WO2012068390A1

WO2012068390A1 - INHIBITORS OF FOOT AND MOUTH DISEASE VIRUS TARGETING THE RNA-DEPENDENT POLYMERASE ACTIVITY OF 3Dpol

Info

Publication number: WO2012068390A1
Application number: PCT/US2011/061219
Authority: WO
Inventors: Stefan G. Sarafianos; Ryan Durk; Elizabeth Rieder; Kamlendra Singh
Original assignee: The Curators Of The University Of Missouri
Priority date: 2010-11-17
Filing date: 2011-11-17
Publication date: 2012-05-24

Abstract

The present invention provides a novel method for treating, preventing or inhibiting Foot and Mouth Disease Virus. Also provided are several new classes of antiviral compounds for the treatment or prevention of Foot and Mouth Disease. The inventive antiviral compounds suppress the replication of Foot and Mouth Disease Virus RNA- Dependent RNA Polymerase (3Dpol) by blocking a preexisting binding pocket proximate to the active site of 3Dpol.

Description

TITLE OF THE INVENTION

INHIBITORS OF FOOT AND MOUTH DISEASE VIRUS TARGETING THE RNA- DEPENDENT POLYMERASE ACTIVITY OF 3Dpol

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of United States Provisional Application No. 61/458,080, filed on November 17, 2010, herein incorporated by reference in its entirety. STATEMENT OF GOVERNMENT SUPPORT

The invention was made with government support under Grant No. USDA-ARS-58- 1940-5-519 by the Department of Agriculture. The U.S. Government has certain rights in the invention. BACKGROUND OF INVENTION

1. Field of the Invention

The present invention relates to the field of antiviral medicine and antiviral therapy, and more specifically to a method of and inhibitors for preventing, treating, or reducing the spread of Foot and Mouth Disease Virus (FMDV) infections.

2. Background of the Invention

Foot and Mouth Disease Virus (FMDV) is a member of the Aphthovirus genus in the Picomavirus family that infects cloven-hoofed and other animals and leads to severe losses in livestock. However, there are currently no approved anti-FMDV drugs or antiviral compounds for the treatment or prevention of FMD.

SUMMARY OF INVENTION

Disclosed herein is a method of inhibiting the replication of Foot and Mouth Disease Virus (FMDV) by directly targeting the binding pocket of 3Dpol. The inhibitor-binding pocket is highly conserved among all 60 FMDV subtypes, and the binding site interface includes the residues such as V55, 156, S58, K59, R168, G176, K177, T178, R179 and 1180, whereas the hydrophobic residues are V55, 156, and 1180, and the aliphatic chains of residues are K59, K177, R168, and R179. The inventive method of inhibiting the replication of FMDV includes the step of providing an antiviral compound that is capable of binding or interacting with one or multiple residues lining the binding pocket of 3Dpol. In another aspect, the invention provides a method of treating an animal comprising administering an antiviral compound capable of binding or interacting with one or more residues within the binding pocket of 3Dpol. In certain embodiments, the animal to be treated is either infected with FMDV or is not infected with FMDV. For instance, an animal not infected with FMDV may potentially be exposed to FMDV. In another embodiment, the animal to be treated is selected from the group consisting of a cow, bison, water buffalo, sheep, goat, pig, deer, hedgehog, elephant, llama, and alpaca.

Further disclosed herein are several new classes of antiviral compounds for the suppression of FMDV by directly blocking the binding pocket of 3Dpol. According to one embodiment of the invention, the inventive antiviral compounds may contain a phenyl ring unit and a benzenesulfonyl with a linker that is based on hydrazide and sulfone moieties having the formula (I)

whereas R may be selected from the group consisting of halo, alkylcarbonyl, and secondary or tertiary alcohol groups. One exemplary inhibitor in this class is 4-chloro-N'-thieno[2,3- d]pyrimidin-4-ylbenzenesulfonohydrazide.

According to another embodiment of the invention, the inventive antiviral compounds may contain a phenyl unit and a nitro-benzoxadiazole unit having the formula (II):

rr

whereas R' and R" may be independently selected from the group consisting of halo, hydroxyl, amino, carboxyl, secondary or tertiary alcohol, and alkylcarbonyl groups. The X bridging atom may be either a Sulfur or Oxygen. One exemplary inhibitor in this class is 4- (2,4-dichlorophenyl)sulfanyl-7-nitro-2, 1 ,3-benzoxadiazole.

According to yet another embodiment of the invention, the inventive antiviral compounds may contain a benzothiophene-dioxide unit having the formula (III):

(III) whereas R may be selected from the group consisting of halo, amino, or hydroxyl groups; Y may either be an oxygen or a sulfur atom; and Z may be either oxygen, sulfur, or nitrogen.

One exemplary inhibitor in this class is 5-chloro-3-(thiophen-2-ylsulfanylmethyl)-l- benzothiophene 1,1 -dioxide.

In another aspect of the invention, the above antiviral compounds of the invention may be used in a method of the invention. For instance, the above antiviral compounds may be administered in a method of inhibiting the replication of FMDV or a method of treating an animal as provided by the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1(A) is an image of the surface representation of the complex of 3Dpol with

RNA and UTP.

FIG. 1(B) shows the binding sites on the pocket binding interface.

FIG. 2(A) shows the relative inhibitions of 3Dpol by compounds from a typical 96- well plate: 1.7μΜ FMDV 3Dpol was incubated in the presence of 20μΜ compounds, 25 mM Tris-HCl, pH 7.8, 40 nM poly-rA/5'-Cy3-dT₁₈, 10μΜ UTP, 25 mM KC1, and 1 mM MnCl₂. After 60 minutes, ATP sulfurylase and luciferase assay components were introduced to the reactions as described in (reference Lescer Malcom et.al; see Scheme 1). Luminescence was measured with a 96-well plate luminometer. Data are presented as percent luminescence inhibition (1 - [luminescence of a reaction / maximum luminescence] X 100). The tallest three bars indicate compounds that inhibited by at least 88%. Compounds meeting this threshold were selected for further evaluation. FIG. 2(B) shows the frequency distribution of percent luminescence. Percent luminescence was calculated by dividing relative luminescence values of individual wells by the luminescence maximum of that plate.

FIG. 3 depicts the chemical structures, names, and inhibition constants of 3Dpol inhibitors N' l-thieno[2,3-d]pyrimidin-4-yl-4-chloro-l-benzenesulfonohydrazide (labeled 5D9), 5-chloro-3-(thiophen-2-yl-sulfanylmethyl)-l-benzothiophene 1,1, -dioxide (labeled 7F8), 3'-deoxy 5 -methyl-uridine- 5 'triphosphate (labeled DMUT), 4-(2,4- dichlorophenyl)sulfanyl-7-nitro-2,l,3-benzoxadiazole (labeled 1A8), 2-(2- bromophenyl)sulfanyl-3-chloronaphthalene-l,4-dione (labeled 8C5), 3-(2-chloroanilino)-2- cyano-3-sulfanylidene-N-[3-(trifluoromethyl)phenyl]-propanamide (labeled 3A11), S- thiophen-2-yl5-(2-phenylethynyl)pyridine-3-carbothioate (labeled 4H6), and ethyl(2E)-2-[(4- bromophenyl)hydrazinylidene]-3-oxo-4-thiocyanobutanoate (labeled 9A3).

FIG. 4 illustrates the gel-based validation of 3Dpol inhibition by different compounds. Representative results depicting the dose-dependent inhibition of RNA synthesis by 5D9, 7F8, and 8C5. RNA synthesis by 1 μΜ 3Dpol on 250 nM poly-rA/dT₁₈ was carried out in the presence of varying concentrations of compounds (0-40 μΜ 5D9 and 7F8, and 0-100 μΜ 8C5) and 500 μΜ UTP in a buffer containing 50 mM Tris-HCl pH 7.8, 60 mM KC1, 0.01% BSA, 1 mM DTT and 0.1% NP40. Lanes labeled as P contain only free dT₁₈ primer. To calculate the IC₅₀, the amount of extended dT₁₈ primer was plotted against the varying concentration of hits. The data points were fit to dose-response curves by GraphPad Prizm 4.0 (experiments were repeated at least 4 times).

FIG. 5(A) and FIG. 5(B) show close-ups of the molecular surface areas of 5D9 and 1A8, respectively, docked at the inhibitor binding site of 3Dpol. Potential hydrogen bond interactions that involve the ε-ΝΗ₂ of K59 and the inhibitors are indicated with dotted lines. Some 3Dpol side chains have been removed for clarity.

FIG. 5(C) shows all seven selected inhibitors docked at the inhibitor binding pocket, proximal to the UTP binding site.

FIG. 6(A) is a graph illustrating the light production as a function of exogenously added pyrophosphate (PPi): The amount of light generated by the coupled ATP sulfurylase and luciferase reactions is directly proportional to the amount of PPi added over a range of at least 2 logs (l-ΙΟΟμΜ PPi).

FIG. 6(B) shows the assay validation using 3'-deoxy 5-methyl-uridine-5' triphosphate (DMUT) as an inhibitor of FMDV 3Dpol (DMUT lacks a 3ΌΗ required for RNA synthesis). Varying concentrations of DMUT incubated with 1.7 μΜ FMDV 3Dpol, 40 nM poly-rA/5'- Cy3-dT₁₈, and 10 μΜ UTP at 37 °C for one hour prior to the addition of the ATP sulfurylase and luciferase assay components (n=3, error bars are standard deviation from the mean).

FIG. 7 shows the effects of compounds on enzymatic activity of Klenow fragment and HIV reverse transcriptase: 10 nM KF or 20 nM HIV-1 RT were incubated with 250 nM poly-rA/dTi₈ and 20 μΜ of a 3Dpol inhibitor (1A8, 4H6, 6B11, 5D9, 7F8, 8C5, or 9A3) in a buffer containing 50 mM Tris-HCl, ni l 7.8, 60 mM C1, 1 in DTT, 0.01% BSA, 0.1 % NP40. and 4% DMSO. DNA synthesis was initiated by the addition of 1 mM MaCh and 500 μ M dT'i^'P final concentrations. No significant inhibition of KF or HIV RT is observed under these conditions.

FIG. 8(A) is the valuation of anti-FMDV activity of 3Dpol inhibitors. Treatment with 15 μΜ of various compounds was performed following virus adsorption on BHK-21. The cells were further incubated in the presence of compound for another 24 hours and virus titer was determined by plaque assays (PFUs, plaque forming units) as described in the Examples below. Experiments were done in duplicates.

FIG. 8(B) shows the dose-dependence of FMDV inhibition by 5D9. Compound 5D9 was administered to BHK-21 cells in a dose-dependent manner prior to infection with FMDV. Post- infection, the compound was re-administered and incubated for another 24 hours. Following this incubation, samples were taken and plaque assays were performed. Results are reported as percent inhibition compared to a sample without inhibitor, and demonstrate a dose-dependent inhibition of virus replication. At the highest concentrations of 5D9 (20μΜ) there was a greater than 90% inhibition of virus replication. Error bars represent standard error of the mean for two experiments. At these inhibitor concentrations no decrease in cell viability was observed.

DETAILED DESCRIPTION OF INVENTION

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any use of a singular term, such as the number one (1), is intended to encompass numerical values greater than one, such as represented by the phrase "one or more." Any use of inclusive terms such as "including" or "such as" and the like is intended to be open ended, with a meaning similar to "including, but not limited to." All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

The present invention provides a novel method of inhibiting the replication of Foot and Mouth Disease Virus (FMDV) by directly targeting the binding pocket of 3Dpol. The Examples below describe the newly identified but pre-existing inhibitor-binding pocket of 3Dpol, wherein the inhibitor-binding pocket is proximal to the active site, but not overlapping with the NTP binding site. In one embodiment, the pocket's binding interface is lined with several residues, such as V55, 156, S58, K59, R168, G176, K177, T178, R179 and 1180. The newly identified inhibitor-binding pocket is highly conserved among all 60 FMDV subtypes, thus represents a excellent target for designing anti-viral compounds for suppressing the replication of 3Dpol. The present inventive method of inhibiting the replication of FMDV includes the step of providing an antiviral compound that is capable of binding or interacting with one or multiple residues lining the binding pocket of 3Dpol.

There are seven presently known serotypes of FMDV: A, O, C, Asia 1, and Southern African Territories (SAT) 1, 2 and 3 (Belsham, 1993). Within these serotypes, over 60 subtypes have also been reported. Because of this diversity there is no universal vaccine, thus presenting challenges in the selection of vaccine strains (Paton et al., 2005). The most effective FMD vaccines consist of chemically inactivated FMDV and can only offer complete protection after seven days of vaccination because of the time needed to trigger an immune response (Grubman, 2005). It has been proposed that a combination of vaccine and antivirals can be a more efficacious strategy to treat FMD-infected animals, contain the spreading of the disease, and reduce the number of animals that need to be slaughtered during outbreaks (Grubman, 2005).

Additionally, a further drawback to vaccination with a chemically inactivated virus is that there is typically at least a 7-day delay period between administration of the vaccine material and the triggering of an effective immune response. In the event of an outbreak of FMD, the delay can leave livestock dangerously exposed to infection and disease during the period. Conversely, antiviral drugs can provide prophylactic and/or therapeutic effects almost immediately upon administration. However, there is presently no approved antiviral compound for FMD treatment, prevention, or inhibition.

The FMDV genome is an 8.5kb uncapped, single-stranded RNA. It is translated as a single polyprotein, which in turn is cleaved into structural and non-structural proteins (Ryan, Belsham, & King, 1989). The non-structural protein that carries out RNA synthesis during transcription and replication is an RNA-dependent RNA polymerase (RdRp or 3Dpol). Because of their pivotal roles in the viral life cycle, viral polymerases have been a primary target for the development of antiviral agents. In fact, there are nearly 35 approved antiviral drugs that target polymerases of various pathogens (De Clercq, 2002, 2005) (Menendez- Arias, 2008; Parniak & Sluis-Cremer, 2000; Shehu-Xhilaga, Tachedjian, Crowe, & Kedzierska, 2005) (Singh, Marchand, Kirby, Michailidis, & Sarafianos, 2010).

FIG. 1(A) shows a surface representation of the complex of 3Dpol with RNA (ribbons for template and primer are shown) and UTP (only the gamma phosphate of UTP is seen) (PDB code 2E9Z). The binding site is shown proximal to the UTP binding site. FIG. 1(B) further shows the position of the inhibitor binding pocket and the binding sites lined with several residues.

Among the compounds that target FMDV 3Dpol is ribavirin, a mutagenic nucleoside analogue, known to exhibit antiviral activity against a broad range of both DNA and RNA viruses (Agudo et al., 2008; Graci & Cameron, 2006; McCormick et al., 1986; McHutchison et al., 1998; Pariente, Sierra, & Airaksinen, 2005; Smith et al., 1991). Suppression of FMDV replication in cell cultures requires relatively high concentrations of ribavirin (ECso=970 μΜ). In addition, a resistant mutation in 3Dpol (M296I) has been shown to decrease FMDV susceptibility to ribavirin (Ferrer-Orta et al., 2010; Sierra et al., 2007). Recently, another compound, 2'-C-methylcytidine, has been shown to inhibit FMDV at low μΜ concentrations, most likely through inhibition of 3Dpol (Goris et al., 2007). Similarly, the pyrazinecarboxamide derivative T1106 (Furuta et al., 2009) has been shown to be effective against FMDV. T1106 is converted to a triphosphate form by host enzymes and likely inhibits the replicase activity of FMDV 3Dpol, although the exact mechanism of FMDV inhibition by Tl 106 is unknown.

The present invention overcomes the diffenincies in the presently available methods of treating, preventing or inhibiting FMDV by directly targeting the binding pocket of 3Dpol. The present invention further provides several classes of compounds inhibiting the replication of RNA-dependent RNA polymerase (RdRp) activity.

The principle of the method employed to identify FMDV 3Dpol inhibitors has been described previously (Lahser & Malcolm, 2004) and is shown in Scheme 1 below. Briefly, the PP; released from the polymerase reaction is converted to ATP in a reaction that uses adenosine 5 '-phospho sulfate and is catalyzed by ATP sulfurylase. The ATP product provides the energy for the luciferase-catalyzed conversion of D-luciferin to oxyluciferin, with a concomitant release of photons. Scheme 1

{RNA)_n + _NTP^-^{RNA)_n+l + PP_t

APS + PP.—— >ATP + sulfate

Luci ^Jferin + ATP + 0₇— L—uciferase > Ox ^yluci ^jferin + AMP + PR ι + C0₇ z

As described in the Examples below, the conditions for the light- generating reaction were optimized and improved efficiency of the overall assay was observed. Approximately 2,000 compounds from the Maybridge HitFinder library were screened in the assay. Representative inhibition data from a single 96-well plate are shown in FIGs. 2(A) and (B). As shown in FIG. 2(A), typically, two to three compounds per plate suppressed FMDV 3Dpol activity by -90%. Cumulative inhibition data are shown in FIG. 2B.

Based on these results, 30 compounds were selected that suppressed luminescence >90% (FIG. 2B). These hits included compounds that inhibited the enzymatic activity of luciferase and/or ATP sulfurylase and consequently appeared as false positives. To exclude false positives the compounds' ability to specifically suppress 3Dpol extension was validated with fluorescently labeled primer-extension assays. Most of the initial hits did not inhibit RNA synthesis by 3Dpol, as assessed by gel-based assays. Instead, they were shown to interfere with the secondary luciferase assay reactions. However, seven compounds did inhibit RNA synthesis by 3Dpol in vitro and their chemical structures, the IC₅₀ values, and the names of these compounds are listed in FIG. 3. Two compounds, 1A8 and 3A11, had IC₅₀S in the very low micromolar range (~2 μΜ), whereas the other five (4H6, 7F8, 8C5, 9A3, 5D9) had values between 8 and 12 μΜ. Concentration-dependent analysis is shown in FIG. 4 with more detail for three of the compounds.

All seven potential inhibitors were docked with the newly identified inhibitor-binding pocket and the docking at the pocket was favorable with significant glide scores, requiring only small adjustments to the side chains of protein residues. The energetically most favorable binding conformers of compounds 5D9 and 1A8 docked in FMDV 3Dpol are shown in FIGs. 5 A and 5B, respectively. The seven 3Dpol-inhibitors docked at the inhibitor- binding site are shown in FIG. 5C. The inhibitors appear to interact with residues of the binding pocket through both hydrophobic and electrostatic interactions. For example, the ε- NH₂ group of K59 is shown to form hydrogen-bonds with the 5D9 and 1A8 inhibitors (FIG. 5A and 5B). Similarly, residues K177 and R168 of 3Dpol are also within interacting distance with the inhibitors. Based on the in vitro assay data and the docking modeling for inhibiting the replication of 3Dpol, described above and in detail below in the Examples, several classes of inhibitors are provided in the present invention. The potential 3Dpol inhibitory compounds optionally comprise two hydrophobic or aromatic components that are connected through a linker that can contain sulfonyl, propanamide, carbothioate, sulfanylidene, sulfonohydrazide, sulfonamide, hydrazine, hydrazinylidene or related groups. The hydrophobic components can be aromatic ring systems that may be substituted in one or more positions with groups that can participate in hydrogen bond interactions with 3Dpol residues such as K59. The ring substituents may also be halogens, arylamines, alkylamines, hydroxyl, alkoxy, alcoholic, alkyl, or aryl groups.

According to one embodiment of the invention, the inventive antiviral compounds may contain a phenyl ring unit and a benzenesulfonyl with a linker that is based on hydrazide and sulfone moieties having the formula (I)

(I)

wherein R may be selected from the group consisting of halo, alkylcarbonyl, and secondary or tertiary alcohol groups. One exemplary inhibitor in this class is 4-chloro-N'-thieno[2,3- d]pyrimidin-4-ylbenzenesulfonohydrazide, also referred to here in as 5D9.

(Π)

wherein R' and R" may be independently selected from the group consisting of halo, hydroxyl, amino, carboxyl, secondary or tertiary alcohol, and alkylcarbonyl groups. The X bridging atom may be either a Sulfur or Oxygen. One exemplary inhibitor in this class is 4- (2,4-dichlorophenyl)sulfanyl-7-nitro-2,l,3-benzoxadiazole, also referred to herein as 1A8.

(HI)

wherein R may be selected from the group consisting of halo, amino, or hydroxyl groups; Y may either be an oxygen or a sulfur atom; and Z may be either oxygen, sulfur, or nitrogen. One exemplary inhibitor in this class is 5-chloro-3-(thiophen-2-ylsulfanylmethyl)-l- benzothiophene 1,1 -dioxide, also referred to herein as 7F8.

In one embodiment of the invention, the inventive 3Dpol inhibitory compounds suppress viral replication by selectively inhibiting the target enzyme without affecting the activity of other related enzymes, such as, for example, human nucleic acid polymerases. If the inhibitory compounds lack specificity, they can injure or negatively affect the organism to which they are administered. As shown in the Examples detailed herein, the disclosed compounds and methods successfully inhibit FMDV 3Dpol activity with specificity, thereby presenting little or no danger to the organism to which they are administered.

In embodiments, the inventive antiviral composition may comprise one or more 3Dpol inhibitory compounds incorporated into the antiviral composition to be administered as a prophylactic or therapeutic treatment for an organism infected with, or potentially exposed to, FMDV. Such organisms include any organism that can contract FMD or carry FMDV, including domestic and undomesticated animals such as cattle, bison, water buffalo, sheep, goats, pigs, deer, hedgehogs, elephants, llamas, and alpacas.

The antiviral composition including one or more 3Dpol inhibitory compounds may optionally include one or more additional components, such as carriers, stabilizers, immune system stimulating materials, disinfectants, chemically or otherwise inactivated FMDV or other viral material, or additional viral inhibitory compounds.

The antiviral composition including one or more 3Dpol inhibitory compounds may be administered to the receiving organism in any medically effective manner, including enteral, parenteral, topical, transmucosal, intramuscular, intravenous, and inhalation delivery methods.

EXAMPLES

Example 1 : Screening Assay

Expression and Purification of FMDV RdRp (3Dpol) Materials: The Maybridge- Hitfinder chemical library of compounds (version 6) was purchased from Maybridge, (Thermo Fisher Scientific, Cornwall, United Kingdom). Screening reactions were carried out in Microfluor 2 black U-bottom 96-well plates (Fisher Scientific, Pittsburg, PA). Oligonucleotides were purchased from Fermentas, (Glen Burnie, Maryland). Ultrapure nucleotides were purchased from Novagen (Madison, Wisconsin) and 3'-deoxy 5-methyl- uridine 5 '-triphosphate (DMUT) was obtained from TriLink BioTech (San Diego, CA).

Expression and Purification of F DV RdRp (3Dpol): Plasmid pET-28a containing the FMDV 3Dpol coding sequence with an AAALE linker followed by 6 histidines at the carboxyl terminus was obtained from Drs. Nuria Venaguer (Institut de Biologia Molecular de Barcelona) and Esteban Domingo (Centra de Biologia Molecular "Severe Ochoa", Madrid). It was transformed into the Rosetta 2 expression strain (Novagen, Madison, Wisconsin). Kanamycin-resistant colonies were grown at 37°C and induced at A₆oo of 0.9-1.0 by the addition of 1 mM isopropyl β-D-l-thiogalactopyranoside (IPTG). Bacteria were pelleted by centrifugation (4500 g, 20 min) and stored at -20°. Frozen cell pellets were resuspended in Buffer A (25 mM Tris-HCl pH 8, 500 mM NaCl and 5% glycerol). The protein was purified by nickel- affinity chromatography with a gradient of a buffer containing 25 mM-500 mM imidazole. Fractions containing pure protein (-95%) were pooled and dialyzed against the storage buffer containing 12.5 mM Tris-HCl pH 8, 100 mM NaCl and 50% glycerol. The protein concentration was determined by Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) and coomassie brilliant blue staining against standard Bovine Serum Albumin solution. The FMDV 3Dpol used in all polymerization assays was more than 95% pure.

Screening Assay Materials: Maybridge-Hitfinder library compounds were supplied in the form of lyophilized films in a 96-well format. A Precision Microplate Pipetting system (Winooski, VT) was used to suspend the lyophilized films in dimethyl sulfoxide (DMSO) to a final concentration of 10 mM ("mother plates"). From these plates, several sets of "daughter" plates containing 500 μΜ of each compound (in 100% DMSO) were generated and stored at 4°C.

Screening Assay: For the screening assay, reaction components were added into wells of 96-well plates in the following order: 10 μΐ_^ of a master mixture consisting of 4.3 μΜ FMDV RdRp, in 62.5 mM Tris-HCl pH 7.5, 62.5 mM NaCl, 5 mM MnCl₂, and 25 μΜ UTP; 1 μΐ, of 500 μΜ inhibitor solutions (final concentration of 20 μΜ in 4% DMSO). After a 10 minute pre-incubation period at room temperature, reactions were initiated by the addition of 14 μL· 400 nM of poly-rA/oligo-dT₁₈ (40 nM final reaction concentration), incubated at 37°C for 1 hour, and then cooled on ice for 10 minutes. The polymerase reaction volume was 25 μΐ_^. The released pyrophosphate (PP from the above mentioned polymerase reaction was subsequently quantitated by adding an equal volume (25 μί) of reaction mix containing 1 nM luciferase and 0.06 units^L adenosine 5 '-triphosphate sulfurylase (Sigma Aldrich, St. Louis MO), 5 μΜ adenosine-5'-phosphosulfate, 310 μΜ d- luciferin, 0.5 mM coenzyme-A, 25 mM Tris pH7.5, and 50 mM NaCl (final concentrations of all components). Luminescence was measured immediately with a Veritas microplate luminometer (Turner BioSystems Sunnyvale, CA).

Example 2: Primer Extension Gel-Based Assay for Validation of FMDV Inhibitors.

Materials and Methods: Compounds from the screening of the library were validated by determining their ability to inhibit the RNA-dependent RNA polymerase (RdRp) activity of 3Dpol by a gel-based primer extension assay. Conditions for the primer extension were optimized by varying the concentration of 3Dpol, type and concentration of substrates, buffer, divalent metal, and temperature. The final conditions were: 30 minute incubations (37° C) of reaction mixtures containing 25 mM Tris-HCl, pH 7.8, 25 mM KC1, 1.7 μΜ 3Dpol, 10 μΜ UTP, 1 mM MnCl₂ and 20 μΜ inhibitor. The polymerase reaction was initiated by the addition of 40 nM of poly-rA/dT₁₈. For calculation of IC₅₀S varying amounts of inhibitors were used (0.25-25 μΜ). The inhibition of RNA synthesis was monitored by resolving the primer extension products on 16% polyacrylamide 8 M urea gels followed by scanning of gels on a Fuji FLA-5000 Fluorometer. Bands corresponding to full extension products were quantified using FUJIFILM MultiGauge (Stamford, CT) and IC₅₀S were obtained using GraphPad Prism 4.

Development of High-Throughput Assay for the Detection of Inhibitors of FMDV 3Dpol: PP; released from the polymerase reaction is converted to ATP in a reaction that uses adenosine 5 '-phospho sulfate and is catalyzed by ATP sulfurylase. The ATP product provides the energy for the luciferase-catalyzed conversion of D-luciferin to oxyluciferin, with a concomitant release of photons, according to the reaction Scheme I.

Scheme 1

{RNA)_n + NTP 3Dpol >{RNA)_n+1 + PP,

APS + PP ATP, » ATP + sulfate

Luciferin + ATP + 0₂ Luciferase » Oxyluciferin + AMP + PP_t + C0₂

To demonstrate that the produced luminescence is in direct proportion to the amount of PP; present, 1-100 μΜ PP; was exogenously added to luciferase, ATP sulfurylase and 100 μΜ adenosine 5 '-phospho sulfate (APS) in the absence of RNA and 3Dpol. Luminescence measured within a spectral response range of 350nm to 650nm was directly proportional to PPi concentration, as illustrated in Scheme I.

To improve the cost-efficiency of this assay, reagent concentrations of the secondary

(light-producing) reaction were decreased, while ensuring that they did not become rate- limiting for the processing of the PP; product of the primary (polymerase) reaction. Hence, the concentration of ATP sulfurylase was reduced from 0.3 Units (5) to 0.03 milliUnits, APS to 20 μΜ and the reaction volume was also decreased four-fold to 25μL·. These changes did not affect the luminescence signal significantly. To validate the assay, decrease in luminescence was monitored when in presence of a positive control, 3'-deoxy-5-methyl uridine-5' -triphosphate (DMUT), which is a ribonucleoside analog that blocks RNA synthesis because it lacks a 3' OH group. FIG. 6A shows that increasing amounts of DMUT suppress the production of light. The results from dose response experiments were plotted using Prism 4 (GraphPad Software Inc., CA) and an IC₅₀ value of 0.68 μΜ was obtained for DMUT at midpoint concentrations. The effect of DMUT on light produced by 1 μΜ of exogenously added PPj, which was incubated with varying concentrations of the inhibitor, was also evaluated to ensure that DMUT did not interfere with the ATP sulfurylase and lucif erase reactions. DMUT was found to have no effect on the production of light.

FMDV Inhibitory Compound Screening: Using the above-described high- throughput assay for the detection of inhibitors of FMDV 3Dpol, -2,000 compounds from the Maybridge HitFinder library were evaluated. Representative inhibition data from a single 96-well plate are shown in FIG. 2A. Typically, two to three compounds per plate suppressed FMDV 3Dpol activity by -90% (FIG. 2A). The cumulative inhibition data are summarized in FIG. 2B. Based on these results, 30 compounds that suppressed luminescence ^ 88 (first six bars in FIG. 2B) were selected. Notably, the relatively large frequency of hits (up to 3%) was due to the presence of compounds that inhibited the enzymatic activity of luciferase and/or ATP sulfurylase and consequently appeared as false positives. Therefore, to exclude false positives the compounds' ability to specifically suppress 3Dpol extension was evaluated with fluorescently labeled primer-extension assays. Twenty compounds were found not to inhibit the RNA synthesis of 3Dpol using gel-based assays and instead were shown to interfere with the secondary luciferase assay reactions. Example 3: Assessment Of Specificity Of FMDV Inhibitory Compounds:

Antiviral inhibitors are expected to suppress viral replication by selectively inhibiting the target enzyme without affecting the activity of other related enzymes, such as, for example, nucleic acid polymerases. Therefore, the specificity of the FMDV 3Dpol inhibitory compounds towards FMDV 3Dpol was tested by evaluating their ability to inhibit DNA synthesis by other polymerases: a viral RNA dependent DNA polymerase (HIV reverse transcriptase; RT) and a bacterial DNA dependent DNA polymerase (Klenow fragment of E.coli DNA pol I). The results illustrated in FIG. 7 show that under these conditions the compounds did not affect the ability of HIV RT to extend fluorescently labeled primers. Similar results were obtained with the bacterial enzyme.

Example 4: Assessment Of Cytotoxicity: Materials and Methods: Cytotoxicity studies were performed with a colorimetric

XTT tetrazolium salt {2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide} assay kit (Roche Diagnostics, Indianapolis, IN). Succinate dehydrogenase, which retains activity in metabolically active cells, reduces XTT to a soluble orange-colored formazan product. The amount of this product is proportional to the number of living cells and can be spectrophotometrically quantified. The assay was performed in triplicate as follows: Baby hamster kidney (BHK-21) cells were seeded to 90% confluency in a 96-well plate. Cells were incubated for 24 hours with 1, 5, 10, and 20 μΜ of inhibitors in a final volume of 100 μL· After the 24-hour incubation period media was removed and replaced with phenol red-free media. 20 μΐ_^ of XTT was added per well and cells were incubated in the presence of XTT for an additional 3.5 hours. Cells treated with Triton were used a control for no viability. After incubation, cells were titrated and absorption was read at A450 by a plate reader. Cytotoxicity of the compounds was also evaluated using the cyto-glo kit (Promega) as per manufacturer's instructions.

Assessment of Cytotoxicity: Inhibitors were initially evaluated for their effect on cell viability by the MTT cytotoxicity assay and confirmed using the XTT cytotoxicity and cyto- glo assays. Uninfected cells were incubated in the presence of various doses of compounds (1, 5, 10, 265 and 20 μΜ) for 24 and 48 hours. Little to no toxicity was demonstrated for these compounds at the indicated concentrations. Cytotoxicity at higher concentrations of the compounds was also evaluated using the cyto-glo assay (1, 10, and 100 μΜ of inhibitors) as described by the manufacturer (Promega, Madison, WI). Using this assay, it is estimated that more than 90% of the cells were viable after 1 hour exposure to even the highest concentrations of the inhibitors.

Example 5: In vivo assessment of inhibition: The ability of the FMDV 3Dpol inhibitory compounds to inhibit viral replication was assessed through evaluation of BHK-21 cells post-infection with FMDV. The infected cells were incubated with various inhibitor concentrations of and for different times. The effect on virus growth was assessed by plaque assay (FIG. 8A and 8B). Inhibition of viral replication was observed to be dose-dependent both at the 6- and at the 24-hour time-points. To further investigate potential antiviral effects of FMDV 3Dpol inhibitory compound 5D9 (N' l- thieno[2,3-d]pyrimidin-4-yl-4-chloro-l-benzenesulfonohydrazide), BHK-21 cells were pretreated with the FMDV 3Dpol inhibitory compound prior to infection with FMDV A24 Cruzeiro. When the BHK-21 cells were pre-treated with 5D9 and subsequently challenged with FMDV, viral growth was suppressed in a dose dependent manner and by as much as 90% at the 20 μΜ concentration after 24 hours of viral growth. Notably, 5D9 did not exhibit significant cytotoxicity at these concentrations in parallel assays using the XTT or MTT methods. The results demonstrate a dose-dependent inhibition of virus replication as determined by plaque assay in the presence of 5D9. At the highest concentrations of the compound, 20 μΜ, there was a greater than 90% inhibition of virus replication. No decrease in cell viability was observed at these concentrations.

Example 6: Molecular Modeling: Materials and Methods:

The crystal structure coordinates of the FMDV 3Dpol complex with RNA, UTP and PPi (PDB file 2E9Z) were used to search for potential binding sites of the inhibitors, using the Q-siteFinder program (Laurie & Jackson, 2005). The potential inhibitor binding sites were initially evaluated by size (if they were large enough to allow docking of the inhibitors). For those sites that were of the right size additional docking studies of the various inhibitors were performed. For this purpose molecular models of the compounds were generated based on structure data files (sdf) using LigPrep, a ligand preparation tool that is interfaced with Maestro (Schrodinger Inc. NY). The structures generated by LigPrep were docked into the ternary complex of FMDV 3Dpol with RNA and UTP (PDB file 2E9Z) using the software 'Glide' with extra precision (XP) and 'Induced Fit Docking' workflow incorporated in Maestro (Schrodinger Inc. NY).

Molecular Modeling: Q-siteFinder identified 10 possible ligand-binding pockets. Almost all of these were smaller in size than the 3Dpol inhibitors, or were located within shallow surface crevices, very distant from the polymerase site. However, one potential ligand-binding pocket was in close proximity to the 3Dpol active site (FIG. 1A). Docking of all seven compounds at this pocket was favorable and with significant glide scores. Interestingly, the binding pocket is pre-existing, and docking of the molecules required only small adjustments to the side chains of protein residues. Hence, using the 'induced fit docking' protocol, which permits more structural changes during the docking process, resulted in negligible changes in the binding mode of the inhibitors.

The inhibitor-binding pocket is proximal to, but not overlapping with the NTP binding site. A close-up of the inhibitor-binding pocket with respect to the UTP-binding site and the possible NTP entry channel. The pocket is formed by residues V55, 156, S58, K59, R168, G176, K177, T178, R179 and 1180. The energetically most favorable binding conformers of compounds 5D9 and 1A8 were also docked in FMDV 3Dpol (FIG. 5A and FIG. 5B). The seven 3Dpol-inhibitors docked at the inhibitor-binding site (FIG. 5C). The inhibitors appear to interact with residues of the binding pocket through both hydrophobic and electrostatic interactions. For example, the ε-ΝΗ₂ group of K59 is shown to form hydrogen-bonds with the 5D9 and 1A8 inhibitors (FIG. 5A and FIG. 5B). Similarly, residues K177 and R168 of 3Dpol are also within interacting distance with the inhibitors.

While the invention has been described in connection with specific embodiments thereof, it will be understood that the inventive methodology is capable of further modifications. This patent application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features herein before set forth and as follows in scope of the appended claims.

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Claims

Claim 1. A method of inhibiting the replication of Foot and Mouth Disease Virus (FMDV) comprising the step of directly targeting the binding pocket of 3Dpol.

Claim 2. The method of Claim 1, wherein the binding pocket comprises residues V55, 156, S58, K59, R168, G176, K177, T178, R179 and 1180.

Claim 3. The method of Claim 1, further comprising the step of providing an antiviral compound capable of binding at least one residue within the binding pocket of 3Dpol.

Claim 4. The method of claim 3, wherein the antiviral compound is selected from the group consisting of: a) a compound comprising a phenyl ring unit and a benzenesulfonyl with a linker that is based on hydrazide and sulfone moieties having the formula (I)

(I)

wherein R may be selected from the group consisting of halo, alkylcarbonyl, and secondary or tertiary alcohol groups;

a compound comprising a phenyl unit and a nitro-benzoxadiazole unit having the formula (II):

wherein R' and R" may be independently selected from the group consisting of halo, hydroxyl, amino, carboxyl, secondary or tertiary alcohol, and alkylcarbonyl groups and wherein the X bridging atom may be either a Sulfur or Oxygen; and a compound comprising a benzothiophene-dioxide unit having the formula

(III) wherein R may be selected from the group consisting of halo, amino, or hydroxyl groups; Y may either be an oxygen or a sulfur atom; and Z may be either oxygen, sulfur, or nitrogen.

Claim 5. The method of claim 3, wherein the antiviral compound is selected from the group consisting of 4-chloro-N' -thieno[2,3-d]pyrimidin-4-ylbenzenesulfonohydrazide, 4- (2,4-dichlorophenyl)sulfanyl-7-nitro-2, 1 ,3-benzoxadiazole and 5-chloro-3-(thiophen-2- ylsulfanylmethyl)- 1 -benzothiophene 1 , 1 -dioxide.

Claim 6. A method of treating an animal comprising administering an antiviral compound capable of binding at least one residue within the binding pocket of 3Dpol.

Claim 7. The method of Claim 6, wherein the binding pocket comprises residues V55,

156, S58, K59, R168, G176, K177, T178, R179 and 1180.

Claim 8. The method of claim 6, wherein the animal is infected with FMDV.

Claim 9. The method of claim 6, wherein the animal is not infected with FMDV.

Claim 10. The method of claim 6, wherein the animal is selected from the group consisting of cow, bison, water buffalo, sheep, goat, pig, deer, hedgehog, elephant, llama, and alpaca.

Claim 11. A compound for inhibiting the replication of Foot and Mouth Disease Virus (FMDV) comprising a phenyl ring unit and a benzenesulfonyl with a linker that is based on hydrazide and sulfone moieties having the formula (I)

(I)

wherein R may be selected from the group consisting of halo, alkylcarbonyl, and secondary or tertiary alcohol groups.

Claim 12. The compound of Claim 11 comprising 4-chloro-N'-thieno[2,3-d]pyrimidin- 4-ylbenzenesulfonohydrazide.

Claim 13. A compound for inhibiting the replication of Foot and Mouth Disease Virus (FMDV) comprising a phenyl unit and a nitro-benzoxadiazole unit having the formula (II):

wherein R' and R" may be independently selected from the group consisting of halo, hydroxyl, amino, carboxyl, secondary or tertiary alcohol, and alkylcarbonyl groups and wherein the X bridging atom may be either a Sulfur or Oxygen.

Claim 14. The compound of Claim 13 comprising 4-(2,4-dichlorophenyl)sulfanyl-7- nitro-2, 1 ,3-benzoxadiazole.

Claim 15. A compound for inhibiting the replication of Foot and Mouth Disease Virus (FMDV) comprising a benzothiophene-dioxide unit having the formula (III):

Claim 16. The compound of Claim 15 comprising 5-chloro-3-(thiophen-2- ylsulfanylmethyl)- 1 -benzothiophene 1 , 1 -dioxide.