# US 2013/0157894 A1 · Methods to identify combinations of ns5a targeting compound that act synergistically to inhibit hepatitis c virus replication

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- Number: US 2013/0157894 A1
- Document ID: US20130157894A1
- Status: Published application
- Kind: A1
- Application number: 13/809,635
- Filed: 2011-07-13
- Published: 2013-06-20
- Earliest claimed: 2010-07-16
- Inventors: Jin-Hua Sun; Min Gao; Donald R. O'Boyle, II; Julie A. Lemm; Susan B. Roberts; Makonen Belema; Nicholas A. Meanwell
- Web page: https://patentgrep.com/patent/US20130157894A1

## CPC

- C12Q 1/025: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics (antimicrobial activity C12Q1/18)}
- C12Q 1/18: Testing for antimicrobial activity of a material
- G01N 33/5008: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics}
- G01N 2333/186: Hepatitis C; Hepatitis NANB
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present invention is based on the surprising finding that pairs of HCV NS5A-targeting inhibitors can be identified which display similar resistance profiles yet, when combined, exhibit synergistic inhibition of wild type replicons and/or replicons carrying mutations conferring resistance to the HCV NS5A-targeting inhibitor. In addition, combinations of these molecules result in a higher genetic barrier to resistance, demonstrating their potential utility as novel combination therapies for treatment of HCV.

## Claims

### Claim 1

A method for identifying NS5A-targeting compounds that in combinations demonstrate a synergistic inhibitory interaction toward wild-type and/or variants that reduce the potency of the NS5A-targeting inhibitors alone comprising the steps of:
(a) screening a compound library for NS5A targeting compounds and
(b) comparing the amount of HCV inhibition of a NS5A-targeting inhibitor in the presence and absence of a fixed concentration of a NS5A-targeting compound and
wherein the second NS5A-targeting compound may or may not demonstrate HCV NS5A inhibitory activity when assayed alone and wherein the combination of the NS5A-targeting compounds demonstrate a synergistic inhibition of HCV replicon or virus replication.

### Claim 2 (depends on 1)

The method of claim 1 wherein the compound library is screened in cells containing a HCV sequence derived from any genotype.

### Claim 3 (depends on 1)

The method of claim 1 wherein the HCV inhibitory activity is determined by using a cell-based method or a biochemical surrogate.

### Claim 4 (depends on 2)

The method of claim 2 wherein the HCV sequence is selected from the group consisting of wild-type or sequences carrying NS5A resistant variants.

### Claim 5 (depends on 2)

The method of claim 2 wherein said HCV sequence is genotype 1b carrying a Y93H single amino acid substitution in NS5A.

### Claim 6 (depends on 2)

The method of claim 2 wherein said HCV sequence is genotype 1b carrying a L31V single amino acid substitution in NS5A.

### Claim 7 (depends on 2)

The method of claim 2 wherein said HCV sequence is genotype 1a carrying a M28T single amino acid substitution in NS5A.

### Claim 8 (depends on 2)

The method of claim 2 wherein said HCV sequence is genotype 1a carrying a Q30R single amino acid substitution in NS5A.

### Claim 9 (depends on 2)

The method of claim 2 wherein said HCV sequence is genotype 1a carrying a L31V single amino acid substitution in NS5A.

### Claim 10 (depends on 2)

The method of claim 2 wherein said HCV sequence is genotype 1a carrying a Y93H single amino acid substitution in NS5A.

### Claim 11 (depends on 2)

The method of claim 2 wherein said HCV sequence is genotype 1a carrying a Q30H single amino acid substitution in NS5A.

### Claim 12 (depends on 2)

The method of claim 2 wherein said HCV sequence is genotype 1a carrying a Q30E single amino acid substitution in NS5A.

### Claim 13 (depends on 2)

The method of claim 2 wherein said HCV sequence is genotype 1a carrying a Y93N single amino acid substitution in NS5A.

### Claim 14 (depends on 2)

The method of claim 2 wherein said HCV sequence is genotype 1a carrying amino acid substitutions at both L31V and Y93H in NS5A.

### Claim 15 (depends on 2)

The method of claim 2 wherein said HCV sequence is genotype 1a carrying amino acid substitutions at both M28T and Q30H in NS5A.

### Claim 16 (depends on 2)

The method of claim 2 wherein said HCV sequence is genotype 1a carrying amino acid substitutions at both Q30R and H58D in NS5A.

### Claim 17 (depends on 2)

The method of claim 2 wherein said HCV sequence is genotype 1a carrying amino acid substitutions at both Q30H and Y93H in NS5A.

### Claim 18 (depends on 2)

The method of claim 2 wherein said HCV sequence is genotype 1a carrying amino acid substitutions at both Q30R and E62D in NS5A.

### Claim 19 (depends on 2)

The method of claim 2 wherein said HCV sequence is genotype 1b carrying amino acid substitutions at both L31V and Y93H in NS5A.

### Claim 20 (depends on 3)

The method of claim 3, wherein said cell-based method is a transient replication assay.

### Claim 21 (depends on 3)

The method of claim 3, wherein said cell-based method is a FRET assay.

### Claim 22 (depends on 3)

The method of claim 3, wherein said cell-based method is a luciferase assay.

### Claim 23 (depends on 3)

The method of claim 3, wherein said cell-based method is a colony formation assay.

### Claim 24 (depends on 3)

The method of claim 3, wherein said cell-based method is a Western blot assay.

### Claim 25 (depends on 3)

The method of claim 3, wherein said cell-based method is a Taqman assay.

### Claim 26 (depends on 3)

The method of claim 3, wherein said cell-based method is an ELISA assay.

### Claim 27 (depends on 3)

The method of claim 3, wherein said biochemical surrogate is a NS5A hyperphosphorylation assay.

### Claim 28 (depends on 1)

The method of claim 1, wherein the HCV inhibitory activity is increased by at least 3 fold.