Methods And Compositions For Treating Viral Diseases Using Combination Of Drugs

Abstract

The present invention describes methods and compositions for treating viral diseases through novel combinational therapy of one or more anti-viral and anti-kinase drugs. The composition and method of the present invention are aimed at targeting a NiRAN domain of RNA dependent RNA polymerase (RdRp) as RdRp is key enzyme for virus replication of the viral pathogens. The compositions of the present invention comprise one or more kinase inhibitors in combination with one or more anti-viral agents. The combination therapy of these agents was found to exhibit synergistic effecta in inhibiting viral activity against several lethal viruses including but not limited to SARS-CoV-2.

Claims

1. A method of inhibiting viral multiplication and treating a viral disease comprising: co-administering, to a subject, a therapeutically effective amount of one or more anti-viral drugs and a therapeutically effective amount of at least one or more anti-kinase drugs, wherein the method inhibits kinase activity of a NiRAN domain of RNA dependent RNA polymerase of a target virus.

2. The method according to claim 1, wherein the one or more anti-viral drugs is selected from Sinefungin, Ribavirin 5′-triphosphate, m7GTP, Nordihydroguaiaretic acid, Remdesivir and 6-MADTP.

3. The method according to claim 1, wherein the one or more anti-kinase drugs is selected from Sunitinib, Sorafenib and SU6656.

4. The method according to claim 1, wherein the one or more anti-viral drugs and the one or more anti-kinase drugs are administered sequentially or simultaneously in combination.

5. The method according to claim 1, wherein the target virus is selected from a family of nidoviruses and any other virus having the NiRAN domain.

6. The method according to claim 1, wherein the target virus is SARS-CoV-2 and the viral disease is COVID-19.

7. The method according to claim 1, wherein the subject is a mammal.

8. The method according to claim 7, wherein the mammal is a human.

9. A composition comprising: a therapeutically effective amount of at least one or more anti-viral drugs selected from Sinefungin, Ribavirin 5′-triphosphate, m7GTP, Nordihydroguaiaretic acid, Remdesivir and 6-MADTP; and a therapeutically effective amount of at least one or more anti-kinase drugs selected from Sunitinib, Sorafenib and SU6656, wherein the one or more anti-kinase drugs augments an antiviral efficacy of the one or more anti-viral drugs.

10. A system comprising: a therapeutically effective amount of at least one or more anti-viral drugs including Sinefungin, Ribavirin 5′-triphosphate, m7GTP, Nordihydroguaiaretic acid, Remdesivir and 6-MADTP; a therapeutically effective amount of at least one or more anti-kinase drugs including Sunitinib, Sorafenib and SU6656; and a target virus.

11. The system according to claim 10, wherein the target vines is selected from a family of nidoviruses and any other virus having a NiRAN domain.

12. The system according to claim 10, wherein the target virus is SARS-CoV-2.

13. The system of claim 10, wherein the target virus resides in a mammal.

14. The system according to claim 13, wherein the mammal is a human.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0023] The foregoing has broadly outlined the features and technical advantages of the present disclosure in order that the detailed description of the disclosure that follows may be better understood. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present disclosure.

[0024] The novel features which are believed to be characteristic of the disclosure, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present disclosure.

ABBREVIATIONS USED

[0025] SARS-CoV-2: Severe Acute Respiratory Syndrome Coronavirus-2 [0026] RdRp:RNA dependent RNA polymerase [0027] NiRAN: Nidovirus RdRp associated nucleotidyltransferase domain [0028] COVID-19: Coronavirus Disease 2019 [0029] EAV: Equine Arterivirus [0030] ATP: Adenosine triphosphate [0031] ADP: Adenosine diphosphate [0032] GTP: Guanosine triphosphate [0033] UTP: Uridine triphosphate

[0034] The present invention provides methods and compositions for treating viral diseases through combinational therapy of one or more anti-viral and anti-kinase drugs or a pharmaceutically acceptable derivative thereof The methods and compositions of the present invention are aimed at targeting NiRAN domain of RNA dependent RNA polymerase (RdRp) as RdRp is key enzyme for virus replication of the viral pathogens. The compositions of the present invention comprise one or more kinase inhibitors in combination with one or more anti-viral agents.

EXAMPLES

[0035] 1.1 Structural Identification of the NiRAN domain of RNA dependent RNA polymerase (RdRp) and its kinase like activity

[0036] The NiRAN and the interface domains span over residues 1-365 of the SARS-CoV-2 RdRp polypeptide sequence. These two domains form an arrow-head like structure, which acts as a base for the RdRp region of protein (FIG. 1A).). The overall topology suggests that the two distinct domains are connected by a small linker of 1 residue (FIG. 1B). A multiple sequence alignment of 17 RdRp polypeptide sequences from coronaviruses across multiple host species (human, bat, cow, pigs and rodents) revealed an absolute conservation of the residues- K73, R116, T123, D126, D218 and F219. A visualization of the conserved residues of the NiRAN domain shows that a significant majority of these residues are localized between the 4 stranded β-sheet and the following helix bundle (FIG. 1B). The understanding of the overall topology, localization of the conserved residues prompted an investigation on understanding the enzymatic function of the NiRAN domain. For this, two different approaches were considered with the Protein data bank as the target database. The first approach utilized the prediction of Hidden Markov Models(16) while the second approach predicted the presence of similar folds(17).Notably, both approaches predicted a variety of kinase and kinase like transferase (phosphotransferase molecules). A previous report had evidenced at the similarity of the

[0037] NiRAN domain to that of a known pseudokinase molecule SelO(5); however further characterization was not feasible due to absence of the NiRAN 3D structure. The inventors of the present invention compared the topologies of the NiRAN domain with that of the canonical kinase fold. Similar to the canonical kinase fold, the NiRAN domain comprises of an antiparallel β-sheet flanked by alpha helices. The canonical kinase domain exhibits a 5 stranded antiparallel β-sheet, which is flanked by two helices running parallel and one helix running perpendicular to the β-sheet (FIG. 1C, middle panel) (18, 19). However, the NiRAN domain shows a 4 stranded antiparallel β-sheet (S1-S4) flanked by one parallel (H2) and two perpendicular helices (H1 and H3) (FIG. IC, left panel). Further investigation into the available literature confirmed the presence of many non-canonical kinase folds, one of which presents a 4 stranded antiparallel β-sheet flanked by three parallel and one perpendicular helix (FIG. 1C, left panel) (20).Taken together, these results suggest that the NiRAN domain assumes a kinase like fold, possibly functioning either as a kinase or a phosphotransferase.

[0038] The earlier study with EAV-RdRp experimentally demonstrated the binding of GTP and UTP nucleotides to the NiRAN domain (8). A docking analysis of these nucleotides with the NiRAN domain was performed to delineate the probable active site. Kinase domains in general possess an active site between the antiparallel β-sheet and the subsequent helix bundle (18, 21, 22). Both GIP (FIG. 2A& A′) and UTP (FIG. 2 B & B′) docked well within the probable active site region with docking scores of -9.84 kcal/mol and -6.59 kcal/mol, respectively. Besides, end-point binding free energy calculation with MM/GBSA suggest a strong interaction between the nucleotides and the residues in the probable active site (-15.77 kcal/mol and -17.67 kcal/mol for GTP and UTP, respectively). An overview of the active site pocket depicting the electrostatic surface potential indicates the localization of charged residues at the entry points to the site, while the interior of the pocket remains lined with largely non-polar residues (FIG. 2C).

[0039] 1.2 Analysis of the kinase activity of NiRAN domain

[0040] In order to further explore the kinase like catalytic nature of NiRAN domain, three broad specificity kinase inhibitors-Sunitinib, Sorafenib and SU6656 were randomly selected and docked into the predicted active site of the NiRAN domain. All three kinase inhibitors show strong binding at the predicted active site as evident from the low free energy of binding (FIG. 3A, A′, B, B′, C and C′, Table 1). Interestingly, these inhibitors also demonstrate potential H-bond with residues lining the active site.

[0041] As mentioned earlier, this domain might be involved in GTP induced protein phosphorylation, thus enabling a primer independent RNA replication (G-capping) (8). Also, the domain may be involved in phosphorylation of UTP, thus functioning as a terminal nucleotidyltransferase. A wide range of viruses, both with DNA and RNA genomes are known to possess either multifunctional or dedicated proteins exhibiting the aforementioned activities. After careful examination, a list of 77 compounds with experimentally demonstrated inhibitory potential against members of Flaviviridae, Togaviridae; Human cytomegalovirus, Herpes simplex virus, and few gram negative bacterial species were selected for docking against the CoV-2-RdRp NiRAN domain active site (23-30).The five inhibitors with the best docking in scores in a decreasing order are- 65482,122108,135659024,4534, and23673624 (numbers represent the PubChem IDs). All these five inhibitors occupy varying regions within the active site pocket in a manner that their aromatic rings align with the uncharged/non-polar regions, while the charged moieties fit in the extremities of the active site pocket (FIG. 4A-E, Table 2).The molecular interactions of these inhibitors with the CoV-2-RdRp NiRAN domain active site are presented in FIG. 5A′-E′.

[0042] In order to determine any putative kinase like activity being harboured by the CoV-2 RdRp, the protein was overexpressed, purified (FIG. 5A & B) and its identity was confirmed by mass spectrometry (FIG. 5C). An ADP-Glo Kinase assay kit was utilized to determine the kinase activity of the CoV-2 RdRp. The efficiency of the assay in accurately determining a kinase activity was verified using a known kinase—human Akt2. The enzyme exhibited significant activity with a Km value of˜300 μM for ATP (FIG. 6A), which is very similar to the K.sub.m values (˜ 350 μM) determined in previously reported studies. Also, Bovine Serum Albumin, a protein that binds ATP exhibited negligible activity (FIG. 6A), further verifying the specificity and selectivity of the protocol. Notably, incubation of the CoV-2 RdRp with varying concentrations of ATP exhibited significant activity akin to that of the human Akt2, with a K.sub.m of˜500 μM (FIG. 5A).

[0043] 1.3 Confirming kinase activity of RdRp

[0044] To further ascertain the possible kinase like activity of CoV-2 RdRp, both CoV-2 RdRp and human Akt2 were incubated with excess of ATP and treated with 500 nM of the each of the three kinase inhibitors- Sorafenib, Sunitinib and SU6656. Interestingly, all the three kinases inhibitors significantly abrogated the kinase like activity of CoV-2 RdRp (FIG. 6B). For human Akt2, Sorafenib and SU6656 significantly inhibited its kinase activity, while Sunitinib treatment demonstrated a mild inhibition (FIG. 6B). The inventors were unable to procure the compound 23673624(6-MADTP) commercially, thus the four remaining compounds-135659024(m7GTP), 122108(Ribavirin 5′-triphosphate), 65482(Sinefungin) and 4534(Nordihydroguaiaretic acid) were used. The first three compounds were observed to have very little effects on the kinase like activity of CoV-2 RdRp at a concentration of 500 nM (FIG. 6C). However, these compounds which are nucleoside analogs/derivatives exhibited conspicuous inhibitions at a concentration of 1000 nM (FIG. 6C). The fourth compound-4534, a catechol derivative failed to inhibit the CoV-2 RdRp kinase like activity at any of the two concentrations (FIG. 6C).

[0045] 2. Ex-vivo studies and results

[0046] To assess the effect of the kinase inhibitors in an ex-vivo infection, epithelial cells infected with SARS-CoV-2 were treated with concentrations of these compounds ranging from 1-50 μM. The approved anti-COVID-19 drug, Remdesivir was used as the control. The anti-kinase drug Sorafenib exhibited significant reduction in the infection, similar to that of Remdesivir (FIG. 6D). Further, a combination of Sorafenib and Remdesivir in variable concentration has synergistic effect on the controlling the infection by clearing the virus completely (FIG. 7).

TABLE-US-00001 TABLE 1 Docking analysis of kinase inhibitors at the proposed active site of NiRAN domain. Docking Binding PubChem Score Energy H-bond No. Name ID (Kcal/Mol) (ΔG bind) Interactions 1 Sunitinib 5329102 −3.50 −28.07 Asp36, Asp218 and Asp221 2 Sorafenib 216239 −3.06 −36.06 Asp36 3 SU6656 5312137 −2.61 −25.07 Lys73

TABLE-US-00002 TABLE 2 Docking analysis of best 5 proposed inhibitors at the proposed active site of NiRAN domain. Docking Binding PubChem Score Energy H-bond Other No. ID (Kcal/Mol) (ΔG bind) Interactions Interactions 1 65482 −10.35 −25.38 Asp36, Phe35 (Sinefungin) Asn52, (Pi-Pi), Lys73, Phe48 Asp218, (Pi-Pi), Asp221 Glu83 (Salt Bridge) 2 122108 −7.42 −10.03 Lys73, Lys73 (Ribavirin 5′- Asp36, (Salt Bridge) triphosphate) Asn52, Asp218, Asp221 3 135659024 −6.94 −24.33 Asn79, Lys73 (m7GTP) Thr76, (Salt bridge), Arg74, Asp218 Asn52, (Salt bridge) Val204, Asn209, 4 4534 −6.84 −32.07 Lys73, NA (Nordihydroguaiaretic Asn52, acid) Asp218 5 23673624 −6.76 −34.18 Arg33, Lys73 (6-MADTP) Thr51, (Salt bridge), Asn52, Arg116 (Salt Bridge)

[0047] Using computational docking and simulation, the present invention predicts possible inhibitor compounds against the NiRAN domain and provides compositions and methods employing use of such inhibitors for augmenting anti-viral action against various viral pathogens possessing NiRAN domain.

[0048] The compound Sunitinib is an inhibitor of the tyrosine family kinases (34); Sorafenib inhibits activities of both serine/threonine and tyrosine family kinases (35, 36); while SU6656 inhibits the Src family kinases (36, 37). While Sorafenib and Sunitinib are approved for medical use in cases of renal, hepatocellular and gastro-intestinal cancers, the compound SU6656 is an experimental molecule used to study the role of Src kinases in cell cycle (38, 39).

[0049] In addition to the kinase inhibitors, the compound 65482 (Sinefungin), is broad specificity microbial nucleotidyitransferase inhibitor, and is known to inhibit RNA replication in flaviviruses and herpes viruses (40, 41). The compound 122108 (Ribavirin 5′-triphosphate), inhibits the formation of g-capping of RNA in a wide range of viruses, such as Dengue virus, Hantaan virus and Hepatitis C virus (11, 42, 43). Officially known as m7GTP, the compound with the PubChem Id 135659024, interferes with the RNA/DNA g-capping activity in many viral pathogens such Rift valley fever virus, Influenza virus, Zika virus and Dengue virus, to name a few (44-46).

[0050] The results suggest that the NiRAN domain of the SARS-CoV-2 RdRp possesses a kinase/phosphotransferase like enzymatic activity which is inhibited significantly by anti-cancer drugs and various anti-microbials targeting the NiRAN active site. In addition, the anti-kinase drug Sorafenib significantly reduces viral load in an ex-vivo SARS-CoV-2 infection in cell line infection.