Class I HDAC imaging using [ (3)H]CI-994 autoradiography.

Epigenetics
Authors
Keywords
Abstract

[ (3)H]CI-994, a radioactive isotopologue of the benzamide CI-994, a class I histone deacetylase inhibitor (HDACi), was evaluated as an autoradiography probe for ex vivo labeling and localizing of class I HDAC (isoforms 1-3) in the rodent brain. After protocol optimization, up to 80% of total binding was attributed to specific binding. Notably, like other benzamide HDACi, [ (3)H]CI-994 exhibits slow binding kinetics when measured in vitro with isolated enzymes and ex vivo when used for autoradiographic mapping of HDAC1-3 density. The regional distribution and density of HDAC1-3 was determined through a series of saturation and kinetics experiments. The binding properties of [ (3)H]CI-994 to HDAC1-3 were characterized and the data were used to determine the regional Bmax of the target proteins. Kd values, determined from slice autoradiography, were between 9.17 and 15.6 nM. The HDAC1-3 density (Bmax), averaged over whole brain sections, was of 12.9 picomol · mg(-1) protein. The highest HDAC1-3 density was found in the cerebellum, followed by hippocampus and cortex. Moderate to low receptor density was found in striatum, hypothalamus and thalamus. These data were correlated with semi-quantitative measures of each HDAC isoform using western blot analysis and it was determined that autoradiographic images most likely represent the sum of HDAC1, HDAC2, and HDAC3 protein density. In competition experiments, [ (3)H]CI-994 binding can be dose-dependently blocked with other HDAC inhibitors, including suberoylanilide hydroxamic acid (SAHA). In summary, we have developed the first known autoradiography tool for imaging class I HDAC enzymes. Although validated in the CNS, [ (3)H]CI-994 will be applicable and beneficial to other target tissues and can be used to evaluate HDAC inhibition in tissues for novel therapies being developed. [ (3)H]CI-994 is now an enabling imaging tool to study the relationship between diseases and epigenetic regulation.

Year of Publication
2013
Journal
Epigenetics
Volume
8
Issue
7
Pages
756-64
Date Published
2013 Jul
ISSN
1559-2308
DOI
10.4161/epi.25202
PubMed ID
23803584
PubMed Central ID
PMC3781195
Links
Grant list
R01DA028301 / DA / NIDA NIH HHS / United States
R01 DA030321 / DA / NIDA NIH HHS / United States
P30 DA028800 / DA / NIDA NIH HHS / United States
P30DA028800 / DA / NIDA NIH HHS / United States
5R01DA030321 / DA / NIDA NIH HHS / United States
S10 RR023385 / RR / NCRR NIH HHS / United States
R01 DA028301 / DA / NIDA NIH HHS / United States