Rescuing compound bioactivity in a secondary cell-based screening by using γ-cyclodextrin as a molecular carrier

Abstract

In vitro primary screening for identifying bioactive compounds (inhibitors, activators or pharmacological chaperones) against a protein target results in the discovery of lead compounds that must be tested in cell-based efficacy secondary screenings. Very often lead compounds do not succeed because of an apparent low potency in cell assays, despite an excellent performance in primary screening. Primary and secondary screenings differ significantly according to the conditions and challenges the compounds must overcome in order to interact with their intended target. Cellular internalization and intracellular metabolism are some of the difficulties the compounds must confront and different strategies can be envisaged for minimizing that problem. Using a novel screening procedure we have identified 15 compounds inhibiting the hepatitis C NS3 protease in an allosteric fashion. After characterizing biophysically the interaction with the target, some of the compounds were not able to inhibit viral replication in cell assays. In order to overcome this obstacle and potentially improve cellular internalization three of these compounds were complexed with γ-cyclodextrin. Two of them showed a five- and 16-fold activity increase, compared to their activity when delivered as free compounds in solution (while γ-cyclodextrin did not show antiviral activity by itself). The most remarkable result came from a third compound that showed no antiviral activity in cell assays when delivered free in solution, but its γ-cyclodextrin complex exhibited a 50% effective concentration of 5 μM. Thus, the antiviral activity of these compounds can be significantly improved, even completely rescued, using γ-cyclodextrin as carrier molecule.

Keywords:

• primary and secondary screenings
• drug activity
• antiviral compounds
• cyclodextrins
• vehiculization
• drug delivery
• hepatitis C
• NS3 protease
• virus replicon system

Acknowledgments

This work was supported by Spanish Ministerio de Ciencia e Innovación (BFU2010-19451 to AVC, PTA2009-2341-I to SV), Spanish Ministerio de Economía y Competitividad (BFU2013-47064-P to AVC), Spanish Ministerio de Educación, Cultura y Deporte (Grant FPU13/3870 to RCG), Miguel Servet Program from Instituto de Salud Carlos III (CP07/00289 to OA), Fondo de Investigaciones Sanitarias (PI10/00186 to OA, PI11/02578 to AL), grant ERC-Starting Grant (239931-NANOPUZZLE project to JML), Diputación General de Aragón (Grant B136/13 to RCG, Protein Targets Group B89 to AVC, Digestive Pathology Group B01 to OA, RCG, and AL, and Nanotherapy and Nanobiosensors Group E93 to JMF), Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd) to AL and OA, and Fondo Social Europeo to JMF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Disclosure

The authors declare no conflicts of interest in this work.