The treatment of malaria, the most common parasitic disease worldwide and the third deadliest infection after HIV and tuberculosis, is currently compromised by the dramatic increase and diffusion of drug resistance among the various species of Plasmodium, especially P. falciparum (Pf). In this view, the development of new antiplasmodial agents that are able to act via innovative mechanisms of action, is crucial to ensure efficacious antimalarial treatments. In one of our previous communications, we described a novel class of compounds endowed with high antiplasmodial activity, characterized by a pharmacophore never described before as antiplasmodial and identified by their 4,4’‐oxybisbenzoyl amide cores. Here, through a detailed structure‐activity relationship (SAR) study, we thoroughly investigated the chemical features of the reported scaffolds and successfully built a novel antiplasmodial agent active on both chloroquine (CQ)‐sensitive and CQ‐resistant Pf strains in the low nanomolar range, without displaying cross‐resistance. Moreover, we conducted an in silico pharmacophore mapping.

The aim of this study was to develop new antiplasmodial compounds acting through distinct mechanisms during both the liver and the blood stages of the parasite life cycle. Compounds were designed on the basis of the “double-drug” approach:  primaquine, which has been linked to statine-based inhibitors of plasmepsins (PLMs), the plasmodial aspartic proteases involved in degradation of hemeoglobin. The compounds were tested in vitro for anti-PLM I/PLM II activities and against chloroquine-sensitive (D10) and chloroquine-resistant (W2) strains of P. falciparum. An antiplasmodial activity (IC50) as low as 0.1 μM was obtained, an excellent improvement in comparison with inhibitors previously reported (IC50 = 2−20 μM). The killing activity was equally directed against both P. falciparum strains and was correlated to lipophilicity (calculated as ALogP), for all compounds but one (9). All compounds inhibited PLM I and PLM II in the nanomolar range (K i = 1−700 nM). The most promising compounds (2, 6, 10) were not cytotoxic against human fibroblasts at 100 μM and were highly selective for PLMs vs human cathepsin D.

Our previous studies showed that (À)-epigallocatechin-3-gallate (EGCG) inhibits signal transducer activator of transcription 1 (STAT1) activation. Since EGCG may be a promising lead compound for new anti-STAT1 drug design, 15 synthetic catechins, characterized by the (À)-gallocatechin-3-gallate stereochemistry, were studied in the human mammary MDA-MB-231 cell line to identify the minimal structural features that preserve the anti-STAT1 activity. We demonstrate that the presence of three hydroxyl groups of B ring and one hydroxyl group in D ring is essential to preserve their inhibitory action. Moreover, a possible molecular target of these compounds in the STAT1 pathway was investigated. Our results demonstrate a direct interaction between STAT1 protein and catechins displaying anti-STAT1 activity. In particular, surface plasmon resonance (SPR) analysis and molecular modeling indicate the presence of two putative binding sites (a and b) with different affinity. Based on docking data, site-directed mutagenesis was performed, and interaction of the most active catechins with STAT1 was studied with SPR to test whether Gln518 on site a and His568 on site b could be important for the catechin-STAT1 interaction. Data indicate that site b has higher affinity for catechins than site a as the highest affinity constant disappears in the H568A-STAT1 mutant. Furthermore, Janus kinase 2 (JAK2) kinase assay data suggest that the contemporary presence in vitro of STAT1 and catechins inhibits JAK2-elicited STAT1 phosphorylation. The very tight catechin-STAT1 interaction prevents STAT1 phosphorylation and represents a novel, specific and efficient molecular mechanism for the inhibition of STAT1 activation. Structured digital abstract• Jak2 phosphorylates STAT1 by protein kinase assay (View interaction)

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