Impact of Resistance Mutations on Inhibitor Binding to HIV‑1 Integrase

HIV-1 integrase (IN) is essential for HIV-1 replication, catalyzing two key reaction steps termed 3′ processing and strand transfer. Therefore, IN has become an important target for antiviral drug discovery. However, mutants have emerged, such as E92Q/N155H and G140S/Q148H, which confer resistance to raltegravir (RAL), the first IN strand transfer inhibitor (INSTI) approved by the FDA, and to the recently approved elvitegravir (EVG). To gain insights into the molecular mechanisms of ligand binding and drug resistance, we performed molecular dynamics (MD) simulations of homology models of the HIV-1 IN and four relevant mutants complexed with viral DNA and RAL. The results show that the structure and dynamics of the 140s’ loop, comprising residues 140 to 149, are strongly influenced by the IN mutations. In the simulation of the G140S/Q148H double mutant, we observe spontaneous dissociation of RAL from the active site, followed by an intrahelical swing-back of the 3′-OH group of nucleotide A17, consistent with the experimental observation that the G140S/Q148H mutant exhibits the highest resistance to RAL compared to other IN mutants. An important hydrogen bond between residues 145 and 148 is present in the wild-type IN but not in the G140S/Q148H mutant, accounting for the structural and dynamical differences of the 140s’ loop and ultimately impairing RAL binding in the double mutant. End-point free energy calculations that broadly capture the experimentally known RAL binding profiles elucidate the contributions of the 140s’ loop to RAL binding free energies and suggest possible approaches to overcoming drug resistance

<i>In vitro</i> anticancer activities of JKA97 against breast cancer cells.

<p>(A) Chemical structure of JKA97. (B) Concentrations of JKA97 inducing 50% growth inhibition (IC₅₀) in breast cancer cells, relative to the corresponding controls, based on the MTT assay. MCF7, MDA-MB-468, and MCF7 p53KD cells were exposed to various concentrations of JKA97 for 72 hrs. (C) Anti-proliferative effects of JKA97 on breast cancer cells. Cells were exposed to various concentrations of JKA97 for 48 hrs, followed by the BrdUrd incorporation assay. The proliferation index was calculated against untreated control cells (*P<0.05). (D) Induction of apoptosis in breast cancer cells by JKA97. Cells were exposed to various concentrations of JKA97 for 24 hrs, followed by measurement of apoptosis by Annexin V assay. The apoptotic index was calculated against untreated control cells (*P<0.05). (E) Effects of JKA97 on the cell cycle distribution of breast cancer cells. Cells were exposed to various concentrations of JKA97 for 24 hrs, followed by measurement DNA contents by flow cytometry. The cell cycle distribution was evaluated by comparing with that of control cells (*P<0.05). All the assays were performed in triplicate. Results were from at least three separate, repeated experiments.</p

Effects of JKA97 on the expression of cell cycle related proteins.

<p>(A) MCF7, MDA-MB-468, and MCF7 p53KD cells were exposed to various concentrations of JKA97 for 24 hrs, and the expression of p53 and p21 proteins was determined by Western blot analysis. (B) Cells were exposed to 10 µM of JKA97 for the indicated time, and the expression of p53 and p21 proteins was determined by Western blot analysis. β-actin was used as an equal-loading control of samples.</p

Effects of JKA97 on p21 expression.

<p>(A1) MCF7, MDA-MB-468, and MCF7 p53KD cells were exposed to various concentrations of JKA97 or vehicle for 24 hrs, followed by exposure to protein synthesis inhibitor cycloheximide (CHX, 10 µg/mL). p21 protein expression was detected by Western blotting at different times after exposure of CHX. (A2) The graph shows the quantification of the Western blotting data. MCF7 (B1), MDA-MB-468 (B2) and MCF7 p53KD (B3) Cells were exposed to various concentrations of JKA97 or vehicle for 24 hrs, and total RNA were extracted followed by reverse transcription, and detection mRNA level of p21 by Real-time quantification PCR and Quantification RT-PCR, normalized by mRNA level of GAPDH. (C) Cells were transfected with p21 promoter luciferase reporter plasmid and a Renilla luciferase reporter together for 12 hrs, followed by treatment of 10 µM JKA97 or vehicle for an additional 24 hrs. The reporter activity was normalized to the corresponding Renilla luciferase reporter. The luciferase assay was performed in triplicate. Statistical significance was determined compared with control (*P<0.05).</p

<i>In vivo</i> anticancer activity of JKA97 against breast cancer cells.

<p>JKA97 was administered by i.p. injection to nude mice bearing MCF7 (A1) or MDA-MB-468 (B1) xenograft tumors. For mice bearing MCF7 xenograft tumor, treatment groups received JKA97 at doses of 5 mg/kg/day or 25 mg/kg/day, 5 days/week, for 6 weeks; for mice bearing MDA-MB-468 xenograft tumor, treatment groups received doses at 5 mg/kg/day or 10 mg/kg/day, 5 days/week, for 18 days. Control groups received vehicle only. Animals were also monitored for changes in body weight as a surrogate marker for toxicity when it was administered to nude mice bearing (A2) MCF7 or (B2) MDA-MB-468 xenograft tumors. At the end of the experiments, xenograft tumors were removed and taken a photograph (A3 and B3).</p