The Substitutions L50F, E166A, and L167F in SARS-CoV-2 3CLpro Are Selected by a Protease Inhibitor In Vitro and Confer Resistance To Nirmatrelvir.

The SARS-CoV-2 main protease (3CLpro) has an indispensable role in the viral life cycle and is a therapeutic target for the treatment of COVID-19. The potential of 3CLpro-inhibitors to select for drug-resistant variants needs to be established. Therefore, SARS-CoV-2 was passaged in vitro in the presence of increasing concentrations of ALG-097161, a probe compound designed in the context of a 3CLpro drug discovery program. We identified a combination of amino acid substitutions in 3CLpro (L50F E166A L167F) that is associated with a >20× increase in 50% effective concentration (EC50) values for ALG-097161, nirmatrelvir (PF-07321332), PF-00835231, and ensitrelvir. While two of the single substitutions (E166A and L167F) provide low-level resistance to the inhibitors in a biochemical assay, the triple mutant results in the highest levels of resistance (6× to 72×). All substitutions are associated with a significant loss of enzymatic 3CLpro activity, suggesting a reduction in viral fitness. Structural biology analysis indicates that the different substitutions reduce the number of inhibitor/enzyme interactions while the binding of the substrate is maintained. These observations will be important for the interpretation of resistance development to 3CLpro inhibitors in the clinical setting. IMPORTANCE Paxlovid is the first oral antiviral approved for treatment of SARS-CoV-2 infection. Antiviral treatments are often associated with the development of drug-resistant viruses. In order to guide the use of novel antivirals, it is essential to understand the risk of resistance development and to characterize the associated changes in the viral genes and proteins. In this work, we describe for the first time a pathway that allows SARS-CoV-2 to develop resistance against Paxlovid in vitro. The characteristics of in vitro antiviral resistance development may be predictive for the clinical situation. Therefore, our work will be important for the management of COVID-19 with Paxlovid and next-generation SARS-CoV-2 3CLpro inhibitors.

ALG-097111, a potent and selective SARS-CoV-2 3-chymotrypsin-like cysteine protease inhibitor exhibits in vivo efficacy in a Syrian Hamster model.

There is an urgent need for antivirals targeting the SARS-CoV-2 virus to fight the current COVID-19 pandemic. The SARS-CoV-2 main protease (3CLpro) represents a promising target for antiviral therapy. The lack of selectivity for some of the reported 3CLpro inhibitors, specifically versus cathepsin L, raises potential safety and efficacy concerns. ALG-097111 potently inhibited SARS-CoV-2 3CLpro (IC50 = 7 nM) without affecting the activity of human cathepsin L (IC50 > 10 µM). When ALG-097111 was dosed in hamsters challenged with SARS-CoV-2, a robust and significant 3.5 log10 (RNA copies/mg) reduction of the viral RNA copies and 3.7 log10 (TCID50/mg) reduction in the infectious virus titers in the lungs was observed. These results provide the first in vivo validation for the SARS-CoV-2 3CLpro as a promising therapeutic target for selective small molecule inhibitors.

Dual inhibition of SARS-CoV-2 and human rhinovirus with protease inhibitors in clinical development.

The 3-chymotrypsin-like cysteine protease (3CLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is considered a major target for the discovery of direct antiviral agents. We previously reported the evaluation of SARS-CoV-2 3CLpro inhibitors in a novel self-assembled monolayer desorption ionization mass spectrometry (SAMDI-MS) enzymatic assay (Gurard-Levin et al., 2020). The assay was further improved by adding the rhinovirus HRV3C protease to the same well as the SARS-CoV-2 3CLpro enzyme. High substrate specificity for each enzyme allowed the proteases to be combined in a single assay reaction without interfering with their individual activities. This novel duplex assay was used to profile a diverse set of reference protease inhibitors. The protease inhibitors were grouped into three categories based on their relative potency against 3CLpro and HRV3C including those that are: equipotent against 3CLpro and HRV3C (GC376 and calpain inhibitor II), selective for 3CLpro (PF-00835231, calpain inhibitor XII, boceprevir), and selective for HRV3C (rupintrivir). Structural analysis showed that the combination of minimal interactions, conformational flexibility, and limited bulk allows GC376 and calpain inhibitor II to potently inhibit both enzymes. In contrast, bulkier compounds interacting more tightly with pockets P2, P3, and P4 due to optimization for a specific target display a more selective inhibition profile. Consistently, the most selective viral protease inhibitors were relatively weak inhibitors of human cathepsin L. Taken together, these results can guide the design of cysteine protease inhibitors that are either virus-specific or retain a broad antiviral spectrum against coronaviruses and rhinoviruses.

An allosteric binding site of the α7 nicotinic acetylcholine receptor revealed in a humanized acetylcholine-binding protein.

Nicotinic acetylcholine receptors (nAChRs) belong to the family of pentameric ligand-gated ion channels and mediate fast excitatory transmission in the central and peripheral nervous systems. Among the different existing receptor subtypes, the homomeric α7 nAChR has attracted considerable attention because of its possible implication in several neurological and psychiatric disorders, including cognitive decline associated with Alzheimer's disease or schizophrenia. Allosteric modulators of ligand-gated ion channels are of particular interest as therapeutic agents, as they modulate receptor activity without affecting normal fluctuations of synaptic neurotransmitter release. Here, we used X-ray crystallography and surface plasmon resonance spectroscopy of α7-acetylcholine-binding protein (AChBP), a humanized chimera of a snail AChBP, which has 71% sequence similarity with the extracellular ligand-binding domain of the human α7 nAChR, to investigate the structural determinants of allosteric modulation. We extended previous observations that an allosteric site located in the vestibule of the receptor offers an attractive target for receptor modulation. We introduced seven additional humanizing mutations in the vestibule-located binding site of AChBP to improve its suitability as a model for studying allosteric binding. Using a fragment-based screening approach, we uncovered an allosteric binding site located near the ß8-ß9 loop, which critically contributes to coupling ligand binding to channel opening in human α7 nAChR. This work expands our understanding of the topology of allosteric binding sites in AChBP and, by extrapolation, in the human α7 nAChR as determined by electrophysiology measurements. Our insights pave the way for drug design strategies targeting nAChRs involved in ion channel-mediated disorders.

Treatment of Intestinal Fibrosis in Experimental Inflammatory Bowel Disease by the Pleiotropic Actions of a Local Rho Kinase Inhibitor.

BACKGROUND: Intestinal fibrosis resulting in (sub)obstruction is a common complication of Crohn's disease (CD). Rho kinases (ROCKs) play multiple roles in TGFß-induced myofibroblast activation that could be therapeutic targets. Because systemic ROCK inhibition causes cardiovascular side effects, we evaluated the effects of a locally acting ROCK inhibitor (AMA0825) on intestinal fibrosis. METHODS: Fibrosis was assessed in mouse models using dextran sulfate sodium (DSS) and adoptive T-cell transfer. The in vitro and ex vivo effects of AMA0825 were studied in different cell types and in CD biopsy cultures. RESULTS: ROCK is expressed in fibroblastic, epithelial, endothelial, and muscle cells of the human intestinal tract and is activated in inflamed and fibrotic tissue. Prophylactic treatment with AMA0825 inhibited myofibroblast accumulation, expression of pro-fibrotic factors, and accumulation of fibrotic tissue without affecting clinical disease activity and histologic inflammation in 2 models of fibrosis. ROCK inhibition reversed established fibrosis in a chronic DSS model and impeded ex vivo pro-fibrotic protein secretion from stenotic CD biopsies. AMA0825 reduced TGFß1-induced activation of myocardin-related transcription factor (MRTF) and p38 mitogen-activated protein kinase (MAPK), down-regulating matrix metalloproteinases, collagen, and IL6 secretion from fibroblasts. In these cells, ROCK inhibition potentiated autophagy, which was required for the observed reduction in collagen and IL6 production. AMA0825 did not affect pro-inflammatory cytokine secretion from other ROCK-positive cell types, corroborating the selective in vivo effect on fibrosis. CONCLUSIONS: Local ROCK inhibition prevents and reverses intestinal fibrosis by diminishing MRTF and p38 MAPK activation and increasing autophagy in fibroblasts. Overall, our results show that local ROCK inhibition is promising for counteracting fibrosis as an add-on therapy for CD.

Rho kinase inhibitors: a patent review (2014 - 2016).

INTRODUCTION: The Rho-kinases (ROCK), ROCK1 and ROCK2, are potent, widespread biochemical modulators which have been extensively studied. Due to the involvement of ROCKs in multiple biological processes, ROCK inhibitors have pleiotropic actions and may be of relevance for a number of therapeutic applications. The drawback is however that their use might be limited by occurrence of side effects. Areas covered: Since the publication of the latest review in 2014, there have been significant advances in the field of ROCK inhibitors. In this paper we reviewed the patents published between September 2013 and September 2016. Recent novel molecules will be described. and progress from the compounds series described in the previous review as well as any new expected therapeutic uses for ROCK inhibitors that popped up in the last three years will be examined. Expert opinion: While a number of potential applications in human for ROCK inhibitors have been reported, very few molecules are currently available to patients. In addition to fasudil, ripasudil (K-115, Kowa) was only recently approved in Japan for the treatment of glaucoma (2014). Notwithstanding some failures and subsequent discontinuation, the Pipeline of preclinical and clinical ROCK inhibitors remains significant.

Design, synthesis and biological characterization of selective LIMK inhibitors.

Inhibitors of LIM kinases are considered of interest for several indications, including elevated intraocular pressure (IOP), cancer, or infection by HIV-1. LX-7101 (Lexicon Pharmaceuticals) was advanced to Phase-I clinical trials as an IOP-lowering agent for treatment of glaucoma. We here discuss the design, synthesis and evaluation of LIMK inhibitors based on a pyrrolopyrimidine scaffold, which represent close analogs of LX-7101. Exploration of structure-activity relationships revealed that many of such compounds, including LX-7101, cause potent inhibition of LIMK1 and LIMK2, and also ROCK2 and PKA. Molecular variations around the various structural elements of LX-7101 were attempted. Substitution on position 6 of the pyrrolopyrimidine scaffold led to the identification of LX-7101 analogs displaying good selectivity versus ROCK, PKA and Akt.

Design, synthesis, and biological evaluation of novel, highly active soft ROCK inhibitors.

ROCK1 and ROCK2 play important roles in numerous cellular functions, including smooth muscle cell contraction, cell proliferation, adhesion, and migration. Consequently, ROCK inhibitors are of interest for treating multiple indications including cardiovascular diseases, inflammatory and autoimmune diseases, lung diseases, and eye diseases. However, systemic inhibition of ROCK is expected to result in significant side effects. Strategies allowing reduced systemic exposure are therefore of interest. In a continuing effort toward identification of ROCK inhibitors, we here report the design, synthesis, and evaluation of novel soft ROCK inhibitors displaying an ester function allowing their rapid inactivation in the systemic circulation. Those compounds display subnanomolar activity against ROCK and strong differences of functional activity between parent compounds and expected metabolites. The binding mode of a representative compound was determined experimentally in a single-crystal X-ray diffraction study. Enzymes responsible for inactivation of these compounds once they enter systemic circulation are also discussed.

Kinase profiling in early stage drug discovery: sorting things out.

Protein kinases represent one of the largest superfamilies of drugable targets and a major research area for both the pharmaceutical industry and academic groups. This has resulted in the emergence of numerous screening technologies and services dedicated to kinase profiling. In spite of this plentiful offering, the field is not without its own pitfalls, as the profusion of reported conditions and data can ultimately complicate interpretation of project results. Here, we discuss how kinase profiling was used in our early stage drug discovery efforts, from the perspective of a smaller biotech relying largely on assay outsourcing.

Novel Roflumilast analogs as soft PDE4 inhibitors.

PDE4 inhibitors are of high interest for treatment of a wide range of inflammatory or autoimmune diseases. Their potential however has not yet been realized due to target-associated side effects, resulting in a low therapeutic window. We herein report the design, synthesis and evaluation of novel PDE4 inhibitors containing a γ-lactone structure. Such molecules are designed to undergo metabolic inactivation when entering circulation, thereby limiting systemic exposure and reducing the risk for side effects. The resulting inhibitors were highly active on both PDE4B1 and PDE4D2 and underwent rapid degradation in human plasma by paraoxonase 1. In contrast, their metabolites displayed markedly reduced permeability and/or on-target activity.

3-[2-(Aminomethyl)-5-[(pyridin-4-yl)carbamoyl]phenyl] benzoates as soft ROCK inhibitors.

Clinical development of ROCK inhibitors has so far been limited by systemic or local ROCK-associated side effects. A soft drug approach, which involves predictable metabolic inactivation of an active compound to a nontoxic metabolite, could represent an attractive way to obtain ROCK inhibitors with improved tolerability. We herein report the design and synthesis of a new series of soft ROCK inhibitors structurally related to the ROCK inhibitor Y-27632. These inhibitors contain carboxylic ester moieties which allow inactivation by esterases. While the parent esters display strong activity in enzymatic (ROCK2) and cellular (MLC phosphorylation) assays, their corresponding carboxylic acid metabolites have negligible functional activity. Compound 32 combined strong efficacy (ROCK2 IC50=2.5 nM) with rapid inactivation in plasma (t1/2 <5'). Compound 32 also demonstrated in vivo efficacy when evaluated as an IOP-lowering agent in ocular normotensive New-Zealand White rabbits, without ocular side effects.

New pyridinone derivatives as potent HIV-1 nonnucleoside reverse transcriptase inhibitors.

Several 5-ethyl-6-methyl-4-cycloalkyloxy-pyridin-2(1H)-ones were synthesized and evaluated for their anti HIV-1 activities against wild-type virus and clinically relevant mutant strains. A racemic mixture (10) with methyl substituents at positions 3 and 5 of the cyclohexyloxy moiety had potent antiviral activity against wild-type HIV-1. Subsequent stereoselective synthesis of a stereoisomer displaying both methyl groups in equatorial position was found to have the best EC(50). Further modulations focused on position 3 of the pyridinone ring improved the antiviral activity against mutant viral strains. Compounds bearing a 3-ethyl (22) or 3-isopropyl group (23) had the highest activity against wild-type HIV-1 and displayed low-nanomolar potency against several clinically relevant mutant strains.