An optimised assay for quantitative, high-throughput analysis of polysialyltransferase activity

YesThe polysialyltransferases are biologically important glycosyltransferase enzymes responsible for the biosynthesis of polysialic acid, a carbohydrate polymer that plays a critical role in the progression of several diseases, notably cancer. Having improved the chemical synthesis and purification of the fluorescently-labelled DMB-DP3 acceptor, we report optimisation and validation of a highly sensitive cell-free high-throughput HPLC-based assay for assessment of human polysialyltransferase activity

Enzymatic Analysis of Recombinant Japanese Encephalitis Virus NS2B(H)-NS3pro Protease with Fluorogenic Model Peptide Substrates

Background Japanese encephalitis virus (JEV), a member of the Flaviviridae family, causes around 68,000 encephalitis cases annually, of which 20–30% are fatal, while 30–50% of the recovered cases develop severe neurological sequelae. Specific antivirals for JEV would be of great importance, particularly in those cases where the infection has become persistent. Being indispensable for flaviviral replication, the NS2B-NS3 protease is a promising target for design of anti-flaviviral inhibitors. Contrary to related flaviviral proteases, the JEV NS2B-NS3 protease is structurally and mechanistically much less characterized. Here we aimed at establishing a straightforward procedure for cloning, expression, purification and biochemical characterization of JEV NS2B(H)-NS3pro protease. Methodology/Principal Findings The full-length sequence of JEV NS2B-NS3 genotype III strain JaOArS 982 was obtained as a synthetic gene. The sequence of NS2B(H)-NS3pro was generated by splicing by overlap extension PCR (SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro, expressed in E. coli as soluble protein, was purified to >95% purity by a single-step immobilized metal affinity chromatography. SDS-PAGE and immunoblotting of the purified enzyme demonstrated NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 36, 21, and 10 kDa bands, respectively. Kinetic parameters, Km and kcat, for fluorogenic protease model substrates, Boc-GRR-amc, Boc-LRR-amc, Ac-nKRR-amc, Bz-nKRR-amc, Pyr-RTKR-amc and Abz-(R)4SAG-nY-amide, were obtained using inner filter effect correction. The highest catalytic efficiency kcat/Km was found for Pyr-RTKR-amc (kcat/Km: 1962.96±85.0 M−1 s−1) and the lowest for Boc-LRR-amc (kcat/Km: 3.74±0.3 M−1 s−1). JEV NS3pro is inhibited by aprotinin but to a lesser extent than DEN and WNV NS3pro. Conclusions/Significance A simplified procedure for the cloning, overexpression and purification of the NS2B(H)-NS3pro was established which is generally applicable to other flaviviral proteases. Kinetic parameters obtained for a number of model substrates and inhibitors, are useful for the characterization of substrate specificity and eventually for the design of high-throughput assays aimed at antiviral inhibitor discovery

A new antiviral scaffold for human norovirus identified with computer-aided approaches on the viral polymerase

Human norovirus is the leading cause of acute gastroenteritis worldwide, affecting every year 685 million people. In about one third of cases, this virus affects children under five years of age, causing each year up to 200,000 child deaths, mainly in the developing countries. Norovirus outbreaks are associated with very significant economic losses, with an estimated societal cost of 60 billion dollars per year. Despite the marked socio-economic consequences associated, no therapeutic options or vaccines are currently available to treat or prevent this infection. One promising target to identify new antiviral agents for norovirus is the viral polymerase, which has a pivotal role for the viral replication and lacks closely homologous structures in the host. Starting from the scaffold of a novel class of norovirus polymerase inhibitors recently discovered in our research group with a computer-aided method, different new chemical modifications were designed and carried out, with the aim to identify improved agents effective against norovirus replication in cell-based assays. While different new inhibitors of the viral polymerase were found, a further computer-aided ligand optimisation approach led to the identification of a new antiviral scaffold for norovirus, which inhibits human norovirus replication at low-micromolar concentrations.status: Published onlin

Biochemical and structural characterization of the putative dihydropteroate synthase ortholog Rv1207 of Mycobacterium tuberculosis.

Dihydropteroate synthase (DHPS) is involved in de novo biosynthesis of the essential cofactor folate by catalyzing the condensation of para-aminobenzoic acid (pABA) and 6-hydroxymethyl-7,8-dihydropterin-pyrophosphate (H2PtPP). Mycobacterium tuberculosis possesses a functional DHPS (MtDHPS, Rv3608c, folP1) and, based on sequence similarities, a putative ortholog (Rv1207, folP2). Here, we demonstrate that Rv1207 shows a low H2PtPP substrate affinity and lacks enzymatic DHPS activity. However, we found dapsone, a structural analog of pABA and clinically used DHPS inhibitor, to weakly bind both proteins. To gain insights into the lack of DHPS activity of Rv1207, its three-dimensional structure was determined at 2.64 A. The overall fold of both, MtDHPS (1EYE) and Rv1207, is highly conserved and conforms to a classical triosephosphate isomerase barrel arrangement. The predicted H2PtPP-binding pocket of Rv1207 is occupied by a histidine side chain, relative to a leucine residue in MtDHPS, consistent with the low affinity for this substrate and the lack of DHPS activity. We conclude that folP2 does not encode a DHPS and therefore cannot act as bypass for folP1. The metabolic function of Rv1207 remains to be defined

Identification of Residues in the Dengue Virus Type 2 NS2B Cofactor That Are Critical for NS3 Protease Activation

Proteolytic processing of the dengue virus polyprotein is mediated by host cell proteases and the virus-encoded NS2B-NS3 two-component protease. The NS3 protease represents an attractive target for the development of antiviral inhibitors. The three-dimensional structure of the NS3 protease domain has been determined, but the structural determinants necessary for activation of the enzyme by the NS2B cofactor have been characterized only to a limited extent. To test a possible functional role of the recently proposed Φx(3)Φ motif in NS3 protease activation, we targeted six residues within the NS2B cofactor by site-specific mutagenesis. Residues Trp62, Ser71, Leu75, Ile77, Thr78, and Ile79 in NS2B were replaced with alanine, and in addition, an L75A/I79A double mutant was generated. The effects of these mutations on the activity of the NS2B(H)-NS3pro protease were analyzed in vitro by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of autoproteolytic cleavage at the NS2B/NS3 site and by assay of the enzyme with the fluorogenic peptide substrate GRR-AMC. Compared to the wild type, the L75A, I77A, and I79A mutants demonstrated inefficient autoproteolysis, whereas in the W62A and the L75A/I79A mutants self-cleavage appeared to be almost completely abolished. With exception of the S71A mutant, which had a k(cat)/K(m) value for the GRR-AMC peptide similar to that of the wild type, all other mutants exhibited drastically reduced k(cat) values. These results indicate a pivotal function of conserved residues Trp62, Leu75, and Ile79 in the NS2B cofactor in the structural activation of the dengue virus NS3 serine protease

Probing the substrate specificity of the dengue virus type 2 NS3 serine protease by using internally quenched fluorescent peptides

The NS3 (dengue virus non-structural protein 3) serine protease of dengue virus is an essential component for virus maturation, thus representing an attractive target for the development of antiviral drugs directed at the inhibition of polyprotein processing. In the present study, we have investigated determinants of substrate specificity of the dengue virus NS3 protease by using internally quenched fluorogenic peptides containing Abz (o-aminobenzoic acid; synonymous to anthranilic acid) and 3-nitrotyrosine (nY) representing both native and chimaeric polyprotein cleavage site sequences. By using this combinatorial approach, we were able to describe the substrate preferences and determinants of specificity for the dengue virus NS2B(H)–NS3pro protease. Kinetic parameters (k(cat)/K(m)) for the hydrolysis of peptide substrates with systematic truncations at the prime and non-prime side revealed a length preference for peptides spanning the P4–P3′ residues, and the peptide Abz-RRRRSAGnY-amide based on the dengue virus capsid protein processing site was discovered as a novel and efficient substrate of the NS3 protease (k(cat)/K(m)=11087 M(−1)·s(−1)). Thus, while having confirmed the exclusive preference of the NS3 protease for basic residues at the P1 and P2 positions, we have also shown that the presence of basic amino acids at the P3 and P4 positions is a major specificity-determining feature of the dengue virus NS3 protease. Investigation of the substrate peptide Abz-KKQRAGVLnY-amide based on the NS2B/NS3 polyprotein cleavage site demonstrated an unexpected high degree of cleavage efficiency. Chimaeric peptides with combinations of prime and non-prime sequences spanning the P4–P4′ positions of all five native polyprotein cleavage sites revealed a preponderant effect of non-prime side residues on the K(m) values, whereas variations at the prime side sequences had higher impact on k(cat)