Ophiobolin A Covalently Targets Mitochondrial Complex IV Leading to Metabolic Collapse in Cancer Cells.

Ophiobolin A (OPA) is a sesterterpenoid fungal natural product with broad anticancer activity. While OPA possesses multiple electrophilic moieties that can covalently react with nucleophilic amino acids on proteins, the proteome-wide targets and mechanism of OPA remain poorly understood in many contexts. In this study, we used covalent chemoproteomic platforms to map the proteome-wide reactivity of the OPA in a highly sensitive lung cancer cell line. Among several proteins that OPA engaged, we focused on two targets: lysine-72 of cytochrome c oxidase subunit 5A (COX5A) and cysteine-53 of mitochondrial hypoxia induced gene 1 domain family member 2A (HIGD2A). These two subunit proteins are part of complex IV (cytochrome C oxidase) within the electron transport chain and contributed significantly to the antiproliferative activity of OPA. OPA activated mitochondrial respiration in a COX5A- and HIGD2A-dependent manner, leading to an initial spike in mitochondrial ATP and heightened mitochondrial oxidative stress. OPA compromised mitochondrial membrane potential, ultimately leading to ATP depletion. We have used chemoproteomic strategies to discover a unique anticancer mechanism of OPA through activation of complex IV leading to compromised mitochondrial energetics and rapid cell death.

Oxidative cyclization reagents reveal tryptophan cation-π interactions.

Methods for selective covalent modification of amino acids on proteins can enable a diverse array of applications, spanning probes and modulators of protein function to proteomics1-3. Owing to their high nucleophilicity, cysteine and lysine residues are the most common points of attachment for protein bioconjugation chemistry through acid-base reactivity3,4. Here we report a redox-based strategy for bioconjugation of tryptophan, the rarest amino acid, using oxaziridine reagents that mimic oxidative cyclization reactions in indole-based alkaloid biosynthetic pathways to achieve highly efficient and specific tryptophan labelling. We establish the broad use of this method, termed tryptophan chemical ligation by cyclization (Trp-CLiC), for selectively appending payloads to tryptophan residues on peptides and proteins with reaction rates that rival traditional click reactions and enabling global profiling of hyper-reactive tryptophan sites across whole proteomes. Notably, these reagents reveal a systematic map of tryptophan residues that participate in cation-π interactions, including functional sites that can regulate protein-mediated phase-separation processes.

Correction to "Rational Chemical Design of Molecular Glue Degraders".

[This corrects the article DOI: 10.1021/acscentsci.2c01317.].

Rational Chemical Design of Molecular Glue Degraders.

Targeted protein degradation with molecular glue degraders has arisen as a powerful therapeutic modality for eliminating classically undruggable disease-causing proteins through proteasome-mediated degradation. However, we currently lack rational chemical design principles for converting protein-targeting ligands into molecular glue degraders. To overcome this challenge, we sought to identify a transposable chemical handle that would convert protein-targeting ligands into molecular degraders of their corresponding targets. Using the CDK4/6 inhibitor ribociclib as a prototype, we identified a covalent handle that, when appended to the exit vector of ribociclib, induced the proteasome-mediated degradation of CDK4 in cancer cells. Further modification of our initial covalent scaffold led to an improved CDK4 degrader with the development of a but-2-ene-1,4-dione ("fumarate") handle that showed improved interactions with RNF126. Subsequent chemoproteomic profiling revealed interactions of the CDK4 degrader and the optimized fumarate handle with RNF126 as well as additional RING-family E3 ligases. We then transplanted this covalent handle onto a diverse set of protein-targeting ligands to induce the degradation of BRD4, BCR-ABL and c-ABL, PDE5, AR and AR-V7, BTK, LRRK2, HDAC1/3, and SMARCA2/4. Our study undercovers a design strategy for converting protein-targeting ligands into covalent molecular glue degraders.

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.

Ophiobolin A Covalently Targets Complex IV Leading to Mitochondrial Metabolic Collapse in Cancer Cells.

Ophiobolin A (OPA) is a sesterterpenoid fungal natural product with broad anti-cancer activity. While OPA possesses multiple electrophilic moieties that can covalently react with nucleophilic amino acids on proteins, the proteome-wide targets and mechanism of OPA remain poorly understood in many contexts. In this study, we used covalent chemoproteomic platforms to map the proteome-wide reactivity of OPA in a highly sensitive lung cancer cell line. Among several proteins that OPA engaged, we focused on two targets-cysteine C53 of HIG2DA and lysine K72 of COX5A-that are part of complex IV of the electron transport chain and contributed significantly to the anti-proliferative activity. OPA activated mitochondrial respiration in a HIG2DA and COX5A-dependent manner, led to an initial spike in mitochondrial ATP, but then compromised mitochondrial membrane potential leading to ATP depletion. We have used chemoproteomic strategies to discover a unique anti-cancer mechanism of OPA through activation of complex IV leading to compromised mitochondrial energetics and rapid cell death.

Targeted Protein Degradation through E2 Recruitment.

Targeted protein degradation (TPD) with proteolysis targeting chimeras (PROTACs), heterobifunctional compounds consisting of protein targeting ligands linked to recruiters of E3 ubiquitin ligases, has arisen as a powerful therapeutic modality to induce the proximity of target proteins with E3 ligases to ubiquitinate and degrade specific proteins in cells. Thus far, PROTACs have primarily exploited the recruitment of E3 ubiquitin ligases or their substrate adapter proteins but have not exploited the recruitment of more core components of the ubiquitin-proteasome system (UPS). In this study, we used covalent chemoproteomic approaches to discover a covalent recruiter against the E2 ubiquitin conjugating enzyme UBE2D─EN67─that targets an allosteric cysteine, C111, without affecting the enzymatic activity of the protein. We demonstrated that this UBE2D recruiter could be used in heterobifunctional degraders to degrade neo-substrate targets in a UBE2D-dependent manner, including BRD4 and the androgen receptor. Overall, our data highlight the potential for the recruitment of core components of the UPS machinery, such as E2 ubiquitin conjugating enzymes, for TPD, and underscore the utility of covalent chemoproteomic strategies for identifying novel recruiters for additional components of the UPS.

Chemoproteomics-enabled discovery of a covalent molecular glue degrader targeting NF-κB.

Targeted protein degradation has arisen as a powerful therapeutic modality for degrading disease targets. While proteolysis-targeting chimera (PROTAC) design is more modular, the discovery of molecular glue degraders has been more challenging. Here, we have coupled the phenotypic screening of a covalent ligand library with chemoproteomic approaches to rapidly discover a covalent molecular glue degrader and associated mechanisms. We have identified a cysteine-reactive covalent ligand EN450 that impairs leukemia cell viability in a NEDDylation and proteasome-dependent manner. Chemoproteomic profiling revealed covalent interaction of EN450 with an allosteric C111 in the E2 ubiquitin-conjugating enzyme UBE2D. Quantitative proteomic profiling revealed the degradation of the oncogenic transcription factor NFKB1 as a putative degradation target. Our study thus puts forth the discovery of a covalent molecular glue degrader that uniquely induced the proximity of an E2 with a transcription factor to induce its degradation in cancer cells.

An Activity-Based Oxaziridine Platform for Identifying and Developing Covalent Ligands for Functional Allosteric Methionine Sites: Redox-Dependent Inhibition of Cyclin-Dependent Kinase 4.

Activity-based protein profiling (ABPP) is a versatile strategy for identifying and characterizing functional protein sites and compounds for therapeutic development. However, the vast majority of ABPP methods for covalent drug discovery target highly nucleophilic amino acids such as cysteine or lysine. Here, we report a methionine-directed ABPP platform using Redox-Activated Chemical Tagging (ReACT), which leverages a biomimetic oxidative ligation strategy for selective methionine modification. Application of ReACT to oncoprotein cyclin-dependent kinase 4 (CDK4) as a representative high-value drug target identified three new ligandable methionine sites. We then synthesized a methionine-targeting covalent ligand library bearing a diverse array of heterocyclic, heteroatom, and stereochemically rich substituents. ABPP screening of this focused library identified 1oxF11 as a covalent modifier of CDK4 at an allosteric M169 site. This compound inhibited kinase activity in a dose-dependent manner on purified protein and in breast cancer cells. Further investigation of 1oxF11 found prominent cation-π and H-bonding interactions stabilizing the binding of this fragment at the M169 site. Quantitative mass-spectrometry studies validated 1oxF11 ligation of CDK4 in breast cancer cell lysates. Further biochemical analyses revealed cross-talk between M169 oxidation and T172 phosphorylation, where M169 oxidation prevented phosphorylation of the activating T172 site on CDK4 and blocked cell cycle progression. By identifying a new mechanism for allosteric methionine redox regulation on CDK4 and developing a unique modality for its therapeutic intervention, this work showcases a generalizable platform that provides a starting point for engaging in broader chemoproteomics and protein ligand discovery efforts to find and target previously undruggable methionine sites.

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.

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.

Discovery of a Functional Covalent Ligand Targeting an Intrinsically Disordered Cysteine within MYC.

MYC is a major oncogenic transcriptional driver of most human cancers that has remained intractable to direct targeting because much of MYC is intrinsically disordered. Here, we have performed a cysteine-reactive covalent ligand screen to identify compounds that could disrupt the binding of MYC to its DNA consensus sequence in vitro and also impair MYC transcriptional activity in situ in cells. We have identified a covalent ligand, EN4, that targets cysteine 171 of MYC within a predicted intrinsically disordered region of the protein. We show that EN4 directly targets MYC in cells, reduces MYC and MAX thermal stability, inhibits MYC transcriptional activity, downregulates multiple MYC transcriptional targets, and impairs tumorigenesis. We also show initial structure-activity relationships of EN4 and identify compounds that show improved potency. Overall, we identify a unique ligandable site within an intrinsically disordered region of MYC that leads to inhibition of MYC transcriptional activity.

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.

The Most Common Functional Groups in Bioactive Molecules and How Their Popularity Has Evolved over Time.

The concept of functional groups (FGs), sets of connected atoms that can determine the intrinsic reactivity of the parent molecule and in part are responsible for the overall properties of the molecule, form a foundation within modern medicinal chemistry. In this Article, we analyze the occurrence of various FGs in molecules described in the medicinal chemistry literature over the last 40 years and show how their development and utilization over time has varied. The popularity of various FGs has not evolved randomly, but instead, clear patterns of use are evident. Various factors influencing these patterns, including the introduction of new synthetic methods, novel techniques, and strategies applied in drug discovery and the better knowledge of molecular properties affecting the success of candidate development, are discussed.

Manumycin polyketides act as molecular glues between UBR7 and P53.

Molecular glues are an intriguing therapeutic modality that harness small molecules to induce interactions between proteins that typically do not interact. However, such molecules are rare and have been discovered fortuitously, thus limiting their potential as a general strategy for therapeutic intervention. We postulated that natural products bearing one or more electrophilic sites may be an unexplored source of new molecular glues, potentially acting through multicovalent attachment. Using chemoproteomic platforms, we show that members of the manumycin family of polyketides, which bear multiple potentially reactive sites, target C374 of the putative E3 ligase UBR7 in breast cancer cells, and engage in molecular glue interactions with the neosubstrate tumor-suppressor TP53, leading to p53 transcriptional activation and cell death. Our results reveal an anticancer mechanism of this natural product family, and highlight the potential for combining chemoproteomics and multicovalent natural products for the discovery of new molecular glues.

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.