Covalent and noncovalent strategies for targeting Lys102 in HIV-1 reverse transcriptase

Giavana R. Prucha, Sean Henry, Klarissa Hollander, Zachary J. Carter, Krasimir A. Spasov, William L. Jorgensen, Karen S. Anderson,

European Journal of Medicinal Chemistry, 2023, 262, 115894, 

https://doi.org/10.1016/j.ejmech.2023.115894

Reverse transcriptase (RT) is one of three key proteins responsible for the replication cycle of HIV-1 in the host. Several classes of inhibitors have been developed to target the enzyme, with non-nucleoside reverse transcriptase inhibitors forming first-line treatment. Previously, covalent RT inhibitors have been identified and found to bind irreversibly to commonly mutated residues such as Y181C. In this work we aim to circumvent the issue of NNRTI resistance through targeting K102, which has not yet been identified to confer drug resistance. As reported here, 34 compounds were synthesized and characterized biochemically and structurally with wild-type (WT) HIV-1 RT. Two of these inhibitors demonstrate covalent inhibition as evidenced by protein crystallography, enzyme kinetics, mass spectrometry, and antiviral potency in HIV-1 infected human T-cell assays.

DrugMap: A quantitative pan-cancer analysis of cysteine ligandability

Mariko Takahashi, Harrison B. Chong, Siwen Zhang, Matthew Lazarov, Stefan Harry, Michelle Maynard, Ryan White, Brendan Hilbert, Magdy Gohar, Maolin Ge, Junbing Zhang, Benedikt Ralf Durr, Gregory Kryukov, Chih-Chiang Tsou, Natasja Brooijmans, Aliyu Alghali, Karla Rubio, Antonio Vilanueva, Drew Harrison, Ann-Sophie Koglin, Samuel Ojeda, Barbara Karakyriakou, Alexander Healy, Jonathan Assaad, Farah Makram, Inbal Rachimin, Neha Khandelwal, Pei-Chieh Tien, George Popoola, Nicholas Chen, Kira Vordermark, Marianne Richter, Himani Patel, Tzu-yi Yang, Hanna Griesshaber, Tobias Hosp, Sanne van den Ouweland, Toshiro Hara, Lily Bussema, Lei Shi, Martin Rasmussen, Ana Carolina Domingues, Aleigha Lawless, Jacy Fang, Satoshi Yoda, Linh Phuong Nguyen, Sarah Marie Reeves, Farrah Nicole Wakefield, Adam Acker, Sarah Elizabeth Clark, Taronish Dubash, David E Fisher, Shyamala Maheswaran, Daniel Haber, Genevieve Boland, Moshe Sade-Feldman, Russ Jenkins, Aaron Hata, Nabeel Bardeesy, Mario Suva, Brent Martin, Brian Liau, Chris Ott, Miguel Rivera, Michael Lawrence, Liron Bar-Peled

bioRxiv 2023.10.20.563287; 

doi: https://doi.org/10.1101/2023.10.20.563287

Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors of a wide-range of targets in cancer. However, how different oncogenic contexts influence cysteine targeting remains unknown. To address this question, we have developed DrugMap, an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NFκB1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NFkB1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting, and illustrate the use of covalent probes to disrupt oncogenic transcription factor activity.

Covalent Stapling of the Cereblon Sensor Loop Histidine Using Sulfur-Heterocycle Exchange

Justin T. Cruite, Radosław P. Nowak, Katherine A. Donovan, Scott B. Ficarro, Huang Huang, Hu Liu, Yingpeng Liu, Jarrod A. Marto, Rebecca J. Metivier, Eric S. Fischer, and Lyn H. Jones

ACS Medicinal Chemistry Letters 2023

DOI: 10.1021/acsmedchemlett.3c00371

Site-specific modification of amino acid residues in protein binding pockets using sulfonyl exchange chemistry expands the druggable proteome by enabling the development of covalent modulators that target residues beyond cysteine. Sulfonyl fluoride and triazole electrophiles were incorporated previously into the cereblon (CRBN) molecular glue degrader EM12, to covalently engage His353 within the CRBN sensor loop, but these probes had poor human plasma stability. Attenuation of intrinsic reactivity through the development of sulfonyl pyrazoles, imidazoles, and nucleobases enhanced plasma stability, and several compounds retained efficient labeling of His353. For example, sulfonyl imidazole EM12-SO2Im covalently blocked the CRBN binding site and possessed excellent metabolic stability in human plasma, liver microsomes, and hepatocytes. These results highlight the potential suitability of sulfonyl imidazole and related sulfur(VI)-diazole exchange (SuDEx) warheads for covalent drug development and further exemplify the therapeutic promise of site-specific histidine targeting.

Exploiting the Cullin E3 Ligase Adaptor Protein SKP1 for Targeted Protein Degradation

Seong Ho Hong, Akane Osa, Ingrid E Wertz, Daniel Nomura

bioRxiv 2023.10.20.563371; 

doi: https://doi.org/10.1101/2023.10.20.563371

Targeted protein degradation with Proteolysis Targeting Chimeras (PROTACs) is a powerful therapeutic modality for eliminating disease-causing proteins through targeted ubiquitination and proteasome-mediated degradation. Most PROTACs have exploited substrate receptors of Cullin-RING E3 ubiquitin ligases such as cereblon and VHL. Whether core, shared, and essential components of the Cullin-RING E3 ubiquitin ligase complex can be used for PROTAC applications remains less explored. Here, we discovered a cysteine-reactive covalent recruiter EN884 against the SKP1 adapter protein of the SKP1-CUL1-F-box containing SCF complex. We further showed that this recruiter can be used in PROTAC applications to degrade neo-substrate proteins such as BRD4 and the androgen receptor in a SKP1- and proteasome-dependent manner. Our studies demonstrate that core and essential adapter proteins within the Cullin-RING E3 ubiquitin ligase complex can be exploited for targeted protein degradation applications and that covalent chemoproteomic strategies can enable recruiter discovery against these targets.

Defining the Cell Surface Cysteinome using Two-step Enrichment Proteomics

Tianyang Yan, Lisa Boatner, Liujuan Cui, Peter Tontonoz, Keriann Backus

bioRxiv 2023.10.17.562832; 

doi: https://doi.org/10.1101/2023.10.17.562832

The plasma membrane proteome is a rich resource of functional and therapeutically relevant protein targets. Distinguished by high hydrophobicity, heavy glycosylation, disulfide-rich sequences, and low overall abundance, the cell surface proteome remains undersampled in established proteomic pipelines, including our own cysteine chemoproteomics platforms. Here we paired cell surface glycoprotein capture with cysteine chemoproteomics to establish a two-stage enrichment method that enables chemoproteomic profiling of cell Surface Cysteinome. Our Cys-Surf platform captures >2,800 total membrane protein cysteines in 1,046 proteins, including 1,907 residues not previously captured by bulk proteomic analysis. By pairing Cys-Surf with an isotopic chemoproteomic readout, we uncovered 821 total ligandable cysteines, including known and novel sites. Cys-Surf also robustly delineates redox-sensitive cysteines, including cysteines prone to activation-dependent changes to cysteine oxidation state and residues sensitive to addition of exogenous reductants. Exemplifying the capacity of Cys-Surf to delineate functionally important cysteines, we identified a redox sensitive cysteine in the low-density lipoprotein receptor (LDLR) that impacts both the protein localization and uptake of LDL particles. Taken together, the Cys-Surf platform, distinguished by its two-stage enrichment paradigm, represents a tailored approach to delineate the functional and therapeutic potential of the plasma membrane cysteinome. 

Ultra-rapid Electrophilic Cysteine Arylation [@WangGroupURICHM]

Bradley M. Lipka, Daniel S. Honeycutt, Gregory M. Bassett, Taylor N. Kowal, Max Adamczyk, Zachary C. Cartnick, Vincent M. Betti, Jacob M. Goldberg, and Fang Wang

Journal of the American Chemical Society 2023

DOI: 10.1021/jacs.3c10334

Rapid bond-forming reactions are crucial for efficient bioconjugation. We describe a simple and practical strategy for facilitating ultra-rapid electrophilic cysteine arylation. Using a variety of sulfone-activated pyridinium salts, this uncatalyzed reaction proceeds with exceptionally high rate constants, ranging from 9800 to 320,000 M–1·s–1, in pH 7.0 aqueous buffer at 25 °C. Such reactions allow for stoichiometric bioconjugation of micromolar cysteine within minutes or even seconds. Even though the arylation is extremely fast, the chemistry exhibits excellent selectivity, thus furnishing functionalized peptides and proteins with both high conversion and purity.

Structure-based design of a phosphotyrosine-masked covalent ligand targeting the E3 ligase SOCS2

Ramachandran, S., Makukhin, N., Haubrich, K. et al.

 Nat Commun 14, 6345 (2023).

https://doi.org/10.1038/s41467-023-41894-3

The Src homology 2 (SH2) domain recognizes phosphotyrosine (pY) post translational modifications in partner proteins to trigger downstream signaling. Drug discovery efforts targeting the SH2 domains have long been stymied by the poor drug-like properties of phosphate and its mimetics. Here, we use structure-based design to target the SH2 domain of the E3 ligase suppressor of cytokine signaling 2 (SOCS2). Starting from the highly ligand-efficient pY amino acid, a fragment growing approach reveals covalent modification of Cys111 in a co-crystal structure, which we leverage to rationally design a cysteine-directed electrophilic covalent inhibitor MN551. We report the prodrug MN714 containing a pivaloyloxymethyl (POM) protecting group and evidence its cell permeability and capping group unmasking using cellular target engagement and in-cell 19F NMR spectroscopy. Covalent engagement at Cys111 competitively blocks recruitment of cellular SOCS2 protein to its native substrate. The qualified inhibitors of SOCS2 could find attractive applications as chemical probes to understand the biology of SOCS2 and its CRL5 complex, and as E3 ligase handles in proteolysis targeting chimera (PROTACs) to induce targeted protein degradation.

Exploration of the Tunability of BRD4 Degradation by DCAF16 Trans-labelling Covalent Glues

Muhammad Murtaza Hassan, Yen-Der Li, Michelle W. Ma, Mingxing Teng, Woong Sub Byun, Kedar Puvar, Ryan Lumpkin, Brittany Sandoval, Justine C. Rutter, Cyrus Y. Jin, Michelle Y. Wang, Shawn Xu, Anna M. Schmoker, Hakyung Cheong, Brian J. Groendyke, Jun Qi, Eric S Fischer, Benjamin L. Ebert, Nathanael Gray

bioRxiv 2023

https://www.biorxiv.org/content/10.1101/2023.10.07.561308v1

Small molecules that can induce protein degradation by inducing proximity between a desired target and an E3 ligase have the potential to greatly expand the number of proteins that can be manipulated pharmacologically. Current strategies for targeted protein degradation are mostly limited in their target scope to proteins with preexisting ligands. Alternate modalities such as molecular glues, as exemplified by the glutarimide class of ligands for the CUL4CRBN ligase, have been mostly discovered serendipitously. We recently reported a trans-labelling covalent glue mechanism which we named Template-assisted covalent modification, where an electrophile decorated small molecule binder of BRD4 was effectively delivered to a cysteine residue on an E3 ligase DCAF16 as a consequence of a BRD4-DCAF16 protein-protein interaction. Herein, we report our medicinal chemistry efforts to evaluate how various electrophilic modifications to the BRD4 binder, JQ1, affect DCAF16 trans-labeling and subsequent BRD4 degradation efficiency. We discovered a decent correlation between the ability of the electrophilic small molecule to induce ternary complex formation between BRD4 and DCAF16 with its ability to induce BRD4 degradation. Moreover, we show that a more solvent-exposed warhead presentation is optimal for DCAF16 recruitment and subsequent BRD4 degradation. Unlike the sensitivity of CUL4CRBN glue degraders to chemical modifications, the diversity of covalent attachments in this class of BRD4 glue degraders suggests a high tolerance and tunability for the BRD4-DCAF16 interaction. This offers a potential new avenue for a rational design of covalent glue degraders by introducing covalent warheads to known binders.

Biomimetic Synthesis and Chemical Proteomics Reveal the Mechanism of Action and Functional Targets of Phloroglucinol Meroterpenoids [@Abbasov_Cornell]

Bracken, A.; Gekko, C.; Suss, N.; Lueders, E.; Cui, Q.; Fu, Q.; Anderson, E.; Zhang, S.; Abbasov, M. 

ChemRxiv 2023

https://doi.org/10.26434/chemrxiv-2023-snx9h

Natural products perennially serve as prolific sources of drug leads and chemical probes, fueling the development of numerous therapeutics. Despite their scarcity, natural products that modulate protein function through covalent interactions with lysine residues hold immense potential to unlock new therapeutic interventions and advance our understanding of the biological processes governed by these modifications. Phloroglucinol meroterpenoids constitute one of the most expansive classes of natural products, displaying a plethora of biological activities. However, their mechanism of action and cellular targets have, until now, remained elusive. In this study, we detail the concise biomimetic synthesis, computational mechanistic insights, physicochemical attributes, kinetic parameters, molecular mechanism of action, and functional cellular targets of several phloroglucinol meroterpenoids. We harness synthetic clickable analogues of natural products to probe their disparate proteome-wide reactivity and subcellular localization through in-gel fluorescence scanning and cell imaging. By implementing sample multiplexing and a redesigned lysine-targeting probe, we streamline a quantitative activity-based protein profiling, enabling the direct mapping of global reactivity and ligandability of proteinaceous lysines in human cells. Leveraging this framework, we identify numerous lysine-meroterpenoid interactions in breast cancer cells at tractable protein sites across diverse structural and functional classes, including those historically deemed undruggable. We validate that phloroglucinol meroterpenoids perturb biochemical functions through stereoselective and site-specific modification of lysines in proteins vital for breast cancer metabolism, including lipid signaling, mitochondrial respiration, and glycolysis. These findings underscore the broad potential of phloroglucinol meroterpenoids for targeting functional lysines in the human proteome 

Advancing protein therapeutics through proximity-induced chemistry

Linqi Cheng, Yixian Wang, Yiming Guo, Sophie S. Zhang, Han Xiao,

Cell Chemical Biology, 2023

https://doi.org/10.1016/j.chembiol.2023.09.004

Recent years have seen a remarkable growth in the field of protein-based medical treatments. Nevertheless, concerns have arisen regarding the cytotoxicity limitations, low affinity, potential immunogenicity, low stability, and challenges to modify these proteins. To overcome these obstacles, proximity-induced chemistry has emerged as a next-generation strategy for advancing protein therapeutics. This method allows site-specific modification of proteins with therapeutic agents, improving their effectiveness without extensive engineering. In addition, this innovative approach enables spatial control of the reaction based on proximity, facilitating the formation of irreversible covalent bonds between therapeutic proteins and their targets. This capability becomes particularly valuable in addressing challenges such as the low affinity frequently encountered between therapeutic proteins and their targets, as well as the limited availability of small molecules for specific protein targets. As a result, proximity-induced chemistry is reshaping the field of protein drug preparation and propelling the revolution in novel protein therapeutics.

Covalent fragment approaches targeting non-cysteine residues

Noémi Csorba,Péter Ábrányi-Balogh,György M. Keserű

Trends in Pharmacological Sciences, 2023

https://doi.org/10.1016/j.tips.2023.08.014

Covalent fragment approaches combine advantages of covalent binders and fragment-based drug discovery (FBDD) for target identification and validation. Although early applications focused mostly on cysteine labeling, the chemistries of available warheads that target other orthosteric and allosteric protein nucleophiles has recently been extended. The range of different warheads and labeling chemistries provide unique opportunities for screening and optimizing warheads necessary for targeting non-cysteine residues. In this review, we discuss these recently developed amino-acid-specific and promiscuous warheads, as well as emerging labeling chemistries, which includes novel transition metal catalyzed, photoactive, electroactive, and noncatalytic methodologies. We also highlight recent applications of covalent fragments for the development of molecular glues and proteolysis-targeting chimeras (PROTACs), and their utility in chemical proteomics-based target identification and validation.

Chemical Specification of E3 Ubiquitin Ligase Engagement by Cysteine-Reactive Chemistry

Roman C. Sarott, Inchul You, Yen-Der Li, Sean T. Toenjes, Katherine A. Donovan, Pooreum Seo, Martha Ordonez, Woong Sub Byun, Muhammad Murtaza Hassan, Franziska Wachter, Edward T. Chouchani, Mikołaj Słabicki, Eric S. Fischer, Benjamin L. Ebert, Stephen M. Hinshaw, and Nathanael S. Gray

Journal of the American Chemical Society Article ASAP

DOI: 10.1021/jacs.3c06622

Targeted protein degradation relies on small molecules that induce new protein–protein interactions between targets and the cellular protein degradation machinery. Most of these small molecules feature specific ligands for ubiquitin ligases. Recently, the attachment of cysteine-reactive chemical groups to pre-existing small molecule inhibitors has been shown to drive specific target degradation. We demonstrate here that different cysteine-reactive groups can specify target degradation via distinct ubiquitin ligases. By focusing on the bromodomain ligand JQ1, we identify cysteine-reactive functional groups that drive BRD4 degradation by either DCAF16 or DCAF11. Unlike proteolysis-targeting chimeric molecules (PROTACs), the new compounds use a single small molecule ligand with a well-positioned cysteine-reactive group to induce protein degradation. The finding that nearly identical compounds can engage multiple ubiquitination pathways suggests that targeting cellular pathways that search for and eliminate chemically reactive proteins is a feasible avenue for converting existing small molecule drugs into protein degrader molecules.

Assigning functionality to cysteines by base editing of cancer dependency genes [@davidrliu]

Haoxin Li, Tiantai Ma, Jarrett R. Remsberg, Sang Joon Won, Kristen E. DeMeester, Evert Njomen, Daisuke Ogasawara, Kevin T. Zhao, Tony P. Huang, Bingwen Lu, Gabriel M. Simon, Bruno Melillo, Stuart L. Schreiber, Jens Lykke-Andersen, David R. Liu & Benjamin F. Cravatt 

Nat Chem Biol, 2023

https://doi.org/10.1038/s41589-023-01428-w

Covalent chemistry represents an attractive strategy for expanding the ligandability of the proteome, and chemical proteomics has revealed numerous electrophile-reactive cysteines on diverse human proteins. Determining which of these covalent binding events affect protein function, however, remains challenging. Here we describe a base-editing strategy to infer the functionality of cysteines by quantifying the impact of their missense mutation on cancer cell proliferation. The resulting atlas, which covers more than 13,800 cysteines on more than 1,750 cancer dependency proteins, confirms the essentiality of cysteines targeted by covalent drugs and, when integrated with chemical proteomic data, identifies essential, ligandable cysteines in more than 160 cancer dependency proteins. We further show that a stereoselective and site-specific ligand targeting an essential cysteine in TOE1 inhibits the nuclease activity of this protein through an apparent allosteric mechanism. Our findings thus describe a versatile method and valuable resource to prioritize the pursuit of small-molecule probes with high function-perturbing potential.