Unravelling the rate of action of hits in the Leishmania donovani box using standard drugs amphotericin B and miltefosine

In recent years, the neglected diseases drug discovery community has elected phenotypic screening as the key approach for the identification of novel hit compounds. However, when this approach is applied, important questions related to the mode of action for these compounds remain unanswered. One of such questions is related to the rate of action, a useful piece of information when facing the challenge of prioritising the most promising hit compounds. In the present work, compounds of the "Leishmania donovani box" were evaluated using a rate of action assay adapted from a replicative intracellular high content assay recently developed. The potency of each compound was determined every 24 hours up to 96 hours, and standard drugs amphotericin B and miltefosine were used as references to group these compounds according to their rate of action. Independently of this biological assessment, compounds were also clustered according to their minimal chemical scaffold. Comparison of the results showed a complete correlation between the chemical scaffold and the biological group for the vast majority of compounds, demonstrating how the assay was able to bring information on the rate of action for each chemical series, a property directly linked to the mode of action. Overall, the assay here described permitted us to evaluate the rate of action of the "Leishmania donovani box" using two of the currently available drugs as references and, also, to propose a number of fast-acting chemical scaffolds present in the box as starting points for future drug discovery projects to the wider scientific community. The results here presented validate the use of this assay for the determination of the rate of action early in the discovery process, to assist in the prioritisation of hit compounds

Chemical clusters (Cl) obtained for the 73 selected compounds in the INF output.

<p>TCMDC ID: Tres Cantos Medicine Discovery Center Identifier. Chemical structures and more information on all compounds tested in these studies are available at reference 18 as TCMDC IDs (Tres Cantos Medicine Discovery Center Identifiers).</p

Complete set of chemical clusters obtained for the clustering exercise conducted with the 88 selected compounds using the data of the AMMAC output and the 73 selected compounds using the data of the INF output.

<p>Complete set of chemical clusters obtained for the clustering exercise conducted with the 88 selected compounds using the data of the AMMAC output and the 73 selected compounds using the data of the INF output.</p

Effect of the serum used in the assay on the pEC50 values obtained.

<p>Second and third columns represent data obtained for the AM/MAC output and for the INF output respectively. Data shown in the upper four rows correspond to all the compounds tested (<i>Leishmania donovani</i> box) while the ones shown in the lower four rows correspond to the selected sets of compounds (88 compounds for the AM/MAC output and 73 compounds for the INF output).</p

pEC50 values of standard drugs amphotericin B (AB) and miltefosine (MF) at each time point calculated using the AM/MAC and INF outputs.

<p>pEC50 = -log EC₅₀ (M). Values are expressed as mean value of two independent test runs. R² values obtained for all curves studied were >0.85.</p

Comparison of the pEC50 values obtained at 96 hours for the two assay runs using the AM/MAC output (left) and the INF output (right).

<p>The R² value calculated is shown at the top of each representation.</p

Comparison of the results obtained for the biological grouping and the chemistry clustering exercise using AM/MAC and INF outputs for the selected compounds.

<p>Compound TCMDC IDs (Tres Cantos Medicine Discovery Center Identifiers): green (group 1), orange (group 2) and red (group 3). Chemical structures for all compounds tested in these studies are available at reference 18 as TCMDC IDs (Tres Cantos Medicine Discovery Center Identifiers).</p

Chemical clusters (Cl) obtained for the 88 selected compounds with the AM/MAC output.

<p>TCMDC ID: Tres Cantos Medicine Discovery Center Identifier. Chemical structures and more information on all compounds tested in these studies are available at reference 18 as TCMDC IDs (Tres Cantos Medicine Discovery Center Identifiers).</p