The Discovery of Exarafenib (KIN-2787): Overcoming the Challenges of Pan-RAF Kinase Inhibition.

RAF, a core signaling component of the MAPK kinase cascade, is often mutated in various cancers, including melanoma, lung, and colorectal cancers. The approved inhibitors were focused on targeting the BRAFV600E mutation that results in constitutive activation of kinase signaling through the monomeric protein (Class I). However, these inhibitors also paradoxically activate kinase signaling of RAF dimers, resulting in increased MAPK signaling in normal tissues. Recently, significant attention has turned to targeting RAF alterations that activate dimeric signaling (class II and III BRAF and NRAS). However, the discovery of a potent and selective inhibitor with biopharmaceutical properties suitable to sustain robust target inhibition in the clinical setting has proven challenging. Herein, we report the discovery of exarafenib (15), a highly potent and selective inhibitor that intercepts the RAF protein in the dimer compatible αC-helix-IN conformation and demonstrates anti-tumor efficacy in preclinical models with BRAF class I, II, and III and NRAS alterations.

Discovery of KIN-3248, An Irreversible, Next Generation FGFR Inhibitor for the Treatment of Advanced Tumors Harboring FGFR2 and/or FGFR3 Gene Alterations.

Fibroblast growth factor receptor (FGFR) alterations are present as oncogenic drivers and bypass mechanisms in many forms of cancer. These alterations can include fusions, amplifications, rearrangements, and mutations. Acquired drug resistance to current FGFR inhibitors often results in disease progression and unfavorable outcomes for patients. Genomic profiling of tumors refractory to current FGFR inhibitors in the clinic has revealed several acquired driver alterations that could be the target of next generation therapeutics. Herein, we describe how structure-based drug design (SBDD) was used to enable the discovery of the potent and kinome selective pan-FGFR inhibitor KIN-3248, which is active against many acquired resistance mutations. KIN-3248 is currently in phase I clinical development for the treatment of advanced tumors harboring FGFR2 and/or FGFR3 gene alterations.

Exploring antiviral and anti-inflammatory effects of thiol drugs in COVID-19.

The redox status of the cysteine-rich SARS-CoV-2 spike glycoprotein (SARS-2-S) is important for the binding of SARS-2-S to angiotensin-converting enzyme 2 (ACE2), suggesting that drugs with a functional thiol group ("thiol drugs") may cleave cystines to disrupt SARS-CoV-2 cell entry. In addition, neutrophil-induced oxidative stress is a mechanism of COVID-19 lung injury, and the antioxidant and anti-inflammatory properties of thiol drugs, especially cysteamine, may limit this injury. To first explore the antiviral effects of thiol drugs in COVID-19, we used an ACE-2 binding assay and cell entry assays utilizing reporter pseudoviruses and authentic SARS-CoV-2 viruses. We found that multiple thiol drugs inhibit SARS-2-S binding to ACE2 and virus infection. The most potent drugs were effective in the low millimolar range, and IC50 values followed the order of their cystine cleavage rates and lower thiol pKa values. To determine if thiol drugs have antiviral effects in vivo and to explore any anti-inflammatory effects of thiol drugs in COVID-19, we tested the effects of cysteamine delivered intraperitoneally to hamsters infected with SARS-CoV-2. Cysteamine did not decrease lung viral infection, but it significantly decreased lung neutrophilic inflammation and alveolar hemorrhage. We speculate that the concentration of cysteamine achieved in the lungs with intraperitoneal delivery was insufficient for antiviral effects but sufficient for anti-inflammatory effects. We conclude that thiol drugs decrease SARS-CoV-2 lung inflammation and injury, and we provide rationale for future studies to test if direct (aerosol) delivery of thiol drugs to the airways might also result in antiviral effects.

Thiol drugs decrease SARS-CoV-2 lung injury in vivo and disrupt SARS-CoV-2 spike complex binding to ACE2 in vitro.

Neutrophil-induced oxidative stress is a mechanism of lung injury in COVID-19, and drugs with a functional thiol group ("thiol drugs"), especially cysteamine, have anti-oxidant and anti-inflammatory properties that could limit this injury. Thiol drugs may also alter the redox status of the cysteine-rich SARS-CoV-2 spike glycoprotein (SARS-2-S) and thereby disrupt ACE2 binding. Using ACE2 binding assay, reporter virus pseudotyped with SARS-CoV-2 spikes (ancestral and variants) and authentic SARS-CoV-2 (Wuhan-1), we find that multiple thiol drugs inhibit SARS-2-S binding to ACE2 and virus entry into cells. Pseudoviruses carrying variant spikes were less efficiently inhibited as compared to pseudotypes bearing an ancestral spike, but the most potent drugs still inhibited the Delta variant in the low millimolar range. IC50 values followed the order of their cystine cleavage rates and lower thiol pKa values. In hamsters infected with SARS-CoV-2, intraperitoneal (IP) cysteamine decreased neutrophilic inflammation and alveolar hemorrhage in the lungs but did not decrease viral infection, most likely because IP delivery could not achieve millimolar concentrations in the airways. These data show that thiol drugs inhibit SARS-CoV-2 infection in vitro and reduce SARS-CoV-2-related lung injury in vivo and provide strong rationale for trials of systemically delivered thiol drugs as COVID-19 treatments. We propose that antiviral effects of thiol drugs in vivo will require delivery directly to the airways to ensure millimolar drug concentrations and that thiol drugs with lower thiol pKa values are most likely to be effective.

An integrin antagonist (MK-0429) decreases proteinuria and renal fibrosis in the ZSF1 rat diabetic nephropathy model.

Multiple integrins have been implicated in modulating renal function. Modulation of integrin function can lead to pathophysiological processes associated with diabetic nephropathy such as alterations in the glomerular filtration barrier and kidney fibrosis. The complexity of these pathophysiological changes implies that multiple integrin subtypes might need to be targeted to ameliorate the progression of renal disease. To address this hypothesis, we investigated the effects of MK-0429, a compound that was originally developed as an αvß3 inhibitor for the treatment of osteoporosis, on renal function and fibrosis. We demonstrated that MK-0429 is an equipotent pan-inhibitor of multiple av integrins. MK-0429 dose-dependently inhibited podocyte motility and also suppressed TGF-ß-induced fibrosis marker gene expression in kidney fibroblasts. Moreover, in the obese ZSF1 rat model of diabetic nephropathy, chronic treatment with MK-0429 resulted in significant reduction in proteinuria, kidney fibrosis, and collagen accumulation. In summary, our results suggest that inhibition of multiple integrin subtypes might lead to meaningful impact on proteinuria and renal fibrosis in diabetic nephropathy.

GPR120 suppresses adipose tissue lipolysis and synergizes with GPR40 in antidiabetic efficacy.

GPR40 and GPR120 are fatty acid sensors that play important roles in glucose and energy homeostasis. GPR40 potentiates glucose-dependent insulin secretion and demonstrated in clinical studies robust glucose lowering in type 2 diabetes. GPR120 improves insulin sensitivity in rodents, albeit its mechanism of action is not fully understood. Here, we postulated that the antidiabetic efficacy of GPR40 could be enhanced by coactivating GPR120. A combination of GPR40 and GPR120 agonists in db/db mice, as well as a single molecule with dual agonist activities, achieved superior glycemic control compared with either monotherapy. Compared with a GPR40 selective agonist, the dual agonist improved insulin sensitivity in ob/ob mice measured by hyperinsulinemic-euglycemic clamp, preserved islet morphology, and increased expression of several key lipolytic genes in adipose tissue of Zucker diabetic fatty rats. Novel insights into the mechanism of action for GPR120 were obtained. Selective GPR120 activation suppressed lipolysis in primary white adipocytes, although this effect was attenuated in adipocytes from obese rats and obese rhesus, and sensitized the antilipolytic effect of insulin in rat and rhesus primary adipocytes. In conclusion, GPR120 agonism enhances insulin action in adipose tissue and yields a synergistic efficacy when combined with GPR40 agonism.

Design, Synthesis, and Evaluation of Novel and Selective G-protein Coupled Receptor 120 (GPR120) Spirocyclic Agonists.

Type 2 diabetes mellitus (T2DM) is an ever increasing worldwide epidemic, and the identification of safe and effective insulin sensitizers, absent of weight gain, has been a long-standing goal of diabetes research. G-protein coupled receptor 120 (GPR120) has recently emerged as a potential therapeutic target for treating T2DM. Natural occurring, and more recently, synthetic agonists have been associated with insulin sensitizing, anti-inflammatory, and fat metabolism effects. Herein we describe the design, synthesis, and evaluation of a novel spirocyclic GPR120 agonist series, which culminated in the discovery of potent and selective agonist 14. Furthermore, compound 14 was evaluated in vivo and demonstrated acute glucose lowering in an oral glucose tolerance test (oGTT), as well as improvements in homeostatic measurement assessment of insulin resistance (HOMA-IR; a surrogate marker for insulin sensitization) and an increase in glucose infusion rate (GIR) during a hyperinsulinemic euglycemic clamp in diet-induced obese (DIO) mice.

The discovery of novel 5,6,5- and 5,5,6-tricyclic pyrrolidines as potent and selective DPP-4 inhibitors.

Novel potent and selective 5,6,5- and 5,5,6-tricyclic pyrrolidine dipeptidyl peptidase IV (DPP-4) inhibitors were identified. Structure-activity relationship (SAR) efforts focused on improving the intrinsic DPP-4 inhibition potency, increasing protease selectivity, and demonstrating clean ion channel and cytochrome P450 profiles while trying to achieve a pharmacokinetic profile suitable for once weekly dosing in humans.

Discovery of benzimidazole oxazolidinediones as novel and selective nonsteroidal mineralocorticoid receptor antagonists.

Elaboration of the oxazolidinedione series led to replacement of the exocyclic amides with substituted benzimidazoles. The structure-activity relationship (SAR) exploration resulted in the discovery of potent and selective nonsteroidal mineralocorticoid receptor (MR) antagonists with significantly improved microsomal stability and pharmacokinetic (PK) profile relative to the HTS hit 1a. One compound 2p possessed comparable efficacy as spironolactone (SPL) at 100 mg/kg (p.o.) in the rat natriuresis model. As such, this series was validated as a lead series for further optimization.

A Gpr120-selective agonist improves insulin resistance and chronic inflammation in obese mice.

It is well known that the ω-3 fatty acids (ω-3-FAs; also known as n-3 fatty acids) can exert potent anti-inflammatory effects. Commonly consumed as fish products, dietary supplements and pharmaceuticals, ω-3-FAs have a number of health benefits ascribed to them, including reduced plasma triglyceride levels, amelioration of atherosclerosis and increased insulin sensitivity. We reported that Gpr120 is the functional receptor for these fatty acids and that ω-3-FAs produce robust anti-inflammatory, insulin-sensitizing effects, both in vivo and in vitro, in a Gpr120-dependent manner. Indeed, genetic variants that predispose to obesity and diabetes have been described in the gene encoding GPR120 in humans (FFAR4). However, the amount of fish oils that would have to be consumed to sustain chronic agonism of Gpr120 is too high to be practical, and, thus, a high-affinity small-molecule Gpr120 agonist would be of potential clinical benefit. Accordingly, Gpr120 is a widely studied drug discovery target within the pharmaceutical industry. Gpr40 is another lipid-sensing G protein-coupled receptor, and it has been difficult to identify compounds with a high degree of selectivity for Gpr120 over Gpr40 (ref. 11). Here we report that a selective high-affinity, orally available, small-molecule Gpr120 agonist (cpdA) exerts potent anti-inflammatory effects on macrophages in vitro and in obese mice in vivo. Gpr120 agonist treatment of high-fat diet-fed obese mice causes improved glucose tolerance, decreased hyperinsulinemia, increased insulin sensitivity and decreased hepatic steatosis. This suggests that Gpr120 agonists could become new insulin-sensitizing drugs for the treatment of type 2 diabetes and other human insulin-resistant states in the future.

Mineralocorticoid receptor antagonists: identification of heterocyclic amide replacements in the oxazolidinedione series.

Novel potent and selective mineralocorticoid receptor antagonists were identified, utilizing heterocyclic amide replacements in the oxazolidinedione series. Structure-activity relationship (SAR) efforts focused on improving lipophilic ligand efficiency (LLE) while maintaining nuclear hormone receptor selectivity and reasonable pharmacokinetic profiles.

Discovery of novel oxazolidinedione derivatives as potent and selective mineralocorticoid receptor antagonists.

Novel oxazolidinedione analogs were discovered as potent and selective mineralocorticoid receptor (MR) antagonists. Structure-activity relationship (SAR) studies were focused on improving the potency and microsomal stability. Selected compounds demonstrated excellent MR activity, reasonable nuclear hormone receptor selectivity, and acceptable rat pharmacokinetics.

Aminopiperidine-fused imidazoles as dipeptidyl peptidase-IV inhibitors.

A new series of DPP-4 inhibitors derived from piperidine-fused benzimidazoles and imidazopyridines is described. Optimization of this class of DPP-4 inhibitors led to the discovery of imidazopyridine 34. The potency, selectivity, cross-species DMPK profiles, and in vivo efficacy of 34 is reported.

Total synthesis of (-)-2-epi-peloruside A.

A convergent synthesis of (-)-2-epi-peloruside A has been achieved. Highlights include implementation of multicomponent type I anion relay chemistry (ARC) to unite 2-TBS-1,3-dithiane with two epoxides to construct the eastern hemisphere, a late-stage dithiane union to secure the complete, fully functionalized carbon backbone, and Yamaguchi macrolactonization, which led to (-)-2-epi-peloruside A via an unexpected epimerization at C(2).

Discovery of 3-aminopiperidines as potent, selective, and orally bioavailable dipeptidyl peptidase IV inhibitors.

Substituted 3-aminopiperidines 3 were evaluated as DPP-4 inhibitors. The inhibitors showed good DPP-4 potency with superb selectivity over other peptidases (QPP, DPP8, and DPP9). Selected DPP-4 inhibitors were further evaluated for their hERG potassium channel, calcium channel, Cyp2D6, and pharmacokinetic profiles.

Nanoporous microbead supported bilayers: stability, physical characterization, and incorporation of functional transmembrane proteins.

The introduction of functional transmembrane proteins into supported bilayer-based biomimetic systems presents a significant challenge for biophysics. Among the various methods for producing supported bilayers, liposomal fusion offers a versatile method for the introduction of membrane proteins into supported bilayers on a variety of substrates. In this study, the properties of protein containing unilamellar phosphocholine lipid bilayers on nanoporous silica microspheres are investigated. The effects of the silica substrate, pore structure, and the substrate curvature on the stability of the membrane and the functionality of the membrane protein are determined. Supported bilayers on porous silica microspheres show a significant increase in surface area on surfaces with structures in excess of 10 nm as well as an overall decrease in stability resulting from increasing pore size and curvature. Comparison of the liposomal and detergent-mediated introduction of purified bacteriorhodopsin (bR) and the human type 3 serotonin receptor (5HT3R) are investigated focusing on the resulting protein function, diffusion, orientation, and incorporation efficiency. In both cases, functional proteins are observed; however, the reconstitution efficiency and orientation selectivity are significantly enhanced through detergent-mediated protein reconstitution. The results of these experiments provide a basis for bulk ionic and fluorescent dye-based compartmentalization assays as well as single-molecule optical and single-channel electrochemical interrogation of transmembrane proteins in a biomimetic platform.

Total synthesis of gambierol: the generation of the A-C and F-H subunits by using a C-glycoside centered strategy.

Gambierol, a representative of the marine ladder toxin family, consists of eight ether rings, 18 stereocenters, and two challenging pyranyl rings having methyl groups that are in a 1,3-diaxial orientation to one another. Herein we describe the generation of gambierol's A-C and F-H ring systems and demonstrate the versatility of the glycosyl anhydride, enol ether-olefin RCM strategy to fused polycyclic ethers. This work has both enabled us to generate sufficient quantities of the gambierol precursors and has enabled us to better understand the chemical transformations that were key to these efforts. Fundamental work included efforts to C-glycosides and C-ketosides, Claisen rearrangements, and enol ether-olefin RCM reactions.

Localized changes in the gp120 envelope glycoprotein confer resistance to human immunodeficiency virus entry inhibitors BMS-806 and #155.

BMS-806 and the related compound, #155, are novel inhibitors of human immunodeficiency virus type 1 (HIV-1) entry that bind the gp120 exterior envelope glycoprotein. BMS-806 and #155 block conformational changes in the HIV-1 envelope glycoproteins that are induced by binding to the host cell receptor, CD4. We tested a panel of HIV-1 envelope glycoprotein mutants and identified several that were resistant to the antiviral effects of BMS-806 and #155. In the CD4-bound conformation of gp120, the amino acid residues implicated in BMS-806 and #155 resistance line the "phenylalanine 43 cavity" and a water-filled channel that extends from this cavity to the inner domain. Structural considerations suggest a model in which BMS-806 and #155 bind gp120 prior to receptor binding and, upon CD4 binding, are accommodated in the Phe-43 cavity and adjacent channel. The integrity of the nearby V1/V2 variable loops and N-linked carbohydrates on the V1/V2 stem indirectly influences sensitivity to the drugs. A putative binding site for BMS-806 and #155 between the gp120 receptor-binding regions and the inner domain, which is thought to interact with the gp41 transmembrane envelope glycoprotein, helps to explain the mode of action of these drugs.

Small-molecule inhibitors of HIV-1 entry block receptor-induced conformational changes in the viral envelope glycoproteins.

When interacting with the CD4 receptor, the HIV gp120 envelope glycoprotein undergoes conformational changes that allow binding to the chemokine receptor. Receptor binding is proposed to lead to conformational changes in the gp41 transmembrane envelope glycoprotein involving the creation and/or exposure of a coiled coil consisting of three heptad repeat (HR) sequences. The subsequent interaction of the HR2 region of gp41 with this coiled coil results in the assembly of a six-helix bundle that promotes the fusion of the viral and target cell membranes. Here we show that CD4 binding to gp120 induces the formation and/or exposure of the gp41 HR1 coiled coil in a process that does not involve gp120 shedding and that depends on the proteolytic maturation of the gp160 envelope glycoprotein precursor. Importantly, BMS-806 and related HIV-1 entry inhibitors bind gp120 and block the CD4 induction of HR1 exposure without significantly affecting CD4 binding. Moreover, these compounds do not disrupt gp120-chemokine receptor binding or the HR1-HR2 interaction within gp41. These studies thus define a receptor-induced conformational rearrangement of gp120-gp41 that is important for both CD4-dependent and CD4-independent HIV-1 entry and is susceptible to inhibition by low-molecular-weight compounds.

Synthesis of an F-H gambierol subunit using a C-glycoside-centered strategy.

[structure: see text] This manuscript describes our synthesis of the F-H subunit of gambierol. In addition to the synthesis of the tricycle, of note is an interesting protecting group influence on the generation of a C(23) C-glycoside as well as the use of ring-closing metathesis to generate a tetrasubstituted enol ether.