Target Deconvolution Efforts on Wnt Pathway Screen Reveal Dual Modulation of Oxidative Phosphorylation and SERCA2.

Wnt signaling is critical for development, cell proliferation and differentiation, and mutations in this pathway resulting in constitutive signaling have been implicated in various cancers. A pathway screen using a Wnt-dependent reporter identified a chemical series based on a 1,2,3-thiadiazole-5-carboxamide (TDZ) core with sub-micromolar potency. Herein we report a comprehensive mechanism-of-action deconvolution study toward identifying the efficacy target(s) and biological implication of this chemical series involving bottom-up quantitative chemoproteomics, cell biology, and biochemical methods.

Zinc-binding cysteines: diverse functions and structural motifs.

Cysteine residues are known to perform essential functions within proteins, including binding to various metal ions. In particular, cysteine residues can display high affinity toward zinc ions (Zn2+), and these resulting Zn2+-cysteine complexes are critical mediators of protein structure, catalysis and regulation. Recent advances in both experimental and theoretical platforms have accelerated the identification and functional characterization of Zn2+-bound cysteines.

Selective 4,6-O-benzylidene formation of methyl α-D-mannopyranoside using 2,6-dimethylbenzaldehyde.

While methyl α-d-glucopyranosides and α-d-galactopyranosides selectively form 4,6-O-benzylidenes when reacted with excess benzaldehyde in the presence of acid catalyst methyl α-d-mannopyranosides does not exhibit the same selectivity because of the cis-arrangement of the C2 and C3 hydroxyl groups. The selectivity for the 4,6-O-benzylidene is restored by using 2,6-dimethylbenzaldehyde instead of benzaldehyde. In addition the excess 2,6-dimethylbenzaldehyde is easily recovered from the reaction by extraction with petroleum ether and can be reused without further purification.

A competitive chemical-proteomic platform to identify zinc-binding cysteines.

Zinc ions (Zn(2+)) play vital catalytic, structural, and regulatory roles in protein function and are commonly chelated to cysteine residues within the protein framework. Current methods to identify Zn(2+)-binding cysteines rely on computational studies based on known Zn(2+)-chelating motifs, as well as high-resolution structural data. These available approaches preclude the global identification of putative Zn(2+)-chelating cysteines, particularly on poorly characterized proteins in the proteome.

An isotopically tagged azobenzene-based cleavable linker for quantitative proteomics.

Putting a number on it: Cleavable linkers are widely utilized in proteomics applications. In particular, the azobenzene-based linker cleaves under mild conditions that are mass-spectrometry-compatible. Here, we adapt this linker for quantitative proteomic applications by incorporating an isotopic label.

1,3,5-Triazine as a modular scaffold for covalent inhibitors with streamlined target identification.

Small-molecule inhibitors can accelerate the functional annotation and validate the therapeutic potential of proteins implicated in disease. Phenotypic screens provide an effective platform to identify such pharmacological agents but are often hindered by challenges associated with target identification. For many protein targets, these bottlenecks can be overcome by incorporating electrophiles into small molecules to covalently trap interactions in vivo and by employing bioorthogonal handles to enrich the protein targets directly from a complex proteome.

Diverse functional roles of reactive cysteines.

Cysteine residues on proteins play key roles in catalysis and regulation. These functional cysteines serve as active sites for nucleophilic and redox catalysis, sites of allosteric regulation, and metal-binding ligands on proteins from diverse classes including proteases, kinases, metabolic enzymes, and transcription factors. In this review, we focus on a few select examples that serve to highlight the multiple functions performed by cysteines, with an emphasis on cysteine-mediated protein activities implicated in cancer.