Molecular dynamics simulations applied to the study of subtypes of HIV-1 protease common to Brazil, Africa, and Asia

Africa accounts for the majority of HIV-1 infections worldwide caused mainly by the A and C viral subtypes rather than B subtype, which prevails in the United States and Western Europe. In Brazil, B subtype is the major subtype, but F, C, and A also circulate. These non-B subtypes present polymorphisms, and some of them occur at sites that have been associated with drug resistance, including the HIV-1 protease (PR), one important drug target. Here, we report a Molecular Dynamics study of the B and non-B PR complexed with the inhibitor ritonavir to delineate the behavior of each subtype. We compare root mean squared deviation, binding free energy by linear interaction energy approach, hydrogen bonds, and intermolecular contact surface area between inhibitor and PR. From our results, we can provide a basis to understand the molecular mechanism of drug resistance in non-B subtypes. In this sense, we found a decrease of approx 4 kcal/mol in ΔG of binding between B and non-B subtypes. This corresponds to the loss of one hydrogen bond, which is in agreement with our H-bond analysis. Previous experimental affinity studies reported analogous results with inhibition constant values for non-B PR.     

Combining cross-sectional and prospective data methods to improve transition parameter estimation for characterizing the accumulation of HIV-1 drug resistance mutations

The order and rate of acquisition of HIV drug resistance mutations have been estimated from longitudinal and cross-sectional data using Markov models and branching trees, respectively. This article proposes methods that make use of both longitudinal and cross-sectional data simultaneously by employing link functions between the two parameter sets. Most functions that link the two parameter sets also depend on the time on treatment before the start of the study-information that may not be available. Nonetheless, under certain assumptions, some link functions eliminate the dependence on time. Using such functions, the two sources of information can be combined to improve the precision of parameter estimation. The method also accommodates error in the link functions from uncertainty in the assumptions required for the links or other reasons. These methods are applied to data from AIDS Clinical Trial Group protocol 398, a randomized comparison of mono- versus dual-protease inhibitor use in heavily treatment experienced HIV patients. Combining the two sources of information allows detection of differences between rates of transition that are not detectable using prospective data alone.     

A structural and thermodynamic escape mechanism from a drug resistant mutation of the HIV-1 protease

The efficacy of HIV-1 protease inhibition therapies is often compromised by the appearance of mutations in the protease molecule that lower the binding affinity of inhibitors while maintaining viable catalytic activity and substrate affinity. The V82F/I84V double mutation is located within the binding site cavity and affects all protease inhibitors in clinical use. KNI-764, a second-generation inhibitor currently under development, maintains significant potency against this mutation by entropically compensating for enthalpic losses, thus minimizing the loss in binding affinity. KNI-577 differs from KNI-764 by a single functional group critical to the inhibitor response to the protease mutation. This single difference changes the response of the two inhibitors to the mutation by one order of magnitude. Accordingly, a structural understanding of the inhibitor response will provide important guidelines for the design of inhibitors that are less susceptible to mutations conveying drug resistance. The structures of the two compounds bound to the wild type and V82F/I84V HIV-1 protease have been determined by X-ray crystallography at 2.0 A resolution. The presence of two asymmetric functional groups, linked by rotatable bonds to the inhibitor scaffold, allows KNI-764 to adapt to the mutated binding site cavity more readily than KNI-577, with a single asymmetric group. Both inhibitors lose about 2.5 kcal/mol in binding enthalpy when facing the drug-resistant mutant protease; however KNI-764 gains binding entropy while KNI-577 loses binding entropy. The gain in binding entropy by KNI-764 accounts for its low susceptibility to the drug-resistant mutation. The heat capacity change associated with binding becomes more negative when KNI-764 binds to the mutant protease, consistent with increased desolvation. With KNI-577, the opposite effect is observed. Structurally, the crystallographic B factors increase for KNI-764 when it is bound to the drug-resistant mutant. The opposite is observed for KNI-577. Consistent with these observations, it appears that KNI-764 is able to gain binding entropy by a two-fold mechanism: it gains solvation entropy by burying itself deeper within the binding pocket and gains conformational entropy by losing interaction with the protease.     

上海地区HIV毒株基因亚型分布及原发基因耐药的分子流行病学研究

本文通过对未经抗病毒治疗患者的人类免疫缺陷病毒1型（HIV-1）毒株进行检测,了解上海地区HIV-1的亚型分布及原发耐药基因变异现状。对118例未经治疗的HIV感染者其标本中HIV蛋白酶全长和部分反转录酶基因进行反转录-聚合酶链反应（RT-PCR）扩增,经DNA测序后进行系统进化树分析和重组分析,以确定HIV-1基因亚型和重组体,并与斯坦福耐药数据库比对,了解耐药性突变位点。使用斯坦福REGA HIV亚型分型工具和美国国立生物技术信息中心（NCBI）HIV亚型分析工具分析亚型,获得118例患者的HIV基因序列,基因分型分别为CRF01_AE重组体57例(48.3%)、B亚型36例(30.5%)、CRF07_BC 15例 (12.7%)、CRF08_BC 7例(5.9%)、C亚型2例(1.7%),亚型间或重组体间二重重组体(B/CRF01_A E)1例(0.8%)。蛋白酶抑制剂(PI)和反转录酶抑制剂相关的耐药基因突变率达54.2%（64/118）,其中2例（1.7%）发生PI耐药,基因突变位点：M46L、Q58E。5例(4.1%)对反转录酶抑制剂产生耐药,其中对核苷类反转录酶抑制剂（NRTI）和非核苷类反转录酶抑制剂（NNRTI）的耐药率分别为3例(2.4%)和5例(4.1%)。基因突变位点：NRTI为M41L、D67N、T69I/N/S、K70L、L74V、V75L、V118I、M184V、L210W/F/M/S和T215F;NNRTI为V90I、L100V、K103R/N、V106M/P/I/G、E138G/A、V179E/D/T、Y181C、G190A、H221Y、F227L、K238S和Y318F。结果提示,上海地区HIV毒株以CRF01_AE重组亚型为主,且发现新的重组体,可能出现新重组体流行的趋势。PI和反转录酶抑制剂相关的耐药基因突变率较高,且存在高度原发耐药毒株,应加强HIV-1耐药基因变异监测,科学、合理地给予抗病毒治疗。     

2014—2015年大连市伤寒沙门菌耐药性及分子分型研究

目的 了解大连市伤寒沙门菌的药物敏感情况及同源性特点,为临床用药和疾病预防提供科学指导。方法 选择15种抗生素、运用微量肉汤稀释法对65株伤寒沙门菌进行抗生素敏感试验;采用脉冲场凝胶电泳（PFGE）法进行聚类分析。结果 65株伤寒沙门菌对阿奇霉素100.00%敏感,对红霉素100.00%耐药,对其他13种药物有不同程度的耐药率（1.54%~73.85%）。发现1株多重耐药菌株。65株菌共产生41种PFGE带型,其中8株菌表现为同一型别。结论 大连地区临床分离伤寒沙门菌耐药形势严峻;聚类分析结果表明其PFGE型别较多,而且其中存在着优势菌株。     

Overcoming drug resistance in HIV-1 chemotherapy: the binding thermodynamics of Amprenavir and TMC-126 to wild-type and drug-resistant mutants of the HIV-1 protease

Amprenavir is one of six protease inhibitors presently approved for clinical use in the therapeutic treatment of AIDS. Biochemical and clinical studies have shown that, unlike other inhibitors, Amprenavir is severely affected by the protease mutation I50V, located in the flap region of the enzyme. TMC-126 is a second-generation inhibitor, chemically related to Amprenavir, with a reported extremely low susceptibility to existing resistant mutations including I50V. In this paper, we have studied the thermodynamic and molecular origin of the response of these two inhibitors to the I50V mutation and the double active-site mutation V82F/I84V that affects all existing clinical inhibitors. Amprenavir binds to the wild-type HIV-1 protease with high affinity (5.0 x 10(9) M(-1) or 200 pM) in a process equally favored by enthalpic and entropic contributions. The mutations I50V and V82F/I84V lower the binding affinity of Amprenavir by a factor of 147 and 104, respectively. TMC-126, on the other hand, binds to the wild-type protease with extremely high binding affinity (2.6 x 10(11) M(-1) or 3.9 pM) in a process in which enthalpic contributions overpower entropic contributions by almost a factor of 4. The mutations I50V and V82F/I84V lower the binding affinity of TMC-126 by only a factor of 16 and 11, respectively, indicating that the binding affinity of TMC-126 to the drug-resistant mutants is still higher than the affinity of Amprenavir to the wild-type protease. Analysis of the data for TMC-126 and KNI-764, another second-generation inhibitor, indicates that their low susceptibility to mutations is caused by their ability to compensate for the loss of interactions with the mutated target by a more favorable entropy of binding.     

Prevalence and mechanisms of resistance to fluoroquinolones in Helicobacter pylori strains from patients living in Belgium

BACKGROUND: Because of the increasing resistance of Helicobacter pylori against metronidazole and clarithromycin, alternative regimens including newer fluoroquinolones have been developed. We aimed to assess the prevalence as well as the mechanisms of this resistance in clinical isolates originating from patients living in Belgium. METHODS: Minimal inhibitory concentration (MIC) values of ciprofloxacin, levofloxacin, and moxifloxacin were determined by Etest method on 488 H. pylori isolates originating from patients who underwent upper gastrointestinal endoscopy at 10 different centers. Resistant strains (MIC values > 1 microg/ml) were evaluated for the presence of point mutations in the quinolone resistance-determining region (QRDR) of the gyrA by amplification and nucleotide sequence. RESULTS: Eighty-two (16.8%) of the strains were found resistant to all fluoroquinolones and 70 of these were further analyzed. Homogeneous and heterogeneous resistance were observed in 55 (78.6%) and in 15 (21.4%) of the strains, respectively. QRDR sequencing revealed various mutations of the codons corresponding to Asn-87 and Asp-91 in all isolates with homogeneous resistance. However, in 12 of 15 strains displaying heterogenous resistance, mutations were only detected after subcultures of isolated colonies growing within the ellipse inhibition zone of the E-test. Amino acid substitutions in the QRDR of GyrA could not be directly related with the MIC values of the isolates. Fluoroquinolone-resistant mutants were easily selected in vitro at frequencies ranging between 10(-6) and 10(-7). Such selected mutants stably persisted after several serial passage in antibiotic-free agar. CONCLUSIONS: These results suggest that H. pylori resistance to fluoroquinolones is occurring at a high frequency in the Belgian population and that it is essentially mediated through a variety of point mutations occurring in a few loci of GyrA. As a consequence, we strongly suggest to determine the susceptibility of the infecting isolates to fluoroquinolones before administration of an anti-H. pylori regimen including these agents.     

Multi-drug resistance profile of PR20 HIV-1 protease is attributed to distorted conformational and drug binding landscape: molecular dynamics insights

The PR20 HIV-1 protease, a variant with 20 mutations, exhibits high levels of multi-drug resistance; however, to date, there has been no report detailing the impact of these 20 mutations on the conformational and drug binding landscape at a molecular level. In this report, we demonstrate the first account of a comprehensive study designed to elaborate on the impact of these mutations on the dynamic features as well as drug binding and resistance profile, using extensive molecular dynamics analyses. Comparative MD simulations for the wild-type and PR20 HIV proteases, starting from bound and unbound conformations in each case, were performed. Results showed that the apo conformation of the PR20 variant of the HIV protease displayed a tendency to remain in the open conformation for a longer period of time when compared to the wild type. This led to a phenomena in which the inhibitor seated at the active site of PR20 tends to diffuse away from the binding site leading to a significant change in inhibitor–protein association. Calculating the per-residue fluctuation (RMSF) and radius of gyration, further validated these findings. MM/GBSA showed that the occurrence of 20 mutations led to a drop in the calculated binding free energies (ΔG_bind) by ~25.17 kcal/mol and ~5 kcal/mol for p2-NC, a natural peptide substrate, and darunavir, respectively, when compared to wild type. Furthermore, the residue interaction network showed a diminished inter-residue hydrogen bond network and changes in inter-residue connections as a result of these mutations. The increased conformational flexibility in PR20 as a result of loss of intra- and inter-molecular hydrogen bond interactions and other prominent binding forces led to a loss of protease grip on ligand. It is interesting to note that the difference in conformational flexibility between PR20 and WT conformations was much higher in the case of substrate-bound conformation as compared to DRV. Thus, developing analogues of DRV by retaining its key pharmacophore features will be the way forward in the search for novel protease inhibitors against multi-drug resistant strains.     

2006～2010年泌尿生殖道炎症患者支原体感染率检测与耐药性分析

目的 了解杭州市萧山区2006～ 2010年泌尿生殖道炎症患者支原体感染及耐药情况.方法 对4 023例泌尿生殖道炎症患者用支原体鉴定及药敏试剂盒进行支原体培养及药敏试验.结果 支原体总检出率为48.1％,女性检出率51.2％明显高于男性41.8％ (P ＜0.05).支原体培养阳性患者中Uu单独感染1701例(88.0％),Mh单独感染57例(2.9％),Uu+ Mh混合感染176例(9.1％),Uu单独感染明显高于Mh单独感染和Uu+ Mh混合感染(P＜0.05).5年间支原体阳性检出率从2006年的39.4％到2010年的58.1％逐年增高,感染模式观察期内无明显变化.支原体对交沙霉素、原始霉素和强力霉素均敏感(敏感率≥91.4％),对四环素、红霉素、氧氟沙星和环丙沙星的耐药率高,均大于50％.比较2006年至2010年各种支原体对9种药物的耐药率,除四环素外耐药率呈不同程度上升,四环素耐药率2007年和2008年较高,后逐年下降,2010年为53.5％.混合感染总体耐药率比Uu或Mh单独感染耐药率高.结论 泌尿生殖道炎症患者支原体感染Uu比较常见,且女性检出率显著高于男性.临床分离支原体大多具有多重耐药性,临床治疗需根据药敏结果加以选择.     

Autocatalytic maturation, physical/chemical properties, and crystal structure of group N HIV-1 protease: Relevance to drug resistance

The mature protease from Group N human immunodeficiency virus Type 1 (HIV‐1) (PR1_N) differs in 20 amino acids from the extensively studied Group M protease (PR1_M) at positions corresponding to minor drug‐resistance mutations (DRMs). The first crystal structure (1.09 Å resolution) of PR1_N with the clinical inhibitor darunavir (DRV) reveals the same overall structure as PR1_M, but with a slightly larger inhibitor‐binding cavity. Changes in the 10s loop and the flap hinge propagate to shift one flap away from the inhibitor, whereas L89F and substitutions in the 60s loop perturb inhibitor‐binding residues 29–32. However, kinetic parameters of PR1_N closely resemble those of PR1_M, and calorimetric results are consistent with similar binding affinities for DRV and two other clinical PIs, suggesting that minor DRMs coevolve to compensate for the detrimental effects of drug‐specific major DRMs. A miniprecursor (TFR 1 - 54 ‐PR1_N) comprising the transframe region (TFR) fused to the N‐terminus of PR1_N undergoes autocatalytic cleavage at the TFR/PR1_N site concomitant with the appearance of catalytic activity characteristic of the dimeric, mature enzyme. This cleavage is inhibited at an equimolar ratio of precursor to DRV (～6 μM), which partially stabilizes the precursor dimer from a monomer. However, cleavage at L34/W35 within the TFR, which precedes the TFR 1 - 54 /PR1_N cleavage at pH ≤ 5, is only partially inhibited. Favorable properties of PR1_N relative to PR1_M include its suitability for column fractionation by size under native conditions and >10‐fold higher dimer dissociation constant (150 nM). Exploiting these properties may facilitate testing of potential dimerization inhibitors that perturb early precursor processing steps.     

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.     

Human IgM monoclonal antibody to ganglioside GM2 and complement suppress virus propagation in ex vivo cultures of lymphocytes from HIV-1 infected patients.

HIV-1 infection induces aberrant ganglioside GM2 expression on infected cell lines, and human IgM anti-GM2 monoclonal antibody (L55 Ab) together with normal fresh human serum (FHS) as a source of complement causes complement mediated cytolysis of HIV-1 infected cells as well as HIV-1 particles. We report here that high expression of GM2 was also detected on HIV-1 infected lymphocytes from HIV-1 seropositive patients. L55 Ab effectively suppressed the generation of HIV in the presence of FHS in primarily cultured lymphocytes from HIV-1 infected patients in ex vivo experiments, and the suppression was enhanced additively by AZT. These data suggest that L55 Ab may increase the therapeutic effect of chemotherapy.     

Co-occurrence of East and West African kdr mutations suggests high levels of resistance to pyrethroid insecticides in Anopheles gambiae from Libreville, Gabon

Point mutations in the voltage-gated sodium channel gene involved in knockdown resistance to DDT and pyrethroid insecticides have been described in several insect species. In the malaria vector Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) two mutations have been identified. The first, consisting of a leucine-phenylalanine substitution at amino acid position 1014, is widespread in West Africa. The second, a leucine-serine substitution at the same position, has to date only been detected in western Kenya. Analysis of the kdr polymorphism in a sample of 106 An. gambiae s.s. of the rDNA S-form/Type I collected in Libreville (Gabon) surprisingly revealed the presence of both East and West African kdr mutations with frequencies of 63% and 37%, respectively. No wild-type alleles were detected and there was an excess of heterozygous genotypes (P = 0.04). In addition, an inconsistency was found during the kdr genotyping procedures by polymerase chain reaction, which could have lead to an underestimation of resistance alleles. The implications of these findings are discussed.     

HIV-1 protease molecular dynamics of a wild-type and of the V82F/I84V mutant: possible contributions to drug resistance and a potential new target site for drugs

The protease from type 1 human immunodeficiency virus (HIV-1) is a critical drug target against which many therapeutically useful inhibitors have been developed; however, the set of viral strains in the population has been shifting to become more drug-resistant. Because indirect effects are contributing to drug resistance, an examination of the dynamic structures of a wild-type and a mutant could be insightful. Consequently, this study examined structural properties sampled during 22 nsec, all atom molecular dynamics (MD) simulations (in explicit water) of both a wild-type and the drug-resistant V82F/I84V mutant of HIV-1 protease. The V82F/I84V mutation significantly decreases the binding affinity of all HIV-1 protease inhibitors currently used clinically. Simulations have shown that the curling of the tips of the active site flaps immediately results in flap opening. In the 22-nsec MD simulations presented here, more frequent and more rapid curling of the mutant's active site flap tips was observed. The mutant protease's flaps also opened farther than the wild-type's flaps did and displayed more flexibility. This suggests that the effect of the mutations on the equilibrium between the semiopen and closed conformations could be one aspect of the mechanism of drug resistance for this mutant. In addition, correlated fluctuations in the active site and periphery were noted that point to a possible binding site for allosteric inhibitors.