ArticleLiterature Review

Drug–target residence time and its implications for lead optimization

March 2007
Nature Reviews Drug Discovery 6(3)

March 2007
6(3)

DOI:10.1038/nrd2281

Authors:

Robert A Copeland

Accent Therapeutics, Inc.

Thomas Meek

Texas A&M University

Much of drug discovery today is predicated on the concept of selective targeting of particular bioactive macromolecules by low-molecular-mass drugs. The binding of drugs to their macromolecular targets is therefore seen as paramount for pharmacological activity. In vitro assessment of drug–target interactions is classically quantified in terms of binding parameters such as IC50 or Kd. This article presents an alternative perspective on drug optimization in terms of drug–target binary complex residence time, as quantified by the dissociative half-life of the drug–target binary complex. We describe the potential advantages of long residence time in terms of duration of pharmacological effect and target selectivity.

An integrated modelling approach for targeted degradation: insights on optimization, data requirements and PKPD predictions from semi- or fully-mechanistic models and exact steady state solutions

Article

Full-text available

Apr 2023
J PHARMACOKINET PHAR

The value of an integrated mathematical modelling approach for protein degraders which combines the benefits of traditional turnover models and fully mechanistic models is presented. Firstly, we show how exact solutions of the mechanistic models of monovalent and bivalent degraders can provide insight on the role of each system parameter in driving the pharmacological response. We show how on/off binding rates and degradation rates are related to potency and maximal effect of monovalent degraders, and how such relationship can be used to suggest a compound optimization strategy. Even convoluted exact steady state solutions for bivalent degraders provide insight on the type of observations required to ensure the predictive capacity of a mechanistic approach. Specifically for PROTACs, the structure of the exact steady state solution suggests that the total remaining target at steady state, which is easily accessible experimentally, is insufficient to reconstruct the state of the whole system at equilibrium and observations on different species (such as binary/ternary complexes) are necessary. Secondly, global sensitivity analysis of fully mechanistic models for PROTACs suggests that both target and ligase baselines (actually, their ratio) are the major sources of variability in the response of non-cooperative systems, which speaks to the importance of characterizing their distribution in the target patient population. Finally, we propose a pragmatic modelling approach which incorporates the insights generated with fully mechanistic models into simpler turnover models to improve their predictive ability, hence enabling acceleration of drug discovery programs and increased probability of success in the clinic.

Prédiction des constantes cinétiques de complexes protéine-ligand

Thesis

Jan 2019

Sonia Ziada

Le coût de plus en plus élevé de l’ensemble du processus de recherche préclinique et de développement clinique d’un médicament pousse la communauté scientifique à limiter au maximum les causes d’échecs. De nombreuses études démontrent qu’une évaluation préclinique des constantes cinétiques de liaison permet de limiter les taux d’échec en phase II des essais cliniques. L’objectif de ce projet de thèse, financé par l’Institut de Recherche Servier (IdRS) à Croissy-sur-Seine, est de développer un outil informatique visant à prédire ces constantes cinétiques en un temps de calcul acceptable, afin de permettre son utilisation en routine en phase précoce de sélection et d’optimisation des molécules actives. Une première partie de cette thèse a été dévolue à l’étude du jeu de données utilisé pour réaliser ces développements techniques. Il est constitué d’inhibiteurs de la kinase dépendante de la cycline 8 (CDK8), une cible thérapeutique émergente de la famille des protéines kinases impliquée dans le cancer colorectal. La deuxième partie de ce travail a porté sur le développement d’un outil visant à classer les composés en fonction de leurs temps de résidence. Cet outil a été ensuite validé sur un jeu de données interne de l’IdRS. Enfin, dans une troisième partie, un protocole a été initié pour prédire les constantes cinétiques de manière quantitative et non qualitative et pouvoir également identifier les déterminants structuraux responsables des propriétés cinétiques des composés.

Predicting Residence Time of GPCR Ligands with Machine Learning

Article

Jan 2022

Drug-target residence time, the duration of binding at a given protein target, has been shown in some protein families to be more significant for conferring efficacy than binding affinity. To carry out efficient optimization of residence time in drug discovery, machine learning models that can predict that value need to be developed. One of the main challenges with predicting residence time is the paucity of data. This chapter outlines all of the currently available ligand kinetic data, providing a repository that contains the largest publicly available source of GPCR-ligand kinetic data to date. To help decipher the features of kinetic data that might be beneficial to include in computational models for the prediction of residence time, the experimental evidence for properties that influence residence time are summarized. Finally, two different workflows for predicting residence time with machine learning are outlined. The first is a single-target model trained on ligand features; the second is a multi-target model trained on features generated from molecular dynamics simulations.

Pharmacodynamic model of slow reversible binding and its applications in pharmacokinetic/pharmacodynamic modeling: review and tutorial

Article

Full-text available

Aug 2022
J PHARMACOKINET PHAR

Therapeutic responses of most drugs are initiated by the rate and degree of binding to their receptors or targets. The law of mass action describes the rate of drug-receptor complex association (kon) and dissociation (koff) where the ratio koff/kon is the equilibrium dissociation constant (Kd). Drugs with slow reversible binding (SRB) often demonstrate delayed onset and prolonged pharmacodynamic effects. This report reviews evidence for drugs with SRB features, describes previous pharmacokinetic/pharmacodynamic (PK/PD) modeling efforts of several such drugs, provides a tutorial on the mathematics and properties of SRB models, demonstrates applications of SRB models to additional compounds, and compares PK/PD fittings of SRB with other mechanistic models. We identified and summarized 52 drugs with in vitro-confirmed SRB from a PubMed literature search. Simulations with a SRB model and observed PK/PD profiles showed delayed and prolonged responses and that increasing doses/kon or decreasing koff led to greater expected maximum effects and a longer duration of effects. Recession slopes for return of responses to baseline after single doses were nearly linear with an inflection point that approaches a limiting value at larger doses. The SRB model newly captured literature data for the antihypertensive effects of candesartan and antiallergic effects of noberastine. Their PD profiles could also be fitted with indirect response and biophase models with minimal differences. The applicability of SRB models is probably commonplace, but underappreciated, owing to the need for in vitro confirmation of binding kinetics and the similarity of PK/PD profiles to models with other mechanistic determinants. Graphical abstract

BiSim Tool: a binding simulation tool to aid and simplify ligand-binding assay design and development

Article

Full-text available

Apr 2024

Ligand-binding assays (LBAs) rely on the reversible, noncovalent binding between the analyte of interest and the assay reagents, and understanding their dynamic equilibrium is key to building robust LBA methods. Although the dynamic interplay of free and bound fractions can be calculated using mathematical models, these are not routinely applied. This approach is costly in terms of both assay development time and reagents, and can result in an under-exploration of the possible parameter combinations. Therefore, we have created a user-friendly simulation tool to facilitate LBA development (the BiSim Tool). We describe the models driving the mathematical simulations and the main features of our software solution by means of case studies, illustrating the tool's value in drug development. To support drug development for all patients worldwide, the BiSim Tool is now available as an open-source code project and as a free web-based tool at https://proteinbindingsimulation.shinyapps.io/BiSim-ProteinBindingSimulation [ 1 ].

Preclinical evaluation of the SARS-CoV-2 M inhibitor RAY1216 shows improved pharmacokinetics compared with nirmatrelvir

Article

Full-text available

Mar 2024

Although vaccines are available for SARS-CoV-2, antiviral drugs such as nirmatrelvir are still needed, particularly for individuals in whom vaccines are less effective, such as the immunocompromised, to prevent severe COVID-19. Here we report an α-ketoamide-based peptidomimetic inhibitor of the SARS-CoV-2 main protease (Mpro), designated RAY1216. Enzyme inhibition kinetic analysis shows that RAY1216 has an inhibition constant of 8.4 nM and suggests that it dissociates about 12 times slower from Mpro compared with nirmatrelvir. The crystal structure of the SARS-CoV-2 Mpro:RAY1216 complex shows that RAY1216 covalently binds to the catalytic Cys145 through the α-ketoamide group. In vitro and using human ACE2 transgenic mouse models, RAY1216 shows antiviral activities against SARS-CoV-2 variants comparable to those of nirmatrelvir. It also shows improved pharmacokinetics in mice and rats, suggesting that RAY1216 could be used without ritonavir, which is co-administered with nirmatrelvir. RAY1216 has been approved as a single-component drug named ‘leritrelvir’ for COVID-19 treatment in China.

Stereoselective recognition of morphine enantiomers by μ-opioid receptor

Article

Full-text available

Jan 2024

Stereospecific recognition of chiral molecules plays a crucial role in biological systems. The μ-opioid receptor (MOR) exhibits binding affinity towards (-)-morphine, a well-established gold standard in pain management, while it shows minimal binding affinity for the (+)-morphine enantiomer, resulting in a lack of analgesic activity. Understanding how MOR stereo-selectively recognizes morphine enantiomers has remained a puzzle in neuroscience and pharmacology for over half a century due to the lack of direct observation techniques. To unravel this mystery, we constructed the binding and unbinding processes of morphine enantiomers with MOR via molecular dynamics simulations to investigate the thermodynamics and kinetics governing MOR’s stereoselective recognition of morphine enantiomers. Our findings reveal that the binding of (-)-morphine stabilizes MOR in its activated state, exhibiting a deep energy well and a prolonged residence time. In contrast, (+)-morphine fails to sustain the activation state of MOR. Furthermore, the results suggest that specific residues, namely D1142.50 and D1473.32, are deprotonated in the active state of MOR bound to (-)-morphine. This work highlights that the selectivity in molecular recognition goes beyond binding affinities, extending into the realm of residence time.

A miniaturized mode-of-action profiling platform enables high throughput characterization of the molecular and cellular dynamics of EZH2 inhibition

Article

Full-text available

Jan 2024

The market approval of Tazemetostat (TAZVERIK) for the treatment of follicular lymphoma and epithelioid sarcoma has established “enhancer of zeste homolog 2” (EZH2) as therapeutic target in oncology. Despite their structural similarities and common mode of inhibition, Tazemetostat and other EZH2 inhibitors display differentiated pharmacological profiles based on their target residence time. Here we established high throughput screening methods based on time-resolved fluorescence energy transfer, scintillation proximity and high content analysis microscopy to quantify the biochemical and cellular binding of a chemically diverse collection of EZH2 inhibitors. These assays allowed to further characterize the interplay between EZH2 allosteric modulation by methylated histone tails (H3K27me3) and inhibitor binding, and to evaluate the impact of EZH2’s clinically relevant mutant Y641N on drug target residence times. While all compounds in this study exhibited slower off-rates, those with clinical candidate status display significantly slower target residence times in wild type EZH2 and disease-related mutants. These inhibitors interact in a more entropy-driven fashion and show the most persistent effects in cellular washout and antiproliferative efficacy experiments. Our work provides mechanistic insights for the largest cohort of EZH2 inhibitors reported to date, demonstrating that—among several other binding parameters—target residence time is the best predictor of cellular efficacy.

Data Driven Classification of Ligand Unbinding Pathways

Preprint

Full-text available

Aug 2023

Studying the pathways of ligand-receptor binding is essential to understand the mechanism of target recognition by small molecules. The binding free energy and kinetics of protein-ligand complexes can be computed using molecular dynamics (MD) simulations, often in quantitative agreement with experiments. However, only a qualitative picture of the ligand binding/unbinding paths can be obtained through a conventional analysis of the MD trajectories. Besides, the higher degree of manual effort involved in analyzing pathways limits its applicability in large-scale drug discovery. Here we address this limitation by introducing an automated approach for analyzing molecular transition paths with a particular focus on protein-ligand dissociation. Our method is based on the dynamic time-warping (DTW) algorithm, originally designed for speech recognition. We accurately classified molecular trajectories using a very generic descriptor set of contacts or distances. Our approach outperforms manual classification by distinguishing between parallel dissociation channels, within the pathways identified by visual inspection. Most notably, we could compute exit-path-specific ligand-dissociation kinetics. The unbinding timescale along the fastest path agrees with the experimental residence time, providing a physical interpretation to our entirely data-driven protocol. In combination with appropriate enhanced sampling algorithms, this technique can be used for the initial exploration of ligand-dissociation pathways as well as for calculating path-specific thermodynamic and kinetic properties.

Dissociation kinetics of small-molecule inhibitors in Escherichia coli is coupled to physiological state of cells

Article

Full-text available

Feb 2023

Bioactive small-molecule inhibitors represent a treasure chest for future drugs. In vitro high-throughput screening is a common approach to identify the small-molecule inhibitors that bind tightly to purified targets. Here, we investigate the inhibitor-target binding/unbinding kinetics in E. coli cells using a benzimidazole-derivative DNA inhibitor as a model system. We find that its unbinding rate is not constant but depends on cell growth rate. This dependence is mediated by the cellular activity, forming a feedback loop with the inhibitor’s activity. In accordance with this feedback, we find cell-to-cell heterogeneity in inhibitor-target interaction, leading to co-existence of two distinct subpopulations: actively growing cells that dissociate the inhibitors from the targets and non-growing cells that do not. We find similar heterogeneity for other clinical DNA inhibitors. Our studies reveal a mechanism that couples inhibitor-target kinetics to cell physiology and demonstrate the significant effect of this coupling on drug efficacy. This study investigates how binding/unbinding kinetics of a benzimidazole-derivative DNA inhibitor in E. coli cells depends on cell growth and physiology and demonstrates the effect of this coupling on drug efficacy.

The nanomolar affinity of C-phycocyanin from virtual screening of microalgal bioactive as potential ACE2 inhibitor for COVID-19 therapy

Article

Full-text available

Jan 2023

The global pandemic of COVID-19 caused by SARS-CoV-2 has caused more than 400 million infections with more than 5.7 million deaths worldwide, and the number of validated therapies from natural products for treating coronavirus infections needs to be increased. Therefore, the virtual screening of bioactive compounds from natural products based on computational methods could be an interesting strategy. Among many sources of bioactive natural products, compounds from marine organisms, particularly microalgae and cyanobacteria, can be potential antiviral agents. The present study investigates bioactive antiviral compounds from microalgae and cyanobacteria as a potential inhibitor of SARS-CoV-2 by targeting Angiotensin-Converting Enzyme II (ACE2) using integrated in silico and in vitro approaches. Our in silico analysis demonstrates that C-Phycocyanin (CPC) can potentially inhibit the binding of ACE2 receptor and SARS-CoV-2 with the docking score of -9.7 kcal mol⁻¹. This score is relatively more favorable than the native ligand on ACE2 receptor. Molecular dynamic simulation also reveals the stability interaction between both CPC and ACE2 receptor with a root mean square deviation (RMSD) value of 1.5 Å. Additionally, our in vitro analysis using the surface plasmon resonance (SPR) method shows that CPC has a high affinity for ACE2 with a binding affinity range from 5 to 125 µM, with KD 3.37 nM. This study could serve as a reference to design microalgae- or cyanobacteria-based antiviral drugs for prophylaxis in SARS-CoV-2 infections.

Design, synthesis, and characterization of novel fluorogenic substrates of the proprotein convertases furin, PC1/3, PC2, PC5/6, and PC7

Article

Aug 2022
ANAL BIOCHEM

Proprotein convertases (PCs) are involved in the pathogenesis of various diseases, making them promising drug targets. Most assays for PCs have been performed with few standard substrates, regardless of differences in cleavage efficiencies. Derived from studies on substrate-analogue inhibitors, 11 novel substrates were synthesized and characterized with five PCs. H-Arg-Arg-Tle-Lys-Arg-AMC is the most efficiently cleaved furin substrate based on its kcat/KM value. Due to its higher kcat value, acetyl-Arg-Arg-Tle-Arg-Arg-AMC was selected for further measurements to demonstrate the benefit of this improved substrate. Compared to our standard conditions, its use allowed a 10-fold reduction of the furin concentration, which enabled Ki value determinations of previously described tight-binding inhibitors under classical conditions. Under these circumstances, a slow-binding behavior was observed for the first time with inhibitor MI-1148. In addition to furin, four additional PCs were used to characterize these substrates. The most efficiently cleaved PC1/3 substrate was Ac-Arg-Arg-Arg-Tle-Lys-Arg-AMC. The highest kcat/KM values for PC2 and PC7 were found for the N-terminally unprotected analogue of this substrate, although other substrates possess higher kcat values. The highest efficiency for PC5/6A was observed for the substrate Ac-Arg-Arg-Tle-Lys-Arg-AMC. In summary, we have identified new substrates for furin, PC1/3, PC2, and PC7 suitable for improved enzyme-kinetic measurements.

Putative Inhibitors for Six-Helix Bundle Post-Fusion Complex of COVID-19 Disease Virus

Article

Full-text available

Nov 2023

Marium Basheer al-khafaji

The new outbreak caused by one of the coronaviruses (COVID-19) threatens humanity, so most research communities are working to fight this threat. Computational approaches could accelerate in finding drugs, peptides, or other possibilities. In this study structure-based drug design (SBDD) as a computational technology was used to find inhibitors to the viral fusion process which is very important to let the virus enter the cells and continue its life cycle. The modeled structure of the six-helix bundle post-fusion complex (pdb ID 6LXT) was used, hundreds of compounds were found in the first run, but, upon filtration and selection only 7 molecules were obtained ; 2 from the special database, the others from the natural product database and one from the food database. These were docked with the target protein (post -fusion complex) with an acceptable binding affinity at zero value for RMSD through different intermolecular interactions. These molecules/ligands are worth to have real applications.

Molecular determinants of antagonist interactions with chemokine receptors CCR2 and CCR5

Preprint

Full-text available

Nov 2023

By driving monocyte chemotaxis, the chemokine receptor CCR2 shapes inflammatory responses and the formation of tumor microenvironments. This makes it a promising target in inflammation and immuno-oncology. Unfortunately, despite extensive efforts, no CCR2-targeting therapeutics have yet reached the clinic. Cited reasons include the redundancy of the chemokine system, suboptimal properties of compound candidates, and poor agreement of clinical responses with preclinical murine model studies. Structure-based drug design approaches can rationalize and greatly accelerate CCR2 compound discovery and optimization. The prerequisites for such efforts include a good atomic-level understanding of the molecular determinants of action of existing antagonists. In this study, using molecular docking and artificial-intelligence-powered compound library screening, we uncover the structural principles of small molecule antagonism and selectivity towards CCR2 and its sister receptor CCR5. We show that CCR2 orthosteric inhibitors universally occupy an inactive-state-specific tunnel between receptor helices 1 and 7; we also discover an unexpected role for an extra-helical groove accessible through this tunnel, suggesting its potential as a new targetable interface for CCR2 and CCR5 modulation. We implicate a single CCR2 residue, S101 2.63 , as a determinant of CCR2/CCR5 and human/mouse antagonist selectivity, and corroborate its role through experimental gain-of-function mutagenesis. We systematically identify the binding determinants for various chemotypes of allosteric antagonists. We establish a critical role of induced fit in antagonist recognition, reveal strong chemotype selectivity of existing structures, and demonstrate the high predictive potential of a new deep-learning-based compound scoring function. Finally, we expand the available CCR2 structural landscape with computationally generated chemotype-specific models well-suited for structure-based antagonist design.

A Critical Analysis of the FDA Omics Perspective on Pharma-codynamic Biomarkers to Support Biosimilarity, and its Surrogates.

Preprint

Full-text available

Sep 2023

Sarfaraz K. Niazi

Demonstrating biosimilarity entails comprehensive analytical evaluations, clinical pharmacolo-gy profiling, and efficacy testing for at least one medical indication in patients. These require-ments are stipulated by the U.S. Biologics Price Competition and Innovation Act (BPCIA). The costliest element—efficacy testing—can be waived if other compliance benchmarks are satisfied, including comparing functional pharmacodynamic (PD) biomarkers, even when they do not di-rectly correlate with clinical outcomes. Most biological drugs, such as monoclonal antibodies (mAbs), lack identifiable PD biomarkers. The FDA has employed various 'omics' technologies to identify potential PD biomarkers, including proteomics, glycomics, transcriptomics, genomics, epigenomics, and metabolomics. Although these efforts provide a robust scientific basis for estab-lishing biosimilarity, they are neither practical nor necessarily superior to existing functional biomarkers, such as receptor binding and mode-of-action outcomes. As we report for the first time, these functional biomarkers can effectively serve as PD indicators for all FDA-licensed bio-logical drugs. We recommend that the FDA consider officially listing these functional biomarkers to expedite and reduce the cost of biosimilar development, thereby increasing the accessibility of biological drugs. PD surrogates, like the receptor binding and pharmacokinetic profiles, are more robust and offer a rational solution to finding PD markers to compare for establishing biosimi-larity.

An antibiotic from an uncultured bacterium binds to an immutable target

Article

Full-text available

Aug 2023
CELL

Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant Gram-positive bacterial pathogens without detectable resistance. Using biochemical assays, solid-state nuclear magnetic resonance, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C55PP, lipid II, and lipid IIIWTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. This potent antibiotic holds the promise of enabling the design of improved therapeutics that kill bacterial pathogens without resistance development.

An open-label, multicenter, randomized, parallel, single-dose, comparative bioavailability study of two triamcinolone hexacetonide injectable suspensions in patients with knee osteoarthritis

Article

Full-text available

Jul 2023

Background: Triamcinolone hexacetonide (THA), a synthetic glucocorticoid with low solubility, can provide sustained pain relief and less systemic side effects in patients with knee osteoarthritis. This study aimed to characterize pharmacokinetic profile of THA-test product containing 20 mg/ml injectable suspension and compare its bioavailability with the standard reference in Indian patients with knee osteoarthritis. Methods: In this open-label, randomized, multicenter study, 44 adult patients were randomized (1:1; test n=23, reference n=21) to receive a single dose of test or reference products. The primary objective was to characterize the pharmacokinetic profile and compare bioavailability of both products via serum triamcinolone acetonide (TCA) concentration. Secondary objectives included safety and tolerability evaluation, impact on hypothalamic-pituitary-adrenal axis, and efficacy of test and reference products in reducing index knee pain. Results: Both products were absorbed with a median Tmax of 23.9 hours. Comparative bioavailability analysis demonstrated no statistically significant formulation effect for ln-transformed Cmax (1098.052 pg/ml for test, 1333.850 pg/ml for reference) and AUC0-t (159112.561 pg×h/ml for test, 211531.035 pg×h/ml for reference) for TCA. T/R ratio for Cmax was 82.3% and T/R ratio for AUC0-t was 75.2%, with >100% inter-subject variability for both Cmax and AUC0-t. Additionally, recovery time of cortisol levels of test and reference arms was 96 hours and 456 hours, respectively. Both products significantly reduced knee pain (p<0.0001). Conclusions: The test product provided lower systemic exposure and faster recovery of serum cortisol levels than the reference, while still providing similar beneficial effect in sustained index knee pain reduction.

Trends and challenges in microfluidic methods for protein manipulation-A review

Article

May 2023
ELECTROPHORESIS

Proteins are important molecules involved in an immensely large number of biological processes. Being capable of manipulating proteins is critical for developing reliable and affordable techniques to analyze and/or detect them. Such techniques would enable the production of therapeutic agents for the treatment of diseases or other biotechnological applications (e.g., bioreactors or biocatalysis). Microfluidic technology represents a potential solution to protein manipulation challenges because of the diverse phenomena that can be exploited to achieve micro- and nanoparticle manipulation. In this review, we discuss recent contributions made in the field of protein manipulation in microfluidic systems using different physicochemical principles and techniques, some of which are miniaturized versions of already established macro-scale techniques.

Comparative analysis of drug-like EP300/CREBBP acetyltransferase inhibitors

Preprint

Full-text available

May 2023

The human acetyltransferase paralogs EP300 and CREBBP are master regulators of lysine acetylation whose activity has been implicated in various cancers. In the half-decade since the first drug-like inhibitors of these proteins were reported, three unique molecular scaffolds have taken precedent: an indane spiro-oxazolidinedione (A-485), a spiro-hydantoin (iP300w), and an aminopyridine (CPI-1612). Despite increasing use of these molecules to study lysine acetylation, the dearth of data regarding their relative biochemical and biological potencies makes their application as chemical probes a challenge. To address this gap, here we present a comparative study of drug-like EP300/CREBBP acetyltransferase inhibitors. First, we determine the biochemical and biological potencies of A-485, iP300w, and CPI-1612, highlighting the increased potency of the latter two compounds at physiological acetyl-CoA concentrations. Cellular evaluation shows that inhibition of histone acetylation and cell growth closely aligns with the biochemical potencies of these molecules, consistent with an on-target mechanism. Finally, we demonstrate the utility of comparative pharmacology by using it to investigate the hypothesis that increased CoA synthesis caused by knockout of PANK4 can competitively antagonize binding of EP300/CREBBP inhibitors and demonstrate proof-of-concept photorelease of a potent inhibitor molecule. Overall, our study demonstrates how knowledge of relative inhibitor potency can guide the study of EP300/CREBBP-dependent mechanisms and suggests new approaches to target delivery, thus broadening the therapeutic window of these preclinical epigenetic drug candidates.

A novel BRET-based assay to investigate binding and residence time of unmodified ligands to the human lysosomal ion channel TRPML1 in intact cells

Article

Full-text available

May 2023
J BIOL CHEM

Here we report a Bioluminescence Resonance Energy Transfer (BRET) assay as a novel way to investigate the binding of unlabeled ligands to the human Transient Receptor Potential Mucolipin 1 (hTRPML1), a lysosomal ion channel involved in several genetic diseases and cancer progression. This novel BRET assay can be used to determine equilibrium and kinetic binding parameters of unlabeled compounds to hTRPML1 using intact human-derived cells, thus complementing the information obtained using functional assays based on ion channel activation. We expect this new BRET assay to expedite the identification and optimization of cell-permeable ligands that interact with hTRPML1 within the physiologically-relevant environment of lysosomes.

Unveiling the molecular recognition process between USP7 N-terminal TRAF-like domain and β- catenin for small molecule discovery

Preprint

Full-text available

Apr 2023

Protein-protein interactions drive the majority of biological processes, but their poorly characterized molecular recognition mechanism serves as bottleneck for therapeutic interventions. Recognition of substrates via speci c sites induce regulation in the protein's activity which instigates abnormalities in cellular processes. Ubiquitin speci c protease-7 (USP7), a negative regulator of Wnt-signaling pathway, deubiquitinates β-catenin and causes colorectal cancer (CRC). USP7 N-terminal TRAF-like domain's (NTLD) orthosteric site serves as hub for the recognition of substrates, however, its role for β-catenin is still elusive. Furthermore, the pivotal structural determinant and signature residues that drives recognition process between USP7 and β-catenin is unexplored. The NTLD is an unchartered territory of USP7 as no small molecules are reported by targeting its orthosteric site. Here, we underscore the key structural determinants and thermodynamic components that possibly drive the recognition of USP7-β-catenin by using extensive computational approaches. By characterizing this site, six potential hits are identi ed from the library of 1.1 million compounds through structure-guided virtual screening. Among six dynamically stable hits, H1 and H6 mimics the orientation and interaction map with endogenous substrate peptides at an atomic level without using any biased approach. Consequently, our approach put-forth a novel route to design potent USP7 inhibitors by targeting NTLD.

Drug Design in the Exascale Era: A Perspective from Massively Parallel QM/MM Simulations

Preprint

Full-text available

Mar 2023

The initial phases of drug discovery - in silico drug design - could benefit from first principle Quantum Mechanics / Molecular Mechanics (QM/MM) molecular dynamics (MD) simulations in explicit solvent, yet many applications are currently limited by the short time scales that this approach can cover. Developing scalable first principle QM/MM MD interfaces fully exploiting current exascale machines - so far an unmet and crucial goal - will help overcome this problem, opening the way to the study of the thermodynamics and kinetics of ligand binding to protein with first principle accuracy. Here, taking two relevant case studies involving the interactions of ligands with rather large enzymes, we showcase the use of our recently developed massively scalable MiMiC QM/MM framework (currently using DFT to describe the QM region) to investigate reactions and ligand binding in enzymes of pharmacological relevance. We also demonstrate for the first time strong scaling of MiMiC QM/MM MD simulations with parallel efficiency above 70\% with over 40,000 cores. Thus, among many others, the MiMiC interface represents a promising candidate towards exascale applications by combining machine learning with statistical mechanics based algorithms tailored for exascale supercomputers.

Structural basis of selective cannabinoid CB2 receptor activation

Article

Full-text available

Mar 2023

Cannabinoid CB2 receptor (CB2R) agonists are investigated as therapeutic agents in the clinic. However, their molecular mode-of-action is not fully understood. Here, we report the discovery of LEI-102, a CB2R agonist, used in conjunction with three other CBR ligands (APD371, HU308, and CP55,940) to investigate the selective CB2R activation by binding kinetics, site-directed mutagenesis, and cryo-EM studies. We identify key residues for CB2R activation. Highly lipophilic HU308 and the endocannabinoids, but not the more polar LEI-102, APD371, and CP55,940, reach the binding pocket through a membrane channel in TM1-TM7. Favorable physico-chemical properties of LEI-102 enable oral efficacy in a chemotherapy-induced nephropathy model. This study delineates the molecular mechanism of CB2R activation by selective agonists and highlights the role of lipophilicity in CB2R engagement. This may have implications for GPCR drug design and sheds light on their activation by endogenous ligands.

Turn On, Tune In, Turnover! Target Biology Impacts In Vivo Potency, Efficacy, and Clearance

Article

Jan 2023

Even though significant efforts have been spent in recent years to understand and define the determinants of in vivo potency and clearance, important pieces of information are still lacking. By introducing target turnover into the reasoning, we open up to further the understanding of central factors important to the optimization of translational dose-concentration-response predictions. We describe: i) new ('open' model) expressions of the in vivo potency and efficacy parameters which embody target turnover, binding and complex kinetics, also capturing full, partial, and inverse ago-nism, and antagonism, ii) a detailed examination of 'open' models to show what potency and efficacy parameters have in common and how they differ, and, iii) a comprehensive literature review showing that target turnover rate varies with age, species, tissue/subregion, treatment, disease state, hormonal and nutritional state, and day-night cycle. The new 'open' model expression which integrates system- and drug properties, shows that: fractional turnover rates rather than the absolute target or ligand-target complex expression determine necessary drug exposure via in vivo potency; absolute ligand-target expression determines the need of drug, based on the transduction Rho and in vivo efficacy parameters; the free enzyme concentration determines clearance and maximum metabolic rate; the fractional turnover rate determines time to equilibrium between substrate, free enzyme and complex; properties of substrate, target, and complex demonstrate non-saturable metabolic behavior at equilibrium; non-linear processes previously referred to as capacity- and time-dependent kinetics may occasionally have been disequilibria; and, the 'open' model may pinpoint why some subjects differ in their demand of drug. Significance Statement 'Understand the target turnover' is a central tenet in many translational dose-concentration-response predictions. New 'open' model expressions of in vivo potency, efficacy parameter and clearance are derived and anchored onto a comprehensive literature review showing that target turnover rate varies with age, species, tissue/subregion, treatment, disease, hormonal and nutritional state, day-night cycle, and more. Target turnover concepts will therefore significantly impact fundamental aspects of pharmacodynamics and pharmacokinetics, thereby also the basics of drug discovery, development, and optimization of clinical dosing.

Adenosine A 1 receptor agonism protection mechanism in intestinal ischemia/reperfusion injury via activation of PI3K/Akt signaling

Article

Full-text available

Nov 2022

Intestinal ischemia/reperfusion (I/R) injury is a common clinical problem with a high mortality rate, resulting from loss of blood flow to an intestinal segment. Adenosine serves a protective role in intestinal I/R injury; however, its potential mechanism is not completely understood. The present study aimed to investigate the protective effects of adenosine A1 receptor (A1R) agonists CPA and LUF6941 and whether their mechanisms are associated with the PI3K/Akt signaling pathway. To simulate intestinal I/R injury, a cell oxygen-glucose deprivation/reoxygenation (OGD/R) model was established and the human colon cancer cell line (Caco-2) was incubated with A1R agonists before OGD/R treatment. The viability of Caco-2 cells was detected by PI and Cell Counting Kit-8 assay, apoptosis was detected using flow cytometry and western blotting was used to analyze protein expression levels of PI3K, Akt and p53 in Caco-2 cells. A1R agonist pretreatment protected Caco-2 cells against OGD/R-induced cell damage and activated PI3K/Akt signaling. Additionally, apoptosis was inhibited by downregulating phosphorylation of p53 protein, as evidenced by increased cell viability. These findings suggested that A1R agonists decreased OGD/R damage in Caco-2 cells, which may be due to their anti-apoptotic effects and activation of the PI3K/Akt/p53 signal pathway.

Toward high-throughput predictive modeling of protein binding/unbinding kinetics

Preprint

Full-text available

Sep 2015

One of the unaddressed challenges in drug discovery is that drug potency determined in vitro is not a reliable indicator of drug efficacy and toxicity in humans. Accumulated evidences suggest that the in vivo activity is more strongly correlated with the binding/unbinding kinetics than the equilibrium thermodynamics of protein-ligand interactions (PLI) in many cases. However, existing experimental and computational techniques are both insufficient in studying the molecular details of kinetics process of PLI. Consequently, we not only have limited mechanistic understanding of the kinetic process but also lack a practical platform for the high-throughput screening and optimization of drug leads based on their kinetic properties. Here we address this unmet need by integrating energetic and conformational dynamic features derived from molecular modeling with multi-task learning. To test our method, HIV-1 protease drug complexes are used as a model system. Our integrated model provides us with new insights into the molecular determinants of the kinetics of PLI. We find that the coherent coupling of conformational dynamics between protein and ligand may play a critical role in determining the kinetic rate constants of PLI. Furthermore, we demonstrated that Normal Mode Analysis (NMA) is an efficient method to capture conformational dynamics of the binding/unbinding kinetics. Coupled with the multi-task learning, we can predict combined kon and koff accurately with an accuracy of 74.35%. Thus, it is possible to screen and optimize compounds based on their kinetic property. Further development of such computational tools will bridge one of the critical missing links between in vitro drug screening and in vivo drug efficacy and toxicity.

Selectivity and ranking of tight-binding JAK-STAT inhibitors using Markovian milestoning with Voronoi tessellations

Preprint

Nov 2022

Janus kinases (JAK) are a group of proteins in the non-receptor tyrosine kinase (NRTKs) family that play a crucial role in growth, survival, and angiogenesis. They are activated by cytokines through the Janus kinase - signal transducer and activator of transcription (JAK-STAT) signaling pathway. JAK-STAT signaling pathways have significant roles in the regulation of cell division, apoptosis, and immunity. Identification of the V617F mutation in the Janus homology 2 (JH2) domain of JAK2 leading to myeloproliferative disorders has stimulated great interest in the drug discovery community to develop JAK2-specific inhibitors. However, such inhibitors should be selective towards JAK2 over other JAKs and display an extended residence time. Recently, novel JAK2/STAT5 axis inhibitors (N-(1H-pyrazol-3-yl)pyrimidin-2-amino derivatives) have displayed extended residence times (hours or longer) on target and adequate selectivity excluding JAK3. To facilitate a deeper understanding of the kinase-inhibitor interactions and advance the development of such inhibitors, we utilize a multiscale Markovian milestoning with Voronoi tessellations (MMVT) approach within the Simulation-Enabled Estimation of Kinetic Rates v.2 (SEEKR2) program to rank-order these inhibitors based on their kinetic properties and further explain the selectivity of JAK2 inhibitors over JAK3. Our approach investigates the kinetic and thermodynamic properties of JAK-inhibitor complexes in a user-friendly, fast, efficient, and accurate manner compared to other brute force and hybrid enhanced sampling approaches.

Lung penetration and pneumococcal target binding of antibiotics in lower respiratory tract infection

Article

Oct 2022

Objectives To achieve the therapeutic effects, antibiotics must penetrate rapidly into infection sites and bind to targets. This study reviewed updated knowledge on the ability of antibiotics to penetrate into the lung, their physicochemical properties influencing the pulmonary penetration and their ability to bind to targets on pneumococci. Methods A search strategy was developed using PubMED, Web of Science and ChEMBL. Data on serum protein binding, drug concentration, target binding ability, drug transporters, lung penetration, physicochemical properties of antibiotics in low respiratory tract infection (LRTI) were collected. Results It was seen that infection site-to-serum concentration ratios of most antibiotics are greater than 1 at different time points except for ceftriaxone, clindamycin and vancomycin. Most agents have proper physicochemical properties that facilitate antibiotic penetration. In antimicrobial-resistant Streptococcus pneumoniae, the binding affinity of antibiotics to targets mostly decreases compared to that in susceptible strains. The data on binding affinity of linezolid, clindamycin and vancomycin were insufficient. The higher drug concentration at the infection sites compared to that in the blood can be associated with inflammation conditions. Little evidence showed the effect of drug transporters on the clinical efficacy of antibiotics against LRTI. Conclusions Data on antibiotic penetration into the lung in LRTI patients and binding affinity of antibiotics for pneumococcal targets are still limited. Further studies are required to clarify the associations of the lung penetration and target binding ability of antibitotics with therapeutic efficacy to help propose the right antibiotics for LRTI.

Characterizing (un)binding mechanism of USP7 inhibitors to unravel the cause of enhanced binding potencies at allosteric checkpoint

Article

Full-text available

Aug 2022
PROTEIN SCI

The ability to predict the intricate mechanistic behavior of ligands and associated structural determinants during protein–ligand (un)binding is of great practical importance in drug discovery. Ubiquitin specific protease‐7 (USP7) is a newly emerging attractive cancer therapeutic target with bound allosteric inhibitors. However, none of the inhibitors have reached clinical trials, allowing opportunities to examine every aspect of allosteric modulation. The crystallographic insights reveal that these inhibitors have common properties such as chemical scaffolds, binding site and interaction fingerprinting. However, they still possess a broader range of binding potencies, ranging from 22 nM to 1,300 nM. Hence, it becomes more critical to decipher the structural determinants guiding the enhanced binding potency of the inhibitors. In this regard, we elucidated the atomic‐level insights from both interacting partners, that is, protein–ligand perspective, and established the structure–activity link between USP7 inhibitors by using classical and advanced molecular dynamics simulations combined with linear interaction energy and molecular mechanics‐Poisson Boltzmann surface area. We revealed the inhibitor potency differences by examining the contributions of chemical moieties and USP7 residues, the involvement of water‐mediated interactions, and the thermodynamic landscape alterations. Additionally, the dissociation profiles aided in the establishment of a correlation between experimental potencies and structural determinants. Our study demonstrates the critical role of blocking loop 1 in allosteric inhibition and enhanced binding affinity. Comprehensively, our findings provide a constructive expansion of experimental outcomes and show the basis for varying binding potency using in‐silico approaches. We expect this atomistic approach to be useful for effective drug design.

Examining the mechanism of action of small-molecule negative allosteric modulators of the human chemokine receptor CXCR2

Article

Jul 2022

Desislava N. Nesheva

The CXC chemokine receptor 2 (CXCR2) is a G protein-coupled receptor (GPCR) with key functions in neutrophil trafficking and activation both during normal homeostasis, and in acute and chronic inflammation. In addition, CXCR2 signalling promotes tumour survival through the mediation of cell proliferation and metastasis, angiogenesis and immune suppression. Despite the therapeutic potential of inhibiting CXCR2 for the treatment of inflammatory conditions and cancer, there is currently not an approved treatment at the receptor. This is largely due to the challenging task of balancing the successful treatment of inflammation or cancer suppression whilst maintaining the homeostatic function of the immune system intact when blocking CXCR2 (Cheng et al., 2019a). There are a range of structurally distinct negative allosteric modulators (NAMs) of CXCR2 compounds that bind to the receptor at an intracellular pocket overlapping with the site of G protein coupling. Two compounds – navarixin and AZD5069 remain in clinical trials as combination therapies for the treatment of cancer. These compounds, in particular, have been reported to have slow dissociation kinetics at CXCR2. This thesis generated new approaches to explore the in vitro pharmacology of candidate CXCR2 NAMs, in particular to understand their mechanism of action in more depth. A number of key questions were identified to address – first, the ability of NAMs to regulate CXCR2 signalling through different effector proteins (e.g. arrestins as well as G proteins); second, the extent to which different NAMs can regulate CXCR2 conformation and modulate chemokine binding, as well as blocking effector coupling; and third, the extent to which NAM binding kinetics at the intracellular site, as well as the allosteric nature of the mechanism, influenced the functional profile of their antagonism over time. First, we co-expressed the human CXCR2 receptor tagged C- terminally with the LgBiT fragment, and β-arrestin2 and mini Gαo effectors with the SmBit fragment of the Nanoluciferase enzyme to generate a luciferase complementation assay (NanoBiT) for CXCR2-effector interactions in stably transfected HEK293 cells. These assays provided live-cell real time readouts of the agonist chemokine CXCL8 activation, and the effects over time of NAM inhibition. For the range of NAM pharmacophores explored, these approaches demonstrated their equivalent inhibition of both mini G protein and arrestin receptor interactions. We also identified differences among the NAMs in their ability to supress the basal receptor activation and in the surmountability of their effects. Using mathematical modelling approaches and comparison of close homologues (enantiomers) of navarixin, NAMs functional effects were attributed to their binding kinetics properties showing that slow koff NAMs insurmountably supress receptor-effector interactions, due to the insufficient time of binding equilibrium to be established. In contrast, fast koff NAMs promoted rightward shifts in the CXCL8 concentration-response curves likely due to negative binding cooperativity between the NAM and the orthosteric agonist. Next, a commercially available AF647 labelled CXCL8 peptide was used to establish a non-radiolabelled CXCR2 binding assay format via both imaging and TR-FRET methodologies, applicable in whole-cells and in membrane preparations. NAMs fully inhibited tracer binding at CXCR2 in high sodium- conditions suggesting stabilisation of the inactive receptor conformation and apparently mutually exclusive binding of the NAM and chemokine, despite the difference in their topography of binding sites. Under conditions in which receptor transition to an active conformation would be better promoted (low sodium), an allosteric effect of NAM inhibition was demonstrated, and an influence on labelled chemokine dissociation kinetics measured in the real time homogeneous TR-FRET assay. In developing novel receptor-effector interaction and fluorescent ligand approaches applicable to real time studies of binding and signalling, these results provide new information on the action of intracellular NAMs at the CXCR2 receptor. Key findings include the ability of NAMs to prevent CXCR2 coupling with multiple effectors, and a role for NAMs in allosteric modulation of chemokine affinity through conformational selection (supported by recent structural studies) – as well as steric blockade of effector interaction. Finally, our data reveal the importance of slow binding kinetics, as well as non-competitive interactions in generating insurmountable inhibition – a feature of CXCR2 antagonism which may be beneficial under inflammatory conditions involving a cytokine storm. This increased understanding may aid future in vitro optimisation of CXCR2 NAM compounds, to titrate the desire for blockade that is therapeutically effective while managing the risk of side effects.

Teixobactin kills bacteria by a two-pronged attack on the cell envelope

Article

Full-text available

Aug 2022
NATURE

Antibiotics that use novel mechanisms are needed to combat antimicrobial resistance 1–3 . Teixobactin ⁴ represents a new class of antibiotics with a unique chemical scaffold and lack of detectable resistance. Teixobactin targets lipid II, a precursor of peptidoglycan ⁵ . Here we unravel the mechanism of teixobactin at the atomic level using a combination of solid-state NMR, microscopy, in vivo assays and molecular dynamics simulations. The unique enduracididine C-terminal headgroup of teixobactin specifically binds to the pyrophosphate-sugar moiety of lipid II, whereas the N terminus coordinates the pyrophosphate of another lipid II molecule. This configuration favours the formation of a β-sheet of teixobactins bound to the target, creating a supramolecular fibrillar structure. Specific binding to the conserved pyrophosphate-sugar moiety accounts for the lack of resistance to teixobactin ⁴ . The supramolecular structure compromises membrane integrity. Atomic force microscopy and molecular dynamics simulations show that the supramolecular structure displaces phospholipids, thinning the membrane. The long hydrophobic tails of lipid II concentrated within the supramolecular structure apparently contribute to membrane disruption. Teixobactin hijacks lipid II to help destroy the membrane. Known membrane-acting antibiotics also damage human cells, producing undesirable side effects. Teixobactin damages only membranes that contain lipid II, which is absent in eukaryotes, elegantly resolving the toxicity problem. The two-pronged action against cell wall synthesis and cytoplasmic membrane produces a highly effective compound targeting the bacterial cell envelope. Structural knowledge of the mechanism of teixobactin will enable the rational design of improved drug candidates.

Structural dynamics and kinase inhibitory activity of three generations of tyrosine kinase inhibitors against wild-type, L858R/T790M, and L858R/T790M/C797S forms of EGFR

Article

Jun 2022
COMPUT BIOL MED

Mutations in the tyrosine kinase domain of epidermal growth factor receptor (EGFR), including L858R/T790M double and L858R/T790M/C797S triple mutations, are major causes of acquired resistance towards EGFR targeted drugs. In this work, a combination of comprehensive molecular modeling and in vitro kinase inhibition assay was used to unravel the mutational effects of EGFR on the susceptibility of three generations of EGFR tyrosine kinase inhibitors (erlotinib, gefitinib, afatinib, dacomitinib, and osimertinib) in comparison with the wild-type EGFR. The binding affinity of all studied inhibitors towards the double and triple EGFR mutations was in good agreement with the experimental data, ranked in the order of osimertinib > afatinib > dacomitinib > erlotinib > gefitinib. Three hot-spot residues at the hinge region (M790, M793, and C797) were involved in the binding of osimertinib and afatinib, enhancing their inhibitory activity towards mutated EGFRs. Both double and triple EGFR mutations activating erlotinib and gefitinib resistance are mainly caused by the low number of H-bond occupations, the low number of surrounding atoms, and the high number of water molecules accessible to the enzyme active site. According to principal component analysis, the molecular complexation of osimertinib against the two mutated EGFRs was in a closed conformation, whereas that against wild-type EGFR was in an open conformation, resulting in drug resistance. This work paves the way for further design of the novel EGFR inhibitors to overcome drug resistance mechanisms.

International Union of Basic and Clinical Pharmacology. CXII: Adenosine Receptors: A Further UpdateS

Article

Apr 2022

Our previous International Union of Basic and Clinical Pharmacology report on the nomenclature and classification of adenosine receptors (2011) contained a number of emerging developments with respect to this G protein-coupled receptor subfamily, including protein structure, protein oligomerization, protein diversity, and allosteric modulation by small molecules. Since then, a wealth of new data and results has been added, allowing us to explore novel concepts such as target binding kinetics and biased signaling of adenosine receptors, to examine a multitude of receptor structures and novel ligands, to gauge new pharmacology, and to evaluate clinical trials with adenosine receptor ligands. This review should therefore be considered a further update of our previous reports from 2001 and 2011. SIGNIFICANCE STATEMENT: Adenosine receptors (ARs) are of continuing interest for future treatment of chronic and acute disease conditions, including inflammatory diseases, neurodegenerative afflictions, and cancer. The design of AR agonists ("biased" or not) and antagonists is largely structure based now, thanks to the tremendous progress in AR structural biology. The A2A- and A2BAR appear to modulate the immune response in tumor biology. Many clinical trials for this indication are ongoing, whereas an A2AAR antagonist (istradefylline) has been approved as an anti-Parkinson agent.

A guide to enzyme kinetics in early drug discovery

Article

Full-text available

Mar 2022
FEBS J

Bharath Srinivasan

Drugs interact with their target of interest to bring about the desired phenotypic outcome that results in disease alleviation. Traditionally, most lead optimization exercises were driven by affinity measures (like IC50) to inform structure–activity relationship (SAR)‐guided medicinal chemistry. However, an IC50 value is a thermodynamic estimate measured under equilibrium conditions that can vary as a function of substrate concentration and/or time (the latter especially for nonequilibrium modalities). Further, like other thermodynamic estimates, it is a state‐function that is indifferent to the path traversed from the initial state to the final state. This can be a cause for concern in drug discovery given the predominance of nonequilibrium interactions and the open thermodynamic nature of the human system. Under such situations, employing rates along with equilibrium constants (or IC50 values) would be far more relevant to capture the time evolution of the small molecule’s interaction with the target of interest. These rates are generally typified by the rate of association, rate of dissociation and the residence time of the small molecule on the target (target occupancy). These parameters, when combined with the concept of target vulnerability, therapeutic window, pharmacokinetic profile of the small molecule, estimates of endogenous ligand and target turnover, will shed critical insights into the kinetics and dynamics of a small molecule’s interaction with the protein, and allow realistic modelling of the system to enable optimizations and dosing decisions. With that aim, this guide will attempt to introduce the traditional role of mechanistic enzymology within drug discovery and emphasize the importance of kinetics in guiding SAR‐based optimizations. It will also present initial ideas on how kinetic investigation should be positioned relative to the temporal span of a drug‐discovery pipeline to leverage maximal utility from the investment in time and effort.

Decisive role of water and protein dynamics in residence time of p38α MAP kinase inhibitors

Article

Full-text available

Jan 2022

Target residence time plays a crucial role in the pharmacological activity of small molecule inhibitors. Little is known, however, about the underlying causes of inhibitor residence time at the molecular level, which complicates drug optimization processes. Here, we employ all-atom molecular dynamics simulations (~400 μs in total) to gain insight into the binding modes of two structurally similar p38α MAPK inhibitors (type I and type I½) with short and long residence times that otherwise show nearly identical inhibitory activities in the low nanomolar IC 50 range. Our results highlight the importance of protein conformational stability and solvent exposure, buried surface area of the ligand and binding site resolvation energy for residence time. These findings are further confirmed by simulations with a structurally diverse short residence time inhibitor SB203580. In summary, our data provide guidance in compound design when aiming for inhibitors with improved target residence time.

Kinetic Intracellular Assay Measures Compound Binding Kinetics at Intracellular Targets Within Living Cells

Article

Full-text available

Dec 2021

Competitive ligand binding kinetics to linear polymers

Preprint

Full-text available

May 2024

Different types of ligands compete in binding to polymers with different consequences for the physical and chemical properties of the resulting complex. Here, we derive a general kinetic model for the competitive binding kinetics of different types of ligands to a linear polymer, using the McGhee and von Hippel detailed binding site counting procedure. The derived model allows the description of the competitive binding process in terms of the size of the ligand, binding and release rates, and cooperativity parameters. We illustrate the implications of the general theory showing the equations for the competitive binding of two ligands. The size of the ligand, given by the number of monomers occluded, is shown to have a great impact in competitive binding. Ligands requiring a large available gap for binding are strongly inhibited by smaller ligands. Ligand size then has a leading role compared to binding affinity or cooperativity. For ligands that can bind in different modes (i.e., different number of monomers), this implies that they are more effective in covering or passivating the polymer in lower modes, if the different modes have similar binding energies.

Positive allosteric modulation of a GPCR ternary complex

Preprint

Full-text available

Apr 2024

The activation of a G protein-coupled receptor (GPCR) leads to the formation of a ternary complex between agonist, receptor, and G protein that is characterised by high-affinity binding. Allosteric modulators bind to a distinct binding site from the orthosteric agonist and can modulate both the affinity and the efficacy of orthosteric agonists. The influence allosteric modulators have on the high-affinity active state of the GPCR-G protein ternary complex is unknown due to limitations on attempting to characterize this interaction in recombinant whole cell or membrane-based assays. Here, we use purified M 2 muscarinic acetylcholine receptor (mAChR) reconstituted into nanodiscs to show that once the agonist-bound high-affinity state is promoted by the G protein, positive allosteric modulators stabilise the ternary complex that, in the presence of nucleotides leads to an enhanced initial rate of signalling. Our results enhance our understanding of how allosteric modulators influence orthosteric ligand signalling and will aid the design of allosteric therapeutics. Teaser Allostery from top and bottom, the combined influence of positive allosteric modulators on receptor signalling.

Learning protein-ligand unbinding pathways via single-parameter community detection

Preprint

Mar 2024

Understanding the dynamics of biomolecular complexes, e.g., of protein-ligand (un)binding, requires the understanding of paths such systems take between metastable states. In MD simulation data, paths are usually not observable per se, but need to be inferred from simulation trajectories. Here we present a novel approach to cluster trajectories based on a community detection algorithm that requires the definition of only a single free parameter. Using the streptavidin-biotin complex as benchmark system and the A2 a adenosine receptor in complex with the inhibitor ZM241385 as an elaborate application, we demonstrate how such clusters of trajectories correspond to pathways, and how the approach help in the identification of reaction coordinates for a considered (un)binding process.

Label-Free Evanescent Imaging of Cellular Heterogeneity in Membrane Protein Binding Kinetics

Preprint

Full-text available

Feb 2024

Quantifying cellular heterogeneity of membrane protein binding kinetics is challenging but important for exploring drug resistance and screening drugs. Label-free analysis methods have emerged as promising tools for in situ binding kinetics analysis, but they have not been used for high throughput single cell analysis in live cells. Here we show that this is possible with Evanescent Scattering Microscopy (ESM). The ESM permits analyzing the kinetics of ligand binding onto membrane proteins in individual fixed and live cells, and provides a throughput of ~200 cells in a single measurement with a period of ~7 minutes. The statistical analysis further shows that the dissociation rate constant dominates the heterogeneity of cell responses to ligand binding, providing evidence for a long-standing hypothesis that the drug-target residence time may play a critical role in drug treatment. In addition, the ESM reveals that under some conditions the cells have responses to drug binding at the single cell level, whereas the ensemble measurements may average out the individual differences and present false negative results. We anticipate that the new evanescent imaging method will provide a powerful tool to quantify the functions of cellular proteins, especially their cell-to-cell heterogeneity that can provide fuel for drug resistance.

MD Simulations for D rug‐Target (Un)binding Kinetics

Chapter

Jan 2024

Steffen Wolf

Late-stage modification of bioactive compounds: Improving druggability through efficient molecular editing

Article

Full-text available

Nov 2023

Synthetic chemistry plays an indispensable role in drug discovery, contributing to hit compounds identification, lead compounds optimization, candidate drugs preparation, and so on. As Nobel Prize laureate James Black emphasized, “the most fruitful basis for the discovery of a new drug is to start with an old drug”¹. Late-stage modification or functionalization of drugs, natural products and bioactive compounds have garnered significant interest due to its ability to introduce diverse elements into bioactive compounds promptly. Such modifications alter the chemical space and physiochemical properties of these compounds, ultimately influencing their potency and druggability. To enrich a toolbox of chemical modification methods for drug discovery, this review focuses on the incorporation of halogen, oxygen, and nitrogen—the ubiquitous elements in pharmacophore components of the marketed drugs—through late-stage modification in recent two decades, and discusses the state and challenges faced in these fields. We also emphasize that increasing cooperation between chemists and pharmacists may be conducive to the rapid discovery of new activities of the functionalized molecules. Ultimately, we hope this review would serve as a valuable resource, facilitating the application of late-stage modification in the construction of novel molecules and inspiring innovative concepts for designing and building new drugs.

Pseudoirreversible inhibition elicits persistent efficacy of a sphigosine-1-phosphate receptor-1 antagonist

Preprint

May 2023

Sphingosine 1-phosphate receptor 1 (S1PR1), a G protein-coupled receptor, is required for lymphocyte trafficking, and is a promising therapeutic target in inflammatory diseases. To find potent S1PR1 antagonists, identification of the structural basis for drug efficacy is important. Here, we synthesized a novel antagonist, KSI-6666, that persistently inhibits S1PR1 activity and effectively suppresses pathogenic inflammation. Metadynamics simulation suggested that the interaction of a benzene ring moiety in KSI-6666 with a methionine residue in the ligand-binding pocket of S1PR1 inhibits the dissociation of KSI-6666 from S1PR1, generating a metastable binding state. Consistently, in vitro functional and mutational analyses revealed that KSI-6666 causes pseudoirreversible inhibition of S1PR1, dependent on the methionine residue of the protein and substituents on the distal benzene ring of KSI-6666. Moreover, in vivo study suggested that this pseudoirreversible inhibition is responsible for the persistent activity of KSI-6666. These findings will contribute to the rational design of potent S1PR1 antagonists for the treatment of inflammatory disorders.

The Use of Surface Plasmon Resonance to Study the Interactions of Proteins Involved in Conformational Diseases: Experimental Approaches for New Therapeutical Perspectives

Article

Jan 2023
CURR MED CHEM

In recent years, the scientific community has been trying to tackle different diseases by using unifying and holistic approaches based on the concept that it is possible to target apparently very different diseases under a comprehensive general scheme. In other words, various different diseases have been grouped together under the label of "conformational diseases", because the triggering cause for each malady is the misfolding of a specific protein, whose dyshomeostasis and accumulation cause all the other downhill biomolecular events characteristic of each different disease. In a parallel manner, analytical techniques have developed to investigate protein misfolding and accumulation, so as to give a valid technical support to the investigation of conformational diseases. In this scenario, surface plasmon resonance (SPR) has widely contributed to study many different aspects correlated to conformational diseases, offering the advantages of real time investigations, use of small amounts of biological materials and possibility to mimic the cellular environments without recurring to the use of fluorescent tags. In this review, after a brief introduction about conformational diseases and the SPR technique, a thorough description of the various uses of SPR to investigate the biomolecular mechanisms involved in these diseases is given in order to provide the reader with an exhaustive list as well as a critical perspective of the use of SPR for such topic. The case of Alzheimer's disease is discussed at a deeper level. We hope that this work will make the reader aware of all the possible SPR experimental approaches, which can be used to develop new possible therapeutic strategies to tackle conformational diseases.

Milestoning Simulation of Ligand Dissociation from the Glycogen Synthase Kinase 3β

Article

Sep 2022

As drug‐binding kinetics has become an important factor to be considered in modern drug discovery, this work evaluated the ability of the Milestoning method in computing the absolute dissociation rate of a ligand from the serine‐threonine kinase glycogen synthase kinase 3β, which is a target for designing drugs to treat diseases such as neurodegenerative disorders and diabetes. We found that the Milestoning method gave good agreement with experiment with modest computational costs. Although the time scale for dissociation lasted tens of seconds, the collective molecular dynamics simulations total less than 1 μs. Computing the committor function helped to identify the transition states, in which the ligand moved substantially away from the binding pocket. The glycine‐rich loop with a serine residue attaching to its tips was found to undergo large movement from the bound to the transition states and might play a role in controlling drug‐dissociation kinetics. This article is protected by copyright. All rights reserved.

Competition in drug binding and the race to equilibrium

Article

Aug 2022

Background: Binding kinetics has become a popular topic in pharmacology due to its potential contribution to the selectivity- and duration of drug action. Yet, the overall kinetic aspects of complex binding mechanisms are still merely described in terms of elaborate algebraic equations. Interestingly, it has been recommended some ten years ago to examine such mechanisms in terms of binding fluxes instead of the conventional rate constants. Alike the velocity of product formation in enzymology, those fluxes refer to the velocity by which one target species converts into another one. Objectives: Novel binding flux- based approaches are utilized to get a better visual insight into the "competition" between two drugs/ligands for a single target as well as between induced fit- and conformational selection pathways for a single ligand within a thermodynamic cycle. Methods: Differential equation- based simulations. Results: Early on, the ligand- binding steps "race" to equilibrium (i.e. when their forward- and reverse fluxes are equal) at their individual pace. The overall/global equilibrium is only reached later on. For the competition association assays, this parting might produce a transient "overshoot" of one of the bound target species. A similar overshoot may also show up within a thermodynamic cycle and, at first glance, suggest that the induced fit pathway dominates. Yet, present findings show that under certain circumstances it could rather be the other way round. Conclusion: Novel binding flux-based approaches offer visually attractive insights into crucial aspects of "complex" binding mechanisms under non-equilibrium conditions.

Inhibition of NaV1.7: The Possibility of Ideal Analgesics

Article

Aug 2022

The selective inhibition of NaV1.7 is a promising strategy for developing novel analgesic agents with fewer adverse effects. Although the potent selective inhibition of NaV1.7 has been recently achieved, multiple NaV1.7 inhibitors failed in clinical development. In this review, the relationship between preclinical in vivo efficacy and NaV1.7 coverage among three types of voltage-gated sodium channel (VGSC) inhibitors, namely conventional VGSC inhibitors, sulphonamides and acyl sulphonamides, is discussed. By demonstrating the PK/PD discrepancy of preclinical studies versus in vivo models and clinical results, the potential reasons behind the disconnect between preclinical results and clinical outcomes are discussed together with strategies for developing ideal analgesic agents.

Ghost Dark Energy with Variable Gravitational Constant

Article

Full-text available

May 2022

In a bid to resolve lingering problems in modern Cosmology, Cosmologist are turning to models in which Physical constants of nature are not real constants but vary as function of the scale factor of the universe. In this work, a cosmological model is developed by incorporating Ghost Dark Energy model with Barrow's ansatz for varying Gravitational Constant. A model obtained is free from initial Big Bang Singularity and Inflation Problem. A test of Causality showed that the model depends of the constant m and is classically stable at −6 ≥ > −3 1. INTRODUCTION In physical cosmology and astronomy, dark energy is an unknown form of energy that affects the universe on the largest scales. The first observational evidence for its existence came from measurements of supernovae, which showed that the universe does not expand at a constant rate; rather, the expansion of the universe is accelerating [1]. Understanding the evolution of the universe requires knowledge of its starting conditions and its composition. Before these observations, it was thought that all forms of matter and energy in the universe would only cause the expansion to slow down over time. Measurements of the cosmic microwave background suggest the universe began in a hot Big Bang, from which general relativity explains its evolution and the subsequent large-scale motion. Since the 1990s, dark energy has been the most accepted premise to account for the accelerated expansion. Assuming that the lambda cold dark matter (Λ-CDM) model of cosmology is correct, the best current measurements indicate that dark energy contributes 68% of the total energy in the present-day observable universe [2]. The ΛCDM model is a parameterization of the Big Bang cosmological model in which the universe contains three major components: first, a cosmological constant denoted by Lambda (Greek Λ) associated with dark energy; second, the postulated cold dark matter (CDM); and third, ordinary matter. It is frequently referred to as the standard model of Big Bang cosmology because it is the simplest model that provides a reasonably good account of the following properties of the cosmos: the existence and structure of the cosmic microwave background, the large-scale structure in the distribution of Galaxies, the observed abundances of hydrogen (including deuterium), helium, and lithium, the accelerating expansion of the universe observed in the light from distant galaxies and supernovae. The simplest model of DE is the cosmological constant, which is a key ingredient in the ΛCDM model. Although the ΛCDM model is consistent very well with all observational data, it faces the fine-tuning problem

PhD Thesis: Molecular Approaches to Explore Drug-Target Interactions

Thesis

Full-text available

Mar 2019

Improved means to assess the clinical potential of drug candidates can critically influence development of new therapeutic entities, a central aim in medical life science. Drug discovery and development relies on construction and selection of small organic compounds or biological agents that bind targets of interest. This thesis includes new methodology to investigate target engagement - that is the tendency for these drugs and drug candidates to bind their intended target molecules versus any off-targets. This is a matter of great importance and current strong interest in the pharmaceutical industry as well as academically and an important aim for precision medicine. Paper I describes the target engagement-mediated amplification (TEMA) technique, an accurate, selective and physiological relevant techniques to monitor target binding by DNA-conjugated low molecular weight drug molecules. The DNA conjugated forms of the drugs are uniquely suited to accurately and sensitively reveal the binding characteristics of drugs directly in relevant tissues. Paper II describes the evaluation of cellular thermal shift assays (CETSA) by multiplex proximity extension assays (PEA), to sensitively measure binding of drugs to their proper targets and off-targets in minimal samples of cells and tissues, and for many targets and samples in parallel. The technique provides valuable advantages during drug development, and potentially also in clinical care. Paper III describes a high- throughput approach to use in situ proximity ligation assays to investigate protein interactions or modifications along with phenotypic responses to drugs or cytokines. The technique allows responses by large numbers of cells to be evaluated by automated microscopy and computer- based analysis. Our approach expands the scope for combined molecular and morphological profiling, offering an information-rich means to profile cellular responses to drugs and other agents at the single cell level. Keywords: Drug discovery, target engagement, target engagement-mediated amplification, cellular thermal shift assay, proximity extension assay, in situ PLA, high-content imaging

Relative Binding Free Energy Predictions for Inhibitors of Tetrameric Influenza Virus Neuraminidase

Preprint

Full-text available

Sep 2021

Accurate methods to predict the free energies of protein-ligand interactions have great potential to assist rational drug design. In this work, we used molecular dynamics simulations with alchemical perturbation to predict the binding of carbohydrate-based ligands to influenza virus neuraminidase (N2). This specific drug target is a challenging test system for alchemical free energy methods because it has flexible binding site motifs. We use a molecular dynamics protocol that works for longer time scales than are often reported in previous molecular dynamics studies of N2. We demonstrated that N2-ligand complex stability and that accurate N2 150-loop dynamics, on a 350 ns time scale, are both force field-dependent (AMBER99SB-ILDN, GAFF and TIP4P water are required). Further, we showed that crystallographic waters must be retained to maintain stability of N2-ligand complexes over 350 ns. Using our modelling protocol, we were able to determine relative binding free energy values between neuraminidase ligands which correlated strongly with experimental differences in pIC50 values (R = -0.96, ρ = 0.86, N = 13, sig < 0.0001). It is anticipated that the molecular dynamics protocol and the relative binding free energy methods reported here, will both be useful in expediting the discovery of novel therapeutics for N2 and other homologous glycohydrolases.

Analysis of resistance to human immunodeficiency virus type 1 protease inhibitors by using matched bacterial expression and proviral infection vectors

Article

Full-text available

Oct 1995

There are already reports, from clinical trials with human immunodeficiency virus type 1 protease inhibitors, of the emergence of drug-resistant mutants which have one or more point mutations in their protease genes. To examine roles of individual and multiple amino acid substitutions in terms of altered enzyme and virus drug sensitivities, we have produced matched vectors for bacterial expression and virus production. Both vectors accept the same restriction enzyme fragment, produced by PCR or PCR-mutagenesis of the protease gene, allowing parallel expression of mutant enzymes in Escherichia coli and in recombinant viruses. The utility of this vector system was demonstrated by using protease variants glycine to valine at amino acid 48 (G48V) and leucine to methionine at amino acid 90 (L90M) identified after passage of HIV-1 in the Roche phase II clinical trial protease inhibitor Ro 31-8959 (H. Jacobsen, K. Yasargil, D. L. Winslow, J. C. Craig, A. Krohn, I. B. Duncan, and J. Mous, Virology 206:527, 1995). G48V, L90M, and G48V/L90M exhibited successively less processing in vitro than the wild-type enzyme, and the purified enzymes were 220-, 20-, and 720-fold, respectively, less sensitive to Ro 31-8959. The reduced enzyme sensitivity correlated directly with the sensitivities of the matched recombinant viruses, in that individual mutations L90M and G48V conferred 2-fold and 4- to 6-fold increases in 50% inhibitory concentration, respectively, whereas G48V/L90M was 8 to 10 times less sensitive to Ro 31-8959. A proviral vector with the entire protease gene deleted was constructed for use as an in vivo recombination target for an overlapping protease PCR fragment, generating wild-type infectious virus. Finally, direct ligation of restriction fragments, generated from random PCR mutagenesis, into the proviral vector should provide a library of protease mutations that allow extremely rapid selection of highly resistant viral variants.

Human immunodeficiency virus: Mutations in the viral protease that confer resistance to saquinavir increase the dissociation rate constant of the protease-saquinavir complex

Article

Full-text available

Dec 1996

Mutations in the human immunodeficiency virus (HIV) protease (L90M, G48V, and L90M/G48V) arise when HIV is passaged in the presence of the HIV protease inhibitor saquinavir. These mutations yield a virus with less sensitivity to the drug (L90M > G48V > L90M/G48V). L90M, G48V, and L90M/G48V proteases have 1/20, 1/160, and 1/1000 the affinity for saquinavir compared to WT protease, respectively. Therefore, the affinity of mutant protease for saquinavir decreased as the sensitivity of the virus to saquinavir decreased. Association rate constants for WT and mutant proteases with saquinavir were similar, ranging from 2 to 4 x 10(7) M-1 s-1. In contrast, the dissociation rate constants for WT, L90M, G48V, and L90M/G48V proteases complexed with saquinavir were 0.0014, 0.019, 0.128, and 0. 54 s-1, respectively. This indicated that the reduced affinity for mutant proteases and saquinavir is primarily the result of larger dissociation rate constants. The increased dissociation rate constants may be the result of a decrease in the internal equilibrium between the bound inhibitor with the protease flaps up and the bound inhibitor with the flaps down. Interestingly, the affinity of these mutant proteases for VX-478, ABT-538, AG-1343, or L-735,524 was not reduced as much as that for saquinavir. Finally, the catalytic constants of WT and mutant proteases were determined for eight small peptide substrates that mimic the viral cleavage sites in vivo. WT and L90M proteases had similar catalytic constants for these substrates. In contrast, G48V and L90M/G48V proteases had catalytic efficiency (kcat/Km) values with TLNF-PISP, RKIL-FLDG, and AETF-YVDG that were 1/10 to 1/20 the value of WT protease. The decreased catalytic efficiencies were primarily the result of increased Km values. Thus, mutations in the protease decrease the affinity of the enzyme for saquinavir and the catalytic efficiency with peptide substrates.

In Vitro Characterization of A-315675, a Highly Potent Inhibitor of A and B Strain Influenza Virus Neuraminidases and Influenza Virus Replication

Article

Full-text available

Apr 2002
ANTIMICROB AGENTS CH

A-315675 is a novel, pyrrolidine-based compound that was evaluated in this study for its ability to inhibit A and B strain influenza virus neuraminidases in enzyme assays and influenza virus replication in cell culture. A-315675 effectively inhibited influenza A N1, N2, and N9 and B strain neuraminidases with inhibitor constant (K(i)) values between 0.024 and 0.31 nM. These values were comparable to or lower than the K(i) values measured for oseltamivir carboxylate (GS4071), zanamivir, and BCX-1812, except for the N1 enzymes that were found to be the most sensitive to BCX-1812. The time-dependent inhibition of neuraminidase catalytic activity observed with A-315675 is likely due to its very low rate of dissociation from the active site of neuraminidase. The half times for dissociation of A-315675 from B/Memphis/3/89 and A/Tokyo/3/67 (H3N2) influenza virus neuraminidases of 10 to 12 h are significantly slower than the half times measured for oseltamivir carboxylate (33 to 60 min). A-315675 inhibited the replication of several laboratory strains of influenza virus in cell culture with potencies that were comparable or superior to those for oseltamivir carboxylate and BCX-1812, except for the A/H1N1 viruses that were found to be two- to fourfold more susceptible to BCX-1812. A-315675 and oseltamivir carboxylate exhibited comparable potencies against a panel of A/H1N1 and A/H3N2 influenza virus clinical isolates, but A-315675 was found to be significantly more potent than oseltamivir carboxylate against the B strain isolates. The favorable in vitro results relative to other clinically effective agents provide strong support for the further investigation of A-315675 as a potential therapy for influenza virus infections.

A Novel N-Methyl-D-aspartate Receptor Open Channel Blocker with in Vivo Neuroprotectant Activity

Article

Full-text available

Aug 2002

Excitotoxicity has been implicated in the etiology of ischemic stroke, chronic neurodegenerative disorders, and very recently, in glioma growth. Thus, the development of novel neuroprotectant molecules that reduce excitotoxic brain damage is vigorously pursued. We have used an ionic current block-based cellular assay to screen a synthetic combinatorial library of trimers of N-alkylglycines on the N-methyl-D-aspartate (NMDA) receptor, a well known molecular target involved in excitotoxicity. We report the identification of a family of N-alkylglycines that selectively blocked the NMDA receptor. Notably, compound 3,3-diphenylpropyl-N-glycinamide (referred to as N20C) inhibited NMDA receptor channel activity with micromolar affinity, fast on-off blockade kinetics, and strong voltage dependence. Molecule N20C did not act as a competitive glutamate or glycine antagonist. In contrast, saturation of the blocker binding site with N20C prevented dizolcipine (MK-801) blockade of the NMDA receptor, implying that both drugs bind to the same receptor site. The N-alkylglycine efficiently prevented in vitro excitotoxic neurodegeneration of cerebellar and hippocampal neurons in culture. Attenuation of neuronal glutamate/NMDA-induced Ca(2+) overload and subsequent modulation of the glutamate-nitric oxide-cGMP pathway seems to underlie N20C neuroprotection. Noteworthy, this molecule exhibited significant in vivo neuroprotectant activity against an acute, severe, excitotoxic insult. Taken together, these findings indicate that N-alkylglycine N20C is a novel, low molecular weight, moderate-affinity NMDA receptor open channel blocker with in vitro and in vivo neuroprotective activity, which, in due turn, may become a tolerated drug for the treatment of neurodegenerative diseases and cancer.

Norman AW, Mizwicki MT, Norman DP.. Steroid-hormone rapid actions, membrane receptors and a conformational ensemble model. Nat Rev Drug Discov 3: 27-41

Article

Full-text available

Feb 2004

Steroid hormones can act as chemical messengers in a wide range of species and target tissues to produce both slow genomic responses, and rapid non-genomic responses. Although it is clear that genomic responses to steroid hormones are mediated by the formation of a complex of the hormone and its cognate steroid-hormone nuclear receptor, new evidence indicates that rapid responses are mediated by a variety of receptor types associated with the plasma membrane or its caveolae components, potentially including a membrane-associated nuclear receptor. This review summarizes our current knowledge of membrane-associated steroid receptors, as well as details of structure–function relationships between steroid hormones and the ligand-binding domains of their nuclear and membrane-associated receptors. Furthermore, a new receptor conformational ensemble model is presented that suggests how the same receptor could produce both rapid and genomic responses. It is apparent that there is a cornucopia of new drug development opportunities in these areas.

A Unique Structure for Epidermal Growth Factor Receptor Bound to GW572016 (Lapatinib) Relationships among Protein Conformation, Inhibitor Off-Rate, and Receptor Activity in Tumor Cells

Article

Full-text available

Oct 2004

GW572016 (Lapatinib) is a tyrosine kinase inhibitor in clinical development for cancer that is a potent dual inhibitor of epidermal growth factor receptor (EGFR, ErbB-1) and ErbB-2. We determined the crystal structure of EGFR bound to GW572016. The compound is bound to an inactive-like conformation of EGFR that is very different from the active-like structure bound by the selective EGFR inhibitor OSI-774 (Tarceva) described previously. Surprisingly, we found that GW572016 has a very slow off-rate from the purified intracellular domains of EGFR and ErbB-2 compared with OSI-774 and another EGFR selective inhibitor, ZD-1839 (Iressa). Treatment of tumor cells with these inhibitors results in down-regulation of receptor tyrosine phosphorylation. We evaluated the duration of the drug effect after washing away free compound and found that the rate of recovery of receptor phosphorylation in the tumor cells reflected the inhibitor off-rate from the purified intracellular domain. The slow off-rate of GW572016 correlates with a prolonged down-regulation of receptor tyrosine phosphorylation in tumor cells. The differences in the off-rates of these drugs and the ability of GW572016 to inhibit ErbB-2 can be explained by the enzyme-inhibitor structures.

Drug-target residence time and its implications for lead optimization. Nat Rev Drug Discov 5(9):730-739 Erratum in: Nat Rev Drug Discov (2007) 6(3):249

Article

Full-text available

Oct 2006

Much of drug discovery today is predicated on the concept of selective targeting of particular bioactive macromolecules by low-molecular-mass drugs. The binding of drugs to their macromolecular targets is therefore seen as paramount for pharmacological activity. In vitro assessment of drug-target interactions is classically quantified in terms of binding parameters such as IC(50) or K(d). This article presents an alternative perspective on drug optimization in terms of drug-target binary complex residence time, as quantified by the dissociative half-life of the drug-target binary complex. We describe the potential advantages of long residence time in terms of duration of pharmacological effect and target selectivity.

Prospective testing for drug-dependent antibodies reduces the incidence of thrombocytopenia observed with the small molecule glycoprotein IIb/IIIa antagonist roxifiban: Implications for the etiology of thrombocytopenia

Article

Full-text available

Feb 2003

Thrombocytopenia is a relatively common side effect observed during glycoprotein (GP) IIb/IIIa antagonist therapy. With the oral antagonist roxifiban, we observed thrombocytopenia, defined as 50% reduction of platelets over predose values or below 90 000/microL (9 x 10(10)/L), with a frequency of 2% (8 of 386). Thrombocytopenia occurred either early (days 2 to 4) or delayed (days 11 to 16). No additional cases were observed with up to 6 months of treatment. Retrospective analysis provided evidence for drug-dependent antibodies (DDABs) to GP IIb/IIIa in 5 of 6 subjects, suggestive of an immune etiology of thrombocytopenia. The hypothesis that excluding patients based on positive DDAB reaction would reduce the frequency of thrombocytopenia was tested. Patients were screened for DDABs during the study qualification period and, overall, 3.9% of the patients were excluded based on pre-existing DDAB concentrations above a statistically defined medical decision limit. An additional 2.6% were excluded based on therapy-related antibody production during the first 2 weeks. With antibody testing, 0.2% of patients (2 of 1044) developed immune-mediated thrombocytopenia. One case developed a rapidly increasing antibody concentration and presented with thrombocytopenia despite discontinuation of roxifiban therapy. The second case was related to a false-negative test result. The frequency of thrombocytopenia was statistically significantly reduced from 2% to 0.2% (P =.0007) comparing nonscreened and screened patients. Testing for DDABs can reduce the frequency of thrombocytopenia in patients treated with roxifiban and, by analogy, other GP IIb/IIIa antagonists. Thus, DDAB testing may be employed to increase the safety of GP IIb/IIIa antagonists.

Evidence that thrombocytopenia observed in humans treated with orally bioavailable glycoprotein IIb/IIIa antagonists is immune mediated

Article

May 2002

Jeffrey Billheimer

Glycoprotein (GP) IIb/IIIa antagonists are effective therapeutic agents, but elicit thrombocytopenia with a frequency that approaches 2%. Here, we provide evidence that thrombocytopenia in humans treated with the GP IIb/IIIa antagonist roxifiban is immune mediated. Two patients underwent conversion to a highly positive drug-dependent antibody (DDAB) status temporally associated with thrombocytopenia. Despite the continued presence of DDABs, the fall in platelet count was reversed by discontinuation of drug treatment, pointing to the exquisite drug dependency of the immune response. DDABs appear to bind to neoepitopes in GP IIb/IIIa elicited on antagonist binding. This information was used to develop an enzyme-linked immunosorbent assay (ELISA) for DDAB using solid-phase GP IIb/IIIa. A high level of specificity is indicated by the observation that DDAB binding is dependent on the chemical structure of the GP IIb/IIIa antagonist and that only 2% to 5% of human blood donors and 5% of chimpanzees present with pre-existing DDABs. Furthermore, none of 108 nonthrombocytopenic patients from the phase II roxifiban study showed an increase in antibody titer. Absorption of thrombocytopenia plasma with platelets reduced the DDAB ELISA signal, indicating that the test detects physiologically relevant antibodies. Screening patients for pre-existing or increasing DDAB titer during treatment with GP IIb/IIIa antagonists may reduce the incidence of drug-induced thrombocytopenia.

Anatomy of protein pockets and cavities: Measurement of binding site geometry and implications for ligand design

Article

Sep 1998

Identification and size characterization of surface pockets and occluded cavities are initial steps in protein structure-based ligand design. A new program, CAST, for automatically locating and measuring protein pockets and cavities, is based on precise computational geometry methods, including alpha shape and discrete flow theory. CAST identifies and measures pockets and pocket mouth openings, as well as cavities. The program specifies the atoms lining pockets, pocket openings, and buried cavities; the volume and area of pockets and cavities; and the area and circumference of mouth openings. CAST analysis of over 100 proteins has been carried out; proteins examined include a set of 51 monomeric enzyme-ligand structures, several elastase-inhibitor complexes, the FK506 binding protein, 30 HIV-1 protease-inhibitor complexes, and a number of small and large protein inhibitors. Medium-sized globular proteins typically have 10-20 pockets/cavities. Most often, binding sites are pockets with 1-2 mouth openings; much less frequently they are cavities. Ligand binding pockets vary widely in size, most within the range 10(2)-10(3)A3. Statistical analysis reveals that the number of pockets and cavities is correlated with protein size, but there is no correlation between the size of the protein and the size of binding sites. Most frequently, the largest pocket/cavity is the active site, but there are a number of instructive exceptions. Ligand volume and binding site volume are somewhat correlated when binding site volume is < or =700 A3, but the ligand seldom occupies the entire site. Auxiliary pockets near the active site have been suggested as additional binding surface for designed ligands (Mattos C et al., 1994, Nat Struct Biol 1:55-58). Analysis of elastase-inhibitor complexes suggests that CAST can identify ancillary pockets suitable for recruitment in ligand design strategies. Analysis of the FK506 binding protein, and of compounds developed in SAR by NMR (Shuker SB et al., 1996, Science 274:1531-1534), indicates that CAST pocket computation may provide a priori identification of target proteins for linked-fragment design. CAST analysis of 30 HIV-1 protease-inhibitor complexes shows that the flexible active site pocket can vary over a range of 853-1,566 A3, and that there are two pockets near or adjoining the active site that may be recruited for ligand design.

Mechanism-Based Inhibition of Human Steroid 5α-Reductase by Finasteride: Enzyme-Catalyzed Formation of NADP−Dihydrofinasteride, a Potent Bisubstrate Analog Inhibitor

Article

Mar 1996

Finasteride is employed in treatment of benign prostatic hyperplasia in man, where its target enzyme is steroid 5α-reductase. It is a novel, potent mechanism-based inhibitor of the human prostate (type 2) isozyme. Although it is accepted as an alternate substrate and is ultimately reduced to dihydrofinasteride, this proceeds through an enzyme-bound NADP−dihydrofinasteride adduct. Finasteride is processed with a second-order rate constant, ki/Ki = 1 × 106 M-1 s-1, that approaches kcat/Km for reduction of testosterone, 3 × 106 M-1 s-1, and essentially every catalytic event is lethal (partition ratio ≤ 1.07). The membrane-bound enzyme−inhibitor complex formed from [3H]finasteride appears to release [3H]dihydrofinasteride with a half-life of 1 month at 37 °C (k = (2.57 ± 0.03) × 10-7 s-1), as identified by mass spectroscopy. The intermediate NADP−dihydrofinasteride adduct can be recovered intact by denaturation of the enzyme−inhibitor complex and has been purified. Free in solution, it likewise decomposes to dihydrofinasteride (half-life = 11 days). An extremely potent bisubstrate analog inhibitor, this NADP−dihydrofinasteride adduct binds to the free enzyme with a second-order rate constant equal to kcat/Km for turnover of testosterone and has a dissociation constant Ki ≤ 1 × 10-13 M. Finasteride is also a mechanism-based inhibitor of the human skin (type 1) isozyme, but it is processed with a much smaller second-order rate constant, ki/Ki = 3 × 103 M-1 s-1, which attenuates its activity against this isozyme in vivo. The mechanism explains the exceptional potency and specificity of finasteride in treatment of benign prostatic hyperplasia, and the concept may have application to other pyridine nucleotide-linked enzymes.

Improved Structure−Activity Relationship Analysis of HIV-1 Protease Inhibitors Using Interaction Kinetic Data

Article

Nov 2004

Despite the availability of large amounts of data for HIV-protease inhibitors and their effectiveness with wild type and resistant enzyme, there is limited knowledge about how this and other information can be systematically applied to the development of new antiviral compounds. To identify in vitro parameters that correlate with the efficacy of HIV inhibitors in cell culture, the relationships between inhibition, interaction kinetic, and cell culture parameters for HIV-1 protease inhibitors were analyzed. Correlation, cluster, and principal component analysis of data for 37 cyclic and linear compounds revealed that the affinities (K(D)) determined from SPR-biosensor binding studies correlated better to cell culture efficacy (ED(50)) than that of the inhibition constants (K(i)), indicating that the conventional use of K(i) values for structure-activity relationship analysis of HIV-1 inhibitors should be seriously reconsidered. The association and dissociation kinetic rate constants (k(on) and k(off)) alone showed weak correlations with ED(50) values. However, ED(50) values were most related to the free enzyme concentration in the viral particle ([E]), calculated from the rate constants and the total enzyme concentration in a viral particle. A structure-activity relationship analysis of the current data set was found to be valid for all classes of compounds analyzed. In summary, use of affinity, based on interaction kinetic rate constants, rather than inhibition constants, and theoretical consideration of the physiological conditions in the virus particle provide improved structure-activity relationship analysis of HIV-1 protease inhibitors.

A024: Candesartan causes a long-lasting antagonism of the angiotensin II receptor mediated contractile effects in isolated vascular preparations: A comparison with losartan and its active metabolite (EXP 3174)

Article

Apr 1998

Platelet GPIIb/IIIa binding characteristics of small molecule RGD mimetic: Distinct binding profile for Roxifiban

Article

Jul 2001

A number of non-peptide orally active RGD mimetic prodrug such as Orbofiban, Sibrafiban, SR121566, Roxifiban and others entered into the clinical evaluation stage. Some of these agents were terminated and some are still in clinical trials. The present study examined the platelet GPIIb/IIIa binding profiles for the active form of Roxifiban, Sibrafiban, SR121566 and Orbofiban using 3H-Roxifiban active form (XV459), 3H-DMP728, 125I-Echistatin, and 125I-Fibrinogen. Either DMP728, Orbofiban, Sibrafiban, SR121566 or Roxifiban active form as well as other RGD mimetic bind to the same binding site (s) on human platelets as evident from the competitive inhibition of binding of each other to human platelet. Additionally, Roxifiban active form competed with FITC labeled GPIIb/IIIa antagonist cyclic RGD peptidomimetic (XL086) as demonstrated using confocal microscopy technique. Roxifiban active form (XV459) demonstrated the highest potency in inhibiting 3H-XV459, 3H-DMP728, 125I-Echistatin, and 125I-Fibrinogen binding to human platelets as compared to the others. Structure activity relationship within the isoxazoline Roxifiban series showed that substituent at the α-carbon next to the carboxy terminal represents an exosite for the affinity binding to human platelets leading to slow platelet dissociation rate. These data indicated a distinct binding profile for Roxifiban (high affinity to both activated and resting platelets associated with a relatively slow Koff) as compared to others. These differences might determine the pharmacodynamics and pharmackokinetics of the different GPIIb/IIIa antagonists. British Journal of Pharmacology (2001) 133, 331–336; doi:10.1038/sj.bjp.0703943

The Kinetics of Competitive Radioligand Binding Predicted by the Law of Mass Action

Article

Feb 1984

Although equilibrium competitive radioligand binding studies are often used to characterize hormone and neurotransmitter receptors, the kinetics of such experiments have not been extensively explored. The interactions of the radioligand and competitor with the receptors can be described by two differential equations which can be solved to yield a single equation describing the binding of the radioligand as a function of time. This equation has several applications: First, it can be used to simulate competitive binding reactions under defined conditions. Second, fitting experimental data to this equation allows one to determine the association and dissociation rate constants of the competing ligand, parameters that cannot be derived from equilibrium experiments. Furthermore, this method can be used to determine the KI of the competing drug from data acquired before equilibrium is reached. Third, mathematical analysis of the binding equation allowed us to answer two specific questions regarding the kinetics of competitive radioligand binding: how long such an incubation takes to equilibrate, and how the IC50 varies over time. The answers to these questions depended, to a large extent, on the relative values of the dissociation rate constants of the radioligand and competitor, which can be determined as noted above. When the competitor dissociates from the receptors more rapidly than the radioligand, the IC50 first decreases and then increases, but never has a value less than the KI. At low radioligand concentrations, equilibrium is reached in the same amount of time required of the radioligand to dissociate completely from the receptors as determined in an "off-rate experiment." At higher concentrations of radioligand this time is halved. When the competitor dissociates from the receptor more slowly than does the radioligand, then the time required to equilibrate depends only on the dissociation rate constant of the competitor, and the IC50 decreases over time.

In vivo time-dependent inhibition of human steroid 5 alpha-reductase by finasteride

Article

Jan 1996

Gaochao Tian

Finasteride (17 beta-(N-t-butylcarbamoyl)-4-aza-5 alpha-androstan-1-en-3- one), a time-dependent, irreversible inhibitor of human steroid 5 alpha-reductase (5AR), may only reduce dihydrotestosterone levels in humans by approximately 60% at the doses used clinically. A theoretical model was developed to aid in understanding the in vivo efficacy data of finasteride. According to the theory, whether an enzyme can be inhibited in vivo by an irreversible inhibitor is dependent on the value of a ratio of the observed rate of enzyme inhibition over the rate constant for inhibitor elimination. As shown, this ratio should be in excess of 3 for > 95% inhibition of the target in vivo. Subsequent application of the theory to evaluate the in vivo efficacy data of finasteride indicates low effective concentration of finasteride at the inhibition sites and suggests complete inhibition of 5AR 2, but insufficient suppression of 5AR 1 at the clinical doses.

Candesartan (CV-11974) dissociates slowly from the angiotensin AT1 receptor

Article

Feb 1997
EUR J PHARMACOL

The mechanisms of the insurmountable antagonism of 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]1H-benzimid azole -7-carboxylic acid, candesartan (CV-11974), an angiotensin AT1 receptor antagonist, on angiotensin II-induced rabbit aortic contraction were examined in contraction and binding studies. Preincubation of the rabbit aorta with CV-11974 (0.1 nM) for 30 min reduced the maximal contractile response to angiostensin II by approximately 50%. This insurmountable antagonism of CV-11974 was reversed in the presence of losartan (1 microM), a surmountable angiotensin AT1 receptor antagonist. The inhibitory effect of CV-11974 on angiotensin II-induced contraction persisted longer after washing than did that of losartan but was irreversible. Scatchard analysis of [3H]CV-11974 binding in bovine adrenal cortical membranes indicated the existence of a single class of binding sites (Kd = 7.4 nM). Competition binding studies using angiotensin II receptor agonists and antagonists have demonstrated that [3H[CV-11974 binding sites may be identical to angiotensin AT1 receptors. The dissociation rate of [3H]CV-11974 binding (t1/2 = 66 min) was 5 times slower than that of [125I]angiotensin II binding (t1/2 = 12 min). These results suggest that the insurmountable antagonism by CV-11974 is due to its slow dissociation from angiotensin AT1 receptors.

Pharmacologic properties of candesartan cilexetil - Possible mechanisms of long-acting antihypertensive action

Article

Feb 1999
J Hum Hypertens

Candesartan cilexetil has shown potent and long-lasting antihypertensive effects in clinical trials and in several animal models of hypertension. In spontaneously hypertensive rats, the duration of the antihypertensive effect of candesartan cilexetil was compared to those of losartan, valsartan, eprosartan, and irbesartan at the same degree of maximal blood pressure reduction. A single oral dose of candesartan cilexetil at 0.3 mg/kg reduced maximal blood pressure by about 25 mm Hg, and the antihypertensive effect of candesartan cilexetil lasted the longest, continuing for more than 1 week, without an effect on circadian rhythm. In a rabbit aortic preparation, candesartan, active form of candesartan cilexetil, decreased the maximal contractile response of angiotensin II. This inhibitory mode was different from that of other angiotensin II-receptor antagonists, and showed a shift to the right in the angiotensin II-induced contraction curve and/or a small depression of the maximal response. In kinetic studies using bovine adrenal cortical membrane and tritiated candesartan, both receptor association and dissociation were found to be slow. The dissociation rate of tritiated candesartan binding (t1/2 = 66 min) was five times slower than that of radiolabelled angiotensin II binding (t1/2 = 12 min). The insurmountable inhibition of candesartan in vascular contraction is the result of its tight binding and slow dissociation from angiotensin II AT1 receptors. These characteristics are related to the potency and long duration of action in candesartan cilexetil.

Rofecoxib [Vioxx, MK-0966; 4-(4 '-methylsulfonylphenyl)-3-phenyl-2-(5H)-furanone]: A potent and orally active cyclooxygenase-2 inhibitor. Pharmacological and biochemical profiles

Article

Aug 1999

The discoveries that cyclooxygenase (COX)-2 is an inducible form of COX involved in inflammation and that COX-1 is the major isoform responsible for the production of prostaglandins (PGs) in the gastrointestinal tract have provided a rationale for the development of specific COX-2 inhibitors as a new class of anti-inflammatory agents with improved gastrointestinal tolerability. In the present study, the preclinical pharmacological and biochemical profiles of rofecoxib [Vioxx, also known as MK-0966, 4-(4'-methylsulfonylphenyl)-3-phenyl-2-(5H)-furanone], an orally active COX-2 inhibitor, are described. Rofecoxib is a potent inhibitor of the COX-2-dependent production of PGE(2) in human osteosarcoma cells (IC(50) = 26 +/- 10 nM) and Chinese hamster ovary cells expressing human COX-2 (IC(50) = 18 +/- 7 nM) with a 1000-fold selectivity for the inhibition of COX-2 compared with the inhibition of COX-1 activity (IC(50) > 50 microM in U937 cells and IC(50) > 15 microM in Chinese hamster ovary cells expressing human COX-1). Rofecoxib is a time-dependent inhibitor of purified human recombinant COX-2 (IC(50) = 0.34 microM) but caused inhibition of purified human COX-1 in a non-time-dependent manner that could only be observed at a very low substrate concentration (IC(50) = 26 microM at 0.1 microM arachidonic acid concentration). In an in vitro human whole blood assay, rofecoxib selectively inhibited lipopolysaccharide-induced, COX-2-derived PGE(2) synthesis with an IC(50) value of 0.53 +/- 0.02 microM compared with an IC(50) value of 18.8 +/- 0.9 microM for the inhibition of COX-1-derived thromboxane B(2) synthesis after blood coagulation. Using the ratio of the COX-1 IC(50) values over the COX-2 IC(50) values in the human whole blood assay, selectivity ratios for the inhibition of COX-2 of 36, 6.6, 2, 3, and 0.4 were obtained for rofecoxib, celecoxib, meloxicam, diclofenac, and indomethacin, respectively. In several in vivo rodent models, rofecoxib is a potent inhibitor of carrageenan-induced paw edema (ID(50) = 1.5 mg/kg), carrageenan-induced paw hyperalgesia (ID(50) = 1.0 mg/kg), lipopolysaccharide-induced pyresis (ID(50) = 0.24 mg/kg), and adjuvant-induced arthritis (ID(50) = 0.74 mg/kg/day). Rofecoxib also has a protective effect on adjuvant-induced destruction of cartilage and bone structures in rats. In a (51)Cr excretion assay for detection of gastrointestinal integrity in either rats or squirrel monkeys, rofecoxib has no effect at doses up to 200 mg/kg/day for 5 days. Rofecoxib is a novel COX-2 inhibitor with a biochemical and pharmacological profile clearly distinct from that of current nonsteroidal anti-inflammatory drugs and represents a new therapeutic class of anti-inflammatory agents for the treatment of the symptoms of osteoarthritis and rheumatoid arthritis with improved gastrointestinal tolerability.

A comparison of the efficacy and duration of action of candesartan cilexetil and losartan as assessed by clinic and ambulatory blood pressure after a missed dose, in truly hypertensive patients: a placebo-controlled, forced titration study. Candesartan/Losartan Study Investigators

Article

Dec 1999

The purpose of this double-blind, forced titration study was to compare the antihypertensive effect duration of candesartan cilexetil, which has a long-lasting binding to the human AT1-receptor, to that of losartan on ambulatory BP (ABP) not only during the 24-h dosing interval but also during the day of a missed dose intake. After a 4-week placebo lead-in period, 268 patients with sitting diastolic BP 95 to 110 mm Hg and mean awake ambulatory DBP ≥85 mm Hg were randomized to receive either 8 mg of candesartan, 50 mg of losartan, or placebo for a 4-week period. Thereafter, the doses were doubled in all patients for an additional 4-week period. Ambulatory BP monitoring was performed for 36 h after dosing and clinic BP measured 48 h after dosing. Candesartan cilexetil (16 mg) reduced ABP to a significantly greater extent than 100 mg of losartan, particularly for systolic ABP during daytime (P < .05), nighttime (P < .05), and 24-h (P < .01) periods, systolic (P < .01) and diastolic (P < .05) ABP between 0 and 36 h, and both systolic (P < .001) and diastolic (P < 0.001) ABP during the day of a missed dose. Clinic BP at 48 h after dosing was significantly reduced exclusively with 16 mg of candesartan. The differences in BP reduction between 8 mg of candesartan and 50 mg of losartan were statistically significant for systolic ABP during daytime (P < .01), nighttime (P < .05), 24-h (P < .01), 0 to 36 h (P < .05) and during the day of missed dose (P < .05). Moreover, although losartan did not significantly reduce ambulatory BP in a dose-related manner, ambulatory systolic and diastolic BP reductions with 16 mg of candesartan were significantly greater (P < .01 and < .001) than those seen with 8 mg of candesartan during every period at the ABP supporting a dose–response relationship. In conclusion, this forced titration study in ambulatory hypertensive patients demonstrates that candesartan cilexetil provides significant dose-dependent reduction in both clinic and ambulatory BP in doses ranging from 8 to 16 mg once daily. Furthermore, candesartan cilexetil is superior to losartan in reducing systolic ABP and in controlling both systolic and diastolic ABP on the day of a missed dose. The differences observed between both agents are most likely attributable to a tighter binding to, and a slower dissociation from, the receptor binding site with candesartan cilexetil. Am J Hypertens 1999;12:1181–1187 © 1999 American Journal of Hypertension, Ltd.

Disabling ErbB Receptors with Rationally Designed Exocyclic Mimetics of Antibodies: Structure-Function Analysis

Article

Sep 2001

Overexpression of the HER2 receptor is observed in about 30% of breast and ovarian cancers and is often associated with an unfavorable prognosis. We have recently designed an anti-HER2 peptide (AHNP) based on the structure of the CDR-H3 loop of the anti-HER2 rhumAb 4D5 and showed that this peptide can mimic some functions of rhumAb 4D5. The peptide disabled HER2 tyrosine kinases in vitro and in vivo similar to the monoclonal antibody (Park, B.-W. et al. Nat. Biotechnol. 2000, 18, 194--198). AHNP has been shown to selectively bind to the extracellular domain of the HER2 receptor with a submicromolar affinity in Biacore assays. In the present paper, we demonstrate that in addition to being a structural and functional mimic of rhumAb 4D5, AHNP can also effectively compete with the antibody for binding to the HER2 receptor indicating a similar binding site for the peptide and the parental antibody. To further develop AHNP as an antitumor agent useful for preclinical trials and as a radiopharmaceutical to be used for tumor imaging, a number of derivatives of AHNP have been designed. Structure--function relationships have been studied using surface plasmon resonance technology. Some of the AHNP analogues have improved binding properties, solubility, and cytotoxic activity relative to AHNP. Residues in the exocyclic region of AHNP appear to be essential for high-affinity binding. Kinetic and equilibrium analysis of peptide-receptor binding for various AHNP analogues revealed a strong correlation between peptide binding characteristics and their biological activity. For AHNP analogues, dissociation rate constants have been shown to be better indicators of peptide biological activity than receptor-binding affinities. This study demonstrates a possibility of mimicking the well-documented antibody effects and its applications in tumor therapy by much smaller antibody-based cyclic peptides with potentially significant therapeutic advantages. Strategies used to improve binding properties of rationally designed AHNP analogues are discussed.

Rusnak DW, Lackey K, Affleck K, Wood ER, Alligood KJ, Rhodes N, Keith BR, Murray DM, Knight WB, Mullin RJ, Gilmer TMThe effects of the novel, reversible epidermal growth factor receptor/ErbB-2 tyrosine kinase inhibitor, GW2016, on the growth of human normal and tumor-derived cell lines in vitro and in vivo. Mol Cancer Ther 1(2): 85-94

Article

Dec 2001

The epidermal growth factor receptor (EGFR) and ErbB-2 transmembrane tyrosine kinases are currently being targeted by various mechanisms in the treatment of cancer. GW2016 is a potent inhibitor of the ErbB-2 and EGFR tyrosine kinase domains with IC50 values against purified EGFR and ErbB-2 of 10.2 and 9.8 nM, respectively. This report describes the efficacy in cell growth assays of GW2016 on human tumor cell lines overexpressing either EGFR or ErbB-2: HN5 (head and neck), A-431 (vulva), BT474 (breast), CaLu-3 (lung), and N87 (gastric). Normal human foreskin fibroblasts, nontumorigenic epithelial cells (HB4a), and nonoverexpressing tumor cells (MCF-7 and T47D) were tested as negative controls. After 3 days of compound exposure, average IC50 values for growth inhibition in the EGFR- and ErbB-2-overexpressing tumor cell lines were < 0.16 microM. The average selectivity for the tumor cells versus the human foreskin fibroblast cell line was 100-fold. Inhibition of EGFR and ErbB-2 receptor autophosphorylation and phosphorylation of the downstream modulator, AKT, was verified by Western blot analysis in the BT474 and HN5 cell lines. As a measure of cytotoxicity versus growth arrest, the HN5 and BT474 cells were assessed in an outgrowth assay after a transient exposure to GW2016. The cells were treated for 3 days in five concentrations of GW2016, and cell growth was monitored for an additional 12 days after removal of the compound. In each of these tumor cell lines, concentrations of GW2016 were reached where outgrowth did not occur. Furthermore, growth arrest and cell death were observed in parallel experiments, as determined by bromodeoxyuridine incorporation and propidium iodide staining. GW2016 treatment inhibited tumor xenograft growth of the HN5 and BT474 cells in a dose-responsive manner at 30 and 100 mg/kg orally, twice daily, with complete inhibition of tumor growth at the higher dose. Together, these results indicate that GW2016 achieves excellent potency on tumor cells with selectivity for tumor versus normal cells and suggest that GW2016 has value as a therapy for patients with tumors overexpressing either EGFR or ErbB-2.

Elucidation of HIV-1 protease resistance by characterization of interaction kinetics between inhibitors and enzyme variants

Article

Jun 2003
ANTIVIR RES

The kinetics of the interaction between drug-resistant variants of HIV-1 protease (G48V, V82A, L90M, I84V/L90M, and G48V/V82A/I84V/L90M) and clinically used inhibitors (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) were determined using biosensor technology. The enzyme variants were immobilized on a biosensor chip and the association and dissociation rate constants (k(on) and k(off)) and affinities (K(D)) for interactions with inhibitors were determined. A unique interaction kinetic profile was observed for each variant/inhibitor combination. Substitution of single amino acids in the protease primarily resulted in reduced affinity through increased k(off) for the inhibitors. For inhibitors characterized by fast association rates to wild-type protease (ritonavir, amprenavir, and indinavir), additional substitutions resulted in a further reduction of affinity by a combination of decreased k(on) and increased k(off). For inhibitors characterized by slow dissociation rates to wild-type enzyme (saquinavir and nelfinavir), the decrease of affinity conferred by additional mutations was attributed to increased k(off) values. Development of resistance thus appears to be associated with a change of the distinctive kinetic parameter contributing to high affinity. Further inhibitor design should focus on improving the "weak point" of the lead compound, that being either k(on) or k(off).

Characteristic Physical Properties and Structural Fragments of Marketed Oral Drugs

Article

Feb 2004

An increasingly competitive pharmaceutical market demands improvement in the efficiency and probability of drug candidate discovery. Usually these new drug candidates are targeted for oral administration, so a detailed understanding of the molecular-level properties that relate to optimal pharmacokinetics is a critical step toward improving the probability of selecting successful clinical candidates. Although the characteristics of druglike molecules have been previously discussed in the literature, the importance of this topic sustains a continued interest for additional perspective and further detailed statistical analyses. In this contribution, we approach the analysis from the perspective of profiling distinguishing features of orally administered drugs. We have compiled both structural and route-administration information for a total of 1729 marketed drugs to provide a solid basis for developing a new perspective on the characteristics of over 1000 orally administered drugs. The molecular properties and most commonly occurring structural elements are statistically analyzed to capture the differences between routes of administration, as well as between marketed drugs and SAR or clinical compounds. We find that, with respect to other routes of administration, oral drugs tend to be lighter and have fewer H-bond donors, acceptors, and rotatable bonds than drugs with other routes of administration. These differences are particularly pronounced when comparing the mean values for oral vs injectable drugs. We also demonstrate that the mean property values for oral drugs do not vary substantially with respect to launch date, suggesting that the range of acceptable oral properties is independent of synthetic complexity or targeted receptor. Finally, we note that, while these properties are descriptive of each class, they are not necessarily predictive of what class any particular drug will reside in, since there is significant overlap in the acceptable ranges found for each drug class.

Biochemical Mechanisms of Drug Action: What Does It Take For Success?

Article

Oct 2004

David C. Swinney

Drug discovery is extremely difficult. There are many unanticipated scientific, medical and business challenges to every drug discovery programme. It is important to increase our understanding of the fundamental properties of effective drugs so that we can anticipate potential problems in developing new agents. This article addresses potential drug discovery and development risks associated with the biochemical mechanism of drug action, and proposes simple rules to minimize these risks.

Kinetic Exclusion Assay Technology: Characterization of Molecular Interactions

Article

Jan 2005

Characterization of intermolecular interactions in terms of affinity, binding kinetics, stoichiometry, specificity, and thermodynamics can facilitate the selection of lead compounds in the discovery and development of protein therapeutics. KinExA (Sapidyne Instruments, Inc., Boise, ID) is a relatively new technology that is gaining use in characterizing molecular interactions, particularly with respect to antibody therapeutics. KinExA offers a platform that allows the measurement of true equilibrium binding affinity and kinetics using unmodified molecules in solution phase. This is accomplished by using a solid-phase immobilized molecule to probe for free concentration of one interaction component after allowing sufficient time to reach equilibrium (affinity measurements), or under pre-equilibrium conditions (kinetics). In this review, the theory behind KinExA technology is discussed, and examples of applying this technology to antibody characterization are provided. Finally, a comparison among KinExA, Biacore (surface plasmon resonance), and isothermal titration calorimetry is presented, and potential future improvements and applications of KinExA are discussed.

Ehrlich, P. Chemotherapeutics: scientific principles, methods and results. Lancet 182, 445-451

Kenakin, T. Efficacy at G-protein-coupled receptors. Nature Rev. Drug Discov. 1, 103-110

Meltzer, H. Y. Action of atypical antipsychotics (letter to the editor). Am. J. Psychiatry 159, 153-154

Drug–target residence time and its implications for lead optimization

Abstract

No full-text available

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