Cytochromes P450 2C8 and 3A Catalyze the Metabolic Activation of the Tyrosine Kinase Inhibitor Masitinib.

Masitinib is a small molecule tyrosine kinase inhibitor under investigation for the treatment of amyotrophic lateral sclerosis, mastocytosis, and COVID-19. Hepatotoxicity has been reported in some patients while taking masitinib. The liver injury is thought to involve hepatic metabolism of masitinib by cytochrome P450 (P450) enzymes to form chemically reactive, potentially toxic metabolites. The goal of the current investigation was to determine the P450 enzymes involved in the metabolic activation of masitinib in vitro. In initial studies, masitinib (30 µM) was incubated with pooled human liver microsomes in the presence of NADPH and potassium cyanide to trap reactive iminium ion metabolites as cyano adducts. Masitinib metabolites and cyano adducts were analyzed using reversed-phase liquid chromatography-tandem mass spectrometry. The primary active metabolite, N-desmethyl masitinib (M485), and several oxygenated metabolites were detected along with four reactive metabolite cyano adducts (MCN510, MCN524, MCN526, and MCN538). To determine which P450 enzymes were involved in metabolite formation, reaction phenotyping experiments were conducted by incubation of masitinib (2 µM) with a panel of recombinant human P450 enzymes and by incubation of masitinib with human liver microsomes in the presence of P450-selective chemical inhibitors. In addition, enzyme kinetic assays were conducted to determine the relative kinetic parameters (apparent Km and Vmax) of masitinib metabolism and cyano adduct formation. Integrated analysis of the results from these experiments indicates that masitinib metabolic activation is catalyzed primarily by P450 3A4 and 2C8, with minor contributions from P450 3A5 and 2D6. These findings provide further insight into the pathways involved in the generation of reactive, potentially toxic metabolites of masitinib. Future studies are needed to evaluate the impact of masitinib metabolism on the toxicity of the drug in vivo.

An automatic pipeline for the design of irreversible derivatives identifies a potent SARS-CoV-2 Mpro inhibitor.

Designing covalent inhibitors is increasingly important, although it remains challenging. Here, we present covalentizer, a computational pipeline for identifying irreversible inhibitors based on structures of targets with non-covalent binders. Through covalent docking of tailored focused libraries, we identify candidates that can bind covalently to a nearby cysteine while preserving the interactions of the original molecule. We found âˆ¼11,000 cysteines proximal to a ligand across 8,386 complexes in the PDB. Of these, the protocol identified 1,553 structures with covalent predictions. In a prospective evaluation, five out of nine predicted covalent kinase inhibitors showed half-maximal inhibitory concentration (IC50) values between 155 nM and 4.5 µM. Application against an existing SARS-CoV Mpro reversible inhibitor led to an acrylamide inhibitor series with low micromolar IC50 values against SARS-CoV-2 Mpro. The docking was validated by 12 co-crystal structures. Together these examples hint at the vast number of covalent inhibitors accessible through our protocol.

Structure-Based Virtual Screening Reveals Ibrutinib and Zanubrutinib as Potential Repurposed Drugs against COVID-19.

Coronavirus disease (COVID)-19 is the leading global health threat to date caused by a severe acute respiratory syndrome coronavirus (SARS-CoV-2). Recent clinical trials reported that the use of Bruton's tyrosine kinase (BTK) inhibitors to treat COVID-19 patients could reduce dyspnea and hypoxia, thromboinflammation, hypercoagulability and improve oxygenation. However, the mechanism of action remains unclear. Thus, this study employs structure-based virtual screening (SBVS) to repurpose BTK inhibitors acalabrutinib, dasatinib, evobrutinib, fostamatinib, ibrutinib, inositol 1,3,4,5-tetrakisphosphate, spebrutinib, XL418 and zanubrutinib against SARS-CoV-2. Molecular docking is conducted with BTK inhibitors against structural and nonstructural proteins of SARS-CoV-2 and host targets (ACE2, TMPRSS2 and BTK). Molecular mechanics-generalized Born surface area (MM/GBSA) calculations and molecular dynamics (MD) simulations are then carried out on the selected complexes with high binding energy. Ibrutinib and zanubrutinib are found to be the most potent of the drugs screened based on the results of computational studies. Results further show that ibrutinib and zanubrutinib could exploit different mechanisms at the viral entry and replication stage and could be repurposed as potential inhibitors of SARS-CoV-2 pathogenesis.

Heparan Sulfate Proteoglycans in Viral Infection and Treatment: A Special Focus on SARS-CoV-2.

Heparan sulfate proteoglycans (HSPGs) encompass a group of glycoproteins composed of unbranched negatively charged heparan sulfate (HS) chains covalently attached to a core protein. The complex HSPG biosynthetic machinery generates an extraordinary structural variety of HS chains that enable them to bind a plethora of ligands, including growth factors, morphogens, cytokines, chemokines, enzymes, matrix proteins, and bacterial and viral pathogens. These interactions translate into key regulatory activity of HSPGs on a wide range of cellular processes such as receptor activation and signaling, cytoskeleton assembly, extracellular matrix remodeling, endocytosis, cell-cell crosstalk, and others. Due to their ubiquitous expression within tissues and their large functional repertoire, HSPGs are involved in many physiopathological processes; thus, they have emerged as valuable targets for the therapy of many human diseases. Among their functions, HSPGs assist many viruses in invading host cells at various steps of their life cycle. Viruses utilize HSPGs for the attachment to the host cell, internalization, intracellular trafficking, egress, and spread. Recently, HSPG involvement in the pathogenesis of SARS-CoV-2 infection has been established. Here, we summarize the current knowledge on the molecular mechanisms underlying HSPG/SARS-CoV-2 interaction and downstream effects, and we provide an overview of the HSPG-based therapeutic strategies that could be used to combat such a fearsome virus.

Role of JAK/STAT in Interstitial Lung Diseases; Molecular and Cellular Mechanisms.

Interstitial lung diseases (ILDs) comprise different fibrotic lung disorders characterized by cellular proliferation, interstitial inflammation, and fibrosis. The JAK/STAT molecular pathway is activated under the interaction of a broad number of profibrotic/pro-inflammatory cytokines, such as IL-6, IL-11, and IL-13, among others, which are increased in different ILDs. Similarly, several growth factors over-expressed in ILDs, such as platelet-derived growth factor (PDGF), transforming growth factor ß1 (TGF-ß1), and fibroblast growth factor (FGF) activate JAK/STAT by canonical or non-canonical pathways, which indicates a predominant role of JAK/STAT in ILDs. Between the different JAK/STAT isoforms, it appears that JAK2/STAT3 are predominant, initiating cellular changes observed in ILDs. This review analyzes the expression and distribution of different JAK/STAT isoforms in ILDs lung tissue and different cell types related to ILDs, such as lung fibroblasts and alveolar epithelial type II cells and analyzes JAK/STAT activation. The effect of JAK/STAT phosphorylation on cellular fibrotic processes, such as proliferation, senescence, autophagy, endoplasmic reticulum stress, or epithelial/fibroblast to mesenchymal transition will be described. The small molecules directed to inhibit JAK/STAT activation were assayed in vitro and in in vivo models of pulmonary fibrosis, and different JAK inhibitors are currently approved for myeloproliferative disorders. Recent evidence indicates that JAK inhibitors or monoclonal antibodies directed to block IL-6 are used as compassionate use to attenuate the excessive inflammation and lung fibrosis related to SARS-CoV-2 virus. These altogether indicate that JAK/STAT pathway is an attractive target to be proven in future clinical trials of lung fibrotic disorders.

Differential impact of BTK active site inhibitors on the conformational state of full-length BTK.

Bruton's tyrosine kinase (BTK) is targeted in the treatment of B-cell disorders including leukemias and lymphomas. Currently approved BTK inhibitors, including Ibrutinib, a first-in-class covalent inhibitor of BTK, bind directly to the kinase active site. While effective at blocking the catalytic activity of BTK, consequences of drug binding on the global conformation of full-length BTK are unknown. Here, we uncover a range of conformational effects in full-length BTK induced by a panel of active site inhibitors, including large-scale shifts in the conformational equilibria of the regulatory domains. Additionally, we find that a remote Ibrutinib resistance mutation, T316A in the BTK SH2 domain, drives spurious BTK activity by destabilizing the compact autoinhibitory conformation of full-length BTK, shifting the conformational ensemble away from the autoinhibited form. Future development of BTK inhibitors will need to consider long-range allosteric consequences of inhibitor binding, including the emerging application of these BTK inhibitors in treating COVID-19.

Treatments for blood cancers, such as leukemia and lymphoma, rely heavily on chemotherapy, using drugs that target a vulnerable aspect of the cancer cells. B-cells, a type of white blood cell that produces antibodies, require a protein called Bruton's tyrosine kinase, or BTK for short, to survive. The drug ibrutinib (Imbruvica) is used to treat B-cell cancers by blocking BTK. The BTK protein consists of several regions. One of them, known as the kinase domain, is responsible for its activity as an enzyme (which allows it to modify other proteins by adding a 'tag' known as a phosphate group). The other regions of BTK, known as regulatory modules, control this activity. In BTK's inactive form, the regulatory modules attach to the kinase domain, blocking the regulatory modules from interacting with other proteins. When BTK is activated, it changes its conformation so the regulatory regions detach and become available for interactions with other proteins, at the same time exposing the active kinase domain. Ibrutinib and other BTK drugs in development bind to the kinase domain to block its activity. However, it is not known how this binding affects the regulatory modules. Previous efforts to study how drugs bind to BTK have used a version of the protein that only had the kinase domain, instead of the full-length protein. Now, Joseph et al. have studied full-length BTK and how it binds to five different drugs. The results reveal that ibrutinib and another drug called dasatinib both indirectly disrupt the normal position of the regulatory domains pushing BTK toward a conformation that resembles the activated state. By contrast, the three other compounds studied do not affect the inactive structure. Joseph et al. also examined a mutation in BTK that confers resistance against ibrutinib. This mutation increases the activity of BTK by disrupting the inactive structure, leading to B cells surviving better. Understanding how drug resistance mechanisms can work will lead to better drug treatment strategies for cancer. BTK is also a target in other diseases such as allergies or asthma and even COVID-19. If interactions between partner proteins and the regulatory domain are important in these diseases, then they may be better treated with drugs that maintain the regulatory modules in their inactive state. This research will help to design drugs that are better able to control BTK activity.