Identification of GDC-1971 (RLY-1971), a SHP2 Inhibitor Designed for the Treatment of Solid Tumors.

Protein tyrosine phosphatase SHP2 mediates RAS-driven MAPK signaling and has emerged in recent years as a target of interest in oncology, both for treating with a single agent and in combination with a KRAS inhibitor. We were drawn to the pharmacological potential of SHP2 inhibition, especially following the initial observation that drug-like compounds could bind an allosteric site and enforce a closed, inactive state of the enzyme. Here, we describe the identification and characterization of GDC-1971 (formerly RLY-1971), a SHP2 inhibitor currently in clinical trials in combination with KRAS G12C inhibitor divarasib (GDC-6036) for the treatment of solid tumors driven by a KRAS G12C mutation.

A Computational Physics-based Approach to Predict Unbound Brain-to-Plasma Partition Coefficient, Kp,uu.

The blood-brain barrier (BBB) plays a critical role in preventing harmful endogenous and exogenous substances from penetrating the brain. Optimal brain penetration of small-molecule central nervous system (CNS) drugs is characterized by a high unbound brain/plasma ratio (Kp,uu). While various medicinal chemistry strategies and in silico models have been reported to improve BBB penetration, they have limited application in predicting Kp,uu directly. We describe a physics-based computational approach, a quantum mechanics (QM)-based energy of solvation (E-sol), to predict Kp,uu. Prospective application of this method in internal CNS drug discovery programs highlights the utility and accuracy of this new method, which showed a categorical accuracy of 79% and an R2 of 0.61 from a linear regression model.

An allosteric mechanism for potent inhibition of human ATP-citrate lyase.

ATP-citrate lyase (ACLY) is a central metabolic enzyme and catalyses the ATP-dependent conversion of citrate and coenzyme A (CoA) to oxaloacetate and acetyl-CoA1-5. The acetyl-CoA product is crucial for the metabolism of fatty acids6,7, the biosynthesis of cholesterol8, and the acetylation and prenylation of proteins9,10. There has been considerable interest in ACLY as a target for anti-cancer drugs, because many cancer cells depend on its activity for proliferation2,5,11. ACLY is also a target against dyslipidaemia and hepatic steatosis, with a compound currently in phase 3 clinical trials4,5. Many inhibitors of ACLY have been reported, but most of them have weak activity5. Here we report the development of a series of low nanomolar, small-molecule inhibitors of human ACLY. We have also determined the structure of the full-length human ACLY homo-tetramer in complex with one of these inhibitors (NDI-091143) by cryo-electron microscopy, which reveals an unexpected mechanism of inhibition. The compound is located in an allosteric, mostly hydrophobic cavity next to the citrate-binding site, and requires extensive conformational changes in the enzyme that indirectly disrupt citrate binding. The observed binding mode is supported by and explains the structure-activity relationships of these compounds. This allosteric site greatly enhances the 'druggability' of ACLY and represents an attractive target for the development of new ACLY inhibitors.

An Allosteric Inhibitory Site Conserved in the Ectodomain of P2X Receptor Channels.

P2X receptors constitute a gene family of cation channels gated by extracellular ATP. They mediate fast ionotropic purinergic signaling in neurons and non-excitable cell types in vertebrates. The highly calcium-permeable P2X4 subtype has been shown to play a significant role in cardiovascular physiology, inflammatory responses and neuro-immune communication. We previously reported the discovery of a P2X4-selective antagonist, the small organic compound BX430, with submicromolar potency for human P2X4 receptors and marked species-dependence (Ase et al., 2015). The present study investigates the molecular basis of P2X4 inhibition by the non-competitive blocker BX430 using a structural and functional approach relying on mutagenesis and electrophysiology. We provide evidence for the critical contribution of a single hydrophobic residue located in the ectodomain of P2X4 channel subunits, Ile312 in human P2X4, which determines blockade by BX430. We also show that the nature of this extracellular residue in various vertebrate P2X4 orthologs underlies their specific sensitivity or resistance to the inhibitory effects of BX430. Taking advantage of high-resolution crystallographic data available on zebrafish P2X4, we used molecular dynamics simulation to model the docking of BX430 on an allosteric binding site around Ile315 (zebrafish numbering) in the ectodomain of P2X4. We also observed that the only substitution I312D (human numbering) that renders P2X4 silent by itself has also a profound silencing effect on all other P2X subtypes tested when introduced at homologous positions. The generic impact of this aspartate mutation on P2X function indicates that the pre-TM2 subregion involved is conserved functionally and defines a novel allosteric inhibitory site present in all P2X receptor channels. This conserved structure-channel activity relationship might be exploited for the rational design of potent P2X subtype-selective antagonists of therapeutic value.

Medicinal Chemistry Projects Requiring Imaginative Structure-Based Drug Design Methods.

Computational methods for docking small molecules to proteins are prominent in drug discovery. There are hundreds, if not thousands, of documented examples-and several pertinent cases within our research program. Fifteen years ago, our first docking-guided drug design project yielded nanomolar metalloproteinase inhibitors and illustrated the potential of structure-based drug design. Subsequent applications of docking programs to the design of integrin antagonists, BACE-1 inhibitors, and aminoglycosides binding to bacterial RNA demonstrated that available docking programs needed significant improvement. At that time, docking programs primarily considered flexible ligands and rigid proteins. We demonstrated that accounting for protein flexibility, employing displaceable water molecules, and using ligand-based pharmacophores improved the docking accuracy of existing methods-enabling the design of bioactive molecules. The success prompted the development of our own program, Fitted, implementing all of these aspects. The primary motivation has always been to respond to the needs of drug design studies; the majority of the concepts behind the evolution of Fitted are rooted in medicinal chemistry projects and collaborations. Several examples follow: (1) Searching for HDAC inhibitors led us to develop methods considering drug-zinc coordination and its effect on the pKa of surrounding residues. (2) Targeting covalent prolyl oligopeptidase (POP) inhibitors prompted an update to Fitted to identify reactive groups and form bonds with a given residue (e.g., a catalytic residue) when the geometry allows it. Fitted-the first fully automated covalent docking program-was successfully applied to the discovery of four new classes of covalent POP inhibitors. As a result, efficient stereoselective syntheses of a few screening hits were prioritized rather than synthesizing large chemical libraries-yielding nanomolar inhibitors. (3) In order to study the metabolism of POP inhibitors by cytochrome P450 enzymes (CYPs)-for toxicology studies-the program Impacts was derived from Fitted and helped us to reveal a complex metabolism with unforeseen stereocenter isomerizations. These efforts, combined with those of other docking software developers, have strengthened our understanding of the complex drug-protein binding process while providing the medicinal chemistry community with useful tools that have led to drug discoveries. In this Account, we describe our contributions over the past 15 years-within their historical context-to the design of drug candidates, including BACE-1 inhibitors, POP covalent inhibitors, G-quadruplex binders, and aminoglycosides binding to nucleic acids. We also remark the necessary developments of docking programs, specifically Fitted, that enabled structure-based design to flourish and yielded multiple fruitful, rational medicinal chemistry campaigns.

Design, synthesis and RON receptor tyrosine kinase inhibitory activity of new head groups analogs of LCRF-0004.

New heteroarylcarboxamide head groups substituted with two aromatic rings analogs of thieno[3,2-b]pyridine-based kinase inhibitor LCRF-0004 were designed and synthesized. Potent inhibitors of RON tyrosine kinase with various level of selectivity for c-Met RTK were obtained.

Design and synthesis of constrained analogs of LCRF-0004 as potent RON tyrosine kinase inhibitors.

New fused bicyclic lactam head groups as rigidified analogs of thieno[3,2-b]pyridine-based kinase inhibitor LCRF-0004 were designed and synthesized. Depending on the functionalities and the size of these bicyclic head groups, potent inhibitors of RON tyrosine kinase with various level of selectivity against c-Met tyrosine kinase were obtained.

Design and synthesis of close analogs of LCRF-0004, a potent and selective RON receptor tyrosine kinase inhibitor.

New carboxamide head group analogs of thieno[3,2-b]pyridine-based kinase inhibitor LCRF-0004 were designed and synthesized. Potent and selective inhibitors of RON enzyme versus c-Met RTK were obtained.

Discovery of bicyclic pyrazoles as class III histone deacetylase SIRT1 and SIRT2 inhibitors.

A series of bicyclic pyrazole carboxamides was synthesized and tested for inhibitory activity against the class III deacetylase sirtuin enzymes. Moderate to low micromolar inhibitory activities were obtained against SIRT1 and SIRT2. These bicyclic pyrazole compounds represent a new class of sirtuin inhibitors with a preference for SIRT1 over SIRT2.

Docking ligands into flexible and solvated macromolecules. 7. Impact of protein flexibility and water molecules on docking-based virtual screening accuracy.

The use of predictive computational methods in the drug discovery process is in a state of continual growth. Over the last two decades, an increasingly large number of docking tools have been developed to identify hits or optimize lead molecules through in-silico screening of chemical libraries to proteins. In recent years, the focus has been on implementing protein flexibility and water molecules. Our efforts led to the development of Fitted first reported in 2007 and further developed since then. In this study, we wished to evaluate the impact of protein flexibility and occurrence of water molecules on the accuracy of the Fitted docking program to discriminate active compounds from inactive compounds in virtual screening (VS) campaigns. For this purpose, a total of 171 proteins cocrystallized with small molecules representing 40 unique enzymes and receptors as well as sets of known ligands and decoys were selected from the Protein Data Bank (PDB) and the Directory of Useful Decoys (DUD), respectively. This study revealed that implementing displaceable crystallographic or computationally placed particle water molecules and protein flexibility can improve the enrichment in active compounds. In addition, an informed decision based on library diversity or research objectives (hit discovery vs lead optimization) on which implementation to use may lead to significant improvements.

Methods for docking small molecules to macromolecules: a user's perspective. 2. Applications.

A large number of research articles describe novel methodologies of docking and/or scoring methods. An even larger number of publications report the successful use of these methods in the identification of novel hit molecules. What is less documented is the application of docking methods in other areas. We review herein the application of docking methods to not only hit identification but also to de novo design, fragment-based drug discovery, lead optimization, metabolism prediction, off-target binding, selectivity, protein structure prediction and drug-drug interaction.

Methods for docking small molecules to macromolecules: a user's perspective. 1. The theory.

Over the last two decades, computationally docking potential protein ligands (e.g., enzyme inhibitors) has become one of the most widely used strategies in computer aided drug design. While these docking methods were developed, some effort focused on their user-friendliness up to a point where they can be used by non-experts with nearly no training, somewhat hiding the underlying theory. However, basic knowledge is still required to avoid pitfalls and misinterpretations of docking experiments. Over the years, we have collected the common mistakes and necessary information for the proper use of docking programs. In this review, we compiled this data for non-experts in the field. In a first section, we present the theory of docking and scoring approaches as well as their limitations, followed by the most recent progress towards the consideration of protein flexibility, water molecules, metal ions, and covalent drugs. In a second section, we describe what we believe are the necessary steps to ensure optimal docking. More specifically, we present the selection of a docking program, available databases of small molecules, macromolecules and biological data, the necessary steps for the preparation of proteins and small molecules, and finally post docking analysis techniques. In the following sections, we compile the sources of biases and describe docking to nucleic acids.

Docking ligands into flexible and solvated macromolecules. 6. Development and application to the docking of HDACs and other zinc metalloenzymes inhibitors.

Metalloenzymes are ubiquitous proteins which feature one or more metal ions either directly involved in the enzymatic activity and/or structural properties (i.e., zinc fingers). Several members of this class take advantage of the Lewis acidic properties of zinc ions to carry out their various catalytic transformations including isomerization or amide cleavage. These enzymes have been validated as drug targets for a number of diseases including cancer; however, despite their pharmaceutical relevance and the availability of crystal structures, structure-based drug design methods have been poorly and indirectly parametrized for these classes of enzymes. More specifically, the metal coordination component and proton transfers of the process of drugs binding to metalloenzymes have been inadequately modeled by current docking programs, if at all. In addition, several known issues, such as coordination geometry, atomic charge variability, and a potential proton transfer from small molecules to a neighboring basic residue, have often been ignored. We report herein the development of specific functions and parameters to account for zinc-drug coordination focusing on the above-listed phenomena and their impact on docking to zinc metalloenzymes. These atom-type-dependent but atomic charge-independent functions implemented into Fitted 3.1 enable the simulation of drug binding to metalloenzymes, considering an acid-base reaction with a neighboring residue when necessary with good accuracy.

Development of a computational tool to rival experts in the prediction of sites of metabolism of xenobiotics by p450s.

The metabolism of xenobiotics--and more specifically drugs--in the liver is a critical process controlling their half-life. Although there exist experimental methods, which measure the metabolic stability of xenobiotics and identify their metabolites, developing higher throughput predictive methods is an avenue of research. It is expected that predicting the chemical nature of the metabolites would be an asset for designing safer drugs and/or drugs with modulated half-lives. We have developed IMPACTS (In-silico Metabolism Prediction by Activated Cytochromes and Transition States), a computational tool combining docking to metabolic enzymes, transition state modeling, and rule-based substrate reactivity prediction to predict the site of metabolism (SoM) of xenobiotics. Its application to sets of CYP1A2, CYP2C9, CYP2D6, and CYP3A4 substrates and comparison to experts' predictions demonstrates its accuracy and significance. IMPACTS identified an experimentally observed SoM in the top 2 predicted sites for 77% of the substrates, while the accuracy of biotransformation experts' prediction was 65%. Application of IMPACTS to external sets and comparison of its accuracy to those of eleven other methods further validated the method implemented in IMPACTS.

Virtual screening and computational optimization for the discovery of covalent prolyl oligopeptidase inhibitors with activity in human cells.

Our docking program, Fitted, implemented in our computational platform, Forecaster, has been modified to carry out automated virtual screening of covalent inhibitors. With this modified version of the program, virtual screening and further docking-based optimization of a selected hit led to the identification of potential covalent reversible inhibitors of prolyl oligopeptidase activity. After visual inspection, a virtual hit molecule together with four analogues were selected for synthesis and made in one-five chemical steps. Biological evaluations on recombinant POP and FAPα enzymes, cell extracts, and living cells demonstrated high potency and selectivity for POP over FAPα and DPPIV. Three compounds even exhibited high nanomolar inhibitory activities in intact living human cells and acceptable metabolic stability. This small set of molecules also demonstrated that covalent binding and/or geometrical constraints to the ligand/protein complex may lead to an increase in bioactivity.

Integrating medicinal chemistry, organic/combinatorial chemistry, and computational chemistry for the discovery of selective estrogen receptor modulators with Forecaster, a novel platform for drug discovery.

As part of a large medicinal chemistry program, we wish to develop novel selective estrogen receptor modulators (SERMs) as potential breast cancer treatments using a combination of experimental and computational approaches. However, one of the remaining difficulties nowadays is to fully integrate computational (i.e., virtual, theoretical) and medicinal (i.e., experimental, intuitive) chemistry to take advantage of the full potential of both. For this purpose, we have developed a Web-based platform, Forecaster, and a number of programs (e.g., Prepare, React, Select) with the aim of combining computational chemistry and medicinal chemistry expertise to facilitate drug discovery and development and more specifically to integrate synthesis into computer-aided drug design. In our quest for potent SERMs, this platform was used to build virtual combinatorial libraries, filter and extract a highly diverse library from the NCI database, and dock them to the estrogen receptor (ER), with all of these steps being fully automated by computational chemists for use by medicinal chemists. As a result, virtual screening of a diverse library seeded with active compounds followed by a search for analogs yielded an enrichment factor of 129, with 98% of the seeded active compounds recovered, while the screening of a designed virtual combinatorial library including known actives yielded an area under the receiver operating characteristic (AU-ROC) of 0.78. The lead optimization proved less successful, further demonstrating the challenge to simulate structure activity relationship studies.

1,2-Diamines as inhibitors of co-activator associated arginine methyltransferase 1 (CARM1).

We have identified the N(1)-benzyl-N(2)-methylethane-1,2-diamine unit as a substitute for the (S)-alanine benzylamide moiety for the design of co-activator associated arginine methyltransferase 1 (CARM1) inhibitors. The potency of these inhibitors is in the same order of magnitude as their predecessors and their clearance, volume of distribution, and half lives were greatly improved.

From natural products to achiral drug prototypes: potent thrombin inhibitors based on P2/P3 dihydropyrid-2-one core motifs.

A series of dihydropyrid-2-ones was synthesized and tested for inhibitory activity against serine protease enzymes. Moderate to low nanomolar inhibitory activities were obtained against thrombin and excellent selectivity against trypsin was observed.

Synthesis and evaluation of lysine derived sulfamides as histone deacetylase inhibitors.

We have recently reported on a novel class of histone deacetylase (HDAC) inhibitors bearing a sulfamide group as the zinc-binding unit. Herein, we report on the synthesis of sulfamide based inhibitors designed around a lysine scaffold and their structure-activity relationships against HDAC1 and HDAC6 isotypes as well as 293T cells. Our efforts led us to an improvement of the originally disclosed lysine-based sulfamide, 2a to compound 12h which has equal potency in enzyme and cell-based assays as well as enhanced metabolic stability and PK profile.

N-Benzyl-1-heteroaryl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamides as inhibitors of co-activator associated arginine methyltransferase 1 (CARM1).

A series of N-benzyl-1-heteroaryl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamides targeting co-activator associated arginine methyltransferase 1 (CARM1) have been designed and synthesized. The potency of these inhibitors was influenced by the nature of the heteroaryl fragment with the thiophene analogues being superior to thiazole, pyridine, isoindoline and benzofuran based inhibitors.