用分子模拟方法研究HIV-1整合酶突变体的耐药性机理

二酮酸类化合物(DKAs)是目前最有前景的HIV-1整合酶(integrase, IN)抑制剂．为了解DKAs引起的多种耐药株共有的耐药性机理,选择3种S-1360引起的IN耐药突变体,用分子对接和分子动力学模拟,研究了野生型和突变型IN与S-1360的结合模式,基于该结合模式探讨了3种耐药突变体所共有的耐药性机理．结果表明：在突变体中,S-1360结合到耐药突变IN核心区中的位置靠近功能loop 3区却远离与 DNA结合的关键残基,结合位置不同导致S-1360的抑制作用部分丧失;残基138到166区域的柔性对IN发挥生物学功能很重要,S-1360能与DNA结合的关键残基N155及K159形成氢键,这2个氢键作用降低了该区域的柔性,突变体中无类似氢键,因而该区域柔性增高;在突变体中,S-1360的苯环远离病毒DNA结合区,不能阻止病毒DNA末端暴露给宿主DNA;T66I突变导致残基Ⅰ的长侧链占据IN的活性口袋,阻止抑制剂以与野生型中相同的方式结合到活性中心,这均是产生抗药性的重要原因．这些模拟结果与实验结果吻合,可为抗IN的抑制剂设计和改造提供帮助．     

Resistance mutations in human immunodeficiency virus type 1 integrase selected with elvitegravir confer reduced susceptibility to a wide range of integrase inhibitors

Integration of viral DNA into the host chromosome is an essential step in the life cycle of retroviruses and is facilitated by the viral integrase enzyme. The first generation of integrase inhibitors recently approved or currently in late-stage clinical trials shows great promise for the treatment of human immunodeficiency virus (HIV) infection, but virus is expected to develop resistance to these drugs. Therefore, we used a novel resistance selection protocol to follow the emergence of resistant HIV in the presence of the integrase inhibitor elvitegravir (GS-9137). We find the primary resistance-conferring mutations to be Q148R, E92Q, and T66I and demonstrate that they confer a reduction in susceptibility not only to elvitegravir but also to raltegravir (MK-0518) and other integrase inhibitors. The locations of the mutations are highlighted in the catalytic sites of integrase, and we correlate the mutations with expected drug-protein contacts. In addition, mutations that do not confer reduced susceptibility when present alone (H114Y, L74M, R20K, A128T, E138K, and S230R) are also discussed in relation to their position in the catalytic core domain and their proximity to known structural features of integrase. These data broaden the understanding of antiviral resistance against integrase inhibitors and may give insight facilitating the discovery of second-generation compounds.     

Azido-containing diketo acid derivatives inhibit human immunodeficiency virus type 1 integrase in vivo and influence the frequency of deletions at two-long-terminal-repeat-circle junctions

We previously found that azido-containing beta-diketo acid derivatives (DKAs) are potent inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase (IN) (X. Zhang et al., Bioorg. Med. Chem. Lett., 13:1215-1219, 2003). To characterize the intracellular mechanisms of action of DKAs, we analyzed the antiviral activities of two potent azido-containing DKAs with either a monosubstitution or a disubstitution of azido groups, using single- and multiple-replication-cycle assays. Both azido-containing DKAs significantly inhibited HIV-1 infection in 293T, CEM-SS, and H9 cells (50% inhibitory concentration = 2 to 13 micro M) and exhibited low cytotoxicity (50% cytotoxic concentration = 60 to 600 micro M). Inhibition of HIV-1 IN in vivo was demonstrated by the observation that previously described L-708,906 resistance mutations in HIV-1 IN (T66I and T66I/S153Y) also conferred resistance to the azido-group-containing DKAs. In vitro assays and in vivo analysis indicated that the DKAs did not significantly inhibit the 3' processing and selectively inhibited the strand transfer reaction. In addition, quantitative PCR indicated that two-long-terminal-repeat (2-LTR) circles were elevated in the presence of the azido-containing DKAs, confirming that HIV-1 IN was the intracellular target of viral inhibition. To gain insight into the mechanism by which the DKAs increased 2-LTR-circle formation of 3'-processed viral DNAs, we performed extensive DNA sequencing analysis of 2-LTR-circle junctions. The results indicated that the frequency of deletions at the circle junctions was elevated from 19% for the untreated controls to 32 to 41% in the presence of monosubstituted (but not disubstituted) DKAs. These results indicate that the structure of the DKAs can influence the extent of degradation of viral DNA ends by host nucleases and the frequency of deletions at the 2-LTR-circle junctions. Thus, sequencing analysis of 2-LTR-circle junctions can elucidate the intracellular mechanisms of action of HIV-1 IN inhibitors.     

Comparative molecular dynamics simulations of HIV-1 integrase and the T66I/M154I mutant: binding modes and drug resistance to a diketo acid inhibitor

HIV-1 IN is an essential enzyme for viral replication and an interesting target for the design of new pharmaceuticals for use in multidrug therapy of AIDS. L-731,988 is one of the most active molecules of the class of beta-diketo acids. Individual and combined mutations of HIV-1 IN at residues T66, S153, and M154 confer important degrees of resistance to one or more inhibitors belonging to this class. In an effort to understand the molecular mechanism of the resistance of T66I/M154I IN to the inhibitor L-731,988 and its specific binding modes, we have carried out docking studies, explicit solvent MD simulations, and binding free energy calculations. The inhibitor was docked against different protein conformations chosen from prior MD trajectories, resulting in 2 major orientations within the active site. MD simulations have been carried out for the T66I/M154I DM IN, DM IN in complex with L-731,988 in 2 different orientations, and 1QS4 IN in complex with L-731,988. The results of these simulations show a similar dynamical behavior between T66I/M154I IN alone and in complex with L-731,988, while significant differences are observed in the mobility of the IN catalytic loop (residues 138-149). Water molecules bridging the inhibitor to residues from the active site have been identified, and residue Gln62 has been found to play an important role in the interactions between the inhibitor and the protein. This work provides information about the binding modes of L-731,988, as well as insight into the mechanism of inhibitor-resistance in HIV-1 integrase.     

E92A是HIV-1整合酶耐药突变N155S的活性回复突变

HIV-1整合酶是目前抗艾滋病药物研发的重要靶点之一,整合酶的耐药突变是导致整合酶抑制剂类药物治疗失败的主要原因,但突变产生耐药性的机理仍不清楚.本工作通过人工构建突变型整合酶,测试其活性和耐药性,对整合酶的耐药机理进行初步探索.构建整合酶的突变型包括E92A、N155S两种单突变及E92A/N155S双突变.通过基因工程操作引入突变、构建质粒、表达纯化得到整合酶蛋白.用基于磁珠的整合酶链转移ELISA测试整合酶的链转移活性,用S-1360和Raltegravir两种抑制剂测试整合酶的耐药性.另外,用Autodock软件做了S-1360和整合酶核心区(包括野生型和突变型)的分子对接.结果表明,N155S突变使整合酶链转移活性下降约80%,而E92A/N155S双突变仅使活性下降约42%,这表明N155S突变基础上的E92A突变可使整合酶的活性大幅回复.E92A和E92A/N155S对不同的抑制剂可产生不同的耐药性,它们对Raltegravir的耐药性强于对S-1360.突变对整合酶活性和耐药性的影响主要是通过改变整合酶活性中心结构实现的,E92A突变可能导致其与周围残基静电相互作用减弱,间接影响到D64和D116残基,产生活性回复作用.     

Broad antiretroviral activity and resistance profile of the novel human immunodeficiency virus integrase inhibitor elvitegravir (JTK-303/GS-9137)

Integrase (IN), an essential enzyme of human immunodeficiency virus (HIV), is an attractive antiretroviral drug target. The antiviral activity and resistance profile in vitro of a novel IN inhibitor, elvitegravir (EVG) (also known as JTK-303/GS-9137), currently being developed for the treatment of HIV-1 infection are described. EVG blocked the integration of HIV-1 cDNA through the inhibition of DNA strand transfer. EVG inhibited the replication of HIV-1, including various subtypes and multiple-drug-resistant clinical isolates, and HIV-2 strains with a 50% effective concentration in the subnanomolar to nanomolar range. EVG-resistant variants were selected in two independent inductions, and a total of 8 amino acid substitutions in the catalytic core domain of IN were observed. Among the observed IN mutations, T66I and E92Q substitutions mainly contributed to EVG resistance. These two primary resistance mutations are located in the active site, and other secondary mutations identified are proximal to these primary mutations. The EVG-selected IN mutations, some of which represent novel IN inhibitor resistance mutations, conferred reduced susceptibility to other IN inhibitors, suggesting that a common mechanism is involved in resistance and potential cross-resistance. The replication capacity of EVG-resistant variants was significantly reduced relative to both wild-type virus and other IN inhibitor-resistant variants selected by L-870,810. EVG and L-870,810 both inhibited the replication of murine leukemia virus and simian immunodeficiency virus, suggesting that IN inhibitors bind to a conformationally conserved region of various retroviral IN enzymes and are an ideal drug for a range of retroviral infections.     

Structural determinants for HIV-1 integrase inhibition by beta-diketo acids.

Among all the HIV-1 integrase inhibitors, the beta-diketo acids (DKAs) represent a major lead in anti-HIV-1 integrase drug design. These derivatives inhibit the integration reaction in vitro with a strong specificity for the 3'-end joining step. They are also antiviral and inhibit integration in vivo. The aim of the present study has been to investigate the molecular interactions between DKAs and HIV-1 integrase. We have compared 5CITEP with one of the most potent DKAs reported by the Merck group (L-708,906) and found that 5CITEP inhibits 3'-processing at concentrations where L-708,906 is only active on strand transfer. We also report a novel bifunctional DKA derivative that inhibits 3'-processing even more effectively than 5CITEP. The interactions of these inhibitors with the viral DNA donor ends have been studied by performing experiments with oligonucleotides containing defined modifications. We propose that the bifunctional DKA derivative binds to both the acceptor and donor sites of HIV-1 integrase, whereas the monofunctional L-708,906 derivative binds selectively to the acceptor site.     

Selection and analysis of HIV-1 integrase strand transfer inhibitor resistant mutant viruses

This report describes methods for the selection and analysis of antiretroviral resistance to HIV integrase strand transfer inhibitors (InSTIs) in cell culture. The method involves the serial passage of HIV-1 in the presence of increasing concentrations of test inhibitors, followed by the cloning and sequencing of the integrase coding region from the selected viruses. The identified mutations are subsequently re-engineered into a reference wild-type molecular clone, and the resulting replication capacity and level of drug resistance are determined relative to the wild-type virus. Here we describe examples of selection and analysis of InSTI-resistant viruses using four integrase inhibitors from three structurally distinct chemical classes; a diketo acid, two naphthyridines, and a pyrimidinecarboxamide. Each inhibitor selected an independent route to resistance. Interestingly, the shift in the IC₅₀ required to suppress the re-engineered resistant mutant viruses closely matched the concentration of compound used during the selection of drug resistance.     

Molecular dynamics studies of the wild-type and double mutant HIV-1 integrase complexed with the 5CITEP inhibitor: mechanism for inhibition and drug resistance

The human immunodeficiency virus type 1 (HIV-1) integrase (IN) is an essential enzyme in the life cycle of the virus and is an attractive target for the development of new drugs useful in acquired immunodeficiency syndrome multidrug therapy. Starting from the crystal structure of the 5CITEP inhibitor bound to the active site in the catalytic domain of the HIV-1 IN, two different molecular dynamics simulations in water have been carried out. In the first simulation the wild-type IN was used, whereas in the second one the double mutation T66I/M154I, described to lead to drug resistance, was introduced in the protein. Compelling differences have been observed in these two structures during analyses of the molecular dynamics trajectories, particularly in the inhibitor binding modes and in the conformational flexibility of the loop (residues 138-149) located near the three catalytic residues in the active site (Asp(64), Asp(116), Glu(152)). Because the conformational flexibility of this region is important for efficient biological activity and its behavior is quite different in the two models, we suggest a hypothetical mechanism for the inhibition and drug resistance of HIV-1 IN. These results can be useful for the rational design of more potent and selective integrase inhibitors and may allow for the design of inhibitors that will be more robust against known resistance mutations.     

Mutational Analysis of the Binding Pockets of the Diketo Acid Inhibitor L-742,001 in the Influenza Virus PA Endonuclease

The influenza virus PA endonuclease, which cleaves capped host pre-mRNAs to initiate synthesis of viral mRNA, is a prime target for antiviral therapy. The diketo acid compound L-742,001 was previously identified as a potent inhibitor of the influenza virus endonuclease reaction, but information on its precise binding mode to PA or potential resistance profile is limited. Computer-assisted docking of L-742,001 into the crystal structure of inhibitor-free N-terminal PA (PA-Nter) indicated a binding orientation distinct from that seen in a recent crystallographic study with L-742,001-bound PA-Nter (R. M. DuBois et al., PLoS Pathog. 8:e1002830, 2012). A comprehensive mutational analysis was performed to determine which amino acid changes within the catalytic center of PA or its surrounding hydrophobic pockets alter the antiviral sensitivity to L-742,001 in cell culture. Marked (up to 20-fold) resistance to L-742,001 was observed for the H41A, I120T, and G81F/V/T mutant forms of PA. Two- to 3-fold resistance was seen for the T20A, L42T, and V122T mutants, and the R124Q and Y130A mutants were 3-fold more sensitive to L-742,001. Several mutations situated at noncatalytic sites in PA had no or only marginal impact on the enzymatic functionality of viral ribonucleoprotein complexes reconstituted in cell culture, consistent with the less conserved nature of these PA residues. Our data provide relevant insights into the binding mode of L-742,001 in the PA endonuclease active site. In addition, we predict some potential resistance sites that should be taken into account during optimization of PA endonuclease inhibitors toward tight binding in any of the hydrophobic pockets surrounding the catalytic center of the enzyme.     

Structure-based design of a novel peptide inhibitor of HIV-1 integrase: a computer modeling approach

The insertion of viral DNA into the host chromosome is an essential step in the replication of HIV-1, and is carried out by an enzyme, HIV-1 integrase (IN). Since the latter has no human cellular counterpart, it is an attractive target for antiviral drug design. Several IN inhibitors having activities in the micromolar range have been reported to date. However, no clinically useful inhibitors have yet been developed. Recently reported diketo acids represent a novel and selective class of IN inhibitors. These are the only class which appear to selectively target integrase and two of the inhibitors, L-708,906 and L-731,988, are the most potent inhibitors of preintegration complexes described to date.The X-ray crystal structure of the IN catalytic domain complexed with a diketo acid derivative inhibitor, 5CITEP, has recently been determined. Although the structure is of great value as a platform for drug design, experimental data suggest that crystal packing effects influence the observed inhibitor position. This has been confirmed by computational docking studies using the latest version (3.0) of the AutoDock program, which has been shown to give results largely consistent with available experimental data. Using AutoDock 3.0 and SYBYL6.6 we have modeled the complexes of IN with the diketo acid inhibitors so as to identify the enzyme binding site. In the quest for novel, potent and selective small molecule inhibitors, we present here a new approach to peptide inhibitor design using a, b- unsaturated (dehydro) residues, which confer a unique conformation on a peptide sequence. Based on the above models, we selected a tetrapeptide sequence containing a dehydro-Phe residue, which was found to have an open conformation as ascertained from its X-ray crystal structure. Docking results on this peptide led us to propose a modification at the C-terminal end. The modified peptide was found to dock in a similar position as the diketo acid inhibitors and was predicted to have a comparable potency.