SAHA Capture Compound – A novel tool for the profiling of histone deacetylases and the identification of additional vorinostat binders

Abstract

Suberoylanilide hydroxamic acid (SAHA) is a potent histone deacetylase (HDAC) inhibitor. Inhibitors of HDACs are used in cancer therapy based on the role HDACs play in transcription by regulating chromatin compaction and non-histone proteins such as transcription factors. Profiling of HDAC expression is of interest in the functional proteomics analysis of cancer. Also, non-HDAC proteins may interact with HDAC inhibitor drugs and contribute to the drug mode of action. We here present a tool for the unbiased chemical proteomic profiling of proteins that specifically interact with SAHA. We designed and synthesized a trifunctional Capture Compound containing SAHA as selectivity and identified HDACs1, 2, 3 and 6, known and predicted HDAC interactors from human-derived HepG2 cell lysate, as well as a set of new potential non-HDAC targets of SAHA. One of these non-HDAC targets, isochorismatase domain-containing protein 2 (ISOC2) is putative hydrolase associated with the negative regulation of the tumor-suppressor p16(INK4a). We demonstrated the direct and dose-dependent interaction of SAHA to the purified recombinant ISOC2 protein. Using SAHA Capture Compound mass spectrometry, we thus identified potential new SAHA target proteins in an entirely unbiased chemical proteomics approach.

A novel approach to detect resistance mechanisms reveals FGR as a factor mediating HDAC inhibitor SAHA resistance in B‐cell lymphoma

“…We compiled a list of 315 candidate proteins that directly or indirectly interact with SAHA. This list contained previously described bona fide targets of SAHA (HDAC1, 2, 3, 6, 8, members of the HDAC1/2 complexes, ISOC1 and ISOC2) and a large number of novel proteins with unknown relation to HDACs ( Supplementary Table S3A) (Bantscheff et al, 2011;Fischer et al, 2011b). Out of the 315 proteins compiled in our initial list, 129 were either predominantly enriched from SAHA Figure 2 e FGR is a candidate for SAHA resistance in B-cell lymphoma.…”

“…Synthesis and characterization of the SAHA‐CC (Supplemental Figure S1) and CCMS experiments (Supplementary Figure S2) were performed as previously described (Fischer et al., 2011b).…”

“…The extracts were cleared by centrifugation for 30 min at 100,000 Â g. The protein content was determined using the bicinchoninic acid assay (BCA) method (Smith et al, 1985). Synthesis and characterization of the SAHA-CC (Supplemental Figure S1) and CCMS experiments (Supplementary Figure S2) were performed as previously described (Fischer et al, 2011b).…”

How Chemoproteomics Can Enable Drug Discovery and Development

“…Multiple chemoproteomic strategies for profiling HDAC inhibitors have also been introduced, including the creation of activity-based probes [49–51] and immobilized broad-spectrum inhibitors [52]. Clickable, photoreactive activity-based probes have been shown to label HDACs in living cells, facilitating the discovery of HDAC activities that were impaired upon cell lysis [49, 50].…”

“…These ABPP studies also provided some of the first evidence that the HDAC inhibitor SAHA, historically considered a pan-class I and II HDAC inhibitor, shows selectivity for a subset of HDACs (1, 2, 3, and 6) [49], a finding that has been confirmed with advanced substrate assays [53]. Chemoproteomic enrichment of inhibitor-interacting proteins has also identified unexpected specificity within HDAC protein complexes and additional potential targets for SAHA that are outside of the HDAC family [51, 52]. …”