Discovery and Optimization of a Covalent AKR1C3 Inhibitor

R. Justin Grams, Wesley J. Wolfe, Robert J. Seal, James Veccia, and Ku-Lung Hsu

Journal of Medicinal Chemistry 2025

DOI: 10.1021/acs.jmedchem.5c00050

Aldo-keto reductase family 1 member C3 (AKR1C3) is a member of the AKR superfamily of enzymes that metabolize androgen, estrogen, and prostaglandin substrates that drive proliferation in hormone-dependent cancers. Interest in developing selective inhibitors has produced tool compounds for the inactivation or degradation of AKR1C3 with varying degrees of selectivity among the 14 known AKR proteins. Selectivity of AKR1C3 inhibitors across the AKR family is critical since a clinical candidate failed due to hepatotoxicity from off-target inhibition of AKR1D1. Here, we report development of a sulfonyl-triazole (SuTEx) covalent AKR1C3 inhibitor (RJG-2051) that selectively engages a noncatalytic tyrosine residue (Y24) on AKR1C3. Importantly, RJG-2051 exhibited negligible cross-reactivity with AKRs or other proteins across 1800+ tyrosine and lysine sites quantified by chemical proteomics. Our disclosure of a covalent inhibitor for potent AKR1C3 inactivation with proteome-wide selectivity in cells will expedite cell biological studies for testing the therapeutic potential of this metabolic target.

DCAF16-Based Covalent Degradative Handles for the Modular Design of Degraders

Lauren M Orr, Sydney J Tomlinson, Hannah R Grupe, Melissa Lim, Emily Ho, Halime Yilmaz, Grace Zhou, Barbara Leon, James A Olzmann, Daniel K Nomura

bioRxiv 2025.04.25.650514; 

doi: https://doi.org/10.1101/2025.04.25.650514

Targeted protein degradation (TPD) is a powerful strategy for targeting and eliminating disease-causing proteins. While heterobifunctional Proteolysis-Targeting Chimeras (PROTACs) are more modular, the rational design of monovalent or molecular glue degraders remains challenging. In this study, we generated a small library of BET-domain inhibitor JQ1 analogs bearing elaborated electrophilic handles to identify permissive covalent degradative handles and E3 ligase pairs. We identified an elaborated fumaramide handle that, when appended onto JQ1, led to the proteasome-dependent degradation of BRD4. Further characterization revealed that the E3 ubiquitin ligase CUL4(DCAF16), a common E3 ligase target of electrophilic degraders, was responsible for BRD4 loss by covalently targeting C173 on DCAF16. While this original fumaramide handle, when appended onto other protein-targeting ligands, did not accommodate the degradation of other neo-substrates, a truncated version of this handle attached to JQ1 was still capable of degrading BRD4, now through targeting both C173 and C178. This truncated fumaramide handle, when appended on various protein targeting ligands, and was also more permissive in degrading other neo-substrates, including CDK4/6, SMARCA2 and SMARCA4, and the androgen receptor (AR). We further demonstrated that this optimized truncated fumaramide handle, when transplanted onto an AR DNA binding domain-targeting ligand, could degrade both AR and the undruggable truncation variant of AR, AR-V7, in androgen-independent prostate cancer cells in a DCAF16-dependent manner. Overall, we have identified a unique DCAF16-targeting covalent degradative handle that can be transplanted across several protein-targeting ligands to induce the degradation of their respective targets for the modular design of monovalent or bifunctional degraders.

Proteomic Ligandability Maps of Phosphorus(V) Stereoprobes Identify Covalent TLCD1 Inhibitors

Hayden A. Sharma, Michael Bielecki, Meredith A. Holm, Ty M. Thompson, Yue Yin, Jacob B. Cravatt, Timothy B. Ware, Alex Reed, Molhm Nassir, Tamara El-Hayek Ewing, Bruno Melillo, J. Fernando Bazan, Phil S. Baran, and Benjamin F. Cravatt

Journal of the American Chemical Society 2025

DOI: 10.1021/jacs.5c01944

Activity-based protein profiling (ABPP) of stereoisomerically defined sets of electrophilic compounds (‘stereoprobes’) offers a versatile way to discover covalent ligands for proteins in native biological systems. Here we report the synthesis and chemical proteomic characterization of stereoprobes bearing a P(V)-oxathiaphospholane (OTP) reactive group. ABPP experiments identified numerous proteins in human cancer cells that showed stereoselective reactivity with OTP stereoprobes, and we confirmed several of these liganding events with recombinant proteins. OTP stereoprobes engaging the poorly characterized transmembrane protein TLCD1 impaired the incorporation of monounsaturated fatty acids into phosphatidylethanolamine lipids in cells, a lipidomic phenotype that mirrored genetic disruption of this protein. Using AlphaFold2, we found that TLCD1 structurally resembles the ceramide synthase and fatty acid elongase families of coenzyme A-dependent lipid processing enzymes. This structural similarity included conservation of catalytic histidine residues, the mutation of which blocked the OTP stereoprobe reactivity and lipid remodeling activity of recombinant TLCD1. Taken together, these data indicate that TLCD1 acts as a lipid acyltransferase in cells, and that OTP stereoprobes function as inhibitors of this enzymatic activity. Our findings thus illuminate how the chemical proteomic analysis of electrophilic compounds can facilitate the functional annotation and chemical inhibition of a key lipid metabolic enzyme in human cells.

Discovery and Optimization of a Covalent AKR1C3 Inhibitor

R. Justin Grams, Wesley J. Wolfe, Robert J. Seal, James Veccia, and Ku-Lung Hsu

Journal of Medicinal Chemistry 2025

https://doi.org/10.1021/acs.jmedchem.5c00050

Aldo-keto reductase family 1 member C3 (AKR1C3) is a member of the AKR superfamily of enzymes that metabolize androgen, estrogen, and prostaglandin substrates that drive proliferation in hormone-dependent cancers. Interest in developing selective inhibitors has produced tool compounds for the inactivation or degradation of AKR1C3 with varying degrees of selectivity among the 14 known AKR proteins. Selectivity of AKR1C3 inhibitors across the AKR family is critical since a clinical candidate failed due to hepatotoxicity from off-target inhibition of AKR1D1. Here, we report development of a sulfonyl-triazole (SuTEx) covalent AKR1C3 inhibitor (RJG-2051) that selectively engages a noncatalytic tyrosine residue (Y24) on AKR1C3. Importantly, RJG-2051 exhibited negligible cross-reactivity with AKRs or other proteins across 1800+ tyrosine and lysine sites quantified by chemical proteomics. Our disclosure of a covalent inhibitor for potent AKR1C3 inactivation with proteome-wide selectivity in cells will expedite cell biological studies for testing the therapeutic potential of this metabolic target.

Discovery of RNA-Reactive Small Molecules Guides Design of Electrophilic Modules for RNA-Specific Covalent Binders

Noah A. Springer, Patrick R. A. Zanon, Amirhossein Taghavi, Kisu Sung, Matthew D. Disney

bioRxiv 2025.04.22.649986; 

doi: https://doi.org/10.1101/2025.04.22.649986

RNA is a key drug target that can be modulated by small molecules, however covalent binders of RNA remain largely unexplored. Using a high-throughput mass spectrometry screen of 2,000 electrophilic compounds, we identified ligands that react with RNA in a binding-dependent manner. RNA reactivity was influenced by both the reactive group and the RNA-binding scaffold. Electrophilic modules such as 3-chloropivalamide, bis(2-chloroethyl)amine, chloroacetamide, and N-acylimidazole that react with proteins also cross-linked to RNA, especially when paired with aromatic heterocycles, particularly those with a thieno[3,2-c]pyridinium core. These results suggest that electrophiles commonly used for protein targeting can also covalently modify RNA, potentially contributing to both on- and off-target effects. This insight enabled the design of an RNA-specific covalent compound by modifying a Hoechst scaffold, originally identified to bind DNA, to react selectively with the expanded triplet repeat RNA, r(CUG)exp, that causes myotonic dystrophy type 1 (DM1). Selectivity appears to arise from binding to the RNA major groove near the reactive site. Overall, this study highlights the potential of rationally designing covalent RNA-targeting small molecules.

Identification of a phenyl ester covalent inhibitor of caseinolytic protease and analysis of the ClpP1P2 inhibition in mycobacteria

Genhui Xiao, Yumeng Cui, Liangliang Zhou, Chuya Niu, Bing Wang, Jinglan Wang, Shaoyang Zhou, Miaomiao Pan, Chi Kin Chan, Yan Xia, Lan Xu, Yu Lu, Shawn Chen

mLife, 2025

DOI: https://doi.org/10.1002/mlf2.12169

The caseinolytic protease complex ClpP1P2 is crucial for protein homeostasis in mycobacteria and stress response and virulence of the pathogens. Its role as a potential drug target for combating tuberculosis (TB) has just begun to be substantiated in drug discovery research. We conducted a biochemical screening targeting the ClpP1P2 using a library of compounds phenotypically active against Mycobacterium tuberculosis (Mtb). The screening identified a phenyl ester compound GDI-5755, inhibiting the growth of Mtb and M. bovis BCG, the model organism of mycobacteria. GDI-5755 covalently modified the active-site serine residue of ClpP1, rendering the peptidase inactive, which was delineated through protein mass spectrometry and kinetic analyses. GDI-5755 exerted antibacterial activity by inhibiting ClpP1P2 in the bacteria, which could be demonstrated through a minimum inhibitory concentration (MIC) shift assay with a clpP1 CRISPRi knockdown (clpP1-KD) mutant GH189. The knockdown also remarkably heightened the mutant's sensitivity to ethionamide and meropenem, but not to many other TB drugs. On the other hand, a comparative proteomic analysis of wild-type cells exposed to GDI-5755 revealed the dysregulated proteome, specifically showing changes in the expression levels of multiple TB drug targets, including EthA, LdtMt2, and PanD. Subsequent evaluation confirmed the synergistic activity of GDI-5755 when combined with the TB drugs to inhibit mycobacterial growth. Our findings indicate that small-molecule inhibitors targeting ClpP1P2, when used alongside existing TB medications, could represent novel therapeutic strategies.

Substrate Trapping in Polyketide Synthase Thioesterase Domains: Structural Basis for Macrolactone Formation

Tyler M. McCullough, Vishakha Choudhary, David L. Akey, Meredith A. Skiba, Steffen M. Bernard, Jeffrey D. Kittendorf, Jennifer J. Schmidt, David H. Sherman, and Janet L. Smith

ACS Catalysis 2024 14 (16), 12551-12563

DOI: 10.1021/acscatal.4c03637

Emerging antibiotic resistance requires continual improvement in the arsenal of antimicrobial drugs, especially the critical macrolide antibiotics. Formation of the macrolactone scaffold of these polyketide natural products is catalyzed by a modular polyketide synthase (PKS) thioesterase (TE). The TE accepts a linear polyketide substrate from the terminal PKS acyl carrier protein to generate an acyl-enzyme adduct that is resolved by attack of a substrate hydroxyl group to form the macrolactone. Our limited mechanistic understanding of TE selectivity for a substrate nucleophile and/or water has hampered development of TEs as biocatalysts that accommodate a variety of natural and non-natural substrates. To understand how TEs direct the substrate nucleophile for macrolactone formation, acyl-enzyme intermediates were trapped as stable amides by substituting the natural serine OH with an amino group. Incorporation of the unnatural amino acid, 1,3-diaminopropionic acid (DAP), was tested with five PKS TEs. DAP-modified TEs (TEDAP) from the pikromycin and erythromycin pathways were purified and tested with six full-length polyketide intermediates from three pathways. The erythromycin TE had permissive substrate selectivity, whereas the pikromycin TE was selective for its native hexaketide and heptaketide substrates. In a crystal structure of a native substrate trapped in pikromycin TEDAP, the linear heptaketide was curled in the active site with the nucleophilic hydroxyl group positioned 4 Å from the amide-enzyme linkage. The curled heptaketide displayed remarkable shape complementarity with the TE acyl cavity. The strikingly different shapes of acyl cavities in TEs of known structure, including those reported here for juvenimicin, tylosin and fluvirucin biosynthesis, provide insights to facilitate TE engineering and optimization.

Covalent Modification of Glutamic Acid Inspired by HaloTag Technology

Waldmann H, Zhang R, liu J, Gasper R, Janning P.

 ChemRxiv. 2025

 doi:10.26434/chemrxiv-2025-70x40 . 

https://chemrxiv.org/engage/chemrxiv/article-details/67ecf55e81d2151a02ab0682

For targeted covalent protein modification at low reactivity aspartates and glutamates, new methods are in high demand. Inspired by the HaloTag technology we have developed a new technique which employs a reaction between chloroalkane-functionalised ligands and a specific glutamate residue. The lipoprotein chaperone PDEδ shuttles prenylated lipoproteins between cellular membranes and, thereby, mediates their activity. In cells, reversible PDEδ inhibition is efficiently counterbalanced by Arl2/3-mediated inhibitor release calling for covalent inhibitor development. However, the hydrophobic ligand binding site contains only Glu88 as accessible nucleophile. Inspired by the HaloTag technology, we have developed a novel covalent PDEδ inhibitor chemotype with alkyl bromide warheads which targets glutamate E88. The best covalent inhibitor, termed DeltaTag, overcomes Arl2-mediated release, modulates signal transduction through the mTOR pathway and inhibits cancer cell proliferation. The design strategy promises to be applicable also to other proteins with carboxylate residues embedded in hydrophobic binding sites, such as other lipoprotein chaperones.

Factors affecting irreversible inhibition of EGFR and influence of chirality on covalent binding

Pasquale A. Morese, Ayaz Ahmad, Mathew P. Martin, Richard A. Noble, Sara Pintar, Lan Z. Wang, Shangze Xu, Andrew Lister, Richard A. Ward, Agnieszka K. Bronowska, Martin E. M. Noble, Hannah L. Stewart & Michael J. Waring

Commun Chem 8, 111 (2025). 

https://doi.org/10.1038/s42004-025-01501-6

The discovery of targeted covalent inhibitors is of increasing importance in drug discovery. Finding efficient covalent binders requires modulation of warhead reactivity and optimisation of warhead geometry and non-covalent interactions. Uncoupling the contributions that these factors make to potency is difficult and best practice for a testing cascade that is pragmatic and informative is yet to be fully established. We studied the structure-reactivity-activity relationships of a series of analogues of the EGFR inhibitor poziotinib with point changes in two substructural regions as well as variations in warhead reactivity and geometry. This showed that a simple probe displacement assay that is appropriately tuned in respect of timing and reagent concentrations can reveal structural effects on all three factors: non-covalent affinity, warhead reactivity and geometry. These effects include the detection of potency differences between an enantiomeric pair that differ greatly in their activity and their capacity to form a covalent bond. This difference is rationalised by X-ray crystallography and computational studies and the effect translates quantitatively into cellular mechanistic and phenotypic effects.

Molecular Pharmacology of the Antibiotic Fosfomycin, an Inhibitor of Peptidoglycan Biosynthesis

Dennis H. Kim and Watson J. Lees

Biochemistry 2025

DOI: 10.1021/acs.biochem.4c00522

The antibiotic fosfomycin is an epoxy-phosphonate natural product with a broad spectrum of antibacterial activity and distinct mechanism of action that has been in clinical use for 50 years. Fosfomycin is an irreversible covalent inhibitor of UDP-GlcNAc enolpyruvyl transferase (MurA), which catalyzes the first committed step in bacterial peptidoglycan biosynthesis. Fosfomycin binds to the active site of MurA in competition with substrate phosphoenolpyruvate (PEP) and undergoes the ring-opening nucleophilic attack of an active-site cysteine. MurA and its related enolpyruvyl transferase, 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase (AroA), are the only known enzymes to catalyze the unusual enolpyruvyl transfer from PEP, and each is the target of an important inhibitor. Specifically, MurA is inactivated by fosfomycin, and EPSP synthase (AroA) of the shikimate pathway is the target of the herbicide glyphosate. Commonalities and differences in enzymatic reaction mechanisms of MurA and EPSP synthase provide a molecular rationale for the specificity of their respective inhibitors. With its distinct mode of molecular action and clinical activity against multidrug-resistant bacteria, fosfomycin continues to motivate the discovery and development of novel inhibitors of MurA.

A predictive model for thiol reactivity of N-heteroaryl α-methylene–γ-lactams—a medicinally relevant covalent reactive group

Meehan, M.; Scofield, G.; Stahl, C.; Wolfe, J.; Horne, W. S.; Liu, P.; Brummond, K. 

ChemRxiv 2025. 
https://doi.org/10.26434/chemrxiv-2025-64qqs

Herein, we present a systematic study on the effects of electronically diverse heteroarenes on the rate of glutathione (GSH) addition to novel N-heteroaryl α–methylene–γ-lactam covalent reactive groups (CRGs). Despite their unique electronic and drug-like properties, heteroarenes have not been extensively studied as handles for systematically tuning the reactivity of CRGs. Informed by mechanistic insights, we evaluated 16 substrate parameters, including a new heteroaryl Hammett-type substituent constant (σHet), for their correlation with experimental reactivity (DG‡exp) as determined by 1H NMR kinetics studies. Of these parameters, electron affinity represents a robust single-parameter predictive model of CRG reactivity with thiols, as demonstrated by test sets of additional N-heteroaryl lactams (MUE = 0.4 kcal/mol) and other α,β-unsaturated amide CRGs (MUE = 0.3 kcal/mol). These N-heteroaryl lactams were subse-quently shown to inhibit cysteine protease activity (i.e., papain enzyme) to varying degrees that correlate with both the experimentally observed and predicted reactivity with GSH.

Covalent adduct Grob fragmentation underlies LSD1 demethylase-specific inhibitor mechanism of action and resistance

Amanda L. Waterbury, Jonatan Caroli, Olivia Zhang, Paloma R. Tuttle, Chao Liu, Jiaming Li, Ji Sung Park, Samuel M. Hoenig, Marco Barone, Airi Furui, Andrea Mattevi & Brian B. Liau

Nat Commun 16, 3156 (2025). 

https://doi.org/10.1038/s41467-025-57477-3

Chromatin modifiers often work in concert with transcription factors (TFs) and other complex members, where they can serve both enzymatic and scaffolding functions. Due to this, active site inhibitors targeting chromatin modifiers may perturb both enzymatic and nonenzymatic functions. For instance, the antiproliferative effects of active-site inhibitors targeting lysine-specific histone demethylase 1A (LSD1) are driven by disruption of a protein-protein interaction with growth factor independence 1B (GFI1B) rather than inhibition of demethylase activity. Recently, next-generation precision LSD1 covalent inhibitors have been developed, which selectively block LSD1 enzyme activity by forming a compact N-formyl flavin adenine dinucleotide (FAD) adduct that spares the GFI1B interaction. However, the mechanism accounting for N-formyl-FAD formation remains unclear. Here we clarify the mechanism of these demethylase-specific inhibitors of LSD1, demonstrating that the covalent inhibitor-FAD adduct undergoes a Grob fragmentation. Using inhibitor analogs and structural biology, we identify structure-activity relationships that promote this transformation. Furthermore, we unveil an unusual drug resistance mechanism whereby distal active-site mutations can promote inhibitor-adduct Grob fragmentation even for previous generation compounds. Our study uncovers the unique Grob fragmentation underlying the mechanism of action of precision LSD1 enzyme inhibitors, offering insight into their reactivity with broader implications for drug resistance.

Size-Dependent Target Engagement of Covalent Probes

László Petri, Ronen Gabizon, György G. Ferenczy, Nikolett Péczka, Attila Egyed, Péter Ábrányi-Balogh, Tamás Takács, and György M. Keserű

Journal of Medicinal Chemistry 2025 68 (6), 6616-6632

DOI: 10.1021/acs.jmedchem.5c00017

Labeling proteins with covalent ligands is finding increasing use in proteomics applications, including identifying nucleophilic residues amenable for labeling and in the development of targeted covalent inhibitors (TCIs). Labeling efficiency is measured by the covalent occupancy of the target or by biochemical activity. Here, we investigate how these observed quantities relate to the intrinsic parameters of complex formation, namely, noncovalent affinity and covalent reactivity, and to experimental conditions, including incubation time and ligand concentration. It is shown that target engagement is beneficially driven by noncovalent recognition for lead-like compounds, which are appropriate starting points for targeted covalent inhibitors owing to their easily detectable occupancy and fixed binding mode, facilitating optimization. In contrast, labeling by fragment-sized compounds is inevitably reactivity-driven as their small size limits noncovalent affinity. They are well-suited for exploring ligandable nucleophilic residues, while small fragments are less appropriate starting points for TCI development.

O-Cyanobenzaldehydes Irreversibly Modify Both Buried and Exposed Lysine Residues in Live Cells

Huan Ling, Lin Li, Liping Duan, Weixue Huang, Jiangnan Zheng, Shijie Zhang, Xinling Li, Xiaorong Qiu, Yang Zhou, Nan Ma, Xiaomei Ren, Jinwei Zhang, Zhen Wang, Yujun Zhao, Ruijun Tian, Zhi-Min Zhang, and Ke Ding

Journal of the American Chemical Society 2025

DOI: 10.1021/jacs.4c18006

Lysine residue represents an attractive site for covalent drug development due to its high abundance (5.6%) and critical functions. However, very few lysines have been characterized to be accessible to covalent ligands and perturb the protein functions, owing to their protonation state and adjacent steric hindrance. Herein, we report a new lysine bioconjugation chemistry, O-cyanobenzaldehyde (CNBA), that enables selective modification of the lysine ε-amine to form iso-indolinones under physiological conditions. Activity-based proteome profiling enabled the mapping of 3451 lysine residues and 85 endogenous kinases in live cells, highlighting its potential for modifying hyper-reactive lysines within the proteome or buried catalytic lysines within the kinome. Further protein crystallography and mass spectrometry confirmed that K271_ABL1 and K162_AURKA are covalently targetable sites in kinases. Leveraging a structure-based drug design, we incorporated CNBA into the core structure of Nutlin-3 to irreversibly inhibit the MDM2-p53 interaction by targeting an exposed lysine K94 on the surface of murine double minute 2. Importantly, we have demonstrated the potential application of CNBA as a lysine-recognized bioconjugation agent for developing new antibody-drug conjugates. The results collectively validate CNBA as a new selective and efficient modifying agent with broad applications for both buried and exposed lysine residues in live cells.