Metalloprotoporphyrin Inhibition of HCV NS3-4A Protease: Structure–Activity Relationships

Abstract

Background

Antiviral actions of tetrapyrroles have been described in a number of systems. Our goal was to evaluate antagonism of the HCV NS3-4A protease by a variety of common porphyrins and characterize structure–activity relationships that may be useful for future drug design of HCV and related Flaviviruses.

Methods

Using fluorometric assays, common metalloprotoporphyrins (MPP) all inhibited NS3-4A protease with IC₅₀ values in low micromolar ranges [CoPP (1.4 µM) < ZnPP = MnPP = SnPP < CuPP < FePP (6.5 µM) = protoporphyrin].

Results

Lineweaver–Burk plots confirmed that MPP: NS3 inhibition was basically competitive. All tested MPPs inhibited HCV genotype 1A, 1B, 2A and 3A recombinant proteases with the same fidelity suggesting wide antagonistic capabilities. However, when the MPPs were tested in cellular incubations with HCV replicons only Zn, Fe and free-base protoporphyrin showed comparable EC₅₀ and IC₅₀ values suggesting that there may be critical differences in MPP uptake and intracellular availability. Meso, deutero, and isohematoporphyrin derivatives, with or without metal substitution, all showed less anti-protease and antiviral activities as compared to protoporphyrins, suggesting that the planar, vinyl side chains are important for protease active site binding. MPPs were also active against three common protease mutants (T54A, A156T, and V36M) with equivalent or better IC₅₀ values as compared to wild type enzyme.

Conclusion

These findings document the versatility of MPPs as antiviral agents with an expanded sensitivity for HCV genotypes and resistance to some common viral mutations. The results also suggest that further study of MPP structure and function will be useful for the development of new antiviral agents.

Keywords:

• porphyrins
• antiviral agents
• hepatitis C virus
• hepatitis C genotype

Acknowledgment

This study was supported by Merit Review grant (2I01 BX000159-09A2) from the Veterans Affairs, University of Iowa Biological Sciences Funding Program of the University of Iowa (WNS), and the Doriann Foundation for hepatitis research University of Iowa (WNS).

Abbreviations

abs, absorbance; BR, bilirubin; BV, biliverdin; EC_50, concentration of agent to inhibit HCV replication by 50%; FRET, fluorescence energy transfer; HCV, Hepatitis C virus; HCVcc, Hepatitis C virus cell culture; HO, heme oxygenase; IC_50, concentration agent to inhibit NS3-4A protease activity by 50%; MP, metalloporphyrin; MPP, metallo-protoporphyrin; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); NS, nonstructural; PCR, polymerase chain reaction; “X”MP, X-mesoporphyrin (where X = heavy metal); “X”PP, X-protoporphyrin (where X = heavy metal).

Disclosure

JB is an employee and shareholder of Frontier Scientific Inc. CAT is an employee of Curza Global LLC. The authors report no other conflicts of interest in this work.