Design and exploration of C-3 benzoic acid bioisosteres and alkyl replacements in the context of GSK3532795 (BMS-955176) that exhibit broad spectrum HIV-1 maturation inhibition.

GSK3532795 (formerly BMS-955176) is a second-generation HIV-1 maturation inhibitor that has shown broad spectrum antiviral activity and preclinical PK predictive of once-daily dosing in humans. Although efficacy was confirmed in clinical trials, the observation of gastrointestinal intolerability and the emergence of drug resistant virus in a Phase 2b clinical study led to the discontinuation of GSK3532795. As part of the effort to further map the maturation inhibitor pharmacophore and provide additional structural options, the evaluation of alternates to the C-3 phenyl substituent in this chemotype was pursued. A cyclohexene carboxylic acid provided exceptional inhibition of wild-type, V370A and ΔV370 mutant viruses in addition to a suitable PK profile following oral dosing to rats. In addition, a novel spiro[3.3]hept-5-ene was designed to extend the carboxylic acid further from the triterpenoid core while reducing side chain flexibility compared to the other alkyl substituents. This modification was shown to closely emulate the C-3 benzoic acid moiety of GSK3532795 from both a potency and PK perspective, providing a non-traditional, sp3-rich bioisostere of benzene. Herein, we detail additional modifications to the C-3 position of the triterpenoid core that offer effective replacements for the benzoic acid of GSK3532795 and capture the interplay between these new C-3 elements and C-17 modifications that contribute to enhanced polymorph coverage.

Epitope and Paratope Mapping of PD-1/Nivolumab by Mass Spectrometry-Based Hydrogen-Deuterium Exchange, Cross-linking, and Molecular Docking.

Programmed cell death-1 (PD-1), an antigen co-receptor on cell surfaces, is one of the conspicuous immune checkpoints. Nivolumab, a monoclonal antibody therapeutic approved by the FDA, binds to PD-1 and efficiently blocks its pathways. In this study, an integrated approach was developed to map the epitope/paratope of PD-1/nivolumab. The approach includes hydrogen-deuterium exchange mass spectrometry (HDX-MS) followed by electron-transfer dissociation (ETD), chemical cross-linking, and molecular docking. HDX-ETD offers some binding-site characterization with amino acid resolution. Chemical cross-linking provides complementary information on one additional epitope (i.e., the BC-loop) and a potential paratope at the N-terminus of the heavy chain. Furthermore, cross-linking identifies another loop region (i.e., the C'D-loop) that undergoes a remote conformational change. The distance restraints derived from the cross-links enable building high-confidence models of PD-1/nivolumab, evaluated with respect to a resolved crystal structure. This integrated strategy is an opportunity to characterize comprehensively other antigen-antibody interactions, to enable the understanding of binding mechanisms, and to design future antibody therapeutics.

An Integrated Approach for Determining a Protein-Protein Binding Interface in Solution and an Evaluation of Hydrogen-Deuterium Exchange Kinetics for Adjudicating Candidate Docking Models.

We describe an integrated approach of using hydrogen-deuterium exchange mass spectrometry (HDX-MS), chemical cross-linking mass spectrometry (XL-MS), and molecular docking to characterize the binding interface and to predict the three-dimensional quaternary structure of a protein-protein complex in solution. Interleukin 7 (IL-7) and its α-receptor, IL-7Rα, serving as essential mediators in the immune system, are the model system. HDX kinetics reports widespread protection on IL-7Rα but shows no differential evidence of binding-induced protection or remote conformational change. Cross-linking with reagents that differ in spacer lengths and targeting residues increases the spatial resolution. Using five cross-links as distance restraints for protein-protein docking, we generated a high-confidence model of the IL-7/IL-7Rα complex. Both the predicted binding interface and regions with direct contacts agree well with those in the solid-state structure, as confirmed by previous X-ray crystallography. An additional binding region was revealed to be the C-terminus of helix B of IL-7, highlighting the value of solution-based characterization. To generalize the integrated approach, protein-protein docking was executed with a different number of cross-links. Combining cluster analysis and HDX kinetics adjudication, we found that two intermolecular cross-link-derived restraints are sufficient to generate a high-confidence model with root-mean-square distance (rmsd) value of all alpha carbons below 2.0 Å relative to the crystal structure. The remarkable results of binding-interface determination and quaternary structure prediction highlight the effectiveness and capability of the integrated approach, which will allow more efficient and comprehensive analysis of interprotein interactions with broad applications in the multiple stages of design, implementation, and evaluation for protein therapeutics.

Orthogonal Mass Spectrometry-Based Footprinting for Epitope Mapping and Structural Characterization: The IL-6 Receptor upon Binding of Protein Therapeutics.

Higher-order structure (HOS) is a crucial determinant for the biological functions and quality attributes of protein therapeutics. Mass spectrometry (MS)-based protein footprinting approaches play an important role in elucidating the relationship between protein biophysical properties and structure. Here, we describe the use of a combined method including hydrogen-deuterium exchange (HDX), fast photochemical oxidation of proteins (FPOP), and site-specific carboxyl group footprinting to investigate the HOS of protein and protein complexes. The work focuses on implementing complementary solution-phase footprinting approaches that differ in time scale, specificity for protein residue side chains vs backbone as well as selectivity for different residue types to map integratively the epitope of human interleukin-6 receptor (IL-6R) for two adnectins with distinct affinities (Kd, Adnectin1 ∼ 6.2 pM vs Kd, Adnectin2 ∼ 46 nM). Furthermore, the study evaluates the resultant conformation/dynamic change of IL-6R. The suggested epitope, which is conserved for adnectin1 and adnectin2 binding, is a flexible loop that connects two ß-strands in the cytokine-binding domain (DII) of IL-6R. We also found that adnectin1, the more strongly binding ligand, induces structural perturbations on two unstructured loops that are distally located beyond the epitope. Those changes are either attenuated or not detected for the case of adnectin2 binding. In addition to providing credibility in epitope determination, utilization of those combined approaches reveals the structural effects that can differentiate protein therapeutics with apparently similar biophysical properties.

Discovery and initial optimization of alkoxyanthranilic acid derivatives as inhibitors of HCV NS5B polymerase.

Alkoxyanthranilic acid derivatives have been identified to inhibit HCV NS5B polymerase, binding in an allosteric site located at the convergence of the palm and thumb regions. Information from co-crystal structures guided the structural design strategy. Ultimately, two independent structural modifications led to a similar shift in binding mode that when combined led to a synergistic improvement in potency and the identification of inhibitors with sub-micromolar HCV NS5B binding potency.

Synthesis and SAR of calcitonin gene-related peptide (CGRP) antagonists containing substituted aryl-piperazines and piperidines.

Calcitonin gene-related peptide (CGRP) is a potent neuropeptide implicated in the pathophysiology of migraine. In the course of seeking CGRP antagonists with improved oral bioavailability, metabolic stability, and pharmacokinetic properties, lower molecular weight, structurally simpler piperidine and piperazine analogs of BMS-694153 were prepared. Several were found to have nM binding affinity in vitro. The synthesis and SAR of these substituted piperidine and piperazine CGRP antagonists are discussed.

C-3 benzoic acid derivatives of C-3 deoxybetulinic acid and deoxybetulin as HIV-1 maturation inhibitors.

A series of C-3 phenyl- and heterocycle-substituted derivatives of C-3 deoxybetulinic acid and C-3 deoxybetulin was designed and synthesized as HIV-1 maturation inhibitors (MIs) and evaluated for their antiviral activity and cytotoxicity in cell culture. A 4-subsituted benzoic acid moiety was identified as an advantageous replacement for the 3'3'-dimethylsuccinate moiety present in previously disclosed MIs that illuminates new aspects of the topography of the pharmacophore. The new analogs exhibit excellent in vitro antiviral activity against wild-type (wt) virus and a lower serum shift when compared with the prototypical HIV-1 MI bevirimat (1, BVM), the first MI to be evaluated in clinical studies. Compound 9a exhibits comparable cell culture potency toward wt virus as 1 (WT EC50=16 nM for 9a compared to 10nM for 1). However, the potency of 9a is less affected by the presence of human serum, while the compound displays a similar pharmacokinetic profile in rats to 1. Hence 9a, the 4-benzoic acid derivative of deoxybetulinic acid, represents a new starting point from which to explore the design of a 2nd generation MI.

Discovery of D1 Dopamine Receptor Positive Allosteric Modulators: Characterization of Pharmacology and Identification of Residues that Regulate Species Selectivity.

The present studies represent the first published report of a dopamine D1 positive allosteric modulator (PAM). D1 receptors have been proposed as a therapeutic target for the treatment of cognitive deficits associated with schizophrenia. However, the clinical utility of orthosteric agonist compounds is limited by cardiovascular side effects, poor pharmacokinetics, lack of D1 selectivity, and an inverted dose response. A number of these challenges may be overcome by utilization of a selective D1 PAM. The current studies describe two chemically distinct D1 PAMs: Compound A [1-((rel-1S,3R,6R)-6-(benzo[d][1,3]dioxol-5-yl)bicyclo[4.1.0]heptan-3-yl)-4-(2-bromo-5-chlorobenzyl)piperazine] and Compound B [rel-(9R,10R,12S)-N-(2,6-dichloro-3-methylphenyl)-12-methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxamide]. Compound A shows pure PAM activity, with an EC50 of 230 nM and agonist activity at the D2 receptor in D2-expressing human embryonic kidney cells. Compound B shows superior potency (EC50 of 43 nM) and selectivity for D1 versus D2 dopamine receptors. Unlike Compound A, Compound B is selective for human and nonhuman primate D1 receptors, but lacks activity at the rodent (rat and mouse) D1 receptors. Using molecular biology techniques, a single amino acid was identified at position 130, which mediates the species selectivity of Compound B. These data represent the first described D1-selective PAMs and define critical amino acids that regulate species selectivity.

Synthesis and SAR studies of novel heteroaryl fused tetracyclic indole-diamide compounds: potent allosteric inhibitors of the hepatitis C virus NS5B polymerase.

Presented here are initial structure-activity relationship (SAR) studies on a series of novel heteroaryl fused tetracyclic indole-based inhibitors of the hepatitis C viral polymerase, NS5B. The introduction of alternative heterocyclic moieties into the indolo-fused inhibitor class significantly expands the reported SAR and resulted in the identification of pyridino analogs, typified by compounds 44 and 45 that displayed excellent potency against the NS5B polymerase of both HCV 1a and HCV 1b genotypes.

SAR studies on a series of N-benzyl-4-heteroaryl-1-(phenylsulfonyl)piperazine-2-carboxamides: potent inhibitors of the polymerase enzyme (NS5B) of the hepatitis C virus.

Described herein is the initial optimization of (+/-) N-benzyl-4-heteroaryl-1-(phenylsulfonyl)piperazine-2-carboxamide (1), a hit discovered in a high throughput screen run against the NS5B polymerase enzyme of the hepatitis C virus. This effort resulted in the identification of (S)-N-sec-butyl-6-((R)-3-(4-(trifluoromethoxy)benzylcarbamoyl)-4-(4-(trifluoromethoxy)phenylsulfonyl)piperazin-1-yl)pyridazine-3-carboxamide (2), that displayed potent replicon activities against HCV genotypes 1b and 1a (EC(50) 1b/1a=7/89 nM).

The synthesis and evaluation of a novel class of (E)-3-(1-cyclohexyl-1H-pyrazol-3-yl)-2-methylacrylic acid hepatitis C virus polymerase NS5B inhibitors.

Herein we report the identification and evaluation of a novel series of (E)-3-(1-cyclohexyl-1H-pyrazol-3-yl)-2-methylacrylic acid derivatives identified from a deannulation study performed on the reported benzimidazole NS5B inhibitor, 1. This resulted in the identification of (E)-3-(2-(4-((4'-cyano-4-(4-hydroxypiperidine-1-carbonyl)biphenyl-2-yl)methoxy)phenyl)-1-cyclohexyl-1H-imidazol-4-yl)-2-methylacrylic acid (11) as a potent inhibitor of NS5B. Potential pathways for the further optimization of this series are suggested.

Investigation of the mode of binding of a novel series of N-benzyl-4-heteroaryl-1-(phenylsulfonyl)piperazine-2-carboxamides to the hepatitis C virus polymerase.

Structure based rationales for the activities of potent N-benzyl-4-heteroaryl-1-(phenylsulfonyl)piperazine-2-carboxamide inhibitors of the hepatitis C viral polymerase are described herein. These compounds bind to the hepatitis C virus non-structural protein 5B (NS5B), and co-crystal structures of select examples from this series with NS5B are reported. Comparison of co-crystal structures of a potent analog with both NS5B genotype 1a and genotype 1b provides a possible explanation for the genotype-selectivity observed with this compound class and suggests opportunities for the further optimization of the series.

Syntheses and initial evaluation of a series of indolo-fused heterocyclic inhibitors of the polymerase enzyme (NS5B) of the hepatitis C virus.

Herein, we present initial SAR studies on a series of bridged 2-arylindole-based NS5B inhibitors. The introduction of bridging elements between the indole N1 and the ortho-position of the 2-aryl moiety resulted in conformationally constrained heterocycles that possess multiple additional vectors for further exploration. The binding mode and pharmacokinetic (PK) properties of select examples, including: 13-cyclohexyl-6-oxo-6,7-dihydro-5H-indolo[2,1-d][1,4]benzodiazepine-10-carboxylic acid (7) (IC(50)=0.07 µM, %F=18), are reported.

Synthesis, Structure-Activity Relationships, and In Vivo Evaluation of Novel Tetrahydropyran-Based Thiodisaccharide Mimics as Galectin-3 Inhibitors.

Galectin-3 is a member of a family of ß-galactoside-binding proteins. A substantial body of literature reports that galectin-3 plays important roles in cancer, inflammation, and fibrosis. Small-molecule galectin-3 inhibitors, which are generally lactose or galactose-based derivatives, have the potential to be valuable disease-modifying agents. In our efforts to identify novel galectin-3 disaccharide mimics to improve drug-like properties, we found that one of the monosaccharide subunits can be replaced with a suitably functionalized tetrahydropyran ring. Optimization of the structure-activity relationships around the tetrahydropyran-based scaffold led to the discovery of potent galectin-3 inhibitors. Compounds 36, 40, and 45 were selected for further in vivo evaluation. The synthesis, structure-activity relationships, and in vivo evaluation of novel tetrahydropyran-based galectin-3 inhibitors are described.

MORPH: a new tool for ligand design.

A frequently employed strategy in drug discovery efforts is to replace aromatic rings in known active compounds with alternative aromatic moieties to create novel compounds with improved potency and/or adsorption, distribution, metabolism, excretion, and toxicity properties. Here we introduce MORPH, which is a simple software tool for systematically modifying aromatic rings in three-dimensional models of molecules without altering the coordinates of the nonhydrogen atoms in the rings. MORPH works on individual rings as well as fused ring systems and additionally provides the ability to filter out modified compounds which do not contain hydrogen-bond donors or acceptors at specific positions on the rings or contain more or less than the desired number of heteroatoms. The MORPH program and its application to two ligands extracted from cocrystal structures with cyclin-dependent kinase 2 (CDK2)/cyclin A and CDK2 are discussed below.

Resistance profile of the HIV-1 maturation inhibitor GSK3532795 in vitro and in a clinical study.

GSK3532795 (formerly BMS955176) is a second-generation maturation inhibitor (MI) that progressed through a Phase 2b study for treatment of HIV-1 infection. Resistance development to GSK3532795 was evaluated through in vitro methods and was correlated with information obtained in a Phase 2a proof-of-concept study in HIV-1 infected participants. Both low and high concentrations of GSK3532795 were used for selections in vitro, and reduced susceptibility to GSK3532795 mapped specifically to amino acids near the capsid/ spacer peptide 1 (SP1) junction, the cleavage of which is blocked by MIs. Two key substitutions, A364V or V362I, were selected, the latter requiring secondary substitutions to reduce susceptibility to GSK3532795. Three main types of secondary substitutions were observed, none of which reduced GSK3532795 susceptibility in isolation. The first type was in the capsid C-terminal domain and downstream SP1 region (including (Gag numbering) R286K, A326T, T332S/N, I333V and V370A/M). The second, was an R41G substitution in viral protease that occurred with V362I. The third was seen in the capsid N-terminal domain, within the cyclophilin A binding domain (V218A/M, H219Q and G221E). H219Q increased viral replication capacity and reduced susceptibility of poorly growing viruses. In the Phase 2a study, a subset of these substitutions was also observed at baseline and some were selected following GSK35323795 treatment in HIV-1-infected participants.

Hydrogen-Deuterium Exchange and Hydroxyl Radical Footprinting for Mapping Hydrophobic Interactions of Human Bromodomain with a Small Molecule Inhibitor.

Mass spectrometry (MS)-based protein footprinting, a valuable structural tool in mapping protein-ligand interaction, has been extensively applied to protein-protein complexes, showing success in mapping large interfaces. Here, we utilized an integrated footprinting strategy incorporating both hydrogen-deuterium exchange (HDX) and hydroxyl radical footprinting (i.e., fast photochemical oxidation of proteins (FPOP)) for molecular-level characterization of the interaction of human bromodomain-containing protein 4 (BRD4) with a hydrophobic benzodiazepine inhibitor. HDX does not provide strong evidence for the location of the binding interface, possibly because the shielding of solvent by the small molecule is not large. Instead, HDX suggests that BRD4 appears to be stabilized by showing a modest decrease in dynamics caused by binding. In contrast, FPOP points to a critical binding region in the hydrophobic cavity, also identified by crystallography, and, therefore, exhibits higher sensitivity than HDX in mapping the interaction of BRD4 with compound 1. In the absence or under low concentrations of the radical scavenger, FPOP modifications on Met residues show significant differences that reflect the minor change in protein conformation. This problem can be avoided by using a sufficient amount of proper scavenger, as suggested by the FPOP kinetics directed by a dosimeter of the hydroxyl radical.

Structure-Property Basis for Solving Transporter-Mediated Efflux and Pan-Genotypic Inhibition in HCV NS5B Inhibitors.

In solving the P-gp and BCRP transporter-mediated efflux issue in a series of benzofuran-derived pan-genotypic palm site inhibitors of the hepatitis C virus NS5B replicase, it was found that close attention to physicochemical properties was essential. In these compounds, where both molecular weight (MW >579) and TPSA (>110 Å2) were high, attenuation of polar surface area together with weakening of hydrogen bond acceptor strength of the molecule provided a higher intrinsic membrane permeability and more desirable Caco-2 parameters, as demonstrated by trifluoroacetamide 11 and the benchmark N-ethylamino analog 12. In addition, the tendency of these inhibitors to form intramolecular hydrogen bonds potentially contributes favorably to the improved membrane permeability and absorption. The functional group minimization that resolved the efflux problem simultaneously maintained potent inhibitory activity toward a gt-2 HCV replicon due to a switching of the role of substituents in interacting with the Gln414 binding pocket, as observed in gt-2a NS5B/inhibitor complex cocrystal structures, thus increasing the efficiency of the optimization. Noteworthy, a novel intermolecular S=O···C=O n → π* type interaction between the ligand sulfonamide oxygen atom and the carbonyl moiety of the side chain of Gln414 was observed. The insights from these structure-property studies and crystallography information provided a direction for optimization in a campaign to identify second generation pan-genotypic NS5B inhibitors.

Design, Synthesis, and SAR of C-3 Benzoic Acid, C-17 Triterpenoid Derivatives. Identification of the HIV-1 Maturation Inhibitor 4-((1 R,3a S,5a R,5b R,7a R,11a S,11b R,13a R,13b R)-3a-((2-(1,1-Dioxidothiomorpholino)ethyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(prop-1-en-2-yl)-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1 H-cyclopenta[ a]chrysen-9-yl)benzoic Acid (GSK3532795, BMS-955176).

GSK3532795, formerly known as BMS-955176 (1), is a potent, orally active, second-generation HIV-1 maturation inhibitor (MI) that advanced through phase IIb clinical trials. The careful design, selection, and evaluation of substituents appended to the C-3 and C-17 positions of the natural product betulinic acid (3) was critical in attaining a molecule with the desired virological and pharmacokinetic profile. Herein, we highlight the key insights made in the discovery program and detail the evolution of the structure-activity relationships (SARs) that led to the design of the specific C-17 amine moiety in 1. These modifications ultimately enabled the discovery of 1 as a second-generation MI that combines broad coverage of polymorphic viruses (EC50 <15 nM toward a panel of common polymorphisms representative of 96.5% HIV-1 subtype B virus) with a favorable pharmacokinetic profile in preclinical species.

The discovery of a pan-genotypic, primer grip inhibitor of HCV NS5B polymerase.

The development of a series of novel 7-azabenzofurans exhibiting pan-genotype inhibition of HCV NS5B polymerase via binding to the primer grip site is presented. Many challenges, including poor oral bioavailability, high clearance, bioactivation, high human serum shift, and metabolic stability were encountered and overcome through SAR studies. This work culminated in the selection of BMS-986139 (43) as a preclinical candidate.