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.

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.

Although steady progress has been achieved toward upcycling waste CO2 through diverse catalytic strategies, each approach has distinct limitations, hampering the generation of complex products like sugars. Here, we provide a roadmap that evaluates the feasibility associated with state-of-the-art electrochemical processes eligible for converting CO2 into glycolaldehydes and formaldehydes, both essential components for sugar generation through the formose reaction. We establish that even in low concentrations, glycolaldehyde plays a crucial role as an autocatalytic initiator during sugar formation and identify formaldehyde production as a bottleneck. Our study demonstrates the chemical resilience of the formose reaction successfully carried out in the chemically complex CO2 electrolysis product stream. This work reveals that CO2-initiated sugars constitute an adequate feedstock for fast-growing and genetically modifiable Escherichia coli. Altogether, we introduce a roadmap, supported by experimental evidence, that pushes the boundaries of product complexity achievable from CO2 electroconversion while integrating CO2 into life-sustaining sugars.

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.

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.

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.

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.

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.

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.