Discovery of Potent Pyrazoline-Based Covalent SARS-CoV-2 Main Protease 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.

Photo-Brook rearrangement of acyl silanes as a strategy for photoaffinity probe design.

Photoaffinity labeling (PAL) is a powerful tool for the identification of non-covalent small molecule-protein interactions that are critical to drug discovery and medicinal chemistry, but this approach is limited to only a small subset of robust photocrosslinkers. The identification of new photoreactive motifs capable of covalent target capture is therefore highly desirable. Herein, we report the design, synthesis, and evaluation of a new class of PAL warheads based on the UV-triggered 1,2-photo-Brook rearrangement of acyl silanes, which hitherto have not been explored for PAL workflows. Irradiation of a series of probes in cell lysate revealed an iPr-substituted acyl silane with superior photolabeling and minimal thermal background labeling compared to other substituted acyl silanes. Further, small molecule (+)-JQ1- and rapamycin-derived iPr acyl silanes were shown to selectively label recombinant BRD4-BD1 and FKBP12, respectively, with minimal background. Together, these data highlight the untapped potential of acyl silanes as a novel, tunable scaffold for photoaffinity labeling.

Deubiquitinase-targeting chimeras for targeted protein stabilization.

Many diseases are driven by proteins that are aberrantly ubiquitinated and degraded. These diseases would be therapeutically benefited by targeted protein stabilization (TPS). Here we present deubiquitinase-targeting chimeras (DUBTACs), heterobifunctional small molecules consisting of a deubiquitinase recruiter linked to a protein-targeting ligand, to stabilize the levels of specific proteins degraded in a ubiquitin-dependent manner. 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 showed that a DUBTAC consisting of our EN523 OTUB1 recruiter linked to lumacaftor, a drug used to treat cystic fibrosis that binds ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR), robustly stabilized ΔF508-CFTR protein levels, leading to improved chloride channel conductance in human cystic fibrosis bronchial epithelial cells. We also demonstrated stabilization of the tumor suppressor kinase WEE1 in hepatoma cells. Our study showcases covalent chemoproteomic approaches to develop new induced proximity-based therapeutic modalities and introduces the DUBTAC platform for TPS.

Discovery of a Covalent FEM1B Recruiter for Targeted Protein Degradation Applications.

Proteolysis-targeting chimeras (PROTACs), heterobifunctional compounds that consist of protein-targeting ligands linked to an E3 ligase recruiter, have arisen as a powerful therapeutic modality for targeted protein degradation (TPD). Despite the popularity of TPD approaches in drug discovery, only a small number of E3 ligase recruiters are available for the >600 E3 ligases that exist in human cells. Here, we have discovered a cysteine-reactive covalent ligand, EN106, that targets FEM1B, an E3 ligase recently discovered as the critical component of the cellular response to reductive stress. By targeting C186 in FEM1B, EN106 disrupts recognition of the key reductive stress substrate of FEM1B, FNIP1. We further establish that EN106 can be used as a covalent recruiter for FEM1B in TPD applications by demonstrating that a PROTAC linking EN106 to the BET bromodomain inhibitor JQ1 or the kinase inhibitor dasatinib leads to the degradation of BRD4 and BCR-ABL, respectively. Our study showcases a covalent ligand that targets a natural E3 ligase-substrate binding site and highlights the utility of covalent ligand screening in expanding the arsenal of E3 ligase recruiters suitable for TPD applications.

Screening a Library of FDA-Approved and Bioactive Compounds for Antiviral Activity against SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has emerged as a major global health threat. The COVID-19 pandemic has resulted in over 168 million cases and 3.4 million deaths to date, while the number of cases continues to rise. With limited therapeutic options, the identification of safe and effective therapeutics is urgently needed. The repurposing of known clinical compounds holds the potential for rapid identification of drugs effective against SARS-CoV-2. Here, we utilized a library of FDA-approved and well-studied preclinical and clinical compounds to screen for antivirals against SARS-CoV-2 in human pulmonary epithelial cells. We identified 13 compounds that exhibit potent antiviral activity across multiple orthogonal assays. Hits include known antivirals, compounds with anti-inflammatory activity, and compounds targeting host pathways such as kinases and proteases critical for SARS-CoV-2 replication. We identified seven compounds not previously reported to have activity against SARS-CoV-2, including B02, a human RAD51 inhibitor. We further demonstrated that B02 exhibits synergy with remdesivir, the only antiviral approved by the FDA to treat COVID-19, highlighting the potential for combination therapy. Taken together, our comparative compound screening strategy highlights the potential of drug repurposing screens to identify novel starting points for development of effective antiviral mono- or combination therapies to treat COVID-19.

Reimagining Druggability Using Chemoproteomic Platforms.

One of the biggest bottlenecks in modern drug discovery efforts is in tackling the undruggable proteome. Currently, over 85% of the proteome is still considered undruggable because most proteins lack well-defined binding pockets that can be functionally targeted with small molecules. Tackling the undruggable proteome necessitates innovative approaches for ligand discovery against undruggable proteins as well as the development of new therapeutic modalities to functionally manipulate proteins of interest. Chemoproteomic platforms, in particular activity-based protein profiling (ABPP), have arisen to tackle the undruggable proteome by using reactivity-based chemical probes and advanced quantitative mass spectrometry-based proteomic approaches to enable the discovery of "ligandable hotspots" or proteome-wide sites that can be targeted with small-molecule ligands. These sites can subsequently be pharmacologically targeted with covalent ligands to rapidly discover functional or nonfunctional binders against therapeutic proteins of interest. Chemoproteomic approaches have also revealed unique insights into ligandability such as the discovery of unique allosteric sites or intrinsically disordered regions of proteins that can be pharmacologically and selectively targeted for biological modulation and therapeutic benefit. Chemoproteomic platforms have also expanded the scope of emerging therapeutic modalities for targeted protein degradation and proteolysis-targeting chimeras (PROTACs) through the discovery of several new covalent E3 ligase recruiters. Looking into the future, chemoproteomic approaches will unquestionably have a major impact in further expansion of existing efforts toward proteome-wide ligandability mapping, targeted ligand discovery efforts against high-value undruggable therapeutic targets, further expansion of the scope of targeted protein degradation platforms, the discovery of new molecular glue scaffolds that enable unique modulation of protein function, and perhaps most excitingly the development of next-generation small-molecule induced-proximity-based therapeutic modalities that go beyond degradation. Exciting days lie ahead in this field as chemical biology becomes an increasingly major driver in drug discovery, and chemoproteomic approaches are sure to be a mainstay in developing next-generation therapeutics.

Chemoproteomics-enabled discovery of covalent RNF114-based degraders that mimic natural product function.

The translation of functionally active natural products into fully synthetic small-molecule mimetics has remained an important process in medicinal chemistry. We recently discovered that the terpene natural product nimbolide can be utilized as a covalent recruiter of the E3 ubiquitin ligase RNF114 for use in targeted protein degradation-a powerful therapeutic modality within modern-day drug discovery. Using activity-based protein profiling-enabled covalent ligand-screening approaches, here we report the discovery of fully synthetic RNF114-based recruiter molecules that can also be exploited for PROTAC applications, and demonstrate their utility in degrading therapeutically relevant targets, such as BRD4 and BCR-ABL, in cells. The identification of simple and easily manipulated drug-like scaffolds that can mimic the function of a complex natural product is beneficial in further expanding the toolbox of E3 ligase recruiters, an area of great importance in drug discovery and chemical biology.

De novo Design of SARS-CoV-2 Main Protease Inhibitors.

The COVID-19 pandemic prompted many scientists to investigate remedies against SARS-CoV-2 and related viruses that are likely to appear in the future. As the main protease of the virus, MPro, is highly conserved among coronaviruses, it has emerged as a prime target for developing inhibitors. Using a combination of virtual screening and molecular modeling, we identified small molecules that were easily accessible and could be quickly diversified. Biochemical assays confirmed a class of pyridones as low micromolar non-covalent inhibitors of the viral main protease.

Bardoxolone conjugation enables targeted protein degradation of BRD4.

Targeted protein degradation (TPD) has emerged as a powerful tool in drug discovery for the perturbation of protein levels using heterobifunctional small molecules. E3 ligase recruiters remain central to this process yet relatively few have been identified relative to the ~ 600 predicted human E3 ligases. While, initial recruiters have utilized non-covalent chemistry for protein binding, very recently covalent engagement to novel E3's has proven fruitful in TPD application. Herein we demonstrate efficient proteasome-mediated degradation of BRD4 by a bifunctional small molecule linking the KEAP1-Nrf2 activator bardoxolone to a BRD4 inhibitor JQ1.

Unbiased Proteomic Profiling Uncovers a Targetable GNAS/PKA/PP2A Axis in Small Cell Lung Cancer Stem Cells.

Using unbiased kinase profiling, we identified protein kinase A (PKA) as an active kinase in small cell lung cancer (SCLC). Inhibition of PKA activity genetically, or pharmacologically by activation of the PP2A phosphatase, suppresses SCLC expansion in culture and in vivo. Conversely, GNAS (G-protein α subunit), a PKA activator that is genetically activated in a small subset of human SCLC, promotes SCLC development. Phosphoproteomic analyses identified many PKA substrates and mechanisms of action. In particular, PKA activity is required for the propagation of SCLC stem cells in transplantation studies. Broad proteomic analysis of recalcitrant cancers has the potential to uncover targetable signaling networks, such as the GNAS/PKA/PP2A axis in SCLC.

A Nimbolide-Based Kinase Degrader Preferentially Degrades Oncogenic BCR-ABL.

Targeted protein degradation (TPD) and proteolysis-targeting chimeras (PROTACs) have arisen as powerful therapeutic modalities for degrading specific proteins in a proteasome-dependent manner. However, a major limitation of TPD is the lack of E3 ligase recruiters. Recently, we discovered the natural product nimbolide as a covalent recruiter for the E3 ligase RNF114. Here, we show the broader utility of nimbolide as an E3 ligase recruiter for TPD applications. We demonstrate that a PROTAC linking nimbolide to the kinase and BCR-ABL fusion oncogene inhibitor dasatinib, BT1, selectively degrades BCR-ABL over c-ABL in leukemia cancer cells, compared to previously reported cereblon or VHL-recruiting BCR-ABL degraders that show opposite selectivity or, in some cases, inactivity. Thus, we further establish nimbolide as an additional general E3 ligase recruiter for PROTACs, and we demonstrate the importance of expanding upon the arsenal of E3 ligase recruiters, as such molecules confer differing selectivity for the degradation of neo-substrate proteins.

Harnessing the anti-cancer natural product nimbolide for targeted protein degradation.

Nimbolide, a terpenoid natural product derived from the Neem tree, impairs cancer pathogenicity; however, the direct targets and mechanisms by which nimbolide exerts its effects are poorly understood. Here, we used activity-based protein profiling (ABPP) chemoproteomic platforms to discover that nimbolide reacts with a novel functional cysteine crucial for substrate recognition in the E3 ubiquitin ligase RNF114. Nimbolide impairs breast cancer cell proliferation in-part by disrupting RNF114-substrate recognition, leading to inhibition of ubiquitination and degradation of tumor suppressors such as p21, resulting in their rapid stabilization. We further demonstrate that nimbolide can be harnessed to recruit RNF114 as an E3 ligase in targeted protein degradation applications and show that synthetically simpler scaffolds are also capable of accessing this unique reactive site. Our study highlights the use of ABPP platforms in uncovering unique druggable modalities accessed by natural products for cancer therapy and targeted protein degradation applications.

Covalent Ligand Discovery against Druggable Hotspots Targeted by Anti-cancer Natural Products.

Many natural products that show therapeutic activities are often difficult to synthesize or isolate and have unknown targets, hindering their development as drugs. Identifying druggable hotspots targeted by covalently acting anti-cancer natural products can enable pharmacological interrogation of these sites with more synthetically tractable compounds. Here, we used chemoproteomic platforms to discover that the anti-cancer natural product withaferin A targets C377 on the regulatory subunit PPP2R1A of the tumor-suppressor protein phosphatase 2A (PP2A) complex leading to activation of PP2A activity, inactivation of AKT, and impaired breast cancer cell proliferation. We developed a more synthetically tractable cysteine-reactive covalent ligand, JNS 1-40, that selectively targets C377 of PPP2R1A to impair breast cancer signaling, proliferation, and in vivo tumor growth. Our study highlights the utility of using chemoproteomics to map druggable hotspots targeted by complex natural products and subsequently interrogating these sites with more synthetically tractable covalent ligands for cancer therapy.