Selective Covalent Inhibiting JNK3 by Small Molecules for Parkinson's Diseases

Liang Ouyang, Wen Shuai, Panpan Yang, Huan Xiao, Yumeng Zhu, Faqian Bu, Aoxue Wang, Qiu Sun, Guan Wang

Angewandte Chemie 2024 e202411037

https://doi.org/10.1002/ange.202411037

c-Jun N-terminal kinases (JNKs) including JNK1/2/3 are key members of mitogen-activated protein kinase family. Wherein JNK3 is specifically expressed in brain and emerges as therapeutic target, especially for neurodegenerative diseases. However, developing JNK3 selective inhibitors as chemical probes to investigate its therapeutic potential in diseases remains challenging. Here, we adopted the covalent strategy for identifying JNK3-selective covalent inhibitorJC16I, with high inhibitory activity against JNK3. Despite targeting a conserved cysteine the vicinity of ATP pocket in JNK family, JC16I exerted a greater than 160-fold selectivity for JNK3 over JNK1/2. Importantly, even at low concentration, JC16I showed enhanced and long-lasting inhibition against cellular JNK3. In addition, its alkyne-containing probe JC-P1 could label JNK3 in SH-SY5Y cell lysate and living cells, with goodproteome-wide selectivity. Furthermore, JC16I selectively suppressed the abnormal activation of JNK3 signaling and sufficiently exhibited neuroprotective effect in Parkinson's diseases (PD) models. Overall, our findings highlight the potential of developing isoform-selective and cell-active JNK3 inhibitors by covalent drug design strategy targeting a conserved cysteine. This work not only provides a valuable chemical probe for JNK3-targeted investigations in vitro and in vivo but also opens new avenues for the treatment of PD.

Development of ketalized unsaturated saccharides as multifunctional cysteine-targeting covalent warheads

Dong, S., Huang, H., Li, J. et al.

Commun Chem 7, 201 (2024). 

https://doi.org/10.1038/s42004-024-01279-z

Multi-functional cysteine-targeting covalent warheads possess significant therapeutic potential in medicinal chemistry and chemical biology. Herein, we present novel unsaturated and asymmetric ketone (oxazolinosene) scaffolds that selectively conjugate cysteine residues of peptides and bovine serum albumin under normal physiological conditions. This unsaturated saccharide depletes GSH in NCI-H1299 cells, leading to anti-tumor effects in vitro. The acetyl group of the ketal moiety on the saccharide ring can be converted to other carboxylic acids in a one-pot synthesis. In this way, the loaded acid can be click-released during cysteine conjugation, making the oxazolinosene a potential multifunctional therapeutic agent. The reaction kinetic model for oxazolinosene conjugation to GSH is well established and was used to evaluate oxazolinosene reactivity. The aforementioned oxazolinosenes were stereoselectively synthesized via a one-step reaction of nitriles with saccharides and conveniently converted into a series of α, β-unsaturated ketone N-glycosides as prevalent synthetic building blocks. The reaction mechanisms of oxazolinosene synthesis were investigated through calculations and validated with control experiments. Overall, these oxazolinosenes can be easily synthesized and developed as cysteine-targeted covalent warheads carrying useful click-releasing groups.

From Mechanism-Based Retaining Glycosidase Inhibitors to Activity-Based Glycosidase Profiling

 Marta Artola, Johannes M. F. G. Aerts, Gijsbert A. van der Marel, Carme Rovira, Jeroen D. C. Codée, Gideon J. Davies, and Herman S. Overkleeft

Journal of the American Chemical Society 2024

DOI: 10.1021/jacs.4c08840

Activity-based protein profiling (ABPP) is an effective technology for the identification and functional annotation of enzymes in complex biological samples. ABP designs are normally directed to an enzyme active site nucleophile, and within the field of Carbohydrate-Active Enzymes (CAZymes), ABPP has been most successful for those enzymes that feature such a residue: retaining glycosidases (GHs). Several mechanism-based covalent and irreversible retaining GH inhibitors have emerged over the past sixty years. ABP designs based on these inhibitor chemistries appeared since the turn of the millennium, and we contributed to the field by designing a suite of retaining GH ABPs modeled on the structure and mode of action of the natural product, cyclophellitol. These ABPs enable the study of both exo- and endo-acting retaining GHs in human health and disease, for instance in genetic metabolic disorders in which retaining GHs are deficient. They are also finding increasing use in the study of GHs in gut microbiota and environmental microorganisms, both in the context of drug (de)toxification in the gut and that of biomass polysaccharide processing for future sustainable energy and chemistries. This account comprises the authors’ view on the history of mechanism-based retaining GH inhibitor design and discovery, on how these inhibitors served as blueprints for retaining GH ABP design, and on some current and future developments on how cyclophellitol-based ABPs may drive the discovery of retaining GHs and their inhibitors.

SuFEx Chemistry Enables Covalent Assembly of a 280-kDa 18-Subunit Pore-Forming Complex

Lee Schnaider, Sophia Tan, Pratik R. Singh, Floriana Capuano, Alistair J. Scott, Richard Hambley, Lei Lu, Hyunjun Yang, E. Jayne Wallace, Hyunil Jo, and William F. DeGrado

Journal of the American Chemical Society 2024

DOI: 10.1021/jacs.4c07920

Proximity-enhanced chemical cross-linking is an invaluable tool for probing protein–protein interactions and enhancing the potency of potential peptide and protein drugs. Here, we extend this approach to covalently stabilize large macromolecular assemblies. We used SuFEx chemistry to covalently stabilize an 18-subunit pore-forming complex, CsgG:CsgF, consisting of nine CsgG membrane protein subunits that noncovalently associate with nine CsgF peptides. Derivatives of the CsgG:CsgF pore have been used for DNA sequencing, which places high demands on the structural stability and homogeneity of the complex. To increase the robustness of the pore, we designed and synthesized derivatives of CsgF-bearing sulfonyl fluorides, which react with CsgG in very high yield to form a covalently stabilized CsgG:CsgF complex. The resulting pores formed highly homogeneous channels when added to artificial membranes. The high yield and rapid reaction rate of the SuFEx reaction prompted molecular dynamics simulations, which revealed that the SO2F groups in the initially formed complex are poised for nucleophilic reaction with a targeted Tyr. These results demonstrate the utility of SuFEx chemistry to structurally stabilize very large (here, 280 kDa) assemblies.

Targeted Protein Localization by Covalent 14–3–3 Recruitment

 Qian Shao, Tuong Nghi Duong, Inji Park, Lauren M. Orr, and Daniel K. Nomura

Journal of the American Chemical Society 2024

DOI: 10.1021/jacs.3c12389

14–3–3 proteins have a unique ability to bind and sequester a multitude of diverse phosphorylated signaling proteins and transcription factors. Many previous studies have shown that interactions of 14–3–3 with specific phosphorylated substrate proteins can be enhanced through small-molecule natural products or fully synthetic molecular glue interactions. However, enhancing 14–3–3 interactions with both therapeutically intractable transcription factor substrates and potential neo-substrates to sequester and inhibit their function remains elusive. One of the 14–3–3 proteins, 14–3–3σ or SFN, has cysteine C38 at the substrate-binding interface, near the sites where previous 14–3–3 molecular glues have been found to bind. In this study, we screen a fully synthetic cysteine-reactive covalent ligand library to identify molecular glues that enhance the interaction of 14–3–3σ with not only druggable transcription factors such as estrogen receptor (ERα) but also challenging oncogenic transcription factors such as YAP and TAZ, which are part of the Hippo transducer pathway. We identify a hit EN171 that covalently targets both C38 and C96 on 14–3–3 to enhance 14–3–3 interactions with ERα, YAP, and TAZ, leading to impaired estrogen receptor and Hippo pathway transcriptional activity. We further demonstrate that EN171 could not only be used as a molecular glue to enhance native protein interactions but could also be used as a covalent 14–3–3 recruiter in heterobifunctional molecules to sequester nuclear neo-substrates such as BRD4 and BLC6 into the cytosol. Overall, our study reveals a covalent ligand that acts as a novel 14–3–3 molecular glue for challenging transcription factors such as YAP and TAZ and demonstrates that these glues can be potentially utilized in heterobifunctional molecules to sequester nuclear neo-substrates out of the nucleus and into the cytosol to enable targeted protein localization.

Aminomethyl Salicylaldehydes Lock onto a Surface Lysine by Forming an Extended Intramolecular Hydrogen Bond Network

Jacqueline Weaver, Gregory B. Craven, Linh Tram, Hao Chen, and Jack Taunton

Journal of the American Chemical Society 2024

DOI: 10.1021/jacs.4c04314

The development of electrophilic ligands that rapidly modify specific lysine residues remains a major challenge. Salicylaldehyde-based inhibitors have been reported to form stable imine adducts with the catalytic lysine of protein kinases. However, the targeted lysine in these examples is buried in a hydrophobic environment. A key unanswered question is whether this strategy can be applied to a lysine on the surface of a protein, where rapid hydrolysis of the resulting salicylaldimine is more likely. Here, we describe a series of aminomethyl-substituted salicylaldehydes that target a fully solvated lysine on the surface of the ATPase domain of Hsp90. By systematically varying the orientation of the salicylaldehyde, we discovered ligands with long residence times, the best of which engages Hsp90 in a quasi-irreversible manner. Crystallographic analysis revealed a daisy-chain network of intramolecular hydrogen bonds in which the salicylaldimine is locked into position by the adjacent piperidine linker. This study highlights the potential of aminomethyl salicylaldehydes to generate conformationally stabilized, hydrolysis-resistant imines, even when the targeted lysine is far from the ligand binding site and is exposed to bulk solvent.

Discovery of a Covalent Inhibitor of Pro-Caspase-1 Zymogen Blocking NLRP3 Inflammasome Activation and Pyroptosis

Dongyi Cao, Ruiying Xi, Hongye Li, Zhonghui Zhang, Xiaoke Shi, Shanshan Li, Yujie Jin, Wanli Liu, Guolin Zhang, Xiaohua Liu, Shunxi Dong, Xiaoming Feng, and Fei Wang

Journal of Medicinal Chemistry 2024

DOI https://doi.org/10.1021/acs.jmedchem.4c01558

Caspase-1 plays a central role in innate immunity, as its activation by inflammasomes induces the production of proinflammatory cytokines and pyroptosis. However, specific inhibition of the enzymatic activity of this protease is not effective in suppressing inflammation, owing to its enzyme-independent function. Herein, we identified a cyclohexenyl isothiocyanate compound (CIB-1476) that potently inhibited caspase-1 activity and suppressed the assembly and activation of the NLRP3 inflammasome and gasdermin-D-mediated pyroptosis. Mechanistically, CIB-1476 directly targeted pro-caspase-1 as an irreversible covalent inhibitor by binding to Cys285 and Cys397, resulting in more durable anti-inflammatory effects in the suppression of enzyme-dependent IL-1β production and enzyme-independent nuclear factor κB activation. Chemoproteomic profiling demonstrated the engagement of CIB-1476 with caspase-1. CIB-1476 showed potent therapeutic effects by suppressing inflammasome activation in mice, which was abolished in Casp1–/– mice. These results warrant further development of CIB-1476 along with its analogues as a novel strategy for caspase-1 inhibitors.

Discovering Covalent Cyclic Peptide Inhibitors of 2 Peptidyl Arginine Deiminase 4 (PADI4) Using mRNA3 Display with a Genetically Encoded Electrophilic 4 Warhead

 Isabel Mathiesen, Ewen Calder, Simone Kunzelmann, Louise Walport 

ChemRxiv, 2024

https://doi.org/10.26434/chemrxiv-2024-w8nbl

Covalent drugs can achieve high potency with long dosing intervals. However, concerns remain about side-effects associated with off-target reactivity. Combining macrocyclic peptides with covalent warheads provides a solution to minimise off-target reactivity: the peptide enables highly specific target binding, positioning a weakly reactive warhead proximal to a suitable residue in the target. Here we demonstrate direct discovery of covalent cyclic peptides using encoded libraries containing a weakly electrophilic cysteine-reactive fluoroamidine warhead. We combine direct incorporation of the warhead into peptide libraries using the flexible in vitro translation system with a peptide selection approach that identifies only covalent target binders. Using this approach, we identify potent covalent inhibitors of the peptidyl arginine deiminase, PADI4 or PAD4, that react exclusively at the active site cysteine. We envisage this approach will enable covalent peptide inhibitor discovery for a range of related enzymes and expansion to alternative warheads in the future.

Electrophilic proximity-inducing synthetic adapters enhance universal T cell function by covalently enforcing immune receptor signaling [@RulloLab]

Nickolas J. Serniuck, Eden Kapcan, Duane Moogk,Allyson E. Moore,Benjamin P.M. Lake, Galina Denisova,Joanne A. Hammill,Jonathan L. Bramson, Anthony F. Rullo

Molecular Therapy 2024

DOI: https://doi.org/10.1016/j.omton.2024.200842

Proximity-induction of cell-cell interactions via small molecules represents an emerging field in basic and translational sciences. Covalent anchoring of these small molecules represents a useful chemical strategy to enforce proximity; however, it remains largely unexplored for driving cell-cell interactions. In immunotherapeutic applications, bifunctional small molecules are attractive tools for inducing proximity between immune effector cells like T cells and tumor cells to induce tumoricidal function. We describe a two-component system composed of electrophilic bifunctional small molecules and paired synthetic antigen receptors (SARs) that elicit T cell activation. The molecules, termed covalent immune recruiters (CIRs), were designed to affinity label and covalently engage SARs. We evaluated the utility of CIRs to direct anti-tumor function of human T cells engineered with three biologically distinct classes of SAR. Irrespective of the electrophilic chemistry, tumor-targeting moiety, or SAR design, CIRs outperformed equivalent non-covalent bifunctional adapters, establishing a key role for covalency in maximizing functionality. We determined that covalent linkage enforced early T cell activation events in a manner that was dependent upon each SARs biology and signaling threshold. These results provide a platform to optimize universal SAR-T cell functionality and more broadly reveal new insights into how covalent adapters modulate cell-cell proximity-induction.

Multi-tiered chemical proteomic maps of tryptoline acrylamide–protein interactions in cancer cells [@EvertNjomen]

Njomen, E., Hayward, R.E., DeMeester, K.E. et al. Multi-tiered chemical proteomic maps of tryptoline acrylamide–protein interactions in cancer cells. 

Nat. Chem. 2024

• DOI https://doi.org/10.1038/s41557-024-01601-1

• Covalent chemistry is a versatile approach for expanding the ligandability of the human proteome. Activity-based protein profiling (ABPP) can infer the specific residues modified by electrophilic compounds through competition with broadly reactive probes. However, the extent to which such residue-directed platforms fully assess the protein targets of electrophilic compounds in cells remains unclear. Here we evaluate a complementary protein-directed ABPP method that identifies proteins showing stereoselective reactivity with alkynylated, chiral electrophilic compounds—termed stereoprobes. Integration of protein- and cysteine-directed data from cancer cells treated with tryptoline acrylamide stereoprobes revealed generally well-correlated ligandability maps and highlighted features, such as protein size and the proteotypicity of cysteine-containing peptides, that explain gaps in each ABPP platform. In total, we identified stereoprobe binding events for >300 structurally and functionally diverse proteins, including compounds that stereoselectively and site-specifically disrupt MAD2L1BP interactions with the spindle assembly checkpoint complex leading to delayed mitotic exit in cancer cells.

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Exploring 2-Sulfonylpyrimidine Warheads as Acrylamide Surrogates for Targeted Covalent Inhibition: A BTK Story

Ruxandra Moraru, Beatriz Valle-Argos, Annabel Minton, Lara Buermann, Suyin Pan, Thomas E. Wales, Raji E. Joseph, Amy H. Andreotti, Jonathan C. Strefford, Graham Packham, and Matthias G. J. Baud

Journal of Medicinal Chemistry 2024

DOI: 10.1021/acs.jmedchem.3c01927

Targeted covalent inhibitors (TCIs) directing cysteine have historically relied on a narrow set of electrophilic “warheads”. While Michael acceptors remain at the forefront of TCI design strategies, they show variable stability and selectivity under physiological conditions. Here, we show that the 2-sulfonylpyrimidine motif is an effective replacement for the acrylamide warhead of Ibrutinib, for the inhibition of Bruton’s tyrosine kinase. In a few iterations, we discovered new derivatives, which inhibit BTK both in vitro and in cellulo at low nanomolar concentrations, on par with Ibrutinib. Several derivatives also displayed good plasma stability and reduced off-target binding in vitro across 135 tyrosine kinases. This proof-of-concept study on a well-studied kinase/TCI system highlights the 2-sulfonylpyrimidine group as a useful acrylamide replacement. In the future, it will be interesting to investigate its wider potential for developing TCIs with improved pharmacologies and selectivity profiles across structurally related protein families.

Discovery of Thioether-Cyclized Macrocyclic Covalent Inhibitors by mRNA Display

Tong Lan, Cheng Peng, Xiyuan Yao, Rachel Shu Ting Chan, Tongyao Wei, Anuchit Rupanya, Aleksandar Radakovic, Sijie Wang, Shiyu Chen, Scott Lovell, Scott A. Snyder, Matthew Bogyo, and Bryan C. Dickinson

Journal of the American Chemical Society 2024

DOI: 10.1021/jacs.4c07851

Macrocyclic peptides are promising scaffolds for the covalent ligand discovery. However, platforms enabling the direct identification of covalent macrocyclic ligands in a high-throughput manner are limited. In this study, we present an mRNA display platform allowing selection of covalent macrocyclic inhibitors using 1,3-dibromoacetone-vinyl sulfone (DBA-VS). Testcase selections on TEV protease resulted in potent covalent inhibitors with diverse cyclic structures, among which cTEV6-2, a macrocyclic peptide with a unique C-terminal cyclization, emerged as the most potent covalent inhibitor of TEV protease described to-date. This study outlines the workflow for integrating chemical functionalization─installation of a covalent warhead─with mRNA display and showcases its application in targeted covalent ligand discovery.

Co-targeting SOS1 enhances the antitumor effects of KRASG12C inhibitors by addressing intrinsic and acquired resistance

Thatikonda, V., Lyu, H., Jurado, S. et al.

Nat Cancer (2024). 

https://doi.org/10.1038/s43018-024-00800-6

Combination approaches are needed to strengthen and extend the clinical response to KRASG12C inhibitors (KRASG12Ci). Here, we assessed the antitumor responses of KRASG12C mutant lung and colorectal cancer models to combination treatment with a SOS1 inhibitor (SOS1i), BI-3406, plus the KRASG12C inhibitor, adagrasib. We found that responses to BI-3406 plus adagrasib were stronger than to adagrasib alone, comparable to adagrasib with SHP2 (SHP2i) or EGFR inhibitors and correlated with stronger suppression of RAS-MAPK signaling. BI-3406 plus adagrasib treatment also delayed the emergence of acquired resistance and elicited antitumor responses from adagrasib-resistant models. Resistance to KRASG12Ci seemed to be driven by upregulation of MRAS activity, which both SOS1i and SHP2i were found to potently inhibit. Knockdown of SHOC2, a MRAS complex partner, partially restored response to KRASG12Ci treatment. These results suggest KRASG12C plus SOS1i to be a promising strategy for treating both KRASG12Ci naive and relapsed KRASG12C-mutant tumors.

Predicting the Intravenous Pharmacokinetics of Covalent Drugs in Animals and Humans

Rowan Stringer and Tobias Kaster

Journal of Medicinal Chemistry 2024

DOI: 10.1021/acs.jmedchem.4c00776

30 covalent drugs were used to assess clearance (CL) prediction reliability in animals and humans. In animals, marked CL underprediction was observed using cryopreserved hepatocytes or liver microsomes (LMs) supplemented for cytochrome P450 activity. Improved quantitative performance was observed by combining metabolic stability data from LMs and liver S9 fractions, the latter supplemented with reduced glutathione for glutathione transferase activity. While human LMs provided reliable human CL predictions, prediction statistics were improved further by incorporating S9 stability data. CL predictions with allometric scaling were less robust compared to in vitro drug metabolism methods; the best results were obtained using the fu-corrected intercept model. Human volume of distribution (Vd) was well predicted using allometric scaling of animal pharmacokinetic data; the most reliable results were achieved using simple allometric scaling of unbound Vd values. These results provide a quantitative framework to guide appropriate method selection for human PK prediction with covalent drugs.

Graph neural networks for identifying protein-reactive compounds

Victor Hugo Cano Gil and Christopher N. Rowley

Digital Discovery, 2024

https://pubs.rsc.org/en/content/articlelanding/2024/dd/d4dd00038b

The identification of protein-reactive electrophilic compounds is critical to the design of new covalent modifier drugs, screening for toxic compounds, and the exclusion of reactive compounds from high throughput screening. In this work, we employ traditional and graph machine learning (ML) algorithms to classify molecules being reactive towards proteins or nonreactive. For training data, we built a new dataset, ProteinReactiveDB, composed primarily of covalent and noncovalent inhibitors from the DrugBank, BindingDB, and CovalentInDB databases. To assess the transferability of the trained models, we created a custom set of covalent and noncovalent inhibitors, which was constructed from the recent literature. Baseline models were developed using Morgan fingerprints as training inputs, but they performed poorly when applied to compounds outside the training set. We then trained various Graph Neural Networks (GNNs), with the best GNN model achieving an Area Under the Receiver Operator Characteristic (AUROC) curve of 0.80, precision of 0.89, and recall of 0.72. We also explore the interpretability of these GNNs using Gradient Activation Mapping (GradCAM), which shows regions of the molecules GNNs deem most relevant when making a prediction. These maps indicated that our trained models can identify electrophilic functional groups in a molecule and classify molecules as protein-reactive based on their presence. We demonstrate the use of these models by comparing their performance against common chemical filters, identifying covalent modifiers in the ChEMBL database and generating a putative covalent inhibitor based on an established noncovalent inhibitor.

Sophisticated natural products as antibiotics

Lewis, K., Lee, R.E., Brötz-Oesterhelt, H. et al. 

Nature 632, 39–49 (2024). 

https://doi.org/10.1038/s41586-024-07530-w

In this Review, we explore natural product antibiotics that do more than simply inhibit an active site of an essential enzyme. We review these compounds to provide inspiration for the design of much-needed new antibacterial agents, and examine the complex mechanisms that have evolved to effectively target bacteria, including covalent binders, inhibitors of resistance, compounds that utilize self-promoted entry, those that evade resistance, prodrugs, target corrupters, inhibitors of ‘undruggable’ targets, compounds that form supramolecular complexes, and selective membrane-acting agents. These are exemplified by β-lactams that bind covalently to inhibit transpeptidases and β-lactamases, siderophore chimeras that hijack import mechanisms to smuggle antibiotics into the cell, compounds that are activated by bacterial enzymes to produce reactive molecules, and antibiotics such as aminoglycosides that corrupt, rather than merely inhibit, their targets. Some of these mechanisms are highly sophisticated, such as the preformed β-strands of darobactins that target the undruggable β-barrel chaperone BamA, or teixobactin, which binds to a precursor of peptidoglycan and then forms a supramolecular structure that damages the membrane, impeding the emergence of resistance. Many of the compounds exhibit more than one notable feature, such as resistance evasion and target corruption. Understanding the surprising complexity of the best antimicrobial compounds provides a roadmap for developing novel compounds to address the antimicrobial resistance crisis by mining for new natural products and inspiring us to design similarly sophisticated antibiotics.

Robust proteome profiling of cysteine-reactive fragments using label-free chemoproteomics

George S. Biggs, Emma E. Cawood, Aini Vuorinen, William J. McCarthy, Harry Wilders, Ioannis G. Riziotis, Antonie J. van der Zouwen, Jonathan Pettinger, Luke Nightingale, Peiling Chen, Andrew J. Powell, David House, Simon J. Boulton, J. Mark Skehel, Katrin Rittinger, Jacob T. Bush

bioRxiv 2024.07.25.605137; 

doi: https://doi.org/10.1101/2024.07.25.605137

Identifying pharmacological probes for human proteins represents a key opportunity to accelerate the discovery of new therapeutics. High-content screening approaches to expand the ligandable proteome offer the potential to expedite the discovery of novel chemical probes to study protein function. Screening libraries of reactive fragments by chemoproteomics offers a compelling approach to ligand discovery, however, optimising sample throughput, proteomic depth, and data reproducibility remains a key challenge.

We report a versatile, label-free quantification proteomics platform for competitive profiling of cysteine-reactive fragments against the native proteome. This high-throughput platform combines SP4 plate-based sample preparation with rapid chromatographic gradients. Data-independent acquisition performed on a Bruker timsTOF Pro 2 consistently identified ∼23,000 cysteine sites per run, with a total of ∼32,000 cysteine sites profiled in HEK293T and Jurkat lysate. Crucially, this depth in cysteinome coverage is met with high data completeness, enabling robust identification of liganded proteins.

In this study, 80 reactive fragments were screened in two cell lines identifying >400 ligand-protein interactions. Hits were validated through concentration-response experiments and the platform was utilised for hit expansion and live cell experiments. This label-free platform represents a significant step forward in high-throughput proteomics to evaluate ligandability of cysteines across the human proteome.

Template-assisted covalent modification underlies activity of covalent molecular glues

Yen-Der Li, Michelle W. Ma, Muhammad Murtaza Hassan, Moritz Hunkeler, Mingxing Teng, Kedar Puvar, Justine C. Rutter, Ryan J. Lumpkin, Brittany Sandoval, Cyrus Y. Jin, Anna M. Schmoker, Scott B. Ficarro, Hakyung Cheong, Rebecca J. Metivier, Michelle Y. Wang, Shawn Xu, Woong Sub Byun, Brian J. Groendyke, Inchul You, Logan H. Sigua, Isidoro Tavares, Charles Zou, Jonathan M. Tsai, Paul M. C. Park, Hojong Yoon, Felix C. Majewski, Haniya T. Sperling, Jarrod A. Marto, Jun Qi, Radosław P. Nowak, Katherine A. Donovan, Mikołaj Słabicki, Nathanael S. Gray, Eric S. Fischer & Benjamin L. Ebert

 Nat Chem Biol, 2024

https://doi.org/10.1038/s41589-024-01668-4

Molecular glues are proximity-inducing small molecules that have emerged as an attractive therapeutic approach. However, developing molecular glues remains challenging, requiring innovative mechanistic strategies to stabilize neoprotein interfaces and expedite discovery. Here we unveil a trans-labeling covalent molecular glue mechanism, termed ‘template-assisted covalent modification’. We identified a new series of BRD4 molecular glue degraders that recruit CUL4DCAF16 ligase to the second bromodomain of BRD4 (BRD4BD2). Through comprehensive biochemical, structural and mutagenesis analyses, we elucidated how pre-existing structural complementarity between DCAF16 and BRD4BD2 serves as a template to optimally orient the degrader for covalent modification of DCAF16Cys58. This process stabilizes the formation of BRD4–degrader–DCAF16 ternary complex and facilitates BRD4 degradation. Supporting generalizability, we found that a subset of degraders also induces GAK–BRD4BD2 interaction through trans-labeling of GAK. Together, our work establishes ‘template-assisted covalent modification’ as a mechanism for covalent molecular glues, which opens a new path to proximity-driven pharmacology.

Direct RAS inhibitors turn 10

 Ostrem, J.M.L., Peters, U. & Shokat, K.M

 Nat Chem Biol (2024). 

https://doi.org/10.1038/s41589-024-01691-5

RAS proteins, central drivers of cancer, appeared ‘undruggable’ for almost 30 years. Here we provide a personal perspective on the effort leading to our initial report of KRASG12C inhibitors in 2013, and the decade of discoveries that followed.

Direct RAS inhibitors turn 10

Restoring susceptibility to β-lactam antibiotics in methicillin-resistant Staphylococcus aureus

Van T. Nguyen, Biruk T. Birhanu, Vega Miguel-Ruano, Choon Kim, Mayte Batuecas, Jingdong Yang, Amr M. El-Araby, Eva Jiménez-Faraco, Valerie A. Schroeder, Alejandra Alba, Neha Rana, Safaa Sader, Caitlyn A. Thomas, Rhona Feltzer, Mijoon Lee, Jed F. Fisher, Juan A. Hermoso, Mayland Chang & Shahriar Mobashery 

Nat Chem Biol 2024

https://www.nature.com/articles/s41589-024-01688-0

Infections by Staphylococcus aureus have been treated historically with β-lactam antibiotics. However, these antibiotics have become obsolete in methicillin-resistant S. aureus by acquisition of the bla and mec operons. The presence of the β-lactam antibiotic is detected by the sensor domains of BlaR and/or MecR, and the information is transmitted to the cytoplasm, resulting in derepression of the antibiotic-resistance genes. We hypothesized that inhibition of the sensor domain would shut down this response system, and β-lactam susceptibility would be restored. An in silico search of 11 million compounds led to a benzimidazole-based hit and, ultimately, to the boronate 4. The X-ray structure of 4 is covalently engaged with the active-site serine of BlaR. Compound 4 potentiates by 16- to 4,096-fold the activities of oxacillin and of meropenem against methicillin-resistant S. aureus strains. The combination of 4 with oxacillin or meropenem shows efficacy in infected mice, validating the strategy.

Structural Dynamics of the Ubiquitin Specific Protease USP30 in Complex with a Cyanopyrrolidine-Containing Covalent Inhibitor

Darragh P O’Brien, Hannah BL Jones, Yuqi Shi, Franziska Guenther, Iolanda Vendrell, Rosa Viner, Paul E Brennan, Emma Mead, Tryfon Zarganes-Tzitzikas, John B Davis, Adán Pinto-Fernández, Katherine S England, Emma J Murphy, Andrew P Turnbull, Benedikt M Kessler

bioRxiv 2024.07.20.604388; 

doi: https://doi.org/10.1101/2024.07.20.604388

Inhibition of the mitochondrial deubiquitinating enzyme USP30 is neuroprotective and presents therapeutic opportunities for the treatment of idiopathic Parkinson’s Disease and mitophagy-related disorders. We have integrated structural and quantitative proteomics with biochemical assays to decipher the mode of action of covalent USP30 inhibition by a small molecule containing a cyanopyrrolidine reactive group, USP30-I-1. The inhibitor demonstrated high potency and selectivity for endogenous USP30 in neuroblastoma cells. Enzyme kinetics and Hydrogen Deuterium eXchange mass spectrometry (HDX-MS) infers that the inhibitor binds tightly to regions surrounding the USP30 catalytic cysteine and positions itself to form a binding pocket along the thumb and palm domains of the protein, thereby interfering its interaction with ubiquitin substrates. A comparison to a non-covalent USP30 inhibitor containing a benzosulfonamide scaffold revealed a slightly different binding mode closer to the active site Cys77, which may provide the molecular basis for improved selectivity towards USP30 against other members of the DUB enzyme family. Our results highlight advantages in developing covalent inhibitors, such as USP30-I-1, for targeting USP30 as treatment of disorders with impaired mitophagy.

DCAF16-Based Covalent Handle for the Rational Design of Monovalent Degraders

Melissa Lim, Thang Do Cong, Lauren M. Orr, Ethan S. Toriki, Andrew C. Kile, James W. Papatzimas, Elijah Lee, Yihan Lin, and Daniel K. Nomura

ACS Central Science 2024 10 (7), 1318-1331

DOI: 10.1021/acscentsci.4c00286

Targeted protein degradation with monovalent molecular glue degraders is a powerful therapeutic modality for eliminating disease causing proteins. However, rational design of molecular glue degraders remains challenging. In this study, we sought to identify a transplantable and linker-less covalent handle that could be appended onto the exit vector of various protein-targeting ligands to induce the degradation of their respective targets. Using the BET family inhibitor JQ1 as a testbed, we synthesized and screened a series of covalent JQ1 analogs and identified a vinylsulfonyl piperazine handle that led to the potent and selective degradation of BRD4 in cells. Through chemoproteomic profiling, we identified DCAF16 as the E3 ligase responsible for BRD4 degradation─an E3 ligase substrate receptor that has been previously covalently targeted for molecular glue-based degradation of BRD4. Interestingly, we demonstrated that this covalent handle can be transplanted across a diverse array of protein-targeting ligands spanning many different protein classes to induce the degradation of CDK4, the androgen receptor, BTK, SMARCA2/4, and BCR-ABL/c-ABL. Our study reveals a DCAF16-based covalent degradative and linker-less chemical handle that can be attached to protein-targeting ligands to induce the degradation of several different classes of protein targets.

Contribution of Noncovalent Recognition and Reactivity to the Optimization of Covalent Inhibitors: A Case Study on KRasG12C

Nikolett Péczka, Ivan Ranđelović, Zoltán Orgován, Noémi Csorba, Attila Egyed, László Petri, Péter Ábrányi-Balogh, Márton Gadanecz, András Perczel, József Tóvári, Gitta Schlosser, Tamás Takács, Levente M. Mihalovits, György G. Ferenczy, László Buday, and György M. Keserű

ACS Chemical Biology 2024

DOI: 10.1021/acschembio.4c00217

Covalent drugs might bear electrophiles to chemically modify their targets and have the potential to target previously undruggable proteins with high potency. Covalent binding of drug-size molecules includes a noncovalent recognition provided by secondary interactions and a chemical reaction leading to covalent complex formation. Optimization of their covalent mechanism of action should involve both types of interactions. Noncovalent and covalent binding steps can be characterized by an equilibrium dissociation constant (KI) and a reaction rate constant (kinact), respectively, and they are affected by both the warhead and the scaffold of the ligand. The relative contribution of these two steps was investigated on a prototypic drug target KRASG12C, an oncogenic mutant of KRAS. We used a synthetically more accessible nonchiral core derived from ARS-1620 that was equipped with four different warheads and a previously described KRAS-specific basic side chain. Combining these structural changes, we have synthesized novel covalent KRASG12C inhibitors and tested their binding and biological effect on KRASG12C by various biophysical and biochemical assays. These data allowed us to dissect the effect of scaffold and warhead on the noncovalent and covalent binding event. Our results revealed that the atropisomeric core of ARS-1620 is not indispensable for KRASG12C inhibition, the basic side chain has little effect on either binding step, and warheads affect the covalent reactivity but not the noncovalent binding. This type of analysis helps identify structural determinants of efficient covalent inhibition and may find use in the design of covalent agents.

High-Throughput Covalent Modifier Screening with Acoustic Ejection Mass Spectrometry

Xiujuan Wen, Chang Liu, Kiersten Tovar, Patrick Curran, Matthew Richards, Sony Agrawal, Richard Johnstone, Ryan E. Loy, Joey L. Methot, My Sam Mansueto, Markus Koglin, Mary Jo Wildey, Lyle Burton, Thomas R. Covey, Kevin P. Bateman, Michael Kavana, and David G. McLaren

Journal of the American Chemical Society 2024 

https://pubs.acs.org/doi/abs/10.1021/jacs.4c02377

Interests in covalent drugs have grown in modern drug discovery as they could tackle challenging targets traditionally considered “undruggable”. The identification of covalent binders to target proteins typically involves directly measuring protein covalent modifications using high-resolution mass spectrometry. With a continually expanding library of compounds, conventional mass spectrometry platforms such as LC–MS and SPE-MS have become limiting factors for high-throughput screening. Here, we introduce a prototype high-resolution acoustic ejection mass spectrometry (AEMS) system for the rapid screening of a covalent modifier library comprising ∼10,000 compounds against a 50 kDa-sized target protein─Werner syndrome helicase. The screening samples were arranged in a 1536-well format. The sample buffer containing high-concentration salts was directly analyzed without any cleanup steps, minimizing sample preparation efforts and ensuring protein stability. The entire AEMS analysis process could be completed within a mere 17 h. An automated data analysis tool facilitated batch processing of the sample data and quantitation of the formation of various covalent protein–ligand adducts. The screening results displayed a high degree of fidelity, with a Z′ factor of 0.8 and a hit rate of 2.3%. The identified hits underwent orthogonal testing in a biochemical activity assay, revealing that 75% were functional antagonists of the target protein. Notably, a comparative analysis with LC–MS showcased the AEMS platform’s low risk of false positives or false negatives. This innovative platform has enabled robust high-throughput covalent modifier screening, featuring a 10-fold increase in library size and a 10- to 100-fold increase in throughput when compared with similar reports in the existing literature.

Covalent isothiocyanate inhibitors of macrophage migration inhibitory factor as potential colorectal cancer treatments

Joel Tyndall, Lohitha Putha, Liang K. Kok, Matthias Fellner, Malcolm T. Rutledge, Allan B. Gamble, Sigurd M. Wilbanks, Andrea J. Vernall,

 ChemMedChem 2024, e202400394.

https://doi.org/10.1002/cmdc.202400394

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with roles in innate and adaptive human immune responses, as well as inflammation. MIF exerts its biological activity by binding to the cell surface receptor CD74 as well as intracellular signalling proteins. MIF also possesses keto-enol tautomerase activity. Inhibition of the tautomerase activity has been associated with loss of biological activity of MIF and a potential anticancer target. Isothiocyanates (ITCs) are a class of compounds present in cruciferous vegetables that inhibit the MIF tautomerase activity via covalent modification of the N-terminal proline. A range of substituted ITCs featuring benzyl, phenethyl and phenyl propyl isothiocyanates were designed, synthesised and tested to determine any structure activity relationship for inhibiting MIF. Crystal structures of covalent compounds 8 and 9 in complex with rhMIF revealed key hydrogen bonding and edge-to-face π stacking interactions. Compound 9 and 11 with sub micromolar activity were tested in the NCI60 cancer cell lines panel. Both compounds showed tissue-specific reduced growth in colon and renal cancer cell lines, while one of these showed potent, dose-dependent inhibition of growth against all seven colon cancer cell lines (GI50 < 2.5 µM) and all eight renal cancer cell lines (GI50 < 2.2 µM).

Recruitment of FBXO22 for targeted degradation of NSD2 [@CherylArrowsmi1]

Nie, D.Y., Tabor, J.R., Li, J. et al.

Nat Chem Biol (2024). 

https://doi.org/10.1038/s41589-024-01660-y

Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain-of-function mutation p.E1099K, resulting in growth suppression, apoptosis and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 to recruit the SCFFBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCFFBXO22. Overall, we present a potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a new FBXO22-recruitment strategy for TPD.

Evaluation of a Covalent Library of Diverse Warheads (CovLib) Binding to JNK3, USP7, or p53

Klett, T., Schwer, M., Ernst, L. N., Engelhardt, M. U., Jaag, S. J., Masberg, B., … Boeckler, F. M.

Drug Design, Development and Therapy, 2024 18, 2653–2679. https://doi.org/10.2147/DDDT.S466829

Purpose

Over the last few years, covalent fragment-based drug discovery has gained significant importance. Thus, striving for more warhead diversity, we conceived a library consisting of 20 covalently reacting compounds. Our covalent fragment library (CovLib) contains four different warhead classes, including five α-cyanoacacrylamides/acrylates (CA), three epoxides (EO), four vinyl sulfones (VS), and eight electron-deficient heteroarenes with a leaving group (SNAr/SN).

Methods

After predicting the theoretical solubility of the fragments by LogP and LogS during the selection process, we determined their experimental solubility using a turbidimetric solubility assay. The reactivities of the different compounds were measured in a high-throughput 5,5'-dithiobis-(2-nitrobenzoic acid) DTNB assay, followed by a (glutathione) GSH stability assay. We employed the CovLib in a (differential scanning fluorimetry) DSF-based screening against different targets: c-Jun N-terminal kinase 3 (JNK3), ubiquitin-specific protease 7 (USP7), and the tumor suppressor p53. Finally, the covalent binding was confirmed by intact protein mass spectrometry (MS).

Results

In general, the purchased fragments turned out to be sufficiently soluble. Additionally, they covered a broad spectrum of reactivity. All investigated α-cyanoacrylamides/acrylates and all structurally confirmed epoxides turned out to be less reactive compounds, possibly due to steric hindrance and reversibility (for α-cyanoacrylamides/acrylates). The SNAr and vinyl sulfone fragments are either highly reactive or stable. DSF measurements with the different targets JNK3, USP7, and p53 identified reactive fragment hits causing a shift in the melting temperatures of the proteins. MS confirmed the covalent binding mode of all these fragments to USP7 and p53, while additionally identifying the SNAr-type electrophile SN002 as a mildly reactive covalent hit for p53.

Conclusion

The screening and target evaluation of the CovLib revealed first interesting hits. The highly cysteine-reactive fragments VS004, SN001, SN006, and SN007 covalently modify several target proteins and showed distinct shifts in the melting temperatures up to +5.1 °C and −9.1 °C.

Prolonged Activation of the GLP-1 Receptor via Covalent Capture

Özge Ünsal, Z. Selin Bacaksiz, Vladislav Khamraev, Vittorio Montanari, Martin Beinborn, and Krishna Kumar

ACS Chemical Biology 2024

DOI: https://doi.org/10.1021/acschembio.4c00039

The incretin gut hormone glucagon-like peptide-1 (GLP-1) has become a household name because of its ability to induce glucose-dependent insulin release with accompanying weight loss in patients. Indeed, derivatives of the peptide exert numerous pleiotropic actions that favorably affect other metabolic functions, and consequently, such compounds are being considered as treatments for a variety of ailments. The ability of native GLP-1 to function as a clinical drug is severely limited because of its short half-life in vivo. All of the beneficial effects of GLP-1 come from its agonism at the cognate receptor, GLP-1R. In our quest for long-lived activation of the receptor, we hypothesized that an agonist that had the ability to covalently cross-link with GLP-1R would prove useful. We here report the structure-guided design of peptide analogues containing an electrophilic warhead that could be covalently captured by a resident native nucleophile on the receptor. The compounds were evaluated using washout experiments, and resistance to such washing serves as an index of prolonged activation and covalent capture, which we use to tabulate longevity and robust long-lived GLP-1R agonism. The addition of SulF (cross-linkable warhead), an N-terminal trifluoroethyl group (for protease protection), and a C18 diacid lipid (protractor) all contributed to the increased wash resistance of GLP-1. The most effective compound based on the wash resistance metric, C2K26DAC18_K34SulF, has all three elements outlined and may serve as a blueprint and a proof-of-concept scaffold for the design of clinically useful molecules.