STUDIES ON ALPHAVIRUS CYSTEINE PROTEASE FOR ANTIVIRAL DRUG DISCOVERY

Abstract

Alphavirus are enveloped, plus-stranded RNA virus which belongs to the Togaviridae family and are transmitted by mosquitoes to the humans and animal. They possesses an emerging public health threat to the global community due to their capacity of inducing larger outbreaks of infectious disease which is characterized by a variety of symptoms and disorders in infected individuals. These viruses causes severe illness in humans ranging from fever, rash and incapacitating arthritis, as witnessed in the case of chikungunya virus (CHIKV) outbreaks, to potentially lethal encephalitis and associated neurological complication as observed in the periodic outbreaks of Venezuelan Equine Encephalitis Virus (VEEV) and Western Equine Encephalitis Virus (WEEV). In particular, the resurgence of CHIKV in many parts of the world including Indian subcontinent, South-East Asian region, Caribbean islands and African provinces in the recent years has posed a momentous threat to the world population. However, despite the severity of alphaviral disease and the widespread geographical presence, no approved treatment therapy or antiviral drug is available in the market to effectively treat alphavirus infection. Therefore, it has become imperative to discover antiviral drugs or inhibitors against CHIKV and other alphavirus. CHIKV genome is a single-stranded RNA of positive polarity, which is directly translated into the nonstructural polyprotein precursor P1234. This polyprotein precursor is efficiently processed by the virus specific nsP2 cysteine protease into the individual nonstructural proteins. These nonstructural proteins in turn, form replication complexes for the synthesis of viral genomic and subgenomic RNA. The nsP2 protein is also crucial for the inactivation of host cell immune response by shutting off the host cellular translational machinery. Thus, nsP2 protease by the virtue of its indispensable role in virus survival constitutes a promising target for anti-CHIKV drug discovery. Therefore, we have targeted this protein for developing structure-based inhibitors which could potentially hinder the CHIKV infection. This thesis encompasses the structural, biophysical & biochemical characterization of CHIKV nsP2 protease (CHIKV nsP2pro) and the structure based identification of a series of peptidomimetic compounds against nsP2 protease and in vitro validation of nsP2pro inhibition by the identified peptidomimetic protease inhibitors. The recombinant CHIKV nsP2pro cloned in pET28c vector was purified by Ni+2 affinity and size-exclusion chromatography. Purified protein was incubated with small substrate peptides and inhibitors and used for crystallization with an aim to crystallize enzyme-substrate/inhibitor II complexes for determining the crystal structures of nsP2pro complexes. However, obtained crystals for nsP2pro complexes did not diffract or diffracted to very low resolution. With an aim to identify nsP2pro inhibitors, 4ZTB pdb co-ordinates of the available crystal structure of CHIKV nsP2pro in pdb database were used for detailed molecular and structural investigations. Comparative structural analysis of nsP2pro (4ZTB) with the previously reported crystal structure of other alphavirus nsP2pro was performed to identify substrate binding and active site residues. Further, the functional significance of residue Asn547 which is proposed to be important for substrate binding was confirmed by mutagenic studies. The binding efficiency of CHIKV nsP2pro with substrate peptides corresponding to the three cleavage sites of nonstructural polyprotein P1234 was determined with purified nsP2pro by doing thermodynamics experiments using Isothermal titration calorimetery (ITC). This biophysical investigation of enzyme binding suggests that the enzyme binds with the substrate peptide corresponding to the 3/4 cleavage site with more efficiency than the substrate peptide 1/2. Interestingly, the binding of purified nsP2pro with the substrate peptide corresponding to 2/3 was not discerned using ITC. Additionally, a robust and sensitive flouroscence resonance energy transfer (FRET) based nsP2pro protease activity assay was developed, which corroborated the result of binding and catalytic efficacy of the enzyme obtained through the biophysical studies. In the wake of the unavailability of crystal structure of CHIKV nsP2pro in complex with substrate/inhibitor, molecular docking of substrate peptide 3/4 in the active site of enzyme was carried out. The docking study revealed molecular insights into the preferable binding mode of the substrate peptide in the active site and molecular interactions crucial for the enzyme-substrate binding. CHIKV nsP2pro active site accommodates a five residue (P4-P1ʹ) long peptide in extended conformation with a Glide score of −6.04 and Emodel score of −71.16. These details were further harnessed for designing and identification of peptidomimetic compounds by employing the computer-aided structure based inhibitor screening. The inhibitor compounds were identified by taking two approaches in consideration; conformer-based approach through which the small molecules that mimic the conformation of junction residues of peptide 3/4 and the second approach in which peptidomimetics compounds were identified by screening the online databases on the basis of pharmacophoric features derived from nsP2pro-peptide 3/4 interactions. The binding and conformational stability of identified peptidomimetic compounds was furthermore validated by molecular docking and molecular dynamics simulation. Subsequent comparative analysis of enzyme-substrate and enzyme-peptidomimetic docking complexes has revealed that two molecules Pep-I and Pep-II docks into the enzyme active site III in a similar manner as the 3/4 substrate peptide. Both the compounds have shown comparable docking score and glide Emodel score to the enzyme-peptide complex. The glide score and glide Emodel score for Pep-I-enzyme complex was observed to be −6.47 and −74.03 respectively and for Pep-II-enzyme complex −6.21 and −65.93 respectively. Furthermore, these two compounds were selected to investigate their in vitro inhibitory potential against CHIKV nsP2pro using the developed FRET based protease assay. Interestingly, both the peptidomimetic compounds demonstrated the inhibition of CHIKV nsP2pro activity within micro molar concentrations. The IC50 values of CHIKV nsP2pro activity inhibition by Pep-I and Pep-II were calculated to be 34 μM and 42 μM, respectively. The inhibition kinetic analysis revealed the inhibition constant (Ki) of both the inhibitors which was calculated to be 33.34 μM for Pep-I and 45.89 μM for Pep-II. Our study has concluded that both the compounds are potent inhibitors, which span the P4-P1ʹ binding interaction of substrate peptide 3/4 in the active site of CHIKV nsP2pro. This study strongly suggests that both the tested inhibitors have potential for the development of antiviral drugs or treatment therapy against chikungunya pathogenesis in humans.