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

Identification of allosteric binding sites for PI3Kα oncogenic mutant specific inhibitor design

“…Although rare, inhibitory activities like these by low molecular weight fragments are not without precedent; La et al identified fragments that inhibited HIV reverse transcriptase with IC 50 values of 20–100 μM. 46 The low IC 50 values could also reflect potential assay interference. Fragment 21 has previously been identified as a frequent hitter, and as such is unlikely to prove a good lead for the development of inhibitors.…”

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

“…In the most recently reported FBDD approach targeting HIV-1 RT, the Tachedjian group in Melbourne, Australia [100] used STD-NMR to screen a library of 630 fragments against HIV-1 RT to search for allosteric inhibitors at sites other than the NNIBP. STD-NMR was followed by a secondary screen using a HIV-1 RT polymerase activity assay, where eight fragment hits were identified with micromolar potencies against the DNA polymerase function of wild-type (WT) HIV-1 RT.…”

“…Only three of the hits, 8-10 (Table 3) maintained a similar potency against WT RT and three clinically relevant NNRTI-resistant RT mutants, K103N, Y181C and G190A. In contrast to Bauman et al [98], this group [100] then used biochemical assays to gather evidence about the mechanisms of action and possible binding sites on the enzyme for the top three fragment hits. Two of three fragments, 9 and 10, were shown to compete with the HIV-1 RT substrates, nucleotide and T/P, respectively, suggesting that these two compounds could be inhibiting the enzyme allosterically with fragment 9 likely acting at or near the polymerase active site, such as the NcRTI binding site [101] or another allosteric site.…”

“…While the focus for FBDD is often on biophysical assays and characterization, it can be difficult to judge the appropriate timing to assess the inhibitory potential of allosteric inhibitors or binding sites. In this study [100], activity assays were used early in the screening process while taking steps to minimise false positives due to compound aggregation at high concentrations. Mechanistic assays were also used to characterise and prioritise hits from the fragment screen for further development.…”

Aspernigrins with anti-HIV-1 activities from the marine-derived fungus Aspergillus niger SCSIO Jcsw6F30

“…Existence of several shortcomings of the current ART, including the emergence of resistant virus, severe side effects and the high cost, has posed an urgent need for the discovery and development of potent anti-HIV-1 drugs with novel modes of action. 1 Marine organisms have been proven to be excellent sources of biologically active compounds against HIV-1. Discovery of anti-HIV-1agents, especially with novel modes of action, from marine organisms is becoming more attractive and promising.…”