Exploiting high-energy hydration sites for the discovery of potent peptide aldehyde inhibitors of the SARS-CoV-2 main protease with cellular antiviral activity.

Small-molecule antivirals that prevent the replication of the SARS-CoV-2 virus by blocking the enzymatic activity of its main protease (Mpro) are and will be a tenet of pandemic preparedness. However, the peptidic nature of such compounds often precludes the design of compounds within favorable physical property ranges, limiting cellular activity. Here we describe the discovery of peptide aldehyde Mpro inhibitors with potent enzymatic and cellular antiviral activity. This structure-activity relationship (SAR) exploration was guided by the use of calculated hydration site thermodynamic maps (WaterMap) to drive potency via displacement of waters from high-energy sites. Thousands of diverse compounds were designed to target these high-energy hydration sites and then prioritized for synthesis by physics- and structure-based Free-Energy Perturbation (FEP+) simulations, which accurately predicted biochemical potencies. This approach ultimately led to the rapid discovery of lead compounds with unique SAR that exhibited potent enzymatic and cellular activity with excellent pan-coronavirus coverage.

Discovery of mobocertinib, a potent, oral inhibitor of EGFR exon 20 insertion mutations in non-small cell lung cancer.

In the treatment of non-small cell lung cancer (NSCLC), patients harboring exon 20 insertion mutations in the epidermal growth factor receptor (EGFR) gene (EGFR) have few effective therapies because this subset of mutants is generally resistant to most currently approved EGFR inhibitors. This report describes the structure-guided design of a novel series of potent, irreversible inhibitors of EGFR exon 20 insertion mutations, including the V769_D770insASV and D770_N771insSVD mutants. Extensive structure-activity relationship (SAR) studies led to the discovery of mobocertinib (compound 21c), which inhibited growth of Ba/F3 cells expressing the ASV insertion with a half-maximal inhibitory concentration of 11 nM and with selectivity over wild-type EGFR. Daily oral administration of mobocertinib induced tumor regression in a Ba/F3 ASV xenograft mouse model at well-tolerated doses. Mobocertinib was approved in September 2021 for the treatment of adult patients with advanced NSCLC with EGFR exon 20 insertion mutations with progression on or after platinum-based chemotherapy.

Design and synthesis of aryl-piperidine derivatives as potent and selective PET tracers for cholesterol 24-hydroxylase (CH24H).

Cholesterol 24-hydroxylase (CH24H, CYP46A1) is a cytochrome P450 family enzyme that maintains the homeostasis of brain cholesterol. Soticlestat, a potent and selective CH24H inhibitor, is in development as a therapeutic agent for Dravet syndrome and Lennox-Gastaut syndrome. Herein, we report the discovery of aryl-piperidine derivatives as potent and selective CH24H positron emission tomography (PET) tracers which can be used for dose guidance of a clinical CH24H inhibitor and as a diagnostic tool for CH24H-related pathology. Starting from compound 1 (IC50 = 16 nM, logD = 1.7), which was reported as a CH24H inhibitor with lower lipophilicity, a18F-labeling site (3-fluoroazetidine) was incorporated by structure-based drug design (SBDD) utilizing the co-crystal structure of a compound 1 analog. Subsequent optimization to adjust key parameters for PET tracers, such as potency, lipophilicity, brain penetration, and unbound plasma protein binding, enabled compounds 3f (IC50 = 8.8 nM) and 3g (IC50 = 8.7 nM) as PET imaging candidates. Selectivity of these compounds for CH24H was validated by a brain distribution study using CH24H-WT and KO mice. In non-human primate PET imaging, [18F]3f and [18F]3g showed similar regional uptake in the brain, indicating that these tracers were specific to the CH24H-expressed regions and validated the expression of CH24H in the living brain by different tracers.

Discovery of Novel 3-Piperidinyl Pyridine Derivatives as Highly Potent and Selective Cholesterol 24-Hydroxylase (CH24H) Inhibitors.

Cholesterol 24-hydroxylase (CH24H or CYP46A1) is a brain-specific cytochrome P450 enzyme that metabolizes cholesterol into 24S-hydroxycholesterol (24HC) for regulating brain cholesterol homeostasis. For the development of a novel and potent CH24H inhibitor, we designed and synthesized 3,4-disubstituted pyridine derivatives using a structure-based drug design approach starting from compounds 1 (soticlestat) and 2 (thioperamide). Optimization of this series by focusing on ligand-lipophilicity efficiency value resulted in the discovery of 4-(4-methyl-1-pyrazolyl)pyridine derivative 17 (IC50 = 8.5 nM) as a potent and highly selective CH24H inhibitor. The X-ray crystal structure of CH24H in complex with compound 17 revealed a unique binding mode. Both blood-brain barrier penetration and reduction of 24HC levels (26% reduction) in the mouse brain were confirmed by oral administration of 17 at 30 mg/kg, indicating that 17 is a promising tool for the novel and selective inhibition of CH24H.

Discovery of a Novel and Brain-Penetrant O-GlcNAcase Inhibitor via Virtual Screening, Structure-Based Analysis, and Rational Lead Optimization.

O-GlcNAcase (OGA) has received increasing attention as an attractive therapeutic target for tau-mediated neurodegenerative disorders; however, its role in these pathologies remains unclear. Therefore, potent chemical tools with favorable pharmacokinetic profiles are desirable to characterize this enzyme. Herein, we report the discovery of a potent and novel OGA inhibitor, compound 5i, comprising an aminopyrimidine scaffold, identified by virtual screening based on multiple methodologies combining structure-based and ligand-based approaches, followed by sequential optimization with a focus on ligand lipophilicity efficiency. This compound was observed to increase the level of O-GlcNAcylated protein in cells and display suitable pharmacokinetic properties and brain permeability. Crystallographic analysis revealed that the chemical series bind to OGA via characteristic hydrophobic interactions, which resulted in a high affinity for OGA with moderate lipophilicity. Compound 5i could serve as a useful chemical probe to help establish a proof-of-concept of OGA inhibition as a therapeutic target for the treatment of tauopathies.

Structure-Based Design, Synthesis, and Biological Evaluation of Imidazo[4,5-b]Pyridin-2-one-Based p38 MAP Kinase Inhibitors: Part†2.

We identified novel potent inhibitors of p38 mitogen-activated protein (MAP) kinase using a structure-based design strategy, beginning with lead compound, 3-(butan-2-yl)-6-(2,4-difluoroanilino)-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-one (1). To enhance the inhibitory activity of 1 against production of tumor necrosis factor-α (TNF-α) in human whole blood (hWB) cell assays, we designed and synthesized hybrid compounds in which the imidazo[4,5-b]pyridin-2-one core was successfully linked with the p-methylbenzamide fragment. Among the compounds evaluated, 3-(3-tert-butyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-6-yl)-4-methyl-N-(1-methyl-1H-pyrazol-3-yl)benzamide (25) exhibited potent p38 inhibition, superior suppression of TNF-α production in hWB cells, and also significant in vivo efficacy in a rat model of collagen-induced arthritis (CIA). In this paper, we report the discovery of potent, selective, and orally bioavailable imidazo[4,5-b]pyridin-2-one-based p38 MAP kinase inhibitors.

Discovery of a novel B-cell lymphoma 6 (BCL6)-corepressor interaction inhibitor by utilizing structure-based drug design.

B-cell lymphoma 6 (BCL6) is a transcriptional repressor that can form complexes with corepressors via protein-protein interactions (PPIs). The complexes of BCL6 and corepressors play an important role in the formation of germinal centers (GCs), and differentiation and proliferation of lymphocytes. Therefore, BCL6-corepressor interaction inhibitors would be drug candidates for managing autoimmune diseases and cancer. Starting from high-throughput screening hits 1a and 2a, we identified a novel BCL6-corepressor interaction inhibitor 8c (cell-free enzyme-linked immunosorbent assay [ELISA] IC50=0.10µM, cell-based mammalian two-hybrid [M2H] assay IC50=0.72µM) by utilizing structure-based drug design (SBDD) based on an X-ray crystal structure of 1a bound to BCL6. Compound 8c also showed a good pharmacokinetic profile, which was acceptable for both in vitro and in vivo studies.

Discovery of a B-Cell Lymphoma 6 Protein-Protein Interaction Inhibitor by a Biophysics-Driven Fragment-Based Approach.

B-cell lymphoma 6 (BCL6) is a transcriptional factor that expresses in lymphocytes and regulates the differentiation and proliferation of lymphocytes. Therefore, BCL6 is a therapeutic target for autoimmune diseases and cancer treatment. This report presents the discovery of BCL6-corepressor interaction inhibitors by using a biophysics-driven fragment-based approach. Using the surface plasmon resonance (SPR)-based fragment screening, we successfully identified fragment 1 (SPR KD = 1200 µM, ligand efficiency (LE) = 0.28), a competitive binder to the natural ligand BCoR peptide. Moreover, we elaborated 1 into the more potent compound 7 (SPR KD = 0.078 µM, LE = 0.37, cell-free protein-protein interaction (PPI) IC50 = 0.48 µM (ELISA), cellular PPI IC50 = 8.6 µM (M2H)) by a structure-based design and structural integration with a second high-throughput screening hit.

Structure of Human GIVD Cytosolic Phospholipase A2 Reveals Insights into Substrate Recognition.

Cytosolic phospholipases A2 (cPLA2s) consist of a family of calcium-sensitive enzymes that function to generate lipid second messengers through hydrolysis of membrane-associated glycerophospholipids. The GIVD cPLA2 (cPLA2δ) is a potential drug target for developing a selective therapeutic agent for the treatment of psoriasis. Here, we present two X-ray structures of human cPLA2δ, capturing an apo state, and in complex with a substrate-like inhibitor. Comparison of the apo and inhibitor-bound structures reveals conformational changes in a flexible cap that allows the substrate to access the relatively buried active site, providing new insight into the mechanism for substrate recognition. The cPLA2δ structure reveals an unexpected second C2 domain that was previously unrecognized from sequence alignments, placing cPLA2δ into the class of membrane-associated proteins that contain a tandem pair of C2 domains. Furthermore, our structures elucidate novel inter-domain interactions and define three potential calcium-binding sites that are likely important for regulation and activation of enzymatic activity. These findings provide novel insights into the molecular mechanisms governing cPLA2's function in signal transduction.

Discovery of Potent and Selective Inhibitors of Phosphodiesterase 1 for the Treatment of Cognitive Impairment Associated with Neurodegenerative and Neuropsychiatric Diseases.

A diverse set of 3-aminopyrazolo[3,4-d]pyrimidinones was designed and synthesized. The structure-activity relationships of these polycyclic compounds as phosphodiesterase 1 (PDE1) inhibitors were studied along with their physicochemical and pharmacokinetic properties. Systematic optimizations of this novel scaffold culminated in the identification of a clinical candidate, (6aR,9aS)-2-(4-(6-fluoropyridin-2-yl)benzyl)-5-methyl-3-(phenylamino)-5,6a,7,8,9,9a-hexahydrocyclopenta[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4-(2H)-one phosphate (ITI-214), which exhibited picomolar inhibitory potency for PDE1, demonstrated excellent selectivity against all other PDE families and showed good efficacy in vivo. Currently, this investigational new drug is in Phase I clinical development and being considered for the treatment of several indications including cognitive deficits associated with schizophrenia and Alzheimer's disease, movement disorders, attention deficit and hyperactivity disorders, and other central nervous system (CNS) and non-CNS disorders.

Crystal structure of human stearoyl-coenzyme A desaturase in complex with substrate.

Stearoyl-coenzyme A desaturase-1 (SCD1) has an important role in lipid metabolism, and SCD1 inhibitors are potential therapeutic agents for the treatment of metabolic diseases and cancers. Here we report the 3.25-Å crystal structure of human SCD1 in complex with its substrate, stearoyl-coenzyme A, which defines the new SCD1 dimetal catalytic center and reveals the determinants of substrate binding to provide insights into the catalytic mechanism of desaturation of the stearoyl moiety. The structure also provides a mechanism for localization of SCD1 in the endoplasmic reticulum: human SCD1 folds around a tight hydrophobic core formed from four long α-helices that presumably function as an anchor spanning the endoplasmic reticulum membrane. Furthermore, our results provide a framework for the rational design of pharmacological inhibitors targeting the SCD1 enzyme.

High-resolution structure of the human GPR40 receptor bound to allosteric agonist TAK-875.

Human GPR40 receptor (hGPR40), also known as free fatty-acid receptor 1 (FFAR1), is a G-protein-coupled receptor that binds long-chain free fatty acids to enhance glucose-dependent insulin secretion. Novel treatments for type-2 diabetes mellitus are therefore possible by targeting hGPR40 with partial or full agonists. TAK-875, or fasiglifam, is an orally available, potent and selective partial agonist of hGPR40 receptor, which reached phase III clinical trials for the potential treatment of type-2 diabetes mellitus. Data from clinical studies indicate that TAK-875, which is an ago-allosteric modulator of hGPR40 (ref. 3), demonstrates improved glycaemic control and low hypoglycaemic risk in diabetic patients. Here we report the crystal structure of hGPR40 receptor bound to TAK-875 at 2.3 Å resolution. The co-complex structure reveals a unique binding mode of TAK-875 and suggests that entry to the non-canonical binding pocket most probably occurs via the lipid bilayer. The atomic details of the extensive charge network in the ligand binding pocket reveal additional interactions not identified in previous studies and contribute to a clear understanding of TAK-875 binding to the receptor. The hGPR40-TAK-875 structure also provides insights into the plausible binding of multiple ligands to the receptor, which has been observed in radioligand binding and Ca(2+) influx assay studies. Comparison of the transmembrane helix architecture with other G-protein-coupled receptors suggests that the crystallized TAK-875-bound hGPR40 complex is in an inactive-like state.