The clinical emergence of diverse c-MET mutations with resistance to approved inhibitors has created an urgent demand for next-generation inhibitors with efficacy against c-MET-resistant mutations while maintaining c-MET wild-type (WT) potency, selectivity over other kinases, and brain penetration. Here, we report a novel chemical series discovered through iterative core enumeration and decoration guided by free energy perturbation calculations. Type-III inhibitor 20 demonstrated potent activity against both WT and c-MET with the D1228V resistance mutation with promising physicochemical properties, laying the foundation for the development of brain-penetrant therapies targeting c-MET-driven cancers.

Dual leucine zipper kinase (DLK), expressed primarily in neuronal cells, is a regulator of neuronal degeneration in response to cellular stress from chronic disease or neuronal injury. This makes it an attractive target for the treatment of neurodegenerative diseases such as Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis; and neuronal injury, such as chemotherapy induced peripheral neuropathy. Here we describe the discovery of a potent, selective, brain-penetrant DLK inhibitor, KAI-11101 (59). Throughout the program's progression, medicinal chemistry challenges such as potency, hERG inhibition, CNS penetration, CYP3A time-dependent inhibition, and kinase selectivity were overcome through the implementation of cutting-edge in silico tools. KAI-11101 displayed an excellent in vitro safety profile and showed neuroprotective properties in an ex vivo axon fragmentation assay as well as dose-dependent activity in a mouse PD model.

Despite the success of first, second, and third generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) for non-small cell lung cancer with classical EGFR mutations (L858R or Exon 19 deletions), disease progression occurs due to the acquisition of T790M and C797S resistance. Herein, we report a physics-based computationally driven lead identification approach that identified structurally unique imidazo[3.2-b]pyrazoles as reversible and wild-type-sparing EGFR TKIs of classical mutations bearing both T790M and C797S. During profiling of imidazo[3.2-b]pyrazoles, we elucidated the bioactivation mechanism causing CYP3A4/5 time-dependent inhibition (TDI) and found key modifications to mitigate the TDI. Compound 31 inhibited EGFR L858R/T790M/C797S in biochemical assays with a Ki = 2.1 nM and EGFR del19/T790M/C797S in a Ba/F3 cellular assay with an IC50 = 56.9 nM. The deuterated analogue of 31 (38) demonstrated dose-dependent tumor growth inhibition in a Ba/F3 EGFR del19/T790M/C797S CDX model by 47% at 50 mg/kg BID and 92% at 100 mg/kg BID.

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.

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.

The non-receptor protein tyrosine phosphatase SHP2 (PTPN11) plays an important role in the regulation of RAS/MAPK signal transduction downstream of growth factor receptor activation. Loss of SHP2 activity suppresses tumor cell growth, making SHP2 a potential target for cancer therapy. Here we report the discovery of GDC-1971 (formerly RLY-1971), a highly potent, selective, and orally bioavailable small-molecule SHP2 inhibitor that stabilizes SHP2 in a closed, auto-inhibited conformation. GDC-1971 potently inhibits both wild-type SHP2 (IC50 <1nM) and the E76K activating mutant (IC50 <250nM) in biochemical assays. In standard 2-dimensional and anchorage-independent growth conditions, GDC-1971 inhibits cellular proliferation in models harboring receptor tyrosine kinases (RTKs), SHP2, NF1, KRAS, or BRAF mutations in a dose-dependent manner. GDC-1971 potently inhibits the proliferation of cellular models harboring KRAS G12C or G12A mutations (median IC50 <80 nM) compared to models harboring other KRAS G12, G13 or Q61 mutations (median IC50 >1 uM), indicating a link between KRAS GTP hydrolysis and SHP2 dependency. Despite this trend, some non-KRAS G12C or G12A cell lines harboring other KRAS mutations responded to GDC-1971 in vitro, suggesting some heterogeneity in RTK/SHP2 signaling dependence in subsets of other KRAS mutants. In vivo, GDC-1971 demonstrates dose-dependent RAS/MAPK pathway inhibition and induces significant tumor-growth inhibition in human xenograft models harboring EGFR and KRAS alterations at continuous daily doses that are well tolerated. Given the reported role of SHP2 as a critical mediator of resistance to targeted therapies, we assessed the activity of GDC-1971 combinations in multiple contexts. We observed increased suppression of the MAPK signaling cascade and anti-proliferation synergy when combining GDC-1971 with EGFR, ALK, and KRAS G12C inhibitors in vitro. The observed in vitro synergy translated to dramatic anti-tumor growth effects in vivo. GDC-1971 in combination with the KRAS G12C covalent inhibitor GDC-6036 resulted in significant regressions at doses well below those required for single agent activity in a KRAS G12C-mutant NSCLC xenograft model. In rodent and dog toxicology studies, GDC-1971 is well tolerated at exposures above those required to induce regression in xenograft models. The biochemical and cellular potency and favorable pharmaceutical properties of GDC-1971 support the further clinical development in RTK/MAPK pathway altered tumors using continuous daily dosing alone and in combination with other targeted agents, including the KRAS-G12C inhibitor GDC-6036 (clinical trial NCT04449874). Citation Format: Bret Williams, Alexander Taylor, Olivia Orozco, Christopher Owen, Elizabeth Kelley, Andre Lescarbeau, Kelley Shortsleeves, Randy Kipp, Vy Nguyen, Erin Brophy, Jeremy Wilbur, Yong Tang, David Lanzetta, Nigel Waters, Sherri Smith, Fabrizio Giordanetto, Paul Maragakis, Jack Greismann, Lindsay Willmore, Eric Therrien, Yang Xiao, Marie Evangelista, Luca Gerosa, Eva Lin, Mark Merchant, Alfonso Arrazate, Emily Chan, Pablo Sáenz-López Larrocha, Stefan Chun, Thomas Hunsaker, Gauri Deshmukh, Christine M. Bowman, David E. Shaw, Mark Murcko, Mahesh Padval, W Patrick Walters, James Watters, Donald A. Bergstrom. Discovery and characterization of the potent, allosteric SHP2 inhibitor GDC-1971 for the treatment of RTK/RAS driven tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3327.

WEE1 inhibits the activation of both CDK1 and CDK2 through phosphorylation of Tyr15, allowing DNA damage repair before entering mitosis, thereby regulating the cell cycle in S and G2/M phases. Inhibition of WEE1 could result in premature progression through the G2/M cell cycle checkpoint with unresolved DNA damage, leading to mitotic catastrophe and cell death. Small molecule WEE1 inhibitors, such as AZD1775 and Zn-C3, are currently being evaluated in the clinic and have demonstrated promising efficacy in solid tumors including ovarian, colon, and uterine carcinoma. By applying Schrödinger’s computational platform including Free Energy Perturbation (FEP) and Protein FEP, we have identified novel, potent, and highly selective WEE1 inhibitors with IC50 values in the low nanomolar range in a biochemical kinase activity assay and cellular target engagement (CDK1 pTyr15) IC50s of 100 - 300 nM in A427 and OVCAR3 cell lines. The compounds also show potent anti-proliferative activity in over 20 breast and ovarian tumor cell lines, including cell lines insensitive to PARP inhibitors. The compounds demonstrate superior kinase selectivity compared to AZD1775 and Zn-C3 in a broad kinase panel with >450 kinases (ScanMAX). In addition, the compounds show desirable ADME properties and PK profiles in preclinical species. Based on in vitro CYP3A4 TDI assay performance (kinact/KI), we have reduced the potential for drug-drug interaction liabilities compared to AZD1775. In the A427 xenograft model, our WEE1 inhibitors demonstrate dose-dependent tumor growth inhibition and tumor regression at high doses. Anti-tumor activity is also demonstrated in additional tumor models, including OVCAR3 and HCC1806 xenograft models. The established PK-PD relationship shows sustained target engagement (pCDK1), increased DNA damage (gH2AX) and mitosis (pHH3). We demonstrate that hematological adverse effects can be mitigated by dosing holidays in xenograft tumor models while maintaining anti-tumor activity. Notably, our compound shows more sustained anti-tumor activity with dosing holidays compared to AZD1775, which we believe is attributable to the prolonged and higher exposure in tumor and plasma with our compound. In the A427 non small-cell lung cancer xenograft model, following 3 dosing holiday cycles at high doses, tumor eradication was maintained after treatment was stopped. In summary, we have identified novel, potent and exquisitely selective WEE1 kinase inhibitors that demonstrate robust anti-tumor activity and sustained target engagement in tumor models. The compound’s anti-tumor effects are maintained with dosing holidays while allowing full recovery of mechanism-based hematological effects. Citation Format: Shaoxian Sun, Sarah Silvergleid, Aleksey I. Gerasyuto, Jiashi Wang, Robert D. Pelletier, Andrew Placzek, Jennifer L. Knight, Anthony Clark, Hamish Wright, Wu Yin, Jackson Chief Elk, Jeff Bell, Pieter H. Bos, Nicholas A. Boyles, Eric Therrien, Kristian Jensen, Karen Akinsanya. Discovery of potent, selective, and orally available WEE1 inhibitors that demonstrate increased DNA damage and mitosis in tumor cells leading to tumor regression in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2570.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.