Yellow fever virus NS3 protease: peptide-inhibition studies.
ABSTRACT A recombinant form of yellow fever virus (YFV) NS3 protease, linked via a nonapeptide to the minimal NS2B co-factor sequence (CF40-gly-NS3pro190), was expressed in Escherichia coli and shown to be catalytically active. It efficiently cleaved the fluorogenic tetrapeptide substrate Bz-norleucine-lysine-arginine-arginine-AMC, which was previously optimized for dengue virus NS2B/3 protease. A series of small peptidic inhibitors based on this substrate sequence readily inhibited its enzymic activity. To understand the structure-activity relationship of the inhibitors, they were docked into a homology model of the YFV NS2B/NS3 protease structure. The results revealed that the P1 and P2 positions are most important for inhibitor binding, whilst the P3 and P4 positions have much less effect. These findings indicate that the characteristics of YFV protease are very similar to those reported for dengue and West Nile virus proteases, and suggest that pan-flavivirus NS3 protease drugs may be developed for flaviviral diseases.
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ABSTRACT: Flaviviral NS3 serine proteases require the NS2B cofactor region (cNS2B) to be active. Recent crystal structures of West Nile virus (WNV) protease in complex with inhibitors revealed that cNS2B participates in formation of the protease active site. No crystal structures of ternary complexes are presently available for Dengue virus (DENV) to validate the role of cNS2B in active site formation. In this study a GST fusion protein of DENV-2 cNS2B49-95 was used as bait to pull-down DENV-2 protease domain (NS3pro). The affinity of NS3pro for cNS2B was strong (equilibrium binding constant <200 nM) and the heterodimeric complex displayed similar catalytic efficiency as single chain DENV-2 cNS2B/NS3pro. Various truncations and mutations in the cNS2B sequence showed that conformational integrity of the entire 47 amino acids is critical for protease activity. Furthermore, DENV-2 NS3 protease can be pulled‑down and trans-activated by cNS2B cofactors from DENV-1, -3, -4, and WNV, suggesting that mechanisms for activation are conserved across the flavivirus genus. To validate NS2B as a potential target in allosteric inhibitor development, a cNS2B specific human monoclonal antibody (3F10) was utilized. 3F10 disrupted the interaction between cNS2B and NS3 in vitro and reduced DENV viral replication in HEK293 cells. This provides proof-of-concept for developing assays to find inhibitors that block the interaction between NS2B and NS3 during viral translation.Bioscience Reports 02/2011; · 1.88 Impact Factor
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ABSTRACT: Conventional laparoscopic Roux-en-Y gastric bypass (LRYGB) has been the reference standard for bariatric surgery but requires 5-7 trocar incisions. We have developed a new procedure-single-incision transumbilical LRYGB (SITU-LRYGB)-that results in minimal scarring and is more cosmetically acceptable. To compare the surgical results and patient satisfaction between 5-port LRYGB and the novel SITU-LRYGB at a university hospital. We performed 5-port or SITU-LRYGB on 140 morbidly obese patients; the patients chose the operation method. We used a novel liver traction method and omega-umbilicoplasty specifically designed for SITU-LRYGB. Before surgery, the patients in the 5-port surgery group were more obese than those in the SITU group (120.8 kg versus 108.9 kg, P = .013). The rate of hypertension was also greater in the former group. The operative time was longer for SITU-LRYGB (101.1 versus 81.1 min, P = .001) without increased intraoperative complications. The total morphine dose for the SITU group seemed to be greater but the difference was not statistically significant. No difference in complications was observed. Postoperatively, the percentage of excess body weight lost the SITU and 5-port surgery groups was 21.2% and 20.9%, 40.4% and 39.4%, 55.0% and 55.2%, 64.8% and 75.2%, and 75.4% and 78.2% at 1, 3, 6, 9, and 12 months, respectively. The SITU-LRYGB patients reported greater satisfaction related to scarring than those who had undergone 5-port surgery (score 4.57 versus 3.96, respectively, P = .005). No patient died. Compared with conventional LRYGB, SITU-LRYGB resulted in acceptable complications, the same recovery, comparative weight loss, and better patient satisfaction related to scarring.Surgery for Obesity and Related Diseases 12/2010; 8(2):201-7. · 4.12 Impact Factor
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ABSTRACT: 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. 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, K(m) and k(cat), for fluorogenic protease model substrates, Boc-GRR-amc, Boc-LRR-amc, Ac-nKRR-amc, Bz-nKRR-amc, Pyr-RTKR-amc and Abz-(R)(4)SAG-nY-amide, were obtained using inner filter effect correction. The highest catalytic efficiency k(cat)/K(m) was found for Pyr-RTKR-amc (k(cat)/K(m): 1962.96 ± 85.0 M(-1) s(-1)) and the lowest for Boc-LRR-amc (k(cat)/K(m): 3.74±0.3 M(-1) s(-1)). JEV NS3pro is inhibited by aprotinin but to a lesser extent than DEN and WNV NS3pro. 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.PLoS ONE 01/2012; 7(5):e36872. · 3.73 Impact Factor