Novel high-throughput screen identifies an HIV-1 reverse transcriptase inhibitor with a unique mechanism of action

Sheen, Chih‐Wei; Alptürk, Onur; Sluis‐Cremer, Nicolas

doi:10.1042/bj20140365

Cited by 3 publications

(5 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Activity of RT constructs. The RNA-dependent DNA polymerase activities of the purified and labeled RTs were assessed as described previously (23). The concentration of NNRTI required to inhibit 50% of the RT DNA polymerase activity (IC 50 ) was determined as described previously (19).…”

Section: Methodsmentioning

confidence: 99%

Nonnucleoside Reverse Transcriptase Inhibitor Hypersusceptibility and Resistance by Mutation of Residue 181 in HIV-1 Reverse Transcriptase

Barnard

Huber

Sluis‐Cremer

2019

Antimicrob Agents Chemother

Self Cite

View full text Add to dashboard Cite

Substitutions at residue Y181 in HIV-1 reverse transcriptase (RT), in particular, Y181C, Y181I, and Y181V, are associated with nonnucleoside RT inhibitor (NNRTI) cross-resistance. In this study, we used kinetic and thermodynamic approaches, in addition to molecular modeling, to gain insight into the mechanisms by which these substitutions confer resistance to nevirapine (NVP), efavirenz (EFV), and rilpivirine (RPV). Using pre-steady-state kinetics, we found that the dissociation constant (K d ) values for inhibitor binding to the Y181C and Y181I RT-template/primer (T/P) complexes were significantly reduced. In the presence of saturating concentrations of inhibitor, the Y181C RT-T/P complex incorporated the next correct deoxynucleoside triphosphate (dNTP) more efficiently than the wild-type (WT) complex, and this phenotype correlated with decreased mobility of the RT on the T/P substrate. Interestingly, we found that the Y181F substitution in RT-which represents a transitional mutation between Y181 and Y181I/V, or a partial revertant-conferred hypersusceptibility to EFV and RPV at both the virus and enzyme levels. EFV and RPV bound more tightly to Y181F RT-T/P. Furthermore, inhibitor-bound Y181F RT-T/P was less efficient than the WT complex in incorporating the next correct dNTP, and this could be attributed to increased mobility of Y181F RT on the T/P substrate. Collectively, our data highlight the key role that Y181 in RT plays in NNRTI binding.

show abstract

Section: Methodsmentioning

confidence: 99%

Nonnucleoside Reverse Transcriptase Inhibitor Hypersusceptibility and Resistance by Mutation of Residue 181 in HIV-1 Reverse Transcriptase

Barnard

Huber

Sluis‐Cremer

2019

Antimicrob Agents Chemother

Self Cite

View full text Add to dashboard Cite

show abstract

“…The multiple modes of inhibition shown for p-hydroxyaniline 8 similarly suggest that one or more of its mechanisms are allosteric. Large and hydrophobic T/P-competing molecules that block HIV-1 RT function, such as SY-3E4 (494 Da) (28), KM-1 (823 Da) (29), and APEX57219 (422 Da) (15), are also known; however, their size makes them undesirable as drug development leads. In contrast, p-hydroxyaniline 8 demonstrates exceptional potency for its size, suggesting it may show promise as a candidate for further development.…”

Section: Discussionmentioning

confidence: 99%

“…Our fragment screen and substructure analysis of one hit identified a total of seven active fragments that are chemically dissimilar to all other known HIV-1 RT inhibitors (Binding DB; www.bindingdb.org) (4,7,15), including those HIV-1 RT inhibitors identified in two previously published FBDD programs that used either SPR-based (16) or X-ray crystallography-based (6) screening. Three of the seven scaffolds that we identified have activities consistent with their ability to bind to sites distinct from the NNIBP on the HIV-1 RT, with one of these fragments inhibiting HIV-1 replication in cell culture at the reverse transcription step.…”

Section: Significancementioning

confidence: 99%

“…To determine that the hits represented new chemical scaffolds for HIV-1 RT inhibitors, a Tanimoto similarity search was performed to compare our seven hits with the 1,531 RT inhibitors recorded in the Binding DB (4,15,19,20) and with fragments identified in previous FBDD screens for HIV-1 RT inhibitors: the nine fragments reported by Bauman et al (6) and the bromoindanone NNRTI identified by Geitmann et al (16). A Tanimoto score of 80% or greater is generally regarded as high structural similarity.…”

Section: Chemical Similarity Search Of Hiv-1 Rt Inhibitor Database Rementioning

confidence: 99%

See 1 more Smart Citation

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

Latham

Tinetti

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

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.HIV | reverse transcriptase | fragment-based drug discovery | allosteric inhibitors | STD-NMR

show abstract

“…These properties are remarkable for such a small molecule with few distinctive chemical features, particularly since other known inhibitors of T/P (i.e. SY-3E4 [102], KM-1 [103] and APEX57219 [104], Table 3) are large and undesirable as drug development candidates. A small structure-activity relationship analysis revealed three related compounds with 3-5 fold higher potency than 8-10, and one of these related compounds, 11 (Table 3), was shown to maintain T/P competition and activity against HIV-1 in cell culture, similar to its parent compound, 8.…”

Section: Fbdd Screening Against Hiv-1 Rt To Discover Novel Allostericmentioning

confidence: 99%

Fragment Based Strategies for Discovery of Novel HIV-1 Reverse Transcriptase and Integrase Inhibitors

Latham

Tinetti

et al. 2015

CTMC

View full text Add to dashboard Cite

Human immunodeficiency virus (HIV) remains a global health problem. While combined antiretroviral therapy has been successful in controlling the virus in patients, HIV can develop resistance to drugs used for treatment, rendering available drugs less effective and limiting treatment options. Initiatives to find novel drugs for HIV treatment are ongoing, although traditional drug design approaches often focus on known binding sites for inhibition of established drug targets like reverse transcriptase and integrase. These approaches tend towards generating more inhibitors in the same drug classes already used in the clinic. Lack of diversity in antiretroviral drug classes can result in limited treatment options, as cross-resistance can emerge to a whole drug class in patients treated with only one drug from that class. A fresh approach in the search for new HIV-1 drugs is fragment-based drug discovery (FBDD), a validated strategy for drug discovery based on using smaller libraries of low molecular weight molecules (<300 Da) screened using primarily biophysical assays. FBDD is aimed at not only finding novel drug scaffolds, but also probing the target protein to find new, often allosteric, inhibitory binding sites. Several fragment-based strategies have been successful in identifying novel inhibitory sites or scaffolds for two proven drug targets for HIV-1, reverse transcriptase and integrase. While any FBDD-generated HIV-1 drugs have yet to enter the clinic, recent FBDD initiatives against these two well-characterised HIV-1 targets have reinvigorated antiretroviral drug discovery and the search for novel classes of HIV-1 drugs.

show abstract

Novel high-throughput screen identifies an HIV-1 reverse transcriptase inhibitor with a unique mechanism of action

Cited by 3 publications

References 30 publications

Nonnucleoside Reverse Transcriptase Inhibitor Hypersusceptibility and Resistance by Mutation of Residue 181 in HIV-1 Reverse Transcriptase

Nonnucleoside Reverse Transcriptase Inhibitor Hypersusceptibility and Resistance by Mutation of Residue 181 in HIV-1 Reverse Transcriptase

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

Fragment Based Strategies for Discovery of Novel HIV-1 Reverse Transcriptase and Integrase Inhibitors

Contact Info

Product

Resources

About