Agents for treatment of hcv and methods of use

Abstract

An amphipathic helix at the approximate N-terminus of Hepatitis C virus (HCV) nonstructural proteins mediates the association of these proteins with cytoplasmic membranes in infected cells. This association is essential for replication. Thus, assessing the ability of compounds or protocols to disrupt the association of such helices with cytoplasmic membranes permits identification of compounds and protocols which are useful in the treatment of HCV infection. Also useful in the invention are mimics, or function-disrupting ligands, of an amphipathic helix of the nonstructural proteins described herein and antibodies and fragments thereof immunoreactive with said helix.

Description

FIELD OF THE INVENTION

[0001] The invention relates generally to Hepatitis C virus (HCV) infection, and more specifically to interrupting the mechanism of infection by HCV and to methods to identify agents, which effect this interruption. The invention also relates to interfering with the ability of HCV components to bind with cellular membranes of an infected cell.

BACKGROUND

[0002] Hepatitis C virus (HCV) establishes a chronic infection in a high percentage of infected individuals and is associated with progressive liver pathology, including cirrhosis and hepatocellular carcinoma. Antiviral drugs such as interferon alpha and ribavarin have had limited success in controlling HCV infection. As a result, it has become the leading cause for liver transplantation in the US. The HCV polyprotein comprises, from the amino terminus to the carboxy terminus, the core protein (C), the envelope proteins (E1 and E2), p7, a membrane bound protein, whose function is unknown and the non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) which are believed to be important for replication. C codes for the core nucleocapsid protein, E1 and E2 are envelope proteins that coat the virus, NS2, NS3 and NS4A are involved in proteolytic processing of the HCV polyprotein, and NS5B has RNA polymerase activity. The functions of NS4B and NS5A are unknown.

[0003] Hepatitis C virus is a significant cause of morbidity and mortality, infecting over 100,000,000 people worldwide. Annual HCV related costs in the United States are about $1 billion. Current therapies are clearly inadequate; the best available treatment at present is the combination of interferon and ribavirin, a treatment which is inconveniently lengthy as it typically lasts over one and a half years, difficult to tolerate in that most patients have flu-like symptoms, and extremely expensive as the cost is in the range of thousands of dollars annually. Not only does the present treatment have these disadvantages, but it is also not particularly effective.

[0004] Certain interactions of viral proteins with cell membranes have previously been described. For example, in poliovirus and Hepatitis A virus, the nonstructural mammalian cells, or preferably using adenoviral or retroviral or other suitable viral vectors.

[0005] As described above, however, these competitor peptides need not be the native sequences per se and need not even be peptides per se, but may contain isosteric linkages or other polymeric features that result in similar charge/shape features as compared to the native helices.

[0006] Peptides, or compounds with similar charge/shape features and having the activity of the peptides described herein, can be identified by phage display using wild-type amphipathic helix and a mutant amphipathic helix peptides as positive and negative selectors, respectively.

[0007] The compositions or agents of the invention may comprise, consist essentially of, or consist of the peptide sequences disclosed herein. The phrase “consists essentially of or consisting essentially of” or the like, when applied to anti-HCV peptides encompassed by the present invention refers to peptide sequences like those disclosed herein, but which contain additional amino acids (or analogs or derivatives thereof as discussed above). Such additional amino acids, etc., however, do not materially affect the basic and novel characteristic(s) of these peptides in modulating, attenuating, or inhibiting HCV infection, replication, and/or pathogenesis, including the specific quantitative effects of these peptides, compared to those of the corresponding peptides disclosed herein.

[0008] In one approach, the agent may be a transdominant inhibitor of the membrane association function whereby forms of the amphipathic helix that interfere with the ability of the helix to form oligomers can be used. Thus, by generating or providing a mutant form of the helix containing one or more amino acid substutions, this form may associate with the native helix to provide an inactive form or rendering it unable to dimerize or oligomerize with additional native forms. In one approach, the decoy peptide is mutated to convert hydrophobic amino acids to hydrophilic ones thus destroying the hydrophobic face of the helix. For example, mutated versions of the peptide sequence for NS5A strains would include

SGSWLRDDWDWECEVLSDDKTWLKAK   (SEQ ID NO: 15)
or
SGSWLRDDWDWECTVLTDDKTWLQSKL. (SEQ ID NO: 16)

[0009] SEQ ID NOS: 15 and 16 are used as PEP2 in Example 4, below.

[0010] In another approach, the agent is a competitive inhibitor of the amphipathic helix. These competitive inhibitors may interrupt the binding between the helix and the membrane, achieving the desired effect. Such inhibitors may be fragments of the wild type sequence of the amphipathic sequence or variants or mutants thereof. Fragments of the HCV nonstructural proteins that may be used as competitive inhibitors may include, but are not limited to:

YIEQGMMLAEQFKQKALGLLQTASRHAEV,	(NS4B 6-34)	(SEQ ID NO: 462)
QDVLKEVKAAASKVKANLLSVEE,	(NS5B 65-87)	(SEQ ID NO: 3)
and
DVRCHARKAVAHINSVWKD.	(NS5B 107-125)	(SEQ ID NO: 4)

[0011] Another competitive inhibitor of NS5A would include: SGSWLRDVWDWICTVLTDFKTWLQSKL (SEQ ID NO: 14) and variants and mutants thereof. Variants and mutants of this peptide would include a peptide with one or more of the following amino acid substitutions: substitution of L at amino acid 16 by A or K; or substitution of the T at amino acid 17 by A; or substitution of the D at amino acid 18 by A; or substitution of the F at amino acid 19 by A; or substitution of the K at amino acid 20 by A; or substitution of the W at amino acid 22 by A; or substitution of the L at amino acid 23 by K, and derivatives thereof. Another such mutant of NS5A is: SGSX1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16 X17X18X19X20QSK L, where X1 is W, A or F; X2 is L, A or K; X3 is R, A or N; X4 is D, A or S;X5 is V, I, D or S; X6 is W, A or F; X7 is D, A or S; X8 is W,A or F; X9 is I, E or L; X10 is C, A or S; X11 is T, E, A or W; X12 is V, D or S; X13 is L, A or K; X14is T, S, A or W; X15is D, A or S; X16is F,D, or L; X17is K, A or L; X18 is T, A or W; X19 is W, A or F; and X20 is L, A or K. In another embodiment, X1, X2, X4, X6, X8, X10, X13, X15, X16 or X20may be D or X16 is A. Where the agent is a competitive inhibitor, some of the above mutations would enhance the inhibitory activity of the peptide, others would completely or partially abolish the inhibitory activity of the peptide. Mutations may be observed alone or in combination, for example, any one substitution, X1-20, occurs within the context of the wild type sequence at any one time, a combination of two mutations such as: X5 and X9, X5 and X16, or X9 and X16 or three mutations are found in the same peptide, X5, with X9, and X16. Additional mutations of the above inhibitor may include the sequence: X1X2DVWDWICTX3X4X5X6X7X8X9, wherein when X1 and X2 are present, X1 is L and X2 is R; wherein X1 and X2 are present, X3, X4, X5, X6, X7, X8 and X9 are optionally present, and when X3, X4, X5, X6, X7, X8 and X9 are present, X3 is V, X4 is L, X5 is T, X6 is D, X7 is F, X8 is K and X9 is T; and wherein X6 is present, X3, X4 and X5 are all present, wherein X6 is D, X3 is V, X4 is L, and X5 is T. In particular where the sequence is LRDVWDWICTVLTDFKT (SEQ ID NO: 463), LRDVWDWICT (SEQ ID NO: 464) or DVWDWICTVLTD (SEQ ID NO: 465). In another embodiment, the competitive inhibitor may be SWLRDVWDWIC (SEQ ID NO: 466).

[0012] A mutant, and competitive inhibitor, of NS4B may include the sequence:

X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18X19
X20X21X22X23X24X25X26X27X28X29
where X1 is Y, A or H; X2 is I, E or L; X3 is E,
A or Q; X4 is Q, V or R; X5 is G, A or D; X6 is
M, E or C; X7 is M, E or C; X8 is L, E or Q; X9
is A, D or S; X10 is E, A or Q; X11 is Q, V, or
R; X12 is F, D or L; X13 is K, I or Q; X14 is Q,
V, or R; X15 is K, I or Q; X16 is A, D or G; X17
is L, A or K; X18 is G, E or S; X19 is L, A or K;
X20 is L, A or K; X21 is Q, V or R; X22 is T, A
or W; X23 is A, D or G; X24 is S, D or A; X25 is
R, L or W; X26 is Q, V or R; X27 is A, D or G;
and X28 is E, A or Q.

[0013] While the majority of the above mutations and substitutions are conservative (i.e. wild type hydrophobic residues are substituted with additional hydrophobic residues (for example, A to F), charged residues are substituted with similarly charged residues, etc.), it is noted that nonconservative substitutions may also be performed. For example, where a wild type hydrophobic residue is substituted with a hydrophilic residue, or a negatively charged residue (for example, D) is substituted with a positively charged residue (for example, K).

[0014] Where the agent is a competitive inhibitor, some of the above mutations would enhance the inhibitory activity of the peptide, others would completely or partially abolish the inhibitory activity of the peptide. Mutations may be observed alone or in combination, for example, any one substitution, X1-28, occurs within the context of the wild type sequence at any one time, a combination of two mutations such as: X2 and X5, X2 and X19, or X5 and X19 or three mutations are found in the same peptide, X2, with X5, and X19.

[0015] In another embodiment, the agent may be a complementary peptide to the helix. Complementary peptides may interrupt the binding between the helix and the membrane, achieving the desired effect. Such complementary peptides may also inhibit the formation of the amphipathic helix, may interact or bind to the helix to inhibit binding of the helix to cellular membranes, or may otherwise inhibit the amphipathic helices.

[0016] For example, the HCV genomic RNA sequence that codes for the NS4B amphipathic helix in the HCV genotype 1A sequence is:

UACAUCGAGCAAGGGAUGAUGCUCGCTGAGCAGU (SEQ ID NO: 17)
UCAAGCAGAAGGCCCUCGGCCUCCUGCAGACCGC
GUCCCGCCAAGCAGAG.

[0017] (Kolykhalov, A. A. and Rice, C. M., Science 277 (5325), 570-574 (1997); GenBank Accession No.: AF009606.) The protein translation of the above sequence is: YIEQGMMLAEQFKQKALGLLQTASRQAE (SEQ ID NO: 18). The reverse complement cDNA sequence corresponding to the HCV genomic RNA sequence that codes for the NS4B amphipathic helix (GenBank Accession No.: AF009606) is: CTCTGCTTGGCGGGACGCGGTCTGCAGGAGGCCGAGGGCCTTCTGCTTGA ACTGCTCAGCGAGCATCATCCCTTGCTCGATGTA (SEQ ID NO: 19). The complementary peptide translated from the reverse complement sequence is: LCLAGRGLQEAEGLLLELLSEHHPLLDV (SEQ ID NO: 20). This complementary peptide, as a whole, or in part, may be active as an HCV antiviral or may be useful in the prediction of small molecules that are HCV antivirals. In one embodiment a fragment of this complementary peptide comprising 6-27 amino acids may be used in the discovery of HCV antivirals. Table 1 sets forth exemplary peptides (SEQ ID NOS: 21-295) of this embodiment.

TABLE 1
All smaller NS4B complementary peptides with
6 or more amino acids:
                            Sequence
Sequence                    Identification Number
LQEAEG                      SEQ ID NO: 21
CLAGRG                      SEQ ID NO: 22
LELLSE                      SEQ ID NO: 23
GLQEAE                      SEQ ID NO: 24
LLELLS                      SEQ ID NO: 25
QEAEGL                      SEQ ID NO: 26
RGLQEA                      SEQ ID NO: 27
LAGRGL                      SEQ ID NO: 28
GRGLQE                      SEQ ID NO: 29
ELLSEH                      SEQ ID NO: 30
LLLELL                      SEQ ID NO: 31
LCLAGR                      SEQ ID NO: 32
HPLLDV                      SEQ ID NO: 33
AEGLLL                      SEQ ID NO: 34
AGRGLQ                      SEQ ID NO: 35
EAEGLL                      SEQ ID NO: 36
LSEHHP                      SEQ ID NO: 37
HHPLLD                      SEQ ID NO: 38
EGLLLE                      SEQ ID NO: 39
SEHHPL                      SEQ ID NO: 40
LLSEHH                      SEQ ID NO: 41
EHHPLL                      SEQ ID NO: 42
GLLLEL                      SEQ ID NO: 43
LELLSEH                     SEQ ID NO: 44
ELLSEHH                     SEQ ID NO: 45
LLELLSE                     SEQ ID NO: 46
LLSEHHP                     SEQ ID NO: 47
HHPLLDV                     SEQ ID NO: 48
GLLLELL                     SEQ ID NO: 49
LAGRGLQ                     SEQ ID NO: 50
LSEHHPL                     SEQ ID NO: 51
LCLAGRG                     SEQ ID NO: 52
LQEAEGL                     SEQ ID NO: 53
AGRGLQE                     SEQ ID NO: 54
AEGLLLE                     SEQ ID NO: 55
GRGLQEA                     SEQ ID NO: 56
RGLQEAE                     SEQ ID NO: 57
QEAEGLL                     SEQ ID NO: 58
SEHHPLL                     SEQ ID NO: 59
EHHPLLD                     SEQ ID NO: 60
GLQEAEG                     SEQ ID NO: 61
EGLLLEL                     SEQ ID NO: 62
LLLELLS                     SEQ ID NO: 63
EAEGLLL                     SEQ ID NO: 64
CLAGRGL                     SEQ ID NO: 65
CLAGRGLQ                    SEQ ID NO: 66
QEAEGLLL                    SEQ ID NO: 67
LAGRGLQE                    SEQ ID NO: 68
LCLAGRGL                    SEQ ID NO: 69
AGRGLQEA                    SEQ ID NO: 70
LLLELLSE                    SEQ ID NO: 71
LELLSEHH                    SEQ ID NO: 72
AEGLLLEL                    SEQ ID NO: 73
EGLLLELL                    SEQ ID NO: 74
LLSEHHPL                    SEQ ID NO: 75
GRGLQEAE                    SEQ ID NO: 76
EAEGLLLE                    SEQ ID NO: 77
RGLQEAEG                    SEQ ID NO: 78
LLELLSEH                    SEQ ID NO: 79
SEHHPLLD                    SEQ ID NO: 80
LSEHHPLL                    SEQ ID NO: 81
LQEAEGLL                    SEQ ID NO: 82
ELLSEHHP                    SEQ ID NO: 83
GLLLELLS                    SEQ ID NO: 84
EHHPLLDV                    SEQ ID NO: 85
GLQEAEGL                    SEQ ID NO: 86
LLSEHHPLL                   SEQ ID NO: 87
LELLSEHHP                   SEQ ID NO: 88
RGLQEAEGL                   SEQ ID NO: 89
ELLSEHHPL                   SEQ ID NO: 90
LCLAGRGLQ                   SEQ ID NO: 91
AGRGLQEAE                   SEQ ID NO: 92
GLLLELLSE                   SEQ ID NO: 93
LSEHHPLLD                   SEQ ID NO: 94
EGLLLELLS                   SEQ ID NO: 95
GLQEAEGLL                   SEQ ID NO: 96
AEGLLLELL                   SEQ ID NO: 97
QEAEGLLLE                   SEQ ID NO: 98
SEHHPLLDV                   SEQ ID NO: 99
LLLELLSEH                   SEQ ID NO: 100
GRGLQEAEG                   SEQ ID NO: 101
CLAGRGLQE                   SEQ ID NO: 102
EAEGLLLEL                   SEQ ID NO: 103
LAGRGLQEA                   SEQ ID NO: 104
LLELLSEHH                   SEQ ID NO: 105
LQEAEGLLL                   SEQ ID NO: 106
LSEHHPLLDV                  SEQ ID NO: 107
LELLSEHHPL                  SEQ ID NO: 108
RGLQEAEGLL                  SEQ ID NO: 109
LLSEHHPLLD                  SEQ ID NO: 110
AEGLLLELLS                  SEQ ID NO: 111
GLQEAEGLLL                  SEQ ID NO: 112
EGLLLELLSE                  SEQ ID NO: 113
LCLAGRGLQE                  SEQ ID NO: 114
LLLELLSEHH                  SEQ ID NO: 115
EAEGLLLELL                  SEQ ID NO: 116
GLLLELLSEH                  SEQ ID NO: 117
ELLSEHHPLL                  SEQ ID NO: 118
GRGLQEAEGL                  SEQ ID NO: 119
LQEAEGLLLE                  SEQ ID NO: 120
LLELLSEHHP                  SEQ ID NO: 121
CLAGRGLQEA                  SEQ ID NO: 122
QEAEGLLLEL                  SEQ ID NO: 123
AGRGLQEAEG                  SEQ ID NO: 124
LAGRGLQEAE                  SEQ ID NO: 125
LLELLSEHHPL                 SEQ ID NO: 126
CLAGRGLQEAE                 SEQ ID NO: 127
RGLQEAEGLLL                 SEQ ID NO: 128
LAGRGLQEAEG                 SEQ ID NO: 129
LQEAEGLLLEL                 SEQ ID NO: 130
LLLELLSEHHP                 SEQ ID NO: 131
ELLSEHHPLLD                 SEQ ID NO: 132
AGRGLQEAEGL                 SEQ ID NO: 133
LLSEHHPLLDV                 SEQ ID NO: 134
LCLAGRGLQEA                 SEQ ID NO: 135
GLLLELLSEHH                 SEQ ID NO: 136
LELLSEHHPLL                 SEQ ID NO: 137
QEAEGLLLELL                 SEQ ID NO: 138
EGLLLELLSEH                 SEQ ID NO: 139
GRGLQEAEGLL                 SEQ ID NO: 140
AEGLLLELLSE                 SEQ ID NO: 141
GLQEAEGLLLE                 SEQ ID NO: 142
EAEGLLLELLS                 SEQ ID NO: 143
EGLLLELLSEHH                SEQ ID NO: 144
RGLQEAEGLLLE                SEQ ID NO: 145
GLLLELLSEHHP                SEQ ID NO: 146
LQEAEGLLLELL                SEQ ID NO: 147
GLQEAEGLLLEL                SEQ ID NO: 148
LAGRGLQEAEGL                SEQ ID NO: 149
GRGLQEAEGLLL                SEQ ID NO: 150
QEAEGLLLELLS                SEQ ID NO: 151
AEGLLLELLSEH                SEQ ID NO: 152
AGRGLQEAEGLL                SEQ ID NO: 153
LELLSEHHPLLD                SEQ ID NO: 154
LCLAGRGLQEAE                SEQ ID NO: 155
LLLELLSEHHPL                SEQ ID NO: 156
LLELLSEHHPLL                SEQ ID NO: 157
ELLSEHHPLLDV                SEQ ID NO: 158
EAEGLLLELLSE                SEQ ID NO: 159
CLAGRGLQEAEG                SEQ ID NO: 160
CLAGRGLQEAEGL               SEQ ID NO: 161
RGLQEAEGLLLEL               SEQ ID NO: 162
LLELLSEHHPLLD               SEQ ID NO: 163
GRGLQEAEGLLLE               SEQ ID NO: 164
LCLAGRGLQEAEG               SEQ ID NO: 165
LELLSEHHPLLDV               SEQ ID NO: 166
EGLLLELLSEHHP               SEQ ID NO: 167
EAEGLLLELLSEH               SEQ ID NO: 168
GLQEAEGLLLELL               SEQ ID NO: 169
AGRGLQEAEGLLL               SEQ ID NO: 170
AEGLLLELLSEHH               SEQ ID NO: 171
LAGRGLQEAEGLL               SEQ ID NO: 172
QEAEGLLLELLSE               SEQ ID NO: 173
LLLELLSEHHPLL               SEQ ID NO: 174
LQEAEGLLLELLS               SEQ ID NO: 175
GLLLELLSEHHPL               SEQ ID NO: 176
EAEGLLLELLSEHH              SEQ ID NO: 177
EGLLLELLSEHHPL              SEQ ID NO: 178
AGRGLQEAEGLLLE              SEQ ID NO: 179
LCLAGRGLQEAEGL              SEQ ID NO: 180
GLLLELLSEHHPLL              SEQ ID NO: 181
QEAEGLLLELLSEH              SEQ ID NO: 182
RGLQEAEGLLLELL              SEQ ID NO: 183
LLLELLSEHHPLLD              SEQ ID NO: 184
LQEAEGLLLELLSE              SEQ ID NO: 185
LLELLSEHHPLLDV              SEQ ID NO: 186
GLQEAEGLLLELLS              SEQ ID NO: 187
GRGLQEAEGLLLEL              SEQ ID NO: 188
AEGLLLELLSEHHP              SEQ ID NO: 189
CLAGRGLQEAEGLL              SEQ ID NO: 190
LAGRGLQEAEGLLL              SEQ ID NO: 191
LLLELLSEHHPLLDV             SEQ ID NO: 192
AGRGLQEAEGLLLEL             SEQ ID NO: 193
AEGLLLELLSEHHPL             SEQ ID NO: 194
GLLLELLSEHHPLLD             SEQ ID NO: 195
LQEAEGLLLELLSEH             SEQ ID NO: 196
GLQEAEGLLLELLSE             SEQ ID NO: 197
LCLAGRGLQEAEGLL             SEQ ID NO: 198
EAEGLLLELLSEHHP             SEQ ID NO: 199
GRGLQEAEGLLLELL             SEQ ID NO: 200
QEAEGLLLELLSEHH             SEQ ID NO: 201
LAGRGLQEAEGLLLE             SEQ ID NO: 202
CLAGRGLQEAEGLLL             SEQ ID NO: 203
EGLLLELLSEHHPLL             SEQ ID NO: 204
RGLQEAEGLLLELLS             SEQ ID NO: 205
LAGRGLQEAEGLLLEL            SEQ ID NO: 206
GLLLELLSEHHLPLLDV           SEQ ID NO: 207
GRGLQEAEGLLLELLS            SEQ ID NO: 208
AGRGLQEAEGLLLELL            SEQ ID NO: 209
GLQEAEGLLLELLSEH            SEQ ID NO: 210
RGLQEAEGLLLELLSE            SEQ ID NO: 211
LQEAEGLLLELLSEHH            SEQ ID NO: 212
CLAGRGLQEAEGLLLE            SEQ ID NO: 213
QEAEGLLLELLSEHHP            SEQ ID NO: 214
AEGLLLELLSEHHPLL            SEQ ID NO: 215
EAEGLLLELLSEHHPL            SEQ ID NO: 216
LCLAGRGLQEAEGLLL            SEQ ID NO: 217
EGLLLELLSEHHPLLD            SEQ ID NO: 218
LCLAGRGLQEAEGLLLE           SEQ ID NO: 219
QEAEGLLLELLSEHHPL           SEQ ID NO: 220
EAEGLLLELLSEHHPLL           SEQ ID NO: 221
RGLQEAEGLLLELLSEH           SEQ ID NO: 222
CLAGRGLQEAEGLLLEL           SEQ ID NO: 223
GRGLQEAEGLLLELLSE           SEQ ID NO: 224
LAGRGLQEAEGLLLELL           SEQ ID NO: 225
AGRGLQEAEGLLLELLS           SEQ ID NO: 226
GLQEAEGLLLELLSEHH           SEQ ID NO: 227
EGLLLELLSEHHPLLDV           SEQ ID NO: 228
AEGLLLELLSEHHPLLD           SEQ ID NO: 229
LQEAEGLLLELLSEHHP           SEQ ID NO: 230
CLAGRGLQEAEGLLLELL          SEQ ID NO: 231
LQEAEGLLLELLSEHHPL          SEQ ID NO: 232
GRGLQEAEGLLLELLSEH          SEQ ID NO: 233
EAEGLLLELLSEHHPLLD          SEQ ID NO: 234
AEGLLLELLSEHHPLLDV          SEQ ID NO: 235
GLQEAEGLLLELLSEHHP          SEQ ID NO: 236
RGLQEAEGLLLELLSEHH          SEQ ID NO: 237
QEAEGLLLELLSEHHPLL          SEQ ID NO: 238
LAGRGLQEAEGLLLELLS          SEQ ID NO: 239
LCLAGRGLQEAEGLLLEL          SEQ ID NO: 240
AGRGLQEAEGLLLELLSE          SEQ ID NO: 241
AGRGLQEAEGLLLELLSEH         SEQ ID NO: 242
GLQEAEGLLLELLSEHHPL         SEQ ID NO: 243
GRGLQEAEGLLLELLSEHH         SEQ ID NO: 244
LCLAGRGLQEAEGLLLELL         SEQ ID NO: 245
CLAGRGLQEAEGLLLELLS         SEQ ID NO: 246
RGLQEAEGLLLELLSEHHP         SEQ ID NO: 247
LAGRGLQEAEGLLLELLSE         SEQ ID NO: 248
QEAEGLLLELLSEHHPLLD         SEQ ID NO: 249
LQEAEGLLLELLSEHHPLL         SEQ ID NO: 250
EAEGLLLELLSEHHPLLDV         SEQ ID NO: 251
RGLQEAEGLLLELLSEHHPL        SEQ ID NO: 252
GLQEAEGLLLELLSEHHPLL        SEQ ID NO: 253
AGRGLQEAEGLLLELLSEHH        SEQ ID NO: 254
LAGRGLQEAEGLLLELLSEH        SEQ ID NO: 255
GRGLQEAEGLLLELLSEHHP        SEQ ID NO: 256
LCLAGRGLQEAEGLLLELLS        SEQ ID NO: 257
LQEAEGLLLELLSEHHPLLD        SEQ ID NO: 258
QEAEGLLLELLSEHHPLLDV        SEQ ID NO: 259
CLAGRGLQEAEGLLLELLSE        SEQ ID NO: 260
LCLAGRGLQEAEGLLLELLSE       SEQ ID NO: 261
RGLQEAEGLLLELLSEHHPLL       SEQ ID NO: 262
LQEAEGLLLELLSEHHPLLDV       SEQ ID NO: 263
GRGLQEAEGLLLELLSEHHPL       SEQ ID NO: 264
AGRGLQEAEGLLLELLSEHHP       SEQ ID NO: 265
LAGRGLQEAEGLLLELLSEHH       SEQ ID NO: 266
CLAGRGLQEAEGLLLELLSEH       SEQ ID NO: 267
GLQEAEGLLLELLSEHHPLLD       SEQ ID NO: 268
GLQEAEGLLLELLSEHHPLLDV      SEQ ID NO: 269
AGRGLQEAEGLLLELLSEHHPL      SEQ ID NO: 270
CLAGRGLQEAEGLLLELLSEHH      SEQ ID NO: 271
GRGLQEAEGLLLELLSEHHPLL      SEQ ID NO: 272
RGLQEAEGLLLELLSEHHPLLD      SEQ ID NO: 273
LAGRGLQEAEGLLLELLSEHHP      SEQ ID NO: 274
LCLAGRGLQEAEGLLLELLSEH      SEQ ID NO: 275
CLAGRGLQEAEGLLLELLSEHHP     SEQ ID NO: 276
GRGLQEAEGLLLELLSEHHPLLD     SEQ ID NO: 277
LCLAGRGLQEAEGLLLELLSEHH     SEQ ID NO: 278
LAGRGLQEAEGLLLELLSEHHPL     SEQ ID NO: 279
RGLQEAEGLLLELLSEHHPLLDV     SEQ ID NO: 280
AGRGLQEAEGLLLELLSEHHPLL     SEQ ID NO: 281
AGRGLQEAEGLLLELLSEHHPLLD    SEQ ID NO: 282
CLAGRGLQEAEGLLLELLSEHHPL    SEQ ID NO: 283
LCLAGRGLQEAEGLLLELLSEHHP    SEQ ID NO: 284
GRGLQEAEGLLLELLSEHHPLLDV    SEQ ID NO: 285
LAGRGLQEAEGLLLELLSEHHPLL    SEQ ID NO: 286
AGRGLQEAEGLLLELLSEHHPLLDV   SEQ ID NO: 287
LCLAGRGLQEAEGLLLELLSEHHPL   SEQ ID NO: 288
LAGRGLQEAEGLLLELLSEHHPLLD   SEQ ID NO: 289
CLAGRGLQEAEGLLLELLSEHHPLL   SEQ ID NO: 290
LCLAGRGLQEAEGLLLELLSEHHPLL  SEQ ID NO: 291
LAGRGLQEAEGLLLELLSEHHPLLDV  SEQ ID NO: 292
CLAGRGLQEAEGLLLELLSEHHPLLD  SEQ ID NO: 293
LCLAGRGLQEAEGLLLELLSEHHPLLD SEQ ID NO: 294
CLAGRGLQEAEGLLLELLSEHHPLLDV SEQ ID NO: 295

[0018] In a particular embodiment, the complementary peptides of the invention are used, in whole or in part, in the prediction of small molecules that are HCV antivirals. “Small molecule” as defined above, may include synthetic organic structures typical of pharmaceuticals, and may also include peptides, nucleic acids, peptide nucleic acids, carbohydrates, lipids, and the like. Additionally, small molecules, as used herein, may include chemically synthesized peptidomimetics of the 6-mer to 9-mer peptides of the invention.

[0019] Additionally, the HCV genomic RNA sequence that codes for the NS5A amphipathic helix in the HCV genotype 1A sequence is: UGGCUAAGGGACAUCUGGGACUGGAUAUGCGAGGUGCUGAGCGACUUU AAGACCUGGCUGAAAGCCAAGCUC (SEQ ID NO: 296). (Kolykhalov, A. A. and Rice, C. M., Science 277 (5325), 570-574 (1997); GenBank Accession No.: AF009606.) The protein translation of the above sequence is: WLRDIWDWICEVLSDFKTWLKAKL (SEQ ID NO: 297). The reverse complement cDNA sequence corresponding to the HCV genomic RNA sequence that codes for the NS5A amphipathic helix (GenBank Accession No.: AF009606) is: GAGCTTGGCTTTCAGCCAGGTCTTAAAGTCGCTCAGCACCTCGCATATCCA GTCCCAGATGTCCCTTAGCCA (SEQ ID NO: 298). This reverse complement sequence contains a stop codon at codon 23. The complementary peptide translated from the reverse complement sequence is: ELGFQPGLKVAQHLAYPVPDVP (SEQ ID NO: 299). This complementary peptide, as a whole, or in part, may be active as an HCV antiviral or may be useful in the prediction of small molecules that are HCV antivirals. In one embodiment a fragment of this complementary peptide comprising 6-21 amino acids may is used in the discovery of HCV antivirals. Table 2 sets forth exemplary peptides (SEQ ID NOS: 300-451) of this embodiment.

TABLE 2
NS5A complementary peptides with 6 or more
amino acids:
                      Sequence
Sequence              Identification Number
AQHLAY                SEQ ID NO: 300
LGFQPG                SEQ ID NO: 301
GLKVAQ                SEQ ID NO: 302
ELGFQP                SEQ ID NO: 303
PVPDVP                SEQ ID NO: 304
FQPGLK                SEQ ID NO: 305
GFQPGL                SEQ ID NO: 306
VAQHLA                SEQ ID NO: 307
LKVAQH                SEQ ID NO: 308
HLAYPV                SEQ ID NO: 309
KVAQHL                SEQ ID NO: 310
QPGLKV                SEQ ID NO: 311
LAYPVP                SEQ ID NO: 312
YPVPDV                SEQ ID NO: 313
QHLAYP                SEQ ID NO: 314
PGLKVA                SEQ ID NO: 315
AYPVPD                SEQ ID NO: 316
QPGLKVA               SEQ ID NO: 317
ELGFQPG               SEQ ID NO: 318
HLAYPVP               SEQ ID NO: 319
LAYPVPD               SEQ ID NO: 320
AQHLAYP               SEQ ID NO: 321
FQPGLKV               SEQ ID NO: 322
KVAQHLA               SEQ ID NO: 323
LGFQPGL               SEQ ID NO: 324
AYPVPDV               SEQ ID NO: 325
PGLKVAQ               SEQ ID NO: 326
LKVAQHL               SEQ ID NO: 327
GLKVAQH               SEQ ID NO: 328
VAQHLAY               SEQ ID NO: 329
GFQPGLK               SEQ ID NO: 330
QHLAYPV               SEQ ID NO: 331
YPVPDVP               SEQ ID NO: 332
AYPVPDVP              SEQ ID NO: 333
PGLKVAQH              SEQ ID NO: 334
QHLAYPVP              SEQ ID NO: 335
LAYPVPDV              SEQ ID NO: 336
LKVAQHLA              SEQ ID NO: 337
GLKVAQHL              SEQ ID NO: 338
GFQPGLKV              SEQ ID NO: 339
AQHLAYPV              SEQ ID NO: 340
VAQHLAYP              SEQ ID NO: 341
KVAQHLAY              SEQ ID NO: 342
ELGFQPGL              SEQ ID NO: 343
LGFQPGLK              SEQ ID NO: 344
FQPGLKVA              SEQ ID NO: 345
HLAYPVPD              SEQ ID NO: 346
QPGLKVAK              SEQ ID NO: 347
ELGFQPGLK             SEQ ID NO: 348
VAQHLAYPV             SEQ ID NO: 349
LGFQPGLKV             SEQ ID NO: 350
QPGLKVAQH             SEQ ID NO: 351
KVAQHLAYP             SEQ ID NO: 352
PGLKVAQHL             SEQ ID NO: 353
FQPGLKVAQ             SEQ ID NO: 354
GLKVAQHLA             SEQ ID NO: 355
LAYPVPDVP             SEQ ID NO: 356
HLAYPVPDV             SEQ ID NO: 357
GFQPGLKVA             SEQ ID NO: 358
AQHLAYPVP             SEQ ID NO: 359
QHLAYPVPD             SEQ ID NO: 360
LKVAQHLAY             SEQ ID NO: 361
QPGLKVAQHL            SEQ ID NO: 362
PGLKVAQHLA            SEQ ID NO: 363
HLAYPVPDVP            SEQ ID NO: 364
GLKVAQHLAY            SEQ ID NO: 365
VAQHLAYPVP            SEQ ID NO: 366
KVAQHLAYPV            SEQ ID NO: 367
GFQPGLKVAQ            SEQ ID NO: 368
QHLAYPVPDV            SEQ ID NO: 369
AQHLAYPVPD            SEQ ID NO: 370
FQPGLKVAQH            SEQ ID NO: 371
ELGFQPGLKV            SEQ ID NO: 372
LKVAQHLAYP            SEQ ID NO: 373
LGFQPGLKVA            SEQ ID NO: 374
QHLAYPVPDVP           SEQ ID NO: 375
LKVAQHLAYPV           SEQ ID NO: 376
VAQHLAYPVPD           SEQ ID NO: 377
PGLKVAQHLAY           SEQ ID NO: 378
LGFQPGLKVAQ           SEQ ID NO: 379
KVAQHLAYPVP           SEQ ID NO: 380
ELGFQPGLKVA           SEQ ID NO: 381
GLKVAQHLAYP           SEQ ID NO: 382
AQHLAYPVPDV           SEQ ID NO: 383
FQPGLKVAQHL           SEQ ID NO: 384
QPGLKVAQHLA           SEQ ID NO: 385
GFQPGLKVAQH           SEQ ID NO: 386
LGFQPGLKVAQH          SEQ ID NO: 387
PGLKVAQHLAYP          SEQ ID NO: 388
LKVAQHLAYPVP          SEQ ID NO: 389
GFQPGLKVAQHL          SEQ ID NO: 390
VAQHLAYPVPDV          SEQ ID NO: 391
QPGLKVAQHLAY          SEQ ID NO: 392
ELGFQPGLKVAQ          SEQ ID NO: 393
AQHLAYPVPDVP          SEQ ID NO: 394
KVAQHLAYPVPD          SEQ ID NO: 395
FQPGLKVAQHLA          SEQ ID NO: 396
GLKVAQHLAYPV          SEQ ID NO: 397
LGFQPGLKVAQHL         SEQ ID NO: 398
VAQHLAYPVPDVP         SEQ ID NO: 399
PGLKVAQHLAYPV         SEQ ID NO: 400
GFQPGLKVAQHLA         SEQ ID NO: 401
LKVAQHLAYPVPD         SEQ ID NO: 402
QPGLKVAQHLAYP         SEQ ID NO: 403
ELGFQPGLKVAQH         SEQ ID NO: 404
GLKVAQHLAYPVP         SEQ ID NO: 405
FQPGLKVAQHLAY         SEQ ID NO: 406
KVAQHLAYPVPDV         SEQ ID NO: 407
ELGFQPGLKVAQHL        SEQ ID NO: 408
QPGLKVAQHLAYPV        SEQ ID NO: 409
GFQPGLKVAQHLAY        SEQ ID NO: 410
LGFQPGLKVAQHLA        SEQ ID NO: 411
FQPGLKVAQHLAYP        SEQ ID NO: 412
KVAQHLAYPVPDVP        SEQ ID NO: 413
PGLKVAQHLAYPVP        SEQ ID NO: 414
LKVAQHLAYPVPDV        SEQ ID NO: 415
GLKVAQHLAYPVPD        SEQ ID NO: 416
GLKVAQHLAYPVPDV       SEQ ID NO: 417
PGLKVAQHLAYPVPD       SEQ ID NO: 418
ELGFQPGLKVAQHLA       SEQ ID NO: 419
LGFQPGLKVAQHLAY       SEQ ID NO: 420
GFQPGLKVAQHLAYP       SEQ ID NO: 421
QPGLKVAQHLAYPVP       SEQ ID NO: 422
FQPGLKVAQHLAYPV       SEQ ID NO: 423
LKVAQHLAYPVPDVP       SEQ ID NO: 424
FQPGLKVAQHLAYPVP      SEQ ID NO: 425
GFQPGLKVAQHLAYPV      SEQ ID NO: 426
GLKVAQHLAYPVPDVP      SEQ ID NO: 427
PGLKVAQHLAYPVPDV      SEQ ID NO: 428
QPGLKVAQHLAYPVPD      SEQ ID NO: 429
ELGFQPGLKVAQHLAY      SEQ ID NO: 430
LGFQPGLKVAQHLAYP      SEQ ID NO: 431
GFQPGLKVAQHLAYPVP     SEQ ID NO: 432
FQPGLKVAQHLAYPVPD     SEQ ID NO: 433
QPGLKVAQHLAYPVPDV     SEQ ID NO: 434
PGLKVAQHLAYPVPDVP     SEQ ID NO: 435
LGFQPGLKVAQHLAYPV     SEQ ID NO: 436
ELGFQPGLKVAQHLAYP     SEQ ID NO: 437
ELGFQPGLKVAQHLAYPV    SEQ ID NO: 438
QPGLKVAQHLAYPVPDVP    SEQ ID NO: 439
LGFQPGLKVAQHLAYPVP    SEQ ID NO: 440
FQPGLKVAQHLAYPVPDV    SEQ ID NO: 441
GFQPGLKVAQHLAYPVPD    SEQ ID NO: 442
FQPGLKVAQHLAYPVPDVP   SEQ ID NO: 443
LGFQPGLKVAQHLAYPVPD   SEQ ID NO: 444
GFQPGLKVAQHLAYPVPDV   SEQ ID NO: 445
ELGFQPGLKVAQHLAYPVP   SEQ ID NO: 446
LGFQPGLKVAQHLAYPVPDV  SEQ ID NO: 447
ELGFQPGLKVAQHLAYPVPD  SEQ ID NO: 448
GFQPGLKVAQHLAYPVPDVP  SEQ ID NO: 449
ELGFQPGLKVAQHLAYPVPDV SEQ ID NO: 450
LGFQPGLKVAQHLAYPVPDVP SEQ ID NO: 451

[0020] The peptides that mimic the helices and functional fragments thereof are administered in formulations and by routes well understood. A variety of methods for introducing such substances are known, typically, by injection, aerosol administration, suppository, and, with proper design, oral administration. This general statement is true as well with respect to providing expression systems for peptides represented by the helix mimics.

[0021] In addition to the peptides or other compounds of the invention, combination therapies including known HCV inhibitors can be utilized in the present invention. For example, it may be desirable to administer both a peptide or peptides of the invention in combination with interferon to a subject infected with HCV. Other drugs or compounds known in the art to be effective against HCV, can also be used.

[0022] In one embodiment, the invention provides a method of screening compounds, to identify those that selectively inhibit the binding of HCV nonstructural proteins (for example, NS4B or NS5A) and cellular membranes. Methods known to those of skill in the art, can be readily adapted to detect interference with the binding of these components. The method of screening may involve high-throughput techniques. For example, to screen for compounds that selectively inhibit the binding of HCV nonstructural proteins and cellular membranes, a synthetic reaction mix, a viral fragment or component, or a preparation of any thereof, comprising an HCV nonstructural protein and a labeled substrate or ligand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may inhibit the binding of the HCV nonstructural proteins and the cellular membrane. The ability of the candidate molecule to inhibit the binding of the HCV nonstructural protein and the cellular membrane is reflected in decreased binding of the labeled ligand or decreased production of product from such substrate.

[0023] In another aspect, the screening can be performed by adding the candidate compound to intact cells that have been infected by HCV, or that contain an HCV replicon, and then examining the component of interest to demonstrate the effect on this component, or the effect on viral or replicon replication. An exemplary cell-based in vitro assay for this purpose is disclosed in PCT International Publication WO 02/18369. Alternatively, the screening can be performed by adding the test agent to in vitro translation reactions and then proceeding with the established analysis. As another alternative, purified or partially purified components which have been determined to interact with one another by the methods described above can be placed under conditions in which the interaction between them would normally occur, with and without the addition of the candidate compound, and the procedures previously established to analyze the interaction can be used to assess the impact of the candidate compound. However their anti-HCV activity is initially assayed, peptide or other inhibitors of the present invention should cause inhibition of infection, replication, or pathogenesis of Hepatitis C Virus in vitro or in vivo when introduced into a host cell containing the virus, and exhibit an IC50 in the range of from about 0.0001 nM to 100 μM in an in vitro assay for at least one step in infection, replication, or pathogenesis of HCV, more preferably from about 0.0001 nM to 75 μM, more preferably from about 0.0001 nM to 50 μM, more preferably from about 0.0001 nM to 25 μM, more preferably from about 0.0001 nM to 10 μM, and even more preferably from about 0.0001 nM to 1 μM.

[0024] In another embodiment of the invention, the method of screening may be by phage display. A method of obtaining selective ligands that bind a chosen target is to select from a library of proteins or peptides displayed on phage. In order to obtain a novel binding protein against a chosen target, such as an amphipathic helix region of an HCV component, DNA molecules, each encoding a protein or peptide fragment thereof, and a structural signal calling for the display of the protein on the outer surface of a chosen genetic package (bacterial cell, bacterial spore or phage) are introduced into a genetic package. The protein is expressed and the potential binding domain is displayed on the outer surface of the package. The package is then exposed to the target. If the genetic package binds to the target, then it is confirmed that the corresponding binding domain is indeed displayed by the genetic package. Packages which display the binding domain (and thereby bind the target) are separated from those which do not. For example, in the present invention, the target may be the amphipathic helix or a mutated amphipathic helix. Potential peptides, which may then be used as anti-HCV agents, are screened by determination of which will bind to the amphipathic helix or a mutated amphipathic helix. Preferred peptides are those that not only bind to the amphipathic helix, but in addition, block or inhibit the amphipathic helix from binding to its receptor or binding site, thereby inhibiting infectivity. Examples of peptides identified by phage display are set forth in Table 3.

TABLE 3
Peptides identified by phage display as ligands
of the amphipathic helix of NS5A:
              Sequence
Sequence      Identification Number
HDSFANATGRFWP SEQ ID NO: 454
QGTSPSRLAVPLA SEQ ID NO: 455
ISSKTGMSSEPPS SEQ ID NO: 456
ILSSIDALGSDSH SEQ ID NO: 457
LDDRSVPTVISQR SEQ ID NO: 458
YPSKPGNVTPKAP SEQ ID NO: 459
QAQGERALK     SEQ ID NO: 460
TDKRASPLTVQAR SEQ ID NO: 461

[0025] In all of the embodiments of the invention, the active agent which will interact, generally, either with the sites on the cytoplasmic membrane to which the amphipathic helix binds or will interact with the amphipathic helix itself, may be derivatized or coupled to additional components. By “derivatives” of these agents is meant modifications thereof that do not interfere with their ability to interact with the sites or the helix, but may optionally confer some additional useful property. One particularly useful property is the ability to penetrate cell membranes, and preferred among the derivatives or conjugated forms are those coupled to such facilitators. An additional desired coupled component may be a labeling component such as a fluorescent dye, an enzyme, or a radioisotope. One particularly useful label is, for example, green fluorescent protein in any of its many colors and forms. Green fluorescent protein thus, includes, not only forms of this fluorescent protein that fluoresce green, but also those that fluoresce various other colors such as red and blue. These forms of the protein are commercially available as are recombinant materials for their production.

[0026] The compositions of the invention can be administered as conventional HCV therapeutics. The compositions of the invention may include more than one ingredient which interrupts the binding of the amphipathic helix to the membranes and more than one peptide of the invention.

[0027] The precise formulations and modes of administration of the anti-HCV compositions of the invention will depend on the nature of the anti-HCV agent, the condition of the subject, and the judgment of the practitioner. Design of such administration and formulation is routine optimization generally carried out without difficulty by the practitioner.

[0028] In addition to the assay methods, methods to identify compounds or protocols against HCV infection, and methods to treat HCV infections as set forth above, the invention provides compositions which are effective to elicit immunological responses to HCV in appropriate subjects, such as humans or other animals subject to HCV infection.

[0029] Administration may be performed using conventional methods, typically by injection. The elicited immunological response is helpful in general HCV prophylaxis.

[0030] The following examples are intended to illustrate but not to limit the invention.

EXAMPLES

Example 1

Effect of Helix Disruption on Membrane Association

[0031] The bottom panels of FIGS. 3A-3C show the structures of the relevant portion of NS5A used in this example. FIG. 3A shows the amino acid sequence and helix formed at positions 4-27 in the NS5A protein of Hepatitis C virus genotype I a. FIG. 3B shows the deletion of positions 7-27, thus deleting the helix, as shown by the brackets. This deletion was conducted by PCR mutagenesis essentially as described by Glenn, J. S., et al., J. Virol (1998) 72:9303-9306 on plasmid pBRTM/HCV 827-3011 which encodes the HCV nonstructural proteins as described by Grakoui, A., et al., J. Virol. (1993) 67:1385-1395. This vector encodes the NS proteins under the T7 promoter and encephalomyocarditis internal ribosome entry site control elements and directs the synthesis and processing of proteins NS3, NS4A, NS4B, NS5A and NS5B. FIG. 3C shows a mutant which was obtained using PCR mutagenesis as described above using a primer

(5′-TCCGGCTCCTGGCTAAGGGACGACTGGGA- (SEQ ID NO: 452)
CTGGGAATGCGAGGTGCTGAGCGACGATAAGAC-
C-3′)

[0032] in which codons isoleucine-8, isoleucine-12, and phenylalanine-19 of NS5A were changed to encode aspartate, glutamate, and aspartate, respectively. This results in disruption of the hydrophobic region of the helix.

[0033] Each of these plasmids was expressed and distribution of the NSSA protein, which is produced by the plasmids in cells, was observed. A liver derived cell line, Huh-7, was first infected with recombinant vaccinia virus encoding T7-RNA polymerase and then transfected with pBRTM/HCV 827-3001, or by this plasmid modified as described in FIGS. 3B and 3C.

[0034] After incubation to effect protein production, the cells were fixed with formaldehyde and immunostained with a monoclonal antibody against NS5A and a Texas Red-labeled donkey anti-mouse secondary antibody essentially as described by Glenn, J. S., et al., J. Virol. (1990) 64:3104-3107. As shown in the upper panel of FIG. 3A, perinuclear punctate vesicular staining suggestive of a Golgi-like intracellular distribution pattern was readily observed, as was, occasionally, a somewhat reticulin chicken-wire-like staining pattern, characteristic of the ER. Both patterns have been reported previously when NS5A is expressed either alone or in combination with other HCV NS proteins using a variety of expression systems. See, for example, Kim, J. E., et al., Arch. Virol. (1999) 144:329-343; Huang, Y., et al., Biochem. Biophys. Res. Commun. (2001) 281:732-740.

[0035] However, when the construct of FIG. 3B, containing a deleted portion of NS5A was expressed, as shown in the upper panel of FIG. 3B, cytoplasmic membrane localization was abolished and a nuclear localization pattern was obtained. A cryptic nuclear localization signal in the NS5A C-terminal domain has previously been reported by Ide, Y., et a., Gene (1996) 182:203-211.

[0036] When the construct described in FIG. 3C, which disrupts the hydrophobic face of the helix, is expressed in these cells, again, as shown in the upper panel of FIG. 3C, the cytoplasmic membrane localization pattern was lost and localization to the nucleus was observed.

[0037] FIGS. 3A-3C show these results at two magnification levels. The uppermost panel in each case shows results for a single cell and the intermediate panel shows results for a number of cells. As shown in the intermediate panels in FIGS. 3B and 3C, occasionally distribution of the dye throughout the cell was observed rather than localization to the nucleus. However, even in these cases, no specific localization to the cytoplasmic membrane could be detected.

Example 2

Intracellular Location of GFP-Labeled Helix

[0038] Three constructs were made. First, as a control, the HCV encoding sequences of pBRTM/HCV 827-3011 were replaced with the coding sequence for jellyfish Aequorea Victoria green fluorescent protein (GFP) using a PCR cloned insert from plasmid C109 which contains E-GFP (Clontech), to obtain “T7-GFP.” A fragment of the NS5A gene encoding the amphipathic helix was fused into frame with the sequence encoding the N-terminus of GFP in T7-GFP by creating a common PstI site using PCR mutagenesis. This permits junction of the first 31 amino acids of NS5A to the 5′ side of serine-2 of the GFP-encoding sequence in T7-GFP. The resulting vector was labeled “T7-5AGFP.” A similar vector, designated “T7-5ANHGFP” was constructed in a manner similar to “T7-5AGFP” except that the corresponding segment from the mutated form of NS5A set forth in FIG. 3C was employed in place of the wildtype helix. Thus, “T7-5ANHGFP” is similar to “T7-5AGFP” except that in the NS5A helix, Ile at position 8 is converted to Asp, Ile at position 12 is converted to Glu, and Phe at position 19 is converted to Asp.

[0039] These constructs were used to effect expression of GFP or of the GFP fusions in Huh-7 cells as described in Example I and the distribution of fluorescence was evaluated with the results as shown in FIGS. 4A-4C. As shown in FIG. 4A, cells expressing GFP alone show a wide diffuse pattern including concentration in the nucleus. Cells expressing 5AGFP show distribution of fluorescence similar to that of the NS5A protein itself with a Golgi-like intracellular distribution pattern and ER staining. Cells expressing 5ANHGFP failed to show this pattern but instead mimic the distribution pattern of GFP.

Example 3

Effect of Helix Disruption on HCV RNA Replication

[0040] Comparative high efficiency, second generation, bicistronic, subgenomic RNA replicons of HCV described by Blight, K. J., et al., Science (2001) 290:1972-1974 were constructed with a neomycin-resistance gene. Similar constructs were made with wildtype NS5A and with NS5A altered in the amphipathic helix as described in Examples 1 and 2. Diagrams of these constructs are shown in the upper panels of FIGS. 5A and 5B.

[0041] The construct containing the wildtype NS5A, Bart79I, was made by PCR mutagenesis of HCVreplbBartMan/AvaII (Blight, supra) such that nucleotide 5336 was changed from a G to T resulting in a change in NS5A codon 1179 from serine to isoleucine. This mutation results in a dramatic increase in replication efficiency of the HCV subgenomic replicon. Bart5X79I (containing the modified helix) was made by PCR mutagenesis of Bart79I using a primer

(5′-GATTGGGATTGGGAATGCACGGTGTTGACT (SEQ ID NO: 453)
GATGACAAGACCTGG-3′)

[0042] in which codons valine-8, isoleucine-12, and phenylalanine-19 of NS5A were changed to encode aspartate, glutamate, and aspartate, respectively. All mutations were confirmed by DNA sequencing.

[0043] The RNA replication efficiency was then assayed by ability to establish G418-resistant colonies after transfection of Huh-7 cells as follows: Replicon RNA's were prepared by in vitro transcription with T7-RNA polymerase of ScaI-linearized plasmids (Bart79I or Bart5X79I) followed by DNase treatment and purification. Replicons were then electroporated into Huh-7 cells and neomycin-resistant colonies selected by inclusion of 1 mg/ml G418 in the culture media. Methods were essentially as described by Blight (supra). Colonies were detected by staining with cresyl violet. Multiple independent preparations of replicons and replication assays yielded similar results.

[0044] As shown in FIG. 5A, lower panel, the replicon with wildtype NS5A N-terminus gave rise to numerous colonies while disrupting the amphipathic nature of the N-terminal helix results in a dramatic inhibition of HCV genome replication as shown in the lower panel of FIG. 5B.

[0045] There was no decreased transfection efficiency or any decrease in the ability to establish colonies when control experiments using plasmids encoding drug resistance marker along with only the wildtype or mutant NS5A proteins were used in place of the replicons above, thus establishing that the results were not due to an increased cytotoxicity associated with mutant NS5A.

Example 4

Floatation Assay

[0046] In this membrane floatation assay, the ability of the NS5A amphipathic helix to bind to a preparation of microsomal membranes was tested. NS5A proteins, both the NS5A wildtype or mutant amphipathic helix (described in Example 1), were in vitro translated and [35S]-labeled using the Promega TNT reticulocyte lysate kit. Plasmids pC5A or pC5ANH were used as transcription templates. Aliquots of the reactions were combined with a membrane fraction derived from Huh-7 cells, with or without synthetic peptides, and overlayed with a 5-40% OptiPrep step gradient. After centrifugation for 4 hrs at 40,000×g in a SW60 rotor, 500 ml fractions were collected from the top and the proteins in each gradient fraction were precipitated and analyzed by SDS-PAGE. The percentage of protein “floating” to the top of the gradient with membranes (fraction 2 from the top) was then quantified using a Molecular Dynamics phosphorimager.

[0047] FIGS. 6A and 6B show a comparison of the results for wildtype and mutant forms. The numbers correspond to Optiprep gradient (5-40%) fraction number. Non-membrane associated proteins remain at the bottom (right side) of the gradient, whereas membranes—and associated proteins—“float” towards less dense gradient fractions present at the top (left side). As shown, in the reaction mixture containing wildtype, the synthesized NS5A floated toward the top of the gradient coupled with the membrane. This was not found in the case of the mutant form. In both cases, there was a degree of non-specific binding associated with fractions that settle to the bottom of the gradient. An advantage of this method is elimination of the artifacts caused by non-specific binding, as illustrated by the labeling of high density fractions in both mixtures.

[0048] FIG. 6C shows the percentage of NS5A wildtype or mutant protein that float with the membrane fractions as a percentage of the total synthesized. As seen, 25% of the wildtype, but only 0.8% of the mutant, was associated with the membrane.

[0049] FIGS. 7A and 7B show the results of a similar floatation experiment in the presence and absence of a test peptide that is a competitive inhibitor of the NS5A helix. This peptide contains amino acids 1-27 of NS5A with a C-terminal “flag” DYKD. The assay described in the preceding paragraph was repeated in the presence and absence of this peptide. As shown in FIG. 7A, the percentage of NS5A floated in the absence of peptide is arbitrarily normalized to 100%; in comparison with this, the presence of the test peptide lowered the percentage to about 21%.

[0050] Similarly, the assay was modified by using, in place of NS5A, a fusion protein consisting of the amphipathic helix of NS5A with green fluorescent protein. As can be seen in FIG. 7B, the amount of labeled NS5A floated with the membrane was set in the absence of test peptide at 100%. In comparison, the presence of the test peptide lowered the percentage floated to about 21%.

[0051] Moreover, as shown in FIG. 6C, a synthetic peptide designed to mimic the wild type AH not only competitively inhibited membrane association of NSSA, but did so in a dose-dependent manner. The maximal extent of inhibition achieved pharmacologically was comparable to that obtained by genetic mutation of the AH. In addition, the synthetic peptide appears equally effective against NS5A derived from different genotypes (data not shown), including those most refractory to current therapies. This convenient membrane floatation assay has also proven well-suited for current efforts focused on studying the mechanistic details of the AH membrane-targeting domain, and ideal for guiding ongoing development of peptidomimetic compounds designed to resemble key elements, or bind to specific features, of the AH. Such compounds represent an exciting potential addition to current anti-HCV combination therapy regimens.

[0052] FIG. 8 shows Pharmacologic inhibition of NS5A membrane association. Huh-7 cells-derived membranes were treated with PEPI (a peptide mimicking the wild type amphipathic helix of NS5A), PEP2 (a control peptide) or mock-treated with water. PEP1 (a peptide that corresponds to the wild type N-terminal amphipathic helix of NS5A (amino acids 1-26) with a C-terminal FLAG tag) and PEP2 (which is identical to PEP1 except amino acids isoleucine-8, isoleucine-12, and phenylalanine-19 of NS5A were changed to aspartate, glutamate, and aspartate, respectively) were synthesized by AnaSpec Inc. In-vitro translated NS5A (or NS5ANH) was incubated with membranes and various concentrations of peptides or water prior to analysis by membrane floatation assays set forth above. The amount of NS5A floating with no peptide treatment typically is˜30% of the total in-vitro translated NS5A. For comparison between treatment conditions, results were normalized to the no-treatment control. The percent of NS5A (or NS5ANH) floating with the membranes under the indicated conditions was determined as in FIG. 6A and expressed relative to mock-treated control. Error bars represent standard error of the mean.

[0053] While the invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of that which is described and claimed.

[0054] References

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Claims

1. A method to identify a compound useful to treat Hepatitis C virus (HCV) infection comprising assessing the ability of a candidate compound to interfere with the binding of an amphipathic helix present in the N-terminal region of an HCV nonstructural protein with cytoplasmic membranes of a eukaryotic cell, where a compound which interferes with the binding is identified as being useful in treating HCV infection.

2. The method of claim 1, wherein the assessing is performed extracellularly.

3. The method of claim 2, wherein the assessing comprises determining the ability of a compound to bind to the amphipathic helix, wherein a compound which binds to the amphipathic helix with high affinity is identified as a compound useful in treating HCV infection.

4. The method of claim 2, wherein the assessing comprises determining the binding of the amphipathic helix to a cell membrane preparation in the presence of a candidate compound and in the absence of a candidate compound, wherein a decrease in the binding of the amphipathic helix to the cell membrane preparation in the presence of the compound as compared to binding in the absence of the compound identifies the compound as being useful in treating HCV infection.

5. The method of claim 1, wherein the method is performed intracellularly in a eukaryotic cell.

6. The method of claim 5, wherein the amphipathic helix is coupled to a detectable label and the intracellular distribution of the label is assessed in the presence of a candidate compound, wherein the candidate compound is identified as being useful in the treatment of HCV infection when its presence results in an intracellular distribution of the label which does not show binding to cytoplasmic membranes in comparison to the distribution of the label in the absence of the compound.

7. The method of claim 6, wherein the detectable label is green fluorescent protein.

8. A compound useful to treat HCV infection identified by the method of any one of claims 1 to 7.

9. A method to treat HCV infection comprising administering to a subject in need of such treatment an anti-HCV effective amount of a compound identified by the method of any one of claims 1 to 7.

10. A method to treat HCV infection in a subject comprising administering to a subject in need of such treatment an anti-HCV effective amount of at least one compound which inhibits the binding of an amphipathic helix present in the N-terminal region of an HCV nonstructural protein with cytoplasmic membranes.

11. The method of claim 10, wherein the at least one compound is a compound that interacts with one or more sites on the cytoplasmic membrane to which the amphipathic helix binds.

12. The method of claim 11, wherein the at least one compound is a compound which mimics the charge/shape configuration of an amphipathic helix present in the N-terminal region of an HCV nonstructural protein.

13. The method of claim 12, wherein the compound comprises the amphipathic helix present in the N-terminal region of an HCV nonstructural protein or is a peptide having at least about 80% sequence identity with the amphipathic helix.

14. The method of claim 10, wherein at least one compound interacts with the amphipathic helix.

15. A composition for eliciting an immunological response against HCV in a subject comprising an HCV replicon, wherein the nucleotide sequence encoding an amphipathic helix in the N-terminal region of an HCV nonstructural protein is deleted or altered to disrupt the amphipathic nature of the amphipathic helix.

16. The composition of claim 15, wherein the HCV nonstructural protein is NS5A, NS5B or NS4B.

17. The composition of claim 16, wherein the HCV non-structural protein is NS5A.

18. A method to elicit an immunological response against HCV in a subject comprising administering to the subject an anti-HCV effective amount of the composition of claim 17 sufficient to elicit the response.

19. An isolated peptide of from about 4 to 60 amino acid residues, which comprises an amphipathic helix present in the N-terminal region of an HCV nonstructural protein, the peptide optionally comprising non-peptide isosteric linkages, wherein the peptide is optionally coupled to an additional heterologous second peptide or to an additional component.

20. The peptide of claim 19, which is coupled to a membrane-penetrating facilitator.

21. The method of claim 20, wherein the membrane-penetrating facilitator is a membrane transport sequence or a TAT peptide.

22. The peptide of claim 19, which is coupled to a detectable label.

23. An isolated peptide consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 to 13 and 462.

24. The peptide of claim 23, which is coupled to a membrane-penetrating facilitator.

25. The peptide of claim 23, which is coupled to a detectable label.

26. The compound of claim 8, which is a peptide having the following amino acid sequence:

27. The peptide of claim 26, wherein the peptide comprises one or more of the following amino acid substitutions: substitution of L at amino acid position 16 by A or K; or substitution of T at amino acid position 17 by A; or substitution of D at amino acid position 18 by A; or substitution of F at amino acid position 19 by A; or substitution of K at amino acid position 20 by A; or substitution of W at amino acid position 22 by A; or substitution of L at amino acid position 23 by K.

28. The peptide of claim 26 or 27, wherein the peptide inhibits binding of HCV nonstructural proteins with a cytoplasmic membrane of a eukaryotic cell.

29. The peptide of claim 26 or 27, wherein the HCV nonstructural protein is NS5A.

30. The compound of claim 8, which is a peptide having the following amino acid sequence:

31. The compound of claim 30, wherein the peptide inhibits binding of HCV nonstructural proteins with the cytoplasmic membrane.

32. The compound of claim 31, wherein the HCV nonstructural protein is NS5A.

33. An isolated peptide having the following amino acid sequence:

34. The compound of claim 33, wherein the peptide comprises one or more of the following amino acid substitutions: substitution of L at amino acid position 16 by A or K; or substitution of T at amino acid position 17 by A; or substitution of D at amino acid position 18 by A; or substitution of F at amino acid position 19 by A; or substitution of K at amino acid position 20 by A; or substitution of W at amino acid position 22 by A; or substitution of L at amino acid position 23 by K.

35. An isolated peptide having the following amino acid sequence:

36. The peptide of claim 35, having the amino acid sequence:

37. An isolated peptide having the amino acid sequence:

38. An isolated peptide having the amino acid sequence: LCLAGRGLQEAEGLLLELLSEHHPLLDV (SEQ ID NO: 20) or any functional fragment thereof.

39. The isolated peptide of claim 38, wherein the functional fragment is 6 to 27 amino acids in length.

40. An isolated peptide having the amino acid sequence ELGFQPGLKVAQHLAYPVPDVP (SEQ ID NO: 299) or any functional fragment thereof

41. The isolated peptide of claim 40, wherein the functional fragment is 6 to 21 amino acids in length.

42. A method of identifying a peptide that inhibits the binding of an HCV nonstructural protein to a cytoplasmic membrane of a eukaryotic cell, comprising contacting an amphipathic helix obtained from the N-terminal region of an HCV nonstructural protein with cytoplasmic membranes of a eukaryotic cell in the presence and absence of a test peptide, wherein a decreased level of binding of the amphipathic helix to the cytoplasmic membrane in the presence of the test peptide as compared to the level of binding of the amphipathic helix to the cytoplasmic membrane in the absence of the test peptide identifies the test peptide as one that inhibits the binding of an HCV nonstructural protein to a cytoplasmic membrane of a eukaryotic cell.

43. The method of claim 42, wherein the peptide is selected from SEQ ID NOS: 1-16, 18, 20-295, 297, 299-451 and 454-466.

44. An isolated peptide selected from the group consisting of SEQ ID NOS: 1-16, 18, 20-295, 297, 299-451, and 454-466, wherein the isolated peptide causes inhibition of infection, replication, or pathogenesis of Hepatitis C Virus in vitro or in vivo when introduced into a host cell containing the virus, and wherein the isolated peptide exhibits an IC50 in the range of from about 0.0001 nM to 100 μM in an in vitro assay for at least one step in infection, replication, or pathogenesis of the virus.

45. A composition, comprising: one or more isolated peptides of claim 44, and a carrier, diluent, excipient, or buffer.

46. A pharmaceutical composition, comprising: one or more isolated peptides of claim 44, and a pharmaceutically acceptable carrier, diluent, excipient, or buffer.

47. A method of preventing or treating HCV infection in a patient in need thereof, comprising administering to the patient an anti-HCV effective amount of one or more isolated peptides of claim 44.

48. Use of an isolated peptide of claim 44 to prepare a medicament for the prevention or treatment of HCV infection in a human patient in need thereof.

49. A method of treatment of HCV infection in a patient in need thereof, comprising administering to the patient an anti-HCV effective amount of an isolated peptide comprising the NS5A N-terminal amphipathic helix.

50. The compound of claim 8, which is a peptide having the following amino acid sequence: