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

Figure 1

Experimental screening for ligands binding to the Zn⁺²-free NS3 protease. Notes: Thermal denaturation curves of Zn⁺²-free NS3 protease followed by ANS fluorescence in the presence of different compounds (100 mM sodium acetate, pH 5, 2 mM EDTA). Free protein with no compound (line with error bars), compound 1 (dash-dotted line), compound 2 (dotted line), and compound 3 (solid line) are shown. Abbreviations: EDTA, ethylenediaminetetraacetic acid; au, arbitrary units; ANS, 8-anilinonaphthalene-1-sulfonic acid.

Figure 2

Compounds selected from experimental screening and exhibiting antiviral activity in cell assays. Notes: Chemical structures of the selected compounds by experimental screening were further tested in cell-based, calorimetric and enzymatic assays.

Figure 3

In vitro enzymatic inhibition of NS3 protease. Notes: NS3 protease activity was determined as the initial slope of the curves. The percentage of activity is calculated as the quotient between the activity of NS3 protease in the presence (25 μM) and the absence of a given compound (compound 1, closed triangles; compound 2, closed circles; compound 3, closed squares). Experiments were carried out in triplicate and the relative errors were 12%. Physiological Zn⁺² concentration lies in the nanomolar-to-picomolar region, where the competitive effect of Zn⁺² is greatly diminished. V₀, initial velocity of hydrolysis reaction catalyzed by NS3 protease in the presence of inhibitor at concentration [I].

Figure 4

Docking of compound/γ-CD complexes. Notes: (A) Compound 1; (B) Compound 2; (C) Compound 3. γ-CD: blue ball and stick; compounds: ball functional groups representation. Abbreviation: γ-CD, γ-cyclodextrin.

Figure 5

Inhibition of HCV replicon in cell assays. Notes: (A) Evaluation of activity and cytotoxicity of the selected compounds in cell assays. HCV replicon replication rates (solid lines and solid symbols) and cell survival curves (dash lines and open symbols) are shown for: γ-CD-free compound 3 (squares), compound 3:γ-CD 1:2.5 complex (circles); compound 3:γ-CD 1:5 complex (triangles); compound 3:γ-CD 1:10 complex (diamonds). (B) Viral replication inhibition level as a function of γ-CD concentration (maximal loss in luminescence percentage observed in [A]). Abbreviations: γ-CD, γ-cyclodextrin; HCV, hepatitis C virus; UTC, untreated controls.