Screening a library of 5,292 hydrazone/hydrazine compounds for inhibition of HIV reverse transcriptase-associated ribonuclease H (RNH) activity identified fluoren-9-yildene hydrazines as highly active inhibitors. The 33 fluoren-9-yildene hydrazines in this library were expanded to 118 compounds, 65 (55%) of which showed validated inhibition of RT RNH activity (IC50 values < 10 uM). These inhibitors were mainly monofunctional for RNH activity, since only 25 (21%) also inhibited RT RNA-dependent DNA polymerase activity. The two most potent RNH inhibitors (RNHIs) were compounds 15 and 25, which inhibited wild type RT-RNH activity with IC50 values of 0.34 ± 0.07 uM and 0.4 ± 0.03 uM, respectively. Similar inhibition was noted with two clinically relevant NNRTI resistant mutants, Y181C and K103N/L100V. Biochemical studies showed that these compounds preferentially inhibited non-directed and DNA 3'-end directed RNH cleavages. These compounds also inhibited the activity of the p15-EC RT RNH domain fragment with IC50 values of 0.43 ± 0.04 uM and 0.032 ± 0.004 uM, respectively. Furthermore, both compounds had antiviral activity against HIV-1 with EC50 values of 10 ± 3 uM and 1.4 ± 0.6 uM for compounds 15 and 25, respectively. Order of addition experiments showed that potent inhibition required pre-incubation of the enzyme with the inhibitor; inhibitory potency substantially decreased if the RNA/DNA substrate was present prior to inhibitor addition. Furthermore, inhibition was competitive with respect to the RNA/DNA substrate, suggesting an active site binding mode. 1H-15N HSQC protein NMR studies with the p15-EC RT RNH domain fragment further suggested that the inhibitor binds to the RNH active site. Molecular docking studies with compound 25 were consistent with an active site binding mode in which the hydrazine functionality hydrogen bonds with essential catalytic metal coordinating residues E52 (RT: 478) and D72 (RT: 498). A sulfonamido-phenyl ring substituent on compound 25 makes edge on-Ï interactions with H127 (RT: 539), another residue essential for RNH catalysis. We therefore propose that the fluoren-9-ylidene hydrazine RNHIs act by preventing access of RNH essential catalytic residues to the RNA/DNA substrate.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-07132011-153632 |
| Date | 17 August 2011 |
| Creators | LaBarge, Krystal Marion |
| Contributors | Pei Tang, Ph.D, Gorden Rule, Ph.D, Michael A. Parniak, Ph.D, Rieko Ishmia, Ph.D |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-07132011-153632/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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