Hematin crystal and Malaria parasite

The broad long-term goal is to optimize critical quinoline and artemisinin malaria drug combinations for maximum heme crystal inhibition in the digestive vacuole of the P. falciparumparasite. In the setting of ongoing drug resistance to currently deployed drugs, this work will investigate a novel quinoline like mechanism of action for the heme-artemisinin adducts, define reversible or near irreversible heme crystal inhibition also related to drug resistance and explore optimum heme crystal inhibition related to parasite killing with quinoline-artemisinin combinations. Preliminary data validate an additional mechanism of action for the artemisinins- activation with formation of the abundant heme-artemisinin adduct which inhibits with near irreversible action heme crystallization. Exogenous heme-artemisinin adducts inhibit artemisinin ring-resistant mutant Kelch13 P. falciparumparasites with low 5 to 50 nM IC₅₀s. The experimental approach employs the synergy between experimental investigations with P. falciparum drug-sensitive and resistant parasites in vitro, murine malaria in vivoand physicochemical insights obtained by observations of crystal growth by atomic force microscopy in the presence of different drug combinations.

The hypothesis is that the heme-artemisinin adduct action on heme crystal formation will render trophozoites of any Plasmodium isolate sensitive, which overcomes the artemisinin ring-stage resistance. We also hypothesize that certain combinations of the quinolines, artemisinins and heme-artemisinins adducts are superior in pharmacodynamic killing of parasites correlated to heme crystal inhibition as well as separately to the degree of reversible/near-irreversible heme crystal inhibition. Towards these objectives, we will pursue three specific aims: Aim 1. Establish the inhibition concentrations and mechanism of actionof exogenous as well as bio-activated heme-artemisinin adducts in drug sensitive and resistant Plasmodium. Aim 2. Establish reversibility of both in vitroheme-artemisinin adduct heme crystal inhibition correlated to competitive drug hemozoin uptake in P. falciparum and in heme crystallization assaysor in vivowith the drug pulse transfer mouse malaria model.Aim 3. Establish if double combinations of antimalarial quinolines, artemisinins and heme-artemisinin adducts enhance, weaken, or are indifferent to their partner’s action on pharmacodynamic parasite killing and the rate of hematin crystallization.

This work will forge a novel path to the heme-artemisinin adduct as a potential safer, effective drug. The work will also inform fundamental knowledge regarding optimum combinations of malaria drugs based upon interaction effects as well as degree of reversibility of hematin crystal growth coupled to a mouse model of Plasmodiumkilling.

This work will improve the drug therapies against malaria, the third most deadly infectious disease. Hemozoin crystal growth is a proven drug target. Here we explore the quantitative parasite killing contribution of heme-artemisinin adducts alone and in combination with the quinolines. An outcome of this work may be a safer effective novel malaria drug based on the heme-artemisinin adduct.

Protein Clusters

Protein misfolding followed by aggregation is the major cause of neurodegenerative diseases such as Alzheimer’s, Parkinson’s, familial amyloid poly neuropathy (FAP), Huntington’s, type-II diabetes, etc. Common aspect of all protein aggregation diseases is the altered protein conformation known as partially unfolded amyloidogenic intermediate that is capable of assembly into amyloid structures. Recently discovered mesoscopic protein-rich clusters may act as crucial precursors for the nucleation of ordered protein solids, such as crystals, sickle hemoglobin polymers, and amyloid fibrils. These clusters challenge settled paradigms of protein condensation as the constituent protein molecules present features characteristic of both partially misfolded and native proteins. Some of their unusual features include the kinetically determined size, thermodynamically controlled number, and their distinct nature from aggregation triggered by reduction of the intramolecular S−S bonds and amyloid aggregates.      We investigated the role of protein structural flexibility on its ability to induce formation of mesoscopic clusters for multiple proteins including the p53, known as guardian of genome, which contains multi dis-ordered and b-sheet rich domains; hemoglobin A, which is the major component of red blood cells and contains a compact structure rich in a-helices; antimicrobial enzyme lysozyme which is a robust model in study of protein aggregation. Whereas lysozyme and hemoglobin A demonstrate mesoscopic clusters at high protein concentrations, p53, whose aggregation is tied to cancer development, exhibits clustering at physiological temperatures for low concentrations of the protein. These findings suggest that the clusters are a product of limited protein structural flexibility. Furthermore, we discovered that the crowding environment of the inside cell significantly promotes clustering of intrinsic disordered proteins (IDPs) such as p53. About half of human cancers are associated with mutations of the tumor suppressor p53. Mutated p53 emerges as a powerful oncogene, which blocks the activity of wild-type p53 and several distinct anticancer pathways. The gained functions of the mutant have been related to the aggregation behaviors of wild-type and mutant p53. Our data reveals that in presence of crowders, the p53 clusters can capture some of the crowder molecules, which causes steric hindrance effects and raises the nucleation barrier of the aggregation. Thus these clusters can potentially act as storage of proteins and protect them from formation of toxic amyloid aggregates by providing sufficient time for the proteomic and chaperonin machinery to clear out or refold the misfolded aggregated species in the cell. The nucleation of p53 fibrils deviates from the accepted mechanism of sequential association of single solute molecule. We find the mesoscopic clusters serve as a pre-assembled precursor of high p53 concentration that facilitate fibril assembly. Fibril nucleation hosted by precursors represents a novel biological pathway, which awards unexplored avenues to suppression of protein fibrillation in aggregation diseases.

Pathological Crystallization

We established the mechanisms of action in blocking hematin crystallization of several classes of antimalarial drugs and related compounds. The results of my colleagues on the mechanism of hematin crystallization, concurrent with studies of parasite physiology, suggest that hemozoin crystals form in organic sub-phases comprised of a blend of several lipids, which may be suspended in the digestive vacuole or lining its walls. They have demonstrated that the formation of hematin crystals follows a classical mechanism whereby new crystal layers are nucleated on top of existing ones and grow by the association of solute molecules that leads to the spreading of the layers until they cover the entire face. They have shown that quinoline-class antimalarials possess an extremely efficient pathway of inhibition of hematin crystallization by binding to specific crystal surface sites and impeding the incorporation of soluble hematin.