Inhibition of Plasmodium falciparum Hsp70-Hop partnership by 2-phenylthynesulfonamide.

Plasmodium falciparum Hsp70-1 (PfHsp70-1; PF3D7_0818900) and PfHsp90 (PF3D7_0708400) are essential cytosol localized chaperones of the malaria parasite. The two chaperones form a functional complex via the adaptor protein, Hsp90-Hsp70 organizing protein (PfHop [PF3D7_1434300]), which modulates the interaction of PfHsp70-1 and PfHsp90 through its tetracopeptide repeat (TPR) domains in a nucleotide-dependent fashion. On the other hand, PfHsp70-1 and PfHsp90 possess C-terminal EEVD and MEEVD motifs, respectively, which are crucial for their interaction with PfHop. By coordinating the cooperation of these two chaperones, PfHop plays an important role in the survival of the malaria parasite. 2-Phenylthynesulfonamide (PES) is a known anti-cancer agent whose mode of action is to inhibit Hsp70 function. In the current study, we explored the antiplasmodial activity of PES and investigated its capability to target the functions of PfHsp70-1 and its co-chaperone, PfHop. PES exhibited modest antiplasmodial activity (IC50 of 38.7 ± 0.7 µM). Furthermore, using surface plasmon resonance (SPR) analysis, we demonstrated that PES was capable of binding recombinant forms of both PfHsp70-1 and PfHop. Using limited proteolysis and intrinsic fluorescence-based analysis, we showed that PES induces conformational changes in PfHsp70-1 and PfHop. In addition, we demonstrated that PES inhibits the chaperone function of PfHsp70-1. Consequently, PES abrogated the association of the two proteins in vitro. Our study findings contribute to the growing efforts to expand the arsenal of potential antimalarial compounds in the wake of growing parasite resistance against currently used drugs.

Adsorption to the Surface of Hemozoin Crystals: Structure-Based Design and Synthesis of Amino-Phenoxazine ß-Hematin Inhibitors.

In silico adsorption of eight antimalarials that inhibit ß-hematin (synthetic hemozoin) formation identified a primary binding site on the (001) face, which accommodates inhibitors via formation of predominantly π-π interactions. A good correlation (r2 =0.64, P=0.017) between adsorption energies and the logarithm of ß-hematin inhibitory activity was found for this face. Of 53 monocyclic, bicyclic and tricyclic scaffolds, the latter yielded the most favorable adsorption energies. Five new amino-phenoxazine compounds were pursued as ß-hematin inhibitors based on adsorption behaviour. The 2-substituted phenoxazines show good to moderate ß-hematin inhibitory activity (<100 µM) and Plasmodium falciparum blood stage activity against the 3D7 strain. N1 ,N1 -diethyl-N4 -(10H-phenoxazin-2-yl)pentane-1,4-diamine (P2a) is the most promising hit with IC50 values of 4.7±0.6 and 0.64±0.05 µM, respectively. Adsorption energies are predictive of ß-hematin inhibitory activity, and thus the in silico approach is a beneficial tool for structure-based development of new non-quinoline inhibitors.

Toward a Stable and Potent Coenzyme A-Targeting Antiplasmodial Agent: Structure-Activity Relationship Studies of N-Phenethyl-α-methyl-pantothenamide.

Pantothenamides (PanAms) are potent antiplasmodials with low human toxicity currently being investigated as antimalarials with a novel mode of action. These structural analogues of pantothenate, the vitamin precursor of the essential cofactor coenzyme A, are susceptible to degradation by pantetheinase enzymes present in serum. We previously discovered that α-methylation of the ß-alanine moiety of PanAms increases their stability in serum and identified N-phenethyl-α-methyl-pantothenamide as a pantetheinase-resistant PanAm with potent, on-target, and selective antiplasmodial activity. In this study, we performed structure-activity relationship investigations to establish whether stability and potency can be improved further through alternative modification of the scissile amide bond and through substitution/modification of the phenyl ring. Additionally, for the first time, the importance of the stereochemistry of the α-methyl group was evaluated in terms of stability versus potency. Our results demonstrate that α-methylation remains the superior choice for amide modification, and that while monofluoro-substitution of the phenyl ring (that often improves ADME properties) was tolerated, N-phenethyl-α-methyl-pantothenamide remains the most potent analogue. We show that the 2S,2'R-diastereomer is far more potent than the 2R,2'R-diastereomer and that this cannot be attributed to preferential metabolic activation by pantothenate kinase, the first enzyme of the coenzyme A biosynthesis pathway. Unexpectedly, the more potent 2S,2'R-diastereomer is also more prone to pantetheinase-mediated degradation. Finally, the results of in vitro studies to assess permeability and metabolic stability of the 2S,2'R-diastereomer suggested species-dependent degradation via amide hydrolysis. Our study provides important information for the continued development of PanAm-based antimalarials.