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Hydroxamic Acid-Modified Peptide Microarrays for Profiling Isozyme-Selective Interactions and Inhibition of Histone Deacetylases

preprint
submitted on 04.01.2020 and posted on 06.01.2020 by Carlos Moreno-Yruela, Adela-Eugenie Vrsanova, Hans M. Maric, Christian Adam Olsen
Histones control gene expression by regulating chromatin structure and function. The posttranslational modifications (PTMs) on the side chains of histones form the epigenetic landscape, which is tightly controlled through the enzymatic action of epigenetic effector proteins and protein complexes. Further, various PTM signatures or combinations are recognized by so-called reader domains. Histone microarrays have been widely applied to investigate such histone–reader interactions. So far, however, these could not be used to directly study the fast and transient interactions of Zn2+-dependent histone deacetylase (HDAC) enzymes. Here, we describe the synthesis of hydroxamic acid-modified histone-derived peptides and their use in femtomolar microarrays for the direct capture and detection of the four class I HDAC isozymes. We further demonstrate their suitability to discover and map HDAC isozyme-specific substrates. Functional assays confirmed the prediction of HDAC–peptide binding requirements and the conversion of acetylated substrates in response to PTMs and mutations. Subsequent analysis of the hydroxamic acid-containing peptides identified compounds with nanomolar potency and unanticipated selectivity. Follow-up analyses confirmed a major contribution of the peptide sequence to substrate turnover, inhibitor potency, and isozyme selectivity. We conclude that similar hydroxamic acid-modified histone peptide microarrays and libraries could find broad application to identify class I HDAC isozyme-specific substrates and facilitate the development of isozyme- or protein complex-selective HDAC inhibitors and affinity probes.

Funding

Carlsbergfondet (2013-01-0333)

Carlsbergfoncet (CF18-0442)

European Research Council (ERC-CoG-725172 – SIRFUNCT)

History

Email Address of Submitting Author

cao@sund.ku.dk

Institution

University of Copenhagen

Country

Denmark

ORCID For Submitting Author

0000-0002-2953-8942

Declaration of Conflict of Interest

The authors declare no conflicts of interest

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