Carboxylesterases represent a large class of hydrolytic enzymes that play important roles in the metabolism of endogenous lipids, pharmacological determination of therapeutic agents and detoxication of organophosphorus insecticides. The focus of the current grant period has been on molecular cloning, enzyme kinetics and xenobiotic regulation. Enzymatic characterization and molecular modeling have revealed that carboxylesterases contain several functional subsites (structures) that likely play determinant roles in substrate recognition and inhibitor reactivity. Carboxylesterases, with the same functional subsites exhibit the same substrate preference and a similar sensitivity to serine enzyme inhibitors. Studies on xenobiotic regulation have demonstrated that dexamethasone suppresses the expression of rat hydrolase A, B/C and S, whereas the expression of human HCE-1 and HCE-2 is induced by this drug. Suppression requires only nanomolar whereas induction requires micromolar levels. The proposed studies are designed to test the hypotheses that non-conserved residues assumed to form functional subsites among carboxylesterases contribute significantly to the observed differences on the substrate preference and that the dexamethasone-directed suppression of rat carboxylesterases is mediated by the glucocorticoid receptor whereas the induction of human carboxylesterases is mediated by the pregnane X receptor.
The specific aims of the proposed studies are: (1) to characterize functional subsites determining substrate and inhibitor selectivity; and (2) to elucidate molecular basis for species-dependent regulation by dexamethasone. Site-direct mutagenesis will be performed to replace a single residue or an entire putative subsite, and the mutants will be tested for the altered hydrolytic activity toward structurally dissimilar substrates. Studies on cell proliferation and toxicity will be performed to determine whether changes in catalytic properties translate into actual perturbations in biological functions. In order to specify the receptor involved in the species-dependent regulation by dexamethasone, antiglucocorticoids and dominant regulators will be used to selectively block or alter either pathway and the role of each receptor will be established in regulating carboxylesterase expression. Establishment of the importance of the subsites for substrate recognition will provide information to elucidate the catalytic action of carboxylesterases and to ascertain isoform-based pharmacological and toxicological relevance. Specification of a receptor involved in dexamethasone-mediated regulation will provide molecular mechanisms to predict potential drug-drug interactions as a result of altered expression of carboxylesterases. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES007965-08
Application #
6915684
Study Section
Alcohol and Toxicology Subcommittee 4 (ALTX)
Program Officer
Balshaw, David M
Project Start
1997-02-01
Project End
2007-06-30
Budget Start
2005-07-01
Budget End
2006-06-30
Support Year
8
Fiscal Year
2005
Total Cost
$307,800
Indirect Cost
Name
University of Rhode Island
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
796475382
City
Kingston
State
RI
Country
United States
Zip Code
02881
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Vachirayonsti, Thaveechai; Ho, Karen W; Yang, Dongfang et al. (2015) Suppression of the pregnane X receptor during endoplasmic reticulum stress is achieved by down-regulating hepatocyte nuclear factor-4? and up-regulating liver-enriched inhibitory protein. Toxicol Sci 144:382-92
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Guo, Liangran; Yan, Daisy D; Yang, Dongfang et al. (2014) Combinatorial photothermal and immuno cancer therapy using chitosan-coated hollow copper sulfide nanoparticles. ACS Nano 8:5670-81
Song, Xiulong; Vasilenko, Alexander; Chen, Yuan et al. (2014) Transcriptional dynamics of bile salt export pump during pregnancy: mechanisms and implications in intrahepatic cholestasis of pregnancy. Hepatology 60:1993-2007
Xiao, Da; Shi, Deshi; Yang, Dongfang et al. (2013) Carboxylesterase-2 is a highly sensitive target of the antiobesity agent orlistat with profound implications in the activation of anticancer prodrugs. Biochem Pharmacol 85:439-47
Xiao, Da; Yang, Dongfang; Guo, Liangran et al. (2013) Regulation of carboxylesterase-2 expression by p53 family proteins and enhanced anti-cancer activities among 5-fluorouracil, irinotecan and doxazolidine prodrug. Br J Pharmacol 168:1989-99
Song, Xiulong; Chen, Yuan; Valanejad, Leila et al. (2013) Mechanistic insights into isoform-dependent and species-specific regulation of bile salt export pump by farnesoid X receptor. J Lipid Res 54:3030-44
Chen, Yuan; Song, Xiulong; Valanejad, Leila et al. (2013) Bile salt export pump is dysregulated with altered farnesoid X receptor isoform expression in patients with hepatocellular carcinoma. Hepatology 57:1530-41

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