Development of nsP2 protease based cell free high throughput screening assay for evaluation of inhibitors against emerging Chikungunya virus

Abstract

Chikungunya virus has emerged as one of the most important global arboviral threats over the last decade. Inspite of large scale morbidity, with long lasting polyarthralgia, so far no licensed vaccine or antiviral is available. CHIKV nsP2 protease is crucial for processing of viral nonstructural polypeptide precursor to release enzymes required for viral replication, thus making it a promising drug target. In this study, high cell density cultivation (HCDC) of Escherichia coli in batch process was carried out to produce rCHIKV nsP2pro in a cost-effective manner. The purified nsP2pro and fluorogenic peptide substrate have been adapted for fluorescence resonance energy transfer (FRET) based high throughput screening (HTS) assay with Z’ value and CV of 0.67 ± 0.054 and <10% respectively. We used this cell free HTS system to screen panel of metal ions and its conjugate which revealed zinc acetate as a potential candidate, which was further found to inhibit CHIKV in Vero cells. Scale-up process has not been previously reported for any of the arboviral nonstructural enzymes. The successful scale-up method for viral protease together with a HTS assay could lead to the development of industrial level large-scale screening platform for identification of protease inhibitors against emerging and re-emerging viruses.

Hepatitis E Virus Cysteine Protease Has Papain Like Properties Validated by in silico Modeling and Cell-Free Inhibition Assays

“…Twelve inhibitors were used to study the inhibition and it was found that the enzyme was completely resistant to the inhibitors of serine proteases (PMSF, AEBSF, aprotinin), aspartic proteases (pepstatin) and metalloproteases (EDTA) while it was moderately inhibited by cysteine protease inhibitor leupeptin, chymostatin and E-64. HEV protease was strongly inhibited by E-64 (99%) and chymostatin (98%), which had been reported to inhibit papain like cysteine protease (Saha et al, 2018). To confirm these results, modeled HEV protease structure has been docked with above inhibitors which shows that cysteine protease inhibitors E64, Chymostatin, Leupeptin, and ALLN have good docking score and strong binding affinity whereas remaining inhibitors PMSF, Phosparamidon, AEBSF, Bestatin and Pepstatin showed no or weak interactions and EDTA and Antipain remain undocked.…”

Development of Processes for Recombinant L-Asparaginase II Production by Escherichia coli Bl21 (De3): From Shaker to Bioreactors

“…To determine the best concentration of each inductor, three concentrations were chosen based on previous studies. For IPTG, concentration often ranges from 0.10 mmol L −1 to 1.00 mmol L −1 [ 21 , 22 , 23 , 24 ] while lactose concentration varies from 10 g L −1 to 18 g L −1 [ 25 , 26 , 27 ]. Considering the reported concentrations, IPTG was evaluated at 0.10 mmol L −1 , 0.45 mmol L −1 and 1.00 mmol L −1 and lactose was evaluated at 10 g L −1 , 14 g L −1 and 18 g L −1 .…”

Advances in structure-assisted antiviral discovery for animal viral diseases

“…Various inhibitors such as sinefungin, aurintricarboxylic acid, and ribavirin were assessed and their inhibitory effect against nsP1 was reported. In addition, nsP2 protease-based cell-free high-throughput screening assay for evaluation of inhibitors against emerging CHIKV has been developed (Saha et al, 2018). These successful methods for identifying antiprotease molecules together with a highthroughput screening assay can lead to the development of industrial level largescale screening platform for identification of protease inhibitors against emerging and reemerging viruses.…”