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Hcv ns3-ns4a protease inhibition

一种NS3-NS4A、蛋白酶的技术，应用在治疗HCV感染,抑制丙型肝炎病毒NS3-NS4A蛋白酶活性的化合物领域，能够解决抗HCV疫苗前景不明朗等问题

Inactive Publication Date: 2007-11-14
VERTEX PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the prospects for an effective vaccine against HCV remain uncertain

Method used

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  • Hcv ns3-ns4a protease inhibition
  • Hcv ns3-ns4a protease inhibition
  • Hcv ns3-ns4a protease inhibition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] HCV NS3 Protease HPLC Peptide Cleavage Assay

[0108] This method is a modification of the method described by Landro et al. [Landro JA et al. Biochemistry, 1997, 36:9340-9348]. For all proteases, a peptide substrate based on the NS5A / NS5B split site of HCV genotype la (NS5AB) was used. 25 mM substrate stocks were prepared in DMSO containing 0.2 M DTT and stored at -20°C. The central core region of NS4A was replaced by a synthetic peptide cofactor (KK4A) appropriate for each genotype. See below for peptide sequences. Hydrolysis reactions were performed on 96-well microtiter plates with 25nM-50nM HCV NS3 protease in a buffer containing 50mM HEPES (pH 7.8), 100mM NaCl, 20% glycerol, 5mM DTT and 25μM KK4A. The final concentration of DMSO was no more than 2% (v / v). The reaction was terminated by adding 10% trifluoroacetic acid (TFA) to make the final concentration of TFA 2.5%. Substrate and product were separated on a reverse-phase microbore HPLC column (Phenomenex Jup...

Embodiment 2

[0113] Potency Determination by HPLC Peptide Fractionation

[0114] In order to determine the apparent Ki value, all components except the test compound and substrate were pre-incubated at room temperature for 5 min. Then the test compound dissolved in DMSO was added to the mixture and incubated at room temperature for 15 min. Add NS5AB peptide at a concentration equal to Km (11 μM-70 μM), and incubate at 30° C. for 15 min to initiate the reaction. Enzyme inhibition activity was titrated with 7 to 8 concentrations of compound. Via non-linear regression, the activity and corresponding inhibitor concentration data points were fitted to the Morrison equation [SculleyMJ & Morrison JF, Biochim Biophys Acta, 1986, 874: 44-53] describing the inhibition reaction of competitive strong binding enzymes.

[0115] VX-950 pairs measured by peptide cleavage

Embodiment 3

[0117] HCV NS3 Protease Fluorescent Peptide Assay

[0118]Enzyme activity was determined by a modification of the method described by Taliani et al [Taliani M et al. Anal Biochem, 1997, 240:66-67]. All reactions used RET-S1 fluorescent peptide (AanSpec, San Jose, CA) as substrate in buffer A (buffer containing 50 mM HEPES (pH 7.8), 100 mM NaCl, 20% glycerol, 5 mM DTT and 25 μM KK4A) conduct. The final concentration of DMSO was maintained at 1-2% (v / v). Unless otherwise stated, reactions were monitored serially using a fluorescent microtiter plate reader thermostatted at 30°C with excitation and emission filters of 355 nm and 495 nm, respectively.

[0119] In order to determine the kinetic parameters Km and Vmax, the concentration of substrate RET-S1 in buffer A was varied between 6 μM and 200 μM, and it was reacted with 5 nM~10 nM HCV NS3 protease for 5~10 min. Add 25 μL of 10% TFA to stop the reaction. The substrate and product were separated on a reverse-phase microbore ...

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Abstract

The present invention relates to inhibiting the activity of non-genotype 1 hepatitis C virus (HCV) NS3-NS4A protease activity. More particularly, the invention relates to inhibiting the activity of the protease from HCV genotype-2 or HCV genotype-3. The methods of the invention emply inhibitors that act by interfering with the life cycle of the HCV and are also useful as antiviral agents. The invention further relates to compositions comprising such compounds either for ex vivo use or for administration to a patient suffering from genotype-2 or genotype-3 HCV infection. The invention also relates to methods of treating an HCV infection in a patient by administering a composition comprising a compound of this invention.

Description

technical field [0001] The present invention relates to compounds that inhibit the activity of serine proteases, especially hepatitis C virus NS3-NS4A proteases. These compounds work by interfering with the HCV life cycle and are useful antivirals. The present invention further relates to compositions which can be used both ex vivo and administered to HCV-infected patients. The invention also relates to methods of administering the compositions of the invention to a patient for the treatment of HCV infection. Background technique [0002] Hepatitis C virus ("HCV") infection is a human medical problem of great concern. It is estimated that 3% of the people's serum is prevalent in the whole world, so HCV is considered to be the pathogenic factor of most non-A non-B hepatitis cases [Alberti A etc. The natural history of hepatitis C. J Hepatology.1999, 31 (Suppl.1 ): 17-24]. About 4 million people may be infected in the United States alone [Alter MJ et al. Epidemiology of vi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/00A61K31/4965A61K31/42A61K31/425
CPCA61K31/7056A61K31/42A61K45/06A61K38/21A61K31/425A61K31/497A61K38/07A61P31/00A61P31/12A61P31/14A61P31/16A61P43/00A61K2300/00A61K38/05A61K31/4965
Inventor C·林W·P·泰勒
Owner VERTEX PHARMA INC
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