Bisindolylmaleimide IX: a Novel Anti-SARS-CoV2 Agent Targeting Viral Main Protease 3CLpro Demonstrated by Virtual Screening Pipeline and In-Vitro Validation Assays

SARS-CoV-2, the virus that causes COVID-19 consists of several enzymes with essential functions within its proteome. Here, we focused on repurposing approved and investigational drugs/compounds. We targeted seven proteins with enzymatic activities known to be essential at different stages of the viral cycle including PLpro, 3CLpro, RdRP, Helicase, ExoN, NendoU, and 2′-O-MT. For virtual screening, energy minimization of a crystal structure of the modeled protein was carried out using the Protein Preparation Wizard (Schrodinger LLC 2020-1). Following active site selection based on data mining and COACH predictions, we performed a high-throughput virtual screen of drugs and investigational molecules (n = 5903). The screening was performed against viral targets using three sequential docking modes (i.e., HTVS, SP, and XP). Virtual screening identified ∼290 potential inhibitors based on the criteria of energy, docking parameters, ligand, and binding site strain and score. Drugs specific to each target protein were further analyzed for binding free energy perturbation by molecular mechanics (prime MM-GBSA) and pruning the hits to the top 32 candidates. The top lead from each target pool was further subjected to molecular dynamics simulation using the Desmond module. The resulting top eight hits were tested for their SARS-CoV-2 anti-viral activity in-vitro. Among these, a known inhibitor of protein kinase C isoforms, Bisindolylmaleimide IX (BIM IX), was found to be a potent inhibitor of SARS-CoV-2. Further, target validation through enzymatic assays confirmed 3CLpro to be the target. This is the first study that has showcased BIM IX as a COVID-19 inhibitor thereby validating our pipeline

The Development of Acrylamide-based Inhibitors of DHHC Family Proteins to Regulate ERK Acylation and Activity

Protein S-acylation, an enzymatically modulated lipid post-translational modification, is frequent and consequential; at the cellular level, it regulates protein subcellular trafficking and signaling activity, and at the organismal level, has been implicated in cancer and neurologic and inflammatory disease. However, the impact of dynamic S-acylation for many proteins remains unknown, in part due to lack of inhibitors for the enzymes that catalyze lipid addition, the DHHC family proteins. Not only is the most commonly used pan-DHHC inhibitor, 2-bromopalmitate (2BP), toxic and poorly potent, it also concurrently inhibits the S-acylation eraser proteins, the acyl protein thioesterases (APTs). Here, we report the synthesis and characterization of CMA a DHHC inhibitor with improved potency and selectivity. We then use it to establish the dynamics and functional consequences of S-acylation for a previously undescribed acylated protein, the extracellular signal-regulated kinase (ERK1/2), whose S¬-acylation is resistant to current DHHC inhibitors. As an α-halo fatty acid, 2BP reacts with a range of nucleophilic amino acids and forms reactive acyl CoA intermediates in cellulo. We therefore proposed to exchange the α-halo with an acrylamide, a warhead known to react with cysteine over serine residues that will not undergo metabolic conversion. Synthesis and screening of a panel of acrylamide-containing lipids revealed CMA as a potent DHHC20 inhibitor in vitro, with the ability to significantly decease both substrate and global protein S-acylation in cellulo. Competitive activity-based protein profiling (ABPP) demonstrated that SA8 engages directly with a panel of DHHCs in cells. Moreover, CMA is less toxic than 2BP, does not inhibit eraser APT activity, and is able to effect cellular responses previously reported to result from loss of S-acylation. These results position the acrylamide as an inhibitory scaffold for DHHC family proteins. We next CMA to probe the effects of perturbing palmitoylation on epidermal growth factor (EGF)-triggered signal transduction. Characterization of disruptions along this signaling cascade enabled the identification of a previously undescribed acylated protein, the extracellular signal-regulated kinase (ERK1/2), whose S-acylation is resistant to 2BP but responsive to CMA. We describe the molecular determinants of ERK1/2 acylation and cross-talk between ERK1/2 phosphorylation and palmitoylation, suggesting the criticality of S-acylation in regulating the activation and downstream behavior of this key effector protein. We then conduct a structure-activity study of CMA, which demonstrates the criticality of the lipid tail and the potential of scaffold extension, but underscores the need for novel inhibitors. To meet this need, we design and validate a fluorescence-based high-throughput assay for DHHC activity in vitro using a panel of fluorogenic probes. These palmitoyl-CoA mimetic probes are recognized and cleaved by the DHHC family proteins, providing a direct, sensitive, and simple readout of zDHHC2, 3, and 20 activities. After establishing its suitability for a high throughput screen, we screen a library of ~1,000 molecules against zDHHC20, identifying new acrylamide-based inhibitory scaffolds for zDHHC20. In sum, this work reveals the potential of acrylamide-based DHHC family inhibitors, introduces a novel platform for the identification of new inhibitors, and emphasizes that much remains unknown about the significance of dynamic S-acylation in regulating protein activity and cellular events

A system for the evolution of protein-protein interaction inducers

Molecules that induce interactions between proteins, often referred to as “molecular glues”, are increasingly recognized as important therapeutic modalities and as entry points for rewiring cellular signaling networks. Here, we report a new PACE-based method to rapidly select and evolve molecules that mediate interactions between otherwise non-interacting proteins: rapid evolution of Protein-Protein Interaction Glues (rePPI-G). Proof-of-concept evolutions demonstrated that rePPI-G reduces the “hook” effect of the engineered molecular glues, due at least in part to tuning the interaction affinities of each individual component of the bifunctional molecule. Altogether, this work validates rePPI-G as a continuous, phage-based evolutionary technology for optimizing molecular glues, providing a strategy for developing molecules that reprogram protein-protein interactions

Regulation of ERK2 activity by dynamic S-acylation

Extracellular signal-regulated kinases (ERK1/2) are key effector proteins of the mitogen-activated protein kinase pathway, choreographing essential processes of cellular physiology. Here, we discover that ERK1/2 are subject to S-acylation, a reversible lipid modification of cysteine residues, at C271/C254. The levels of ERK1/2 S-acylation are modulated by epidermal growth factor (EGF) signaling, mirroring its phosphorylation dynamics, and acylation-deficient ERK2 displays altered phosphorylation patterns. We show that ERK1/2 S-acylation is mediated by “writer” protein acyl transferases (PATs) and “eraser” acyl protein thioesterases (APTs) and that chemical inhibition of either lipid addition or removal alters ERK1/2’s EGF-triggered transcriptional program. Finally, in a mouse model of metabolic syndrome, we find that ERK1/2 lipidation levels correlate with alterations in ERK1/2 lipidation writer/eraser expression, solidifying a link between ERK1/2 activity, ERK1/2 lipidation, and organismal health. This study describes how lipidation regulates ERK1/2 and offers insight into the role of dynamic S-acylation in cell signaling more broadly

A High-Throughput Fluorescent Turn-On Assay for Inhibitors of DHHC Family Proteins

As the “writer” enzymes of protein S-acylation, a dynamic and functionally significant post-translational modification (PTM), DHHC family proteins have emerged in the past decade as both key modulators of cellular homeostasis and as drivers of neoplastic, autoimmune, metabolic, and neurological pathologies. Currently, biological and clinical discovery is hampered by the limitations of existing DHHC family inhibitors, which possess poor physicochemical properties and off-target profiles. However, progress in identifying new inhibitory scaffolds has been meager, in part due to a lack of robust in vitro assays suitable for high-throughput screening (HTS). Here, we report the development of palmitoyl transferase probes (PTPs), a novel family of turn-on pro-fluorescent molecules that mimic the palmitoyl-CoA substrate of DHHC proteins. We use the PTPs to develop and validate an assay with an excellent Z′-factor for HTS. We then perform a pilot screen of 1687 acrylamide-based molecules against zDHHC20, establishing the PTP-based HTS assay as a platform for the discovery of improved DHHC family inhibitors

The glymphatic system and cerebral small vessel disease

Objectives: Cerebral small vessel disease is a group of pathologies in which alterations of the brain's blood vessels contribute to stroke and neurocognitive changes. Recently, a neurotoxic waste clearance system composed of perivascular spaces abutting the brain's blood vessels, termed the glymphatic system, has been identified as a key player in brain homeostasis. Given that small vessel disease and the glymphatic system share anatomical structures, this review aims to reexamine small vessel disease in the context of the glymphatic system and highlight novel aspects of small vessel disease physiology. Materials and methods: This review was conducted with an emphasis on studies that examined aspects of small vessel disease and on works characterizing the glymphatic system. We searched PubMed for relevant articles using the following keywords: glymphatics, cerebral small vessel disease, arterial pulsatility, hypertension, blood-brain barrier, endothelial dysfunction, stroke, diabetes. Results: Cerebral small vessel disease and glymphatic dysfunction are anatomically connected and significant risk factors are shared between the two. These include hypertension, type 2 diabetes, advanced age, poor sleep, obesity, and neuroinflammation. There is clear evidence that CSVD hinders the effective functioning of glymphatic system. Conclusion: These shared risk factors, as well as the model of cerebral amyloid angiopathy pathogenesis, hint at the possibility that glymphatic dysfunction could independently contribute to the pathogenesis of cerebral small vessel disease. However, the current evidence supports a model of cascading dysfunction, wherein concurrent small vessel and glymphatic injury hinder glymphatic-mediated recovery and promote the progression of subclinical to clinical disease.</p

Charting the Chemical Space of Acrylamide-Based Inhibitors of zDHHC20

Protein S-acylation is a dynamic and reversible lipid post-translational modification that can affect the activity, stability, localization, and interactions of target proteins. Lipid modification occurs on cysteine residues via a thioester bond and in humans is mediated by 23 Asp-His-His-Cys domain-containing protein acyltransferases (DHHC-PATs). The DHHC-PATs have well-known roles in physiology and disease, but much remains to be discovered about their biological function and therapeutic potential. We recently developed cyanomyracrylamide (CMA), an acrylamide-based DHHC inhibitor with key improvements over existing inhibitors. Here we conduct a structure–activity relationship (SAR) study of CMA and its acrylamide derivatives against zDHHC20, the most structurally characterized member of the human DHHC family, and validate the results against the homologous zDHHC2. This SAR maps out the limitations and potential of the acrylamide scaffold, underscoring the need for a bivalent inhibitor and identifying along the way three molecules with activity on par with CMA but with an improved logP

Development of an Acrylamide-Based Inhibitor of Protein S-Acylation

Protein S-acylation is a dynamic lipid post-translational modification that can modulate the localization and activity of target proteins. In humans, the installation of the lipid onto target proteins is catalyzed by a family of 23 Asp-His-His-Cys domain-containing protein acyltransferases (DHHC-PATs). DHHCs are increasingly recognized as critical players in cellular signaling events and in human disease. However, progress elucidating the functions and mechanisms of DHHC “writers” has been hampered by a lack of chemical tools to perturb their activity in live cells. Herein, we report the synthesis and characterization of cyano-myracrylamide (CMA), a broad-spectrum DHHC family inhibitor with similar potency to 2-bromopalmitate (2BP), the most commonly used DHHC inhibitor in the field. Possessing an acrylamide warhead instead of 2BP’s α-halo fatty acid, CMA inhibits DHHC family proteins in cellulo while demonstrating decreased toxicity and avoiding inhibition of the S-acylation eraser enzymes – two of the major weaknesses of 2BP. Our studies show that CMA engages with DHHC family proteins in cells, inhibits protein S-acylation, and disrupts DHHC-regulated cellular events. CMA represents an improved chemical scaffold for untangling the complexities of DHHC-mediated cell signaling by protein S-acylation.</p

Structure of papain-like protease from SARS-CoV-2 and its complexes with non-covalent inhibitors

The SARS-CoV-2 papain-like protease (PLpro) is of interest as an antiviral drug target. Here, the authors synthesize and characterise naphthalene-based inhibitors for PLpro and present the crystal structures of PLpro in its apo state and with the bound inhibitors, which is of interest for further structure-based drug design efforts