High-Throughput Screening Identifies Novel Antileishmanial Scaffolds and Reveals Cross-Species Activity of Structurally Diverse Compounds.

Abstract

Background: Leishmaniasis remains a neglected tropical disease with limited therapeutic options and a continued need for new and effective treatments. High-throughput phenotypic screening provides an approach for identifying chemically diverse compounds with antileishmanial activity that can serve as a starting point for drug development. This study further characterized compounds identified through a previous high-throughput phenotypic screen to prioritize promising antileishmanial chemical scaffolds and evaluate their potential for continued drug development.

Methods: Compounds belonging to two enriched scaffolds, 1,4-diaryl-pyrazolo-pyridinone (1,4-DAPP) and pyrazolo-[5,1-c][1,2,4]triazine (PTZ), along with structurally diverse singleton compounds, were evaluated for antileishmanial activity across Leishmania parasite life stages by determining half-maximal inhibitory concentrations (IC₅₀). Host-cell cytotoxicity was evaluated against THP-1 macrophages by determining half-maximal cytotoxic concentrations (CC₅₀). The IC₅₀ and CC₅₀ values were determined by nonlinear regression analysis. Selected lead compounds underwent preliminary in vitro absorption, distribution, metabolism, and excretion (ADME) profiling. Synthesized analogs of both scaffolds were evaluated to investigate structure-activity relationships and opportunities for chemical optimization.

Results: 1,4-DAPP #9279817 and PTZ #9234527 were prioritized as lead compounds based on their combined antileishmanial activity and host-cell selectivity, exhibiting intracellular amastigote IC₅₀ values of 3.50 and 3.12 μM and selectivity indices (SI) of 14.29 and 16.03, respectively. Singleton compounds exhibited variable parasite stage-specific activity, with host-cell cytotoxicity limiting the selectivity of several candidates. Both lead scaffolds retained activity across multiple Leishmania species, although compound susceptibility varied among species. Preliminary ADME profiling revealed distinct properties of the two leads, highlighting strengths and limitations for further optimization. Structural modifications of both scaffolds generated analogs with improved intracellular antileishmanial activity and selectivity.

Conclusions: The 1,4-DAPP and PTZ scaffolds represent promising candidates for continued antileishmanial development. These findings support continued optimization of both scaffolds while emphasizing the importance of evaluating antileishmanial activity in disease-relevant parasite models.