Date of Award

2017

Embargo Period

8-1-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Drug Discovery and Biomedical Sciences

College

College of Graduate Studies

First Advisor

Stuart Parnham

Second Advisor

Yuri Peterson

Abstract

The acetylation status of lysine residues on histone proteins has long been attributed to a balance struck between the catalytic activity of Histone Acetyl Transferases and Histone Deacetylases (HDAC). HDACs were identified as the sole removers of acetyl post-translational modifications (PTM) of histone lysine residues. Studies into the biological role of HDACs have also elucidated their role as removers of acetyl PTMs from lysine residues of non-histone proteins. These findings, coupled with high-resolution mass spectrometry studies that revealed the presence of acyl-group PTMs on lysine residues of non-histone proteins, brought forth the possibility of HDACs acting as removers of both acyl- and acetyl-based PTMs. We posited that HDACs fulfill this dual role, and sought to investigate their specificity. Utilizing a fluorescence- based assay and biologically relevant acyl-substrates, the selectivity of zinc-dependent HDACs toward these acyl-based PTMs were identified. These findings were further validated using cellular models and molecular biology techniques. As a proof of principal, an HDAC3 selective inhibitor was designed using HDAC3’s substrate preference. This resulting inhibitor demonstrates nanomolar activity and >30 fold selectivity toward HDAC3 compared to the other class I HDACs. This inhibitor is capable of increasing p65 acetylation, attenuating NF-κB activation and thereby preventing downstream nitric oxide signaling. Additionally, this selective HDAC3 inhibition allows for control of HMGB-1 secretion from activated macrophages without altering the acetylation status of histones or tubulin. In addition to this substrate-driven design of a novel HDAC3 selective inhibitor, we sought to tackle one of the biggest hurdles yet to be overcome for the continued improvement of HDAC inhibitors. First generation HDAC inhibitors frequently utilize a metal binding hydroxamic acid moiety. The N-hydroxyl group of this motif is highly subject to sulfation/glucuronidation-based inactivation in humans; compounds containing this motif require much higher dosing in clinic to achieve therapeutic concentrations. With the goal of developing a second generation of HDAC inhibitors, lacking this hydroxamate, we designed a series of potent and selective class I HDAC inhibitors using a hydrazide motif. These inhibitors are impervious to glucuronidation and demonstrate allosteric inhibition. In vitro and ex vivo characterization of our lead analogs’ efficacy, selectivity, and toxicity profiles demonstrate they possess low nanomolar activity against models of Acute Myeloid Leukemia (AML) and are at least 100-fold more selective for AML than solid immortalized cells such as Hek293 or human peripheral blood mononuclear cells. Further, these compounds seem to kill through a non-caspase-mediated mechanism with possible involvement of p53. Lead analogs demonstrate favorable half lives in vivo (>3 hours) and are possess promising bioavailabilty profiles. Lastly, these compounds are non-inferior to current FDA approved HDAC inhibitors, vorinostat and panobinostat, in causation of mutagenesis.

Rights

All rights reserved. Copyright is held by the author.

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