Exploration of the P1 residue in 3CL protease inhibitors leading to the discovery of a 2-tetrahydrofuran P1 replacement.

The virally encoded 3C-like protease (3CLpro) is a well-validated drug target for the inhibition of coronaviruses including Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Most inhibitors of 3CLpro are peptidomimetic, with a γ-lactam in place of Gln at the P1 position of the pseudopeptide chain. An effort was pursued to identify a viable alternative to the γ-lactam P1 mimetic which would improve physicochemical properties while retaining affinity for the target. Discovery of a 2-tetrahydrofuran as a suitable P1 replacement that is a potent enzymatic inhibitor of 3CLpro in SARS-CoV-2 virus is described herein.

Novel Bent Conformation of CD4 Induced by HIV-1 Inhibitor Indirectly Prevents Productive Viral Attachment.

GSK3732394 is a multi-specific biologic inhibitor of HIV entry currently under clinical evaluation. A key component of this molecule is an adnectin (6940_B01) that binds to CD4 and inhibits downstream actions of gp160. Studies were performed to determine the binding site of the adnectin on CD4 and to understand the mechanism of inhibition. Using hydrogen-deuterium exchange with mass spectrometry (HDX), CD4 peptides showed differential rates of deuteration (either enhanced or slowed) in the presence of the adnectin that mapped predominantly to the interface of domains 2 and 3 (D2-D3). In addition, an X-ray crystal structure of an ibalizumab Fab/CD4(D1-D4)/adnectin complex revealed an extensive interface between the adnectin and residues on CD4 domains D2-D4 that stabilize a novel T-shaped CD4 conformation. A cryo-EM map of the gp140/CD4/GSK3732394 complex clearly shows the bent conformation for CD4 while bound to gp140. Mutagenic analyses on CD4 confirmed that amino acid F202 forms a key interaction with the adnectin. In addition, amino acid L151 was shown to be a critical indirect determinant of the specificity for binding to the human CD4 protein over related primate CD4 molecules, as it appears to modulate CD4's flexibility to adopt the adnectin-bound conformation. The significant conformational change of CD4 upon adnectin binding brings the D1 domain of CD4 in proximity to the host cell membrane surface, thereby re-orienting the gp120 binding site in a direction that is inaccessible to incoming virus due to a steric clash between gp160 trimers on the virus surface and the target cell membrane.

Anti-tumor Activity of the Type I PRMT Inhibitor, GSK3368715, Synergizes with PRMT5 Inhibition through MTAP Loss.

Type I protein arginine methyltransferases (PRMTs) catalyze asymmetric dimethylation of arginines on proteins. Type I PRMTs and their substrates have been implicated in human cancers, suggesting inhibition of type I PRMTs may offer a therapeutic approach for oncology. The current report describes GSK3368715 (EPZ019997), a potent, reversible type I PRMT inhibitor with anti-tumor effects in human cancer models. Inhibition of PRMT5, the predominant type II PRMT, produces synergistic cancer cell growth inhibition when combined with GSK3368715. Interestingly, deletion of the methylthioadenosine phosphorylase gene (MTAP) results in accumulation of the metabolite 2-methylthioadenosine, an endogenous inhibitor of PRMT5, and correlates with sensitivity to GSK3368715 in cell lines. These data provide rationale to explore MTAP status as a biomarker strategy for patient selection.

Author Correction: Design of amidobenzimidazole STING receptor agonists with systemic activity.

Change history: In this Letter, author Ana Puhl was inadvertently omitted; this error has been corrected online.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Design of amidobenzimidazole STING receptor agonists with systemic activity.

Stimulator of interferon genes (STING) is a receptor in the endoplasmic reticulum that propagates innate immune sensing of cytosolic pathogen-derived and self DNA1. The development of compounds that modulate STING has recently been the focus of intense research for the treatment of cancer and infectious diseases and as vaccine adjuvants2. To our knowledge, current efforts are focused on the development of modified cyclic dinucleotides that mimic the endogenous STING ligand cGAMP; these have progressed into clinical trials in patients with solid accessible tumours amenable to intratumoral delivery3. Here we report the discovery of a small molecule STING agonist that is not a cyclic dinucleotide and is systemically efficacious for treating tumours in mice. We developed a linking strategy to synergize the effect of two symmetry-related amidobenzimidazole (ABZI)-based compounds to create linked ABZIs (diABZIs) with enhanced binding to STING and cellular function. Intravenous administration of a diABZI STING agonist to immunocompetent mice with established syngeneic colon tumours elicited strong anti-tumour activity, with complete and lasting regression of tumours. Our findings represent a milestone in the rapidly growing field of immune-modifying cancer therapies.

Discovery of renin inhibitors containing a simple aspartate binding moiety that imparts reduced P450 inhibition.

Discovery of potent renin inhibitors which contain a simplified alkylamino Asp-binding group and exhibit improved selectivity for renin over Cyp3A4 is described. Structure-function results in this series are rationalized based on analysis of selected compounds bound to renin, and the contribution of each molecular feature leading to the reduced P450 inhibition is quantified.

Discovery and Characterization of a Class of Pyrazole Inhibitors of Bacterial Undecaprenyl Pyrophosphate Synthase.

Undecaprenyl pyrophosphate synthase (UppS) is an essential enzyme in bacterial cell wall synthesis. Here we report the discovery of Staphylococcus aureus UppS inhibitors from an Encoded Library Technology screen and demonstrate binding to the hydrophobic substrate site through cocrystallography studies. The use of bacterial strains with regulated uppS expression and inhibitor resistant mutant studies confirmed that the whole cell activity was the result of UppS inhibition, validating UppS as a druggable antibacterial target.

New IDH1 mutant inhibitors for treatment of acute myeloid leukemia.

Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are driver mutations in acute myeloid leukemia (AML) and other cancers. We report the development of new allosteric inhibitors of mutant IDH1. Crystallographic and biochemical results demonstrated that compounds of this chemical series bind to an allosteric site and lock the enzyme in a catalytically inactive conformation, thereby enabling inhibition of different clinically relevant IDH1 mutants. Treatment of IDH1 mutant primary AML cells uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block and induction of granulocytic differentiation at the level of leukemic blasts and more immature stem-like cells, in vitro and in vivo. Molecularly, treatment with the inhibitors led to a reversal of the DNA cytosine hypermethylation patterns caused by mutant IDH1 in the cells of individuals with AML. Our study provides proof of concept for the molecular and biological activity of novel allosteric inhibitors for targeting different mutant forms of IDH1 in leukemia.

Long-Range Inhibitor-Induced Conformational Regulation of Human IRE1α Endoribonuclease Activity.

Activation of the inositol-requiring enzyme-1 alpha (IRE1α) protein caused by endoplasmic reticulum stress results in the homodimerization of the N-terminal endoplasmic reticulum luminal domains, autophosphorylation of the cytoplasmic kinase domains, and conformational changes to the cytoplasmic endoribonuclease (RNase) domains, which render them functional and can lead to the splicing of X-box binding protein 1 (XBP 1) mRNA. Herein, we report the first crystal structures of the cytoplasmic portion of a human phosphorylated IRE1α dimer in complex with (R)-2-(3,4-dichlorobenzyl)-N-(4-methylbenzyl)-2,7-diazaspiro(4.5)decane-7-carboxamide, a novel, IRE1α-selective kinase inhibitor, and staurosporine, a broad spectrum kinase inhibitor. (R)-2-(3,4-dichlorobenzyl)-N-(4-methylbenzyl)-2,7-diazaspiro(4.5)decane-7-carboxamide inhibits both the kinase and RNase activities of IRE1α. The inhibitor interacts with the catalytic residues Lys599 and Glu612 and displaces the kinase activation loop to the DFG-out conformation. Inactivation of IRE1α RNase activity appears to be caused by a conformational change, whereby the αC helix is displaced, resulting in the rearrangement of the kinase domain-dimer interface and a rotation of the RNase domains away from each other. In contrast, staurosporine binds at the ATP-binding site of IRE1α, resulting in a dimer consistent with RNase active yeast Ire1 dimers. Activation of IRE1α RNase activity appears to be promoted by a network of hydrogen bond interactions between highly conserved residues across the RNase dimer interface that place key catalytic residues poised for reaction. These data implicate that the intermolecular interactions between conserved residues in the RNase domain are required for activity, and that the disruption of these interactions can be achieved pharmacologically by small molecule kinase domain inhibitors.

A DNA Hypomethylation Signature Predicts Antitumor Activity of LSD1 Inhibitors in SCLC.

Epigenetic dysregulation has emerged as an important mechanism in cancer. Alterations in epigenetic machinery have become a major focus for targeted therapies. The current report describes the discovery and biological activity of a cyclopropylamine containing inhibitor of Lysine Demethylase 1 (LSD1), GSK2879552. This small molecule is a potent, selective, orally bioavailable, mechanism-based irreversible inactivator of LSD1. A proliferation screen of cell lines representing a number of tumor types indicated that small cell lung carcinoma (SCLC) is sensitive to LSD1 inhibition. The subset of SCLC lines and primary samples that undergo growth inhibition in response to GSK2879552 exhibit DNA hypomethylation of a signature set of probes, suggesting this may be used as a predictive biomarker of activity.

Structure of the BTB domain of Keap1 and its interaction with the triterpenoid antagonist CDDO.

The protein Keap1 is central to the regulation of the Nrf2-mediated cytoprotective response, and is increasingly recognized as an important target for therapeutic intervention in a range of diseases involving excessive oxidative stress and inflammation. The BTB domain of Keap1 plays key roles in sensing environmental electrophiles and in mediating interactions with the Cul3/Rbx1 E3 ubiquitin ligase system, and is believed to be the target for several small molecule covalent activators of the Nrf2 pathway. However, despite structural information being available for several BTB domains from related proteins, there have been no reported crystal structures of Keap1 BTB, and this has precluded a detailed understanding of its mechanism of action and interaction with antagonists. We report here the first structure of the BTB domain of Keap1, which is thought to contain the key cysteine residue responsible for interaction with electrophiles, as well as structures of the covalent complex with the antagonist CDDO/bardoxolone, and of the constitutively inactive C151W BTB mutant. In addition to providing the first structural confirmation of antagonist binding to Keap1 BTB, we also present biochemical evidence that adduction of Cys 151 by CDDO is capable of inhibiting the binding of Cul3 to Keap1, and discuss how this class of compound might exert Nrf2 activation through disruption of the BTB-Cul3 interface.

Mutant IDH1 enhances the production of 2-hydroxyglutarate due to its kinetic mechanism.

The human, cytosolic enzyme isocitrate dehydrogenase 1 (IDH1) reversibly converts isocitrate to α-ketoglutarate (αKG). Cancer-associated somatic mutations in IDH1 result in a loss of this normal function but a gain in a new or neomorphic ability to convert αKG to the oncometabolite 2-hydroxyglutarate (2HG). To improve our understanding of the basis for this phenomenon, we have conducted a detailed kinetic study of wild-type IDH1 as well as the known 2HG-producing clinical R132H and G97D mutants and mechanistic Y139D and (newly described) G97N mutants. In the reductive direction of the normal reaction (αKG to isocitrate), dead-end inhibition studies suggest that wild-type IDH1 goes through a random sequential mechanism, similar to previous reports on related mammalian IDH enzymes. However, analogous experiments studying the reductive neomorphic reaction (αKG to 2HG) with the mutant forms of IDH1 are more consistent with an ordered sequential mechanism, with NADPH binding before αKG. This result was further confirmed by primary kinetic isotope effects for which saturating with αKG greatly reduced the observed isotope effect on (D)(V/K)NADPH. For the mutant IDH1 enzyme, the change in mechanism was consistently associated with reduced efficiencies in the use of αKG as a substrate and enhanced efficiencies using NADPH as a substrate. We propose that the sum of these kinetic changes allows the mutant IDH1 enzymes to reductively trap αKG directly into 2HG, rather than allowing it to react with carbon dioxide and form isocitrate, as occurs in the wild-type enzyme.

A tale of two subunits: how the neomorphic R132H IDH1 mutation enhances production of αHG.

Heterozygously expressed single-point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2, respectively) render these dimeric enzymes capable of producing the novel metabolite α-hydroxyglutarate (αHG). Accumulation of αHG is used as a biomarker for a number of cancer types, helping to identify tumors with similar IDH mutations. With IDH1, it has been shown that one role of the mutation is to increase the rate of conversion from αKG to αHG. To improve our understanding of the function of this mutation, we have detailed the kinetics of the normal (isocitrate to αKG) and neomorphic (αKG to αHG) reactions, as well as the coupled conversion of isocitrate to αHG. We find that the mutant IDH1 is very efficient in this coupled reaction, with the ability to form αHG from isocitrate and NADP(+). The wild type/wild type IDH1 is also able to catalyze this conversion, though it is much more sensitive to concentrations of isocitrate. This difference in behavior can be attributed to the competitive binding between isocitrate and αKG, which is made more favorable for αKG by the neomorphic mutation at arginine 132. Thus, each partial reaction in the heterodimer is functionally isolated from the other. To test whether there is a cooperative effect resulting from the two subunits being in a dimer, we selectively inactivated each subunit with a secondary mutation in the NADP/H binding site. We observed that the remaining, active subunit was unaffected in its associated activity, reinforcing the notion of each subunit being functionally independent. This was further demonstrated using a monomeric form of IDH from Azotobacter vinelandii, which can be shown to gain the same neomorphic reaction when a homologous mutation is introduced into that protein.

Kinetic mechanism and rate-limiting steps of focal adhesion kinase-1.

Steady-state kinetic analysis of focal adhesion kinase-1 (FAK1) was performed using radiometric measurement of phosphorylation of a synthetic peptide substrate (Ac-RRRRRRSETDDYAEIID-NH(2), FAK-tide) which corresponds to the sequence of an autophosphorylation site in FAK1. Initial velocity studies were consistent with a sequential kinetic mechanism, for which apparent kinetic values k(cat) (0.052 +/- 0.001 s(-1)), K(MgATP) (1.2 +/- 0.1 microM), K(iMgATP) (1.3 +/- 0.2 microM), K(FAK-tide) (5.6 +/- 0.4 microM), and K(iFAK-tide) (6.1 +/- 1.1 microM) were obtained. Product and dead-end inhibition data indicated that enzymatic phosphorylation of FAK-tide by FAK1 was best described by a random bi bi kinetic mechanism, for which both E-MgADP-FAK-tide and E-MgATP-P-FAK-tide dead-end complexes form. FAK1 catalyzed the betagamma-bridge:beta-nonbridge positional oxygen exchange of [gamma-(18)O(4)]ATP in the presence of 1 mM [gamma-(18)O(4)]ATP and 1.5 mM FAK-tide with a progressive time course which was commensurate with catalysis, resulting in a rate of exchange to catalysis of k(x)/k(cat) = 0.14 +/- 0.01. These results indicate that phosphoryl transfer is reversible and that a slow kinetic step follows formation of the E-MgADP-P-FAK-tide complex. Further kinetic studies performed in the presence of the microscopic viscosogen sucrose revealed that solvent viscosity had no effect on k(cat)/K(FAK-tide), while k(cat) and k(cat)/K(MgATP) were both decreased linearly at increasing solvent viscosity. Crystallographic characterization of inactive versus AMP-PNP-liganded structures of FAK1 showed that a large conformational motion of the activation loop upon ATP binding may be an essential step during catalysis and would explain the viscosity effect observed on k(cat)/K(m) for MgATP but not on k(cat)/K(m) for FAK-tide. From the positional isotope exchange, viscosity, and structural data it may be concluded that enzyme turnover (k(cat)) is rate-limited by both reversible phosphoryl group transfer (k(forward) approximately 0.2 s(-1) and k(reverse) approximately 0.04 s(-1)) and a slow step (k(conf) approximately 0.1 s(-1)) which is probably the opening of the activation loop after phosphoryl group transfer but preceding product release.

2,3,5-Trisubstituted pyridines as selective AKT inhibitors-Part I: Substitution at 2-position of the core pyridine for ROCK1 selectivity.

2,3,5-Trisubstituted pyridines have been designed as potent AKT inhibitors that are selective against ROCK1 based on the comparison between AKT and ROCK1 structures. Substitution at the 2-position of the core pyridine is the key element to provide selectivity against ROCK1. An X-ray co-crystal structure of 9p in PKA supports the proposed rationale of ROCK1 selectivity.

Substituted benzothiadizine inhibitors of Hepatitis C virus polymerase.

The synthesis and optimisation of HCV NS5B polymerase inhibitors with improved potency versus the existing compound 1 is described. Substitution in the benzothiadiazine portion of the molecule, furnishing improvement in potency in the high protein Replicon assay, is highlighted, culminating in the discovery of 12h, a highly potent oxyacetamide derivative.

Discovery of 5-pyrrolopyridinyl-2-thiophenecarboxamides as potent AKT kinase inhibitors.

A pyrrolopyridinyl thiophene carboxamide 7 was discovered as a tractable starting point for a lead optimization effort in an AKT kinase inhibition program. SAR studies aided by a co-crystal structure of 7 in AKT2 led to the identification of AKT inhibitors with subnanomolar potency. Representative compounds showed antiproliferative activity as well as inhibition of phosphorylation of the downstream target GSK3beta.

Aminofurazans as potent inhibitors of AKT kinase.

AKT inhibitors containing an imidazopyridine aminofurazan scaffold have been optimized. We have previously disclosed identification of the AKT inhibitor GSK690693, which has been evaluated in clinical trials in cancer patients. Herein we describe recent efforts focusing on investigating a distinct region of this scaffold that have afforded compounds (30 and 32) with comparable activity profiles to that of GSK690693.

Identification of 4-(2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-7-{[(3S)-3-piperidinylmethyl]oxy}-1H-imidazo[4,5-c]pyridin-4-yl)-2-methyl-3-butyn-2-ol (GSK690693), a novel inhibitor of AKT kinase.

Overexpression of AKT has an antiapoptotic effect in many cell types, and expression of dominant negative AKT blocks the ability of a variety of growth factors to promote survival. Therefore, inhibitors of AKT kinase activity might be useful as monotherapy for the treatment of tumors with activated AKT. Herein, we describe our lead optimization studies culminating in the discovery of compound 3g (GSK690693). Compound 3g is a novel ATP competitive, pan-AKT kinase inhibitor with IC 50 values of 2, 13, and 9 nM against AKT1, 2, and 3, respectively. An X-ray cocrystal structure was solved with 3g and the kinase domain of AKT2, confirming that 3g bound in the ATP binding pocket. Compound 3g potently inhibits intracellular AKT activity as measured by the inhibition of the phosphorylation levels of GSK3beta. Intraperitoneal administration of 3g in immunocompromised mice results in the inhibition of GSK3beta phosphorylation and tumor growth in human breast carcinoma (BT474) xenografts.

3-(1,1-dioxo-2H-(1,2,4)-benzothiadiazin-3-yl)-4-hydroxy-2(1H)-quinolinones, potent inhibitors of hepatitis C virus RNA-dependent RNA polymerase.

Recently, we disclosed a new class of HCV polymerase inhibitors discovered through high-throughput screening (HTS) of the GlaxoSmithKline proprietary compound collection. This interesting class of 3-(1,1-dioxo-2H-1,2,4-benzothiadiazin-3-yl)-4-hydroxy-2(1H)-quinolinones potently inhibits HCV polymerase enzymatic activity and inhibits the ability of the subgenomic HCV replicon to replicate in Huh-7 cells. This report will focus on the structure-activity relationships (SAR) of substituents on the quinolinone ring, culminating in the discovery of 1-(2-cyclopropylethyl)-3-(1,1-dioxo-2H-1,2,4-benzothiadiazin-3-yl)-6-fluoro-4-hydroxy-2(1H)-quinolinone (130), an inhibitor with excellent potency in biochemical and cellular assays possessing attractive molecular properties for advancement as a clinical candidate. The potential for development and safety assessment profile of compound 130 will also be discussed.