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.