GB2527364A - Treatment - Google Patents

Abstract

The present invention provides a protein kinase D (PKD) inhibitor for use in a method of treating or preventing picomavirus infection such as a rhinovirus, polio virus, foot and mouth disease virus, hepatitis A virus, coxsackievirus or an enterovirus. Any PKD inhibitor may be use in the present invention, but the PKD inhibitor is typically selected from the group consisting of a small molecule, a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), an antisense RNA, a nbozyme, a DNAzyme and a cDNA encoding an inactive mutant of PKD. The examples are particularly directed to the use of the compounds CRT0066101 and CID2011756 in the prevention of of replication of human rhinovirus, specifically HRV16.

Description

Intellectual Property Office Application No. GB1411027.4 RTTVI Date:3 March 2015 The following terms are registered trade marks and should be read as such wherever they occur in this document: Tween Intellectual Property Office is an operating name of the Patent Office www.gov.uk/ipo

TREATMENT

Field of the Invention

The present invention relates to the use of protein kinase D (PKD) inhibitors in the treatment or prevention of picornavirus infection, for example for the treatment or prevention of human rhinovirus (HRV) infection.

Background to the Invention

Viral respiratory tract infections represent a major healthcare burden with an estimated 500 million cases and a cost of $40 billion per year in the USA alone. It has been suspected since the 1970s that respiratory viral infections are a major trigger for asthma exacerbations in children and adults and with the development of more sensitive and specific diagnostics it is now clear that around 80% of wheezing episodes in school aged children and between half to three quarters of wheezing in adults can be attributed to respiratory viral infections. Although there are a number of viruses implicated, human rhinoviruses (HRV5) are the most frequently detected pathogens and are found in -65% of these cases. HRV infection is also known to be a major contributory factor in exacerbations of chronic obstructive pulmonary disease (COPD). However, anti-viral therapies and vaccines for HRV have failed to date, largely because of the wide number of viral serotypes and the ability of the virus to rapidly mutate, thus leading to drug resistance.

HRV5 are members of the positive-sense single-strand RNA Picornaviridae family for which the best studied prototype is poliovirus (PV) and which includes other important pathogens such as foot and mouth disease virus (FMDV), hepatitis A and coxsackievirus (CV). Upon entering a host cell, the RNA genome of these viruses is translated into a polyprotein that is post-translationally cleaved by the encoded viral proteases to generate the structural and non-structural proteins required for viral replication. The developing virus forms replication complexes on the surface of intracellular membranes, believed to be derived either from the Endoplasmic Reticulum (ER)/Golgi secretory apparatus or from autophagosomes, and greatly remodels their morphology and lipid composition (Richards AL, Jackson WT. 2013 Behind closed membranes: the secret lives of picornaviruses? PL0S Pathog. 9: e1003262). The viral proteins 2B, 2C, 3A and the intermediates 2BC and 3AB have been shown to be membrane associated and these membranous replication complexes have been intensely investigated by microscopy, molecular and biochemical studies (Belov GA, Ehrenfeld E. 2007 Involvement of Cellular Membrane Traffic Proteins in Poliovirus Replication, Cell Cycle 6: 36-38), although their exact origins remain unclear.

Summary of the Invention

The inventor has identified a novel method of inhibiting viral replication by inhibiting a host cell process which is crucial to replication of the virus, thereby providing a novel anti-viral therapy.

Accordingly, in a first aspect, the present invention provides a protein kinase D (PKD) inhibitor for use in a method of treating or preventing picornavirus infection.

Detailed Description of the Invention

Protein Kinase D (PKD, also referred to herein as PRKD) is part of the calmodulin-dependent kinase family. PKD is related to the PKC family and is directly activated by Diacylglycerol (DAG) and PKC. There are three isoforms of PKD in mammals: PKD1, PKD2 and PKD3. The three mammalian isoforms of PKD share a conserved C-terminal kinase domain and N-terminal regulatory domains. PKD1 and PKD2 contain an N-terminal hydrophobic stretch of amino acids that could potentially insert into the membranes. The domain structure of PKD1, PKD2 and PKD3 is shown in Figure 1. Representative nucleic acid and amino acid sequences of human PKD1, PKD2 and PKD3 are shown in Figures 2, 3, 4, 5, 6 and 7 respectively.

Studies have shown that PKD is activated by PKCc by phosphorylation at Ser744 and Ser748 in the kinase activation loop which triggers autophosporylation at Ser916. PKD has been implicated in several biological functions which include the proliferation, apoptosis and invasion of cancer cells, which has made PKD an attractive target for cancer drug discovery. PKD also appears to play an important role in the architecture and function of the Golgi apparatus. However, the present invention is based on the novel finding that PKD inhibitors block picornavirus replication, in particular HRV replication.

A protein kinase D (PKD) inhibitor' as used herein is any compound or substance that inhibits the activity or expression of any one or more isoforms of PKD, to any degree. For example, a PKD inhibitor as defined herein inhibits the activity or expression of mammalian, typically human, PKD1, PKD2 and/or PKD3. A PKD inhibitor for use in the invention can completely inhibit the activity or expression of one or more isoforms of PKD.

Alternatively, a PKD inhibitor for use in the invention can reduce the activity or expression of PKD by more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98% or 99%, for example in comparison to a control cell, tissue or subject which has not been exposed to a PKD inhibitor. Assays for identifying and testing PKD inhibitors for use in the invention are described in detail herein.

Any protein kinase D (PKD) inhibitor can be used in the present invention. PKD inhibitors include, for example, small molecules, siRNAs, shRNAs, microRNAs, antisense SNAs, ribozymes, DNAzymes and cDNA5 encoding an inactive mutant of FKD, which will now be described in detail.

A "small molecule PKD inhibitor" is an organic compound, whether naturally-occurring or artificially created (e.g., via chemical synthesis) that has relatively low molecular weight and that is not a protein, polypeptide, or nucleic acid. Small molecules typically have multiple carbon-carbon bonds, as well as other bonds. Small molecules may have a molecular weight of less than about 2000 g/mol. In some embodiments, small molecules may have a molecular weight of less than about 1500 g/mol, less than about 1000 g/mol or less than about 500 g/mol, for example in the range of 300 g/mol to 1200 g/mol or in the range of 500 glmol to 1000 g/mol.

Small molecule PKD inhibitors include compounds as described in WO 2012/078859, which is incorporated herein by reference in its entirety. Such PKD inhibitors include compounds according to Formula I: yXiThR \\ Formula I or a pharmaceutically acceptable salt, stereoisomer, tautomer, or prodrug thereof, wherein: R1, R2 R3, and R4 are each independently selected from the group consisting of hydrogen, straight or branched chain (C1-C5)alkyl, (C2-C5)alkene, halogen, -OH, -OR', -OC(O)CH3, (C1-C5)alkoxy, -N3, -NR'R", isocyanate, isothiocyanate, straight or branched (C1-C6)haloalkyl and straight or branched (C1-C6)haloalkoxy; S5 is selected from the group consisting of hydrogen, a straight or branched chain (C1C6)alkyl, (C1-C5)alkylene-NH2, (C1-C5)alkoxy, and -C(O)-(C1-C6)alkyl; each X is independently a -CR'-, or-N-; Y is selected from the group consisting of -0-, -S-, -S(0) and _NRa; Z is selected from the group consisting of C(Rd)2, -0-, -3-and _NRb; and n is an integer from 0 to 3; wherein R', R", R, Rb and Rd are each independently selected from the group consisting of H, straight or branched (C1-C6)alkyl, (CC6)alkene, (C2-C6)alkenyloxy, halogen, -OH, -OC(0)CH3, C(0)CH3, C(0)CHhalide, straight or branched (C1-C5)haloalkyl, and benzyl.

Exemplary PKD inhibitors according to Formula I include: 7-hydroxy-2,3,4,5-tetrahydro-1 H-Ij'IEIIII'IIIL OH [1]benzofuro[2,3-c]azepin-1 -one 9-hydroxy-3,4-dihydro-2H-[1]-benzothiolo[2,3-f][1,4]thiazepin-5-one * . 9-hydroxy-1 0-iodo-3,4-dihydro-2H-[1]-benzothiolo[2,3-f][1,4]thiazepin-5-one 9-methoxy-3,4-dihydro-2H-[1]-benzothiolo[2,3-f][1,4]thiazepin-5-one 2,3,4,5-tetrahydro-lO-

S

HO) hydroxybenzo[b]thieno[2,3-f]-1,5-thiazocin-6-one 2,3,4,5-tetrahydro-lO-or a pharmaceutically acceptable salt, stereoisomer, tautomer, or prodrug thereof.

Other PKD inhibitors which are derivatives of compounds of Formula I are described in K. M. George etaL, Pharmaceutics, 2011,3, 186-228, which is incorporated herein by reference in its entirety. Such PKD inhibitors include: 0 7-hydroxy-3,4-dihydro-[1]benzoxolo[2,3-HO.%.%.....\NH c]azepine-1,5(2H)-dione PhHN 7-hydroxy-5-(2-phenylhydrazono)-2,3,4,5- tetrahydro-[1]benzoxolo[2,3-c]azepin-1 -one TsH 7-hydroxy-5-[2-{(4- HO \ methylphenyl)sulfonyl}hydiazono)-2,3,4,5- _%.__% NH tetrahydro-[1]benzoxolo[2,3-c]azepin-1-one 0% ,,, 3,4-dihydro-9-methoxy-1-oxide- S [1]benzothieno[2,3-tj-1,4-thiazepin-5(2H)-H one OH methyl 3-(3-hydroxypropylamino)-5-

HN

methoxybenzo[b]thiophene-2-carboxylate and pharmaceutically acceptable salts, stereoisomers, tautomers, or prodrugs thereof.

Another known FKD inhibitor is:

HO NH

9-hydroxy-1,2,3,4-tetrahydro-chromeno[3,4-b]pyridin-5-one. This compound is disclosed in K. M. George eta!., Pharmaceutics, 2011, 3, 186-228 and WO 2012/078859.

Small molecule PKD inhibitors also include compounds as described in WO 2008/074997, which is incorporated herein by reference in its entirety. Such PKD inhibitors include compounds according to Formula II: :xxç° RB4f' RB6 R B5 Formula II and pharmaceutically acceptable salts, solvates, chemically protected forms, and prodrugs thereof; wherein: X is independently C(RA3) or N; R is independently: -H or-NRR"M2; wherein: each R11 is independently -H or each R12 is independently -H or wherein: each R is independently C1.3alkyl or cyclopropyl; and wherein additionally, each -NRR2 may be azetidino, pyrrolidino, imidazolidino, N-(C13a1ky1)-imidazolidino, pyrazolidino, N-(C13a1ky1)-pyrazolidino, piperidino, N-(C13a1ky1)-piperidino, piperizino, morpholino, azepino, diazepino, or N-(C13a1ky1)-diazepino, each of which is optionally substituted with one or more C13a1ky1 groups; each of RAS, RAS, RB2, R, RB5, and RBS, if present, is independently selected from: -H, Rz2, -F, -Cl, -Br, -OH, ORz2, SRz2 -CF3, -OCF3, -CN, -NRR2, -C(=O)-N RNZI RNZ2, and -NR C(=O) Rz2; wherein: each RNZI is independently -H or each RNZ2 is independently -H or Rz2; each RNZ3 is independently -H or wherein: each RZ2 is independently C13a1ky1 or cyclopropyl; and wherein additionally each -N R RNZ2 may be azetidino, pyrrolidino, imidazolidino, N-(C13a1ky1)-imidazolidino, pyrazolidino, N-(C13a1ky1)-pyrazolidino, piperidino, N-(C13alky-piperidino, piperizino, morpholino, azepino, diazepino, or N-(013a1ky1)-diazepino, each of which is optionally substituted with one or more Cialkyl groups; 0 is independently -NH2, NRN RNQ2, or -W; wherein: RNQ1 is independently C14a1ky1; RN02 is independently -H or C14a1ky1; and additionally, NRrJdl RNQ2 may be azetidino, pyrrolidino, imidazolidino, N-(C1 3alkyl)-imidazolidino, pyrazolidino, N-(C13a1ky1)-pyrazolidino, piperidino, N-(C13a1ky1)-piperidino, piperizino, morpholino, azepino, diazepino, or N-(C13alky-diazepino, each of which is optionally substituted with one or more C13a1ky1 groups; W is the following group: 0C1A 0C2A 0C3A 0C4A n F F' RNW N" I I I I I NW3 R1 RC1B pC2B RC3B RC4B p q wherein: p is 0 and q is 0 or p is 1 and q is 0; or pislandqisl; RNWI is independently -H or C13a1ky1; each of RNW2 and RNWZ is independently -H or C14a1ky1; and additionally: -NRNWRN may be azetidino, pyrrolidino, imidazolidino, N-(C13alky-imidazolidino, pyrazolidino, N-(C13a1ky1)-pyrazolidino, piperidino, N-(C13a1ky1)-piperidino, piperizino, morpholino, azepino, diazepino, or N-(C13a1ky1)-diazepino, each of which is optionally substituted with one or more C13a1ky1 groups; each of RC1A,RC1B, RC2A, and RC2B is independently -H or C13a1ky1; each of RC3A and RC3B, if present, is independently -H or C13a1ky1; and each of RC4A and R046, if present, is independently -H or C13a1ky1; and additionally: if p is 0 and q is 0, then: (al) RN and one of RN and RN may together form: -(CH2)2-or -(CH2)3-; or (a2) one of R0 and RCIB and one of RN and RNWZ may together form: -(OH2)2-or -(OH2)4-; or (a3) one of RO2A and RO2B and one of RN and RNWS may together form: -(CH2)4-or -(CH2)5-; if p is 1 and q is 0, then: (bi) RNWI and one of RN and RNWS may together form: -OH2-or -(OH2)2-; or (b2) one of R01A and R0 and one of RN and RNWS may together form: -(OH2)2-or -(OH2)3-; or (b3) one of pC2A and R°26 and one of RN and RNWS may together form: -(OH2)3-or -(OH2)4-; (b4) one of ROSA and ROSB and one of RN and RNWS may together form: -(OH2)4-or -(OH2)5-; and if p is 1 and q is 1, then: (ci) RN and one of RN and RNWS may together form: -OH2-; or (c2) one of ROM and ROIB and one of RN and RNWS may together form: -OH2-or -(OH2)2-; or (c3) one of RC2A and Rc2B and one of RN and RNWS may together form: -(OH2)2-or -(OH2)3-; or (c4) one of ROSA and RCSB and one of RN and RNWS may together form: -(OH2)3-or -(OH2)4-; or (c5) one of RO4A and RO4B and one of RN and RNWS may together form: -(OH2)4-or -(OH2)5-; RA2 is independently O610carboaryl or O514heteroaryl; and is independently A subclass of compounds of Formula II includes compounds wherein pAS, R°2, R°4, RBS and RBB are H, RAS, if present, is H and Q is W, preferably wherein W is -NH-(02H4)-N (OH3)2.

Exemplary PKD inhibitors according to Formula II include: N* N-(2-dimethylamino-ethy-3-[6-(4-hydroxy- 3-methoxy-pheny-pyrazin-2-yl]-benzamide

HO 0 NH 4.

H N N 3-[5-amino-6-(4-hydioxy-3-methoxy- I phenyl)-pyrazin-2-yl]-N-(2-dimethylamino-HO N ethy-benzamide

C NH

HN 3-[6-am ino-5-(4-hydroxy-3-methoxy- I phenyl)-pyridin-3-yl]-N-(2-dimethylamino- 0 NH ethy-benzamide 3-[6-amino-5-(6-ethoxy-napthalen-2-y-k_N pyridin-3-yl]-N-(2-dimethylamino-ethyl)-

-

benzamide or a pharmaceutically acceptable salt, solvate, hydrate, ether, ester, chemically protected form, or prodrugs thereof.

Small molecule PKD inhibitors also include compounds as described in WO 2007/125331, which is incorporated herein by reference in its entirety. Such PKD inhibitors include compounds according to Formula Ill:

RI R3 N

5Kw "R2 R4fl R5 :h1

R

Formula Ill and pharmaceutically acceptable salts, solvates, hydrates, ethers, esters, chemically protected forms, and prodrugs thereof: wherein: J is independently N or OH; and wherein: (1) each of R8 and R9 is independently -H or a Ring B substituent; or: (2) R8 and R°, taken together with the atoms to which they are attached, form an aromatic Ring C having exactly 5 ring atoms or exactly 6 ring atoms, wherein each ring atom is a carbon ring atom or a nitrogen ring atom, wherein Ring C has exactly 0, exactly 1, or exactly 2 ring nitrogen atoms, and wherein Ring C is fused to Ring B; and wherein: (1) each of R10, R11, R12, and R13 is independently -H or a Ring A substituent; or: (2) each of R12 and R13 is independently -H or a Ring A substituent; and R1° and R11, taken together with the atoms to which they are attached, form an aromatic Ring D having exactly 6 ring atoms, wherein each ring atom is a carbon ring atom, and wherein Ring D is fused to Ring A; or: (3) each of R1° and R13 is independently -H or a Ring A substituent; and R11 and R12, taken together with the atoms to which they are attached, form an aromatic Ring E having exactly 6 ring atoms, wherein each ring atom is a carbon ring atom, and wherein Ring E is fused to Ring A; or: (4) each of R1° and R11 is independently -H or a Ring A substituent; and R12 and R13, taken together with the atoms to which they are attached, form an aromatic Ring F having exactly 6 ring atoms, wherein each ring atom is a carbon ring atom, and wherein Ring F is fused to Ring A; and wherein: each of R1, R2, R3, R4, R5, R6, and R7 is independently -H or a group G; and additionally wherein: each of R3, R4, R5, and R6 may be a group Y; each of R1, R2, and R7 may be a group Z; R3 and R4, taken together, may form a group =0; R5 and R6, taken together, may form a group =0; and wherein: R14 is independently -H or a group W; wherein: each Ring A substituent, if present, is independently R' or R"; each Ring B substituent, if present, is independently R' or R"; the group W, if present, is independently a R'; each group G, if present, is independently a R'; each goup Y, if present, is independently R"; each group 7, if present, is independently a k' selected from: (H-lU), (H-12), (H- 13), and (H-iS); wherein: each R' is independently selected from: (C-i) C17a1ky1, (C-2) C27alkenyl, (C-3) C27alkynyl, (C-4) C37cycloalkyl, (C-5) C37cycloalkenyl, (C-6) C314heterocyclyl, (C-7) C514carboaryl, (C-8) C514heteroaryl, (C-9) C614carboaryl-C17a1ky1, and (C-lU) C514heteroaryl-C17a1ky1; wherein each C17a1ky1, C27alkenyl, C27alkynyl, C37cycloalkyl, and C37 cycloalkenyl, is independently unsubstituted or substituted with one or more substituents selected from k'; and wherein each C314heterocyclyl, C614carboaryl, and C514heteroaryl is independently unsubstituted or substituted with one or more substituents selected from R" and R"; each k" is independently as defined for R', except that: ii each C17a1ky1, C27alkenyl, C27alkynyl, C37cycloalkyl, and C37cycloalkenyl, is independently unsubstituted or substituted with one or more substituents selected from R"; and each C314heterocyclyl, C514carboaryl, and C514heteroaryl is independently unsubstituted or substituted with one or more substituents selected from Rv and R"; each is independently as defined for R', except that: each C17a1ky1, C27alkenyl, C27alkynyl, C37cycloalkyl, and C37cycloalkenyl, is unsubstituted; and each C314heterocyclyl, C14carboaryl, and C514heteroaryl is each R" is independently selected from: (H-i) -F, -Cl -Br, -I; (H-2) -OH; (H-3) ORM, wherein R is independently a (H-4) -SH; (H-5) SRA2, wherein RA2 is independently a R"; (H-6) -NH2, NHRA3, NRA4RAS, wherein each of RA3, RA4, and RA5 is independently a R"; or RA4 and RA5 taken together with the nitrogen atom to which they are attached form a ring having from 3 to 7 ring atoms; (H-7) -NHC(=O)R"6, NRAJC(=O)RA6, wherein each of pA6 and RAY is independently R"; (H-8) -NHC(=O)OR'°, NRMoC(=O)ORAo, wherein each of RAD and RAIO is independently a R"; (H-9) -NHC(=O)NH2, NRMoC(=O)NH2, -NHC(=O)NHR, NRMoC(=O)NHR, -NHC(O)NR RAI2, -NR °C(O)NHRRM2, wherein each of R0, R, and RM2 is independently R"; or R and RAI2 taken together with the nitrogen atom to which they are aftached form a ring having from 3 to 7 ring atoms; (H-b) C(=O)RM3, wherein RM3 is independently R"; (H-il) -C(O)OH; (H-12) C(=O)ORM4, wherein RM4 is independently R"; H-b3) -C(=O)NH2, -C(=O)NHRM5, C(=O)NRM5RM6, wherein each of R"5 and RMG is independently R"; or RAI5 and RM6 taken together with the nitrogen atom to which they are attached form a ring having from 3 to 7 ring atoms; (H-14) OC(=O)RM7, wherein RAI? is independently R"; (H-b5) -OC(O)NH2, OC(O)NHRMa, OC(O)NRMBRM9, wherein each of R3 and RAI9 is independently R"; or RM8 and RAIQ taken together with the nitrogen atom to which they are attached form a ring having from 3 to 7 ring atoms; (H-16) -S(=O)2NH2, -S(=O)2NHR'20, S(=O)2NRA2oRI\2l, wherein each of RA2O and RA2I is independently R"; or R° and R"21 taken together with the nitrogen atom to which they are attached form a ring having from 3 to 7 ring atoms; (H-17) -NHS(=O)2R2, NRA23S(=O)2R2, wherein each of RA22 and RA23 is independently R"; (H-18) -S(=O)2R4, wherein RA24 is independently R"; (H-19) -S(0)20H; (H-20) S(=O)2ORA25, OS(=O)2RA2s, wherein each of RA25 and R6 is independently R"; (H-21) -NO2; each R" is independently as defined for R", except that: each R" is a A subclass of compounds of Formula Ill includes compounds wherein: R1 and R2 are independently either H or C1-C5alkyl; R3, R4, R5 and R6 are each independently H or C1-C6alkyl, preferably wherein one of R3 or S4 is C1-C6alkyl and the remainder of 3, 4, 5 and 6 are H; 7 is H or C1-C6alkyl; and R14 is H. In some embodiments, Ring A is a phenol ring (i.e. 14 is H) and has one or two additional substituents selected from chloro, fluoro, bromo, iodo, 1-methyl- 1H-pyrazolyl, 1H-pyrazolyl, trifluoromethyl, methyl, trifluoromethoxy and hydroxy, or Ring Exemplary PKD inhibitors according to Formula Ill include: 2-[4-((R)-2-amino-butylamino)-quinazolin-L.. .NH2 T 2-yl]-4-chloro-phenol RN2

HO

2-[4-(2-amino-ethylamino)-quinazolin-2-yl]-4-chloro-phenol

H

cII±N OH

V

2-[4-((R)-2-amino-butylamino)-pyrimidin-2-J yl]-4-(1-methyl-1 /

LI

-.1.. N'1

2-{4-[((R)-2-amino-buty-methyl-amino]-pyrimidin-2-yl}-4-chloro-phenol or a pharmaceutically acceptable salt, solvate, hydrate, ether, ester, chemically protected form, or prodrug thereof.

Another known and available FKD inhibitor is: Cl°HOH0 5-(3-chlorophenyl)-N-[4-(4-morpholinylmethyl)phenyl]-2-furancarboxamide. This compound is disclosed in E. R. Sharlow eta!., Discovery of Diverse Small Molecule Chemotypes with Cell-Based PKD1 Inhibitory Activity, 2011, PLoS ONE 6(10): e25134, which is incorporated herein by reference in its entirety.

Small molecule PKD inhibitors also include compounds as described in WO 2008/122615, which is incorporated herein by reference in its entirety. Such PKD inhibitors include compounds according to Formula IV: NR3 Formula IV wherein R1 and R2 are independently hydrogen, alkyl, cycloalkyl, heterocyclyl, each of which is optionally substituted by one to two R3, wherein S8 is hydrogen, halogen, alkyl, -O-Rg, -N(R10)(R11), -C(O)-N(R12)(R13), aryl, or heterocyclyl or heteroaryl, said heterocyclyl and heteroaryl are optionally substituted by one or two alkyl groups; R and R2 taken together with the nitrogen atom to which they are attached to optionally form a 4-7 membered ring; R3 is -N(R14)(R15), or halogen; 54, 55, R6 and 57 are independently hydrogen, halogen, alkyl, (C3-C7) cycloalkyl, aryl-alkyl, aryl, or alkoxy; Rg, R10, R1 and are independently hydrogen, -C(O)-O-alkyl, -C(O)-N(H)-alkyl, -0(O)-N H-C(O)-alkyl, cycloalkyl, cycloalkyl-alkyl, -SOR16, -C(O)-R17, heterocyclyl or alkyl, said heterocyclyl is further optionally substituted by one or two cycloalkyl-alkyl groups, and said alkyl is further optionally substituted by one or two groups selected from hydroxy, alkoxy, alkylamine, dialkylamine, or heteroaryl; and taken together with the nitrogen atom to which they are attached to optionally form a 5-7 membered ring; S12 and taken together with the nitrogen atom to which they are attached to optionally form a 5-7 membered ring; R14 and R15 are independently hydrogen, alkyl, aryl, cycloalkyl, aryl-alkyl, heterocyclyl or heteroaryl, said alkyl, cycloalkyl, aryl and heteroaryl are further optionally substituted by one or two groups selected from alkyl, alkoxy, hydroxy, halogen, haloalkyl, cyano, or-C(O)-NHR18; is aryl or heteroaryl; S17 is heterocyclyl, or alkyl optionally substituted by one or two groups selected from -NH2, aryl-alkyl, or-NH-C(O)-alkyl; R18 is heterocyclyl-alkyl; or and pharmaceutically acceptable salts thereof, optical isomers thereof and mixtures of optical isomers thereof.

A subclass of compounds of Formula IV includes compounds wherein: R4, R5, R6 and S7 are each hydrogen.

Exemplary PKD inhibitors according to Formula IV include: 1-{3-[2-(tetrahydropyran-4-ylamino)pyridine- 4-yl]-[2,6]napthyridin-1-yl}-piperidine-4-H carboxylic acid isopropylamide

P 0 NH

cyclohexyl-[4-(1-piperazin-1 -yl- [2,6]napthyridin-3-ypyridine-2-yl]amine and pharmaceutically acceptable salts thereof, optical isomers thereof and mixtures of optical isomers thereof.

PKD inhibitors may also be compounds of Formula IV, above, where R is hydrogen. For example, another known PKD inhibitor is: N-[3-pyridine-4-yl-[2,6]napthyridin-1-yl]-ethane-1,2-diamine. This compound is disclosed in E. L. Meredith etaL, J. Med. Chem., 2010, 53, 5400-542, which is incorporated herein by reference in its entirety.

Small molecule PKD inhibitors also include compounds as described in WO 2009/150230, which is incorporated herein by reference in its entirety. Such PKD inhibitors include compounds according to Formula V: RLjR3 (v) n Form ula V wherein R1, R2, and R3 are each independently hydrogen, halogen, cyano, nitro, hydroxy, alkyl, alkoxy, alkoxycarbonyl, -C(O)NR7R8, hydroxycarbonyl, -NR9R10, alkylsulfonyl, heterocyclyl, heteroaryl, or aryl; or may be linked with R1 to form a lactam ring, or may be linked with R3 to form a lactam ring; X is hydrogen, nitrogen, or unsubstituted or substituted carbon; R4 and R5 are each independently hydrogen, heterocyclyl, alkyl, or R4 and R5 are absent when X is hydrogen, or R4 and R5 are linked together to form a heterocyclic or heteroaryl ring; R7and R6 are each independently hydrogen, alkyl, orcycloalkyl; R9 and R1° are each independently hydrogen, alkoxycarbonyl, arylaminocarbonyl, sulfonyl, acyl, or aryl; Y is independently selected for each occurrence from halogen, cyano, nitro, hydroxy, aryl, alkyl, alkoxy, or -NR 11R-12, provided that at least one Y is -NR11R12; S11 and 12 are each independently hydrogen, cycloalkyl, heterocyclyl, aryl, arylamino, heteroaryl, or alkyl; n is an integer selected from 0, 1, 2, 3, or 4; and pharmaceutically acceptable salts, polymorphs, rotamers, prodrugs, enantiomers, hydrates, and solvates thereof.

An exemplary PKD inhibitor according to Formula V includes: 2'-cyclohexylamino-6-pipeiazin-1 -yl- [2,4']bipyridinyl-4-carboxylic acid amide and pharmaceutically acceptable salts, polymorphs, rotamers, prodrugs, enantiomers, hydrates and solvates thereof.

Small molecule PKD inhibitors also include compounds as described in WO 2011/009484, which is incorporated herein by reference in its entirety. Such PKD inhibitors include compounds according to Formula VIa or Formula VIb:

N-O

R18R2a R1aR2a Formula VIa Formula VIb wherein: x is NF or 0; sla is a substituted or unsubstituted aryl; s2a is: or s5a, R6, R7, R8 and R9 are each, independently, absent, hydrogen, halogen, alkyl, aminocarbonyl, carbonyl, heterocyclyl, hydroxyl, alkoxy, amido or imino; R5 and R6 may be linked to form an aryl or heterocyclyl ring; RB and R? may be linked to form an aryl ring; R8 and R9 may be linked to form a lactam; Y, y2a and (3a are each independently, N or C; R11 and R4 are each, independently, hydrogen, halogen or alkyl; and pharmaceutically acceptable salts, polymorphs, rotamers, prodrugs and enantiomers thereof.

A subclass of compounds of Formula VIa or Formula VIb includes compounds represented by Formula Vlai and Formula VIbi: b R1 bb\/R7b Formula Vlai Formula VIbi wherein: Xlb is NRbob or 0; R is heterocyclyl or aminoalkyl; R2b, R3b, R4b, R9b and Rlob are each, independently, hydrogen, alkyl or halogen; R5b, Reb and R?b are each, independently, hydrogen, halogen or alkyl R8b and Reb are each, independently, hydrogen, hydroxy, alkyl, alkoxy, amido or heterocyclyl; RSb and Rob can be linked together to form an aryl or heterocyclyl ring; ROLD and R7b can be linked together to form an aryl ring; Rob' and R9b can be linked together to form a I acta m; and pharmaceutically acceptable salts, polymorphs, rotamers, prodrugs and enantiomers thereof.

Exemplary PKD inhibitors according to Formula VIa or Formula VIb include: o N-isopropyl-3-{5-[4-(4-methyl-piperazin-1 -y-phenyl]-1 H-pyrazol-3-yl}-benzamide o 3-{5-[4-(4-methyl-piperazin-1 -yl)-phenyl]- 1 H-pyrazol-3-yl}-N-propyl-benzamide N-((S)-cyano-methyl-methyl)-3-(5-{4- [(tetrahyd ro-pyran-4-ylam ino)-methyl]- \ / NC phenyl}-isoxazol-3-y-benzamide

QNH ______

and pharmaceutically acceptable salts, polymorphs, rotamers, prodrugs and enantiomers thereof.

Small molecule PKD inhibitors also include compounds as described in WO 2003/082341, which is incorporated herein by reference in its entirety. Such PKD inhibitors include compounds according to Formula VII: NH2

N LJL'N

Formula VII and pharmaceutically active salts and prodrugs, wherein: S is a straight or branched chain C1-C7 alkyl, a 5-, 6-, or 7-membered cycloalkyl, or a 5-, 6-, or 7-membered heterocyclic radical which could be unsubstituted or substituted with one or more of hydroxy, nitro, cyano, amino, halogen, C1C7 alkyl, perfluorinated 01- 04 alkyl, C1C6 alkoxy, mono-or di(C1-C6 alkyl)amino, amino(C1-C5 alkyl) ; and W is m-phenyloxyphenyl, m-benzyloxyphenyl, m-2, 6-dichlorobenzyloxyphenyl, 3-piperonylphenyl, 4-t-butylphenyl, 1-napthoxyylmethyl radical, or a radical having the following structure: ff wherein X is OH or N, and which is unsubstituted, mono-, di-, or trisubstituted with one or more hydroxy, nitro, cyano, amino, halogen, OlC7 alkyl, periluorinated ClC4 alkyl, 01-06 alkoxy, mono-ordi(O1-C5alkyl)amino, oramino(01-06 alkyl) groups.

An exemplary PKD inhibitor according to Formula VII includes: 1-0, 1-dimethylethy-3-(1-ft NH2 / \ naphthalenylmethy-1H-pyrazolo[3,4- / d]pyrimidin-4-amine !1 T

NN

and pharmaceutically active salts and prodrugs thereof.

The term "halogen", "halo" or "halide" as used herein means fluorine, chlorine, bromine or iodine. The term "perhalogenated" (e.g. perfluorinated) as used herein refers to a moiety wherein all hydrogens are replaced by halogen atoms.

The term "heteroatom" as used herein refers to one or more of oxygen, sulfur, nitrogen, phosphorus or silicon.

The term "alkyl" as used herein means optionally substituted saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups and cycloalkyl groups.

Unless otherwise specified, an alkyl comprises 1 to 20 carbon atoms, 1 to 16 carbon atoms, 1 to 10 carbon atoms, 1 to 7 carbon atoms or 1 to 6 carbon atoms. The term "alkenyl" as used herein refers to an alkyl chain containing one or more units of unsaturation, i.e. one or more C=C double bonds. Unless otherwise specified, an alkenyl chain comprises 2 to 20 carbon atoms. The term "alkynyl" as used herein refers to an alkyl chain containing one or more units of unsaturation, i.e. one or more CEC triple bonds. Unless otherwise specified, an alkynyl chain comprises 2 to 20 carbon atoms. An alkyl, alkenyl or alkynyl group may be unsubstituted or may be substituted any group selected from -C(C)OH, -C(O)OW, -C(O)NH2, C(O)NHRa, -C(cJ)NWR8, C(O)NRLR0, - C(O)R3, halo, cyano, nitro, hydroxy, imino, thiol, -SR3, C(S)R3, -OC(O)R2, - OC(O)NH2, -OC(O)NHR3, OC(O)NRaRa, OC(O)NRbRc, -NH2, NHRa, NRaRa, NRbRc, - NHC(O)Ra, NReC(O)RE, -NHC(O)NH2, NHC(O)NHRE, NHC(O)NRaRa, NHC(O)NRbR0, -NRt(O)NH2, -NRt(c)NHw, -NRt(O)NR3R3, NR3C(O)NRLRc, -NHSO2R3, -NWSO2R3, so2Ra, oso2Ra, -SO2NH2, -SO2NHRt1, SO2NRtIRa, SO2NRbR0, oxo and _Rd, wherein Rd and each R is independently selected from hydrogen, C17a1ky1, C27alkenyl, C2 7alkynyl, C3.7cycloalkyl, C37cycloalkenyl, C314heterocyclyl, C6.14carboaryl, C514heteroaryl, C3.7cycloalkyl-C1.7alkylenyl, C3.14heterocyclyl-C1.7alkylenyl, C6.14carboaryl-C1.7alkylenyl and C5.14heteroahl-C1.7alkylenyl. One or more carbon atoms of the backbone of an alkyl, alkenyl or alkynyl group may be replaced by a heteroatom selected from nitrogen, oxygen or sulfur.

The terms "aryl" and "carboaryl" refer to optionally substituted monocyclic or multicyclic aromatic hydrocarbon groups having 6-20 carbon atoms in the ring portion. An aryl may be a (CC10) aryl. An aryl may be a 6-membered single-ring aromatic carbon group, for example, phenyl. The term "aryl" also includes multicyclic aryl groups, e.g., tricyclic, bicyclic, for example, naphthalene, anthracene, phenanthrene or pyrene. Aryl groups can also be fused or bridged with alicyclic or heterocyclic rings which are aromatic or not aromatic so as to form a polycycle (for example, tetralin). An awl may be substituted by any group selected from -C(O)OH, -C(O)OW, -C(O)NH2, c(o)NHRa, c(o)NRaRa, - C(O)NRbRC, c(o)Ra, halo, cyano, nitro, hydroxy, imino, ORa, thiol, sRa, C(S)Ra, -OC(O)R8, -OC(O)NH2, oC(o)NHRa, -OC(O)NR8R8, OC(O)NRbRc, -NH2, -NHR8, NRaRa, NRbR0, NHc(c)Ra, NRac(o)Ra, -NHC(O)NH2, NHc(o)NHRa, NHc(o)NRaRa, - NHC(O)NRbRC, -NR3C(O)NH2, NRac(o)NHRa, NRac(o)NRaRa, NRac(o)NRbR, -NHSO2Ra, NRaso2Ra, SO2Ra, OSO2Ra, -SO2NH2, sO2NHRa, SO2NRaRa, SO2NRbRc, oxo and _pd, wherein Rd and each R is independently selected from hydrogen, C1.7alkyl, C2.7alkenyl, C2.7alkynyl, C3.7cycloalkyl, C3.7cycloalkenyl, C3.14heterocyclyl, C6.14carboaryl, C5.14heteroaryl, C3.7cycloalkyl-C1.7alkylenyl, C3.14heterocyclyl-C1.7alkylenyl, C6.14carboaryl-C1.7alkylenyl and C5.14heteroaryl-C1.7alkylenyl.

The term "heteroaryl" refers to an optionally substituted 5-14 membered monocyclic-or bicyclic-or polycyclic-aromatic ring system, having ito 8 heteroatoms selected from N, 0 or S. A heteroaryl may be a 5-iO-membered ring system. A heteroaryl may be, for example, pyrrole, furan, thiophene, thiazole, isothiaozole, imidazole, triazole, tetrazole, pyrazole, oxazole, isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine.

Furthermore, heteroaryl includes multicyclic heteroaryl groups, e.g., tricyclic, bicyclic. For example, benzoxazole, benzodioxazole, benzothiazole, benzoimidazole, benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole, benzofuran, purine, benzofuran, deazapurine, or indoiizine. The term "heteroaryl" also refers to a group in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings. A heteroaryl may be substituted by any group specified for aryl, above.

A "cycloalkyl" refers to an optionally substituted saturated or partially unsaturated monocyclic, bicyclic or tricyclic hydrocarbon groups of 3 to 20 carbon atoms. A cycloalkyl group may have 3-12 carbon atoms or 3-9, 3-8 or 3-7 carbon atoms. Bi-or tn-cyclic groups may contain fused saturated and/or partially unsaturated rings. A cycloalkyl may be substituted by any group specified for aryl, above. A "cycloalkenyl" as used herein refers to a partially saturated hydrocarbon cyclic group. Preferably, a cycloalkenyl group is a monocyclic, bicyclic or tricyclic containing at least one carbon-carbon double bond as part of the ring(s), and having from 3 to 20 carbon atoms. A cycloalkenyl may be substituted by any group specified for aryl, above.

A "heterocyclyl" as used herein refers to an optionally substituted, saturated or unsaturated non-aromatic ring or ring system, e.g., which is unless otherwise specified, a 4-, 5-, 6-or7-membered monocyclic, 7-, 8-, 9-, 10-, 11-, or 12-membered bicyclicor 10-, 11-, 12-, 13-, 14-or 15-membered tricyclic ring system and contains at least one heteroatom selected from N, S or 0. Examples of heterocycles include tetrahydrofuran (THF), dihydrofurari, 1,4dioxane, morpholine, 1,4-dithiane, piperazine, piperidine, 1,3-dioxolane, imidazolidine, imidazoline, pyrroline, pyrrolidine, tetrahydropyran, dihydropyran, oxathiolane, dithiolane, 1,3-dioxane, 1,3-dithiane, oxathiane or thiomorpholine. A heterocyclyl may be substituted by any group specified for aryl, above.

The terms "aralkyl" and "heteroaralkyl" as used herein refers to an alkyl group as defined above substituted with an aryl or heteroaryl group as defined above. The alkyl component of an "aralkyl" or "heteroaralkyl" group may be substituted with any one or more of the substituents listed above for an alkyl group and the aryl or heteroaryl component of an "aralkyl" or "heteroaralkyl" group may be substituted with any one or more of the substituents listed above for aryl or heteroaryl groups. Preferably, aralkyl is benzyl.

A carbon atom when used in a ring may be substituted with any substituent as specified for cycloalkyl or aryl, above.

The term "alkoxy" as used herein refers to a group of the form -0-alkyl. The term "alkenyloxy" as used herein refers to a group of the form -0-alkene The term "haloalkyl" as used herein refers to any alkyl group substituted with one or more halogen atoms.

The term "carbonyl" or "carboxy" includes moieties which contain a carbon connected with a double bond to an oxygen atom (>C=O). The carbon atom of the carbonyl may be substituted with any substituent described herein. For example, carbonyl moieties may be substituted with alkyls, alkenyls, alkynyls, aryls, alkoxy, aminos, etc. Examples of moieties which contain a carbonyl include aldehydes, ketones, amides, carboxylic acids, esters and anhydrides. The term "thiocarbonyl" includes compounds and moieties which contain a carbon connected with a double bond to a sulfur atom (>C=S).

The term amino" or "amine" as used herein refers to a nitrogen atom substituent bonded to two other atoms (-N<), for example, hydrogen or a substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, aralkyl, heteroaryl, heterocyclyl, heteroaralkyl. An amino may be -NH2, NHR or NR2, wherein R is a substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, aralkyl, heteroaryl, heterocyclyl, heteroaralkyl.

The term "alkylamino" as used herein includes "monoalkylamino" and "dialkylamino", i.e. -NH(alkyl) and -N(alkyl)2, wherein, alkyl is as defined herein and may be substituted.

The term "amide," "amido" or "aminocarbonyl" includes moieties which contain a nitrogen atom (or an amine) bound to the carbon of a carbonyl group (a group of structure -C(O)-NC). The term includes "alkaminocarbonyl" or "alkylaminocarbonyl" groups which include alkyl, alkenyl, aryl or alkynyl groups bound to an amino group bound to a carbonyl group.

It includes arylaminocarbonyl and arylcarbonylamino groups which include aryl or heteroaryl moieties bound to an amino group which is bound to the carbon of a carbonyl.

The terms "alkylaminocarbonyl," "alkenylaminocarbonyl," "alkynylaminocarbonyl," "arylaminocarbonyl," "alkylcarbonylamino," "alkenylcarbonylamino," "alkynylcarbonylamino," and "arylcarbonylamino" are included in the term "amide." Amides also include urea groups (aminocarbonylamino) and carbamates (oxycarbonylamino). The term "amide," "amido" or "aminocarbonyl" also includes substituted moieties from any substituent described herein.

The term "imino" as used herein refers to a group of structure >C=N-R, wherein S is, for example, hydrogen or a substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, aralkyl, heteroaryl, heterocyclyl, heteroaralkyl.

The term "alkylsulfonyl" as used herein refers to a moiety of structure -S(O)R, wherein S is, for example, a substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, aral kyl, heteroaryl, heterocyclyl, heteroaralkyl.

The terms "thioether" or "alkylthio" refer to a group of structure -S-R, wherein S is, for example, a substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, aralkyl, heteroaryl, heterocyclyl, heteroaralkyl.

A lactam is a cyclic amide of structure: (o H wherein n is an integer from 1 to 4.

PKD inhibitors aso include inhibitors that work by inhibiting the expression of a nucleic acid encoding PKD, for exampe a nucleic acid having the sequence shown in any one of Figures 2 to 7 (SEQ ID NOs: 1, 3, 5. 7, 9 and 11).

As described herein, there are 3 isoforms of PKD in humans: PKD1, PKD2 and PKD3.

Representative nucleic acid and amino acid sequences of human PKD1, PKD2 and PKD3 are set out in Figures 2 to 7 respectively. Figure 2A shows the mRNA sequence of 1-lomo sapiens protein kinase Dl (PRKD1) having NCBI Accession No: NM_002742.2 (Gl:1 15529462, SEQ ID NO: 1). Figure 28 shows the amino acid sequence of the translated protein (SEQ ID NO: 2). Figure 3A shows the mRNA sequence of Homo sapiens protein kinase D2 (PRKD2) transcript variant 1 having NCBI Accession No: NM_016457.4 (GI:120659783, SEQ ID NO: 3). Figure 3B shows the amino acid sequence of the translated protein (SEQ ID NO: 4). Figure 4A shows the mRNA sequence of Homo sapiens protein kinase D2 (PRKD2) transcript variant 2 having NCBI Reference Sequence: NM_001079880.1 (GI:120659781, SEQ ID NO: 5). Figure 48 shows the amino acid sequence of the translated protein (SEQ ID NO: 6). Figure 5A shows the mRNA sequence of Homo sapiens protein kinase D2 (PRKD2) transcript variant 3 having NCBI Reference Sequence: NM_001079881.1 (GI:120659784, SEQ ID NO: 7). Figure 5B shows the amino acid sequence of the translated protein (SEQ ID NO: 8). Figure 6A shows the mRNA sequence of Homo sapiens protein kinase D2 (PRKD2) transcript variant 4 having NCBI Reference Sequence: NM_001079882.1 (Gl:120659786, SEQ ID NO: 9). Figure 68 shows the amino acid sequence of the translated protein (SEQ ID NO: 10). Figure 7A shows the mRNA sequence of Homo sapiens protein kinase D3 (PRKD3) having NCBI Accession No: NM_005813.3 (Gl:48255886, SEQ ID NO: 11).

Figure 78 shows the amino acid sequence of the translated protein (SEQ ID NO: 12).

PKD inhibitors that work in this way include, for example, small interfering RNA5 (siRNAs), shod hairpin RNA5 (shRNAs), microRNAs (miRNAs), antisense RNAs, ribozymes and DNAzymes.

A shod interfering SNA (siRNA), also known as silencing RNA, is an RNA molecule that can be used to interfere with the expression of a specific gene in a process known as RNA interference (RNAi). SiRNA5 are typically double stranded, but single-stranded siRNAs are also known (see, e.g., Martinez et al., 2002 Cell 1 10:563-74). s1RNA molecules occur in nature, but can also be used experimentally to suppress the expression of a gene in vitro or in vivo. This can be achieved by targeting an mRNA for degradation, preventing mRNA translation or by establishing regions of silenced chromatin. 5iRNA molecules are usually short, with an average length of 20 to 25 nucleotides, and double stranded, with phosphoryated 5' ends and hydroxylated 3' ends with two overhanging nucleotides. siRNA molecules may be incorporated into vectors when used experimentally to aid the successful transfection of these molecules into living cells. The Dicer enzyme (an RNAase Ill type nuclease) catalyses production of siRNAs from long dsRNAs and short hairpin RNAs.

Short hairpin RNA (shRNA) molecules are another type of RNA molecule which can be used for RNAi. shRNAs include a sequence which binds to itself via a hairpin structure, and are typically expressed in cells through delivery of plasmids, or viral or bacterial vectors.

A microRNA (miRNA) is a shod RNA molecule that binds to complementary sequences on target messenger RNA (mRNA) transcripts. This results in translational repression or target degradation and gene silencing.

An antisense RNA is a single-stranded RNA molecule that is complementary to at least a portion of a particular mRNA. The antisense RNA therefore binds to the mRNA by hybridization, therefore forming a double-stranded RNA molecule and thus inhibiting translation of a gene.

Ribozymes are RNA molecules that are able to cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases, thus inhibiting the expression of the RNA. There are two basic types of ribozymes: tetrahymena-type ribozymes, which recognise sequences of 4 bases, and hammerhead-type ribozymes, which recognise sequences of 11 to 18 bases.

DNAzymes (deoxyribozymes or DNA enzymes) are catalytically active DNA molecules, some of which are able to cleave RNA molecules after appropriate binding.

"Targeting" or "PKD-targeted", as used herein, refers to the ability of a PKD inhibitor to selectively bind to nucleic acids, in particular RNA molecules, of interest. The FKD inhibitors are typically nucleotides that bind to target sequences of interest. For example, an siRNA molecule or antisense RNA molecule may hybridize to nucleic acid, such as an mRNA molecule that encodes PKD protein or even genomic DNA sequences (such as genomic DNA encoding PKD).

"Target sequences" or "target nucleic acids", depending on the context, may refer to nucleic acids, such as mRNA or genomic DNA, encoding a PKD protein. In particular, the target sequence may be a segment of the target nucleic acid, for example an RNA molecule. For example, in some embodiments of the invention, the PKD inhibitor can target any section of the sequence of any one or more PKD gene (or corresponding PKD mRNA), for example a segment of one or more PKD gene (or PKD mRNA), for example the PKD1, PKD2 or PKD3 gene, that is 10 to 50 or 15 to 30 nucleotides in length, optionally 15 to 29 nucleotides in length, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 nucleotides in length, typically 19 to 29 nucleotides. Typically, the target sequence is that of SEQ D NOs: 1, 3, 5, 7, 9 or 11, or a portion, of any thereof.

In order to hybridize to a target nucleic acid, the PKD inhibitor may itself be a nucleic acid (polynucleotide or oligonucleotide, single or double stranded) that is complementary, or substantially complementary, to the target sequence or segment thereof. If the PKD inhibitor is double stranded (for example, an 5iRNA), at least one of the strands may be complementary, or substantially complementary, to the target sequence or segment thereof. "Substantially complementary" refers to at least 50% identity to a sequence that is complementary to the target nucleotide or target mRNA (or segment thereof), for example at least 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% identical, typically along the whole length of the target sequence. For example, a PKD inhibitor that is a nucleic acid may have at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% identity to a to 30 residue nucleotide sequence that is complementary to a segment of PKD mRNA.

Therefore, PKD inhibitors that are nucleic acids may have a sequence that is the reverse complement (antisense) to the target nucleic acid or segment thereof. PKD inhibitors for use in the present invention may comprise a sequence that is the reverse complement of any one of the sequences in SEQ ID NOs 1, 3, 5, 7, 9 or 11, or the reverse complement of a segment of any one of the sequences in SEQ ID NOs 1, 3, 5, 7, 9 or 11.

Nucleic acid inhibitors for use in the present invention can either be obtained commercially or can be prepared using a variety of techniques known to a person skilled in the art, for example by amplification of a cDNA prepared from a suitable cell or tissue type using PCR, by solid phase chemical synthesis, or by transcription of suitable DNA sequences from a vector.

Any part of any of the nucleic acid sequences shown in Figures 2 to 7 may be targeted in the present invention. Typically, the PKD inhibitors used in the present invention target a part of the sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9or SEQ ID NO: 11.

For example, siRNA oligonucleotides targeting the 5' untranslated region of PKD2 (5'-cuggguucuagauccgcgguu-3') and PKD3 (5'-aaggugaaauccucuucguuu-3') are commercially available from Dharmacon RNA Technologies (Lafayette, CO). Bisbal et al. PKD1 and Trafficking of Dendritic Membrane Proteins J. Neurosci., September 10, 2008 28(37):9297-9308 describe a PKD1 short hairpin RNA (5hRNA) plasmid targeting the sequences 5'-GGTTCTGGACAGTTCGGAATAAGCTTATTCCGAACTGTCCAGAACCCTTTTTTG and 5'-

AATTCAMMGGGTTCTGGACAGTTCGGAATAAGCTTATTCCGAACTGTCCAGAACC

In some embodiments of the invention, the PKD inhibitor targets a nucleotide sequence (DNA or mRNA) or segment thereof that encodes the protein sequence of one or more isoforms of FKD, for example the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12.

Accordingly, PKD inhibitors for use in the present invention may comprise a sequence that is the reverse complement of a nucleotide sequence (such as a DNA sequence of mRNA sequence) that encodes SEQ ID NO: 2, 4, 6, 8, 10 or 12, or the reverse complement of a nucleotide sequence (such as a DNA sequence or mRNA sequence) that encodes a segment of SEQ ID NO: 2,4,6,8, lOorl2.

Preferably the PKD inhibitor does not substantially hybridize with other nucleic acids in the cell, in particular mRNA other than the target mRNA or genomic DNA molecules. When PKD inhibitor hybridizes to target mRNA, it can therefore effectively silence the corresponding gene or reduce expression of that gene (in this case, a PKD gene).

In some embodiments of the invention, the PKD inhibitor can be modified to increase its stability and/or uptake by cells. For example, the PKD inhibitor such as an siRNA or 5hRNA can be incorporated into a vector, such as a plasmid or viral vector, such as a lentivirus or adenovirus vector.

Targeting of nucleic acids, such as mRNA, using 5iRNA may be achieved by exploitation of a cell's RNA-inducing silencing complex (RISC). In such an approach, without wishing to be bound by theory, double-stranded RNA is cleaved by the enzyme Dicer. The siRNAs are loaded on to pre-RISC complex and the two strands separated. The RISC complex selectively hybridises to the target RNA and can induce cleavage or inhibit translation, and hence inhibition of gene expression. The siRNAs used in the present invention may or may not require processing by Dicer before being loaded onto a pre-RISC complex. The siRNAs can be exogenously applied and/or artificial siRNAs.

siRNA molecules are typically between 15 and 30 nucleotides in length, optionally 15 and nucleotides in length, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 nucleotides in length, typically 19 to 29 nucleotides. The target nucleotides and/or the nucleotides of an 5iRNA are generally contiguous, although 5iRNA molecules may undergo processing or chemical modification prior to delivery to increase their stability and/or efficacy. Double stranded RNA molecules may also be used that are later processed into smaller siRNA molecules in vivo, for example by action of the enzyme Dicer.

The 5iRNA used in the present invention can be any siRNA that targets PKD.

Accordingly, any siRNA designed to target PKD mRNA can be used in the present invention. Typically, the 5iRNA targets one or more of the sequences of SEQ ID NO:s 1, 3, 5, 7, 9 and 11. However, siRNA molecules that target one or more other PKD sequence can be used, and it is within the ability of the skilled person to select a suitable siRNA according to known protocols and requirements.

siRNAs can be designed to target specific DNA (or mRNA) sequences according to a number of algorithms known to the skilled person. Such methods are described in, for example, Walton et el., FEBS, 2010, 277(23):4806-13 and Pei et at, Nat Methods, 2006, 3(9)670-6. The 5iRNA molecules may be chemically modified, for example to increase potency, half-life or stability (for example by optionally adding one or more thymine residues to the end of the 5iRNA duplex). Such chemical modification is described in, for example, Bramsen & Kjems, Methods Mo! 8101, 2011, 721:77-103.

siRNAs that inhibit expression of PKD are known in the art and are commercially available, for example from Dharmacon RNA Technologies (Lafayette, CO) (now part of GE Healthcare) and Ambion (Life Technologies, Inc.) (for example the 5iRNA PKD3 and the siRNA PKD2, which are Silencer-validated siRNAs). Other siRNAs that can be used as PKD inhibitors are the following oligonucleotides: 5'-AUGCUGUGGGGGCUGGUACdTdT-3' and 5'-GUACCAGCCCCCACAGCAUdTdT-3' (nucleotides 496±516 in human PKD), as described by Peter Storz and Alex Toker.

(Protein kinase D mediates a stress-induced NF-kappaB activation and survival pathway.

EMBO J. 2003 Jan 2. 22(1):109-20). 5,-

GATCCCCATGCTGTGGGGGCTGGTACTTCAAGAGAGTACCAGCCCCCACAGCATTTT

TTGGAAA-3' and 5,-

AGCTTTTCCAAAAAATGCTGTGGGGGCTGGTACTCTCTTGAAGTACCAGCCCCCACA

GCATGGG-3'.

(as described in Brummelkamp et al., A system for stable expression of short interfering RNAs in mammalian cells. Science. 2002 Apr 19; 296(5567):550-3. Epub 2002 Mar 21) Other PKD inhibitors include those which act by inactivating and/or sequestering PKD.

For example, inactivacted forms of PKD can be expressed in a cell by transfecting the cells with a cDNA construct that encodes an inactivated mutant of PKD. Suitable mutants of PKD1 include the activation loop active site mutants S738E and S742E, the PH domain deleted cDNA, the Y463F inactive mutant and the activation loop inactive mutants: 5738A and S742A mutants.

The activity and efficacy of PKD inhibitors can be determined using PKD activity assays.

Such assays allow the effect of the PKD inhibitor on PKD activity to be observed and/or quantified.

Assays for determining the activity of PKD inhibitors include whole cell assays.

One such assay includes the steps of: contacting cells with an agent that activates PKD; contacting cells with a PKD inhibitor; making extracts of the cells and analysing the cell extracts by SDS-PAGE and Western blotting with an antibody that detects activation of PKD. Suitably, a dose-dependent inhibition of PKD activity will be observed for PKD inhibitors. In some embodiments, the cells are pre-treated with the PKD inhibitor. The cells will be contacted with the PKD inhibitor at a suitable concentration and for a suitable amount of time, which will be readily determined by a person skilled in the art.

Suitable cells for use in such an assay include HeLa cells, bronchial epithelial cells (BEC5) and cell lines such as BEAS2B. Agents that activate PKD include phorbol ester.

Antibodies that detect activation of FKD include anti-phosphoPKD-Ser916 antibody.

An example of such an assay is given in the Example herein and the results are shown in Figure 11.

Another whole cell assay for PKD activity involves the use of a PKD reporter, for example to visualise site-specific FKD activation. A molecular reporter can comprise, for example a PKD-specific substrate sequence fused to enhanced green fluorescent protein (EGFP).

The EGFP can be targeted to a specific site by means of a sequence, for example the p230 GRIP domain for targeting to the trans-Golgi network (TGN). Quantitative analysis can then be carried out using an antibody that detects phosporylation of a specfic residue in the sequence of PKD, such as the anti-phosphoFKD-5er916 antibody, and ratiometric fluorescence imaging to determine the effect of a PKD inhibitor on phosphorylation at a particular location. Such an assay can be used to measure the activity of small molecule PKD inhibitors and other PKD inhibitors such as siRNAs.

Such an assay is described in Fuchs et al Traffic 2009; 10: 858-867 An alternative assay involves the use of a genetically encoded FRET reporter. An example of this is the reporter G-PKD rep-live described in Stephan A. Eisler et al, Biotechnol. J. 2012, 7, 148-154, which is used to measure PKD activity at the trans-Golgi-network (TGN).

Another cell based PKD assay is described in: Kunkel MT, Toker A, Tsien RY, Newton AC. J Biol Chem. 2007 Mar 2;282(9):6733-42. Epub 2006 Dec 21. Calcium-dependent regulation of protein kinase D revealed by a genetically encoded kinase activity reporter.

Suitable in vitro assays include kinase assays, which have the advantage of being high throughput.

Typically this involves making purified PKD by expression of human PKD isoforms (such as PKD1, PKD2 and/or PKD3, having the sequences shown in Figures 2 to 7 herein), for example by expression in insect cells (using e.g. baculovirus expression systems) or in mammalian cells. The kinase can have a "tag" to assist in purification. Common tags include myc, HA, poly-Histidine or GST, but any convenient tag can be used and will be known to the person skilled in the art. The kinase can be activated by contacting the producing cells with an agent that activates PKD, such as phorbol ester, or by activating the purified kinase in vitro with protein kinase C. Kinase activity can be measured, for example, by FP (fluorescence polarisation) of a fluorescently labelled substrate peptide.

Typically, the assay includes ATP at a concentration equal to the Km for ATP for this kinase. In addition, the assay typically also includes MgCI2, usually at a concentration of 2-10mM.

An example of such an assay is that disclosed in Manuj Tandon et al New Pyrazolopyrimidine Inhibitors of Protein Kinase D as Potent Anticancer Agents for Prostate Cancer Cells Pbs One September 2013 Volume 8 Issue 9 e75601. Another example is that disclosed in Elizabeth R. Sharlow et al Potent and Selective Disruption of Protein Kinase D Functionality by a Benzoxoloazepinolone J Biol Chem 283, NO. 48, pp. 33516-33526, November 28, 2008. However, many other assay formats are available and will be well known to those skilled in the art.

George et aL, Pharmaceutics 2011, 3, 186-228 describes the design, synthesis and biological evaluation of PKD inhibitors.

Such assays can be used to test compounds for PKD inhibition in accordance with the first aspect of the invention or to screen for compounds that are useful in the treatment or prevention of picornavirus infection, in accordance with the second aspect of the invention described below.

The PKD inhibitor for use in the invention is typically administered as part of a pharmaceutical composition.

The pharmaceutical composition can be formulated for use by any convenient route. The pharmaceutical composition will normally include a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, vehicle, buffer or stabiliser in addition to a protein kinase 0 inhibitor as defined herein. Such carriers include, but are not limited to, saline, buffered saline such as phosphate buffered saline (PBS), dextrose, liposomes, water, glycerol, polyethylene glycol, ethanol and combinations thereof. This pharmaceutical composition may be in any suitable form depending upon the desired method of administering it to a patient.

It can be provided in unit dosage form, will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It can include a plurality of said unit dosage forms.

The pharmaceutical composition can be adapted for administration by any appropriate route, for example by inhalation or by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intraperitoneal or intradermal) route, although it will typically be adapted for administration by inhalation. Such compositions can be prepared by any method known in the art of pharmacy, for example by admixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.

Pharmaceutical compositions adapted for administration by inhalation include fine particle dusts, dry powders or mists that can be generated by means of various types of metered dose pressurised aerosols, nebulizers or insufflators.

Pharmaceutical compositions adapted for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 2 to 500 microns, for example 10 to 300 microns, for example 20 to 250 microns or 50 to 100 microns, which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable compositions wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.

Pharmaceutical compositions adapted for oral administration can be presented as discrete units such as capsules or tablets; as powders or granules; as solutions, syrups or suspensions (in aqueous or non-aqueous liquids; or as edible foams or whips; or as emulsions). Suitable excipients for tablets or hard gelatine capsules include lactose, maize starch or derivatives thereof, stearic acid or salts thereof. Suitable excipients for use with soft gelatine capsules include for example vegetable oils, waxes, fats, semi-solid, or liquid polyols etc. For the preparation of solutions and syrups, excipients which can be used include for example water, polyols and sugars. For the preparation of suspensions, oils (e.g. vegetable oils) can be used to provide oil-in-water or water in oil suspensions.

Pharmaceutical compositions adapted for transdermal administration can be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient can be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research, 3(6):318 (1986).

Pharmaceutical compositions adapted for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.

Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solution which can contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation substantially isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which can include suspending agents and thickening agents. Excipients which can be used for injectable solutions include water, alcohols, polyols, glycerine and vegetable oils, for example. The compositions can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carried, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and

tablets.

The pharmaceutical compositions can contain preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifiers, sweeteners, colourants, odourants, salts, buffers, coating agents or antioxidants.

The pharmaceutical compositions for use in the invention can also contain one or more other therapeutically active agents in addition to the PKD inhibitor as defined herein. Other therapeutically active agents that may be useful in the present invention include, for example, other antiviral agents, such as brefeldin A, and anti-inflammatory agents such as but not limited to glucocorticoid receptor agonists, for example beclomethasone, fluticasone, mometasone, budesonide and ciclesonide.

Dosages of the PKD inhibitor and/or pharmaceutical composition for use in the present invention can vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used.

This dosage can be repeated as often as appropriate. If side effects develop the amount and/or frequency of the dosage can be reduced, in accordance with normal clinical practice.

For administration to mammals, and particularly humans, it is expected that the daily dosage of the active agent will be from lpg/kg to 10mg/kg body weight, typically around lOpg/kg to 1mg/kg body weight. The physician in any event will determine the actual dosage which will be most suitable for an individual, which will be dependent on factors including the age, weight, sex and response of the individual. The above dosages are exemplary of the average case. There can, of course, be instances where higher or lower dosages are merited, and such are within the scope of this invention.

The FKD inhibitors and/or pharmaceutical compositions as defined herein are used in the treatment or prevention of picornavirus infection.

The invention therefore also extends to the use of a PKD inhibitor or a pharmaceutical composition as defined herein in the manufacture of a medicament for use in the treatment or prevention of picornavirus infection, or alternatively to the use of a FKD inhibitor or a pharmaceutical composition as defined herein in the manufacture of a medicament for the treatment or prevention of picornavirus infection.

The invention also includes a method for the treatment or prevention of picornavirus infection in a subject, typically a subject in need thereof, comprising administering to the subject a PKD inhibitor or a pharmaceutical composition as defined herein. The method of treatment can be of a human or an animal subject and the invention extends equally to uses in both human and/or veterinary medicine. The PKD inhibitor and/or pharmaceutical composition is preferably administered to an individual in a "therapeutically effective amount", this being sufficient to show benefit to the individual and/or to ameliorate, eliminate or prevent one or more symptoms of picornavirus infection. As used herein, "treatment" includes any regime that can benefit a human or non-human animal, preferably a mammal, for example an economically important mammals such as cattle, sheep, goats and pigs. The treatment may be in respect of an existing condition or may be prophylactic (preventative treatment).

Picornavirus infection includes infection with one or more of rhinovirus, polio virus, foot and mouth disease virus (FMDV), hepatitis A virus, coxsackievirus (CV) and enteroviruses. In one embodiment, the picornavirus is rhinovirus. In this embodiment, the subject may be one suffering from asthma and/or chronic obstructive pulmonary disease (COPD). In an embodiment, the invention is useful for the treatment of HRV induced asthma, COPD, cystic fibrosis (CE) or interstitial lung disease exacerbations.

In another embodiment, where the picornavirus is polio virus or FMDV, the invention is useful for the prophylaxis of viral infection. For example, prophylactic treatment may be useful to protect family members of an infected individual or the rest of a herd of animals to stop the spread of disease.

As described herein, the present invention is based on the novel finding that PKD inhibitors block picornavirus replication, in particular HRV replication. Accordingly, the present invention also extends to methods of screening for compounds that can be used for the treatment or prevention of picornavirus infection by determining whether such compounds are PKD inhibitors.

In a second aspect, the present invention therefore provides a method of identifying a compound useful for the treatment or prevention of picornavirus infection, comprising determining whether said compound is a PKD inhibitor. This can be done using any of the PKD activity assays described herein in relation to the first aspect of the invention. For example, a typical method according to the second aspect of the invention comprises the steps of: contacting a cell in which PKD has been activated with a candidate compound; incubating the cell with the candidate compound for a suitable period of time; and determining whether PKD activation is inhibited. If PKD activation is inhibited, the candidate compound is identified as a PKD inhibitor and is therefore useful for the treatment or prevention of picornavirus infection, in accordance with the first aspect of the invention.

Once a compound has been identifed as a PKD inhibitor it can further be tested for its activity and/or efficacy in inhibiting picornavirus replication. The method of the second aspect of the invention can therefore include a further step of determining whether the candidate compound inhibits picornavirus replication. If the compound inhibits picornavirus replication then it is a suitable candidate for the treatment or prevention of picornavirus infection, for example human rhinovirus (HRV) infection.

Suitable assays for inhibitors of HRV replication include an "in cell ELISA" which can be carried out using various commercially available kits such as those available from Thermo Fisher Scientific, R&D Systems, AbCam and RayBiotech.

The methodology for such an assay typically involves infecting a cell line such as an epithelial cell line (for example HeLa, A549, BEAS2B or primary human bronchial epithelial cells (BECs)) with HRV at a multiplicity of infection (MOl) of between 1-20 in a multiwall plate and allowing time for replication (for example between 6-24 hours). The cells are then washed and fixed (e.g. with a standard fixative such as gluteraldehyde), permeabilised with a detergent (such as TritonXlOO) and then an antibody to an HRV protein such as a rabbit anti-HRV 20 antibody or a rabbit anti-HRV 3A antibody (primary antibody) is added. The primary antibody is revealed with a labelled anti-rabbit secondary antibody. The label could be fluorescent or enzyme linked. The secondary antibody is then detected in an appropriate plate reader.

As an alteinative, replication can be detected by immunofluorescence microscopy of infected cells.

As an alternative to the in cell ELISA, instead of processing the cells for an ELISA, the infected cells can be detached from the plate (by standard protocols such as EDTA treatment), and HRV 20 or HRV 3A protein is detected by rabbit primary antibody followed by fluorescent secondary antibody and analysed by FACS.

As a further alternative, at the end of the infection in the in cell ELISA, cells can be lysed in an appropriate lysis buffer such as RIPA buffer and extracts run on SDS-PAGE and HRV 20 and 3A revealed by Western blotting with anti-HRV 20 or 3A antibodies.

Other cell based assays can be built that measure the expression of the HRV 3C protease, as described by Lonneke van der Linden et al Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases. Antiviral Research 103 (2014) 17-24 (and references therein). In an alternative to the described method, cells can be infected with virus rather than transfected with 30 protease.

A modification of the "molecular beacon" assay described by Jeong Hee Kim et al In-Cell Protease Assay Systems Based on Trans-Localizing Molecular Beacon Proteins Using HCV Protease as a Model System PlosOne March 2013 Volume 8, Issue 3, e59710 could also be configured to measure HRV replication.

Alternatively, a "split GFP" complementation assay could be constructed, as described in Yutaka Kodama Bimolecular fluorescence complementation (BiFC): A 5-year update and future perspectives. BioTechniques 53:285-298 (November 2012) doi 10.2144/000113943. A cell line (such as HeLa, A549 etc) is stably transfected with a large fragment of GFP and a recombinant HRV constructed that has inserted into its RNA genome the complementary small fragment of the GFF. Infection of the cells with this recombinant virus would bring together the two fragments of GFP and so generate fluorescence.

HRV replicons can be constructed where the capsid encoding RNA sequences (or pads thereof) are replaced with a reporter such as luciferase and cloned into a plasmid. These plasmids are translated in vitro and the viral replicon encoding mRNA is transfected into cells. Replication of the viral mRNA produces the reporter (luciferase) which can be detected but not intact virus. Such an approach is described, for example, in Puig-Basagoiti F et al High-throughput assays using a luciferase-expressing leplicon, virus-like particles, and full-length virus for West Nile virus drug discovery. Antimicrob Agents Chemother. 2005 Dec;49(12):4980-8. A corresponding approach for a picornavirus is described by Hilde M. van der Schaar et al A Novel, Broad-Spectrum Inhibitor of Enterovirus Replication That Targets Host Cell Factor Phosphatidylinositol 4-Kinase Ill Antimicrobial Agents and Chemotherapy p. 4971-498lOctober 2013 Volume 57 Number 10.

A further alternative is a split luciferase bioluminescence assay, for example as described by Zhiwei Guo A 3Cpro-dependent bioluminescence imaging assay for in vivo evaluation of anti-enterovirus 71 agents Antiviral Research 101 (2014) 82-92 A further alternative is to infect cells with HRV and to measure replication by measuring viral RNA by quantitative PCR.

A further alternative is to infect cells with HRV, then to lyse cells by freeze thawing to release viral particles and then determining the number of viral particles (titre) by measuring the tissue culture infective dose required to give 50% cell lysis (TCID50) as described in the Materials and Methods section of the Example herein.

Preferred features for the second aspect of the invention are as for the first aspect mutatis mutandis.

The present invention will now be described by way of illustration only with reference to the following Examples and Figures, in which: Figure 1 is a schematic of domain organisation of human Protein Kinase D 1,2 and 3 (PRKD). Each isoform has two diacylglycerol (DAG) binding domains (Cia and Cib), a Pleckstrin Homology (PH) domain and a kinase domain. Important regulatory Tyrosine and Serine phosphorylation sites are shown.

Figure 2 shows (A) a representative nucleic acid sequence and (B) a representative amino acid sequence of human PKD1.

Figure 3 shows (A) a representative nucleic acid sequence and (B) a representative amino acid sequence of human PKD2, transcript variant 1.

Figure 4 shows (A) a representative nucleic acid sequence and (B) a representative amino acid sequence of human PKD2, transcript variant 2.

Figure 5 shows (A) a representative nucleic acid sequence and (B) a representative amino acid sequence of human PKD2, transcript variant 3.

Figure 6 shows (A) a representative nucleic acid sequence and (B) a representative amino acid sequence of human PKD2, transcript variant 4.

Figure 7 shows (A) a representative nucleic acid sequence and (B) a representative amino acid sequence of human PKD3.

Figure 8. HeLa cells infected with HRV16 for lh followed by a time course of 0-7 hours. Cells were fixed and stained for HRV 2C protein expression. Top panels are phase contrast and the corresponding lower panels show immunofluorescence.

Figure 9. HeLa cells were infected with HRV1S at an MCI of 20 for one hour. The cells were washed and replication allowed to proceed for various times up to 7 hours. At each time point cell lysates were prepared and analysed by SDS-PAGE and Western blotting. The blot was revealed with a rabbit anti-H RV2C antibody and with anti-beta actin as a loading control.

Figure 10. HeLa cells were infected with HRV16 at a MOl of 20 for the indicated times. As controls, cells were either uninfected (Oh) or uninfected but treated with Phorbol Ester (PDBu). Cell lysates were prepared, analysed by SDS-PAGE and Western blotted with the indicated antibodies.

Figure 11. HeLa cells were either untreated (Oh) or treated with 200 nM Phorbol Ester (PDBu) alone for 15 mm or with Phorbol ester and increasing concetrations of the PKD inhibitor CR10066101.

Figure 12. HeLa cells were infected with HRV16 at a MOl of 20 and incubated for 6h in the presence of increasing concentraions of CRTOO661 01. Controls were either uninfected (Oh) or treated with 200 nM Phorbol Ester (PDBu) or with DMSO vehicle alone. Cell lysates were prepared and analysed by SDS-PAGE and Western blotting with anti-phosphoPKD-Ser916, anti-PKD and with anti-HRV 2C antibodies.

Figure 13. HeLa cells were infected with HRV16 for 6 hours in the presence of increasing concentraions of CRTOO661O1 or a DMSO control. Cells were lysed by freeze/thawing and viral yield was determined as the median tissue culture infective dose (1C1D50) as described in Materials & Methods.

Figure 14. HeLa cells were infected with HRV16 at a MCI of 20 and incubated for 6h in the presence of increasing concentraions of Cl D201 1756. Controls were either uninfected (Oh) or treated with 200 nM Phorbol ester (PDBu) or with DMSO vehicle alone. Cell lysates were prepared and analysed by SDS-PAGE and Western blotting with anti-phosphoPKD-Ser916, anti-PKD and with anti-HRV 2C antibodies.

Examples

Materials and Methods Cell culture. The cervical epithelial HeLa Ohio (ECACC 93021013) and HeLa Hi (ATCC CRL-i958) cell lines were maintained in exponential growth in high glucose DMEM supplemented with Glutamine, 1% Sodium Bicarbonate, 25mM HEPES, and 10% foetal bovine serum (FBS).

HRV16 stock production and infections. HRV16 viral stocks (ATCC VR-283) were produced by infecting HeLa Hi cells. The viral titre was determined by TCID50 in HeLa Ohio cells. HeLa Ohio cells were infected with HRV16 at a multiplicity of infection (MOl) of 20 at different time points. Infections were synchronized by incubating the virus for 1 h at room temperature (RT), followed by a wash with phosphate buffer saline (PBS) and addition of fresh media before incubating the cells at 37°C for the different time points.

PKD inhibitors. Cells were pre-treated with different PKD inhibitors (CRTOO66iO1, R&D Systems; Cl 02011756, Tocris) or with OMSO vehicle alone for i h at 37°C and either incubated with PDBu at 200 nM in the presence of the inhibitor for 15 mm or infected with HRV16 at an MOI of 20 for the indicated time points. 3HC

is)OLuHo CRTOO661O1 C10201 1756 TCID50 determination. HeLa Ohio cells were incubated at a concentration of lO5cells/ml in DMEM supplemented with Glutamine, 1% Sodium Bicarbonate, 25mM HEPES, 2% FBS and i%Penicillin/Streptomycin, with 8 10-fold serial dilutions of the virus in six replicates for 4 days using 96-well plates. Titration was assessed by the presence or absence of cytopathic effect (CPE) in each well, using as a positive control HRV16 stock. TCID50 was calculated using the Reed & Muench calculator. To measure the viral replication after PKD inhibitors treatment, media and cells from infected cultures were collected at the indicated time points. Cells were lysed by freeze/thaw cycles and centrifuged to remove cellular debris. The supernatant containing the virus was titrated as the viral stock.

Immunofluorescence microscopy. Cells grown on glass coverslips were washed with PBS, fixed for 1 5mm with 4% formaldehyde and washed with PBS. After quenching residual formaldehyde with DiM glycine, cells were washed with PBS and permeabilized for 10 mm at RT with 0.1% Triton X-i00 and then washed with PBS. After blocking in 5% EBS, cells were sequentially incubated with primary and secondary antibodies diluted in 1% bovine serum albumin (BSA). Rabbit anti-HRV16 2C antibody (this study, 1:500) was incubated for lb and the secondary antibody (Jackson lmmunoResearch, 1:200) was incubated for 45mm, both of them at room temperature (RI). Cells were washed in PBS between antibody incubations. Coverslips were mounted in ProLong Gold antifade reagent (Invitrogen) and analyzed using a LSM 5 PASCAL Laser Scanning microscope (Carl Zeiss).

Western blotting. Cells were lysed in Laerrmli sample buffer and their DNA content was measured with a Nanodrop. Equivalent amount of DNA was loaded for each sample and proteins were separated by SDS-PAGE. After the transference to PVDF membranes, membranes were blocked with TBS1XI5% BSA/0.1% Iween-20 for lh at RT. Primary antibodies were incubated overnight at 4°C and secondary antibodies were incubated for 1 h at ST. The following primary antibodies were used at the indicated dilution: anti-phosphoPKDl Ser744174° (Cell Signaling, 1:1000), anti-phosphoPKDl 5er916 (Cell Signaling, 1:1000), anti-PKD1 (1:2000, Cell Signaling), HRV16 2C antiserum (this study, 1:15000) and anti-13-actin antibody (Bio Vision, 1:1000). Secondary antibodies conjugated to HRP were obtained from Jackson lmmunoResearch and were revealed using ECL reagent (Geneflow) and analysed on a Fusion FX7 machine (Vilber Lourmat).

Generation of antibodies. A full length cDNA sequence for HRV16 2C with an N-terminal 6-His tag was codon optimised and cloned into the bacterial expression plasmid pET-26b (Novagen) using the Ndel/Xhol sites. The full length 2C protein was expressed in E.coli BL21 (DE3) and purified from inclusion bodies. The identity of the purified protein was confirmed by peptide mass fingerprinting. Purified HRV1S 2C protein was used to immunize two rabbits using standard protocols (Covalab, France. www.covalab.com). All antisera titres were quantified by ELISA and their specificity was tested by Western blotting.

Results We first established a replication assay for Human Rhinovirus (HRV) in HeLa Ohio cells.

Cells were grown on glass coverslips and were infected with HRV16 at a multiplicity of infection (MOl) 20. Infections were synchronized by virus adsorption to the cells for lh at RI, followed by one wash with PBS and addition of fresh media before incubating the cells at 37°C for 2 to 7h, as indicated. At the end of each time point, cells were fixed, stained for HRV 2C and processed for Imniunofluorescence confocal microscopy. As seen in Figure 8, 20 expression (indicative of viral replication) can begin to be seen between 3-4 hours post infection.

In a similar experimental study, HeLa cells were infected with HRV16 and at the end of each time point cell lysates were prepared. HRV 20 expression was analysed by Western Blotting (Fig 9).

We next went on to examine if HRV16 infection and replication in HeLa cells activated PKD (Figure 10). As controls, cells were either untreated (Oh) or stimulated with Phorbol Ester (PDBu at 200nM for 15 mm). In parallel, cells were infected with HRV16 at a MOI of for 0-7 hours. Cell extracts were prepared and analysed by Western blotting. Viral replication was confirmed by 20 and 2BC expression which can clearly be seen at 4 hours post infection. As loading controls the blot was stained for -actin and with an antibody to PKD1. The blot was also stained with antibodies that specifically recognise PKD1 phosphorylated at Ser7441748 and Ser916. The 3er7441748 phosphorylation site is within the activation loop of the kinase and is indicative of activation of the kinase by upstream effectors such as FKCc. The Ser916 phosphorylation site is in the C-terminus of PKD and is an autophosphorylation site indicative that PKD has been activated. It is clear that PDBu treatment causes phosphorylation at both sites and it is also clear that HRV16 replication also causes phosphorylation at both sites. This is the first time this has been reported for a Picornavirus.

Having established that HRV16 activates PKD and having assay conditions to measure viral replication we went on to test PKD inhibitors. The first inhibitor we tested was 0RT0066101. We confirmed that it was a PKD inhibitor by activating HeLa cells with PDBu (200nM) with increasing concentrations of the CRTOO661O1 inhibitor. Cells were pre-treated with the inhibitor for 1 h and the incubated with PDBu in the presence of the inhibitor for a further 15 mm. Cell lysates were prepared and analysed by Western Blofting with the anti-phosphoPKD-Ser°16 antibody to detect PKD activity. It is clear that the compound produced a dose-dependent inhibition of PKD activity. Total PKD was also revealed as a loading control (Figure 11).

Having confirmed that CRTOO661O1 is an effective PKD inhibitor and establishing a suitable concentration-response curve, we went on to test if CRTOO661O1 would inhibit HRV16 replication. We tested this in two ways. Firstly we infected HeLa cells with HRV16 for 6h at a MCI of 20 in the presence of increasing concentrations of the drug from 0.1 pM to 50 pM (Figure 12). Cell lysates were prepared and analysed by Western blotting with the anti-phosphoPKD-Ser916 antibody to demonstate PKD activation, with anti-HRV 20 to detect viral replication and with anti-PKD as a loading control. As additional controls, cells were either uninfected (Oh) or uninfected but treated with Phorbol Ester (PDBu) to activate PKD. The drug at low concentrations produced a small activation of PKD before inhibiting the kinase at higher concentrations. This is consistent with the theory of ATP pocket occupancy (in this case by an ATP competitive drug) priming kinases for phosphorylation/maturation as proposed for the AGO kinase family. The PKD loading control confirms equivalent amounts of cell lysate were loaded in each lane, and the HRV 2C staining clearly reveal viral replication is completely blocked at drug concentraions above2pM.

We went on to confirm the antiviral effect by quantifying viral replication (Figure 13). HeLa cells were infected with HRV16 at a MOl of 20 for 6 hours with increasing concentrations of drug. The cells were then lysed by freeze thawing and the amount of virus produced determined by titration in a TCID50 assay as described. This result confirmed the 2C Western blot showing viral replication is totally blocked at concentrations of drug above 2pM In order to confirm the role of PKD in HRV16 replication, we tested a structurally unrelated PKD inhibitor: 002011756 (Figure 14). In an identical experimental design to Figure 12, we can clearly see that C1D2011756 also inhibits HRV16 replication, albeit less potently than CRTOO661O1.

Claims (14)

1. CLAIMS1. A protein kinase D (PKD) inhibitor for use in a method of treating or preventing picornavirus infection.
2. 2. A PKD inhibitor for use according to claim 1, wherein the PKD inhibitor is selected from the group consisting of a small molecule, a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), an antisense RNA, a ribozyme, a DNAzyme and a cDNA encoding an inactive mutant of PKD.
3. 3. A PKD inhibitor for use according to claim 1, wherein the picornavirus is selected from the group consisting of rhinovirus, polio virus, foot and mouth disease virus, hepatitis A virus, coxsackievirus (CV) and an enterovirus.
4. 4. A PKD inhibitor for use according to claim 2, wherein the picornavirus is rhinovirus.
5. 5. A PKD inhibitor for use according to claim 3, wherein the subject being treated is also suffering from asthma and/or chronic obstructive pulmonary disease (COPD).
6. 6. A PKD inhibitor for use according to any one of the preceding claims, wherein the PKD inhibitor is administered as part of a pharmaceutical composition.
7. 7. A PKD inhibitor for use according to claim 6, wherein said pharmaceutical composition further comprises another therapeutically active agent.
8. 8. A PKD inhibitor for use according to claim 7, wherein said therapeutically active agent is an antiviral agent or an anti-inflammatory agent.
9. 9. A FKD inhibitor for use according to claim 8, wherein said antiviral agent is brefeldin A or wherein said anti-inflammatory agent is a glucocorticoid receptor agonist.
10. 10. A PKD inhibitor for use according to claim 9, wherein said glucocorticoid receptor agonist is selected from the group consisting of beclomethasone, fluticasone, mometasone, budesonide and ciclesonide.
11. 11. Use of a PKD inhibitor in the manufacture of a medicament for the treatment or prevention of picornavirus infection.
12. 12. A method of treating or preventing picornavirus infection, comprising administering a PKD inhibitor to a subject in need thereof.
13. 13. A method of identifying a compound useful for the treatment or prevention of picornavirus infection, comprising determining whether said compound is a PKD inhibitor.
14. 14. A method according to claim 14, further comprising determining whether said compound inhibits picornavirus replication.