Inhibition of the precursor and mature forms of HIV-1 protease as a tool for drug evaluation.
Sci Rep
; 8(1): 10438, 2018 Jul 11.
Article
en En
| MEDLINE
| ID: mdl-29992979
ABSTRACT
HIV-1 protease (PR) is a homodimeric enzyme that is autocatalytically cleaved from the Gag-Pol precursor. Known PR inhibitors bind the mature enzyme several orders of magnitude more strongly than the PR precursor. Inhibition of PR at the precursor level, however, may stop the process at its rate-limiting step before the proteolytic cascade is initiated. Due to its structural heterogeneity, limited solubility and autoprocessing, the PR precursor is difficult to access by classical methods, and limited knowledge regarding precursor inhibition is available. Here, we describe a cell-based assay addressing precursor inhibition. We used a reporter molecule containing the transframe (TFP) and p6* peptides, PR, and N-terminal fragment of reverse transcriptase flanked by the fluorescent proteins mCherry and EGFP on its N- and C- termini, respectively. The level of FRET between EGFP and mCherry indicates the amount of unprocessed reporter, allowing specific monitoring of precursor inhibition. The inhibition can be quantified by flow cytometry. Additionally, two microscopy techniques confirmed that the reporter remains unprocessed within individual cells upon inhibition. We tested darunavir, atazanavir and nelfinavir and their combinations against wild-type PR. Shedding light on an inhibitor's ability to act on non-mature forms of PR may aid novel strategies for next-generation drug design.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Contexto en salud:
1_ASSA2030
Problema de salud:
1_medicamentos_vacinas_tecnologias
Asunto principal:
Precursores de Proteínas
/
VIH-1
/
Inhibidores de la Proteasa del VIH
/
Fármacos Anti-VIH
Límite:
Humans
Idioma:
En
Revista:
Sci Rep
Año:
2018
Tipo del documento:
Article
País de afiliación:
República Checa