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.
authors.affiliation: Retroviral Biology and Antivirals Laboratory, Centre for Biomedical Research, Burnet Institute, Melbourne, VIC 3004, Australia; Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia;
authors.forename: Jennifer
authors.initials: J
authors.lastname: La
authors.affiliation: Retroviral Biology and Antivirals Laboratory, Centre for Biomedical Research, Burnet Institute, Melbourne, VIC 3004, Australia;
authors.forename: Catherine F
authors.initials: CF
authors.lastname: Latham
authors.affiliation: Retroviral Biology and Antivirals Laboratory, Centre for Biomedical Research, Burnet Institute, Melbourne, VIC 3004, Australia; Department of Microbiology, Monash University, Clayton, VIC 3168, Australia;
authors.forename: Ricky N
authors.initials: RN
authors.lastname: Tinetti
authors.affiliation: Retroviral Biology and Antivirals Laboratory, Centre for Biomedical Research, Burnet Institute, Melbourne, VIC 3004, Australia;
authors.forename: Adam
authors.initials: A
authors.lastname: Johnson
authors.affiliation: Retroviral Biology and Antivirals Laboratory, Centre for Biomedical Research, Burnet Institute, Melbourne, VIC 3004, Australia;
authors.forename: David
authors.initials: D
authors.lastname: Tyssen
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261;
authors.forename: Kelly D
authors.initials: KD
authors.lastname: Huber
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261;
authors.forename: Nicolas
authors.initials: N
authors.lastname: Sluis-Cremer
authors.affiliation: Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia;
authors.forename: Jamie S
authors.initials: JS
authors.lastname: Simpson
authors.affiliation: Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia;
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Headey
authors.affiliation: Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia;
authors.forename: David K
authors.initials: DK
authors.lastname: Chalmers
authors.affiliation: Retroviral Biology and Antivirals Laboratory, Centre for Biomedical Research, Burnet Institute, Melbourne, VIC 3004, Australia; Department of Microbiology, Monash University, Clayton, VIC 3168, Australia; Department of Infectious Diseases, Monash University, Melbourne, 3004, Australia; Department of Microbiology and Immunology, University of Melbourne, Parkville, VIC 3010, Australia gildat@burnet.edu.au.
authors.forename: Gilda
authors.initials: G
authors.lastname: Tachedjian
mesh_terms: D017931:DNA Primers / Q000235:genetics; D055808:Drug Discovery / Q000379:methods*; D024202:Electrophoretic Mobility Shift Assay; D015497:HIV-1 / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D011355:Prodrugs / Q000032:analysis / Q000302:isolation & purification*; D018894:Reverse Transcriptase Inhibitors / Q000032:analysis / Q000302:isolation & purification* / Q000494:pharmacology*; D016914:Ribonuclease H / Q000037:antagonists & inhibitors; D054852:Small Molecule Libraries; D014779:Virus Replication / Q000187:drug effects
chemical_list: D017931:DNA Primers; D011355:Prodrugs; D018894:Reverse Transcriptase Inhibitors; D054852:Small Molecule Libraries; D016914:Ribonuclease H
keywords: HIV; STD-NMR; allosteric inhibitors; fragment-based drug discovery; reverse transcriptase