Paresh C. Sanghani

Kinetic and Cellular Characterization of Novel Inhibitors of S-Nitrosoglutathione Reductase

S-Nitrosoglutathione reductase (GSNOR) is an alcohol dehydrogenase involved in the regulation of S-nitrosothiols (SNOs) in vivo. Knock-out studies in mice have shown that GSNOR regulates the smooth muscle tone in airways and the function of β-adrenergic receptors in lungs and heart. GSNOR has emerged as a target for the development of therapeutic approaches for treating lung and cardiovascular diseases. We report three compounds that exclude GSNOR substrate, S-nitrosoglutathione (GSNO) from its binding site in GSNOR and cause an accumulation of SNOs inside the cells. The new inhibitors selectively inhibit GSNOR among the alcohol dehydrogenases. Using the inhibitors, we demonstrate that GSNOR limits nitric oxide-mediated suppression of NF-κB and activation of soluble guanylyl cyclase. Our findings reveal GSNOR inhibitors to be novel tools for regulating nitric oxide bioactivity and assessing the role of SNOs in vivo.

Expression and Characterization of a Human Carboxylesterase 2 Splice Variant

CPT-11 [7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin or Irinotecan] is a carbamate prodrug that is activated in vivo by carboxylesterase (CES)-2 to SN-38 (7-ethyl-10-hydroxycamptothecin), a potent topoisomerase I inhibitor. There is high interindividual variation when CPT-11 is used in the treatment of colorectal cancer. Several splice variants of CES2 are reported in the expressed sequence tag database. Real-time polymerase chain reaction was used to determine the abundance of the CES2 and splice variant of human carboxylesterase 2 (CES2Δ458–473) transcripts in 10 paired samples of human tumor and normal colon tissue. The results showed that the CES2Δ458–473 transcript accounts for an average of 6% of total CES2 transcripts in colon tissue, and there is large interindividual variation in CES2 expression in both tumor and normal colon samples. The carboxylesterase activity of the colon samples was determined by 4-methylumbelliferyl acetate hydrolysis assays and nondenaturing polyacrylamide gel electrophoresis followed by activity staining. Significant, positive correlations were found between CES2 expression levels and both measures of carboxylesterase activity. We cloned and expressed the CES2Δ458–473 protein in Sf9 insect cells. The purification profiles and preliminary characterization of the CES2Δ458–473 protein indicated that the expressed protein is folded and glycosylated like CES2. However, in vitro assays show that the CES2Δ458–473 protein lacks 4-methylumbelliferyl acetate and irinotecan hydrolase activities. In conclusion, we found that the CES2Δ458–473 protein is an inactive splice variant of CES2 and that its transcript is spliced at a relatively constant rate in tumor and normal colon tissue.

Ethanol Metabolism by HeLa Cells Transduced With Human Alcohol Dehydrogenase Isoenzymes: Control of the Pathway by Acetaldehyde Concentration

BACKGROUND Human class I alcohol dehydrogenase 2 isoenzymes (encoded by the ADH1B locus) have large differences in kinetic properties; however, individuals inheriting the alleles for the different isoenzymes exhibit only small differences in alcohol elimination rates. This suggests that other cellular factors must regulate the activity of the isoenzymes. METHODS The activity of the isoenzymes expressed from ADH1B*1, ADH1B*2, and ADH1B*3 cDNAs was examined in stably transduced HeLa cell lines, including lines which expressed human low K(m) aldehyde dehydrogenase (ALDH2). The ability of the cells to metabolize ethanol was compared with that of HeLa cells expressing rat class I alcohol dehydrogenase (ADH) (HeLa-rat ADH cells), rat hepatoma (H4IIEC3) cells, and rat hepatocytes. RESULTS The isoenzymes had similar protein half-lives in the HeLa cells. Rat hepatocytes, H4IIEC3 cells, and HeLa-rat ADH cells oxidized ethanol much faster than the cells expressing the ADH1B isoenzymes. This was not explained by high cellular NADH levels or endogenous inhibitors; but rather because the activity of the β1 and β2 ADHs was constrained by the accumulation of acetaldehyde, as shown by the increased rate of ethanol oxidation by cell lines expressing β2 ADH plus ALDH2. CONCLUSION The activity of the human β2 ADH isoenzyme is sensitive to inhibition by acetaldehyde, which likely limits its activity in vivo. This study emphasizes the importance of maintaining a low steady-state acetaldehyde concentration in hepatocytes during ethanol metabolism.

Structure-function relationships in human Class III alcohol dehydrogenase (formaldehyde dehydrogenase)

Human Class III alcohol dehydrogenase (ADH), also known as glutathione-dependent formaldehyde dehydrogenase plays an important role in the formaldehyde detoxification and reduction of the nitric oxide metabolite s-nitrosoglutathione (GSNO). It follows a random bi bi kinetic mechanism and prefers bulkier substrates like long chain primary alcohols and glutathione adducts like s-hydroxymethylglutathione and GSNO over smaller alcohols like ethanol. The structure of the FDH.NAD(H) binary complex reported here, in conjunction with the other complexes of FDH, provide the structural basis of the kinetic observations. These structures show that the apoenzyme has a semi-open domain conformation that permits random random addition of alcohol or NAD(H). Moreover, there is no significant domain movement upon binding of the coenzyme or the substrate, 12-hydroxydodecanoic acid. Interestingly, two active site zinc coordination environments are observed in FDH. In the apoenzyme, the active site zinc is coordinated to Cys44, His66, Cys173 and a water molecule. In the FDH.NAD(H) binary complex reported here, Glu67 is added to the coordination environment of the active site zinc and the distance between the water molecule and zinc is increased. This change in the zinc coordination, brought about by the displacement of zinc of about 2 A towards Glu67 could promote substrate exchange at the active site metal during catalysis.