Covalent drugs have been used to treat diseases for more than a century, but tools that facilitate the rational design of covalent drugs have emerged more recently. The purposeful addition of reactive functional groups to existing ligands can enable potent and selective inhibition of target proteins, as demonstrated by the covalent epidermal growth factor receptor (EGFR) and Bruton’s tyrosine kinase (BTK) inhibitors used to treat various cancers. Moreover, the identification of covalent ligands through ‘electrophile-first’ approaches has also led to the discovery of covalent drugs, such as covalent inhibitors for KRAS(G12C) and SARS-CoV-2 main protease. In particular, the discovery of KRAS(G12C) inhibitors validates the use of covalent screening technologies, which have become more powerful and widespread over the past decade. Chemoproteomics platforms have emerged to complement covalent ligand screening and assist in ligand discovery, selectivity profiling and target identification. This Review showcases covalent drug discovery milestones with emphasis on the lessons learned from these programmes and how an evolving toolbox of covalent drug discovery techniques facilitates success in this field.
ngaging the mostly undruggable proteome to uncover new disease therapies not only requires technological innovations that facilitate rapid discovery of ligandable hotspots across the proteome but also demands new therapeutic modalities that alter protein function through novel mechanisms 1,2 . Targeted protein degradation (TPD) tackles the undruggable proteome by targeting specific proteins for ubiquitination and proteasomal degradation. One major class of small-molecule effectors of TPD, proteolysis-targeting chimeras (PROTACs), are heterobifunctional molecules that consist of an E3 ligase recruiter linked to a protein-targeting ligand to induce the formation of ternary complexes that bring together an E3 ubiquitin ligase and the target protein as a neo-substrate [3][4][5] . PROTACs have enabled the targeted and specific degradation of numerous disease-causing proteins in cells 3,6 . New approaches for TPD have also arisen that exploit endosomal and lysosomal degradation pathways with lysosome-targeting chimeras or autophagy with autophagy-targeting chimeras 7,8 . New approaches for chemically induced proximity beyond degradation have also been developed in recent years, including targeted phosphorylation with phosphorylation-inducing chimeric small molecules and targeted dephosphorylation, but no small-molecule-based induced proximity approaches exist for targeted deubiquitination and subsequent stabilization of proteins 9,10 .Active ubiquitination and degradation of proteins is the root cause of several classes of diseases, including many tumor suppressors in cancer (for example, TP53, CDKN1A, CDN1C and BAX), and mutated and misfolded proteins, such as ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR) in cystic fibrosis or glucokinase in pancreatic cells in maturity-onset diabetes of the young type 2. In these cases, a TPS therapeutic strategy, rather than degradation, would be beneficial [11][12][13][14] . Analogous to TPD, we hypothesized that TPS could be enabled by the discovery of a small-molecule recruiter of a deubiquitinase (DUB) that could be linked to a protein-targeting ligand to form a chimeric molecule, which would induce the deubiquitination and stabilization of proteins of interest. We call this heterobifunctional stabilizer a DUBTAC (Fig. 1a). In this study, we report the discovery of a covalent recruiter for the K48-ubiquitin chain-specific DUB OTUB1, which when linked to a protein-targeting ligand stabilizes an actively degraded target protein to demonstrate proof of concept for the DUBTAC platform. ResultsIdentifying allosteric ligandable sites within DUBs. To enable the DUBTAC platform, our first goal was to identify a small-molecule recruiter that targeted an allosteric site on a DUB without inhibiting DUB function, as the recruitment of a functional DUB would be required to deubiquitinate and stabilize the target protein. While many DUBs possess well-defined active sites bearing a catalytic and highly nucleophilic cysteine, there have not yet been systematic evaluations of ...
Targeted protein degradation is a powerful therapeutic modality that uses heterobifunctional small-molecules to induce proximity between E3 ubiquitin ligases and target proteins to ubiquitinate and degrade specific proteins of interest. However, many proteins are ubiquitinated and degraded to drive disease pathology; in these cases targeted protein stabilization (TPS), rather than degradation, of the actively degraded target using a small-molecule would be therapeutically beneficial. Here, we present the Deubiquitinase-Targeting Chimera (DUBTAC) platform for TPS of specific proteins. Using chemoproteomic approaches, we discovered the covalent ligand EN523 that targets a non-catalytic allosteric cysteine C23 in the K48 ubiquitin-specific deubiquitinase OTUB1. We then developed a heterobifunctional DUBTAC consisting of our EN523 OTUB1 recruiter linked to lumacaftor, a drug used to treat cystic fibrosis that binds ΔF508-CFTR. We demonstrated proof-of-concept of TPS by showing that this DUBTAC robustly stabilized ΔF508-CFTR in human cystic fibrosis bronchial epithelial cells in an OTUB1-dependent manner. Our study underscores the utility of chemoproteomics-enabled covalent ligand discovery approaches to develop new induced proximity-based therapeutic modalities and introduces the DUBTAC platform for TPS.Editorial summaryWe have developed the Deubiquitinase Targeting Chimera (DUBTAC) platform for targeted protein stabilization. We have discovered a covalent recruiter against the deubiquitinase OTUB1 that we have linked to the mutant ΔF508-CFTR targeting cystic fibrosis drug Lumacaftor to stabilize mutant CFTR protein in cells.
A convenient enzymatic strategy is reported for the modification of cell surfaces. Using a tyrosinase enzyme isolated from Agaricus bisporus, unique tyrosine residues introduced at the C-termini of nanobodies can be site-selectively oxidized to reactive o-quinones. These reactive intermediates undergo rapid modification with nucleophilic thiol, amine, and imidazole residues present on cell surfaces, producing novel nanobody−cell conjugates that display targeted antigen binding. We extend this approach toward the synthesis of nanobody−NK cell conjugates for targeted immunotherapy applications. The resulting NK cell conjugates exhibit targeted cell binding and elicit targeted cell death.
While vaccines and antivirals are now being deployed for the current SARS-CoV-2 pandemic, we require additional antiviral therapeutics to not only effectively combat SARS-CoV-2 and its variants, but also future coronaviruses. All coronaviruses have relatively similar genomes that provide a potential exploitable opening to develop antiviral therapies that will be effective against all coronaviruses. Among the various genes and proteins encoded by all coronaviruses, one particularly druggable or relatively easy-to-drug target is the coronavirus Main Protease (3CLpro or Mpro), an enzyme that is involved in cleaving a long peptide translated by the viral genome into its individual protein components that are then assembled into the virus to enable viral replication in the cell. Inhibiting Mpro with a small-molecule antiviral would effectively stop the ability of the virus to replicate, providing therapeutic benefit. In this study, we have utilized activity-based protein profiling (ABPP)-based chemoproteomic approaches to discover and further optimize cysteine-reactive pyrazoline-based covalent inhibitors for the SARS-CoV-2 Mpro. Structure-guided medicinal chemistry and modular synthesis of di- and tri-substituted pyrazolines bearing either chloroacetamide or vinyl sulfonamide cysteine-reactive warheads enabled the expedient exploration of structure-activity relationships (SAR), yielding nanomolar potency inhibitors against Mpro from not only SARS-CoV-2, but across many other coronaviruses. Our studies highlight promising chemical scaffolds that may contribute to future pan-coronavirus inhibitors.
While vaccines and antivirals are now being deployed for the current SARS‐CoV‐2 pandemic, we require additional antiviral therapeutics to not only effectively combat SARS‐CoV‐2 and its variants, but also future coronaviruses. All coronaviruses have relatively similar genomes that provide a potential exploitable opening to develop antiviral therapies that will be effective against all coronaviruses. Among the various genes and proteins encoded by all coronaviruses, one particularly “druggable” or relatively easy‐to‐drug target is the coronavirus Main Protease (3CLpro or Mpro), an enzyme that is involved in cleaving a long peptide translated by the viral genome into its individual protein components that are then assembled into the virus to enable viral replication in the cell. Inhibiting Mpro with a small‐molecule antiviral would effectively stop the ability of the virus to replicate, providing therapeutic benefit. In this study, we have utilized activity‐based protein profiling (ABPP)‐based chemoproteomic approaches to discover and further optimize cysteine‐reactive pyrazoline‐based covalent inhibitors for the SARS‐CoV‐2 Mpro. Structure‐guided medicinal chemistry and modular synthesis of di‐ and tri‐substituted pyrazolines bearing either chloroacetamide or vinyl sulfonamide cysteine‐reactive warheads enabled the expedient exploration of structure‐activity relationships (SAR), yielding nanomolar potency inhibitors against Mpro from not only SARS‐CoV‐2, but across many other coronaviruses. Our studies highlight promising chemical scaffolds that may contribute to future pan‐coronavirus inhibitors.
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