Demonstrating
intracellular protein target engagement is an essential step in the
development and progression of new chemical probes and potential small
molecule therapeutics. However, this can be particularly challenging
for poorly studied and noncatalytic proteins, as robust proximal biomarkers
are rarely known. To confirm that our recently discovered chemical
probe 1 (CCT251236) binds the putative transcription
factor regulator pirin in living cells, we developed a heterobifunctional
protein degradation probe. Focusing on linker design and physicochemical
properties, we generated a highly active probe 16 (CCT367766)
in only three iterations, validating our efficient strategy for degradation
probe design against nonvalidated protein targets.
Phenotypic screens, which focus on
measuring and quantifying discrete
cellular changes rather than affinity for individual recombinant proteins,
have recently attracted renewed interest as an efficient strategy
for drug discovery. In this article, we describe the discovery of
a new chemical probe, bisamide (CCT251236), identified using an unbiased
phenotypic screen to detect inhibitors of the HSF1 stress pathway.
The chemical probe is orally bioavailable and displays efficacy in
a human ovarian carcinoma xenograft model. By developing cell-based
SAR and using chemical proteomics, we identified pirin as a high affinity
molecular target, which was confirmed by SPR and crystallography.
Nitrile reductase QueF catalyzes the reduction of 2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one (preQ0) to 2-amino-5-aminomethylpyrrolo[2,3-d]pyrimidin-4-one (preQ1) in the biosynthetic pathway of the hypermodified nucleoside queuosine. It is the only enzyme known to catalyze a reduction of a nitrile to its corresponding primary amine and could therefore expand the toolbox of biocatalytic reactions of nitriles. To evaluate this new oxidoreductase for application in biocatalytic reactions, investigation of its substrate scope is prerequisite. We report here an investigation of the active site binding properties and the substrate scope of nitrile reductase QueF from Escherichia coli. Screenings with simple nitrile structures revealed high substrate specificity. Consequently, binding interactions of the substrate to the active site were identified based on a new homology model of E. coli QueF and modeled complex structures of the natural and non-natural substrates. Various structural analogues of the natural substrate preQ0 were synthesized and screened with wild-type QueF from E. coli and several active site mutants. Two amino acid residues Cys190 and Asp197 were shown to play an essential role in the catalytic mechanism. Three non-natural substrates were identified and compared to the natural substrate regarding their specific activities by using wild-type and mutant nitrile reductase.
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