoring function, was chosen on the basis of previous studies demonstrating the higher reliability of HINT in respect to other scoring functions and the efficacy as re-scoring function to predict ligands interaction with several protein targets [20][21][22][23][24][25], including estrogen [26,27] and androgen receptors [28]. The HINT scores provide empirical and quantitative evaluation of protein-ligand interaction, as a sum of all single atomic contributions. ...
... where bij is the interaction score between atoms i and j, a is the hydrophobic atomic constant, S represents the solvent accessible surface area, Tij is a logic function assuming +1 or −1 values, depending on the nature of the interatomic interaction, Rij and rij are functions of the distance between atoms i and j. Positive and high HS correlates with favorable binding free energy, thus allowing evaluation of the thermodynamic benefit of predicted complexes [20,[22][23][24]. We termed this procedure "rescoring" because GOLD results are intrinsically ranked by its internal scoring functions. ...
... The more rotational entropy the water loses due to charge-charge interactions, the harder it is to displace with ligand groups. We observed a similar phenomenon when examining the water molecules of HIV-1 protease in 2004 [42]-while displacing water 301 did gain binding energy for the cyclic ligands [32,33]-that gain appeared to be largely attributable to the binding energy of the water; that is, these ligands made the same interactions that the water did. ...
... Second, the water/polar network is responsive to changes in ionization state: any neighboring water molecules need to be optimized for each of the models conceived during a CT run. Third, while the protocol we described here and elsewhere [42,[61][62][63] is based on the HINT force field, the concepts and strategy could be simply translated to another paradigm. ...
The importance of water molecules in biological interactions is not debatable, but the various diverse and specific roles that water can play are not as well understood on a molecular scale. In this methods report, the theoretical basis for a computational framework that focuses on water is described. The framework is HINT (for Hydropathic INTeractions) and is a related series of algorithms and methods for probing and modeling the hydrophobic effect, solvation and desolvation, ionization of acids and bases, and tautomerism. HINT is derived from the experimental measurement of the partition coefficient for solute transfer between water and 1-octanol, stylized as log P
o/w, which is a free energy term. Discussion of computational approaches to quantitating the hydrophobic effect, scoring biological associations with a free energy force field, evaluating the conservation of water molecules in complexes, modeling ionization state ensembles in complex environments, enumerating putative small-molecule tautomers in complexes, and predicting the location of important bridging waters are provided. These factors are summarized in terms of their potential effects on drug discovery projects.
... This analysis was carried out without the contribution of bound water molecules to the binding energy. A successive analysis considered such contributions (see Section 4.1) Fornabaio et al., 2004;. To demonstrate that HINT was able to correctly predict the interaction energy between protein and ligands, independently of ligand and protein active site polarity, the selected protein's active sites vary from very apolar, such as retinolbinding proteins, where the hydrophobic contribution and, consequently, the entropic contribution to the binding energy is predominant, to polar, such as penicillopepsin, where the free energy of interaction is mainly associated with contributions from Coulombic interaction between polar or ionizable residues. ...
A long-lasting goal of computational biochemists, medicinal chemists, and structural biologists has been the development of tools capable of deciphering the molecule–molecule interaction code that produces a rich variety of complex biomolecular assemblies comprised of the many different simple and biological molecules of life: water, small metabolites, cofactors, substrates, proteins, DNAs, and RNAs. Software applications that can mimic the interactions amongst all of these species, taking account of the laws of thermodynamics, would help gain information for understanding qualitatively and quantitatively key determinants contributing to the energetics of the bimolecular recognition process. This, in turn, would allow the design of novel compounds that might bind at the intermolecular interface by either preventing or reinforcing the recognition. HINT, hydropathic interaction, was a model and software code developed from a deceptively simple idea of Donald Abraham with the close collaboration with Glen Kellogg at Virginia Commonwealth University. HINT is based on a function that scores atom–atom interaction using LogP, the partition coefficient of any molecule between two phases; here, the solvents are water that mimics the cytoplasm milieu and octanol that mimics the protein internal hydropathic environment. This review summarizes the results of the extensive and successful collaboration between Abraham and Kellogg at VCU and the group at the University of Parma for testing HINT in a variety of different biomolecular interactions, from proteins with ligands to proteins with DNA.
... 46−48 The two important structural water molecules traced in the HIV PR active site that play a crucial role in protease activity are the catalytic water and the flap water. 48 The catalytic water is buried in the active site and forms a bridge between the catalytic aspartates, Asp25-Asp25′, and the incoming substrate ( Figure 3a). 43,49 It then acts as a nucleophile for hydrolyzing the substrate, as shown from the QM/MM studies. ...
Since the emergence of the Human Immunodeficiency Virus (HIV) in the 1980s, strategies to combat HIV-AIDS are continuously evolving. Among the many tested targets to tackle this virus, its protease enzyme (PR) was proven to be an attractive option that brought about numerous research publications and ten FDA-approved drugs to inhibit the PR activity. However, the drug-induced mutations in the enzyme made these small molecule inhibitors ineffective with prolonged usage. The research on HIV PR, therefore, remains a thrust area even today. Through this review, we reiterate the importance of understanding the various structural and functional components of HIV PR in redesigning the structure-based small molecule inhibitors. We also discuss at length the currently available FDA-approved drugs and how these drug molecules induced mutations in the enzyme structure. We then recapitulate the reported mechanisms on how these drug-resistant variants remain sufficiently active to cleave the natural substrates. We end with the future scope covering the recently proposed strategies that show promise to deal with the mutations.
... HINT and expanded its applications to 3D QSAR (Comparative Molecular Field Analysis, CoMFA) [74] , as a scoring function for molecular modeling activities such as docking [75][76][77] and virtual screening [78][79][80] , and in protein structure analysis and prediction ( vide infra ). Kellogg has developed a suite of computational tools for examining the roles of water in liganded and unliganded protein structures [81][82][83] evaluating the ionization states of residues and ligand functional groups [ 84 , 85 ], and have rationalized "hydrophobic interactions " as 3D properties [ 86 , 87 ]. ...
The Department of Medicinal Chemistry, together with the Institute for Structural Biology, Drug Discovery and Development, at Virginia Commonwealth University (VCU) has evolved, organically with quite a bit of bootstrapping, into a unique drug discovery ecosystem in response to the environment and culture of the university and the wider research enterprise. Each faculty member that joined the department and/or institute added a layer of expertise, technology and most importantly, innovation, that fertilized numerous collaborations within the University and with outside partners. Despite moderate institutional support with respect to a typical drug discovery enterprise, the VCU drug discovery ecosystem has built and maintained an impressive array of facilities and instrumentation for drug synthesis, drug characterization, biomolecular structural analysis and biophysical analysis, and pharmacological studies. Altogether, this ecosystem has had major impacts on numerous therapeutic areas, such as neurology, psychiatry, drugs of abuse, cancer, sickle cell disease, coagulopathy, inflammation, aging disorders and others. Novel tools and strategies for drug discovery, design and development have been developed at VCU in the last five decades; e.g., fundamental rational structure-activity relationship (SAR)-based drug design, structure-based drug design, orthosteric and allosteric drug design, design of multi-functional agents towards polypharmacy outcomes, principles on designing glycosaminoglycans as drugs, and computational tools and algorithms for quantitative SAR (QSAR) and understanding the roles of water and the hydrophobic effect.
... Numerous methods are designed for predicting the location of water molecules, and significant improvements have been reported [116][117][118][119][120][121][122]. Although there are some degrees of skepticism and reluctance towards utilizing such information within a drug discovery program as reviewed by Bodnarchuk [121], the situation may change rapidly. ...
It is frequently mentioned that QSARs have not generally lived up to expectations, especially in cases where high predictability is expected yet failed to deliver satisfactory results. Even though outliers can provide an increased opportunity in drug discovery research, outliers in SAR and QSAR can contort predictions and affect the accuracy if proper attention is not given. The percentages of outliers in QSARs have not changed appreciably over the last decade. In our previous studies, we suggested two possible sources of outliers in SAR and QSAR. In this paper, we suggest an additional possible source of outliers in QSAR. We presented several literature examples that show one or more water molecules that play a critical role in protein–ligand binding interactions as observed in their crystal structures. These examples illustrate that failing to account for the effects of water molecules in protein–ligand interactions could mislead interpretation and possibly yield outliers in SAR and QSAR. Examples include cases where QSAR, considering the role of water molecules in protein–ligand crystal structures, provided deeper insight into the understanding and interpretation of the developed QSAR.
... The results of VS showed a minimum FEB ranging from −4.3 to −9.5 kcal/mol. A protein-ligand complex with lowest FEB is considered as potential inhibitor (Fornabaio et al., 2004). Consequently, the four compounds that exhibited the lowest FEB were selected as the potential candidates. ...
The rapid outbreak of Coronavirus Disease 2019 (COVID-19) that was first identified in Wuhan, China is caused by a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The 3CL protease (3CLpro) is the main protease of the SARS-CoV-2, which is responsible for the viral replication and therefore considered as an attractive drug target since to date there is no specific and effective vaccine available against this virus. In this paper, we reported molecular docking-based virtual screening (VS) of 2000 compounds obtained from the ZINC database and 10 FDA-approved (antiviral and anti-malaria) on 3CLpro using AutoDock Vina to find potential inhibitors. The screening results showed that the top four compounds, namely ZINC32960814, ZINC12006217, ZINC03231196, and ZINC33173588 exhibited high affinity at the 3CLpro binding pocket. Their free energy of binding (FEB) were −12.3, −11.9, −11.7, and −11.2 kcal/mol while AutoDock Vina scores were −12.61, −12.32, −12.01, and -11.92 kcal/mol, respectively. These results were better than the co-crystallized ligand N3, whereby its FEB was −7.5 kcal/mol and FDA-approved drugs. Different but stable interactions were obtained between the four identified compounds with the catalytic dyad residues of the 3CLpro. In conclusion, novel 3CLpro inhibitors from the ZINC database were successfully identified using VS and molecular docking approach, fulfilling the Lipinski rule of five, and having low FEB and functional molecular interactions with the target protein. The findings suggests that the identified compounds may serve as potential leads that act as COVID-19 3CLpro inhibitors, worthy for further evaluation and development.
... Careful investigation of water molecules and their energetics can significantly contribute to a successful drug design campaign 103 ; there are many well-known examples, such as nonpeptidic urea inhibitors of HIV protease. 104 Structural biology and computational chemistry are useful to exploit water-binding pockets in structure-based design. 105,106 Displacing a water molecule that mediates protein-ligand binding by adding an appropriate moiety into the ligand to generate an isostere of the ligand-water complex can result in remarkable improvements in activity by establishing new hydrogen bond interactions directly between the protein and binder. ...
Solvent‐exposed regions, or solvent‐filled pockets, within or adjacent to the ligand‐binding sites of drug‐target proteins provide opportunities for substantial modifications of existing small‐molecular drug molecules without serious loss of activity. In this review, we present recent selected examples of exploitation of solvent‐exposed regions of proteins in drug design and development from the recent medicinal‐chemistry literature.
... In the docking study, solvent effect is not considered; however, many studies have shown that the water molecules increase the specificity range of the binding site [44,45]. In the present work, MD simulation was performed in order to further investigate the binding mode and the conformation change of Eg5inhibitor complexes in the presence of solvent. ...
Kinesin Eg5 plays an essential role in the early stages of mitosis, and it is an interesting drug target for the design of potent inhibitors. In this work, combined molecular modeling studies of molecular docking, receptor-guided QSAR methodology, and molecular dynamics (MD) simulation were performed on a series of novel S-trityl-l-cysteine (STLC) analogues as Eg5 inhibitors to understand the structural features and key residues which are involved in the inhibition. Molecular docking study was used to find the actual conformations of STLC analogues in the binding site of Eg5. Multiple linear regression (MLR), artificial neural network (ANN), and support vector machine (SVM) models were developed by the conformation which was obtained by performing docking studies. The satisfactory result of the SVM model (R2 = 0.962, SE = 0.210, RMSE = 0.190, and Q2LOO = 0.930) demonstrated the superiority of this model over other models. Also, the satisfactory agreement between experiment and predicted inhibitory values suggested that the SVM model represents good correlation and predictive power. Molecular docking was used to study the functionalities of active molecular interaction between inhibitors and Eg5. Moreover, molecular dynamics (MD) simulation was performed on the best inhibitor-Eg5 complex to investigate the stability of docked conformation and to study the binding interactions in detail. The MD simulation result showed four hydrogen bond interactions with Eg5 residues including Gly117, Glu116, Gly117, and Glu118. The outcome of this study can be used as a guideline to better interpret the protein-ligand interaction and also can assist in the designing and development of more potent Eg5 inhibitors.
... Nonetheless, well-acknowledged successes related to the HIV-1 protease were accomplished using the information of conserved water molecules experimentally determined at the binding site. [15][16][17][18][19][20] Thus, it is reasonable to consider that the same logic might apply to other systems. Multiple methods have been developed using this philosophy to estimate conserved water positions using information available in public databases (e.g. ...
Background:
The inclusion of direct effects mediated by water during the ligand-receptor recognition is a hot-topic of modern computational chemistry applied to drug discovery and development. Docking or virtual screening with explicit hydration is still debatable, despite the successful cases that have been presented in the last years. Indeed, how to select the water molecules that will be included in the docking process or how the included waters should be treated remain open questions.
Objective:
In this review, we will discuss some of the most recent methods that can be used in computational drug discovery and drug development when the effect of a single water, or of a small network of interacting waters, needs to be explicitly considered.
Results:
Here, we analyse software to aid the selection, or to predict the position, of water molecules that are going to be explicitly considered in later docking studies. We also present software and protocols able to efficiently treat flexible water molecules during docking, including examples of applications. Finally, we discuss methods based on molecular dynamics simulations that can be used to integrate docking studies or to reliably and efficiently compute binding energies of ligands in presence of interfacial or bridging water molecules.
Conclusions:
Software applications aiding the design of new drugs that exploit water molecules, either as displaceable residues or as bridges to the receptor, are constantly being developed. Although further validation is needed, workflows that explicitly consider water will probably become a standard for computational drug discovery soon.
... For example, waters can bridge protein and ligand and license what would otherwise represent unfavorable interactions between two chemically incompatible groups (e.g. two bases). Water molecules can also alter the "shape" and microenvironment of the active site by tightly associating with specific residues and thereby present a steric and electrostatic binding pocket profile that is different to the one presented by an anhydrous active site [23,24]. These varied functional involvements of water define yet another set of important considerations that must be respected in quality docking experiments and in rational design of high affinity lead molecules. ...
Accurate modeling of protein ligand binding is an important step in structure-based drug design, is a useful starting point for finding new lead compounds or drug candidates. The 'Lock and Key' concept of protein-ligand binding has dominated descriptions of these interactions, and has been effectively translated to computational molecular docking approaches. In turn, molecular docking can reveal key elements in protein-ligand interactions-thereby enabling design of potent small molecule inhibitors directed against specific targets. However, accurate predictions of binding pose and energetic remain challenging problems. The last decade has witnessed more sophisticated molecular docking approaches to modeling protein-ligand binding and energetics. However, the complexities that confront accurate modeling of binding phenomena remain formidable. Subtle recognition and discrimination patterns governed by three-dimensional features and microenvironments of the active site play vital roles in consolidating the key intermolecular interactions that mediates ligand binding. Herein, we briefly review contemporary approaches and suggest that future approaches treat protein-ligand docking problems in the context of a 'combination lock' system.
... Following the general procedure for amide synthesis between 5 and pentylamine, compound 19 was obtained as white solid (yield 82%, mp 301−303°C). 1 (E)-3-(4-(((1r,3r,5R,7S)-Adamantan-2-ylidene)(4-hydroxyphenyl)methyl)phenyl)-N-(2-hydroxyethyl)acrylamide (20). Following the general procedure for amide synthesis between 5 and ethanolamine, compound 20 was obtained as white solid (yield 76%, mp 259°C). 1 13 (E)-3-(4-(((1r,3r,5R,7S)-Adamantan-2-ylidene)(4-hydroxyphenyl)methyl)phenyl)-N-(3-hydroxypropyl)acrylamide (21). Following the general procedure for amide synthesis between 5 and 3-amino-1propanol, compound 21 was obtained as white solid (yield 74%, mp 155°C). 1 13 (E)-3-(4-(((1r,3r,5R,7S)-Adamantan-2-ylidene)(4-hydroxyphenyl)methyl)phenyl)-N-(4-hydroxybutyl)acrylamide (22). ...
To search for new antiestrogens more effective in treating breast cancers, we explored alternatives to the acrylic acid side chain used in many antiestrogens. To facilitate our search, we used a simple adamantyl ligand core that by avoiding stereochemical issues enabled rapid synthesis of acrylate ketone, ester, and amide analogs. All compounds were high affinity estrogen receptor-alpha (ERα) ligands, but displayed a range of efficacies and potencies as antiproliferative and ERα-downregulating agents. There were large differences in activity between compounds having minor structural changes, but antiproliferative and ERα-downregulating efficacies generally paralleled one another. Some compounds with side chain polar groups had particularly high affinities. The secondary carboxamides had the best cellular activities, and the 3-hydroxypropylamide was as efficacious as fulvestrant in suppressing cell proliferation and gene expression. This study has produced structurally novel antiestrogens based on a simple adamantyl core structure with acrylate side chains optimized for cellular antagonist activity.
... The software HINT (Hydrophatic INTeraction) [22] was used as the re-scoring function on the basis of previous studies attesting the higher reliability of HINT scoring with respect to other scoring functions, and its successful use in the search for ligands for other targets, as well as in the estimation of ligand binding free energies. More in details, the score provides the evaluation of thermodynamic benefits of protein-ligand interaction, and therefore low/negative scores indicate not appreciable protein-ligand interactions [13][14][15][23][24][25][26][27]. GOLD uses a Lamarckian genetic algorithm and scores may slightly change from run to run. ...
The term Ergot is referred to the protective kernel (i.e. sclerotium) produced during resting stage of
ascomycetes belonging to Claviceps genus that replaces seeds of susceptible cereals and plants
intended for human and animal diet. It contains various composition of tryptophan-derived toxins
defined ergot alkaloids (EAs). Since sclerotia can be harvested and milled together with cereals,
EAs represent a source of food and feed contamination after breakage and spreading of mycotoxins
into the various milling fractions. The effects of EAs have been known since the Middle Ages and,
currently, it has been recognized the occurrence of gangrenous and convulsive ergotism. Nowadays,
it is known that EAs action is manly mediated by the interaction with -adrenergic, dopaminergic
and serotonergic receptor classes. In spite of the wealth of studies on synthetic compounds and
drugs, further data are needed on metabolism and target receptors-binding of common EAs in food,
as recently stated also by the European Food Safety Authority.
Unfortunately, the systematic in-depth analysis of interaction between the entire spectrum of
metabolites and the array of targets is hardly achievable through the unbiased experimental analysis.
The use of in silico techniques may be an effective choice to smartly drive the upstream selection of
strong candidates for more detailed workbench investigations. Focusing on ergotamine as the case
study, the present work was aimed at assessing whether an in silico approach based on docking
simulations and re-scoring procedures can be an effective tool to investigate the interaction between
multiple serotonin receptors and a wide set of ergotamine metabolites. In the effort to profile the
overall effect of human metabolism on ergotamine action all the metabolites identified so far were
collected from the literature (i.e. 10 molecules). Most likely, they do not complete the truly
circulating array of metabolites, thus, with the end to assess more widely the effect of chemical
modification by human metabolism, a series of metabolites has been computed (i.e. 22 molecules).
Starting from crystallographic structure of serotonin receptor 2B (5HT-2B), 5HT-2A and 5HT-2Cmodels were derived by using homology modeling. All the three models underwent validating
procedure and then the capability of both sets of metabolites to interact with the validated binding
sites was computed.
It is worthy to note that the most of metabolites were predicted as able to interact with targets
(notably, glycosylated compounds were not included), albeit they showed a certain degree of
receptor-specificity. Thus, the need for more detailed investigation has been ultimately suggested to
verify the possible retaining of activity for the most of phase-I metabolites. Furthermore, in silico
modeling showed to be a powerful tool for the hierarchical prioritization of molecules and receptors
in the view of supporting the rational design of future workbench experiments.
... The coupling of GOLD (Genetic Optimization for Ligand Docking ), as docking software, and HINT (Hydropathic INTeraction) (Kellog and Abraham, 2000), as rescoring function, was chosen on the basis of previous studies demonstrating the higher reliability of HINT with respect to other scoring functions in estimating the ligand binding free energies and evaluating the bioactivity of small molecules (Dellafiora et al., 2014aDellafiora et al., ,b, 2015aDellafiora et al., ,b, 2013). More in details, HINT score provides the evaluation of thermodynamic benefits of protein-ligand complex formation, and therefore low/negative scores indicate the lack of appreciable protein-ligand interactions (Kellogg and Abraham, 2000; Cozzini et al., 2002; Fornabaio et al., 2003 Fornabaio et al., , 2004). Software setting and rescoring procedures reported by Ehrlich (Ehrlich et al., 2015) were used. ...
... The magnitude and systematic character of these deviations could result from errors in the force field's representation of specific interactions present in the host-guest systems, perhaps amplified by the greater size of these host-guest systems relative to the small molecules in the hydration study. It is also worth noting, however, that neither small molecule hydration data nor the properties of neat liquids probe how accurately water models treat confined water, which is present in the binding sites of host molecules and proteins, and is thought to significantly influence binding thermodynamics [41][42][43][44] . ...
Improving the capability of atomistic computer models to predict the thermodynamics of noncovalent binding is critical for successful structure-based drug design, and the accuracy of such calculations remains limited by non-optimal force field parameters. Ideally, one would incorporate protein-ligand affinity data into force field parametrization, but this would be inefficient and costly. We now demonstrate that sensitivity analysis can be used to efficiently tune Lennard-Jones parameters of aqueous host-guest systems for increasingly accurate calculations of binding enthalpy. These results highlight the promise of a comprehensive use of calorimetric host-guest binding data, along with existing validation data sets, to improve force field parameters for the simulation of noncovalent binding, with the ultimate goal of making protein-ligand modeling more accurate and hence speeding drug discovery.
... (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) binding free energy; thus HINT allows the evaluation of overall thermodynamic benefits of complex formation (Cozzini et al., 2002; Fornabaio, Cozzini, Mozzarelli, Abraham, & Kellogg, 2003; Fornabaio et al., 2004; Marabotti et al., 2008). ...
... Asp 25 , Asp 125 , Ile 50 and Ile 150 in addition to water 300 and water 301 are the main components of the active site of the homodimeric HIV-1 protease. Water 301 is located on the interfacial symmetry axis and is conserved with longer residence time (Wang et al., 1996) and Ile 50 and Ile 150 donate two H-bonds to inhibitor, Kynostatin 272 (KNI-272), and accept two H-bonds from the backbone residues of HIV-1 protease (Baldwin et al., 1995;Fornabaio et al., 2004). Interactions of DMP450 to HIV-1 protease and substrate to Cytochrome P450cam lead to displacement of water molecule and this displacement thermodynamically favours for this inhibitor binding (Li & Lazaridis, 2003;Poulos, Finzel, & Howard, 1986). ...
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... High HS (best interaction) are associated with low-free-energy levels for protein−ligand complexes. 24,25 The HINT model describes specific atom−atom interactions between two molecules using the equation ...
Thioxanthones and related chemicals are widely used in food packaging as photoinitiator/sensitizers during UV-curing polymerization ink procedures. Despite their good chemical properties, some cases of food contamination were occurred, especially in infant formulae, as reported by EFSA in 2005. Contamination was also occurred in fruits juices, fruit nectars and drinks. ITX lipophilicity suggests rapid hepatic detoxification phenomena but, to date, the few available toxicological information on androgen receptors concern only the synthetic and commercially available 2 and 4-ITX, 2,4-diethyl-TX, 2Cl-TX and 1Chloro-4-propoxy-TX. A more exhaustive and complete definition of the toxicological profile of TX compounds should include also their metabolic panel. In this perspective, a preliminary in silico method to qualitatively provide the binding affinity of all known 2-ITX metabolites for AR ligand-binding pocket (LBP) was developed and compared to the endogenous ligand testosterone. Whether these chemicals proved to be putative ligands for this nuclear receptor, they should undergo further wet investigations, in vitro or/and in vivo. As shown in our previous works, the applied molecular docking procedure has proved to be able to reproduce the experimental data available today for TXs. On the light of this result, we report a preliminary evaluation of endocrine disrupting hazard for ten experimental 2-ITX metabolites and thirteen computationally predicted TX metabolites. Joined to classical in vitro tests, molecular docking methodologies could allow to rapidly testing both the chemical precursor and its metabolic panel, when available, for a more concrete risk assessment.
... After individual inspection, molecules with at least one hydrogen bond acceptor group and a hydrophobic moiety were selected, docked into the binding site of OASS-A with GOLD, rescored with the HINT force field and again individually inspected. A HINT score value of 3000 was chosen as threshold, indicative of an energetically stable complex [111]. On the basis of: i) the generated conformations, ii) the interactions with the surrounding residues and iii) the HINT score value, seven compounds were selected for purchase and assays (Table 1 and Figures 4-5). ...
The last step of cysteine biosynthesis in bacteria and plants is catalyzed by O-acetylserine sulfhydrylase. In bacteria, two isozymes, O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, have been identified that share similar binding sites, although the respective specific functions are still debated. O-acetylserine sulfhydrylase plays a key role in the adaptation of bacteria to the host environment, in the defense mechanisms to oxidative stress and in antibiotic resistance. Because mammals synthesize cysteine from methionine and lack O-acetylserine sulfhydrylase, the enzyme is a potential target for antimicrobials. With this aim, we first identified potential inhibitors of the two isozymes via a ligand- and structure-based in silico screening of a subset of the ZINC library using FLAP. The binding affinities of the most promising candidates were measured in vitro on purified O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B from Salmonella typhimurium by a direct method that exploits the change in the cofactor fluorescence. Two molecules were identified with dissociation constants of 3.7 and 33 µM for O-acetylserine sulfhydrylase-A and O-acetylserine sulfhydrylase-B, respectively. Because GRID analysis of the two isoenzymes indicates the presence of a few common pharmacophoric features, cross binding titrations were carried out. It was found that the best binder for O-acetylserine sulfhydrylase-B exhibits a dissociation constant of 29 µM for O-acetylserine sulfhydrylase-A, thus displaying a limited selectivity, whereas the best binder for O-acetylserine sulfhydrylase-A exhibits a dissociation constant of 50 µM for O-acetylserine sulfhydrylase-B and is thus 8-fold selective towards the former isozyme. Therefore, isoform-specific and isoform-independent ligands allow to either selectively target the isozyme that predominantly supports bacteria during infection and long-term survival or to completely block bacterial cysteine biosynthesis.
Since its introduction about four decades ago, docking and scoring are now the very heart of structure‐based drug design. The past 10–20 years have seen a plethora of docking and scoring tools successfully integrated into drug discovery pipelines. Interestingly, while artificial intelligence is now receiving significant attention in the computational modeling arena, docking and scoring have long utilized these techniques. This comprehensive summary highlights some of the most significant achievements of docking and scoring, reviews the current status, provides descriptions of many of the tools available, comments on some of the outstanding challenges facing the paradigm, and offers perspectives and advice on best practices for users. While significant development is still needed in docking of flexible molecules and accurate Gibb's free energy predictions, docking and scoring are very useful when handled by experienced practitioners, but less so if treated as a “black box.” Lastly, we present a hypothetical case so beginners may appreciate the nuances of setting up a docking study. Focus in docking is now shifting toward parallel applications, i.e. protein–protein, protein–oligosaccharide, protein–DNA, or protein–RNA docking and polypharmacology. In summary, this article is intended to elucidate the nuances of the subject, while providing guidelines for practical implementation of effective workflows in drug discovery and structural biology.
Water molecules are common and usually ignored entity in a protein assembly, however, they play a very critical role in the cellular systems and ligand-protein binding. Both the structure and the thermodynamics of active site waters significantly influence the binding process between the protein and ligands. Structurally conserved waters cannot always be determined experimentally and if observed, it is not clear if they will be replaced upon ligand binding, even if sufficient space is available. Several methods to predict the presence of water in protein-ligand complexes have been disclosed, which are now included in computational drug research. Either as an additional factor in automated docking experiments, or explicitly in detailed molecular dynamics simulations, the effect of water on the quality of the simulations is significant, but not easily predicted. The most detailed calculations involve estimates of the free energy contribution of water molecules to protein-ligand complexes. These calculations are computationally demanding, but give insight into the versatility and importance of water in ligand binding. This chapter will discuss the role of water and its influence on computational structure-based drug design approaches. In particular, we will focus on the structural and thermodynamic properties of water into active sites. There are several well-studied examples of protein-ligand complexes in which water molecules seem to play crucial roles. Special attention should be given to computational methods to first predict the presence of water molecules in protein-ligand complexes and subsequently to include such molecules in existing computational approaches
Molecular modeling and simulation play a central role in academic and industrial research focused on physico-chemical properties and processes. The efforts carried out in this field have crystallized in a variety of models, simulation methods, and computational techniques that are examining the relationship between the structure, dynamics and functional role of biomolecules and their interactions. In particular, there has been a huge advance in the understanding of the molecular determinants that mediate the interaction between small compounds acting as ligands and their macromolecular targets. This book provides an updated description of the advances experienced in recent years in the field of molecular modeling and simulation of biomolecular recognition, with particular emphasis towards the development of efficient strategies in structure-based drug design.
Introducing novel strategies, concepts and technologies that speed up drug discovery and the drug development cycle is of great importance both in the highly competitive pharmaceutical industry as well as in academia. This review aims to present a "big-picture" overview of recent strategic innovations in medicinal chemistry and drug discovery.
Currently, molecular docking is becoming a key tool in drug discovery and molecular modeling applications. The reliability of molecular docking depends on the accuracy of the adopted scoring function, which can guide and determine the ligand poses when thousands of possible poses of ligand are generated. The scoring function can be used to determine the binding mode and site of a ligand, predict binding affinity and identify the potential drug leads for a given protein target. Despite intensive research over the years, accurate and rapid prediction of protein–ligand interactions is still a challenge in molecular docking. For this reason, this study reviews four basic types of scoring functions, physics-based, empirical, knowledge-based, and machine learning-based scoring functions, based on an up-to-date classification scheme. We not only discuss the foundations of the four types scoring functions, suitable application areas and shortcomings, but also discuss challenges and potential future study directions.
The pharmacophore concept is a fundamental cornerstone in drug discovery, playing a critical role in determining the success of in silico techniques, such as virtual screening and 3D-QSAR studies. The reliability of these approaches is influenced by the quality of the physicochemical descriptors used to characterize the chemical entities. In this context, a pivotal role is exerted by lipophilicity, which is a major contribution to host-guest interaction and ligand binding affinity. Several approaches have been undertaken to account for the descriptive and predictive capabilities of lipophilicity in 3D-QSAR modeling. Recent efforts encode the use of quantum mechanical-based descriptors derived from continuum solvation models, which open novel avenues for gaining insight into structure-activity relationships studies.
Molecular alignment is a standard procedure for 3D similarity measurements and pharmacophore elucidation. This process is influenced by several factors, such as the physico-chemical descriptors utilized to account for the molecular determinants of biological activity and the reference templates. Relying on the hypothesis that the maximal achievable binding affinity for a drug-like molecule is largely due to desolvation, we explore a novel strategy for 3D molecular overlays that exploits the partitioning of molecular hydrophobicity into atomic contributions in conjunction with information about the distribution of hydrogen-bond (HB) donor /acceptor groups. A brief description of the method, as implemented in the software package PharmScreen, including the derivation of the fractional hydrophobic contributions within the quantum mechanical version of the MST continuum model, and the procedure utilized for the optimal superposition between molecules, is presented. The computational procedure is calibrated by using a dataset of 402 molecules pertaining to 14 distinct targets taken from the literature, and validated against the AstraZeneca test, which comprises 121 experimentally derived sets of molecular overlays. The results point out the suitability of the MST based-hydrophobic parameters for generating molecular overlays, as correct predictions were obtained for 94%, 79%, and 54% of the molecules classified into easy, moderate and hard sets, respectively. Moreover, the results point out that this accuracy is attained at a much lower degree of identity between the templates used by hydrophobic/HB fields and electrostatic/steric ones. These findings support the usefulness of the hydrophobic/HB descriptors to generate complementary overlays that may be valuable to rationalize structure-activity relationships and for virtual screening campaigns.
Insulin dimerization and aggregation play important roles in the endogenous delivery of the hormone. One of the important residues at the insulin dimer interface is PheB24 which is an invariant aromatic anchor that packs towards its own monomer inside a hydrophobic cavity formed by ValB12, LeuB15, TyrB16, CysB19 and TyrB26. Using molecular dynamics and free energy simulations in explicit solvent, the structural and dynamical consequences of mutations of Phe at position B24 to Gly, Ala, and d-Ala and the des-PheB25 variant are quantified. Consistent with experiments it is found that the Gly and Ala modifications lead to insulin dimers with reduced stability by 4 and 5 kcal/mol from thermodynamic integration and 4 and 8 kcal/mol from results using MM-GBSA, respectively. Given the experimental difficulties to quantify the thermodynamic stability of modified insulin dimers, such computations provide a valuable complement. Interestingly, the Gly-mutant exists as a strongly and a weakly interacting dimer. Analysis of the molecular dynamics simulations shows that this can be explained by water molecules that replace direct monomer-monomer H-bonding contacts at the dimerization interface involving residues B24 to B26. It is concluded that such solvent molecules play an essential role and must be included in future insulin dimerization studies.
The aim of the present study is to understand and analyze the factors that are influencing CK2 ligands and the role of crystal waters. The influence of the crystal waters on the stability of the ligand was deliberated based on the results of interaction energy and two body interaction energy analyses at M062X/def2-QZVP level of theory. The two body interaction energy analyses was carried out to assess whether important waters were contributing to favourable binding of the ligand through hinge region, where it acts as a bridge in linking protein with ligand or having interaction with ligand alone or merely specific polar interactions. Among the halogen ligands 1ZOH and 1ZOE are observed with the highest interaction energy of -13.96 and -13.75 kcal/mol. This is owing to the interaction of four crystal waters in above ligands. Among the non-halogen ligands, 2ZJW is observed to be more stable with huge interaction energy of -42.9 kcal/mol. Tetrabromobenzotriazole (TBB) derivatives of Halogen ligands 5CQU and 3KXM are observed with the highest volume of 294.23 and 266.75 cm3/mol, respectively. The Potential energy surface scan for 3NGA, 2ZJW and 1ZOH disclose the fact that the shorter distance subsist in the X-ray crystal is reliable, owing to the strong interaction. The NBO analysis reveals that the hydrogen/halogen bonds forming interaction with water molecules are found to have reasonable energy, but only those hydrogen/halogen bonds with shorter distance have large stabilization energy.
Water molecules play an important role in the recognition and stabilization of protein-ligand interactions. The water molecules at binding sites bridge the protein and ligand and can make three or more hydrogen bonds when distance and bond angles are used as criteria to define hydrogen-bonding interactions. There exists a distinct structural similarity between purines and between pyrimidine moieties. In the present study, we have analyzed the contribution of the water-mediated hydrogen bonds in the structure wise discrimination of these nucleotide bases. Significant differences in the amino acid residue preferences and the ligand atom preferences were observed in the case of purine moieties, adenine and guanine. In pyrimidine nucleotides, the amino acid residue Serine was preferred in both cytosine and thymine contacts; Asparagine was preferred in both thymine and uracil; Aspartic acid was preferred in both uracil and cytosine water-mediated contacts. Cytosine O 2 atom was highly preferred whereas the O 4 atom was highly preferred in thymine and uracil interactions. Since, the water networks provide increased specificity and affinity, the ability to include water-binding sites into the interface between a drug and its target might prove useful in drug design strategies.
Gabriele Cruciani is full professor of Organic Chemistry and Cheminformatrics at the University of Perugia. He currently leads a team comprising 10 students and three PhD students. In 1992, he spent a year as a postdoc with Prof Peter Goodford in the Laboratory of Molecular Biophysics at Oxford University, UK. In 1994–95 he became team laboratory leader of the Biotechnology European Project BIO2-CT94-3025 to design antidiabetic drug inhibitors of the enzyme glycogen phosphorylase b, together with Prof Louise Johnson, LMB, Oxford University. In 1999, he spent about 5 months with Bernard Testa and Pierre Alain Carrupt's group (Lausanne) working on metabolism and in silico pharmacokinetic prediction. In 2001, he received the Corvin Hansch Award from the Molecular Modeling Society at the Gordon conference in USA for his work on QSAR and molecular modeling. In 2005, he received the Research Award from Società Chimica Italiana, Organic Chemistry Division. Prof Cruciani is the scientific director of Molecular Discovery company, based in London (UK).
Donald J Abraham is Professor and Chair of the Department of Medicinal Chemistry at Virginia Commonwealth University and is the Director of the Institute for Structural Biology and Drug Discovery. He holds a BS degree in chemistry from Pennsylvania State University, an MS in organic chemistry from Marshall University, and a PhD in organic chemistry from Purdue University. Dr Abraham's research is interdisciplinary and focuses on structure-based drug design including x-ray crystallography, molecular modeling, synthetic medicinal chemistry, and structural function studies involving allosteric proteins. Targeted therapeutic areas of research include sickle cell anemia, radiation oncology, ischemic cardiovascular diseases, stroke, cancer, and Alzheimer's disease.
Domantas Motiejunas is currently studying for his PhD in the Faculty of Biosciences at the University of Heidelberg. He is carrying out his doctoral work at EML Research, in the Molecular and Cellular Modeling group led by Dr Rebecca Wade. In 2001, he graduated in molecular biology from Vilnius University where he investigated ASR (acid shock RNA) protein function in Escherichia coli in the Department of Biochemistry and Biophysics under the leadership of Dr Edita Suziedeliene. In 2002, he entered the International Graduate Program in Molecular and Cellular Biology organized by the University of Heidelberg and the German Cancer Research Center. He carried out his work for a Masters degree at EML Research on protein–protein docking assisted by sequence conservation and experimental data, and obtained a Masters degree from the University of Heidelberg in 2004. In his PhD project he focuses on the development and application of computational methods to models of macromolecular interactions.
The bioactivity assessment of foodborne peptides is currently a research area of great relevance, and, in particular, several studies are devoted to the antihypertensive effects through the inhibition of ACE (angiotensin I converting enzyme). In the present work, a straightforward workflow to identify inhibitory peptides from food matrices is proposed, which involves a hybrid in vitro / in silico tandem approach. Parma dry-cured ham was chosen as case study. In particular, the advantage of using the hybrid approach to identify active sequences (in comparison to the experimental trials alone) has been pointed out. Specifically, fractions obtained by in vitro gastrointestinal digestion of ham samples of 18 and 24 months of aging have been assessed for ACE inhibition. At the same time, the released peptidomic profiles - which cannot be entirely evaluated by using in vitro assays - have been screened for the inhibition by using an in silico model. Then, in order to identify novel inhibitory sequences, a series of strong candidates have been synthesized and assessed for their inhibitory activity through in vitro assay. On one hand, the use of computational simulations appeared to be an effective strategy to find active sequences, as confirmed by in vitro analysis. On the other hand, strong inhibitory sequences were identified for the first time in Parma dry-cured ham (e.g. LGL and SFVTT with IC50 values of 145 and 395 M, respectively), which is a product of international dietary and economic relevance. Therefore, our findings demonstrate the usefulness of in silico methodologies coupled to in vitro tests for the identification of potentially bioactive peptides and it gives an important contribution to the study of the overall nutritional value of Parma ham.
Within the framework of reduction, refinement and replacement of animal experiments, new approaches for identification and characterization of chemical hazards have been developed. Grouping and read across has been promoted as a most promising alternative approach. It uses existing toxicological information on a group of chemicals to make predictions on the toxicity of uncharacterized ones. In the present work, the feasibility of applying in vitro and in silico techniques to group chemicals for read across was studied using the food mycotoxin zearalenone (ZEN) and metabolites as a case study. ZEN and its reduced metabolites are known to act through activation of the estrogen receptor α (ERα). The ranking of their estrogenic potencies appeared highly conserved across test systems including binding, in vitro and in vivo assays. This data suggests that activation of ERα may play a role in the molecular initiating event (MIE) and be predictive of adverse effects and provides the rationale to model receptor-binding for hazard identification. The investigation of receptor-ligand interactions through docking simulation proved to accurately rank estrogenic potencies of ZEN and reduced metabolites, showing the suitability of the model to address estrogenic potency for this group of compounds. Therefore, the model was further applied to biologically uncharacterized, commercially unavailable, oxidized ZEN metabolites (6α-, 6β-, 8α-, 8β-, 13- and 15-OH-ZEN). Except for 15-OH-ZEN, the data indicate that in general, the oxidized metabolites would be considered a lower estrogenic concern than ZEN and reduced metabolites.
In April 2013 the mouse antibody serum neutralization test (SNT) was formally incorporated into European Pharmacopoeia monograph 0451 for potency testing of inactivated veterinary rabies vaccines. The SNT is designed to replace the highly variable and pain and distress causing NIH mouse rabies challenge assay. The adoption of the SNT meets the European ambition (i.e., EC and CoE) to replace, reduce and/or refine laboratory animal testing. However, regulatory acceptance and use of 3R models, such as the SNT, remains challenging. This paper aims at clarifying the process of acceptance and use of the SNT. For this purpose it reconstructs the process and reveals barriers and drivers that have been observed by involved stakeholders to have played a role. In addition it extracts lessons to stimulate regulatory acceptance in similar future processes. The incorporation of the SNT into the monographs went relatively quickly due to a thorough test development and pre-validation phase, commitment and cooperation of relevant stakeholders and a strong project coordination of the international validation study. The test was developed by the Paul Ehrlich Institut, a leading European OMCL. This facilitated its European regulatory use. The use by industry is in a critical phase. At this stage product specific validation and the question whether the SNT will be accepted outside Europe are important influencing factors.
The tuberculin skin test is the method of choice for tuberculosis surveillance in livestock ruminants. The exact definition of the biological activity of bovine tuberculin purified protein derivatives (bovine tuberculin PPDs) is essential for the reliability of a test system. PPDs consist of heterogeneous mixtures of mycobacterial antigens, making it difficult to determine their potency in vitro. The commonly used batch potency test is therefore based on the evaluation of skin reactions in mycobacteria-sensitized guinea pigs. The aim of the present study was to test an alternative in vitro method that reliably quantifies tuberculin PPD potency. This novel approach may prevent animal distress in the future.To this end a flow cytometry-based lymphocyte proliferation assay using peripheral blood mononuclear cells (PBMCs) from sensitized guinea pigs was established. Potency estimates for individual PPD preparations were calculated in comparison to an international standard. The comparison with results obtained from the guinea pig skin test revealed that the lymphocyte proliferation assay is more precise but results in systematically higher potency estimates. However, with a manufacturer specific correction factor a correlation of over 85% was achieved, highlighting the potential of this in vitro method to replace the current guinea pig skin test.
Silver nanoparticles (AgNPs) are gaining rapid popularity in many commonly used medical and commercial products for their unique anti-bacterial properties. The molecular mechanisms of effects of AgNPs on stem cell self-renewal and proliferation have not yet been well understood. The aim of the work is to use mouse embryonic stem cells (mESCs) as a cellular model to evaluate the toxicity of AgNPs. mESC is a very special cell type which has self-renewal and differentiation properties. The objective of this project is to determine the effects of AgNPs with different surface chemical compositions on the self-renewal and cell cycle of mESCs. Two different surface chemical compositions of AgNPs, polysaccharide-coated and hydrocarbon-coated, were used to test their toxic effects on self-renewal and proliferation of mESCs. The results indicated that both polysaccharide-coated and hydrocarbon-coated AgNPs changed the cell morphology of mESCs. Cell cycle analysis indicated that AgNPs induced mESCs cell cycle arrest at G1 and S phases through inhibition of the hyperphosphorylation of Retinoblastoma (Rb) protein. Furthermore, AgNPs exposure reduced Oct4A isoform expression which is responsible for the pluripotency of mESCs, and induced the expression of several isoforms OCT4B-265, OCT4B-190, OCT4B-164 which were suggested involved in stem cell stresses responses. In addition, the evidence of reactive oxygen species (ROS) production with two different surface chemical compositions of AgNPs supported our hypothesis that the toxic effect AgNPs exposure is due to overproduction of ROS which altered the gene expression and protein modifications. Polysaccharide coating reduced ROS production, and thus reduced the AgNPs toxicity.
The term Ergot is referred to the sclerotium of ascomycetes–a protective kernel produced during resting stage of some fungi–which replaces seeds of susceptible cereals and plants intended for human and animal diet. It contains various composition of tryptophan-derived toxins defined ergot alkaloids. Since sclerotia can be harvested and milled together with cereals, they represent a source of food and feed contamination after breakage and spreading of mycotoxins into the various milling fractions. The effects of ergot alkaloids, including those adverse for human health, have been known since the Middle Ages. Nevertheless, as recently stated by the European Food Safety Authority, further information is needed on metabolism and target receptors-binding of common alkaloids in food. Unfortunately, the experimental investigation is challenging due to the high costs in terms of time and money. This study was thus aimed at assessing whether the in silico modeling can be an effective tool to investigate the interaction between multiple serotonin receptors and a wide set of ergotamine metabolites, including experimentally detected molecules and predicted derivatives. Validated models provided precious insights about the effects exerted by metabolic modifications on the receptor-ligand interaction. Such structural information may be useful to support the design of further experimental analysis.
Scoring functions are a class of computational methods widely applied in structure-based drug design for evaluating protein-ligand interactions. Dozens of scoring functions have been published since early 1990s. In literature, scoring functions are typically classified as force-field-based, empirical, and knowledge-based. This classification scheme has been quoted for more than a decade and is still repeatedly quoted by some recent publications. Unfortunately, it does not reflect the recent progress in this field. Besides, the naming convention used for describing different types of scoring functions has been somewhat jumbled in literature, which could be confusing for newcomers to this field. In this article, we discuss an up-to-date classification scheme and appropriate naming convention for current scoring functions. We propose that they can be classified into physics-based methods, empirical scoring functions, knowledge-based potentials, and descriptor-based scoring functions. We have also outlined the major difference and connections between different categories of scoring functions.
Topoisomerases are targeted by several drugs in cancer chemotherapy acting as key enzymes in cell viability. Some flavonoids and their glycosides may exert health protective effects through the poisoning of topoisomerases. However, previous studies did not consider the substantial modifications taking place after ingestion neglecting that only metabolites can interact with the internal compartments of the human body. Since the high number of possible metabolites hinders their systematic analysis, an in silico approach can be a valuable tool to prioritize compounds by identifying candidates for further characterization. Specifically focusing on luteolin and quercetin, among the most ubiquitous flavonoids in the human diet, this work reports a computational procedure to model the effect of hepatic phase II conjugative metabolism on poisoning of human Topoisomerase I. As a general effect, glucuronidation and sulphation might enhance and quench poisoning activity, respectively. Among all, quercetin-3-O-glucuronide represents a promising candidate to be analyzed more thoroughly.
The use of molecular simulation to estimate the strength of macromolecular binding free energies is becoming increasingly widespread, with goals ranging from lead optimization and enrichment in drug discovery to personalizing or stratifying treatment regimes. In order to realize the potential of such approaches to predict new results, not merely to explain previous experimental findings, it is necessary that the methods used are reliable and accurate, and that their limitations are thoroughly understood. However, the computational cost of atomistic simulation techniques such as molecular dynamics (MD) has meant that until recently little work has focused on validating and verifying the available free energy methodologies, with the consequence that many of the results published in the literature are not reproducible. Here, we present a detailed analysis of two of the most popular approximate methods for calculating binding free energies from molecular simulations, molecular mechanics Poisson-Boltzmann surface area (MMPBSA) and molecular mechanics generalized Born surface area (MMGBSA), applied to the nine FDA-approved HIV-1 protease inhibitors. Our results show that the values obtained from replica simulations of the same protease-drug complex, differing only in initially assigned atom velocities, can vary by as much as 10 kcal mol(-1), which is greater than the difference between the best and worst binding inhibitors under investigation. Despite this, analysis of ensembles of simulations producing 50 trajectories of 4 ns duration leads to well converged free energy estimates. For seven inhibitors, we find that with correctly converged normal mode estimates of the configurational entropy, we can correctly distinguish inhibitors in agreement with experimental data for both the MMPBSA and MMGBSA methods and thus have the ability to rank the efficacy of binding of this selection of drugs to the protease (no account is made for free energy penalties associated with protein distortion leading to the over estimation of the binding strength of the two largest inhibitors ritonavir and atazanavir). We obtain improved rankings and estimates of the relative binding strengths of the drugs by using a novel combination of MMPBSA/MMGBSA with normal mode entropy estimates and the free energy of association calculated directly from simulation trajectories. Our work provides a thorough assessment of what is required to produce converged and hence reliable free energies for protein-ligand binding.
Water can be considered the most important molecule in living systems.
It can play a variety of roles: (a) the solvent of most biological
molecules, (b) substrate and product of enzyme catalysis, (c) a building
block of macromolecules, or (d) functioning as a "lubricant" via the
formation of networks linking distant residues. In particular,
protein-protein, protein-nucleic acid, and protein-ligand recognition
are water dependent via both enthalpic and entropic contributions. We
present a computational approach based on the natural force field
Hydropathic INTeractions (HINT) that incorporates atomic
LogPoctanol/water data, to evaluate the strength of the interaction
between water molecules and protein, as well as the contribution of
water molecules bridging protein and ligand to the free energy of
binding. We describe the rules for characterizing the binding/energetic
roles of water molecules located at ligand binding sites. As an example,
results from a set of 23 HIV-1 protease ligand complexes are described.
Cannabinoid receptors are a family of G-protein coupled receptors that are involved in a wide variety of physiological processes and diseases. One of the key regulators that are unique to cannabinoid receptors is the cannabinoid receptor interacting proteins (CRIPs). Among them CRIP1a was found to decrease the constitutive activity of the cannabinoid type-1 receptor (CB1R). The aim of this study is to gain an understanding of the interaction between CRIP1a and CB1R through using different computational techniques. The generated model demonstrated several key putative interactions between CRIP1a and CB1R, including the critical involvement of Lys130 in CRIP1a.
Characterizing the nature of interaction between proteins that have not been experimentally co-crystallized requires a computational docking approach that can successfully predict the spatial conformation adopted in the complex. In this work, the Hydropathic INTeractions (HINT) force field model was used for scoring docked models in a data set of 30 high-resolution crystallographically characterized "dry" protein-protein complexes, and was shown to reliably identify native-like models. However, most current protein-protein docking algorithms fail to explicitly account for water molecules involved in bridging interactions that mediate and stabilize the association of the protein partners, so we used HINT to illuminate the physical and chemical properties of bridging waters and account for their energetic stabilizing contributions. The HINT water Relevance metric identified the 'truly' bridging waters at the 30 protein-protein interfaces and we utilized them in "solvated" docking by manually inserting them into the input files for the rigid body ZDOCK program. By accounting for these interfacial waters, a statistically significant improvement of ~24% in the average hit-count within the top-10 predictions the protein-protein dataset was seen, compared to standard "dry" docking. The results also show scoring improvement, with medium and high accuracy models ranking much better than incorrect ones. These improvements can be attributed to the physical presence of water molecules that alter surface properties and better represent native shape and hydropathic complementarity between interacting partners, with concomitantly more accurate native-like structure predictions. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
The importance of protein-protein interactions (PPIs) is becoming increasingly appreciated, as these interactions lie at the core of virtually every biological process. Small molecule modulators that target PPIs are under exploration as new therapies. One of the greatest obstacles faced in crystallographically determining the 3D structures of proteins is coaxing the proteins to form "artificial" PPIs that lead to uniform crystals suitable for X-ray diffraction. This work compares interactions formed naturally, i.e., "biological", with those artificially formed under crystallization conditions or "non-biological". In particular, a detailed analysis of water molecules at the interfaces of high-resolution (≤2.30Å) X-ray crystal structures of protein-protein complexes, where 140 are biological protein-protein complex structures and 112 include non-biological protein-protein interfaces, was carried out using modeling tools based on the HINT forcefield. Surprisingly few and relatively subtle differences were observed between the two types of interfaces: (i) non-biological interfaces are more polar than biological interfaces, yet there is better organized hydrogen bonding at the latter; (ii) biological associations rely more on water-mediated interactions with backbone atoms compared to non-biological associations; (iii) aromatic/planar residues play a larger role in biological associations with respect to water, and (iv) Lys has a particularly large role at non-biological interfaces. A support vector machines (SVMs) classifier using descriptors from this study was devised that was able to correctly classify 84% of the two interface types.
Computational methods available for the calculation of relative and absolute binding affinities (free energy simulations, continuum electrostatics, linear interaction energy approximations, and empirical solvation models) are reviewed together with recent applications to biological systems. The decomposability of the binding free energy into physically meaningful components is examined and results obtained for these components are presented. Some of these components, such as the direct interactions, the translational / rotational entropy loss, and the desolvation free energy are well recognized. Recent calculations have shown that the translational / rotational entropy loss is not as large as some theoretical calculations have previously suggested because of substantial residual movements in the bound complex. Recent work also points to the importance of contributions that are often neglected in binding affinity calculations, such as the protein reorganization energy and, for flexible ligands, the ligand reorganization energy. Future work should concentrate on the improvement of the energy functions and simulation protocols for the achievement of more precise and accurate predictions.
Ten C2-symmetric cyclic urea and sulfamide derivatives have been synthesized from l-mannonic γ-lactone and d-mannitol. The results of experimental measurement of their inhibitory potencies against HIV-1 protease were compared to calculated free energies of binding derived from molecular dynamics (MD) simulations. The compounds were selected, firstly, to enable elucidation of the role of stereochemistry for binding affinity (1a−d) and, secondly, to allow evaluation of the effects of variation in the link to the P1 and P1‘ phenyl groups on affinity (1a and 2−5). Thirdly, compounds with hydrogen bond-accepting or -donating groups attached to the phenyl groups in the P2 and P2‘ side chains (6 and 7) were selected. Binding free energies were estimated by a linear response method, whose predictive power for estimating binding affinities from MD simulations was demonstrated.
A historical perspective on the application of molecular dynamics (MD) to biological macromolecules is presented. Recent developments combining state-of-the-art force fields with continuum solvation calculations have allowed us to reach the fourth era of MD applications in which one can often derive both accurate structure and accurate relative free energies from molecular dynamics trajectories. We illustrate such applications on nucleic acid duplexes, RNA hairpins, protein folding trajectories, and protein−ligand, protein−protein, and protein−nucleic acid interactions.
This work introduces a continuous smooth permittivity function into Poisson–Boltzmann techniques for continuum approaches to modeling the solvation of small molecules and proteins. The permittivity function is derived using a Gaussian method to describe volume exclusion. The new method allows a rigorous determination of solvent forces within a grid-based technology. The generality of approach is demonstrated by considering a range of applications for small molecules and macromolecules. We also present a very complete statistical analysis of grid errors, and show that the accuracy of our Gaussian-based method is improved over standard techniques. The method has been implemented in a new code called ZAP, which is freely available to academic institutions.1 © 2001 John Wiley & Sons, Inc. J Comput Chem 22: 608–640, 2001
The activity of the human immunodeficiency virus (HIV) protease is essential for processing of the gag-pol precursor proteins and maturation of infectious virions. We have prepared a peptidomimetic inhibitor, U-75875, that inhibited HIV-1 gag-pol protein processing in an essentially irreversible manner. Noninfectious virus particles produced in the presence of the drug contained gag precursors and were morphologically immature. In human peripheral blood mononuclear cells and in a continuous cell line, U-75875 completely blocked HIV replication; in the latter case, no spread occurred over a period of 4 weeks. U-75875, on a molar basis, was as potent as 3'-azido-3'-deoxythymidine in blocking HIV-1 replication in human lymphocytes and also inhibited HIV-2 and simian immunodeficiency virus proteases, demonstrating that it has broad activity. These results provide further evidence for the therapeutic potential of protease inhibitors in HIV infection.
The structure of a crystal complex of the chemically synthesized protease of human immunodeficiency virus 1 with a heptapeptide-derived inhibitor bound in the active site has been determined. The sequence of the inhibitor JG-365 is Ac-Ser-Leu-Asn-Phe-psi[CH(OH)CH2N]-Pro-Ile-Val-OMe; the Ki is 0.24 nM. The hydroxyethylamine moiety, in place of the normal scissile bond of the substrate, is believed to mimic a tetrahedral reaction intermediate. The structure of the complex has been refined to an R factor of 0.146 at 2.4-A resolution by using restrained least squares with rms deviations in bond lengths of 0.02 A and bond angles of 4. The bound inhibitor diastereomer has the S configuration at the hydroxyethylamine chiral carbon, and the hydroxyl group is positioned between the active site aspartate carboxyl groups within hydrogen bonding distance. Comparison of this structure with a reduced peptide bond inhibitor-protease complex indicates that these contacts confer the exceptional binding strength of JG-365.
The structure of a complex between a peptide inhibitor with the sequence N-aceti-Thr-Ile-Nle-t[CH2-NH]-Nle-ψ[CH_2-NH]Nel-Gln-Arg.amide (Nle,norleucine) with chemically synthesized HIV-1 (human immunodeficiency virus 1) protease was determined at 2.3 Å resolution (R factor of 0.176). Despite the symmetric nature of the unliganded enzyme, the asymmetric inhibitor lies in a single orientation and makes extensive interactions at the interface between the two subunits of the homodimeric protein. Compared with the unliganded enzyme, the protein molecule underwent substancial changes, particularly in an extended region
corresponding to the "flaps"(residues 35 to 57 in each
chain), where backbone movements as large as 7 Å are
observed.
Three-dimensional structure of an asymmetrically mutated (C95M) tethered human immunodeficiency virus type 1 protease enzyme (HIV-1 PR) has been determined in an unliganded form using X-ray diffraction data to 1.9 Angstrom resolution. The structure, refined using X-PLOR to an R factor of 19.5%, is unexpectedly similar to the ligand-bound native enzyme, rather than to the ligand-free native enzyme. In particular, the two flaps in the tethered dimer are in a closed configuration, The environments around M95 and C1095 are identical, showing no structural effect of this asymmetric mutation at position 95. Oxidation of Cys1095 has been observed for the first time. There is one well-defined water molecule that hydrogen bonds to both carboxyl groups of the essential aspartic acids in the active site, Proteins 2001;43:57-64, (C) 2001 Wiley-Liss, Inc.
The structure of a crystal complex of the chemically synthesized protease of human immunodeficiency virus 1 with a heptapeptide-derived inhibitor bound in the active site has been determined. The sequence of the inhibitor JG-365 is Ac-Ser-Leu-Asn-Phe-ψ[CH(OH)CH_2N]-Pro-Ile-Val-OMe; the K_i is 0.24 nM. The hydroxyethylamine moiety, in place of the normal scissile bond of the substrate, is believed to mimic a tetrahedral reaction intermediate. The structure of the complex has been refined to an R factor of 0.146 at 2.4-Å resolution by using restrained least squares with rms deviations in bond lengths of 0.02 Å and bond angles of 4. The bound inhibitor diastereomer has the S configuration at the hydroxyethylamine chiral carbon, and the hydroxyl group is positioned between the active site aspartate carboxyl groups within hydrogen bonding distance. Comparison of this structure with a reduced peptide bond inhibitor-protease complex indicates that these contacts confer the exceptional binding strength of JG-365.
A novel and robust automated docking method that predicts the bound conformations of flexible ligands to macromolecular targets has been developed and tested, in combination with a new scoring function that estimates the free energy change upon binding. Interestingly, this method applies a Lamarckian model of genetics, in which environmental adaptations of an individual's phenotype are reverse transcribed into its genotype and become . heritable traits sic . We consider three search methods, Monte Carlo simulated annealing, a traditional genetic algorithm, and the Lamarckian genetic algorithm, and compare their performance in dockings of seven protein)ligand test systems having known three-dimensional structure. We show that both the traditional and Lamarckian genetic algorithms can handle ligands with more degrees of freedom than the simulated annealing method used in earlier versions of AUTODOCK, and that the Lamarckian genetic algorithm is the most efficient, reliable, and successful of the three. The empirical free energy function was calibrated using a set of 30 structurally known protein)ligand complexes with experimentally determined binding constants. Linear regression analysis of the observed binding constants in terms of a wide variety of structure-derived molecular properties was performed. The final model had a residual standard y1 y1 .
The computational modeling program HINT (Hydropathic INTeractions), an empirical hydropathic force field that includes hydrogen bonding, Coulombic, and hydrophobic terms, was used to model the free energy of dimer-tetramer association in a series of deoxy hemoglobin beta 37 double mutants. Five of the analyzed mutants (beta 37W -> Y, beta 37W - A, beta 37W - G, beta 37W - E, and beta 37W - R) have been solved crystallographically and characterized thermodynamically and subsequently made a good test set for the calibration of our method as a tool for free energy prediction. Initial free energy estimates for these mutants were conducted without the inclusion of crystallographically conserved water molecules and systematically underestimated the experimentally calculated loss in free energy observed for each mutant dimer-tetramer association. However, the inclusion of crystallographic waters, interacting at the dimer-dimer interface of each mutant, resulted in HINT free energy estimates that were more accurate with respect to experimental data. To evaluate the ability of our method to predict free energies for de novo protein models, the same beta 37 mutants were computationally generated from native deoxy hemoglobin and similarly analyzed. Our theoretical models were sufficiently robust to accurately predict free energy changes in a localized region around the mutated residue. However, our method did not possess the capacity to generate the long-range secondary structural effects observed in crystallographically solved mutant structures. Final method analysis involved the computational generation of structurally and/or thermodynamically uncharacterized beta 37 deoxy hemoglobin mutants. HINT analysis of these structures revealed that free energy predictions for dimer-tetramer association in these models agreed well with previously observed energy predictions for structurally and thermodynamically characterized beta 37 deoxy hemoglobin mutants.
The crystal structure of a complex between chemically synthesized human immunodeficiency virus type 1 (HIV-1) protease and an octapeptide inhibitor has been refined to an R factor of 0.138 at 2.5-angstrom resolution. The substrate-based inhibitor, H-Val-Ser-Gln-Asn-Leu-PSI[CH(OH)CH2]Val-Ile-Val-OH (U-85548e) contains a hydroxyethylene isostere replacement at the scissile bond that is believed to mimic the tetrahedral transition state of the proteolytic reaction. This potent inhibitor has K(i) < 1 nM and was developed as an active-site titrant of the HIV-1 protease. The inhibitor binds in an extended conformation and is involved in beta-sheet interactions with the active-site floor and flaps of the enzyme, which form the substrate/inhibitor cavity. The inhibitor diastereomer has the S configuration at the chiral carbon atom of the hydroxyethylene insert, and the hydroxyl group is within H-bonding distance of the two active-site carboxyl groups in the enzyme dimer. The two subunits of the enzyme are related by a pseudodyad, which superposes them at a 178-degrees rotation. The main difference between the subunits is in the beta turns of the flaps, which have different conformations in the two monomers. The inhibitor has a clear preferred orientation in the active site and the alternative conformation, if any, is a minor one (occupancy of less than 30%). A new model of the enzymatic mechanism is proposed in which the proteolytic reaction is viewed as a one-step process during which the nucleophile (water molecule) and electrophile (an acidic proton) attack the scissile bond in a concerted manner.
A fast and efficient solvation model based on the weighted solvent accessible surface area (WSAS) was developed to predict solvation free energies for organic and biological molecules. To reproduce the experimental solvation free energies, least-squares fittings were applied to optimize the weights of solvent accessible surface area for different atoms. For the 184-molecule set (model I), with experimental solvation free energies in 1-octanol, WSAS achieved an unsigned average error of 0.36 kcal/mole, better than those of SM5.42R universal solvation model. For the 245-molecule set (model II), WSAS and the universal solvation models achieved similar performances in reproducing the experimental aqueous solvation free energies and the unsigned average error was about 0.46 kcal/mole with WSAS. For model III, the whole molecule set was separated into training set (293 molecules) and test set (94 molecules). The overall unsigned average error (0.538 kcal/mole) of model III was very close to that of the 401-molecule set (model IV)(0.536 kcal/mole). WSAS was successfully used in predicting the relative binding free energies for the five binding modes of HIV-1RT/efavirenz. With this model, the correct binding mode had a binding free energy at least 10 kcal/mole favorable than other binding modes. The solvation free energies with WSAS correlated well to the solvation free energies calculated by the Poisson-Boltzmann/surface area model. The solvation free energies of various organic compounds, e.g., methane, ethylene, propyne, BTEX, methanol, chloromethane, acetonitrile, dimethyl sulfide, etc., for the four WSAS models were presented.
We have previously reported on the unexpected flipped conformation in the cyclic sulfamide class of inhibitors. An attempt to induce a symmetric binding conformation by introducing P2/P2' substituents foreseen to bind preferentially in the S2/S2' subsite was unsuccessful. On the basis of the flipped conformation we anticipated that nonsymmetric sulfamide inhibitors, with P2/P2' side chains modified individually for the S1' and S2 subsites, should be more potent than the corresponding symmetric analogues. To test this hypothesis, a set of 18 cyclic sulfamide inhibitors (11 nonsymmetric and 7 symmetric) with different P2/P2' substituents was prepared and evaluated in an enzyme assay. To rationalize the structure-activity relationship (SAR) and enable the alignment of the nonsymmetric inhibitors, i.e., which of the P2/P2' substituents of the nonsymmetric inhibitors interact with which subsite, a CoMFA study was performed. The CoMFA model, constructed from the 18 inhibitors in this study along with seven inhibitors from previous work by our group, has successfully been used to rationalize the SAR of the cyclic sulfamide inhibitors. Furthermore, from the information presented herein, the SAR of the cyclic sulfamide class of inhibitors seems to differ from the SAR of the related cyclic urea inhibitors reported by DuPont and DuPont-Merck.
We have previously reported on the unexpected flipped conformation in the cyclic sulfamide class of inhibitors. An attempt to induce a symmetric binding conformation by introducing P2/P2‘ substituents foreseen to bind preferentially in the S2/S2‘ subsite was unsuccessful. On the basis of the flipped conformation we anticipated that nonsymmetric sulfamide inhibitors, with P2/P2‘ side chains modified individually for the S1‘ and S2 subsites, should be more potent than the corresponding symmetric analogues. To test this hypothesis, a set of 18 cyclic sulfamide inhibitors (11 nonsymmetric and 7 symmetric) with different P2/P2‘ substituents was prepared and evaluated in an enzyme assay. To rationalize the structure−activity relationship (SAR) and enable the alignment of the nonsymmetric inhibitors, i.e., which of the P2/P2‘ substituents of the nonsymmetric inhibitors interact with which subsite, a CoMFA study was performed. The CoMFA model, constructed from the 18 inhibitors in this study along with seven inhibitors from previous work by our group, has successfully been used to rationalize the SAR of the cyclic sulfamide inhibitors. Furthermore, from the information presented herein, the SAR of the cyclic sulfamide class of inhibitors seems to differ from the SAR of the related cyclic urea inhibitors reported by DuPont and DuPont-Merck.
A methodology is presented for calculating relative binding free energies of enzyme−inhibitor associations in aqueous solvent. The methodology uses synthesis of semiempirical quantum chemistry to determine the protonation state of important residues in the enzyme active site, molecular mechanics to determine the gas-phase energetic contributions to the relative binding free energy, and dielectric continuum solvation to calculate electrostatic hydration contributions. The methodology is then applied to the calculation of the relative binding free energy of the inhibitors KNI-272, Ro31-8959, L-735,524, and A-77003 to HIV-1 protease and its I84V mutant. The calculated relative binding free energy is sensitive to the active-site protonation state of the aspartic acid residues of HIV-1 protease. The protonation state is inhibitor dependent. Given a particular protonation state, it was found that quantitatively accurate relative binding free energies could only be achieved when solvent effects were included. Three categories of binding were found. In the first, the change in binding free energy due to mutation is mainly due to the change in enthalpic interactions within the inhibitor−enzyme complex (Ro31-8959). In the second (L-735,524 and A-77003), the change in affinity is caused both by a change in enthalpic interactions within the enzyme and by a change in the hydration energy of the enzyme and inhibitor−enzyme complexes. In the third case (KNI-272), the change in affinity is mainly a solvent effectit is due to changes in hydration of the enzyme only. In all cases, it was found that the I84V mutant enzyme was more stable than the wild-type enzyme. This alone (without consideration of the inhibitor−enzyme complexes) can qualitatively explain the reduction in binding affinity due to mutation.
HIV protease is an attractive therapeutic target for the
treatment of HIV infection and AIDS, due to its obligatory role
in the life cycle of the virus.I Of the many in vitro potent HIV
protease inhibitors described so far, however, only a few have
advanced into clinical trials. Here, we report the structure of
HIV-I protease in complex with VX-478 (amprenavir), a potent, low
molecular weight. orally bioavailable HIV protease inhibitor
currently in advanced preclinical development. VX-478 emerged
from a focused program of structure-based drug design that
sought to maintain sub-nanomolar in vitro potency while
reducing inhibitor size, a tactic supported by our analysis of
the molecular weight distribution of all marketed drugs (Figure
1 ). Historically. in the development of angiotensin convening
enzyme inhibitors, structural information was similarly used
to design smaller, potent inhibitors (like captopril) to specifically
resolve issues of in vivo efficacy. In order to enhance inhibitory potency, we sought to optimize binding to the catalytic aspartate residues of the enzyme and to the critical water molecule that mediates inhibitor interactions with the flap. In addition. we tried to minimize unfavorable strain energy by favoring compounds whose conformation would require minimal reorganization on enzyme binding. Other design goals included synthetic accessibility, high antiviral potency, low cellular toxicity, and aqueous solubility without obligate charges.
The incorporation of C-2 Symmetry has become a useful paradigm in the design of active site inhibitors for HIV-1 protease (HIV PR) and has led to the design of a series of highly potent, C-2 symmetry-based, diol-containing inhibitors of HIV PR, one of which, A-77003, has reached clinical trials. However, the stereochemistry of the diol core influences protease inhibition and antiviral activity in a manner that is not well understood. We analyzed the crystal structures of a diastereomeric series of C-2 symmetry-based diol inhibitors, along with a deshydroxy analogue, bound to HIV PR and found that the stereochemistry of the diol core influences the mode of binding to the active site aspartic acids. Diasteromers with similar binding affinity can bind in different, asymmetric and symmetric, modes, while those with different binding affinities can bind in a similar manner. The positional symmetry of an inhibitor with respect to the enzyme C-2 axis may be distinguished from its conformational symmetry. The structural differences between the inhibitor complexes were mainly confined to the central core portion of the diols, can be described by torsional differences about the central three bonds, and primarily affect interactions within the active site pocket formed by Asp 25/125 and Gly 27/127. Some flexibility in the enzyme backbone at Gly 127 was also apparent. Based on these results, we suggest that the binding mode for central hydroxy-bearing, C-2-symmetric inhibitors will be determined by how well the inhibitor can simultaneously optimize hydrogen bonding with the active site carboxylate groups and van der Waals contacts with the neighboring backbone atoms of the active site ''psi''-loops. A symmetric hydrogen-bonding arrangement with either one or tio symmetrically positioned hydroxy groups appears to be preferred over less symmetric configurations.
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Introduction Theory Computational Details Free Energy Perturbation Calculations for Small Molecules Free Energy Perturbation Calculations for Macromolecules Guide to Structure-Based Ligand Optimization Optimization of Ligands to HIV-1 Protease: Using the FEP Method Conclusions Brief Guide for Free Energy Calculations and Their Use in Ligand Optimization
A practical computational method for the molecular modeling of free-energy changes associated with protein mutations is reported. The de novo generation, optimization, and thermodynamic analysis of a wide variety of deoxy and oxy hemoglobin mutants are described in detail. Hemoglobin is shown to be an ideal candidate protein for study because both the native deoxy and oxy states have been crystallographically determined, and a large and diverse population of its mutants has been thermodynamically characterized. Noncovalent interactions for all computationally generated hemoglobin mutants are quantitatively examined with the molecular modeling program HINT (Hydropathic INTeractions). HINT scores all biomolecular noncovalent interactions, including hydrogen bonding, acid–base, hydrophobic–hydrophobic, acid–acid, base–base, and hydrophobic–polar, to generate dimer–dimer interface “scores” that are translated into free-energy estimates. Analysis of 23 hemoglobin mutants, in both deoxy and oxy states, indicates that the effects of mutant residues on structurally bound waters (and visa versa) are important for generating accurate free-energy estimates. For several mutants, the addition/elimination of structural waters is key to understanding the thermodynamic consequences of residue mutation. Good agreement is found between calculated and experimental data for deoxy hemoglobin mutants (r = 0.79, slope = 0.78, standard error = 1.4 kcal mol−1, n = 23). Less accurate estimates were initially obtained for oxy hemoglobin mutants (r = 0.48, slope = 0.47, standard error = 1.4 kcal mol−1, n = 23). However, the elimination of three outliers from this data set results in a better correlation of r = 0.87 (slope = 0.72, standard error = 0.75, n = 20). These three mutations may significantly perturb the hemoglobin quaternary structure beyond the scope of our structural optimization procedure. The method described is also useful in the examination of residue ionization states in protein structures. Specifically, we find an acidic residue within the native deoxy hemoglobin dimer–dimer interface that may be protonated at physiological pH. The final analysis is a model design of novel hemoglobin mutants that modify cooperative free energy (ΔGc)—the energy barrier between the allosteric transition from deoxy to oxy hemoglobin. Proteins 2001;42:355–377. © 2000 Wiley-Liss, Inc.
A non-covalent interaction force field model derived from the partition coefficient of 1-octanol/water solubility is described. This model, HINT for Hydropathic INTeractions, is shown to include, in very empirical and approximate terms, all components of biomolecular associations, including hydrogen bonding, Coulombic interactions, hydrophobic interactions, entropy and solvation/desolvation. Particular emphasis is placed on: (1) demonstrating the relationship between the total empirical HINT score and free energy of association, G
interaction; (2) showing that the HINT hydrophobic-polar interaction sub-score represents the energy cost of desolvation upon binding for interacting biomolecules; and (3) a new methodology for treating constrained water molecules as discrete independent small ligands. An example calculation is reported for dihydrofolate reductase (DHFR) bound with methotrexate (MTX). In that case the observed very tight binding, G
interaction–13.6kcal mol–1, is largely due to ten hydrogen bonds between the ligand and enzyme with estimated strength ranging between –0.4 and –2.3kcalmol–1. Four water molecules bridging between DHFR and MTX contribute an additional –1.7kcalmol–1 stability to the complex. The HINT estimate of the cost of desolvation is +13.9kcalmol–1.
A novel and robust automated docking method that predicts the bound conformations of flexible ligands to macromolecular targets has been developed and tested, in combination with a new scoring function that estimates the free energy change upon binding. Interestingly, this method applies a Lamarckian model of genetics, in which environmental adaptations of an individual's phenotype are reverse transcribed into its genotype and become . heritable traits sic . We consider three search methods, Monte Carlo simulated annealing, a traditional genetic algorithm, and the Lamarckian genetic algorithm, and compare their performance in dockings of seven protein)ligand test systems having known three-dimensional structure. We show that both the traditional and Lamarckian genetic algorithms can handle ligands with more degrees of freedom than the simulated annealing method used in earlier versions of AUTODOCK, and that the Lamarckian genetic algorithm is the most efficient, reliable, and successful of the three. The empirical free energy function was calibrated using a set of 30 structurally known protein)ligand complexes with experimentally determined binding constants. Linear regression analysis of the observed binding constants in terms of a wide variety of structure-derived molecular properties was performed. The final model had a residual standard y1 y1. error of 9.11 kJ mol 2.177 kcal mol and was chosen as the new energy
The structure of a crystal complex of recombinant human immunodeficiency virus type 1 (HIV-1) protease with a peptide-mimetic inhibitor containing a dihydroxyethylene isostere insert replacing the scissile bond has been determined. The inhibitor is Noa-His-Hch psi [CH(OH)CH(OH)]Vam-Ile-Amp (U-75875), and its Ki for inhibition of the HIV-1 protease is < 1.0 nM (Noa = 1-naphthoxyacetyl, Hch = a hydroxy-modified form of cyclohexylalanine, Vam = a hydroxy-modified form of valine, Amp = 2-pyridylmethylamine). The structure of the complex has been refined to a crystallographic R factor of 0.169 at 2.0 A resolution by using restrained least-squares procedures. Root mean square deviations from ideality are 0.02 A and 2.4 degrees, for bond lengths and angles, respectively. The bound inhibitor diastereomer has the R configurations at both of the hydroxyl chiral carbon atoms. One of the diol hydroxyl groups is positioned such that it forms hydrogen bonds with both the active site aspartates, whereas the other interacts with only one of them. Comparison of this X-ray structure with a model-built structure of the inhibitor, published earlier, reveals similar positioning of the backbone atoms and of the side-chain atoms in the P2-P2' region, where the interaction with the protein is strongest. However, the X-ray structure and the model differ considerably in the location of the P3 and P3' end groups, and also in the positioning of the second of the two central hydroxyl groups. Reconstruction of the central portion of the model revealed the source of the hydroxyl discrepancy, which, when corrected, provided a P1-P1' geometry very close to that seen in the X-ray structure.
Analogues of peptides ranging in size from three to six amino acids and containing the hydroxyethylene dipeptide isosteres Phe psi Gly, Phe psi Ala, Phe psi NorVal, Phe psi Leu, and Phe psi Phe, where psi denotes replacement of CONH by (S)-CH(OH)CH2, were synthesized and studied as HIV-1 protease inhibitors. Inhibition constants (Ki) with purified HIV-1 protease depend strongly on the isostere in the order Phe psi Gly greater than Phe psi Ala greater than Phe psi NorVal greater than Phe psi Leu greater than Phe psi Phe and decrease with increasing length of the peptide analogue, converging to a value of 0.4 nM. Ki values are progressively less dependent on inhibitor length as the size of the P1' side chain within the isostere increases. The structures of HIV-1 protease complexed with the inhibitors Ala-Ala-X-Val-Val-OMe, where X is Phe psi Gly, Phe psi Ala, Phe psi NorVal, and Phe psi Phe, have been determined by X-ray crystallography (resolution 2.3-3.2 A). The crystals exhibit symmetry consistent with space group P6(1) with strong noncrystallographic 2-fold symmetry, and the inhibitors all exhibit 2-fold disorder. The inhibitors bind in similar conformations, forming conserved hydrogen bonds with the enzyme. The Phe psi Gly inhibitor adopts an altered conformation that places its P3' valine side chain partially in the hydrophobic S1' pocket, thus suggesting an explanation for the greater dependence of the Ki value on inhibitor length in the Phe psi Gly series. From the kinetic and crystallographic data, a minimal inhibitor model for tight-binding inhibition is derived in which the enzyme subsites S2-S2' are optimally occupied. The Ki values for several compounds are compared with their potencies as inhibitors of proteolytic processing in T-cell cultures chronically infected with HIV-1 (MIC values) and as inhibitors of acute infectivity (IC50 values). There is a rank-order correspondence, but a 20-1000-fold difference, between the values of Ki and those of MIC or IC50. IC50 values can approach those of Ki but are highly dependent on the conditions of the acute infectivity assay and are influenced by physiochemical properties of the inhibitors such as solubility.
The structure and activity of a protein molecule are strongly influenced by the extent of hydration of its cavities. This is, in turn, related to the free energy change on transfer of a water molecule from bulk solvent into a cavity. Such free energy changes have been calculated for two cavities in a sulfate-binding protein. One of these cavities contains a crystallographically observed water molecule while the other does not. Thermodynamic integration and perturbation methods were used to calculate free energies of hydration for each of the cavities from molecular dynamics simulations of two separate events: the removal of a water molecule from pure water, and the introduction of a water molecule into each protein cavity. From the simulations for the pure water system, the excess chemical potential of water was computed to be -6.4 +/- 0.4 kcal/mol, in accord with experiment and with other recent theoretical calculations. For the protein cavity containing an experimentally observed water molecule, the free energy change on hydrating it with one water molecule was calculated as -10.0 +/- 1.3 kcal/mol, indicating the high probability that this cavity is occupied by a water molecule. By contrast, for the cavity in which no water molecules were experimentally observed, the free energy change on hydrating it with one water molecule was calculated as 0.2 +/- 1.5 kcal/mol, indicating its low occupancy by water. The agreement of these results with experiment suggests that thermodynamic simulation methods may become useful for the prediction and analysis of internal hydration in proteins.
The protein-bound conformations of six new allosteric effectors similar to bezafibrate that markedly decrease the oxygen affinity of hemoglobin have been determined by X-ray crystallography. Comparisons are made with the bound conformations of three urea analogues reported by Lalezari, Perutz, and co-workers. All six new molecules bind at the same site previously observed for bezafibrate and exhibit a wide range of allosteric activity. Unlike the urea derivatives, which show two binding sites for the most potent derivatives, only one of the six new molecules (one with moderate allosteric activity) exhibits a second binding site. A new computer program, HINT (hydrophobic interactions), has been created and utilized to identify the major interactions between small molecules and the protein. The three strongest interactions identified by HINT involve Arg 141 alpha with the acid of the analogues, Lys 99 alpha with the bridging amide carbonyl, and the amide NH of the side chain of Asn 108 beta with the halogenated aromatic ring.
A two-fold (C2) symmetric inhibitor of the protease of human immunodeficiency virus type-1 (HIV-1) has been designed on the basis of the three-dimensional symmetry of the enzyme active site. The symmetric molecule inhibited both protease activity and acute HIV-1 infection in vitro, was at least 10,000-fold more potent against HIV-1 protease than against related enzymes, and appeared to be stable to degradative enzymes. The 2.8 angstrom crystal structure of the inhibitor-enzyme complex demonstrated that the inhibitor binds to the enzyme in a highly symmetric fashion.
The crystal structure of the protease of the human immunodeficiency virus type (HIV-1), which releases structural proteins and enzymes from viral polyprotein products, has been determined to 3 A resolution. Large regions of the protease dimer, including the active site, have structural homology to the family of microbial aspartyl proteases. The structure suggests a mechanism for the autoproteolytic release of protease and a role in the control of virus maturation.
The interaction of a probe group with a protein of known structure is computed at sample positions throughout and around the macromolecule, giving an array of energy values. The probes include water, the methyl group, amine nitrogen, carboxy oxygen, and hydroxyl. Contour surfaces at appropriate energy levels are calculated for each probe and displayed by computer graphics together with the protein structure. Contours at negative energy levels delineate contours also enable other regions of attraction between probe and protein and are found at known ligand binding clefts in particular. The contours also enable other regions of attraction to be identified and facilitate the interpretation of protein-ligand energetics. They may, therefore, be of value for drug design.
We have developed a method for calculating the stability in water of protein structures, starting from their atomic coordinates. The contribution of each protein atom to the solvation free energy is estimated as the product of the accessibility of the atom to solvent and its atomic solvation parameter. Applications of the method include estimates of the relative stability of different protein conformations, estimates of the free energy of binding of ligands to proteins and atomic-level descriptions of hydrophobicity and amphiphilicity.
A program is described for drawing the van der Waal's surface of a protein molecule. An extension of the program permits the accessibility of atoms, or groups of atoms, to solvent or solute molecules of specified size to be quantitatively assessed. As defined in this study, the accessibility is proportional to surface area. The accessibility of all atoms in the twenty common amino acids in model tripeptides of the type Ala-X-Ala are given for defined conformation. The accessibilities are also given for all atoms in ribonuclease-S, lysozyme and myogoblin. Internal cavities are defined and discussed. Various summaries of these data are provided. Forty to fifty per cent of the surface area of each protein is occupied by non-polar atoms. The actual numerical results are sensitive to the values chosen for the van der Waal's radii of the various groups. Since there is uncertainty over the correct values for these radii, the derived numbers should only be used as a qualitative guide at this stage.The average change in accessibility for the atoms of all three proteins in going from a hypothetical extended chain to the folded conformation of the native protein is about a factor of 3. This number applies to both polar (nitrogen and oxygen) and non-polar (carbon and sulfur) atoms considered separately. The larger non-polar amino acids tend to be more “buried” in the native form of all three proteins. However, for all classes and for residues within a given class the accessibility changes on folding tend to be highly variable.
The human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS). Two subtypes of the virus, HIV-1 and HIV-2, have been characterized. The protease enzymes from these two subtypes, which are aspartic acid proteases and have been found to be essential for maturation of the infectious particle, share about 50% sequence identity. Differences in substrate and inhibitor binding between these enzymes have been previously reported.
We report the X-ray crystal structures of both HIV-1 and HIV-2 proteases each in complex with the pseudosymmetric inhibitor, CGP 53820, to 2.2 A and 2.3 A, respectively. In both structures, the entire enzyme and inhibitor could be located. The structures confirmed earlier modeling studies. Differences between the CGP 53820 inhibitory binding constants for the two enzymes could be correlated with structural differences.
Minor sequence changes in subsites at the active site can explain some of the observed differences in substrate and inhibitor binding between the two enzymes. The information gained from this investigation may help in the design of equipotent HIV-1/HIV-2 protease inhibitors.
We have previously reported (Newlander et al., J. Med. Chem. 1993, 36, 2321-2331) the design of human immunodeficiency virus type 1 (HIV-1) protease inhibitors incorporating C7 mimetics that lock three amino acid residues of a peptide sequence into a gamma-turn. The design of one such compound, SB203238, was based on X-ray structures of reduced amide aspartyl protease inhibitors. It incorporates a gamma-turn mimetic in the P2-P1' position, where the carbonyl of the C7 ring is replaced with an sp3 methylene group yielding a constrained reduced amide. It shows competitive inhibition with Ki = 430 nM at pH 6.0. The three-dimensional structure of SB203238 bound to the active site of HIV-1 protease has been determined at 2.3 A resolution by X-ray diffraction and refined to a crystallographic R-factor (R = sigma magnitude of Fo magnitude of - magnitude of Fc magnitude of /sigma magnitude of Fo magnitude of, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively) of 0.177. The inhibitor lies in an extended conformation in the active site; however, because of the constrained geometry of the C7 ring, it maintains fewer hydrogen bonds with the protein than in most other HIV-1 protease-inhibitor complexes. More importantly, the inhibitor binds to the enzyme differently than predicted in its design, by binding with the P2-P1' alpha-carbon atoms shifted by approximately one-half a residue toward the N-terminus from their presumed positions. This study illustrates the importance of structural information in an approach to rational drug design.
Examination of the structural basis for antiviral activity, oral pharmacokinetics, and hepatic metabolism among a series of symmetry-based inhibitors of the human immunodeficiency virus (HIV) protease led to the discovery of ABT-538, a promising experimental drug for the therapeutic intervention in acquired immunodeficiency syndrome (AIDS). ABT-538 exhibited potent in vitro activity against laboratory and clinical strains of HIV-1 [50% effective concentration (EC50) = 0.022-0.13 microM] and HIV-2 (EC50 = 0.16 microM). Following a single 10-mg/kg oral dose, plasma concentrations in rat, dog, and monkey exceeded the in vitro antiviral EC50 for > 12 h. In human trials, a single 400-mg dose of ABT-538 displayed a prolonged absorption profile and achieved a peak plasma concentration in excess of 5 micrograms/ml. These findings demonstrate that high oral bioavailability can be achieved in humans with peptidomimetic inhibitors of HIV protease.
We have observed a high correlation between the intermolecular interaction energy (Einter) calculated for HIV-1 protease inhibitor complexes and the observed in vitro enzyme inhibition. A training set of 33 inhibitors containing modifications in the P1' and P2' positions was used to develop a regression equation which relates Einter and pIC50. This correlation was subsequently employed to successfully predict the activity of proposed HIV-1 protease inhibitors in advance of synthesis in a structure-based design program. This included a precursor, 47, to the current phase II clinical candidate, L-735,524 (51). The development of the correlation, its applications, and its limitations are discussed, and the force field (MM2X) and host molecular mechanics program (OPTIMOL) used in this work are described.
(2R,4S,5S,1'S)-2-Phenylmethyl-4-hydroxy-5-(tert-butoxycarbonyl) amino-6-phenylhexanoyl-N-(1'-imidazo-2-yl)-2'-methylpropanamide (compound 2) is a tripeptide analogue inhibitor of HIV-1 protease in which a C-terminal imidazole substituent constitutes an isoelectronic, structural mimic of a carboxamide group. Compound 2 is a potent inhibitor of the protease (K(i) = 18 nM) and inhibits HIV-1 acute infectivity of CD4+ T-lymphocytes (IC50 = 570 nM). Crystallographic analysis of an HIV-1 protease-compound 2 complex demonstrates that the nitrogen atoms of the imidazole ring assume the same hydrogen-bonding interactions with the protease as amide linkages in other peptide analogue inhibitors. The sole substitution of the C-terminal carboxamide of a hydroxyethylene-containing tripeptide analogue with an imidazole group imparts greatly improved pharmacokinetic and oral bioavailability properties on the compound compared to its carboxamide-containing homologue (compound 1). While the oral bioavailability of compound 1 in rats was negligible, compound 2 displayed oral bioavailabilities of 30% and 14%, respectively, in rats and monkeys.
The rational design and synthesis of a highly potent inhibitor of HIV-1 protease have been accomplished. The inhibitor, SB 206343, is based on a model derived from the structure of the MVT-101/HIV-1 protease complex and contains a 4(5)-acylimidazole ring as an isosteric replacement for the P1'--P2' amide bond. It is a competitive inhibitor with an apparent inhibition constant of 0.6 nM at pH 6.0. The three-dimensional structure of SB 206343 bound in the active site of HIV-1 protease has been determined at 2.3 A resolution by X-ray diffraction techniques and refined to a crystallographic discrepancy factor, R (= sigma parallel Fo magnitude of/Fc parallel/sigma magnitude of), of 0.194. The inhibitor is held in the enzyme by a set of hydrophobic and polar interactions. N-3 of the imidazole ring participates in a novel hydrogen-bonding interaction with the bound water molecule, demonstrating the effectiveness of the imidazole ring as an isosteric replacement for the P1'--P2' amide bond in hydroxyethylene-based HIV-1 protease inhibitors. Also present are hydrogen-bonding interactions between N-1 of the imidazole ring and the carbonyl of Gly-127 as well as between the imidazole acyl carbonyl oxygen and the amide nitrogen of Asp-129, exemplifying the peptidomimetic nature of the 4(5)-acylimidazole isostere. All of these interactions are in qualitative agreement with those predicted by the model.
In the development of a treatment for AIDS, the HIV-1 protease has been identified as a good target enzyme for inhibitor design. We previously reported a series of dimeric penicillin-derived C2-symmetric HIV-1 protease inhibitors [Humber, D., et al. (1993) J. Med. Chem. 36, 3120-3128]. In an attempt to reduce the size and optimize the binding of these C2-symmetric inhibitors, molecular modeling studies led to a novel series of monomeric penicillin-derived inhibitors of HIV-1 protease. The binding modes of these monomeric inhibitors have been characterized by X-ray crystallographic and NMR studies. Crystal structures of HIV-1 protease complexed to three inhibitors (GR123976, GR126045, and GR137615) from this series identify the molecular details of the interactions. The binding of GR123976 (IC50 = 2.3 microM) exhibits good hydrophobic contacts but few electrostatic interactions. A strategy of structure-based design and chemical synthesis led to the elaboration of GR123976 to optimize interactions with the protein. Crystallographic analysis of HIV-1 protease complexed to GR126045 and GR137615 identified these interactions with the catalytic aspartates and the protein binding pockets. The crystal structures of the three complexes confirm the presence of the major interactions modeled in order to optimize potency and reveal details of the molecular recognition by HIV-1 protease of this novel series of nonpeptidic inhibitors.
Assembled class I histocompatibility molecules, consisting of heavy chain, beta 2-microglobulin, and peptide ligand, are transported
rapidly to the cell surface. In contrast, the intracellular transport of free heavy chains or peptide-deficient heavy chain-beta
2-microglobulin heterodimers is impaired. A 90-kilodalton membrane-bound chaperone of the endoplasmic reticulum (ER), termed
calnexin, associates quantitatively with newly synthesized class I heavy chains, but the functions of calnexin in this interaction
are unknown. Class I subunits were expressed alone or in combination with calnexin in Drosophila melanogaster cells. Calnexin
retarded the intracellular transport of both peptide-deficient heavy chain-beta 2-microglobulin heterodimers and free heavy
chains. Calnexin also impeded the rapid intracellular degradation of free heavy chains. The ability of calnexin to protect
and retain class I assembly intermediates is likely to contribute to the efficient intracellular formation of class I-peptide
complexes.
Mechanistic information and structure-based design methods have been used to design a series of nonpeptide cyclic ureas that are potent inhibitors of human immunodeficiency virus (HIV) protease and HIV replication. A fundamental feature of these inhibitors is the cyclic urea carbonyl oxygen that mimics the hydrogen-bonding features of a key structural water molecule. The success of the design in both displacing and mimicking the structural water molecule was confirmed by x-ray crystallographic studies. Highly selective, preorganized inhibitors with relatively low molecular weight and high oral bioavailability were synthesized.
The specificity of interactions between biological macromolecules and their ligands may be partially attributed to the directional properties of hydrogen bonds. We have now extended the GRID method (Goodford, P. J. J. Med. Chem. 1985, 28, 849. Boobbyer, D. N. A.; Goodford, P. J.; McWhinnie, P. M.; Wade, R. C. J. Med. Chem. 1989, 32, 1083), of determining energetically favorable ligand binding sites on molecules of known structure, in order to improve the treatment of groups which can make multiple hydrogen bonds. In this method, the interaction energy between a probe (a small chemical group that may be part of a larger ligand) and a target molecule is calculated using an energy function which includes a hydrogen bond term which is dependent on the length of the hydrogen bond, its orientation at the hydrogen-bonding atoms, and their chemical character. The methods described in the preceding paper (Wade, R. C.; Clark, K. J.; Goodford, P. J. J. Med. Chem., preceding paper in this issue) for probes capable of making two hydrogen bonds are here extended to the following probes which have the ability to make more than two hydrogen bonds: ammonium-NH3+, amine-NH2, sp3-hybridized hydroxyl, and water. Use of the improved GRID procedure is demonstrated by the determination of the conformation of an amino acid side chain at the subunit interface in hemoglobin and of the location of water binding sites in human lysozyme.
The directional properties of hydrogen bonds play a major role in determining the specificity of intermolecular interactions. An energy function which takes explicit account of these properties has been developed for use in the determination of energetically favorable ligand binding sites on molecules of known structure by the GRID method (Goodford, P.J.J. Med. Chem. 1985, 28, 849. Boobbyer, D.N.A.; Goodford, P.J.; McWhinnie, P.M.; Wade, R.C.J. Med. Chem. 1989, 32, 1083). In this method, the interaction energy between a target molecule and a small chemical group (a probe), which may be part of a larger ligand, was calculated using an energy function consisting of Lennard-Jones, electrostatic, and hydrogen bond terms. The latter term was a function of the length of the hydrogen bond, its orientation at the hydrogen-bonding atoms, and their chemical nature. We now describe hydrogen bond energy functions which take account of the spatial distribution of the hydrogen bonds made by probes with the ability to form two hydrogen bonds. These functions were designed so as to model the experimentally observed angular dependence of the hydrogen bonds. We also describe the procedure to locate the position and orientation of the probe at which the interaction energy is optimized. The use of this procedure is demonstrated by examples of biological and pharmacological interest which show that it can produce results that are consistent with other theoretical approaches and with experimental observations.
The interface between protein receptor-ligand complexes has been studied with respect to their binary interatomic interactions. Crystal structure data have been used to catalogue surfaces buried by atoms from each member of a bound complex and determine a statistical preference for pairs of amino-acid atoms. A simple free energy model of the receptor-ligand system is constructed from these atom-atom preferences and used to assess the energetic importance of interfacial interactions. The free energy approximation of binding strength in this model has a reliability of about +/- 1.5 kcal/mol, despite limited knowledge of the unbound states. The main utility of such a scheme lies in the identification of important stabilizing atomic interactions across the receptor-ligand interface. Thus, apart from an overall hydrophobic attraction (Young L, Jernigan RL, Covell DG, 1994, Protein Sci 3:717-729), a rich variety of specific interactions is observed. An analysis of 10 HIV-1 protease inhibitor complexes is presented that reveals a common binding motif comprised of energetically important contacts with a rather limited set of atoms. Design improvements to existing HIV-1 protease inhibitors are explored based on a detailed analysis of this binding motif.
Several molecular modeling techniques were used to generate an all-atom molecular model of a receptor binding site starting only from Ca atom coordinates. The model consists of 48 noncontiguous residues of the non-nucleoside binding site of HIV-1 reverse transcriptase and was generated using a congeneric series of nevirapine analogs as structural probes. On the basis of the receptor-ligand atom contacts, the program HINT was used to develop a 3D quantitative structure activity relationship that predicted the rank order of binding affinities for the series of inhibitors. Electronic profiles of the ligands in their docked conformations were characterized using electrostatic potential maps and frontier orbital calculations. These results led to the development of a 3D stereoelectronic pharmacophore which was used to construct 3D queries for database searches. A search of the National Cancer Institute's open database identified a lead compound that exhibited moderate antiviral activity.
In order to improve the design of HIV-1 protease inhibitors, it is essential to understand how they interact with active site residues, particularly the catalytic Asp25 and Asp125 residues. KNI-272 is a promising, potent HIV-1 protease inhibitor (K(i) approximately 5 pM), currently undergoing phase 1 clinical trials. Because KNI-272 is asymmetric, the complex it forms with the homodimeric HIV-1 protease also lacks symmetry, and the two protease monomers can have distinct NMR spectra. Monomer specific signal assignments were obtained for amino acid residues in the drug binding site as well as for six of the eight Asp residues in the protease/KNI-272 complex. Using these assignments, the ionization states of the Asp carboxyl groups were determined from measurements of (a) the pD dependence of the chemical shifts of the Asp carboxyl carbons and (b) the H/D isotope effect upon the Asp carboxyl carbon chemical shifts. The results of these measurements indicate that the carboxyl of Asp25 is protonated while that of Asp125 is not protonated. These findings provide not only the first experimental evidence regarding the distinct protonation states of Asp25/125 in HIV-1 protease/drug complexes, but also shed light on interactions responsible for inhibitor binding that should form the basis for improved drug designs.