Dataset Information

Chemical proteomics with novel fully functionalized fragments and stringent target prioritization identifies the glutathione-dependent isomerase GSTZ1 as a lung cancer target

ABSTRACT: Photoreactive fragment-like probes have been applied to discover target proteins that constitute novel cellular vulnerabilities and to identify viable chemical hits for drug discovery. Through forming covalent bonds, functionalized probes can achieve stronger target engagement and require less effort for on-target mechanism validation. However, the design of probe libraries, which directly affects the biological target space that is interrogated, and effective target prioritization remain critical challenges of such a chemical proteomic platform. In this study, we designed and synthesized a diverse panel of twenty fragment-based probes with privileged structural motifs containing both natural product and lead-like elements. These probes were fully functionalized with orthogonal diazirine and alkyne moieties and used for protein crosslinking in live lung cancer cells, target enrichment via “click chemistry,” and subsequent target identification through label-free quantitative LC-MS/MS analysis. Pair-wise comparison with a blunted negative control probe and stringent prioritization via individual cross-comparisons against the entire panel identified glutathione S-transferase zeta 1 (GSTZ1) as a specific and unique target candidate. DepMap database query, RNA interference-based gene silencing and proteome-wide tyrosine reactivity profiling suggested that GSTZ1 cooperated with different oncogenic alterations by supporting survival signaling in refractory NSCLC cells. This finding may form the basis for developing novel GSTZ1 inhibitors to improve the therapeutic efficacy of oncogene-directed targeted drugs. In summary, we designed a novel fragment-based probe panel and developed a target prioritization scheme with improved stringency, which allows for identification of unique target candidates, such as GSTZ1 in refractory lung cancer.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Type Ii Pneumocyte

DISEASE(S): Lung Cancer

SUBMITTER: John Koomen

LAB HEAD: Uwe Rix, PhD

PROVIDER: PXD035201 | Pride | 2024-04-05

REPOSITORIES: Pride

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Publications

Chemical Proteomics with Novel Fully Functionalized Fragments and Stringent Target Prioritization Identifies the Glutathione-Dependent Isomerase GSTZ1 as a Lung Cancer Target.

Liao Yi Y Chin Chan Sean S Welsh Eric A EA Fang Bin B Sun Luxin L Schönbrunn Ernst E Koomen John M JM Duckett Derek R DR Haura Eric B EB Monastyrskyi Andrii A Rix Uwe U

ACS chemical biology 20230111 2

Photoreactive fragment-like probes have been applied to discover target proteins that constitute novel cellular vulnerabilities and to identify viable chemical hits for drug discovery. Through forming covalent bonds, functionalized probes can achieve stronger target engagement and require less effort for on-target mechanism validation. However, the design of probe libraries, which directly affects the biological target space that is interrogated, and effective target prioritization remain critic ...[more]

PMID: 36630201

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Project description:Unprecedented bacterial targets are urgently needed for the development of novel antibiotics to overcome the current resistance crisis. Challenges include the limited uptake of compounds as well as prioritizing proteins for their druggability and functional relevance. Especially, the wealth of uncharacterized proteins represents an untapped source for novel targets. However, tools to decipher their function are largely lacking. We here utilize the systematic mining of pyridoxal phosphate dependent enzymes (PLP DEs) in bacteria to focus on a target class, which is known to bind ligands, accesses PLP via active transport from the media and is involved in crucial metabolic processes. For this, we systematically exploited the chemical space of the pyridoxal (PL) scaffold and obtained eight PL probes bearing modifications at various ring positions. These probes were subsequently tested for phosphorylation by cognate kinases and labelling of PLP DEs in clinically relevant Gram-positive (Staphylococcus aureus) as well as Gram-negative (Escherichia coli and Pseudomonas aeruginosa) strains. Overall, the coverage of this diverse set of probes along with refined labelling conditions not only exceeded the performance of a previous probe generation, it also provided a detailed map of binding preferences of certain structural motifs. Although originally conducted in mutant cells devoid of PLP de novo biosynthesis, we here demonstrate efficient PLP DE labelling also in a wild type strain. Overall, the profiling revealed several putative PLP DEs with unknown function, of which we exemplarily characterized five via in-depth enzymatic assays. Finally, we screened a panel of putative PLP binders for antibiotic activity and unravelled the targets of hit molecules via competitive profiling with our probes. Here, an uncharacterized enzyme, essential for bacterial growth, was assigned as PLP dependent cysteine desulfurase and confirmed to be inhibited by the marketed drug phenelzine. Our approach provides a basis for deciphering novel PLP DEs as essential antibiotic targets along with corresponding ways to decipher small molecule inhibitors.

Dataset Information

Chemical proteomics with novel fully functionalized fragments and stringent target prioritization identifies the glutathione-dependent isomerase GSTZ1 as a lung cancer target

Publications

Chemical Proteomics with Novel Fully Functionalized Fragments and Stringent Target Prioritization Identifies the Glutathione-Dependent Isomerase GSTZ1 as a Lung Cancer Target.

Similar Datasets

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