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.