Biomimetic Assay for Hematin Crystallization Inhibitors: A New Platform To Screen Antimalarial Drugs

Abstract

Molecular inhibitors are commonly employed in natural, synthetic, and biological crystallization as a means of regulating crystal size and habit. In pathological crystallization, growth inhibitors are commonly employed as therapies to decrease the rate of crystal growth, thereby reducing the incidence rate of diseases. When designing inhibitors for such purposes, it is often unknown a priori what properties (e.g., structure, functionality) are critical for drug efficacy and specificity. Identification of lead candidates is often accomplished through brute force screening without knowledge of the molecular-level driving force(s) governing favorable inhibitor− crystal interactions. Here, we present a biomimetic assay to characterize the crystallization of hematin, a critical component of Plasmodium parasite survival in malaria. Many antimalarial drugs have been shown to function as inhibitors that suppress hematin crystal growth. The increasing resistance of malaria parasites to current antimalarials has created an impetus to design alternative drugs; however, current approaches to identify lead candidates rely on combinatorial methods employing parasite assays to screen compounds, which are incapable of elucidating the effect(s) of inhibitors on hematin crystallization. In this study, we use citric-buffer saturated octanol (CBSO) as a physiologically relevant growth medium to develop a facile, robust method of screening compounds that inhibit hematin crystal growth. As benchmarks, we selected antimalarials and antibiotics that are commonly used in combination therapies. We assessed drug solubility in CBSO and designed a biomimetic assay to quantify the relative efficiency of hematin growth inhibitors. We demonstrate that this assay can be used as a high-throughput platform to screen libraries of compounds as a more streamlined approach to identify inhibitors of pathological crystallization.

The Effects of Quinoline and Non-Quinoline Inhibitors on the Kinetics of Lipid-Mediated β-Hematin Crystallization

“…34 The data show a qualitative relationship between the β-hematin IC 50 data and the biological potency of the compounds in question, although , as above (for the D10 case), the correlation is not significant. This is most likely due to the limited size of the compound test set, however pharmacokinetic factors would again be relevant.…”

“…While other biomimetic assays, for example the NP-40 detergent assay 36 and recently-reported CBSO assay, 34 do permit determination of inhibitory activity (IC 50 ) as discussed above, to our knowledge this is the first assay that provides a measure of the strength of adsorption of a compound to β-hematin, the proposed mechanism of these compounds’ action. In all cases, we show that low inhibitor concentrations cause a reduction in the rate constant for β-hematin formation without altering the maximal yield.…”

Influences of Crystal Anisotropy in Pharmaceutical Process Development

“…In this context, it was possible to develop a rationale for understanding the action of quinoline based drugs. More in depth studies lead to the development of screening platforms ( 125 ) for the selection of optimum drug candidates based on growth inhibitor activity ( 115 – 117 ).…”

Antimalarial Quinoline Drugs Inhibit β-Hematin and Increase Free Hemin Catalyzing Peroxidative Reactions and Inhibition of Cysteine Proteases

“…The synthesis of hemozoin constitutes a unique system of Plasmodium to detoxify heme; therefore its inhibition is a useful target for antimalarial drugs action 2,18,31,32 . Quinoline drugs ( e.g .…”