Investigating the role of metal chelation in HIV-1 integrase strand transfer inhibitors

HIV-1 integrase (IN) has been validated as an attractive target for the treatment of HIV/AIDS. Several studies have confirmed that the metal binding function is a crucial feature in many of the reported IN inhibitors. To provide new insights on the metal chelating mechanism of IN inhibitors, we prepared a series of metal complexes of two ligands (HL1 and HL2), designed as representative models of the clinically used compounds raltegravir and elvitegravir. Potentiometric measurements were conducted for HL2 in the presence of Mg(II), Mn(II), Co(II), and Zn(II) in order to delineate a metal speciation model. We also determined the X-ray structures of both of the ligands and of three representative metal complexes. Our results support the hypothesis that several selective strand transfer inhibitors preferentially chelate one cation in solution and that the metal complexes can interact with the active site of the enzyme.     

Chromone and chromanone derivatives as strand transfer inhibitors of HIV-1 integrase

HIV-1 integrase catalyzes terminal cleavage at the 3′ end of the proviral DNA, removing a pair of bases and causing strand transfer by joining the 3′ end to 5′-phosphates in the target DNA. Several aryl 1,3-diketo acids that can inhibit the strand transfer reaction of HIV-1 IN have been identified. Here we synthesized a new series of compounds with a chromone or chromanone ring as conformationally constrained scaffolds of 1,3-diketo acids, and then tested their ability to inhibit HIV-1 IN-mediated strand transfer. All compounds moderately inhibited HIV-1 IN activity, indicating that the conformational restriction of one keto group into a chromone or chromanone ring decreases inhibition of the HIV-1 IN strand transfer.     

Clinical pharmacology profile of raltegravir, an HIV-1 integrase strand transfer inhibitor

Raltegravir is an HIV-1 integrase inhibitor approved to treat HIV infection in adults in combination with other antiretrovirals. Data from healthy volunteers demonstrate that raltegravir is rapidly absorbed with a mean half-life of approximately 7 to 12 hours, with steady state achieved in approximately 2 days. Raltegravir is characterized by both high intra- and interindividual variabilities, although neither gender, race, age, body mass index, food intake, nor renal or hepatic insufficiency has a clinically meaningful effect on raltegravir pharmacokinetics. Raltegravir lacks activity as a perpetrator of drug-drug interactions and demonstrates a low propensity to be subject to drug-drug interactions. Raltegravir is metabolized primarily by UGT1A1 and is not affected by P450 inhibitors or inducers. Inhibitors of UGT1A1 (eg, atazanavir) can increase plasma concentrations of raltegravir, although this increase has not been found to be clinically meaningful. Likewise, inducers of UGT1A1 (eg, rifampin) can reduce plasma concentrations of raltegravir, and the clinical significance of this reduction is being investigated in ongoing clinical studies. Raltegravir demonstrates favorable clinical pharmacology and a drug interaction profile that permits administration to a wide, demographically diverse patient population and coadministration with many other therapeutic agents, including antiretroviral agents and supportive medications, without restrictions or dose adjustment.     

Binding mode prediction of strand transfer HIV-1 integrase inhibitors using Tn5 transposase as a plausible surrogate model for HIV-1 integrase

The crystal structure of Tn5 transposase-DNA complex was used in docking experiments to predict binding modes of HIV-1 integrase strand transfer inhibitors (INSTIs). In fact, the identification of HIV-1 integrase inhibitors from an in vitro screen using Tn5 transposase as the target has been recently reported. Our results suggest the utility of this protein as a useful surrogate model for IN and also for in silico screening, in the search for new potential INSTIs.     

Mechanism of HIV-1 integrase inhibition by styrylquinoline derivatives in vitro

Styrylquinoline derivatives (SQ) efficiently inhibit the 3'-processing activity of integrase (IN) with IC50 values of between 0.5 and 5 microM. We studied the mechanism of action of these compounds in vitro. First, we used steady-state fluorescence anisotropy to assay the effects of the SQ derivatives on the formation of IN-viral DNA complexes independently of the catalytic process. The IC50 values obtained in activity and DNA-binding tests were similar, suggesting that the inhibition of 3'-processing can be fully explained by the prevention of IN-DNA recognition. SQ compounds act in a competitive manner, with Ki values of between 400 and 900 nM. In contrast, SQs did not inhibit 3'-processing when IN-DNA complexes were preassembled. Computational docking followed or not by molecular dynamics using the catalytic core of HIV-1 IN suggested a competitive inhibition mechanism, which is consistent with our previous data obtained with the corresponding Rous sarcoma virus domain. Second, we used preassembled IN-preprocessed DNA complexes to assay the potency of SQs against the strand transfer reaction, independently of 3'-processing. Inhibition occurred even if the efficiency was decreased by about 5- to 10-fold. Our results suggest that two inhibitor-binding modes exist: the first one prevents the binding of the viral DNA and then the two subsequent reactions (i.e., 3'-processing and strand transfer), whereas the second one prevents the binding of target DNA, thus inhibiting strand transfer. SQ derivatives have a higher affinity for the first site, in contrast to that observed for the diketo acids, which preferentially bind to the second one.     

第二代整合酶链转移抑制剂dolutegravir

dolutegravir是第二代整合酶链转移抑制剂。与第一代整合酶链转移抑制剂相比,dolutegravir具有更高的耐药发展屏障,可一天1次给药。对已产生对拉替拉韦和elvitegravir耐药的人免疫缺陷病毒感染患者,仍可使用dolutegravir,但用药频率调整为一天2次。dolutegravir用于肝、肾功能损害患者时不需剂量调整。在临床试验中,dolutegravir治疗的最常见不良反应是失眠和头痛,发生率均为2%~3%。作为一种新的整合酶链转移抑制剂, dolutegravir具有疗效显著、耐受性好的特点。     

Fungal phenalenones inhibit HIV-1 integrase

A phenalenone compound, atrovenetinone methyl acetal, was isolated from a culture broth of Penicillium sp. FKI-1463 as an HIV-1 integrase inhibitor, and it showed anti-HIV activity in vitro. HIV-1 integrase inhibition and anti-HIV activity of two other natural phenalenones were also studied. Among the tested compounds, funalenone inhibited HIV-1 integrase with an IC50 value of 10 microM and showed the best selectivity (anti-HIV, IC50=1.7 microM; cytotoxicity, IC50=87 microM).     

High-throughput real-time assay based on molecular beacons for HIV-1 integrase 3＇-processing reaction

Aim： To develop a high-throughput real-time assay based on molecular beacons to monitor the integrase 3＇-processing reaction in vitro and apply it to inhibitor screening. Methods： The recombinant human irnmunodeficiency virus （HIV）-I integrase （IN） is incubated with a 38 mer oligonucleotide substrate, a sequence identical to the U5 end of HIV- 1 long terminal repeats （LTR）. Based on the fluorescence properties of molecular beacons, the substrate is designed to form a stem-loop structure labeled with a fluorophore at the 5＇ end and a quencher at the 3＇ end. IN cleaves the terminal 3＇-dinucleotide containing the quencher, resulting in an increase in fluorescence which can be monitored on a spectrofluorometer. To optimize this assay, tests were performed to investigate the effects of substrates, enzyme and the metal ion concentrations on the IN activity and optimal parameters were obtained. Moreover, 2 IN inhibitors were employed to test the performance of this assay in antiviral compound screening. Results： The fluorescent intensity of the reaction mixture varies linearly with time and is proportional to the velocity of the 3＇-processing reaction. Tests were performed and the results showed that the optimal rate was obtained for a reaction mixture containing 50 mg/L recombinant HIV- 1 IN, 400 nmol/L substrate, and 10 mmol/L Mn^2＋. The IN 3＇-processing reaction under the optimal conditions showed a more than 18-fold increase in the fluorescence intensity compared to the enzyme-free control. The IC50 values of the IN inhibitors obtained in our assay were similar to the values obtained from a radiolabeled substrate assay. Conclusion： Our results demonstrated that this is a fast, reliable, and sensitive method to monitor HIV IN 3＇-processing reaction and that it can be used for inhibitor screening.     

HIV-1 integrase inhibition: binding sites,structure activity relationships and future perspectives

The integrase enzyme encoded by the human immunodeficiency virus plays an integral role in the viral life cycle, but is as yet unexploited as a clinical drug target. Integrase processes the viral DNA in the cytoplasm, translocates to the nucleus, and catalyzes viral DNA insertion into the host genome. A wide variety of chemical structures inhibit integrase in vitro, yet few of these apparently promising compounds have demonstrated similar efficacy in vivo. Multiple binding targets have been identified for different integrase inhibitors. These targets include the integrase enzyme prior to substrate binding, the viral DNA substrate, and the preintegration complex consisting of oligomeric integrase and the viral DNA. Some known inhibitors are effective only in the presence of divalent manganese as the active site metal ion cofactor, whereas others do not discriminate between manganese and magnesium ions. Integrase inhibition in response to ligand binding at one of multiple sites renders derivation of a simple set of structure activity relationships challenging. Progress toward this goal is reviewed in the context of experimental and theoretical structural information about integrase.     

Structure-based inhibitor design targeting HIV-1 integrase

HIV integrase (IN) is a viral-encoded protein that catalyzes the breaking and joining reactions that mediate integration of viral DNA into the host genome. Therefore, IN offers a unique target for the development of novel anti-HIV and anti-AIDS therapeutics. To take advantage of this potential, drug discovery efforts via structure-based design approaches have been undertaken. Presented is a review of computer-aided drug design efforts targeting HIV IN. Included is an overview of the life-cycle of HIV, with emphasis on the mechanism of action of IN, biological assays for measuring IN activity and identifying IN inhibitors, and the appropriate cell-based assays required for determining the antiviral activity of IN inhibitors. This is followed by a review of the available three-dimensional structures of HIV IN. Structure-based drug design efforts are then critiqued, including both ligand-based (e.g. pharmacophore) and target-based (e.g. docking) methods. Results from recent computational chemistry studies of IN are also discussed.     

Styrylquinazoline derivatives as HIV-1 integrase inhibitors

Styrylquinazoline derivatives were prepared by Perkin condensation and evaluated for inhibitory activity against HIV-1 integrase. Among them, compound 5c containing a free catechol ring was the most potent (IC(50)=0.8 +/- 1.9 microM)and showed 6-fold more potency than the corresponding styrylquinoline compound (IC(50) = 130.7 +/- 8.6 microM).