title: Applying small molecule microarrays and resulting affinity probe cocktails for proteome profiling of mammalian cell lysates.
abstract: Small molecule microarrays (SMMs) are proving to be increasingly important tools for assessing protein-ligand interactions, as well as in screening for enzyme substrates and inhibitors, in a high-throughput manner. We previously described an SMM-facilitated screening strategy for the rapid identification of probes against γ-secretase, an aspartic protease. In this article, we extend upon this work with an expanded library of hydroxyethylamine-derived inhibitors which non-exclusively target aspartic proteases. Our library is diversified across P(2), P(1), P(1)', and P(2)' positions. Accordingly, 86 new inhibitors are synthesized using a combinatorial, solid-phase synthetic approach, bringing the total library size to 284-biotinylated compounds, which were arrayed onto avidin slides. In order to elucidate enzymatic activity and profiles within complex biological samples, screening is performed using fluorescently-labeled mammalian cell lysates. This yielded reproducible profiles or binding fingerprints that correspond with interactions from aspartic proteases or accessory proteins as well as other interacting targets that were present in the sample. The brightest microarray hits were converted to affinity-based probes (AfBPs) using convenient, 1-step "click" chemistry with benzophenone from the relevant building blocks. Pull-down/mass spectrometric analysis with these probes (individuals or cocktail) yielded putative protein targets that include well-known aspartic proteases, such as cathepsin D which is a clear marker for breast cancer cell lines, T47D. Many other hits were also identified, which may be secondary or tertiary interactors of aspartic proteases, or yet unreported off-targets of the hydroxyethylamine pharmacophore. Our work herein thus provides a candidate list of biomarkers for further investigations. Taken together, this SMM-facilitated strategy for the discovery of new AfBPs should provide a useful tool for high-throughput development of novel small molecule probes and the identification of new aspartic proteases as well as related biomarkers in the future.
authors: Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543.
authors: Haibin
authors: H
authors: Shi
authors: Mahesh
authors: M
authors: Uttamchandani
authors: Shao Q
authors: SQ
authors: Yao
mesh_terms: D000345:Affinity Labels; D057055:Aspartic Acid Proteases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015415:Biomarkers / Q000032:analysis; D002457:Cell Extracts / Q000737:chemistry*; D045744:Cell Line, Tumor; D057930:Click Chemistry; D005021:Ethylamines / Q000032:analysis / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D046228:Microarray Analysis / Q000379:methods*; D015335:Molecular Probes / Q000032:analysis; D015394:Molecular Structure; D011480:Protease Inhibitors / Q000032:analysis / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D020543:Proteome / Q000032:analysis*; D054852:Small Molecule Libraries / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
chemical_list: D000345:Affinity Labels; D015415:Biomarkers; D002457:Cell Extracts; D005021:Ethylamines; D015335:Molecular Probes; D011480:Protease Inhibitors; D020543:Proteome; D054852:Small Molecule Libraries; D057055:Aspartic Acid Proteases