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Overview of a typical ensemble-based computer-aided drug design strategy Shown here is a simplified workflow of a structure-based computer-aided drug design process involving target selection, binding site identification, high-throughput virtual screening of ligand libraries, experimental validation, and optimization for potency and selectivity.
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A number of computational techniques have been proposed to expedite the process of allosteric ligand binding site identification
in inherently flexible and hence challenging drug targets. Some of these techniques have been instrumental in the discovery
of allosteric ligand binding sites on Ras proteins, a group of elusive anticancer drug targets. T...
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Context 1
... typical modern CADD workflow is shown in Fig. 2. One of the key starting points for CADD is a well-characterized target whose atomic structure has been determined to a sufficiently high resolution [64]. Ras meets this condition with more than 150 high resolution crystal structures available in the protein data bank. Also of key im- portance is knowledge about the potential drug ...
Context 2
... docking runs and different programs to reduce false positives that usually arise from limitations in the scoring func- tions [89,90]. The predicted hits should be validated by experimental methods before being subjected to a series of optimization steps that are required to generate a lead compound with the desired potency and selectivity (Fig. ...
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Citations
... Our previous studies showed that a deeper understanding of protein motion is key to finding ligands for different allosteric sites of KRAS. [22][23][24][25]50 We reported a dynamics-guided drug discovery workflow that allowed us to profile the druggability and identify the chemical fingerprint of ligands targeting four different pockets on KRAS. 35 Specifically, we used molecular dynamics (MD) simulations of wild type (WT) and various KRAS mutants to analyze the potential of all four allosteric sites in each protein to bind drug-like molecules, and virtual screening of pocket-tailored libraries against the MD ensembles to document the chemical diversity of the potential hits. ...
... The orientational dynamics of the two proteins on membrane surfaces is also generally similar. 24,50,57 On the contrary, there is some evidence to suggest that some KRAS mutants stabilize a membrane binding mode in which p1 is more accessible to solvent than in WT KRAS. 58 Taken together, it appears that pan-RAS activity or impact of membrane does not explain the data in Having ruled out effects on nucleotide exchange, effector binding, pan-RAS activity, or membrane binding as possible mechanisms, we considered the difference in the binding affinity of ACA22 for GDP-and GTP-bound WT versus G12D KRAS. ...
Mutations in KRAS account for about 20% of human cancers. Despite the major progress in recent years toward the development of KRAS inhibitors, including the discovery of covalent inhibitors of the G12C KRAS variant for the treatment of non-small-cell lung cancer, much work remains to be done to discover broad-acting inhibitors to treat many other KRAS-driven cancers. In a previous report, we showed that a 308.4 Da small-molecule ligand [(2R)-2-(N'-(1H-indole-3-carbonyl)hydrazino)-2-phenyl-acetamide] binds to KRAS with low micro-molar affinity [Chem. Biol. Drug Des.2019; 94(2):1441-1456]. Binding of this ligand, which we call ACA22, to the p1 pocket of KRAS and its interactions with residues at beta-strand 1 and the switch loops have been supported by data from nuclear magnetic resonance spectroscopy and microscale thermophoresis experiments. However, the inhibitory potential of the compound was not demonstrated. Here, we show that ACA22 inhibits KRAS-mediated signal transduction in cells expressing wild type (WT) and G12D mutant KRAS and reduces levels of guanosine triphosphate-loaded WT KRAS more effectively than G12D KRAS. We ruled out the direct effect on nucleotide exchange or effector binding as possible mechanisms of inhibition using a variety of biophysical assays. Combining these observations with binding data that show comparable affinities of the compound for the active and inactive forms of the mutant but not the WT, we propose conformational selection as a possible mechanism of action of ACA22.
... Several studies reveal the intrinsic allosteric nature of KRAS [106][107][108][109][110] providing insightful ideas for targeting this oncoprotein via modalities other than active site inhibition. Such an approach was implemented by the Shokat group in 2013, targeting G12C GDP-bound KRAS mutant with small inhibitors that bind covalently on a pocket located over the Switch II loop, impairing GTP and Raf binding. ...
K-Ras4B is one the most frequently mutated proteins in cancer, yet mechanistic details of its activation such as its homodimerization on the membrane remain elusive. The structural determinants of K-Ras4B homodimerization have been debated with different conformations being proposed in the literature. Here, we perform microsecond all-atom Molecular Dynamics (MD) simulations on the K-Ras4B monomer in solution, the K-Ras4B monomer on the membrane, and two experimentally-based K-Ras4B dimer models of the α4-α5 interface to investigate the stability of these structures bound to GTP on a model cell membrane. We then evaluate the complexes for their propensity to form stable dimers on the plasma membrane in the presence and absence of Raf[RBD-CRD] effectors. We find that Raf[RBD-CRD] effectors enhance dimer stability, suggesting that the presence of effectors is necessary for K-Ras4B dimers stabilization on the cell membrane. Moreover, we observe, for the first time, a dynamic water channel at the K-Ras4B dimer interface, and identify putative allosteric connections in the K-Ras4B dimer interface. To discover novel K-Ras4B interfaces, we perform coarse-grained MD simulations in two dissociated K-Ras4B monomers on the membrane, which reveal that the dominant dimer interface is the α4-α5 interface. Finally, a druggability analysis is performed in the different K-Ras4B structures in the monomeric states. Strikingly, all known binding pockets of K-Ras4B are identified only in the structure that is membrane-bound, but not in the solution structure. Based on these results, we propose that modulating the protein-membrane interactions can be an alternative strategy for inhibiting K-Ras4B signaling.
... One potential solution to overcome this resistance is targeting proteins upstream of MEK such as RAF or RAS. Unfortunately, RAS proteins are ubiquitous, and the binding Acta Haematol 2022;145:529-536 DOI: 10.1159/000525566 pocket shares structural similarities to many other proteins, making it notoriously difficult to target [22,23]. Sotorasib, a novel KRAS G12C inhibitor, has shown promising results in non-small-cell lung cancer [24]. ...
Introduction:
RAS pathway mutations are common mechanisms of resistance to acute myeloid leukemia (AML) therapies. Trametinib, an oral MEK inhibitor, has been shown to have single-agent activity in relapsed/refractory AML and preclinical synergy with venetoclax.
Methods:
We conducted a single-center, open-label, phase 2 trial of the combination of azacitidine, venetoclax, and trametinib in patients with relapsed or refractory AML harboring a RAS pathway-activating mutation.
Results:
Sixteen patients were treated. The patients were heavily pretreated with a median number of 4 prior therapies; 13 (81%) had received prior hypomethylating agent (HMA) with venetoclax and 8 (50%) had undergone prior stem cell transplant. Four patients (25%) responded (CR, n=1; CRi, n=1; MLFS, n=2). Two of the 3 patients (67%) who had not previously received HMA plus venetoclax responded; in contrast, only 2 of the 13 patients (15%) who had previously received HMA plus venetoclax responded. The median OS was 2.4 months, and the 6-month OS rate was 31%. Related grade 3-4 adverse events occurred in 50% of patients, and 50% of patients required a dose adjustment of trametinib.
Conclusions:
The combination of azacitidine, venetoclax and trametinib had only modest activity in patients with relapsed/refractory AML, with a response rate that was similar to previous reports of trametinib monotherapy. Substantial toxicity was observed with this combination. Given the established role of RAS pathway mutations in mediating resistance to AML therapies, future studies of better tolerated, more active inhibitors of this pathway are still needed.
... d The rates between macrostates from HMM analysis. Thicker arrows correspond to faster rates confirming experimental findings (Chakrabarti et al. 2016;Li et al. 2018b;Mazhab-Jafari et al. 2015;McCarthy et al. 2016;Gorfe 2013, 2014;Prakash et al. 2016;Travers et al. 2018). More specifically, molecular dynamic (MD) simulations suggest that the association of the G-domain to the membrane is lipid-dependent (Cao et al. 2019;Gregory et al. 2017;Prakash et al. 2016), highlighting the role of the C-terminal polybasic hypervariable region (HVR, residues 167-185) (Banerjee et al. 2016;Janosi and Gorfe 2010;Neale and Garcia 2018;Zhou et al. 2017) and its farnesyl group, both of which are involved in the recruitment of KRAS4b within the anionic-rich lipid domains of membranes (Brunsveld et al. 2009;Erwin et al. 2016;Jang et al. 2015;Plowman et al. 2008;Rowat et al. 2004;Vogel et al. 2009). ...
Small GTPase proteins are ubiquitous and responsible for regulating several processes related to cell growth and differentiation. Mutations that stabilize their active state can lead to uncontrolled cell proliferation and cancer. Although these proteins are well characterized at the cellular scale, the molecular mechanisms governing their functions are still poorly understood. In addition, there is limited information about the regulatory function of the cell membrane which supports their activity. Thus, we have studied the dynamics and conformations of the farnesylated KRAS4b in various membrane model systems, ranging from binary fluid mixtures to heterogeneous raft mimics. Our approach combines long time-scale coarse-grained (CG) simulations and Markov state models to dissect the membrane-supported dynamics of KRAS4b. Our simulations reveal that protein dynamics is mainly modulated by the presence of anionic lipids and to some extent by the nucleotide state (activation) of the protein. In addition, our results suggest that both the farnesyl and the polybasic hypervariable region (HVR) are responsible for its preferential partitioning within the liquid-disordered (Ld) domains in membranes, potentially enhancing the formation of membrane-driven signaling platforms.
Graphic Abstract
... sites, usually in cases where the location of binding sites are not yet known, or to identify allosteric sites. Predictive binding tools have been used in conjunction with molecular docking to elucidate potential allosteric sites in various systems (Nayal and Honig, 2006;McCarthy et al., 2015;Kim et al., 2012). As well as a hydrophilic and hydrophobic contour map of the predicted binding site, SiteMap also generates a SiteScore which characterises the binding site in terms of its size, exposure to solvent, tightness of interaction between site points and the receptor, balance between the site hydrophobicity and hydrophilicity, and the degree of hydrogen bond donation and acceptance (Halgren, 2009;Halgren, 2007). ...
The SARS-CoV-2 virus is causing COVID-19 resulting in an ongoing pandemic with serious health, social, and economic implications. Much research is focused in repurposing or identifying new small molecules which may interact with viral or host-cell molecular targets. An important SARS-CoV-2 target is the main protease (Mpro), and the peptidomimetic α-ketoamides represent prototypical experimental inhibitors. The protease is characterised
by the dimerization of two monomers each which contains the catalytic dyad defined by Cys145 and His41 residues (active site). Dimerization yields the functional homodimer. Here, our aim was to investigate small molecules, including lopinavir and ritonavir, α-ketoamide 13b, and ebselen, for their ability to interact with the Mpro. The sirtuin 1 agonist SRT1720 was also used in our analyses. Blind docking to each monomer individually
indicated preferential binding of the ligands in the active site. Site-mapping of the dimeric protease indicated a highly reactive pocket in the dimerization region at the domain III apex. Blind docking consistently indicated a strong preference of ligand binding in domain III, away from the active site. Molecular dynamics simulations indicated that ligands docked both to the active site and in the dimerization region at the apex, formed relatively stable interactions. Overall, our findings do not obviate the superior potency with respect to inhibition of protease activity of covalently-linked inhibitors such as α-ketoamide 13b in the Mpro active site. Nevertheless, along with those from others, our findings highlight the importance of further characterisation of the Mpro active site and any potential allosteric sites.
... Table 1 lists some published 146 3D structure-based LBS prediction methods. 147 The basic idea of LBS prediction methods based on spatial 148 geometry measurements is to locate large or even the largest hol- 149 low or cavity on the protein structure by calculating and simulat- 150 ing some certain geometric measures from the protein structure 151 information. Researchers have come up with many different and 152 creative ways to accomplish this over the past few decades. ...
Proteins participate in various essential processes in vivo via interactions with other molecules. Identifying the residues participating in these interactions not only provides biological insights for protein function studies but also has great significance for drug discoveries. Therefore, predicting protein–ligand binding sites has long been under intense research in the fields of bioinformatics and computer aided drug discovery. In this review, we first introduce the research background of predicting protein–ligand binding sites and then classify the methods into four categories, namely, 3D structure-based, template similarity-based, traditional machine learning-based and deep learning-based methods. We describe representative algorithms in each category and elaborate on machine learning and deep learning-based prediction methods in more detail. Finally, we discuss the trends and challenges of the current research such as molecular dynamics simulation based cryptic binding sites prediction, and highlight prospective directions for the near future.
... The concept of Ras allostery was initially somewhat controversial given its small size and shallow surface that lacks any obvious ligand binding site outside of the canonical nucleotide binding site. This has changed by the identification of up to four allosteric ligand binding sites first using computational approaches [58][59][60][61][62][63][64], and then using NMR or crystallographic studies of ligands bound to these pockets [65][66][67][68] (Figure 2). Many ligands that directly bind to the allosteric sites of K-Ras and modulate its functions have been reported [58,63,65,69,70], including small-molecules [58,70,71], peptidomimetics [72,73], monobodies [74], and even DARPins [75,76]. ...
Activating somatic K-Ras mutations are associated with >15% all human tumors and up to 90% of specific tumor types such as pancreatic cancer. Successfully inhibiting abnormal K-Ras signaling would therefore be a game changer in cancer therapy. However, K-Ras has long been considered an undruggable target for various reasons. This view is now changing by the discovery of allosteric inhibitors that directly target K-Ras and inhibit its functions, and by the identification of new mechanisms to dislodge it from the plasma membrane and thereby abrogate its cellular activities. In this review, we will discuss recent progresses and challenges to inhibiting aberrant K-Ras functions by these two approaches. We will also provide a broad overview of other approaches such as inhibition of K-Ras effectors, and offer a brief perspective on the way forward.
... Recent studies suggest that protein flexibility is associated with several biological activities such as protein stability [86,87], allosteric effects [78,83,88] and protein interactions [89]. Based on these foundations, molecular docking methodologies and computer programmes were developed [90][91][92][93][94][95][96]. For example, recently Agafonov et al. [97] and Wilson et al. [98] showed that the induced-fit (conformational change after binding) is a prerequisite for the anticancer drug Gleevec to bind with Abl kinase protein. ...
Apoptosis is a vital physiological process, which is observed in various biological events. The anti-apoptotic and pro-apoptotic members of Bcl-2 family are the most characterized proteins which are involved in the regulation of apoptotic cell death. The anti-apoptotic proteins such as Bcl-2 and Bcl-xL prevent apoptosis, whereas pro-apoptotic members like Bax and Bak, elicit the release of caspases from death antagonists inducing apoptosis. Thus, the Bcl-2 family of proteins play a vital role in controlling programmed cell death. Over expression of anti-apoptotic Bcl-2 proteins are often directly associated with various kinds of cancer. Developing suitable inhibitors for controlling the elevated levels of these proteins got much attention in last decade. Structural biology techniques such as Nuclear Magnetic Resonance (NMR) spectroscopy, X-ray crystallography, homology modeling and molecular docking play a significant role in identifying the key inhibitors of these proteins. The authors have developed and tested successfully, several series of indole pharmacore containing inhibitors for Bcl-2 and Bcl-xL proteins based on the homology modeling, docking and suitable biochemical and apoptosis assays. This review provides a summary of potential inhibitor molecules developed for Bcl-2 and Bcl-xL proteins, as well as the the key residues of these proteins interacting with potential drug molecules. The present appraisal also focuses on the role of computational algorithms in developing potential drug molecules,with more emphasis on the role of homology modeling and docking studies in developing inhibitors for Bcl- 2, and Bcl-xL proteins in cancer therapy.
... The use of allostery for druggable targets in intervening pathogenesis of many diseases are many, ranging from (i) identifying allosteric targets that influence enzymatic activity; (ii) identifying allosteric epitopes/sites for targeting by antibodies [46,47,48,49] and/or inhibitors [50,51,52,53,54,55] to affect the active site; or (iii) repurposing existing natural enzymes. ...
The reductionist approach is prevalent in biomedical science. However, increasing evidence now shows that biological systems cannot be simply considered as the sum of its parts. With experimental, technological and computational advances, we can now do more than view parts in isolation, thus we propose that an increasing holistic view (where a protein is investigated as much as a whole as possible) is now timely. To further advocate this, we review and discuss several studies and applications involving allostery, where distant protein regions can cross-talk to influence functionality. Therefore, we believe that an increasing big picture approach holds great promise, particularly in the areas of antibody engineering and drug discovery in rational drug design.
... In previous reports, we described four allosteric ligand-binding sites on KRAS using a range of computational approaches (32,33), including molecular dynamics (MD) simulations to sample transient conformations with open allosteric pockets (34)(35)(36). Among these, pocket p1 was the best characterized and is well established as a suitable target with many crystal structures of p1-bound ligand-KRAS complexes available in the protein data bank (PDB). ...
Approximately 15 precent of all human tumors harbor mutant KRAS, a membrane-associated small GTPase and a notorious oncogene. Somatic mutations that render KRAS constitutively active lead to uncontrolled cell growth, survival, proliferation, and eventually cancer. KRAS is thus a critical anticancer drug target. However, despite aggressive efforts in recent years, there is no drug on the market that directly targets KRAS. In the current work, we combined molecular simulation and high-throughput virtual screening with a battery of cell-based and biophysical assays to discover a novel, pyrazolopyrimidine-based allosteric KRAS inhibitor that exhibits promising biochemical properties. The compound selectively binds to active KRAS with submicromolar affinity, slightly modulates exchange factor activity, disrupts effector Raf binding, significantly reduces signal transduction through mutant KRAS and inhibits cancer cell growth. Moreover, by studying two of its analogues, we identified key chemical features of the compound that are critical for affinity, effect on effector binding and mode of action. We propose a set of specific interactions with key residues at the switch regions of KRAS as critical for abrogating effector binding and reducing the rate of nucleotide exchange. Together, these findings not only demonstrate the viability of direct KRAS inhibition and offer guidance for future optimization efforts, but also show that pyrazolopyrimidine-based compounds may represent a first-in-class lead toward a clinically relevant targeting of KRAS by allosteric non-covalent
inhibitors.
