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.

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.

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.

Cannabidiol inhibits SARS-CoV-2 replication through induction of the host ER stress and innate immune responses.

The spread of SARS-CoV-2 and ongoing COVID-19 pandemic underscores the need for new treatments. Here we report that cannabidiol (CBD) inhibits infection of SARS-CoV-2 in cells and mice. CBD and its metabolite 7-OH-CBD, but not THC or other congeneric cannabinoids tested, potently block SARS-CoV-2 replication in lung epithelial cells. CBD acts after viral entry, inhibiting viral gene expression and reversing many effects of SARS-CoV-2 on host gene transcription. CBD inhibits SARS-CoV-2 replication in part by up-regulating the host IRE1α RNase endoplasmic reticulum (ER) stress response and interferon signaling pathways. In matched groups of human patients from the National COVID Cohort Collaborative, CBD (100 mg/ml oral solution per medical records) had a significant negative association with positive SARS-CoV-2 tests. This study highlights CBD as a potential preventative agent for early-stage SARS-CoV-2 infection and merits future clinical trials. We caution against use of non-medical formulations including edibles, inhalants or topicals as a preventative or treatment therapy at the present time.

Antiviral evaluation of hydroxyethylamine analogs: Inhibitors of SARS-CoV-2 main protease (3CLpro), a virtual screening and simulation approach.

The continued toll of COVID-19 has halted the smooth functioning of civilization on a global scale. With a limited understanding of all the essential components of viral machinery and the lack of structural information of this new virus, initial drug discovery efforts had limited success. The availability of high-resolution crystal structures of functionally essential SARS-CoV-2 proteins, including 3CLpro, supports the development of target-specific therapeutics. 3CLpro, the main protease responsible for the processing of viral polypeptide, plays a vital role in SARS-CoV-2 viral replication and translation and is an important target in other coronaviruses. Additionally, 3CLpro is the target of repurposed drugs, such as lopinavir and ritonavir. In this study, target proteins were retrieved from the protein data bank (PDB IDs: 6 M03, 6LU7, 2GZ7, 6 W63, 6SQS, 6YB7, and 6YVF) representing different open states of the main protease to accommodate macromolecular substrate. A hydroxyethylamine (HEA) library was constructed from harvested chemical structures from all the series being used in our laboratories for screening against malaria and Leishmania parasites. The database consisted of ∼1000 structure entries, of which 70% were new to ChemSpider at the time of screening. This in-house library was subjected to high throughput virtual screening (HTVS), followed by standard precision (SP) and then extra precision (XP) docking (Schrodinger LLC 2021). The ligand strain and complex energy of top hits were calculated by Molecular Mechanics Generalized Born Surface Area (MM/GBSA) method. Promising hit compounds (n = 40) specifically binding to 3CLpro with high energy and average MM/GBSA scores were then subjected to (100-ns) MD simulations. Using this sequential selection followed by an in-silico validation approach, we found a promising HEA-based compound (N,N'-((3S,3'S)-piperazine-1,4-diylbis(3-hydroxy-1-phenylbutane-4,2-diyl))bis(2-(5-methyl-1,3-dioxoisoindolin-2-yl)-3-phenylpropanamide)), which showed high in vitro antiviral activity against SARS-CoV-2. Further to reduce the size of the otherwise larger ligand, a pharmacophore-based predicted library of âˆ¼42 derivatives was constructed, which were added to the previous compound library and rescreened virtually. Out of several hits from the predicted library, two compounds were synthesized, tested against SARS-CoV-2 culture, and found to have markedly improved antiviral activity.

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.

Masitinib is a broad coronavirus 3CL inhibitor that blocks replication of SARS-CoV-2.

There is an urgent need for antiviral agents that treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We screened a library of 1900 clinically safe drugs against OC43, a human beta coronavirus that causes the common cold, and evaluated the top hits against SARS-CoV-2. Twenty drugs significantly inhibited replication of both viruses in cultured human cells. Eight of these drugs inhibited the activity of the SARS-CoV-2 main protease, 3CLpro, with the most potent being masitinib, an orally bioavailable tyrosine kinase inhibitor. X-ray crystallography and biochemistry show that masitinib acts as a competitive inhibitor of 3CLpro. Mice infected with SARS-CoV-2 and then treated with masitinib showed >200-fold reduction in viral titers in the lungs and nose, as well as reduced lung inflammation. Masitinib was also effective in vitro against all tested variants of concern (B.1.1.7, B.1.351, and P.1).

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

The pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to expand. Papain-like protease (PLpro) is one of two SARS-CoV-2 proteases potentially targetable with antivirals. PLpro is an attractive target because it plays an essential role in cleavage and maturation of viral polyproteins, assembly of the replicase-transcriptase complex, and disruption of host responses. We report a substantive body of structural, biochemical, and virus replication studies that identify several inhibitors of the SARS-CoV-2 enzyme. We determined the high resolution structure of wild-type PLpro, the active site C111S mutant, and their complexes with inhibitors. This collection of structures details inhibitors recognition and interactions providing fundamental molecular and mechanistic insight into PLpro. All compounds inhibit the peptidase activity of PLpro in vitro, some block SARS-CoV-2 replication in cell culture assays. These findings will accelerate structure-based drug design efforts targeting PLpro to identify high-affinity inhibitors of clinical value.

Drug repurposing screen identifies masitinib as a 3CLpro inhibitor that blocks replication of SARS-CoV-2 in vitro.

There is an urgent need for anti-viral agents that treat SARS-CoV-2 infection. The shortest path to clinical use is repurposing of drugs that have an established safety profile in humans. Here, we first screened a library of 1,900 clinically safe drugs for inhibiting replication of OC43, a human beta-coronavirus that causes the common-cold and is a relative of SARS-CoV-2, and identified 108 effective drugs. We further evaluated the top 26 hits and determined their ability to inhibit SARS-CoV-2, as well as other pathogenic RNA viruses. 20 of the 26 drugs significantly inhibited SARS-CoV-2 replication in human lung cells (A549 epithelial cell line), with EC50 values ranging from 0.1 to 8 micromolar. We investigated the mechanism of action for these and found that masitinib, a drug originally developed as a tyrosine-kinase inhibitor for cancer treatment, strongly inhibited the activity of the SARS-CoV-2 main protease 3CLpro. X-ray crystallography revealed that masitinib directly binds to the active site of 3CLpro, thereby blocking its enzymatic activity. Mastinib also inhibited the related viral protease of picornaviruses and blocked picornaviruses replication. Thus, our results show that masitinib has broad anti-viral activity against two distinct beta-coronaviruses and multiple picornaviruses that cause human disease and is a strong candidate for clinical trials to treat SARS-CoV-2 infection.

Neurological injuries in COVID-19 patients: direct viral invasion or a bystander injury after infection of epithelial/endothelial cells.

A subset of patients with coronavirus 2 disease (COVID-19) experience neurological complications. These complications include loss of sense of taste and smell, stroke, delirium, and neuromuscular signs and symptoms. The etiological agent of COVID-19 is SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), an RNA virus with a glycoprotein-studded viral envelope that uses ACE2 (angiotensin-converting enzyme 2) as a functional receptor for infecting the host cells. Thus, the interaction of the envelope spike proteins with ACE2 on host cells determines the tropism and virulence of SARS-CoV-2. Loss of sense of taste and smell is an initial symptom of COVID-19 because the virus enters the nasal and oral cavities first and the epithelial cells are the receptors for these senses. Stroke in COVID-19 patients is likely a consequence of coagulopathy and injury to cerebral vascular endothelial cells that cause thrombo-embolism and stroke. Delirium and encephalopathy in acute and post COVID-19 patients are likely multifactorial and secondary to hypoxia, metabolic abnormalities, and immunological abnormalities. Thus far, there is no clear evidence that coronaviruses cause inflammatory neuromuscular diseases via direct invasion of peripheral nerves or muscles or via molecular mimicry. It appears that most of neurologic complications in COVID-19 patients are indirect and as a result of a bystander injury to neurons.

Activity-Based Sensing of S-Depalmitoylases: Chemical Technologies and Biological Discovery.

While lipids were first appreciated as a critical hydrophobic barrier, our understanding of their roles at the cellular and organismal levels continues to grow. Not only are they important independent operators, providing a platform for both static and dynamic organization and communication within the cell, they also exert significant effects via the chemical modification of proteins. Addition of a lipid post-translational modification (PTM) alters protein hydrophobicity and behavior, with distinct consequences for subcellular trafficking, localization, intra- and intermolecular interactions, and stability. One of the most abundant and widespread protein lipidation events is S-acylation, installation of a long-chain lipid to the thiol of a cysteine side chain through a thioester linkage. S-Acylation is often referred to as S-palmitoylation, due to the prevalence of palmitate as the lipid modification. Unlike many lipid PTMs, S-acylation is enzymatically reversible, enabling the cell to tune proteome-wide properties through dynamic alterations in protein lipidation status. While much has been uncovered about the molecular effects of S-acylation and its implications for physiology, current biochemical and chemical methods only assess substrate lipidation levels or steady-state levels of enzyme activity. Yet, the writer protein acyl transferases (PATs) and eraser acyl protein thioesterases (APTs) are dynamically active, responsible for sometimes-rapid changes in S-palmitoylation status of target proteins. Thus, to understand the full scope, significance, and subtlety of S-deacylation and its regulation in the cell, it is necessary to observe the timing and cellular geography of regulatory enzyme activities. In this Account, we review the chemical tools developed by our group to selectively visualize and perturb the activity of APTs in live cells, highlighting the biological insights gained from their application. To visualize APT activity, we masked fluorogenic molecules with thioacylated, peptide-based APT substrate mimetics; APT activity and thus thiol deprotection releases a fluorescent product in the turn-on depalmitoylation probes (DPPs), while in ratiometric depalmitoylation probes (RDPs) the emission of the parent fluorophore is altered. Application of these probes in live cells reveals that APT activity is sensitive to cell signaling events and metabolic disturbances. Additionally, as indicated above, the location of regulatory enzymes is critical in lipid signaling, and one organelle of particular interest, due to its role in maintaining cellular homeostasis and its legion of lipidated proteins, is the mitochondria. Therefore, we developed a class of spatially constrained mitoDPPs to visualize mitochondrial APT activity as well as a selective inhibitor of mitochondrial deacylation activity, mitoFP. With these tools, we identify two mitochondrial S-depalmitoylases and connect mitochondrial S-depalmitoylation to redox buffering capacity. Moreover, some of the changes in activity observed are specific to the mitochondria, confirming spatial as well as temporal regulation of eraser protein activity. Overall, this chemical toolkit for S-depalmitoylase activity, imaging reagents and a targeted inhibitor, will continue to illuminate the regulatory mechanisms and roles of S-depalmitoylation within the complex homeostatic networks of the cell.

Construction of homogeneous antibody-drug conjugates using site-selective protein chemistry.

Systemic chemotherapy, the current standard of care for the treatment of cancer, is rarely curative and is often accompanied by debilitating side effects. Targeted drug delivery stands as an alternative to chemotherapy, with the potential to improve upon its low efficacy and systemic toxicity. Among targeted therapeutic options, antibody-drug conjugates (ADCs) have emerged as the most promising. These conjugates represent a new class of biopharmaceuticals that selectively deliver potent cytotoxic drugs to cancer cells, sparing healthy tissue throughout the body. Despite this promise, early heterogenous ADCs suffered from stability, pharmacokinetic, and efficacy issues that hindered clinical development. Recent advances in antibody engineering, linkers for drug-release, and chemical site-selective antibody conjugation have led to the creation of homogenous ADCs that have proven to be more efficacious than their heterogeneous predecessors both in vitro and in vivo. In this minireview, we focus on and discuss recent advances in chemical site-selective modification strategies for the conjugation of drugs to antibodies and the resulting potential for the development of a new generation of homogenous ADCs.