WO2019092510A1

WO2019092510A1 - A highly sensitive nsp1 enzyme assay and kit for determining inhibitors of alphavirus/chikungunya virus

Info

Publication number: WO2019092510A1
Application number: PCT/IB2018/051760
Authority: WO
Inventors: Shailly TOMAR; Pravindra KUMAR; Ramanjit KAUR
Original assignee: Indian Institute Of Technology, Roorkee
Priority date: 2017-11-08
Filing date: 2018-03-16
Publication date: 2019-05-16

Abstract

The present invention is in the field of determining inhibitory compounds against the capping enzyme, nsP1 of alphaviruses/Chikungunya/alphavirus-like superfamily. The invention also provides kit for identifying inhibitors of capping enzyme as drugs for the treatment or prevention of infections caused by Chikungunya/alphavirus.

Description

Ά HIGHLY SENSITIVE NSP1 ENZYME ASSAY AND KIT FOR DETERMINING INHIBITORS OF ALPHA VIRUS/CHIKUNGUNYA VIRUS'

Field of the Invention

The present invention is in the field of determining inhibitory compounds against the capping enzyme, nsPl of alpha viruses/Chikungunya/alphavirus-like superfamily. The invention also provides kit for identifying inhibitors of capping enzyme as drugs for the treatment or prevention of infections caused by Chikungunya/alphavirus.

Background of the Invention

The genus alphavirus of the Togaviridae family comprises 31 species of positive-strand RNA viruses approximately 12 kb in length. The members of alphavirus genus include Chikungunya virus (CHIKV), Aura virus (AV), Semliki Forest virus (SFV), Western, Eastern and Venezuelan equine encephalitis virus (WEEV, EEEV and VEEV), Sindbis virus (SINV), O'nyong'nyong virus (ONNV) and Mayaro virus (MAYV). The reemergence of alphaviruses represents the potential threat to human health. From last 10 years, Chikungunya virus (CHIKV) causes the worldwide epidemics mainly affecting the southwest Indian Ocean islands, the Indian-sub continent, Latin America and the southeast coast of Africa (Charrel et al., 2014; Chevillon et al., 2008). In 1952, CHIKV outbreak was seen in the Tanganyika and Makonde Plateau region (Lumsden, 1955; Robinson, 1955).

In 2005, a major outbreak was seen in Reunion Island, Comoro, Madagascar, India and Southeast Asia (Muniaraj, 2014; Roth et al., 2014; Sergon et al., 2007). The infection symptoms of CHIKV are very similar to dengue fever which includes sudden high fever, severe muscle and joint pain, nausea, vomiting, and rashes. Antiviral drugs or vaccines against alphavirus infection are still not available which makes it very difficult to understand the biology of these viruses during infection of the mammalian host. Antiviral compounds in development against alphaviruses including Chikungunya are still in initial stages of evaluation (Chung et al., 2013; Gould et al., 2010). The capping mechanism of alphavirus has been studied in detail using radioactive isotopes and assays for SFV and SINV have been develped (Laakkonen et al., 1994; Mi and Stollar, 1991 ; Tomar et al., 2011). Recently the enzymatic activity of VEEV nsPl has also been demonstrated (Li et al., 2015). The standard nsPl MTase assays for screening inhibitors are radioactive assays that utilize ³H-SAM as the methyl donor or ³²P labelled GTP, which are hazardous to the environment.

US 20100152200 Al relates to chemical compounds, methods for their discovery, and their therapeutic use. In particular, the present invention provides compounds as inhibitors of alphaviruses.

US 20130267460 Al provides methods of inhibiting alphavirus replication and treating alpha virus infection.

EP 2370455 A2 provides compositions and methods for the prevention or treatment of one or more strains of Chikungunya virus, as well as other alphavirus-mediated diseases, defines few of the area/ studies in the domain of finding inhibitors/treating infection caused by alphavirus-mediated diseases.

Hence, there is a need of a highly sensitive and specific plate based assays for screening inhibitors against virus specific enzymes and kits which do not use radioisotopes.

The present invention is directed towards the development of a plate based immunoassay and a kit which determines the capping enzyme activity of CHIKV nsPl for screening MTase/GTase inhibitors.

Obiect(s) of the Invention

A primary object of the present invention is to overcome the drawbacks associated with the prior art.

Yet another object of the present invention is to provide a non-radioactive method for testing and/or screening of inhibitory compounds against the capping enzyme, nsPl of alphaviruses/Chikungunya/alphavirus-like superfamily.

Yet another object of the present invention is to provide a non-radioactive method for testing and/or screening of inhibitory compounds against the capping enzyme, nsPl of alphaviruses/Chikungunya/alphavirus-like superfamily, which reduces the environment and health risks.

Yet another object of the present invention is to provide an ELISA based nsPl assay for testing and screening inhibitors against Chikungunya/alphavirus methyltransferase (MTase)/guanylyltransferase (GTase) nsPl .

Yet another object of the present invention is to provide an ELISA based assay for testing and screening antiviral drugs/molecules specific for alphaviruses/Chikungunya/alphavirus- like superfamily by targeting the capping enzyme having MTase and GTase activity. Yet another object of the present invention is to provide a plate based immunoassay which determines the enzyme activity of CHIKV nsPl for screening MTase inhibitors.

Yet another object of the present invention is to provide a plate based immunoassay which determines the enzyme activity of CHIKV nsPl for screening GTase inhibitors.

Yet another object of the present invention is to provide a kit based on the non-radioactive method for testing and/or screening of inhibitory compounds against the capping enzyme, nsPl of alphaviruses/Chikungunya/alphavirus-like superfamily.

Summary of the Invention:

In an aspect, the invention provides a plate based non-radioactive method for screening a test compound as the nsPl capping enzyme inhibitors of alphavirus/Chikungunya/alphavirus-like superfamily, comprising the steps of: a. Performing ELISA based methyltransferase and guanylyltransferase assay in the presence of said test compound and recombinant Chikungunya virus nsPl protein by the steps of preparing the reaction mixture followed by starting the reaction with the addition of nsPl protein and incubating at temperature of approximately 25°C for different time intervals where said reaction mixture comprises assay buffer, Guanosine-5 '-triphosphate and S-adenosyl-L-methionine and said different time interval comprises 0, 5, 10, 15, 20, 25, 30, 45 minutes;

b. Treating the reaction mixture resulting from step (a) with stop reaction buffer followed by subjecting said mixture to ELISA plate followed by incubating at temperature of approximately 25 °C for 60 minutes;

c. Treating with a blocking buffer at approximately 25 °C for 90 minutes followed by treating with a primary antibody at approximately 25 °C for 1 hour;

d. Treating with a secondary antibody at approximately 25 °C for 1 hour followed by washing with TBST buffer;

e. Adding a chromogenic substrate followed by addition of stop solution upon sufficient color development and identifying absorbance at 450 nm.

wherein a decreased MTase activity of the nsPl protein or decreased absorbance level in the presence of said test compound indicates that the test compound is an inhibitor of alphavirus/Chikungunya/alphavirus-like superfamily and/or suitable for treatment or prevention of infections caused by alphavirus/Chikungunya/alphavirus-like superfamily.

In another aspect, the invention provides a kit for non-radioactive enzyme linked immunosorbent assay for screening nsPl methyl transferase inhibitors, comprising components: a. Purified recombinant Chikungunya nsPl present in the concentration of 5 mg/ml

b. S-adenosyl-L-methionine present as 10 mM solution in water c. Guanosine-5 '-triphosphate present as 5 mM solution in water d. Assay buffer comprising 50 mM Tris-HCl (pH 7.3), 10 mM NaCl, 5 mM DTT, 2 mM MgCl₂

e. Stop reaction buffer comprising 100 mM EDTA

f. TBST buffer comprising 20 mM Tris (pH 7.5), 150 mM NaCl, 0.1 % Tween-20

g. Blocking solution comprising 3 % skim milk in TBST buffer h. Primary antibody comprising Mouse anti-m⁷G cap primary monoclonal antibody at 1 :5000 dilution in TBST buffer

i. Secondary antibody comprising Horseradish peroxidase (HRP)-conjugated anti- mouse IgGl secondary antibody at 1 :10,000 dilution in TBST buffer j. Chromogenic substrate comprising 20X Tetramethylbenzidine k. Stop solution comprising 1 N H₂SO₄

1. Positive control comprising 1 mM aurintricarboxylic acid solution in water

m. 96-well solid plate with a cover sheet. Brief Description of Figures:

To clarify advantages and features of the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which is illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. The invention will be described and explained with additional specificity and detail with the accompanying drawings in which: Fig. 1 illustrates the enzymatic reaction steps catalyzed by alpha virus nsPl , of the general art.

Fig. 2 illustrates expression and purification of CHIKV nsPl (residues 1-509) from E. coli. SDS-PAGE gel stained with Coomassie brilliant blue (a) Lanes: 1, Uninduced supernatant containing soluble fraction of protein; 2, Uninduced pellet containing insoluble fraction of protein; 3, Induced pellet at 18 °C; 4, Induced supernatant at 18 °C (maximum expression); 5, Induced pellet at 25 °C; 6, Induced supernatant at 25 °C; 7 Induced pellet at 37 °C; 8, Induced supernatant at 37 °C; 9, Molecular weight markers (kDa); (b) Lanes: 1 , Molecular weight markers (kDa); 2, Purified CHIKV nsPl (residues 1-509) ~ 56 kDa. Fig. 3 illustrates nsPl assay (a) Indirect ELISA based determination of MTase activity assay using different concentrations of SAM at different time points. The activity assay was performed by adding 10 to 200 μΜ of SAM in each 50 μΐ reaction mixture of 50 mM Tris- HCl pH 7.3, 10 mM NaCl, 5 mM DTT, 2 mM MgCl₂ and 100 μΜ GTP. The nsPl protein was added and incubated the reaction mixture at 25 °C for different intervals of time and ELISA was performed using anti-m⁷G cap monoclonal antibody and subsequently HRP- conjugated anti-mouse IgGl secondary antibody. The TMB substrate was added and absorbance was measured at 450 nm. An increase in absorbance was observed with the increasing concentration of SAM. The different colored lines represent different concentrations of the substrate (SAM); Blue = 10 μΜ, Red = 25 μΜ, Green =50 μΜ, Purple = 100 μΜ, Light blue = 150 μΜ, Orange= 200 μΜ. Absorbance values represent an average of two readings plotted against time; (b) Indirect ELISA based determination of MTase activity assay using different concentrations of GTP at different time points. Indirect ELISA based activity assay was performed by adding 5 to 150 μΜ of GTP in each 50 μΐ reaction mixture of 50 mM Tris-HCl pH 7.3, 10 mM NaCl, 5 mM DTT, 2 mM MgCl₂ and 200 μΜ SAM. The nsPl protein was added and incubated the reaction mixture at 25 °C for different intervals of time and ELISA was performed using anti-m⁷G cap monoclonal antibody and subsequently HRP-conjugated anti-mouse IgGl secondary antibody. The ELISA substrate was added and absorbance at 450 nm was measured. An increase in absorbance with the increasing concentration of GTP was observed. The different colored lines represent different concentrations of GTP; where Blue = 5 μΜ, Red = 10 μΜ, Green =25 μΜ, Purple = 50 μΜ, Light blue = 100 μΜ, Orange= 150 μΜ lines represent different concentrations of GTP. The absorbance values represent the average of two readings and plotted against time. Fig. 4 illustrates methyltransferase inhibition assay for CHIKV nsPl . CHIKV nsPl was incubated with reaction mixture containing SAM and GTP in the presence of increasing concentration of sinefungin, aurintricarboxylic acid (ATA) and ribavirin and subjected to ELISA based activity assay. The absorbance was measured at 450 nm. IC50 values of (a) sinefungin (b) aurintricarboxylic acid (ATA) (c) ribavirin are shown. The data represent an average of two readings plotted against logistic concentration.

Fig. 5 illustrates schematics of the ELISA assay of the present invention.

Fig. 6 illustrates nucleotide sequence of CHIKV nsPl. Fig. 7 illustrates methyltransferase activity where in case of reaction in which no inhibitor was added to the enzymatic reaction mixture was taken as 100 %. The percent inhibition of (a) sinefungin (b) ATA (c) ribavirin was calculated. The data represent an average of two readings plotted against inhibitor concentration. Detailed Description of the Invention

For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.

It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof. Throughout the patent specification, a convention employed is that in the appended drawings, like numerals denote like components.

The present invention provides an assay method and kit for determining inhibitory compounds against the capping enzyme, nsPl of alphaviruses/Chikungunya/alphavirus-like superfamily. The invention also provides assay method and kit for determining drug/s targeting nsPl enzyme activity for treatment or prevention of infections caused by Chikungunya/alphaviruses/viruses of the alphavirus-like superfamily. The assay is highly sensitive for the identification and/or validation of inhibitors of Chikungunya/alphavirus methyltransferase (MTase)/nsPl and/or drugs for treatment or prevention of infections caused by alphaviruses.

In an embodiment, the assay and kit facilitates screening of nsPl methyltransferase inhibitors.

In an embodiment, the assay and kit facilitates screening of nsPl guanylyltransferase (GTase) inhibitors.

In an embodiment, the assay and kit facilitates detection of nsPl reaction intermediate which is formed only after methyltransferase reaction of nsPl.

In an embodiment, the assay method for screening nsPl methyltransferase inhibitors comprises of the following steps:

(a) determining the MTase and GTase activity of the nsPl protein; and

(b) comparing the enzymatic activity determined in step (a) with the enzymatic activity of the nsPl protein in the absence of the candidate compound or compound to be tested; wherein a decreased MTase activity of the nsPl protein in the presence of the candidate compound indicates that the candidate compound is an inhibitor of alphavirus/Chikungunya virus (CHIKV) and/or suitable for treatment or prevention of infections caused by alphavirus/Chikungunya virus (CHIKV).

The method screens nsPl MTase and GTase activity by detecting methylated GMP moiety conjugated to protein.

The plate based immunoassay of the present invention is a non-radioactive method which determines the enzymatic activity of CHIKV nsPl for screening MTase/GTase inhibitors.

In an embodiment, the invention provides ELISA based method for detection of capping enzyme nsPl inhibitors and particularly nsPl methyltransferase and guanylyltranserase inhibitors. In an embodiment, the method is based on detecting nsPl inhibitors comprising methyltr ansferase , gu anylyltr ansferase .

In an embodiment, the method is based on detecting nsPl inhibitors comprising the viral capping enzyme/replicase complex.

In an embodiment, the assay is ELISA based method to determine the enzymatic activity of the nsPl protein.

In an embodiment, the plate based non-radioactive method for screening a test compound as the nsPl capping enzyme inhibitors of alphavirus/Chikungunya/alphavirus-like superfamily, comprises following steps:

a. Performing ELISA based methyltransferase and guanylyltransferase assay in the presence of said test compound and recombinant Chikungunya virus nsPl protein by the steps of preparing the reaction mixture followed by starting the reaction with the addition of nsPl protein and incubating at temperature of approximately 25°C for different time intervals where said reaction mixture comprises assay buffer, Guanosine-5 '-triphosphate and S-adenosyl-L-methionine and said different time interval comprises 0, 5, 10, 15, 20, 25, 30, 45 minutes;

The decreased MTase activity of the nsPl protein or decreased absorbance level in the presence of said test compound would indicate that the test compound is an inhibitor of alphavirus/Chikungunya/alphavirus-like superfamily and/or suitable for treatment or prevention of infections caused by alphavirus/Chikungunya/alphavirus-like superfamily.

In an embodiment, the assay buffer comprises 50 mM Tris-HCl pH 7.3, 10 mM NaCl, 5 mM DTT, 2 mM MgCl₂.

In an embodiment, the TBST buffer comprises 20 mM Tris at pH 7.5, 150 mM NaCl, 0.1 % Tween-20.

In an embodiment, the stop reaction buffer comprises approximately 5 mM EDTA.

In an embodiment, the blocking buffer comprises 3 % skim milk in said TBST buffer.

In an embodiment, the primary antibody comprises mouse anti-m⁷G cap primary monoclonal antibody present in a ratio of 1 :5000 in TBST.

In an embodiment, the secondary antibody comprises Horseradish peroxidase (HRP)- conjugated anti-mouse IgGl secondary antibody present in a ratio of 1 : 10,000 in TBST. In an embodiment, the chromogenic substrate comprises 20 X Tetramethylbenzidine.

In an embodiment, the alphavirus-like superfamily comprises ssRNA viruses comprising but not limited to Tymovirales, Hordeivirus, Tobravirus, Tobamovirus, Bromovirus, Clostero virus and Calicivirus.

In an embodiment, the method detects the m⁷GMP-nsPl covalent complex formed as a result of coupled MTase and GTase enzymatic activity of nsPl for screening a test compound as the nsPl capping enzyme inhibitors of alphavirus/Chikungunya/ alphavirus-like superfamily.

In an embodiment, the cloned recombinant plasmid containing CHIKV nsPl (residues 1 -509) was transformed into E. coli Rosetta cells (DE3) and protein was expressed at 18°C. Immobilized metal affinity chromatography (IMAC) was used to purify the protein. The purified protein was used to perform methyltransferase activity assay. The present invention provides an ELISA based assay for testing and screening inhibitors against CHIKV capping enzyme, nsPl . The recombinant and purified CHIKV nsPl was incubated with the enzymatic reaction mixture containing SAM and GTP. After incubation, the reaction was stopped and subjected to ELISA plate. Afterwards, primary anti-m⁷G monoclonal antibody was added followed by the addition of horseradish peroxidase (HRP)-conjugated anti-mouse IgGl secondary antibody.

In an embodiment, the immunoreactive proteins were detected using TMB (tetramethylbenzidine)/H202-ELISA substrate and the optical density of resulting yellow color was measured at 450 nm. An increase in absorbance at 450 nm is seen only when nsPl MTase reaction occurs to produce methylated GMP which then gets covalent conjugated to nsPl and forms m⁷GMP-nsPl covalent complex. Anti-m⁷G monoclonal antibody detects the η

formation of m GMP-nsPl covalent complex. However, if MTase reaction does not occur due to the presence of inhibitors, then the transfer of methyl group from SAM to GTP will not be catalyzed and subsequent the formation of m⁷GMP-nsPl covalent complex will not happen. Therefore, the increase in absorbance is not detected in the absence of the formation η

of m GMP-nsPl conjugate. The known SAM-dependent MTase inhibitory compounds namely, sinefungin, ATA and ribavirin were tested for their effects on nsPl using this ELISA based assay.

The details are mentioned below:

CHIKV nsPl expression and purification:

The enzymatic reaction steps catalyzed by alpha virus nsPl are given in Fig.l . For bacterial expression, CHIKV nsPl gene was cloned into expression vector pET28c. To get protein in soluble form, expression was checked at various temperatures (37°C, 25 °C and 18°C). The recombinant nsPl protein with the molecular mass of -56 kDa was produced in soluble form in E. coli expression strain Rosetta (DE3) grown at 18°C for 12 hours after induction with 0.4 mM IPTG (Fig. 2a; Expression and purification of CHIKV nsPl (residues 1-509) from E. coli.). For the production of large-scale (1 liter) recombinant CHIKV nsPl, expression conditions were optimized as discussed in the Materials and Methods section.

An immobilized metal-affinity chromatography (IMAC) method was used to purify the recombinant CHIKV nsPl (residues 1-509) as explained in the Materials and Methods section. Elution of recombinant nsPl was done in 50 mM Tris-HCl pH 7.3, 250 mM imidazole, 5 % glycerol and 200 mM NaCl. The presence of a single band on SDS-PAGE gel suggested the homogeneity and purity of the CHIKV nsPl (residues 1-509) protein. The molecular weight of the purified protein was -56 kDa (Fig. 2b; Expression and purification of CHIKV nsPl (residues 1-509) from E. coli.). The protein was concentrated up to -6 mg ml ¹. ELISA assay:

Indirect ELISA was performed to check the methyltransferase activity of nsPl . Reaction mixture was prepared by adding 10 to 200 μΜ of substrate (SAM) in each 50 μΐ reaction volume of 50 mM Tris-HCl pH 7.3, 5 mM NaCl, 5 mM DTT, 2 mM MgCl₂, 100 μΜ GTP. 20 μΜ of nsPl protein was added and incubated at 25 °C for different intervals of time (0, 5, 10, 15, 20, 25, 30, 45 minutes). The nsPl enzyme reaction was stopped by adding 5 mM η

EDTA and subjected to ELISA using anti-m G cap monoclonal antibody, and subsequently horseradish peroxidase (HRP)-conjugated anti-mouse IgGl secondary antibody was added. The immunoreactive proteins containing m⁷GMP-nsPl complex were detected using TMB/H₂0₂-ELISA substrate (GeNei). After sufficient color development, 1 N H₂S0₄ was added and absorbance at 450 nm was measured. An increase in absorbance was seen with the increasing concentration of the substrate (SAM), indicating the MTase reaction has been occurred (Fig. 3a; Methyltransferase nsPl assay). All these values were subtracted from the readings with reaction buffer having no enzyme for normalization. Similarly, ELISA was performed by keeping the concentration of SAM (200 μΜ) as constant and by increasing the concentration of GTP from 5 to 150 μΜ in each reaction volume of 50 μΐ and same as above reaction was performed. With the increasing concentration of GTP, an increase in the absorbance was observed confirming nsPl MTase activity (Fig. 3b; Methyltransferase nsPl assay). nsPl inhibition:

For inhibitory assays, same as above reactions were used in the presence of increasing concentrations of sinefungin, ATA or ribavirin. The absorbance was measured at 450 nm for each concentration of these inhibitors. The dose-response curve showed IC₅₀ values for sinefungin, ATA and ribavirin as 2.62 μΜ, 5.64 μΜ and 1.2 mM respectively. IC₅₀ values showed sinefungin and ATA as effective inhibitors of CHIKV MTase activity. However, ribavirin with IC₅₀ value of 1.2 mM indicated its poor inhibitory activity against CHIKV MTase as compared to sinefungin and ATA (Fig. 4; Methyltransferase inhibition assay of CHIKV nsPl).

The cloned recombinant plasmid containing CHIKV nsPl (residues 1 -509) was transformed into E. coli Rosetta cells (DE3) and protein was expressed at 18 °C. Immobilized metal affinity chromatography (IMAC) was used to purify the protein. The purified protein was used to perform methyltransferase activity assay. The present invention provides an ELISA based assay for testing and screening inhibitors against CHIKV capping enzyme, nsPl . Recombinant and purified CHIKV nsPl was incubated with the enzymatic reaction mixture containing SAM and GTP. After incubation, the reaction was stopped and subjected to η

ELISA plate. Afterwards, primary anti-m G monoclonal antibody was added followed by the addition of horseradish peroxidase (HRP)-conjugated anti-mouse IgGl secondary antibody. The immunoreactive proteins were detected using TMB/H₂O₂-ELISA substrate. After sufficient color development 1 N H₂SO₄ was added and the optical density of resulting yellow color was measured at 450 nm (Fig. 5; Schematics of the ELISA assay). An increase in absorbance at 450 nm is seen only when nsPl MTase reaction occurs to produce methylated GMP which then gets covalent conjugated to nsPl and forms m⁷GMP-nsPl covalent complex. Anti-m⁷G monoclonal antibody detects the formation of m⁷GMP-nsPl covalent complex. However, if MTase reaction does not occur due to the presence of inhibitors, then the transfer of methyl group from SAM to GTP will not be catalyzed and subsequent the formation of m⁷GMP-nsPl covalent complex will not happen. Therefore, the η increase in absorbance is not detected in the absence of the formation of m GMP-nsPl conjugate. The known SAM-dependent MTase inhibitory compounds namely, sinefungin, ATA and ribavirin were tested for their effects on nsPl using this ELISA based assay. Fig. 6 illustrates nucleotide sequence of CHIKVnsPl .

Fig. 7 illustrates methyltransferase activity where in case of reaction in which no inhibitor was added to the enzymatic reaction mixture was taken as 100 %. The percent inhibition of (a) sinefungin (b) ATA (c) ribavirin was calculated. The data represent an average of two readings plotted against inhibitor concentration.

The assay employs conventional reagents in all the method steps. In an embodiment, the components/reagents used in the assay are following:

(i) Protein expression and purification

(a) Expression

• E. coli Rosetta cells (DE3)

• Luria-Bertani (LB) broth

• 50 μg ml^"1 Kanamycin • 35 μg ml^"1 Chloramphenicol

• 0.4 mM Isopropyl-B-thiogalactopyranoside (IPTG)

• Refrigerated incubator shaker (37 °C, 25 °C and 18 °C) with shaking (200 rpm) (b) Purification

• Nickel- nitrilotriacetic acid (NTA) beads (Qiagen, USA)

• Cell disruptor (Constant Systems, Ltd., Daventry, England)

• Centrifuge (Eppendorf, Germany)

· 3 kDa cutoff Amicon Ultra- 15 microconcentrator (Millipore, Bedford, USA)

• UV- visible light (Vis) spectrophotometer (Eppendorf, Germany)

Lysis Buffer · 50 mM Tris-HCl buffer pH 7.3

• 200 mM NaCl

• 20 mM imidazole

• 5 % glycerol Wash Buffers

• 50 mM Tris-HCl pH 7.3

• 200 mM NaCl

• 60/ 100/ 130 mM imidazole

· 5 % glycerol

Elution Buffer

• 50 mM Tris-HCl pH 7.3

· 200 mM NaCl

• 250 mM imidazole

• 5 % glycerol Dialysis Buffer

• 50 mM Tris-HCl pH 7.3

• 100 mM NaCl

• 5 % glycerol

(ii) ELBA nsPl assay buffer · 50 mM Tris-HCl pH 7.3

• 5 mM NaCl

• 5 mM DTT

• 2 mM MgCl₂

• SAM (10, 25, 50, 100, 150 and 200 uM)

· GTP (5, 10, 25, 50, 100 and 150 μΜ)

Wash buffer/TBST buffer

• 20 mM Tris pH 7.5

• 150 mM NaCl

· 0.1 % Tween-20

Stop buffer

• 5 mM EDTA Blocking buffer

• 3 % skim milk in TBST

Primary antibody

• Anti-m⁷G cap clone H-20 mouse monoclonal antibody (Merck) at a dilution of 1 :5000 in TBST Secondary antibody

• Horseradish peroxidase (HRP)-conjugated anti-mouse IgGl secondary antibody (Affymetrix eBioscience) at a dilution of 1 : 10,000 in TBST

Microplates

• 96 wells microplates MICROLON® (Greiner bio-one, Germany)

Chromogenic substrate

· TMB/H₂0₂-ELISA substrate (GeNei)

Stop solution

• 1 N H₂SO₄ Multi-mode plate reader

• Cytation 3 (BioTek Instruments, Inc.)

In another aspect of the invention, there is provided a kit based on the non-radioactive method for testing and/or screening of inhibitory compounds against the capping enzyme, nsPl of alphaviruses/Chikungunya/alphavirus-like superfamily. The kit comprises of the essential components including nsPl protein along with other conventional reagents.

In an embodiment, the present invention provides a kit which does not use radioisotopes for testing and/or screening of various inhibitory compounds against the capping enzyme, nsPl of alphaviruses/Chikungunya/viruses of the alphavirus-like superfamily.

In an embodiment, the Chikungunya nsPl enzyme assay kit comprises following components: a. Purified recombinant Chikungunya nsPl present in the concentration of 5 mg/ml

b. S-adenosyl-L-methionine present as 10 mM solution in water

c. Guanosine-5 '-triphosphate present as 5 mM solution in water d. Assay buffer comprising 50 mM Tris-HCl (pH 7.3), 10 mM NaCl, 5 mM DTT, 2 mM MgCl₂

e. Stop reaction buffer comprising 100 mM EDTA

f. TBST buffer comprising 20 mM Tris (pH 7.5), 150 mM NaCl, 0.1 % Tween-20

1. Positive control comprising 1 mM aurintricarboxylic acid solution in water

m. 96-well solid plate with a cover sheet.

In an embodiment, the compounds sinefungin, aurintricarboxylic acid (ATA) and ribavirin are tested for their MTase inhibitory activity against CHIKV nsPl. The inhibition results for the compounds were validated against the IC₅₀ values reported for nsPl using a radioactive method for nsPl MTase assay.

In an embodiment, following are the components of kit of the present invention.

S.No. Component Composition Storage

1 Purified recombinant 0.1 to lO mg/ml -80 °C

Chikungunya nsPl

2 S-adenosyl-L- 1-50 mM solution in water -80 °C

methionine (SAM)

3 Guanosine-5'- 0.1 - 5 mM solution in water -80 °C

triphosphate (GTP)

4 Assay buffer 50 mM Tris-HCl (pH 7.3), 10 mM NaCl, 5 -80 °C

mM DTT, 2 mM MgCl₂

5 Stop reaction buffer 1-100 mM EDTA 4 °C

6 TB ST buffer 20 mM Tris (pH 7.5), 150 mM NaCl, 0.1 4 °C

% Tween-20

7 Blocking solution 1-10 % skim milk in TBST buffer 4 °C

8 Primary antibody Mouse anti-m'G cap primary monoclonal -20 °C

antibody at 1:500 to 1:10,000 dilution in

TBST buffer

9 Secondary antibody Horseradish peroxidase (HRP)-conjugated -20 °C

anti-mouse IgGl secondary antibody at

1 :3,000 to 1 :20,000 dilution in TBST

buffer

10 Chromogenic 20X Tetramethylbenzidine (TMB) 4 °C

substrate

11 Stop solution 1 N H₂S0₄ Room temperature

12 Positive control 0.1 to 10 mM Aurintricarboxylic acid -20 °C

solution in water

The Chikungunya nsPl enzyme assay kit further comprises of 96-well solid plate with a cover sheet.

In another embodiment, 384- well solid plate can also be used.

In another embodiment, 1536-well solid plate can also be used.

The invention is further described with the help of non-limiting examples:

Example 1:

Protein Expression and Purification (a) Expression

For the production of protein, the cloned recombinant plasmid containing CHIKV nsPl (residues 1-509) encoding DNA fragment was transformed into E. coli Rosetta cells (DE3) (Inoue, Nojima, and Okayama, 1990). The colonies obtained after the transformation were used to inoculate LB broth containing 50 μg ml^"1 of kanamycin and 35 μg ml^"1 of chloramphenicol and grown at 37 °C/ 200 rpm till the optical density of 0.4 at 600 nm (OD₆oo) was reached. The temperature was reduced at this point to 18 °C. Growth was continued at 18 °C until the optical density of 0.7 at OD₆₀₀ was reached. 0.4 mM isopropyl-β- thiogalactopyranoside (IPTG) was used to induce expression of the protein. The culture after induction with IPTG was further grown for 12 hours at 18 °C/ 200 rpm. Finally, the harvesting of cells was done by centrifugation at 8,000 X g at 4 °C and the obtained cell pellets were stored at -80 °C until further use.

(b) Purification

The frozen cell pellets from 2 liters culture were thawed on ice. The cell pellets were resuspended in 30 ml of lysis buffer (50 mM Tris-HCl pH 7.3, 20 mM imidazole, 5 % glycerol and 200 mM NaCl) and cell lysis was done with the help of a cell disruptor (Constant Systems, Ltd., Daventry, England). The obtained cell lysate was clarified by centrifugation at 12,000 X g for 45 minutes at 4 °C. Ni-nitrilotriacetic acid (NT A) beads (Qiagen, USA) were used to purify the protein. The supernatant was loaded onto the Ni-NTA column and incubated for 30 minutes at 4 °C. Elution of the recombinant nsPl protein was done in 50 mM Tris-HCl pH 7.3, 250 mM imidazole, 5 % glycerol, 200 mM NaCl and the fractions were analyzed by gel electrophoresis (SDS-PAGE) and Coomassie blue staining. The fractions containing purified protein were pooled and dialyzed against the dialysis buffer (50 mM Tris-HCl pH 7.3, 5 % glycerol and 100 mM NaCl) at 4 °C for 3 hours. 3 kDa cutoff Amicon Ultra-15 microconcentrator (Millipore, Bedford, Massachusetts, USA) was used to concentrate the purified protein. UV-visible light (Vis) spectroscopy at 280 nm was used to measure the concentration and yield of the protein using an extinction coefficient method (calculated extinction coefficient of CHIKV nsPl is 66,810 M^"1 cm^"1). Example 2:

ELISA based assay

(a) nsPl enzyme reaction:

The CHIKV nsPl enzymatic reactions were set in a reaction mixture of 50 μΐ containing 50 mM Tris-HCl pH 7.3, 5 mM NaCl, 5 mM DTT, 2 mM MgCl₂, 100 μΜ GTP and varying concentration of SAM (10, 25, 50, 100, 150 and 200 μΜ). The reactions were initiated with the addition of 20 μΜ of nsPl and incubated at 25 °C for different intervals of time (0, 5, 10, 15, 20, 25, 30, 45 minutes). The reactions were stopped by adding 5 mM EDTA and subjected to 96-well ELISA plate followed by incubation of 1 hour at 25 °C. After two washes of 3 minutes each with TBST buffer (20 mM Tris pH 7.5, 150 mM NaCl, 0.1 % Tween-20), blocking was done in 3 % skim milk in TBST at room temperature for 1 hour and 30 minutes. After washing twice with TBST, plate was incubated with anti-m⁷G cap clone H- 20 monoclonal antibody (Merck) at a dilution of 1 :5000 in TBST at 25 °C for 1 hour and subsequently after washing with TBST, plate was incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgGl secondary antibody (Affymetrix eBioscience) at a dilution of 1 :10,000 in TBST at 25 °C for 1 hour. The immunoreactive proteins were detected using TMB/H₂0₂-ELISA substrate (GeNei) (100 μΐ per well). After sufficient color development, stopped the reaction by adding stop solution comprising 1 N H₂SO₄ (100 μΐ per well) and the optical density of resulting yellow color was measured at 450 nm using a multi- mode plate reader Cytation 3 (BioTek Instruments, Inc.). In other set, same procedure was followed in a reaction mixture of 50 μΐ containing 50 mM Tris-HCl pH 7.3, 5 mM NaCl, 5 mM DTT, 2 mM MgCl₂, 200 μΜ SAM with varying concentration of GTP (5, 10, 25, 50, 100 and 150 μΜ).

A negative control reaction for MTase assay was done with all the reaction reagents without SAM because SAM is the methyl donor in nsPl MTase reaction. In the absence of SAM, the methylation of GTP will not take place and thus, the subsequent m⁷GMP-nsPl conjugate will not be formed.

(b) nsPl MTase inhibition:

For inhibition assays, same procedure of nsPl assay and ELISA methods were followed using different concentrations of various inhibitors. The compounds tested for nsPl enzymatic inhibitory activity namely are, sinefungin, ATA and ribavirin. The IC₅₀ (half- maximal inhibitory concentration) values of all these compounds using logistic function were determined using GraphPad prism.

Example 3: CHIKV nsPl (residues 1-509) was expressed in soluble form in E. coli and purified. The mass of the soluble protein is -56 kDa. The MTase activity was determined by using an ELISA based MTase assay. The methyl group was transferred from SAM to the substrate GTP and resulted in m⁷GTP formation which is catalyzed by the nsPl MTase. Subsequently, η nsPl enzyme in the presence of divalent metal then resulted in the formation of m GMP-nsPl covalent complex releasing PPi. Using the present ELISA based nsPl assay, this m⁷GMP- nsPl covalent complex was detected using anti-m⁷G cap monoclonal antibody and then incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgGl secondary antibody. Finally immunoreactive proteins were detected by using TMB/H₂O₂-ELISA substrate. After sufficient color development 1 N H₂SO₄ was added and the optical density at 450 nm was measured. Here, we detected the m⁷GMP-nsPl covalent complex which is dependent on MTase activity of nsPl i.e. methylation of GTP. So using this ELISA based assay MTase activity of CHIKV nsPl was indirectly determined. This ELISA based assay can be used for testing and screening inhibitors against CHIKV. In the present work, sinefungin, ATA and ribavirin were tested for their inhibitory effects. Sinefungin, an analog of SAM was shown to inhibit the MTase activity of nsPl . ATA also showed similar inhibitory effects on CHIKV MTase activity. Ribavirin, a guanosine analog was found to inhibit of nsPl MTase activity to a less extend as compared to sinefungin and ATA. Example 4:

It provides an ELISA based plate method for detecting nsPl enzymatic activity or for testing nsPl MTase or GTase inhibitors. The MTase activity has been determined by using an ELISA based plate method assay. The methyl group is transferred from SAM to the substrate η

GTP and results in m GTP formation which is catalyzed by the nsPl MTase. Subsequently, nsPl enzyme in the presence of divalent metal then results in the formation of m⁷GMP-nsPl covalent complex releasing PPi. In this ELISA based nsPl assay, the m⁷GMP-nsPl covalent complex is formed as a result of methyltransferase activity of purified nsPl , which is detected η

using anti-m G cap monoclonal antibody and horseradish peroxidase (HRP)-conjugated anti- mouse IgGl secondary antibody. In the last step immunoreactive m⁷GMP-nsPl covalent complex formed due to nsPl enzymatic activity were detected using TMB/H₂O₂-ELISA substrate and the optical density at 450 nm was measured. The method detects m⁷GMP-nsPl covalent complex which is dependent on MTase activity of nsPl i.e. methylation of GTP. So using this developed ELISA based assay MTase activity of CHIKV nsPl can be determined.

Claims

Claims:

1. A plate based non-radioactive method for screening a test compound as the nsPl capping enzyme inhibitors of alphavirus/Chikungunya/alphavirus-like superfamily, comprising the steps of:

f. Performing ELISA based methyltransferase and guanylyltransferase assay in the presence of said test compound and recombinant Chikungunya virus nsPl protein by the steps of preparing the reaction mixture followed by starting the reaction with the addition of nsPl protein and incubating at temperature of approximately 25°C for different time intervals where said reaction mixture comprises assay buffer, Guanosine-5 '-triphosphate and S-adenosyl-L-methionine and said different time interval comprises 0, 5, 10, 15, 20, 25, 30, 45 minutes;

g. Treating the reaction mixture resulting from step (a) with stop reaction buffer followed by subjecting said mixture to ELISA plate followed by incubating at temperature of approximately 25 °C for 60 minutes;

h. Treating with a blocking buffer at approximately 25 °C for 90 minutes followed by treating with a primary antibody at approximately 25 °C for 1 hour;

i. Treating with a secondary antibody at approximately 25 °C for 1 hour followed by washing with TBST buffer;

j. Adding a chromogenic substrate followed by addition of stop solution upon sufficient color development and identifying absorbance at 450 nm.

2. The method as claimed in claim 1, wherein said assay buffer comprises 50 mM Tris- HC1 pH 7.3, 10 mM NaCl, 5 mM DTT, 2 mM MgCl_2.

3. The method as claimed in claim 1, wherein said TBST buffer comprises 20 mM Tris at pH 7.5, 150 mM NaCl, 0.1 % Tween-20.

4. The method as claimed in claim 1 , wherein said stop reaction buffer comprises approximately 5 mM EDTA.

5. The method as claimed in claim 1, wherein said blocking buffer comprises 3% skim milk in said TBST buffer.

6. The method as claimed in claim 1 , wherein said primary antibody comprises mouse η

anti-m G cap primary monoclonal antibody present in a ratio of 1 :5000 in TBST.

7. The method as claimed in claim 1, wherein said secondary antibody comprises Horseradish peroxidase (HRP)-conjugated anti-mouse IgGl secondary antibody present in a ratio of 1 : 10,000 in TBST.

8. The method as claimed in claim 1, wherein said chromogenic substrate comprises 20 X Tetramethylbenzidine.

9. The method as claimed in claim 1 , wherein said alphavirus-like superfamily comprises ssRNA viruses including but not limited to Tymovirales, Hordeivirus, Tobravirus, Tobamovirus, Bromovirus, Closterovirus and Calicivirus.

10. The method as claimed in claim 1, detects the m⁷GMP-nsPl covalent complex formed as a result of coupled MTase and GTase enzymatic activity of nsPl for screening a test compound as the nsPl capping enzyme inhibitors of alphavirus/Chikungunya/ alphavirus-like superfamily.

11. A kit for non-radioactive enzyme linked immunosorbent assay for screening nsPl methyltransferase inhibitors, comprising components:

n. Purified recombinant Chikungunya nsPl present in the concentration of 5 mg/ml

o. S-adenosyl-L-methionine present as 10 mM solution in water p. Guanosine-5 '-triphosphate present as 5 mM solution in water

q. Assay buffer comprising 50 mM Tris-HCl (pH 7.3), 10 mM NaCl, 5 mM

DTT, 2 mM MgCl₂

r. Stop reaction buffer comprising 100 mM EDTA

s. TBST buffer comprising 20 mM Tris (pH 7.5), 150 mM NaCl, 0.1 % Tween-20

t. Blocking solution comprising 3 % skim milk in TBST buffer

u. Primary antibody comprising Mouse anti-m⁷G cap primary monoclonal antibody at 1 :5000 dilution in TBST buffer

v. Secondary antibody comprising Horseradish peroxidase (HRP)-conjugated anti- mouse IgGl secondary antibody at 1 :10,000 dilution in TBST buffer w. Chromogenic substrate comprising 20X Tetramethylbenzidine x. Stop solution comprising 1 N H₂SO₄ Positive control comprising 1 mM aurintricarboxylic acid solution water

96-well solid plate with a cover sheet.