Link to the University of Pittsburgh Homepage
Link to the University Library System Homepage Link to the Contact Us Form

Fluoren-9-ylidene Hydrazine Inhibitors of HIV-1 Ribonuclease H

LaBarge, Krystal Marion (2011) Fluoren-9-ylidene Hydrazine Inhibitors of HIV-1 Ribonuclease H. Master's Thesis, University of Pittsburgh. (Unpublished)

Primary Text

Download (6MB) | Preview


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-π 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.


Social Networking:
Share |


Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
LaBarge, Krystal
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairRule,
Committee MemberParniak, Michael Amap167@pitt.eduMAP167
Committee MemberTang, PeiTangP@anes.upmc.eduPTANG
Committee MemberIshmia, Riekoishima@pitt.eduISHIMA
Date: 17 August 2011
Date Type: Completion
Defense Date: 7 July 2011
Approval Date: 17 August 2011
Submission Date: 13 July 2011
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Molecular Biophysics and Structural Biology
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: Fluoren-9-ylidene hdyrazine; fluorene; HIV; HIV reverse transcriptase-associated ribonuclease; Human Immunodeficiency Virus; hydrazine; hydrazone/hydrazine; Inhibitors; Ribonuclease H; RNH; RNHI; RNHIs; RT; hydrazone; Reverse Transcriptase
Other ID:, etd-07132011-153632
Date Deposited: 10 Nov 2011 19:51
Last Modified: 19 Dec 2016 14:36


Monthly Views for the past 3 years

Plum Analytics

Actions (login required)

View Item View Item