Synthesis and in vitro evaluation of West Nile virus protease inhibitors based on the 1,3,4,5-tetrasubstituted 1H-pyrrol-2(5H)-one scaffold.

West Nile virus (WNV), a member of the Flaviviridae family, is a mosquito-borne pathogen that causes a large number of human infections each year. There are currently no vaccines or antiviral therapies available for human use against WNV. Therefore, efforts to develop new chemotherapeutics against this virus are highly desired. In this study, a WNV NS2B-NS3 protease inhibitor with a 1,3,4,5-tetrasubstituted 1H-pyrrol-2(5H)-one scaffold was identified by screening a small library of nonpeptidic compounds. Optimization of this initial hit by the synthesis and screening of a focused library of compounds with this scaffold led to the identification of a novel uncompetitive inhibitor ((-)-1a16, IC50 =2.2±0.7 µM) of the WNV NS2B-NS3 protease. Molecular docking of the chiral compound onto the WNV protease indicates that the R enantiomer of 1a16 interferes with the productive interactions between the NS2B cofactor and the NS3 protease domain and is thus the preferred isomer for inhibition of the WNV NS2B-NS3 protease.

Discovery, synthesis, and in vitro evaluation of West Nile virus protease inhibitors based on the 9,10-dihydro-3H,4aH-1,3,9,10a-tetraazaphenanthren-4-one scaffold.

West Nile virus (WNV), a member of the Flaviviridae family, is a mosquito-borne pathogen that causes a great number of human infections each year. Neither vaccines nor antiviral therapies are currently available for human use. In this study, a WNV NS2B-NS3 protease inhibitor with a 9,10-dihydro-3H,4aH-1,3,9,10a-tetraazaphenanthren-4-one scaffold was identified by screening a small library of non-peptidic compounds. This initial hit was optimized by solution-phase synthesis and screening of a focused library of compounds bearing this scaffold. This led to the identification of a novel, uncompetitive inhibitor (1a40, IC(50) = 5.41±0.45 µM) of WNV NS2B-NS3 protease. Molecular docking of this chiral compound onto the WNV protease indicates that the S enantiomer of 1a40 appears to interfere with the productive interactions between the NS2B cofactor and the NS3 protease domain; (S)-1a40 is a preferred isomer for inhibition of WNV NS3 protease.

[Long-term effectiveness of tendon allograft for repairing tendon defect].

OBJECTIVE: To observe the long-term effectiveness of tendon allograft to repair tendon defect. METHODS: Between October 1996 and September 1999, 24 patients with tendon defect were treated with tendon allograft which was cultured with deoxyguanosine and preserved at low-temperature or ultra-deep-low-temperature. There were 19 males and 5 females, aged from 12 to 46 years with an average of 25.9 years. These patients included 7 cases of total extensor tendon defect of 2nd-5th fingers, 7 cases of index finger extensor tendon defect, 3 cases of deep flexor tendon defect of 2nd-5th fingers, 1 case of ring finger deep flexor tendon defect, 3 cases of long extensor tendon defect of 2nd-5th toes, 2 cases of long extensor hallucis tendon defect, and 1 case of shoulder adduction missing. The sizes of tendon defect ranged from 5 to 15 cm. The mean time from injury to operation was 1.3 months (range, 2 hours to 3 months). RESULTS: Incisions healed by first intention. No deep infection, infectious diseases, and obvious immune rejection occurred. All patients were followed up from 10 to 12 years with an average of 10.8 years. When compared with contralateral sides, at 10 years of follow-up, 1 patient lost 6-100 flexion function; after 10.6 years, flexion tendon releasing was performed; allografted tendon had normal color and elasticity with decreased diameter and with mild and moderate adherence; and after releasing, function was improved. According to Hand Surgery Association assessment standard, the results were excellent in 12 cases, good in 6, and poor in 6; the excellent and good rate was 75%. CONCLUSION: Tendon allograft which is cultured with deoxyguanosine and preserved at low-temperature or ultra-deep-low-temperature is safe to use in clinical, which has good long-term effectiveness in treating tendon defect.

Synthesis and disulfide bond connectivity-activity studies of a kalata B1-inspired cyclopeptide against dengue NS2B-NS3 protease.

Kalata B1 is a plant protein with remarkable thermal, chemical and enzymatic stability. Its potential applications could be centered on the possibility of using its cyclic structure and cystine knot motif as a scaffold for the design of stable pharmaceuticals. To discover potent dengue NS2B-NS3 protease inhibitors, we have prepared various kalata B1 analogues by varying the amino acid sequence. Mass spectrometric and biochemical investigations of these analogues revealed a cyclopeptide whose two fully oxidized forms are substrate-competitive inhibitors of the dengue viral NS2B-NS3 protease. Both oxidized forms showed potent inhibition with K(i) of 1.39+/-0.35 and 3.03+/-0.75 microM, respectively.

Hydrogen peroxide enhances enterokinase-catalysed proteolytic cleavage of fusion protein.

The effects of hydrogen peroxide on enterokinase catalysis were studied using several fusion proteins recombinantly produced from E. coli. It was demonstrated that hydrogen peroxide enhanced the rate of enterokinase cleavage reaction, leading to a faster release of the target peptide as discussed in patent WO07149053. Among the conditions tested, we observed that hydrogen peroxide could exert its effect on the cleavage of fusion proteins over a wide range of pH and temperature. This finding might provide a simple solution for the accelerated enterokinase cleavage of thermolabile fusion proteins at low temperature.

Efficient preparation of an acyclic permutant of kalata B1 from a recombinant fusion protein with thioredoxin.

A new approach to prepare an acyclic permutant of kalata B1, a cysteine-rich plant cyclopeptide with uterotonic activity, is described. The synthetic codon-optimized cDNA sequence encoding this 29-residue peptide was cloned and fused in-frame to the His(6)-tagged thioredoxin gene in the bacterial expression vector pET-32a. The fusion protein was overexpressed in the bacterial host, Escherichia coli strain BL21 (DE3), and isolated by affinity chromatography on a metal-chelating Sepharose column. An enterokinase recognition sequence incorporated immediately upstream of the target peptide allowed the 29-residue peptide to be released without any unwanted residues upon treatment with enterokinase. This peptide was subsequently separated from the larger thioredoxin moiety by ultracentrifugation through a semipermeable membrane. Further purification was achieved using reversed-phase HPLC. Hydrogen peroxide was found to enhance the rate of enterokinase cleavage in a concentration-dependent manner. Thermal stability studies demonstrated that the recombinant acyclic kalata B1 (ac kalata) was exceptionally stable against thermal denaturation. Mass spectrometric analysis revealed that the recombinant ac kalata was obtained in a fully oxidized form, indicating a high reducing potential and a strong tendency of the 29-residue peptide to form a tightly folded structure.

Mapping the functional domain of the prion protein.

Prion diseases such as Creutzfeldt-Jakob disease are possibly caused by the conversion of a normal cellular glycoprotein, the prion protein (PrPc) into an abnormal isoform (PrPSc). The process that causes this conversion is unknown, but to understand it requires a detailed insight into the normal activity of PrPc. It has become accepted from results of numerous studies that PrPc is a Cu-binding protein and that its normal function requires Cu. Further work has suggested that PrPc is an antioxidant with an activity like that of a superoxide dismutase. We have shown in this investigation that this activity is optimal for the whole protein and that deletion of parts of the protein reduce or abolish this activity. The protein therefore contains an active domain requiring certain regions such as the Cu-binding octameric repeat region and the hydrophobic core. These regions show high evolutionary conservation fitting with the idea that they are important to the active domain of the protein.

Analysis of doppel protein toxicity.

The recently described doppel protein (Dpl) is a homologue of the prion protein (PrP(c)). This protein, expressed in the brains of mice that lack the expression of PrP(c), causes neuronal death as the mice age. Previous studies have suggested this neuronal damage is caused by oxidative assault and changes in the activity of NOS proteins. We investigated the toxicity of Dpl in cell culture models and showed that Dpl was toxic to neurons. This toxicity was inhibited by the expression of PrP(c) and possibly involved direct interaction between the two proteins. The mechanism of toxicity involved stimulation of nitric oxide production via activation of the nitric oxide synthases, nNOS and iNOS. This mechanism of toxicity is quite different from that of PrP(Sc) and does not require the protein to change conformation. These results provide the first evidence for the mechanism of Dpl toxicity.