Molecular mechanics analysis of inhibitor binding to HIV-1 protease

Crystallographic structures of HIV protease with three differentpeptide-mimetic inhibitors were subjected to energy minimizationusing molecular mechanics, the minimized structures analyzedand the inhibitor binding energies calculated. Partial chargeassignment for the hydrogen bonded catalytic aspartk acids,Asp25 and -25', was in good agreement with charge calculationsusing semi-empirical molecular orbital methods. Root mean squaredeviations on minimization were small and similar for both subunitsin the protease dimer. The surface loops, which had the largestB factors, changed most on minimization; the hydrophobic coreand the inhibitor binding site showed little change. The distance-dependentdielectric of D(r) = 4r was found to be preferable to D(r) =r. Distance restraints were applied for the intermolecular hydrogenbonds to maintain the conformation of the inhibitor bindingsite. Using the dielectric of D(r) = 4r, the calculated interactionenergy of the three inhibitors with the protease ranged from–53 to –56 kcal/mol. The groups of the inhibitorswere changed to add or remove a ‘transition state analogue’hydroxyl group, and the loss in energy on the removal of thisgroup was calculated to be 0.9–1.7 kcal/mol. This wouldrepresent 19–36% of the total measured difference in bindingenergy between the inhibitors JG365 and MVT-101.     

Pentacycloundecane-diol-based HIV-1 protease inhibitors: biological screening, 2D NMR, and molecular simulation studies

Novel compounds incorporating a pentacycloundecane (PCU) diol moiety were designed, synthesized, and evaluated as inhibitors of the wild-type C-South African (C-SA) HIV-1 protease. Seven compounds are reported herein, three of which displayed IC(50) values in the 0.5-0.6 μM range. The cytotoxicity of PCU cage peptides toward human MT-4 cells appears to be several orders of magnitude less toxic than the current antiviral medications ritonavir and lopinavir. NMR studies based on the observed through-space (1)H,(1)H distances/contacts in the EASY-ROESY spectra of three of the considered PCU peptide inhibitors enabled us to describe their secondary solution structure. Conserved hydrogen bonding interactions were observed between the hydroxy group of the PCU diol inhibitors and the catalytic triad (Asp25, Ile26, Gly27) of HIV protease in docking and molecular dynamics simulations. The biological significance and possible mode of inhibition by PCU-based HIV protease inhibitors discussed herein facilitates a deeper understanding of this family of inhibitors and their potential application to a vast number of alternative diseases related to proteases.     

蛋白酶体抑制剂MG132对肿瘤恶病质的作用及其分子机制

目的探讨蛋白酶体抑制剂MG132对肿瘤恶病质的作用及其可能的分子机制。方法经小鼠腋窝皮下注射结肠腺癌C26细胞,建立肿瘤恶病质模型,并设正常对照组(HC组)。将模型小鼠分为肿瘤恶病质组(CC组)和MG132治疗组(MG组),待小鼠进入恶病质状态后,CC组小鼠经腹腔注射0.1 ml生理盐水,MG组小鼠经腹腔注射0.1 mg/kg的MG132,7 d后处死小鼠,称量小鼠肿瘤、左侧腓肠肌和附睾脂肪的重量,测量腓肠肌纤维横切面积,ELISA法检测血清中炎性因子TNF-α和IL-6的水平,RT-PCR及Western blot法检测腓肠肌中IKBa、P65、MuRF1和MAFbx基因mRNA的转录水平及蛋白的表达水平。结果与CC组相比,MG组小鼠腓肠肌和附睾脂肪组织的重量分别增加了31.6%和39.5%(P<0.05),腓肠肌纤维横切面积增加了36.1%(P<0.05);血清中TNF-α和IL-6的水平分别降低了20.9%和42.0%(P<0.05);腓肠肌组织IKBa基因mRNA的转录水平和蛋白表达水平分别升高了132.7%和56.5%(P<0.05),MuRF1基因mRNA的转录水平和蛋白表达水平分别降低了70.1%和42.6%(P<0.05),MAFbx基因mRNA的转录水平和蛋白表达水平分别降低了76.8%和47.3%(P<0.05),P65基因mRNA的转录水平和蛋白表达水平分别降低了59.1%和53.1%(P<0.05)。结论 MG132改善肿瘤恶病质的分子机制可能与抑制NF-κB途径及MuRF1和MAFbx的表达、抑制炎症反应及肿瘤生长有关。     

Targeting the open-flap conformation of HIV-1 protease with pyrrolidine-based inhibitors

HIV protease is a well-established drug target in antiviral chemotherapy. Immense research efforts have been made to discover effective inhibitors, thus making the enzyme one of the most studied and best characterized proteins. Although the protease exhibits high flexibility, all approved drugs target virtually the same protein conformation. The development of viral cross-resistance demands the generation of inhibitors with novel scaffolds and deviating modes of binding. Herein we report the design and the short, high-yielding stereoselective synthesis of a series of chiral, symmetric pyrrolidine-based inhibitors targeting the open-flap conformation of the protease. The obtained co-crystal structure with one derivative provides a valuable starting point for further inhibitor design.     

Molecular dynamics simulations of HIV-1 protease with peptide substrate

Molecular dynamics simulations of human immunodeficiency virus(HIV)-l protease with a model substrate were used to test ifthere is a stable energy minimum for a proton that is equidistantfrom the four delta oxygen atoms of the two catalytic asparticacids. The crystal structure of HIV-1 protease with a peptidicinhibitor was modified to model the peptide substrate Ser-Gln-Asn-Tyr-Pro-Ile-Val-Glnfor the starting geometry. A proton was positioned between thetwo closest oxygen atoms of the two catalytic aspartic acids,and close to the carbonyl oxygen of the scissile bond in thesubstrate. All crystallographic water molecules were included.Two molecular dynamics simulations were run: 30 ps with united-atompotentials and 40 ps using the more accurate all-atom potentials.The molecular dynamics used a new algorithm that increased thespeed and allowed the elimination of a cut-off for non-bondedinteractions and the inclusion of an 8 shell of water moleculesin the calculations. The overall structure of the protease dimer,including the catalytic aspartic acids, was stable during thecourse of the molecular dynamics simulations. The substrateand a water molecule, that is an important component of thebinding site, were stable during the simulation using all-atompotentials, but more mobile when united-atom potentials wereused. A Poincare map representation showed that the positionsof the proton and its coordinating oxygen atoms were stablefor 93% of both simulations, although many of the buried andpoorly accessible water molecules exchanged with solvent. Theproton has a stable minimum energy position and maintains coordinationwith all four delta oxygen atoms of the two catalytic asparticacids and the carbonyl oxygen of the scissile bond of the substrate.Therefore, a loosely bound hydrogen ion at this position willnot be rapidly exchanged with solvent, and will rebond to eithera catalytic aspartic acid or possibly the substrate. The implicationsfor the reaction mechanism are discussed.     

基于结构的HIV-1整合酶抑制剂的虚拟筛选

目的虚拟筛选基于结构的HIV-1整合酶抑制剂。方法从PDB(Protein Data Bank)下载HIV-1整合酶催化核心结构域与LEDGF/p75整合酶结合结构域(integrase binding domain,IBD)的晶体结构(PDB ID:2B4J),通过AutoDockTools对结构进行处理;从ZINC数据库下载化合物结构,用PyRx处理和转换成pdbqt格式,建立一个处理后的化合物数据库;以HIV整合酶为靶点,通过新的虚拟筛选工具PyRx运行AutoDock Vina,对ZINC数据库的化合物进行虚拟筛选;分析得到的小分子抑制剂与整合酶之间的结合情况,并用PyMol对小分子抑制剂与整合酶的结合模式进行3D建模。结果经3轮筛选,发现5个高活性的HIV-1整合酶抑制剂ZINC9486894、ZINC47636331、ZINC57383520、ZINC68964708、ZINC73549421;5个小分子抑制剂与整合酶之间的结合主要是氢键结合力和疏水相互作用。结论通过PyRx运行AutoDock Vina,从ZINC数据库的化合物中筛选出5个新的HIV-1整合酶抑制剂。     

Examples of peptide-peptoid hybrid serine protease inhibitors based on the trypsin inhibitor SFTI-1 with complete protease resistance at the P1-P1' reactive site

Research in the field of protease inhibitors is focused on obtaining potent, specific and protease-resistant inhibitors. To our knowledge, there are no reports in the literature that consider the application of N-substituted glycine residues (peptoid monomers) for the design of peptidomimetic protease inhibitors. We hereby present the chemical synthesis and kinetic properties of two new analogues of the trypsin inhibitor SFTI-1 modified at the P1 position. Substitution of Lys5 in SFTI-1 by N-(4-aminobutyl)-glycine and N-benzylglycine, which mimic Lys and Phe, respectively, made these analogues completely protease-resistant at their P1-P1' reactive sites. The analogues synthesised appeared to be potent inhibitors of bovine beta-trypsin and alpha-chymotrypsin. These noncovalent, competitive and selective peptide-peptoid hybrid (peptomeric) inhibitors might open the way to targeting unwanted proteolysis.     

Antiviral agent based on the non-structural protein targeting the maturation process of HIV-1: expression and susceptibility of chimeric Vpr as a substrate for cleavage by HIV-1 protease

The processing of precursor proteins (Gag and Gag-pol) by theviral protease is absolutely required in order to generate infectiousparticles. This prompted us to consider novel strategies thattarget viral maturation. Towards this end, we have engineeredan HIV-1 virion associated protein, Vpr, to contain proteasecleavage signal sequences from Gag and Gag-pol precursor proteins.We previously reported that virus particles derived from HIV-1proviral DNA, encoding chimeric Vpr, showed a lack of infectivity,depending on the fusion partner. As an extension of that work,the potential of chimeric Vpr as a substrate for HIV-1 proteasewas tested utilizing an epitope-based assay. Chimeric Vpr moleculeswere modified such that the Flag epitope is removed followingcleavage, thus allowing us to determine the efficiency of proteasecleavage. Following incubation with the protease, the resultantproducts were analyzed by radioimmunoprecipitation using antibodiesdirected against the Flag epitope. Densitometric analysis ofthe autoradiograms showed processing to be both rapid and specific.Further, the analysis of virus particles containing chimericVpr by immunoblot showed reactivities to antibodies againstthe Flag epitope similar to the data observed in vitro. Theseresults suggest that the pseudosubstrate approach may provideanother avenue for developing antiviral agents.     

Modified solvent accessibility free energy prediction analysis of cyclic urea inhibitors binding to the HIV-1 protease

One of the most successful drug targets against AIDS in thelast decade has been the HIV-1 protease (HIV-1 PR), an enzymethat processes the polyprotein gene products into active replicativeviral proteins. In our quest for a wide-ranging, binding freeenergy function we have extended the solvent accessibility freeenergy predictor (SAFE_p) method, recently developed for peptidicHIV-1 PR inhibitors, to the study of the binding of cyclic urea(CU) HIV-1 PR inhibitors. Our results show that there is a needfor a specific term depicting polar contacts to be added tothe original SAFE_p analytical expression, an outcome not seenin our studies of HIV-1 PR peptidic inhibitors. Nevertheless,despite the higher profile of the electrostatic interactionsin the binding of the CU inhibitors, our analysis indicatesthat CU inhibitor binding is still driven by the hydrophobicentropic contribution, as much as for the peptidic inhibitors.     

Free energy perturbation studies on binding of A-74704 and its diester analog to HIV-1 protease

Free energy simulations have been employed to rationalize thebinding differences between A-74704, a pseudo C₂- symmetricinhibitor of HIV-1 protease and its diester analog. The diesteranalog inhibitor, which misses two hydrogen bonds with the enzymeactive site, is surprisingly only 10-fold weaker. The calculatedfree energy difference of 1.7 ± 0.6 kcal/mol is in agreementwith the experimental result. Further, the simulations showthat such a small difference in binding free energies is dueto (1) weaker hydrogen bond interactions between the two (P₁and P₁) NH groups of A-74704 with Gly27/Gly27' carbonyls ofthe enzyme and (2) the higher desolvation free energy of A-74704compared with its ester analog. The results of these calculationsand their implications for design of HIV-1 protease inhibitorsare discussed.     

Molecular modelling investigation of wild-type and the R528H mutated segment IIS4 of human L-type voltage-gated calcium channels

A molecular modelling study was performed in order to investigate the pathologically modified properties of L-type voltage-gated calcium channels caused by the arginine-to-histidine mutation at position 528 (R528H) in segment IIS4. For an appropriate consideration of the ionization state the finite difference Poisson-Boltzmann method was applied to compute the apparent pKa values of all titratable residues using standard conditions and an explicit lipophilic environment, respectively. Restrained molecular dynamics simulations were carried out for the alpha-helical transmembrane segments of the wild-type and the R528H mutant to explore their conformational behaviour. While both structures showed almost the same side-chain flexibility around the conserved residues, only the mutant partially formed a hydrogen bond from H528 to R531 during dynamics simulations. This local interaction not only causes a lower mobility of the directly involved residues but also leads to a global distortion of all positively charged amino acids of the mutant. Mostly affected is the side-chain of R534 that is shifted about 61 degrees closer to R531 and about 5 A in the direction of the cytoplasm. Subsequent examination of the molecular characteristics of this putative voltage sensor of the channel revealed considerable variations with regard to hydrogen bonding and electronic properties. Most obvious are the dramatic loss of a strong positive molecular electrostatic potential and the reduced hydrogen donor activities around position 528 of the mutant. How these results may be interpreted in relation to an enhanced inactivation rate is discussed, considering earlier findings at homologous voltage-gated potassium and sodium channels.      

Heterogeneity in recombinant HIV-1 integrase corrected by site-directed mutagenesis: the identification and elimination of a protease cleavage site

Purified recombinant human immunodeficiency virus type 1 (HIV-1) integrase and certain deletion mutants exhibit heterogeneity consistent with proteolysis at a site close to the C-terminus. Electrospray ionization mass spectrometric analysis indicated that proteolytic cleavage generated a protein missing five residues from the C-terminus. PCR mutagenesis of amino acids on either side of the cleavage site identified two changes which were subsequently shown to prevent clipping when proteins were expressed and purified from Escherichia coli: the substitution of Arg284, the residue on the C-terminal side of the cleavage site, by either glycine or lysine. The introduction of either of these mutations into full-length integrase did not affect in vitro 3' processing or strand transfer activities. Thus, the incorporation of either of these mutations is likely to be beneficial when homogeneity of HIV-1 integrase is a concern, as in crystallographic or nuclear magnetic resonance spectroscopic experiments.      

Ponatinib抑制野生型和T315I突变型BCR-ABL1激酶的分子模拟

药物普纳替尼（Ponatinib）对野生型和T315I突变型BCR-ABL1激酶均有较强抑制作用,然而其抑制机理仍然未知。为了更好地了解Ponatinib与BCR-ABL1激酶的作用机理,本文首先运用分子动力学模拟方法研究Ponatinib对BCR-ABL1激酶构象变化的影响,然后采用MM-PBSA方法计算复合物的结合自由能。结果表明：Ponatinib诱导野生型BCR-ABL1激酶P-loop和铰链区相互靠近,导致野生型激酶结合口袋关闭,有利于激酶与药物结合;而Ponatinib诱导T315I突变型BCR-ABL1激酶P-loop和铰链区相互远离,导致T315I突变型激酶结合口袋打开,对激酶与药物结合不利;Ponatinib与野生型以及T315I突变型BCR-ABL1激酶的结合自由能分别为-58.57 kcal/mol和-43.54kcal/mol,Ponatinib对野生型BCR-ABL1激酶的抑制能力明显强于T315I突变体,与文献报道的实验抑制活性结果一致。研究结果对认识靶点蛋白和抑制剂分子机制以及设计新药有重要意义。     

Effects of hydrophobic amino acid substitution in Pleurotus ostreatus proteinase A inhibitor 1 on its structure and functions as protease inhibitor and intramolecular chaperone

We previously demonstrated that Pleurotus ostreatus proteinase A inhibitor 1 (POIA1) could function as an intramolecular chaperone of subtilisin BPN', as in the case of the propeptide of subtilisin BPN', and that its Phe44 --> Ala mutant, which lost its tertiary structure, could not assist the refolding of subtilisin BPN'. In this study, we examined the effects of hydrophobic amino acid substitutions at other sites and substitutions of Phe44 with other hydrophobic residues on the structure and functions of POIA1. These mutations were introduced into POIA1cm that had been obtained by the substitution of the C-terminal six residues of POIA1 with those of the propeptide of subtilisin BPN'. When Ile32 or Ile64 was substituted with Ala, the tertiary structure of the resultant mutant was markedly destroyed, and the activities as a protease inhibitor and an intramolecular chaperone were significantly lowered. Among the position 44 mutants, the Phe44 --> Val mutant was a much less effective intramolecular chaperone with conversion to a digestible inhibitor, possibly owing to destruction of the tertiary structure. On the other hand, the Phe44 --> Leu or Ile mutant maintained its tertiary structure, and hence could function as a more effective intramolecular chaperone than the Phe44 --> Val mutant. Furthermore, since the Phe44 --> Leu mutant was a more susceptible inhibitor than POIA1cm, the halo formed around a colony of Bacillus cells transformed with a plasmid encoding this mutant was larger than others. These results clearly show the close relationship between the tertiary structure and functions of POIA1 as a protease inhibitor and an intramolecular chaperone, and that a combination of such inhibitory properties and intramolecular chaperone activity of POIA1 might affect the diameter of the halo formed around Bacillus colonies in vivo.