Chiral synthons for ibuprofen and ketoprofen via chloroperoxidase catalysis

Thesis (B.S.)--Univeristy of Illinois at Urbana-Champaign, 1997.Includes bibliographical reference (leaf 17)U of I OnlyTheses restricted to UIUC community onl

Abstract 104: ATF7IP does not alter the substrate specificity of the lysine methyltransferase SETDB1

Abstract The histone methyltransferase (HMT) SETDB1 is a strong candidate oncogene in melanoma and lung carcinomas. SETDB1 methylates lysine 9 of histone 3 (H3K9), utilizing S-adenosylmethionine (SAM) as the methyl donor. Its activity has been shown to be regulated by its partner protein ATF7IP, and here we examine the contribution of ATF7IP to the in vitro activity and substrate specificity of SETDB1. SETDB1 and ATF7IP were co-expressed in Sf21 insect cells and 1:1 stoichiometric complexes were purified for comparison against apo-SETDB1 enzyme. We employed both radiometric flashplate-based and MALDI mass spectrometry assays to follow methylation on histone 3 15-mer peptides where lysine 9 was either unmodified, mono-, or di-methylated. These two methods provide orthogonal readouts on H3K9 methylation: the radiometric flashplate-based assay employs 3H-labeled SAM and provides quantitation of labeled, methylated peptide. The MALDI mass spectrometry assay offers quantitation of consumption and accumulation of each individual H3K9 methylation state (H3K9me0, me1, me2, and me3). Results show that both apo-SETDB1 and SETDB1:ATF7IP complex catalyzed both monomethylation and dimethylation of H3K9 peptide substrates, but were unable to perform H3K9 trimethylation. While ATF7IP did not impact the substrate methylation profile, the activity of the complex was lower than apo-SETDB1 by a factor of 4. This difference was due to a decrease in the value of kcat as the substrate KM values were comparable between apo-SETDB1 and the SETDB1:ATF7IP complex. ATF7IP therefore does not alter SETDB1's substrate specificity. H3K9 monomethylation and dimethylation by SETDB1 occurred in a distributive manner and was unaffected by the presence of ATF7IP. This finding is important as H3K9 can be methylated by HMTs other than SETDB1 and a distributive mechanism would allow for interplay between multiple HMTs on H3K9me1 and H3K9me2. The results presented here indicate that ATF7IP does not alter substrate specificity of SETDB1 in vitro, and though it decreases SETDB1's catalytic activity, our data points to a nonenzymatic function of ATF7IP in regulating SETDB1 function. Citation Format: Aravind Basavapathruni, Jodi Gureasko, Margaret Porter Scott, P. Ann Boriack-Sjodin, Timothy J. Wigle, Thomas V. Riera, Robert A. Copeland. ATF7IP does not alter the substrate specificity of the lysine methyltransferase SETDB1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 104. doi:10.1158/1538-7445.AM2015-104</jats:p

Probing the Mechanistic Consequences of 5-Fluorine Substitution on Cytidine Nucleotide Analogue Incorporation by HIV-1 Reverse Transcriptase

β-D and β-L-enantiomers of 2′,3′-dideoxycytidine analogues are potent chain-terminators and antimetabolites for viral and cellular replication. Seemingly small modifications markedly alter their antiviral and toxicity patterns. This review discusses previously published and recently obtained data on the effects of 5- and 2′-fluorine substitution on the pre-steady state incorporation of 2′-deoxycytidine-5′-monophosphate analogues by HIV-1 reverse transcriptase (RT) in light of their biological activity. The addition of fluorine at the 5-position of the pyrimidine ring altered the kinetic parameters for all nucleotides tested. Only the 5-fluorine substitution of the clinically relevant nucleosides (-)-β-L-2′,3′-dideoxy-3′-thia-5-fluoro-cytidine (L-FTC, Emtriva™), and (+)-β-D-2′,3′-dide-hydro-2′,3′-dideoxy-5-fluorocytidine (D-D4FC, Reverset™), caused a higher overall efficiency of nucleotide incorporation during both DNA- and RNA-directed synthesis. Enhanced incorporation by RT may in part explain the potency of these nucleosides against HIV-1. In other cases, a lack of correlation between RT incorporation in enzymatic assays and antiviral activity in cell culture illustrates the importance of other cellular factors in defining antiviral potency. The substitution of fluorine at the 2′ position of the deoxyribose ring negatively affects incorporation by RT indicating the steric gate of RT can detect electrostatic perturbations. Intriguing results pertaining to drug resistance have led to a better understanding of HIV-1 RT resistance mechanisms. These insights serve as a basis for understanding the mechanism of action for nucleoside analogues and, coupled with studies on other key enzymes, may lead to the more effective use of fluorine to enhance the potency and selectivity of antiviral agents.</jats:p

Modulation of Human Immunodeficiency Virus Type 1 Synergistic Inhibition by Reverse Transcriptase Mutations^†

Synergy between the anti-human immunodeficiency virus type 1 (HIV) nucleoside reverse transcriptase (RT) inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs) results from a general mechanism in which NNRTIs inhibit ATP-mediated removal of NRTIs from chain-terminated primers by decreasing the maximum rate of removal, thus sustaining NRTI chain termination. With this molecular mechanism of synergy, β-d-(+)-3‘-azido-3‘-deoxythymidine monophosphate (AZTMP) removal was examined in the context of clinically relevant RT mutants. The IC50 value for inhibition by nevirapine against wild-type (WT) RT in our removal assay was 3 μM, but this concentration had no effect on removal by the nevirapine-resistant Y181C mutant. Rather, a ∼83-fold increase in nevirapine was required to decrease the rate of removal by 50% for this mutant. Efavirenz displayed a 100 nM IC50 value against WT and the efavirenz-sensitive Y181C mutant, but the efavirenz-resistant mutants K103N and K103N/Y181C required a 6-fold increase in efavirenz concentration to achieve the same effect. A newer generation NNRTI, TMC125, showed potency (55 nM) against WT and all mutants, paralleling the activity of this inhibitor relative to nevirapine and efavirenz in cell culture. When tested against the AZT-resistant mutant, all NNRTIs inhibited removal by greater than 50%, showing that this mutant is hypersensitive to NNRTIs. Altogether these results illustrate that both the NNRTI and NRTI mutations can modulate chain termination. This demonstrates that sustaining synergistic HIV inhibition in combination NRTI/NNRTI therapy requires NNRTIs that are potent against WT virus and possess favorable activity profiles against clinically relevant mutations