الفهرس 
The changing face of medicinal chemistryOnly 14 pages are availabe for public view
 |  | from | 289 |  |  |
| | | from | 289 | | |
Abstract
Histone deacetylases (HDACs) constitute an epigenetic enzyme family that
was implicated in cancer and other diseases. Increasing evidence shows that
by inhibiting a single isoform, one could regulate a focused subset of genes
and achieve the desired therapeutic effect with lower toxicity. The isoform
selectivity of HDACi is typically measured in biochemical assays using
purified isolated recombinant enzymes and artificial substrate mimics, which
doesn’t recapitulate the factors modulating HDAC activity in live settings.
Therefore, development of novel chemical tools and assays in live cells, live
tissues and in vivo is needed.
Photoaffinity labeling uses small molecule trifunctional photoreactive probes
(PRPs) that contain: (1) an affinity group which bind to the target protein
active site, (2) a photoreactive group (PRG) that, upon UV irradiation,
covalently crosslink to the proteins in proximity, and (3) a tagging group that
can be conjugated to a reporter tag using bioorthogonal reactions for
visualization, enrichment, and identification.
In this study, a synthetic strategy to access chemically diverse PRPs based on
both FDA approved and investigational, both non-selective and isoform
selective HDACi is described, and used for synthesis of a set of chemically
diverse HDAC PRPs and control compounds. Tetrafluorophenylazide was the
most efficient among the studied PRG. The synthetic strategy was optimized
to be applicable to the diverse chemical scaffolds. Molecular modeling guided
the placement of the added moieties not to interfere with enzyme-PRP binding.
The PRPs and their parent HDACi were tested for in vitro enzyme inhibitory
activity, using a common HDAC biochemical assay against recombinant
HDAC isoforms. The assay utilizes artificial substrate mimics with a
fluorogenic moiety that produces fluorescence signal after being deacetylated
by the HDAC. Rational incorporation of the PRGs in the structure of PRPs
was found to modestly affect their potency and selectivity determined
biochemically.
Abstract xxiii
S. M. Aboukhatwa, PhD thesis Pharmaceutical Sciences (Pharm. Chemistry), Tanta University 2019
Class I HDACs were identified as targets of the PRPs in MCF-7 cells, using
mass-spectrometry based protein profiling. In triple negative breast cancer
cells, the cell-based HDAC selectivity profiles of the PRPs were skewed
toward HDAC3 irrespective of PRP chemotype and biochemical selectivity,
or cellular HDAC isoform abundance. Unlike the HDAC protein abundance
and the biochemical activity of PRPs, the PRPs chemotype and the cell type
were key in defining HDAC isoform engagement in live SET-2, HepG2,
HuH7 and HEK293T cells. Further, photolabeling studies were done in mice
liver tissue (ex vivo) as a more realistic model of live cells in vivo. The druglike
properties and liver microsomal metabolic stability profile of PRPs
indicate their applicability for in vivo studies.
The findings of the current study stress the importance of revisiting HDACi
selectivity accounting for the multiple roles of HDACs and their regulation by
post-translational modifications and multiprotein complex formation. The
study reinforces that target engagement of HDAC isoforms by HDACi in vivo
is significantly modulated in a cell- and tissue- type dependent manner. The
study also highlights the importance of testing HDACi within the cellular
context, in the presence of multiprotein complex components and posttranslational
modification status. Ultimately, this knowledge can be used to
develop and apply novel rational approaches and experimental tools to
understand the mechanisms underlying the beneficial and adverse effects of
HDACi as a basis for design of more potent and safer therapeutics for cancer.