Wednesday, March 13, 2024

An orally bioavailable SARS-CoV-2 main protease inhibitor exhibits improved affinity and reduced sensitivity to mutations

Michael Westberg et al.

Sci. Transl. Med.16,eadi0979(2024).

DOI:10.1126/scitranslmed.adi0979

Inhibitors of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) such as nirmatrelvir (NTV) and ensitrelvir (ETV) have proven effective in reducing the severity of COVID-19, but the presence of resistance-conferring mutations in sequenced viral genomes raises concerns about future drug resistance. Second-generation oral drugs that retain function against these mutants are thus urgently needed. We hypothesized that the covalent hepatitis C virus protease inhibitor boceprevir (BPV) could serve as the basis for orally bioavailable drugs that inhibit SARS-CoV-2 Mpro more efficiently than existing drugs. Performing structure-guided modifications of BPV, we developed a picomolar-affinity inhibitor, ML2006a4, with antiviral activity, oral pharmacokinetics, and therapeutic efficacy similar or superior to those of NTV. A crucial feature of ML2006a4 is a derivatization of the ketoamide reactive group that improves cell permeability and oral bioavailability. Last, ML2006a4 was found to be less sensitive to several mutations that cause resistance to NTV or ETV and occur in the natural SARS-CoV-2 population. Thus, anticipatory design can preemptively address potential resistance mechanisms to expand future treatment options against coronavirus variants.



Wednesday, March 6, 2024

Strain-release alkylation of Asp12 enables mutant selective targeting of K-Ras-G12D

Qinheng Zheng, Ziyang Zhang, Keelan Z. Guiley & Kevan M. Shokat

Nat Chem Biol 2024

 https://doi.org/10.1038/s41589-024-01565-w

K-Ras is the most commonly mutated oncogene in human cancer. The recently approved non-small cell lung cancer drugs sotorasib and adagrasib covalently capture an acquired cysteine in K-Ras-G12C mutation and lock it in a signaling-incompetent state. However, covalent inhibition of G12D, the most frequent K-Ras mutation particularly prevalent in pancreatic ductal adenocarcinoma, has remained elusive due to the lack of aspartate-targeting chemistry. Here we present a set of malolactone-based electrophiles that exploit ring strain to crosslink K-Ras-G12D at the mutant aspartate to form stable covalent complexes. Structural insights from X-ray crystallography and exploitation of the stereoelectronic requirements for attack of the electrophile allowed development of a substituted malolactone that resisted attack by aqueous buffer but rapidly crosslinked with the aspartate-12 of K-Ras in both GDP and GTP state. The GTP-state targeting allowed effective suppression of downstream signaling, and selective inhibition of K-Ras-G12D-driven cancer cell proliferation in vitro and xenograft growth in mice.



Friday, March 1, 2024

Chemical tools to expand the ligandable proteome: diversity-oriented synthesis-based photoreactive stereoprobes

Daisuke Ogasawara, David Konrad, Zher Yin Tan, Kimberly Carey, Jessica Luo, Sang Joon Won, Haoxin Li, Trever Carter, Kristen DeMeester, Evert Njomen, Stuart Schreiber, Ramnik Xavier, Bruno Melillo, Benjamin Cravatt

bioRxiv 2024.02.27.582206; 

doi: https://doi.org/10.1101/2024.02.27.582206

Chemical proteomics enables the global assessment of small molecule-protein interactions in native biological systems and has emerged as a versatile approach for ligand discovery. The range of small molecules explored by chemical proteomics has, however, been limited. Here, we describe a diversity-oriented synthesis (DOS)-inspired library of stereochemically-defined compounds bearing diazirine and alkyne units for UV light-induced covalent modification and click chemistry enrichment of interacting proteins, respectively. We find that these photo-stereoprobes interact in a stereoselective manner with hundreds of proteins from various structural and functional classes in human cells and demonstrate that these interactions can form the basis for high-throughput screening-compatible nanoBRET assays. Integrated phenotypic analysis and chemical proteomics identified photo-stereoprobes that modulate autophagy by engaging the mitochondrial serine protease CLPP. Our findings show the utility of photo-stereoprobes for expanding the ligandable proteome, furnishing target engagement assays, and discovering and characterizing bioactive small molecules by cell-based screening.


Formaldehyde regulates S-adenosylmethionine biosynthesis and one-carbon metabolism

VANHA N. PHAM, KEVIN J. BRUEMMER, JOEL D. W. TOH, EVA J. GE, LOGAN TENNEY, CARL C. WARD, FELIX A. DINGLER, CHRISTOPHER L. MILLINGTON, CARLOS A. GARCIA-PRIETO  MIA C. PULOS-HOLMES  NICHOLAS T. INGOLIA LUCAS B. PONTEL MANEL ESTELLER  KETAN J. PATEL  DANIEL K. NOMURA AND CHRISTOPHER J. CHANG 

Science 2023 382, 6670

DOI: 10.1126/science.abp9201

INTRODUCTION

One-carbon metabolism manages cellular carbon pools by detoxifying highly reactive carbon species, such as aldehydes, and diverting their carbon toward the biosynthesis of useful products, including amino acids and nucleotides. Formaldehyde (FA) is a major one-carbon unit derived from exogenous environmental exposure and endogenous sources and is quickly scavenged in the cell through enzymatic oxidation to formate and carbon dioxide and/or metabolized through the folate cycle. S-adenosylmethionine (SAM) serves as the primary cellular methyl donor and harbors one-carbon units in a stable and accessible form. The ability to decipher the biochemical interplay between toxic reactive carbon species and stable physiological carbon units is essential for understanding fundamentals of one-carbon metabolism across all kingdoms of life. Especially important is understanding how aberrant carbon imbalances are connected to human diseases such as cancer, liver diseases, and asthma. Although the chronic exposure of toxic aldehydes is correlated to disease states, biological mechanisms of aldehyde signaling and their relation to carbon metabolism remain underexplored.

RATIONALE

Owing to its highly electrophilic nature, we hypothesized that FA could act as a one-carbon signal sensed by privileged cysteine sites across the proteome. FA reacts with cysteines on synthetic peptides, and we designed an unbiased, proteome-wide profiling study to systematically identify FA-sensitive cysteine residues. This work builds a biochemical framework for understanding global FA reactivity as a selective posttranslational modification of target proteins and downstream regulatory effects of such modifications.

RESULTS

Activity-based protein profiling identified FA modification of privileged cysteine sites across the proteome, including several enzymes responsible for FA metabolism, one-carbon metabolism, and amino acid biosynthesis. We focused on biochemical characterization of a key Cys120 residue on the SAM-generating enzyme S-adenosylmethionine synthase isoform type-1 (MAT1A) that is proximal to the MAT1A active site. FA exposure resulted in inhibition of MAT1A activity in an isoform-specific manner, which led to decreased SAM production. Cellular models containing only the MAT1A isoform displayed a reciprocal decrease in SAM levels with increasing doses of FA exposure. Moreover, an Adh5–/– mouse model of chronic FA elevation also showed SAM deficiency accompanied by lower levels of methylation on select histone methyl sinks. The chronic FA model also resulted in a decrease in methylation of the Mat1a promoter region, resulting in increased MAT1A expression as a compensatory mechanism to maintain available carbon units. We deciphered a compensatory biochemical feedback cycle where FA-dependent SAM deficiency led to an increase in MAT1A expression through genetic and epigenetic mechanisms regulated by FA-dependent transcription factors and DNA promoter hypomethylation, respectively.

CONCLUSION

In contrast to the traditional view of FA as an indiscriminate electrophile and toxic metabolite, we show that FA is sensed by specific cysteine sites in the proteome to regulate one-carbon metabolism feedback cycles through SAM biosynthesis. FA reacts with a key cysteine residue on MAT1A to inhibit its activity, resulting in SAM depletion and downstream changes in histone and DNA methylation. Under normal homeostatic conditions, FA is quickly sequestered into the folate cycle for conservation of one-carbon units to maintain balanced SAM biosynthesis. In response to FA overload, reciprocal SAM depletion through isoform-specific MAT1A inhibition results in changes to cellular methylation potential, epigenetic dysregulation, and perturbations in one-carbon metabolism, which in turns leads to compensatory up-regulation of MAT1A expression. This work provides a starting point for further exploration of aldehydes as signaling agents and the nexus between one-carbon metabolism and one-carbon signaling.



Thursday, February 29, 2024

Molecular Bidents with Two Electrophilic Warheads as a New Pharmacological Modality

Zhengnian Li, Jie Jiang, Scott B. Ficarro, Tyler S. Beyett, Ciric To, Isidoro Tavares, Yingde Zhu, Jiaqi Li, Michael J. Eck, Pasi A. Jänne, Jarrod A. Marto, Tinghu Zhang, Jianwei Che, and Nathanael S. Gray
ACS Central Science 2024

DOI: 10.1021/acscentsci.3c01245

A systematic strategy to develop dual-warhead inhibitors is introduced to circumvent the limitations of conventional covalent inhibitors such as vulnerability to mutations of the corresponding nucleophilic residue. Currently, all FDA-approved covalent small molecules feature one electrophile, leaving open a facile route to acquired resistance. We conducted a systematic analysis of human proteins in the protein data bank to reveal ∼400 unique targets amendable to dual covalent inhibitors, which we term “molecular bidents”. We demonstrated this strategy by targeting two kinases: MKK7 and EGFR. The designed compounds, ZNL-8162 and ZNL-0056, are ATP-competitive inhibitors that form two covalent bonds with cysteines and retain potency against single cysteine mutants. Therefore, molecular bidents represent a new pharmacological modality with the potential for improved selectivity, potency, and drug resistance profile.


Tuesday, February 27, 2024

Development of Oxadiazolone Activity-Based Probes Targeting FphE for Specific Detection of Staphylococcus aureus Infections

Jeyun Jo, Tulsi Upadhyay, Emily C. Woods, Ki Wan Park, Nichole J. Pedowitz, Joanna Jaworek-Korjakowska, Sijie Wang, Tulio A. Valdez, Matthias Fellner, and Matthew Bogyo

Journal of the American Chemical Society 2024
DOI: 10.1021/jacs.3c13974

Staphylococcus aureus (S. aureus) is a major human pathogen that is responsible for a wide range of systemic infections. Since its propensity to form biofilms in vivo poses formidable challenges for both detection and treatment, tools that can be used to specifically image S. aureus biofilms are highly valuable for clinical management. Here, we describe the development of oxadiazolone-based activity-based probes to target the S. aureus-specific serine hydrolase FphE. Because this enzyme lacks homologues in other bacteria, it is an ideal target for selective imaging of S. aureus infections. Using X-ray crystallography, direct cell labeling, and mouse models of infection, we demonstrate that oxadiazolone-based probes enable specific labeling of S. aureus bacteria through the direct covalent modification of the FphE active site serine. These results demonstrate the utility of the oxadizolone electrophile for activity-based probes and validate FphE as a target for the development of imaging contrast agents for the rapid detection of S. aureus infections.


Sunday, February 25, 2024

A covalent compound selectively inhibits RNA demethylase ALKBH5 rather than FTO

Gan-Qiang Lai,  Yali Li,  Heping Zhu,   Tao Zhang,  Jing Gao,  Hu Zhou  and  Cai-Guang Yang 

RSC Chem. Biol., 2024DOI: 10.1039/D3CB00230F

N6-Methyladenosine (m6A) is the most prevalent mRNA modification and is required for gene regulation in eukaryotes. ALKBH5, an m6A demethylase, is a promising target, particularly for anticancer drug discovery. However, the development of selective and potent inhibitors of ALKBH5 rather than FTO remains challenging. Herein, we used a targeted covalent inhibition strategy and identified a covalent inhibitor, TD19, which selectively inhibits ALKBH5 compared with FTO demethylase in protein-based and tumor cell-based assays. TD19 irreversibly modifies the residues C100 and C267, preventing ALKBH5 from binding to m6A-containing RNA. Moreover, TD19 displays good anticancer efficacy in acute myeloid leukemia and glioblastoma multiforme cell lines. Thus, the ALKBH5 inhibitor developed in this study, which selectively targets ALKBH5 compared with FTO, can potentially be used as a probe for investigating the biological functions of RNA demethylase and as a lead compound in anticancer research.



An orally bioavailable SARS-CoV-2 main protease inhibitor exhibits improved affinity and reduced sensitivity to mutations

Michael Westberg  et al. Sci. Transl. Med. 16 , eadi0979 (2024). DOI: 10.1126/scitranslmed.adi0979 Inhibitors of the severe acute respirator...