Structure and binding analysis of active site inhibitors targeting HIV-1 RNase H

ABSTRACT HIV/AIDS continues to be a menace to public health. Several drugs currently on the market have successfully improved the ability to manage the viral burden in infected patients. However, new drugs are needed to combat the rapid emergence of mutated forms of the virus that are resistant to existing therapies. Currently, approved drugs target three of the four major enzyme activities encoded by the virus that are critical to the HIV life cycle. Although a number of inhibitors of HIV RNase H activity have been reported, few inhibit by directly engaging the RNase H active site. Here, we describe structures of naphthyridinone-containing inhibitors bound to the RNase H active site. This class of compounds binds to the active site via two metal ions that are coordinated by catalytic site residues, D443, E478, D498, and D549. The directionality of the naphthyridinone pharmacophore is restricted by the ordering of D549 and H539 in the RNase H domain. In addition, one of the naphthyridinone-based compounds was found to bind at a second site close to the polymerase active site and non-nucleoside/nucleotide inhibitor sites in a metal-independent manner. Further characterization, using fluorescence-based thermal denaturation and a crystal structure of the isolated RNase H domain reveals that this compound can also bind the RNase H site and retains the metal-dependent binding mode of this class of molecules. These structures provide a means for structurally guided design of novel RNase H inhibitors.

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Structural and Binding Analysis of Pyrimidinol Carboxylic Acid andN-Hydroxy Quinazolinedione HIV-1 RNase H Inhibitors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01594-10 ◽

2011 ◽

Vol 55 (6) ◽

pp. 2905-2915 ◽

Cited By ~ 58

Author(s):

Eric B. Lansdon ◽

Qi Liu ◽

Stephanie A. Leavitt ◽

Mini Balakrishnan ◽

Jason K. Perry ◽

...

Keyword(s):

Carboxylic Acid ◽

Metal Ions ◽

Active Site ◽

Water Environment ◽

Divalent Metal ◽

Rnase H ◽

Accurate Assessment ◽

Divalent Metal Ions ◽

Substrate State ◽

Hiv 1

ABSTRACTHIV-1 RNase H breaks down the intermediate RNA-DNA hybrids during reverse transcription, requiring two divalent metal ions for activity. Pyrimidinol carboxylic acid andN-hydroxy quinazolinedione inhibitors were designed to coordinate the two metal ions in the active site of RNase H. High-resolution (1.4 Å to 2.1 Å) crystal structures were determined with the isolated RNase H domain and reverse transcriptase (RT), which permit accurate assessment of the metal and water environment at the active site. The geometry of the metal coordination suggests that the inhibitors mimic a substrate state prior to phosphodiester catalysis. Surface plasmon resonance studies confirm metal-dependent binding to RNase H and demonstrate that the inhibitors do not bind at the polymerase active site of RT. Additional evaluation of the RNase H site reveals an open protein surface with few additional interactions to optimize active-site inhibitors.

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Structure for an HIV-1 reverse transcriptase RNase H inhibitor bound at the active site

Acta Crystallographica Section A Foundations of Crystallography ◽

10.1107/s0108767308088880 ◽

2008 ◽

Vol 64 (a1) ◽

pp. C348-C348

Author(s):

D.M. Himmel ◽

T.A. Pauly ◽

J. Bauman ◽

C. Dharia ◽

A.D. Clark ◽

...

Keyword(s):

Reverse Transcriptase ◽

Active Site ◽

Rnase H ◽

Hiv 1

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Targeting Divalent Metal Ions at the Active Site of the HIV-1 RNase H Domain: NMR Studies on the Interactions of Divalent Metal Ions with RNase H and Its Inhibitors

American Journal of Analytical Chemistry ◽

10.4236/ajac.2011.26073 ◽

2011 ◽

Vol 02 (06) ◽

pp. 639-649 ◽

Cited By ~ 5

Author(s):

Jiangli Yan ◽

Haihong Wu ◽

Tiffany Tom ◽

Oleg Brodsky ◽

Karen Maegley

Keyword(s):

Metal Ions ◽

Active Site ◽

Divalent Metal ◽

Rnase H ◽

Divalent Metal Ions ◽

Nmr Studies ◽

Hiv 1

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Structure of HIV-1 Reverse Transcriptase with the Inhibitor β-Thujaplicinol Bound at the RNase H Active Site

Structure ◽

10.1016/j.str.2009.09.016 ◽

2009 ◽

Vol 17 (12) ◽

pp. 1625-1635 ◽

Cited By ~ 101

Author(s):

Daniel M. Himmel ◽

Karen A. Maegley ◽

Tom A. Pauly ◽

Joseph D. Bauman ◽

Kalyan Das ◽

...

Keyword(s):

Reverse Transcriptase ◽

Active Site ◽

Rnase H ◽

Hiv 1

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Structure of a Dihydroxycoumarin Active-Site Inhibitor in Complex with the RNase H Domain of HIV-1 Reverse Transcriptase and Structure–Activity Analysis of Inhibitor Analogs

Journal of Molecular Biology ◽

10.1016/j.jmb.2014.05.006 ◽

2014 ◽

Vol 426 (14) ◽

pp. 2617-2631 ◽

Cited By ~ 23

Author(s):

Daniel M. Himmel ◽

Nataliya S. Myshakina ◽

Tatiana Ilina ◽

Alexander Van Ry ◽

William C. Ho ◽

...

Keyword(s):

Reverse Transcriptase ◽

Active Site ◽

Activity Analysis ◽

Rnase H ◽

Structure Activity ◽

Hiv 1

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Localization of the Active Site of HIV-1 Reverse Transcriptase-associated RNase H Domain on a DNA Template Using Site-specific Generated Hydroxyl Radicals

Journal of Biological Chemistry ◽

10.1074/jbc.273.17.10139 ◽

1998 ◽

Vol 273 (17) ◽

pp. 10139-10146 ◽

Cited By ~ 41

Author(s):

Matthias Götte ◽

Gottfried Maier ◽

Hans J. Gross ◽

Hermann Heumann

Keyword(s):

Reverse Transcriptase ◽

Hydroxyl Radicals ◽

Active Site ◽

Rnase H ◽

Site Specific ◽

Dna Template ◽

Hiv 1

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Inhibition of Foamy Virus Reverse Transcriptase by Human Immunodeficiency Virus Type 1 RNase H Inhibitors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00056-14 ◽

2014 ◽

Vol 58 (7) ◽

pp. 4086-4093 ◽

Cited By ~ 17

Author(s):

Angela Corona ◽

Anna Schneider ◽

Kristian Schweimer ◽

Paul Rösch ◽

Birgitta M. Wöhrl ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Binding Site ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Active Site ◽

Foamy Virus ◽

Rnase H ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACTRNase H plays an essential role in the replication of human immunodeficiency virus type 1 (HIV-1). Therefore, it is a promising target for drug development. However, the identification of HIV-1 RNase H inhibitors (RHIs) has been hampered by the open morphology of its active site, the limited number of available RNase H crystal structures in complex with inhibitors, and the fact that, due to the high concentrations of Mg2+needed for protein stability, HIV-1 RNase H is not suitable for nuclear magnetic resonance (NMR) inhibitor studies. We recently showed that the RNase H domains of HIV-1 and prototype foamy virus (PFV) reverse transcriptases (RTs) exhibit a high degree of structural similarity. Thus, we examined whether PFV RNase H can serve as an HIV-1 RNase H model for inhibitor interaction studies. Five HIV-1 RHIs inhibited PFV RNase H activity at low-micromolar concentrations similar to those of HIV-1 RNase H, suggesting pocket similarity of the RNase H domains. NMR titration experiments with the PFV RNase H domain and the RHI RDS1643 (6-[1-(4-fluorophenyl)methyl-1H-pyrrol-2-yl)]-2,4-dioxo-5-hexenoic acid ethyl ester) were performed to determine its binding site. Based on these results and previous data,in silicodocking analysis showed a putative RDS1643 binding region that reaches into the PFV RNase H active site. Structural overlays were performed with HIV-1 and PFV RNase H to propose the RDS1643 binding site in HIV-1 RNase H. Our results suggest that this approach can be used to establish PFV RNase H as a model system for HIV-1 RNase H in order to identify putative inhibitor binding sites in HIV-1 RNase H.

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Developing and Evaluating Inhibitors against the RNase H Active Site of HIV-1 Reverse Transcriptase

Journal of Virology ◽

10.1128/jvi.02203-17 ◽

2018 ◽

Vol 92 (13) ◽

Cited By ~ 15

Author(s):

Paul L. Boyer ◽

Steven J. Smith ◽

Xue Zhi Zhao ◽

Kalyan Das ◽

Kevin Gruber ◽

...

Keyword(s):

Reverse Transcriptase ◽

Active Site ◽

Nucleoside Analogs ◽

Polymerase Activity ◽

Rnase H ◽

Strand Transfer ◽

Hiv Replication ◽

Content Type ◽

Rnase Hi ◽

Hiv 1

ABSTRACT We tested three compounds for their ability to inhibit the RNase H (RH) and polymerase activities of HIV-1 reverse transcriptase (RT). A high-resolution crystal structure (2.2 Å) of one of the compounds showed that it chelates the two magnesium ions at the RH active site; this prevents the RH active site from interacting with, and cleaving, the RNA strand of an RNA-DNA heteroduplex. The compounds were tested using a variety of substrates: all three compounds inhibited the polymerase-independent RH activity of HIV-1 RT. Time-of-addition experiments showed that the compounds were more potent if they were bound to RT before the nucleic acid substrate was added. The compounds significantly inhibited the site-specific cleavage required to generate the polypurine tract (PPT) RNA primer that initiates the second strand of viral DNA synthesis. The compounds also reduced the polymerase activity of RT; this ability was a result of the compounds binding to the RH active site. These compounds appear to be relatively specific; they do not inhibit either Escherichia coli RNase HI or human RNase H2. The compounds inhibit the replication of an HIV-1-based vector in a one-round assay, and their potencies were only modestly decreased by mutations that confer resistance to integrase strand transfer inhibitors (INSTIs), nucleoside analogs, or nonnucleoside RT inhibitors (NNRTIs), suggesting that their ability to block HIV replication is related to their ability to block RH cleavage. These compounds appear to be useful leads that can be used to develop more potent and specific compounds. IMPORTANCE Despite advances in HIV-1 treatment, drug resistance is still a problem. Of the four enzymatic activities found in HIV-1 proteins (protease, RT polymerase, RT RNase H, and integrase), only RNase H has no approved therapeutics directed against it. This new target could be used to design and develop new classes of inhibitors that would suppress the replication of the drug-resistant variants that have been selected by the current therapeutics.

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