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.

Single-Agent Divarasib (GDC-6036) in Solid Tumors with a KRAS G12C Mutation

N Engl J Med 2023; 389: 710–721
DOI: 10.1056/NEJMoa2303810

BACKGROUND

Divarasib (GDC-6036) is a covalent KRAS G12C inhibitor that was designed to have high potency and selectivity.

METHODS

In a phase 1 study, we evaluated divarasib administered orally once daily (at doses ranging from 50 to 400 mg) in patients who had advanced or metastatic solid tumors that harbor a KRAS G12C mutation. The primary objective was an assessment of safety; pharmacokinetics, investigator-evaluated antitumor activity, and biomarkers of response and resistance were also assessed.

RESULTS

A total of 137 patients (60 with non–small-cell lung cancer [NSCLC], 55 with colorectal cancer, and 22 with other solid tumors) received divarasib. No dose-limiting toxic effects or treatment-related deaths were reported. Treatment-related adverse events occurred in 127 patients (93%); grade 3 events occurred in 15 patients (11%) and a grade 4 event in 1 patient (1%). Treatment-related adverse events resulted in a dose reduction in 19 patients (14%) and discontinuation of treatment in 4 patients (3%). Among patients with NSCLC, a confirmed response was observed in 53.4% of patients (95% confidence interval [CI], 39.9 to 66.7), and the median progression-free survival was 13.1 months (95% CI, 8.8 to could not be estimated). Among patients with colorectal cancer, a confirmed response was observed in 29.1% of patients (95% CI, 17.6 to 42.9), and the median progression-free survival was 5.6 months (95% CI, 4.1 to 8.2). Responses were also observed in patients with other solid tumors. Serial assessment of circulating tumor DNA showed declines in KRAS G12C variant allele frequency associated with response and identified genomic alterations that may confer resistance to divarasib.

CONCLUSIONS

Treatment with divarasib resulted in durable clinical responses across KRAS G12C–positive tumors, with mostly low-grade adverse events. (Funded by Genentech; ClinicalTrials.gov number, NCT04449874. opens in new tab.)

Development of LB244, an Irreversible STING Antagonist

 Leonard Barasa, Sauradip Chaudhuri, Jeffrey Y. Zhou, Zhaozhao Jiang, Shruti Choudhary, Robert Madison Green, Elenore Wiggin, Michael Cameron, Fiachra Humphries, Katherine A. Fitzgerald, and Paul R. Thompson

Journal of the American Chemical Society 2023

DOI: 10.1021/jacs.3c03637

The cGMP-AMP Synthase (cGAS)-Stimulator of Interferon Genes (STING) pathway plays a critical role in sensing dsDNA localized to the cytosol, resulting in the activation of a robust inflammatory response. While cGAS-STING signaling is essential for antiviral immunity, aberrant STING activation is observed in amyotrophic lateral sclerosis (ALS), lupus, and autoinflammatory diseases such as Aicardi-Goutières syndrome (AGS) and STING associated vasculopathy with onset in infancy (SAVI). Significant efforts have therefore focused on the development of STING inhibitors. In a concurrent submission, we reported that BB-Cl-amidine inhibits STING-dependent signaling in the nanomolar range, both in vitro and in vivo. Considering this discovery, we sought to generate analogs with higher potency and proteome-wide selectivity. Herein, we report the development of LB244, which displays nanomolar potency and inhibits STING signaling with markedly enhanced proteome-wide selectivity. Moreover, LB244 mirrored the efficacy of BB-Cl-amidine in vivo. In summary, our data identify novel chemical entities that inhibit STING signaling and provide a scaffold for the development of therapeutics for treating STING-dependent inflammatory diseases.

Phage Display of Two Distinct Warheads to Inhibit Challenging Proteins

Mengmeng Zheng and Jianmin Gao

ACS Chemical Biology 2023

DOI: 10.1021/acschembio.3c00297

Falling in between traditional small molecules and antibodies in size, peptides are emerging as a privileged therapeutic modality, one that can harness the benefits of both small molecule and antibody drugs. To discover potential peptide therapeutics, it is highly desirable to have high throughput screening platforms that can assess peptides with diverse and non-natural functional motifs. With this contribution, we present a novel phage library that incorporates two distinct designer groups. As an example, a pair of reversible covalent warheads was installed onto phage-displayed peptides to target a cysteine and a lysine. The double modification is realized by sequential modification of an N-terminal cysteine and then an internal cysteine using chemoselective chemistry. Screening of this double-warhead-presenting library against TEV protease readily revealed peptide inhibitors with single-digit micromolar potency. Importantly, our structure–activity studies demonstrate that both covalent warheads make important contributions to TEV protease inhibition. We envision that our strategy of double phage modification can be readily extended to build phage libraries with diverse structural motifs, allowing facile expansion of the chemical space coverable by phage display.

Structure-Based Optimization of Covalent, Small-Molecule Stabilizers of the 14-3-3σ/ERα Protein–Protein Interaction from Nonselective Fragments

Markella Konstantinidou, Emira J. Visser, Edmee Vandenboorn, Sheng Chen, Priyadarshini Jaishankar, Maurits Overmans, Shubhankar Dutta, R. Jeffrey Neitz, Adam R. Renslo, Christian Ottmann, Luc Brunsveld, and Michelle R. Arkin

Journal of the American Chemical Society 2023

DOI: 10.1021/jacs.3c05161

The stabilization of protein–protein interactions (PPIs) has emerged as a promising strategy in chemical biology and drug discovery. The identification of suitable starting points for stabilizing native PPIs and their subsequent elaboration into selective and potent molecular glues lacks structure-guided optimization strategies. We have previously identified a disulfide fragment that stabilized the hub protein 14-3-3σ bound to several of its clients, including ERα and C-RAF. Here, we show the structure-based optimization of the nonselective fragment toward selective and highly potent small-molecule stabilizers of the 14-3-3σ/ERα complex. The more elaborated molecular glues, for example, show no stabilization of 14-3-3σ/C-RAF up to 150 μM compound. Orthogonal biophysical assays, including mass spectrometry and fluorescence anisotropy, were used to establish structure–activity relationships. The binding modes of 37 compounds were elucidated with X-ray crystallography, which further assisted the concomitant structure-guided optimization. By targeting specific amino acids in the 14-3-3σ/ERα interface and locking the conformation with a spirocycle, the optimized covalent stabilizer 181 achieved potency, cooperativity, and selectivity similar to the natural product Fusicoccin-A. This case study showcases the value of addressing the structure, kinetics, and cooperativity for molecular glue development.

Cyanotriazoles are selective topoisomerase II poisons that rapidly cure trypanosome infections

Srinivasa P. S. Rao et al.

Science 2023, 380, 1349-1356

DOI:10.1126/science.adh0614

Millions who live in Latin America and sub-Saharan Africa are at risk of trypanosomatid infections, which cause Chagas disease and human African trypanosomiasis (HAT). Improved HAT treatments are available, but Chagas disease therapies rely on two nitroheterocycles, which suffer from lengthy drug regimens and safety concerns that cause frequent treatment discontinuation. We performed phenotypic screening against trypanosomes and identified a class of cyanotriazoles (CTs) with potent trypanocidal activity both in vitro and in mouse models of Chagas disease and HAT. Cryo–electron microscopy approaches confirmed that CT compounds acted through selective, irreversible inhibition of trypanosomal topoisomerase II by stabilizing double-stranded DNA:enzyme cleavage complexes. These findings suggest a potential approach toward successful therapeutics for the treatment of Chagas disease.

Discovery of AZD4747, a Potent and Selective Inhibitor of Mutant GTPase KRASG12C with Demonstrable CNS Penetration

Jason G. Kettle, Sharan K. Bagal, Derek Barratt, Michael S. Bodnarchuk, Scott Boyd, Erin Braybrooke, Jason Breed, Doyle J. Cassar, Sabina Cosulich, Michael Davies, Nichola L. Davies, Chao Deng, Andrew Eatherton, Laura Evans, Lyman J. Feron, Shaun Fillery, Emma S. Gleave, Frederick W. Goldberg, Miguel A. Cortés González, Carine Guerot, Afreen Haider, Stephanie Harlfinger, Rachel Howells, Anne Jackson, Peter Johnström, Paul D. Kemmitt, Alex Koers, Mikhail Kondrashov, Gillian M. Lamont, Scott Lamont, Hilary J. Lewis, Libin Liu, Megan Mylrea, Samuel Nash, Michael J. Niedbala, Alison Peter, Christopher Phillips, Kurt Pike, Piotr Raubo, Graeme R. Robb, Sarah Ross, Matthew G. Sanders, Magnus Schou, Iain Simpson, and Oliver Steward

Journal of Medicinal Chemistry 2023 66 (13), 9147-9160

DOI: 10.1021/acs.jmedchem.3c00746

The glycine to cysteine mutation at codon 12 of Kirsten rat sarcoma (KRAS) represents an Achilles heel that has now rendered this important GTPase druggable. Herein, we report our structure-based drug design approach that led to the identification of 14, AZD4747, a clinical development candidate for the treatment of KRASG12C-positive tumors, including the treatment of central nervous system (CNS) metastases. Building on our earlier discovery of C5-tethered quinazoline AZD4625, excision of a usually critical pyrimidine ring yielded a weak but brain-penetrant start point which was optimized for potency and DMPK. Key design principles and measured parameters that give high confidence in CNS exposure are discussed. During optimization, divergence between rodent and non-rodent species was observed in CNS exposure, with primate PET studies ultimately giving high confidence in the expected translation to patients. AZD4747 is a highly potent and selective inhibitor of KRASG12C with an anticipated low clearance and high oral bioavailability profile in humans.