RESUMEN
Botulinum neurotoxins (BoNTs) are known as the most toxic natural substances. Synaptic vesicle protein 2 (SV2) has been proposed to be a protein receptor for BoNT/A. Recently, two short peptides (BoNT/A-A2 and SV2C-A3) were designed to inhibit complex formation between the BoNT/A receptor-binding domain (BoNT/A-RBD) and the synaptic vesicle protein 2C luminal domain (SV2C-LD). In this article, the two peptide complex systems are studied by molecular dynamics (MD) simulations. The structural stability analysis indicates that BoNT/A-A2 system is more stable than SV2C-A3 system. The conformational analysis implies that the ß-sheet in BoNT/A-A2 system maintains its secondary structure but the two ß-strands in SV2C-A3 system have remarkable conformational changes. Based on the calculation of hydrogen bonds, hydrophobic interactions and cation-π interactions, it is found that the internal hydrogen bonds play crucial roles in the structural stability of the peptides. Because of the stable secondary structure, the ß-sheet in BoNT/A-A2 system establishes effective interactions at the interface and inhibits BoNT/A-RBD binding to SV2C-LD. In contrast, without other ß-strands forming internal hydrogen bonds, the two isolated ß-strands in SV2C-A3 system become the random coil. This conformational change breaks important hydrogen bonds and weakens cation-π interaction in the interface, so the complex formation is only partially inhibited by the two ß-strands. These results are consistent with experimental studies and may be helpful in understanding the inhibition mechanisms of peptide inhibitors.
Asunto(s)
Toxinas Botulínicas Tipo A/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Simulación de Dinámica Molecular , Péptidos/química , Péptidos/metabolismo , Sitios de Unión , Toxinas Botulínicas Tipo A/química , Enlace de Hidrógeno , Unión ProteicaRESUMEN
HIV-1 integrase (IN) is a key enzyme for the viral replication. The protein-protein interaction (PPI) between HIV-1 IN and a cellular cofactor lens epithelium-derived growth factor (LEDGF/p75) is a validated target for anti-HIV drug discovery. In order to build the platform for screening inhibitor against PPI between IN and LEDGF/p75, the vector containing the LEDGF/p75 protein cDNA was constructed and expressed in Escherichia coli and the function of the LEDGF/p75 protein was assayed. The LGDGF/p75 encoding gene optimized according to the preference codon usage of E. coli, was synthesized and cloned into the expression vector pGEX-4T-1 to form a recombined plasmid, then transformed into host cell E. coli BL21 (DE3). The recombined clones were identified and confirmed by BamH I/Sal I digestion and sequencing, the successfully recombined plasmid in the host cell was induced by IPTG and the condition of the expression was optimized. The expressed protein was purified by the Ni2+ affinity chromatography column and SDS-PAGE was used to analyze the molecular weight and specificity. In addition, ELISA assay was used to analyze the function of the recombinant protein. The recombinant LGDGF/p75 was soluble, and expressed highly and stably in E. coli. The protein was proved to enhance HIV-1 IN strand transfer activity in vitro by ELISA. It will be helpful to build the platform of screening inhibitors against PPI between IN and LEDGF/p75.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Clonación Molecular , Escherichia coli/metabolismo , Integrasa de VIH/metabolismo , VIH-1/fisiología , Humanos , Unión Proteica , Replicación ViralRESUMEN
HIV-1 integrase is a key enzyme for retroviral replication. The integrase can perform a palindrome cleavage reaction. In this work, a hairpin DNA probe with a palindromic DNA sequence mimicking the HIV-1 LTR-LTR junction of the 2-LTR circles in its long stem was designed for fluorescence detection of specific restriction-like cleavage activity of HIV-1 integrase. Results showed that the designed probe could be recognized and cleaved by HIV-1 integrase. The palindrome cleavage reaction can be monitored according to the increase in fluorescent signal. The assay can be applied to real-time detection of palindrome cleavage of HIV-1 integrase with advantages of simplicity, high sensitivity, and specificity.
Asunto(s)
Sondas de ADN/genética , Sondas de ADN/metabolismo , Pruebas de Enzimas/métodos , Integrasa de VIH/metabolismo , VIH-1/enzimología , Secuencias Invertidas Repetidas , Secuencia de Bases , Duplicado del Terminal Largo de VIH , VIH-1/genética , Espectrometría de FluorescenciaRESUMEN
HIV-1 integrase, an essential enzyme for retroviral replication, is a validated target for anti-HIV therapy development. The catalytic core domain of integrase (IN-CCD) is capable of catalyzing disintegration reaction. In this work, a hairpin-shaped disintegration substrate was designed and validated by enzyme-linked immunosorbent assay; a molecular beacon-based assay was developed for disintegration reaction of IN-CCD. Results showed that the disintegration substrate could be recognized and catalyzed by IN-CCD, and the disintegration reaction can be monitored according to the increase of fluorescent signal. The assay can be applied to real-time detection of disintegration with advantages of simplicity, high sensitivity, and excellent specificity.
Asunto(s)
Dominio Catalítico , Pruebas de Enzimas/métodos , Integrasa de VIH/química , Integrasa de VIH/metabolismo , VIH-1/enzimología , Sondas de Oligonucleótidos/metabolismo , Secuencia de Bases , Sondas de Oligonucleótidos/genética , Factores de TiempoRESUMEN
Human immunodeficiency virus type 1 (HIV-1) integrase (IN) is an important drug target for anti-acquired immune deficiency disease (AIDS) treatment and diketo-acid (DKA) inhibitors are potent and selective inhibitors of HIV-1 IN. Due to lack of three-dimensional structures including detail interactions between HIV-1 IN and its substrate viral DNA, the drug design and screening platform remains incompleteness and deficient. In addition, the action mechanism of DKA inhibitors with HIV-1 IN is not well understood. In view of the high homology between the structure of prototype foamy virus (PFV) IN and that of HIV-1 IN, we used PFV IN as a surrogate model for HIV-1 IN to investigate the inhibitory mechanism of raltegravir (RLV) and the binding modes with a series of DKA inhibitors. Firstly, molecular dynamics simulations of PFV IN, IN-RLV, IN-DNA, and IN-DNA-RLV systems were performed for 10 ns each. The interactions and inhibitory mechanism of RLV to PFV IN were explored through overall dynamics behaviors, catalytic loop conformation distribution, and hydrogen bond network analysis. The results show that the coordinated interactions of RLV with IN and viral DNA slightly reduce the flexibility of catalytic loop region of IN, and remarkably restrict the mobility of the CA end of viral DNA, which may lead to the partial loss of the inhibitory activity of IN. Then, we docked a series of DKA inhibitors into PFV IN-DNA receptor and obtained the IN-DNA-inhibitor complexes. The docking results between PFV IN-DNA and DKA inhibitors agree well with the corresponding complex of HIV-1 IN, which proves the dependability of PFV IN-DNA used for the anti-AIDS drug screening. Our study may help to make clear some theoretical questions and to design anti-AIDS drug based on the structure of IN.
Asunto(s)
Fármacos Anti-VIH/química , ADN Viral/metabolismo , Inhibidores de Integrasa VIH/química , Integrasa de VIH/química , Cetoácidos/química , Spumavirus/enzimología , Secuencia de Aminoácidos , Fármacos Anti-VIH/metabolismo , Fármacos Anti-VIH/farmacología , Sitios de Unión , ADN Viral/química , Diseño de Fármacos , Integrasa de VIH/metabolismo , Inhibidores de Integrasa VIH/metabolismo , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Conformación Proteica , Spumavirus/efectos de los fármacos , Spumavirus/metabolismoRESUMEN
The signal recognition particle (SRP) and its receptors (SR) mediate the cotranslational targeting of the membrane and secretory proteins in all cells. In Escherichia coli, SRP is composed of the Ffh protein and the 4.5S SRP RNA. Ffh is a multidomain protein comprising a methionine-rich (M) domain, a helical N domain, and a Ras-like guanine triphosphatase (GTPase) (G) domain. The N and G domains are commonly referred to as one structural unit, the NG domain. In this article, the complex structure of SRP and SR is investigated with the Gaussian network model (GNM) and anisotropic network model (ANM). GNM provides the information of structure stability. It is found that the intermolecular interactions between SRP and SR can obviously decrease the fluctuation of NG domains. Nevertheless, the large structural rearrangement will take place during the cotranslational protein targeting cycle. Hence, the moving directions of fluctuation regions are further ascertained by using cross-correlation analysis and the ANM. The NG domain of Ffh undergoes a clockwise rotation around the GM linker and the M domain of Ffh shows an opposite direction to the NG domain. These functional movements will facilitate the SRP structure to transform into the free form and the sequence-bound form. These simple coarse-grained analyses can be used as a general and quick method for the mechanism studies of protein assembly and supramolecular systems.
Asunto(s)
Partícula de Reconocimiento de Señal/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Distribución Normal , Conformación Proteica , Partícula de Reconocimiento de Señal/metabolismoRESUMEN
Styrylquinoline derivatives are demonstrated to be HIV-1 integrase inhibitors. On the basis of our previous CoMFA analysis of a series of styrylquinoline derivatives, N-[(2-substituted-styryl)-5-chloro-8-hydroxyquinolin-7-yl]-benzenesulfonamide derivatives were designed and synthesized,and their possible HIV IN inhibitory activity was evaluated.
Asunto(s)
Inhibidores de Integrasa VIH/síntesis química , Inhibidores de Integrasa VIH/farmacología , Sulfonamidas/síntesis química , Sulfonamidas/farmacología , Evaluación Preclínica de Medicamentos , Inhibidores de Integrasa VIH/química , Espectroscopía de Resonancia Magnética , Espectrometría de Masa por Ionización de Electrospray , Sulfonamidas/químicaRESUMEN
AIM: To develop a novel high-throughput format assay to monitor the integrase (IN) strand transfer (ST) reaction in vitro and apply it to a reaction character study and the identification of antiviral drugs. METHODS: The donor DNA duplex, with a sequence identical to the U5 end of HIV-1 long terminal repeats, is labeled at its 5' end with biotin (BIO). The target DNA duplex is labeled at its 3' end with digoxin (DIG). IN mediates the integration of donor DNA into target DNA and results in a 5' BIO and 3' DIG-labeled duplex DNA product. Streptavidin-coated magnetic beads were used to capture the product, and the amount of DIG was measured as the ST reaction product. The assay was optimized in 96-well microplate format for high-throughput screening purpose. Moreover, the assay was applied in a ST reaction character study, and the efficiency of the assay in the identification of antiviral compounds was tested. RESULTS: The end-point values, measured as absorbance at 405 nm was approximately 1.5 for the IN-mediated ST reaction as compared with no more than 0.05 of background readings. The ST reaction character and the half maximal inhibitory concentration (IC50) values of 2 known IN inhibitors obtained in our assay were similar to previously reported results using other assays. The evaluation parameter Z' factor for this assay ranged from 0.6 to 0.9. CONCLUSION: The assay presented here has been proven to be rapid, sensitive, and specific for the detection of IN ST activity, the reaction character study, as well as for the identification of antiviral drugs targeting IN.
Asunto(s)
Bioensayo/métodos , Inhibidores de Integrasa VIH/farmacología , VIH-1/enzimología , Magnetismo , Microesferas , Biotina/metabolismo , ADN/genética , Digoxina/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/metabolismo , Humanos , Concentración 50 Inhibidora , Sensibilidad y Especificidad , Estreptavidina/metabolismo , Factores de Tiempo , Complejo Vitamínico B/metabolismoRESUMEN
Protein-protein docking is usually exploited with a two-step strategy, i.e., conformational sampling and decoy scoring. In this work, a new filter enhanced sampling scheme was proposed and added into the RosettaDock algorithm to improve the conformational sampling efficiency. The filter term is based on the statistical result that backbone hydrogen bonds in the native protein structures are wrapped by more than nine hydrophobic groups to shield them from attacks of water molecules (Fernandez and Scheraga, Proc Natl Acad Sci USA 2003;100:113-118). A combinatorial scoring function, ComScore, specially designed for the other-type protein-protein complexes was also adopted to select the near native docked modes. ComScore was composed of the atomic contact energy, van der Waals, and electrostatic interaction energies, and the weight of each item was fit through the multiple linear regression approach. To analyze our docking results, the filter enhanced sampling scheme was applied to targets T12, T20, and T21 after the CAPRI blind test, and improvements were obtained. The ligand least root mean square deviations (L_rmsds) were reduced and the hit numbers were increased. ComScore was used in the scoring test for CAPRI rounds 9-12 with good success in rounds 9 and 11.
Asunto(s)
Biología Computacional/métodos , Simulación por Computador , Mapeo de Interacción de Proteínas , Proteínas/química , Proteómica/métodos , Algoritmos , Interpretación Estadística de Datos , Bases de Datos de Proteínas , Genómica , Ligandos , Conformación Molecular , Unión Proteica , Conformación Proteica , Programas InformáticosRESUMEN
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 immunodeficiency virus (HIV)-1 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.