Screening and rank ordering of reversible mechanism‐based inhibitors of Hepatitis C virus NS3 protease using electrospray ionization mass spectrometry

An affinity‐selection study using size exclusion chromatography (SEC) combined with off‐line electrospray ionization mass spectrometry (ESI‐MS) was performed on libraries of peptidic α‐ketoamide inhibitors directed against the hepatitis C virus (HCV) NS3 protease. A limiting amount of HCV NS3 protease (25 µM ) was incubated with equimolar amounts (100 µM ) of 49 reversible mechanism‐based ketoamide inhibitors, previously grouped into seven sets to ensure clearly distinguishable mass differences of the enzyme‐inhibitor complexes (>10 Da). The unbound compounds were separated rapidly from the protease and the protease‐inhibitor complexes by SEC spin columns. The eluate of the SEC was immediately analyzed by direct‐infusion ESI‐MS. An enzyme‐inhibitor complex, with a molecular mass corresponding to the NS3 protease binding to the preferred inhibitor, SCH212986, was the only molecular species detected. By increasing the molar ratio of HCV NS3 protease to inhibitors to 1:2 while keeping the inhibitors' concentration constant, the complex of the second most tightly bound inhibitor, SCH215426, was also identified. Although the potencies of these inhibitors were virtually un‐measurable by kinetic assays, a rank order of CVS4441 > SCH212986 > SCH215426 was deduced for their inhibition potencies by direct competition experiment with CVS4441 ( M ). As discussed in the article, through judicious application of this strategy, even large libraries of fairly weak, reversible and slow‐binding inhibitors could be rapidly screened and rank ordered to provide critical initial structure‐activity insights. Copyright © 2011 John Wiley & Sons, Ltd.     

High-Throughput Fluorescent Assay for Inhibitor Screening of Proteases from RNA Viruses

Spanish flu, polio epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases caused by RNA viruses. The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands affordable and reliable assays for testing antivirals. To test inhibitors of viral proteases, we have developed an inexpensive high-throughput assay based on fluorescent energy transfer (FRET). We assayed an array of inhibitors for papain-like protease from SARS-CoV-2 and validated it on protease from the tick-borne encephalitis virus to emphasize its versatility. The reaction progress is monitored as loss of FRET signal of the substrate. This robust and reproducible assay can be used for testing the inhibitors in 96- or 384-well plates.     

Small Angle X-ray Scattering,Molecular Modeling,and Chemometric Studies from a Thrombin-Like (Lmr-47) Enzyme of Lachesis m. rhombeata Venom

Introduction: Snakebite envenomation is considered a neglected tropical disease, and SVTLEs critical elements are involved in serious coagulopathies that occur on envenoming. Although some enzymes of this group have been structurally investigated, it is essential to characterize other proteins to better understand their unique properties such as the Lachesis muta rhombeata 47 kDa (Lmr-47) venom serine protease. Methods: The structure of Lmr-47 was studied in solution, using SAXS, DLS, CD, and in silico by homology modeling. Molecular docking experiments simulated 21 competitive inhibitors. Results: At pH 8.0, Lmr-47 has an Rg of 34.5 ± 0.6 Å, Dmax of 130 Å, and SR of 50 Å, according to DLS data. Kratky plot analysis indicates a rigid shape at pH 8.0. Conversely, the pH variation does not change the center of mass’s intrinsic fluorescence, possibly indicating the absence of fluorescent amino acids in the regions affected by pH variation. CD experiments show a substantially random coiled secondary structure not affected by pH. The low-resolution model of Lmr-47 presented a prolate elongated shape at pH 8.0. Using the 3D structure obtained by molecular modeling, docking experiments identified five good and three suitable competitive inhibitors. Conclusion: Together, our work provided insights into the structure of the Lmr-47 and identified inhibitors that may enhance our understanding of thrombin-like family proteins.     

抑制剂G4G与HIV-1蛋白酶作用模式的结合自由能计算研究

本文采用分子动力学模拟和结合自由能计算研究了抑制剂G4G与HIV-1蛋白酶的作用机制。研究结果表明范德华作用主导了抑制剂G4G与HIV-1蛋白酶的结合。基于残基的自由能计算表明残基Gly49、Ile50、Val82、Ile84、Gly27′、Ala28′、Ile50′和Ile84′与G4G产生了较强的相互作用,同时研究也表明CH-π,CH-O相互作用和氢键是其主要的作用形式。该研究能为以HIV-1蛋白酶为靶标的抗艾滋病药物设计提供理论上的指导。     

HIV-1蛋白酶与抑制剂BEG相互作用的分子对接计算研究

本文使用分子对接方法和MM/PBSA方法计算抑制剂BEG与HIV-1蛋白酶的结合自由能,结果表明范德华相互作用主宰了BEG与HIV-1蛋白酶的结合,同时计算还证明分子对接采用的分子力场相比于Amber力场更适合于BEG-HIV蛋白酶系统。这篇论文还采用基于残基的自由能分解方法计算了抑制剂-残基相互作用,结果表明残基Leu23、Gly49、Ile50、Val82、Ile84、Asp25′、Gly27′、Ala28′、Gly49′、Ile50′和Ile84′与BEG产生了较强的相互作用,同时也证明CH-π和CH-O相互作用驱动了抑制剂BEG与HIV-1蛋白酶的结合。我们期望这个研究能为艾滋病治疗药物的合理化设计提供理论上的指导和实验上的参考。     

嗜热蛋白酶PH1704别构中心量子化学计算与突变体动力学简

通过量子化学计算,确定嗜热菌Pyrococcus horikoshii OT3的PH1704蛋白酶别构位点的关键残基为Arg113,Tyr120和Asn129. 其中,Arg113及Asn129与别构抑制剂结合,参与别构调控. Tyr120残基位于亚基交界面附近,并与亲核残基Cys100之间以氢键相连,可通过影响亚基聚合来影响酶的亲核催化. DJ-1超家族的4种构建蛋白的结构显示,120位点位于亚基交界面处,影响亚基的聚合,进而影响蛋白酶的活力,并间接参与别构调控. 分子生物学实验显示,突变体R113T/Y120P/N129D的kcat/km(L·μmol-1·min-1)值是野生型kcat/km值的6倍,h系数由野生型的0.86转变为1.3,负协同效应消失. 113和129位点处阴离子别构剂脱离,从而破坏113,120和129位点间的封闭环结构,使AC交界面α7螺旋(124~129,524~529)间聚合度增强;120位点残基由Tyr转变为Pro,与Cys100间氢键断裂,亲核进攻的阻力减小,从而使酶活力提高,别构负调控消失.     

An ultra-fast and highly efficient multiple proteases digestion strategy using graphene-oxide-based immobilized protease reagents

Highly efficient and rapid proteolytic digestion of proteins into peptides is a crucial step in shotgun-based proteome-analysis strategy.Tandem digestion by two or more proteases is demonstrated to be helpful for increasing digestion efficiency and decreasing missed cleavages,which results in more peptides that are compatible with mass-spectrometry analysis.Compared to conventional solution digestion,immobilized protease digestion has the obvious advantages of short digestion time,no self-proteolysis,and reusability.We proposed a multiple-immobilized proteases-digestion strategy that combines the advantages of the two digestion strategies mentioned above.Graphene-oxide(GO)-based immobilized trypsin and endoproteinase Glu-C were prepared by covalently attaching them onto the GO surface.The prepared GO-trypsin and GO-Glu-C were successfully applied in standard protein digestion and multiple immobilized proteases digestion of total proteins of Thermoanaerobacter tengcongensis.Compared to 12-hour solution digestion using trypsin or Glu-C,14%and 7%improvement were obtained,respectively,in the sequence coverage of BSA by one-minute digestion using GO-trypsin and GO-Glu-C.Multiple immobilized-proteases digestion of the total proteins of Thermoanaerobacter tengcongensis showed 24.3%and 48.7%enhancement in the numbers of identified proteins than was obtained using GO-trypsin or GO-Glu-C alone.The ultra-fast and highly efficient digestion can be contributed to the high loading capacity of protease on GO,which leads to fewer missed cleavages and more complete digestion.As a result,improved protein identification and sequence coverage can be expected.     

Role of structural water molecule in HIV protease-inhibitor complexes: a QM/MM study

Structural water molecule 301 found at the interface of HIV protease-inhibitor complexes function as a hydrogen bond (H-bond) donor to carbonyl groups of the inhibitor as well as H-bond acceptor to amide/amine groups of the flap region of the protease. In this study, six systems of HIV protease-inhibitor complexes were analyzed, which have the presence of this "conserved" structural water molecule using a two-layer QM/MM ONIOM method. The combination of QM/MM and QM method enabled the calculation of strain energies of the bound ligands as well as the determination of their binding energies in the ligand-water and ligand-water-protease complexes. Although the ligand experiences considerable strain in the protein bound structure, the H-bond interactions through the structural water overcomes this strain effect to give a net stability in the range of 16-24 kcal/mol. For instance, in 1HIV system, the strain energy of the ligand was 12.2 kcal/mol, whereas the binding energy associated with the structural water molecule was 20.8 kcal/mol. In most of the cases, the calculated binding energy of structural water molecule showed the same trend as that of the experimental binding free energy values. Further, the classical MD simulations carried out on 1HVL system with and without structural water 301 showed that this conserved water molecule enhances the H-bond dynamics occurring at the Asp-bound active site region of the protease-inhibitor system, and therefore it will have a direct influence on the mechanism of drug action.     

An Inhibitive Determination Method for Heavy Metals Using Bromelain,A Cysteine Protease

A heavy-metal assay has been developed using bromelain, a protease. The enzyme is assayed using casein as a substrate with Coomassie dye to track completion of hydrolysis of casein. In the absence of inhibitors, casein is hydrolysed to completion, and the solution is brown. In the presence of metal ions such as Hg²⁺ and Cu²⁺, the hydrolysis of casein is inhibited, and the solution remains blue. Exclusion of sulfhydryl protective agent and ethylenediaminetetraacetic in the original assay improved sensitivity to heavy metals several fold. The assay is sensitive to Hg²⁺ and Cu²⁺, exhibiting a dose–response curve with an IC₅₀ of 0.15 mg l⁻¹ for Hg²⁺ and a one-phase binding curve with an IC₅₀ of 0.23 mg l⁻¹ for Cu²⁺. The IC₅₀ value for Hg²⁺ is found to be lower to several other assays such as immobilized urease and papain assay, whilst the IC₅₀ value for Cu²⁺ is lower than immobilized urease, 15-min Microtox™, and rainbow trout.