Identification of mechanistically distinct inhibitors of HIV-1 reverse transcriptase through fragment screening

Jennifer La, Catherine Frances Mary Latham, Ricky Nathan Tinetti, Adam Peter Johnson, David P Tyssen, Kelly Dawn Huber, Nicolas Sluis-Cremer, Jamie Scott Simpson, Stephen James Headey, David Kenneth Chalmers, Gilda Tachedjian

Research output: Contribution to journalArticleResearchpeer-review

14 Citations (Scopus)

Abstract

Fragment-based screening methods can be used to discover novel active site or allosteric inhibitors for therapeutic intervention. Using saturation transfer difference (STD) NMR and in vitro activity assays, we have identified fragment-sized inhibitors of HIV-1 reverse transcriptase (RT) with distinct chemical scaffolds and mechanisms compared to nonnucleoside RT inhibitors (NNRTIs) and nucleoside/nucleotide RT inhibitors (NRTIs). Three compounds were found to inhibit RNA- and DNA-dependent DNA polymerase activity of HIV-1 RT in the micromolar range while retaining potency against RT variants carrying one of three major NNRTI resistance mutations: K103N, Y181C, or G190A. These compounds also inhibit Moloney murine leukemia virus RT but not the Klenow fragment of Escherichia coli DNA polymerase I. Steady-state kinetic analyses demonstrate that one of these fragments is a competitive inhibitor of HIV-1 RT with respect to deoxyribonucleoside triphosphate (dNTP) substrate, whereas a second compound is a competitive inhibitor of RT polymerase activity with respect to the DNA template/primer (T/P), and consequently also inhibits RNase H activity. The dNTP competing RT inhibitor retains activity against the NRTI-resistant mutants K65R and M184V, demonstrating a drug resistance profile distinct from the nucleotide competing RT inhibitors indolopyridone-1 (INDOPY-1) and 4-dimethylamino-6-vinylpyrimidine-1 (DAVP-1). In antiviral assays, the T/P competing compound inhibits HIV-1 replication at a step consistent with an RT inhibitor. Screening of additional structurally related compounds to the three fragments led to the discovery of molecules with improved potency against HIV-1 RT. These fragment inhibitors represent previously unidentified scaffolds for development of novel drugs for HIV-1 prevention or treatment.
Original languageEnglish
Pages (from-to)6979-6984
Number of pages6
JournalProceedings of the National Academy of Sciences
Volume112
Issue number22
DOIs
Publication statusPublished - 2015

Cite this

La, Jennifer ; Latham, Catherine Frances Mary ; Tinetti, Ricky Nathan ; Johnson, Adam Peter ; Tyssen, David P ; Huber, Kelly Dawn ; Sluis-Cremer, Nicolas ; Simpson, Jamie Scott ; Headey, Stephen James ; Chalmers, David Kenneth ; Tachedjian, Gilda. / Identification of mechanistically distinct inhibitors of HIV-1 reverse transcriptase through fragment screening. In: Proceedings of the National Academy of Sciences. 2015 ; Vol. 112, No. 22. pp. 6979-6984.
@article{3ec56b42339146b09a092a3aee4575c4,
title = "Identification of mechanistically distinct inhibitors of HIV-1 reverse transcriptase through fragment screening",
abstract = "Fragment-based screening methods can be used to discover novel active site or allosteric inhibitors for therapeutic intervention. Using saturation transfer difference (STD) NMR and in vitro activity assays, we have identified fragment-sized inhibitors of HIV-1 reverse transcriptase (RT) with distinct chemical scaffolds and mechanisms compared to nonnucleoside RT inhibitors (NNRTIs) and nucleoside/nucleotide RT inhibitors (NRTIs). Three compounds were found to inhibit RNA- and DNA-dependent DNA polymerase activity of HIV-1 RT in the micromolar range while retaining potency against RT variants carrying one of three major NNRTI resistance mutations: K103N, Y181C, or G190A. These compounds also inhibit Moloney murine leukemia virus RT but not the Klenow fragment of Escherichia coli DNA polymerase I. Steady-state kinetic analyses demonstrate that one of these fragments is a competitive inhibitor of HIV-1 RT with respect to deoxyribonucleoside triphosphate (dNTP) substrate, whereas a second compound is a competitive inhibitor of RT polymerase activity with respect to the DNA template/primer (T/P), and consequently also inhibits RNase H activity. The dNTP competing RT inhibitor retains activity against the NRTI-resistant mutants K65R and M184V, demonstrating a drug resistance profile distinct from the nucleotide competing RT inhibitors indolopyridone-1 (INDOPY-1) and 4-dimethylamino-6-vinylpyrimidine-1 (DAVP-1). In antiviral assays, the T/P competing compound inhibits HIV-1 replication at a step consistent with an RT inhibitor. Screening of additional structurally related compounds to the three fragments led to the discovery of molecules with improved potency against HIV-1 RT. These fragment inhibitors represent previously unidentified scaffolds for development of novel drugs for HIV-1 prevention or treatment.",
author = "Jennifer La and Latham, {Catherine Frances Mary} and Tinetti, {Ricky Nathan} and Johnson, {Adam Peter} and Tyssen, {David P} and Huber, {Kelly Dawn} and Nicolas Sluis-Cremer and Simpson, {Jamie Scott} and Headey, {Stephen James} and Chalmers, {David Kenneth} and Gilda Tachedjian",
year = "2015",
doi = "10.1073/pnas.1423900112",
language = "English",
volume = "112",
pages = "6979--6984",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
publisher = "National Academy of Sciences",
number = "22",

}

La, J, Latham, CFM, Tinetti, RN, Johnson, AP, Tyssen, DP, Huber, KD, Sluis-Cremer, N, Simpson, JS, Headey, SJ, Chalmers, DK & Tachedjian, G 2015, 'Identification of mechanistically distinct inhibitors of HIV-1 reverse transcriptase through fragment screening', Proceedings of the National Academy of Sciences, vol. 112, no. 22, pp. 6979-6984. https://doi.org/10.1073/pnas.1423900112

Identification of mechanistically distinct inhibitors of HIV-1 reverse transcriptase through fragment screening. / La, Jennifer; Latham, Catherine Frances Mary; Tinetti, Ricky Nathan; Johnson, Adam Peter; Tyssen, David P; Huber, Kelly Dawn; Sluis-Cremer, Nicolas; Simpson, Jamie Scott; Headey, Stephen James; Chalmers, David Kenneth; Tachedjian, Gilda.

In: Proceedings of the National Academy of Sciences, Vol. 112, No. 22, 2015, p. 6979-6984.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Identification of mechanistically distinct inhibitors of HIV-1 reverse transcriptase through fragment screening

AU - La, Jennifer

AU - Latham, Catherine Frances Mary

AU - Tinetti, Ricky Nathan

AU - Johnson, Adam Peter

AU - Tyssen, David P

AU - Huber, Kelly Dawn

AU - Sluis-Cremer, Nicolas

AU - Simpson, Jamie Scott

AU - Headey, Stephen James

AU - Chalmers, David Kenneth

AU - Tachedjian, Gilda

PY - 2015

Y1 - 2015

N2 - Fragment-based screening methods can be used to discover novel active site or allosteric inhibitors for therapeutic intervention. Using saturation transfer difference (STD) NMR and in vitro activity assays, we have identified fragment-sized inhibitors of HIV-1 reverse transcriptase (RT) with distinct chemical scaffolds and mechanisms compared to nonnucleoside RT inhibitors (NNRTIs) and nucleoside/nucleotide RT inhibitors (NRTIs). Three compounds were found to inhibit RNA- and DNA-dependent DNA polymerase activity of HIV-1 RT in the micromolar range while retaining potency against RT variants carrying one of three major NNRTI resistance mutations: K103N, Y181C, or G190A. These compounds also inhibit Moloney murine leukemia virus RT but not the Klenow fragment of Escherichia coli DNA polymerase I. Steady-state kinetic analyses demonstrate that one of these fragments is a competitive inhibitor of HIV-1 RT with respect to deoxyribonucleoside triphosphate (dNTP) substrate, whereas a second compound is a competitive inhibitor of RT polymerase activity with respect to the DNA template/primer (T/P), and consequently also inhibits RNase H activity. The dNTP competing RT inhibitor retains activity against the NRTI-resistant mutants K65R and M184V, demonstrating a drug resistance profile distinct from the nucleotide competing RT inhibitors indolopyridone-1 (INDOPY-1) and 4-dimethylamino-6-vinylpyrimidine-1 (DAVP-1). In antiviral assays, the T/P competing compound inhibits HIV-1 replication at a step consistent with an RT inhibitor. Screening of additional structurally related compounds to the three fragments led to the discovery of molecules with improved potency against HIV-1 RT. These fragment inhibitors represent previously unidentified scaffolds for development of novel drugs for HIV-1 prevention or treatment.

AB - Fragment-based screening methods can be used to discover novel active site or allosteric inhibitors for therapeutic intervention. Using saturation transfer difference (STD) NMR and in vitro activity assays, we have identified fragment-sized inhibitors of HIV-1 reverse transcriptase (RT) with distinct chemical scaffolds and mechanisms compared to nonnucleoside RT inhibitors (NNRTIs) and nucleoside/nucleotide RT inhibitors (NRTIs). Three compounds were found to inhibit RNA- and DNA-dependent DNA polymerase activity of HIV-1 RT in the micromolar range while retaining potency against RT variants carrying one of three major NNRTI resistance mutations: K103N, Y181C, or G190A. These compounds also inhibit Moloney murine leukemia virus RT but not the Klenow fragment of Escherichia coli DNA polymerase I. Steady-state kinetic analyses demonstrate that one of these fragments is a competitive inhibitor of HIV-1 RT with respect to deoxyribonucleoside triphosphate (dNTP) substrate, whereas a second compound is a competitive inhibitor of RT polymerase activity with respect to the DNA template/primer (T/P), and consequently also inhibits RNase H activity. The dNTP competing RT inhibitor retains activity against the NRTI-resistant mutants K65R and M184V, demonstrating a drug resistance profile distinct from the nucleotide competing RT inhibitors indolopyridone-1 (INDOPY-1) and 4-dimethylamino-6-vinylpyrimidine-1 (DAVP-1). In antiviral assays, the T/P competing compound inhibits HIV-1 replication at a step consistent with an RT inhibitor. Screening of additional structurally related compounds to the three fragments led to the discovery of molecules with improved potency against HIV-1 RT. These fragment inhibitors represent previously unidentified scaffolds for development of novel drugs for HIV-1 prevention or treatment.

UR - http://www.pnas.org.ezproxy.lib.monash.edu.au/content/112/22/6979.full.pdf

U2 - 10.1073/pnas.1423900112

DO - 10.1073/pnas.1423900112

M3 - Article

VL - 112

SP - 6979

EP - 6984

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 22

ER -