Heme Crystallization Research Articles

α-Glucosidase isoform G contributes to heme detoxification in Rhodnius prolixus and its knockdown affects Trypanosoma cruzi metacyclogenesis

The triatomine bug Rhodnius prolixus is a hematophagous hemipteran and a primary vector of Trypanosoma cruzi, the causative agent of Chagas' disease (CD), in Central America and Northern South America. Blood-feeding poses significant challenges for hematophagous organisms, particularly due to the release of high doses of pro-oxidant free heme during hemoglobin digestion. In this arthropod, most of the free heme in the gut is aggregated into hemozoin (Hz), an inert and non-oxidative biocrystal. Two major components present in the perimicrovillar membranes (PMM) of triatomine insects have been previously implicated in heme crystallization: lipids and the biochemical marker of the PMM, the enzyme α-glucosidase. In this study, we investigated the role of R. prolixus α-glucosidase isoform G (Rp-αGluG) in heme detoxification and the effects of its knockdown on the insect physiology. The effect of α-glucosidase isoform G (αGluG) knockdown on T. cruzi proliferation and metacyclogenesis was also investigated. Initially, a 3D structure of Rp-αGluG was predicted by comparative modeling and then subjected to molecular docking with the heme molecule, providing in silico support for understanding the process of Hz biocrystallization. Next, adult females of R. prolixus were challenged with RNAi against Rp-αGluG (dsαGluG) to assess physiological and phenotypic changes caused by its knockdown. Our data show that the group challenged with dsαGluG produced less Hz, resulting in more intact hemoglobin available in the digestive tract. These animals also laid fewer eggs, which had a lower hatching rate. In addition, T. cruzi metacyclogenesis was significantly lower in the dsαGluG group. The present work demonstrates the importance of Rp-αGluG in heme detoxification, the digestive and reproductive physiology of R. prolixus, as well as its influence on the life cycle of T. cruzi. Since heme neutralization is a vital process for hematophagous bugs, our study provides useful information for the development of new strategies targeting the Hz formation and potentially affecting the vectorial transmission of Chagas disease.

Nonclassical mechanisms to irreversibly suppress β-hematin crystal growth

Hematin crystallization is an essential element of heme detoxification of malaria parasites and its inhibition by antimalarial drugs is a common treatment avenue. We demonstrate at biomimetic conditions in vitro irreversible inhibition of hematin crystal growth due to distinct cooperative mechanisms that activate at high crystallization driving forces. The evolution of crystal shape after limited-time exposure to both artemisinin metabolites and quinoline-class antimalarials indicates that crystal growth remains suppressed after the artemisinin metabolites and the drugs are purged from the solution. Treating malaria parasites with the same agents reveals that three- and six-hour inhibitor pulses inhibit parasite growth with efficacy comparable to that of inhibitor exposure during the entire parasite lifetime. Time-resolved in situ atomic force microscopy (AFM), complemented by light scattering, reveals two molecular-level mechanisms of inhibitor action that prevent β-hematin growth recovery. Hematin adducts of artemisinins incite copious nucleation of nonextendable nanocrystals, which incorporate into larger growing crystals, whereas pyronaridine, a quinoline-class drug, promotes step bunches, which evolve to engender abundant dislocations. Both incorporated crystals and dislocations are known to induce lattice strain, which persists and permanently impedes crystal growth. Nucleation, step bunching, and other cooperative behaviors can be amplified or curtailed as means to control crystal sizes, size distributions, aspect ratios, and other properties essential for numerous fields that rely on crystalline materials.

Optimization of the synthesis, in silico ADME/Tox profiling studies, and evaluation of the antimalarial activity of (7-chloroquinolin-4-ylthio)alkylbenzoate derivatives

Optimized reaction conditions are developed to obtain a series of [(7-chloroquinolin-4-yl)sulfanyl] alcohol derivatives as intermediates to prepare a range of (7-chloroquinolin-4-ylthio) alkylbenzoate derivatives. The structures of all the synthesized compounds are confirmed from their infrared and nuclear magnetic resonance spectral data, and by elemental analysis. In silico ADME/Tox profiling studies of the synthesized molecules are undertaken, and the potential antimalarial activity of the products is determined. In vitro, all the prepared compounds significantly reduce heme crystallization with IC50 values of < 10 µM. In vivo, the reduction in parasitemia levels and survival time increases are marginal.

Synthesis and in silico ADME/Tox profiling studies of heterocyclic hybrids based on chloroquine scaffolds with potential antimalarial activity.

A series of heterocyclic chloroquine hybrids containing either a β-phenethylamine fragment or a 2-aminoindane moiety were synthesized and screened in vitro as inhibitors of β-hematin formation and in vivo for their antimalarial activity against chloroquine-sensitive strains of Plasmodium berghei ANKA. Although these new compounds were not found to be more active than chloroquine in vivo, all new compounds significantly reduced heme crystallization with IC50 values < 1 μM. Compounds 12 and 13 were able to inhibit heme crystallization with IC50 values of 0.39 ± 0.09 and 0.48 ± 0.02 μM, respectively, and these values were comparable to that of chloroquine with an IC50 value of 0.18 ± 0.03. It was also determined that the physicochemical and pharmacokinetic properties were moderately favorable after in silico evaluation, derivatives 8 and 10 did not present hepatotoxicity, and the in vitro hemolytic activity against red blood cells was found to be low. Spectral (infrared, nuclear magnetic resonance, and elemental analysis) data for all final compounds were consistent with the proposed structures.

Solvent Structure and Dynamics near the Surfaces of β-Hematin Crystals.

Hematin crystallization, which is an essential component of the physiology of malaria parasites and the most successful target for antimalarial drugs, proceeds in mixed organic-aqueous solvents both in vivo and in vitro. Here we employ molecular dynamics simulations to examine the structuring and dynamics of a water-normal octanol mixture (a solvent that mimics the environment hosting hematin crystallization in vivo) in the vicinity of the typical faces in the habit of a hematin crystal. The simulations reveal that the properties of the solvent in the layer adjacent to the crystal are strongly impacted by the distinct chemical and topological features presented by each crystal face. The solvent organizes into at least three distinct layers. We also show that structuring of the solvent near the different faces of β-hematin strongly impacts the interfacial dynamics. The relaxation time of n-octanol molecules is longest in the contact layers and correlates with the degree of structural ordering at the respective face. We show that the macroscopically homogeneous water-octanol solution holds clusters of water and n-octanol connected by hydrogen bonds that entrap the majority of the water but are mostly smaller than 30 water molecules. Near the crystal surface the clusters anchor on hematin carboxyl groups. These results provide a direct example that solvent structuring is not restricted to aqueous and other hydrogen-bonded solutions. Our findings illuminate two fundamental features of the mechanisms of hematin crystallization: the elongated shapes of natural and synthetic hematin crystals and the stabilization of charged groups of hematin and antimalarials by encasing in water clusters. In addition, these findings suggest that hematin crystallization may be controlled by additives that disrupt or reinforce solvent structuring.

High-Content Phenotypic Screen of a Focused TCAMS Drug Library Identifies Novel Disruptors of the Malaria Parasite Calcium Dynamics.

The search for new antimalarial drugs with unexplored mechanisms of action is currently one of the main objectives to combat the resistance already in the clinic. New drugs should target specific mechanisms that once initiated lead inevitably to the parasite's death and clearance and cause minimal toxicity to the host. One such new mode of action recently characterized is to target the parasite's calcium dynamics. Disruption of the calcium homeostasis is associated with compromised digestive vacuole membrane integrity and release of its contents, leading to programmed cell death-like features characterized by loss of mitochondrial membrane potential and DNA degradation. Intriguingly, chloroquine (CQ)-treated parasites were previously reported to exhibit such cellular features. Using a high-throughput phenotypic screen, we identified 158 physiological disruptors (hits) of parasite calcium distribution from a small subset of approximately 3000 compounds selected from the GSK TCAMS (Tres Cantos Anti-Malarial Set) compound library. These compounds were then extensively profiled for biological activity against various CQ- and artemisinin-resistant Plasmodium falciparum strains and stages. The hits were also examined for cytotoxicity, speed of antimalarial activity, and their possible inhibitory effects on heme crystallization. Overall, we identified three compounds, TCMDC-136230, -125431, and -125457, which were potent in inducing calcium redistribution but minimally inhibited heme crystallization. Molecular superimposition of the molecules by computational methods identified a common pharmacophore, with the best fit assigned to TCMDC-125457. There were low cytotoxicity or CQ cross-resistance issues for these three compounds. IC50 values of these three compounds were in the low micromolar range. In addition, TCMDC-125457 demonstrated high efficacy when pulsed in a single-dose combination with artesunate against tightly synchronized artemisinin-resistant ring-stage parasites. These results should add new drug options to the current armament of antimalarial drugs as well as provide promising starting points for development of drugs with non-classical modes of action.

Light optical and Ultrastructural Characteristics of the Maral Liver with Chronic Dicrocoeliasis

BACKGROUND: Dicrocoeliasis is caused by trematode Dicrocoelium lanceatum from the family Dicrocoeliidae, a parasite in the bile ducts of the liver of domestic and wild animals. Dicrocoeliasis mainly affects sheep, cattle, camels, zebu, deer, fallow deer, argali, less often – horses, donkeys, dogs, rabbits, hares and bears, as well as humans. Dicrocoeliasis of ruminants is widespread across the whole Kazakhstan. Invasive diseases represent a significant obstacle in the development of domestic maral breeding, among which trematodoses, and particularly dicrocoeliasis of maral play a major role. AIM: The aim of the research was to study the influence of dicrocoelia on the ultrastructural organization of the liver of maral. MATERIALS AND METHODS: For examination under electron microscope, biopsy pieces of liver tissue of maral were fixed in 2.5% solution of glutaraldehyde with post-fixation in 1% solution of osmium tetroxide, conducted according to a conventional method, and enclosed in epon. Semi-thin and ultra-thin sections were prepared on the ultra-microtome Leica. Semi-thin sections were stained with methylene blue, azure 2, and studied at a high-resolution light optical level. The ultra-thin sections were contrasted with uranyl acetate and lead citrate according Reynolds method and examined under electron microscope Libra 120 (C. Zeiss). RESULTS: The light optical examination of half-thin sections revealed that the morphological pattern of pathological changes in liver tissue was polymorphic, even within a single hepatic lobe. CONCLUSIONS: In the liver of maral infected with chronic dicrocoeliasis, dystrophic and destructive pathological changes developed in all the cellular structures of the hepatic lobules: In the form of plethora and vast enlargement of sinusoids, vacuolar and lipodegeneration of hepatocytes, destruction of the hepatic tissue with edema, hemorrhages, in the appearance of cells associated with inflammation, and the deposition of hematin crystals.

A second mechanism employed by artemisinins to suppress Plasmodium falciparum hinges on inhibition of hematin crystallization

Malaria is a pervasive disease that affects millions of lives each year in equatorial regions of the world. During the erythrocytic phase of the parasite life cycle, Plasmodium falciparum invades red blood cells, where it catabolizes hemoglobin and sequesters the released toxic heme as innocuous hemozoin crystals. Artemisinin (ART)-class drugs are activated in vivo by newly released heme, which creates a carbon-centered radical that markedly reduces parasite density. Radical damage to parasite lipids and proteins is perceived to be ARTs’ dominant mechanism of action. By contrast, quinoline-class antimalarials inhibit the formation of hemozoin and in this way suppress heme detoxification. Here, we combine malaria parasite assays and scanning probe microscopy of growing β-hematin crystals to elucidate an unexpected mechanism employed by two widely administered antimalarials, ART, and artesunate to subdue the erythrocytic phase of the parasite life cycle. We demonstrate that heme–drug adducts, produced after the radical activation of ARTs and largely believed to be benign bystanders, potently kills P. falciparum at low exogenous concentrations. We show that these adducts inhibit β-hematin crystallization and heme detoxification, a pathway which complements the deleterious effect of radicals generated via parent drug activation. Our findings reveal an irreversible mechanism of heme–ART adduct inhibition of heme crystallization, unique among antimalarials and common crystal growth inhibitors, that opens new avenues for evaluating drug dosing regimens and understanding growing resistance of P. falciparum to ART.

A lipocalin mediates unidirectional heme biomineralization in malaria parasites

During blood-stage development, malaria parasites are challenged with the detoxification of enormous amounts of heme released during the proteolytic catabolism of erythrocytic hemoglobin. They tackle this problem by sequestering heme into bioinert crystals known as hemozoin. The mechanisms underlying this biomineralization process remain enigmatic. Here, we demonstrate that both rodent and human malaria parasite species secrete and internalize a lipocalin-like protein, PV5, to control heme crystallization. Transcriptional deregulation of PV5 in the rodent parasite Plasmodium berghei results in inordinate elongation of hemozoin crystals, while conditional PV5 inactivation in the human malaria agent Plasmodium falciparum causes excessive multidirectional crystal branching. Although hemoglobin processing remains unaffected, PV5-deficient parasites generate less hemozoin. Electron diffraction analysis indicates that despite the distinct changes in crystal morphology, neither the crystalline order nor unit cell of hemozoin are affected by impaired PV5 function. Deregulation of PV5 expression renders P. berghei hypersensitive to the antimalarial drugs artesunate, chloroquine, and atovaquone, resulting in accelerated parasite clearance following drug treatment in vivo. Together, our findings demonstrate the Plasmodium-tailored role of a lipocalin family member in hemozoin formation and underscore the heme biomineralization pathway as an attractive target for therapeutic exploitation.

Pharmacophore Modeling, Synthesis, Scaffold Hopping and Biological β- Hematin Inhibition Interaction Studies for Anti-malaria Compounds.

A series of chloroquine analogues were designed using a repositioning approach to develop new anticancer compounds. Protein-ligand interaction fingerprints and ADMET descriptors were used to assess docking performance in virtual screenings to design chloroquine hybrid β-hematin inhibitors. A PLS algorithm was applied to correlate the molecular descriptors to IC50 values. The modeling presented excellent predictive power with correlation coefficients for calibration and cross-validation of r2 = 0.93 and q2 = 0.72. Using the model, a series of 4-aminoquinlin hybrids were synthesized and evaluated for their biological activity as an external test series. These compounds were evaluated for cytotoxic cell lines and β-hematin inhibition. The target compounds exhibited high β-hematin inhibition activity and were 3-9 times more active than the positive control. Furthermore, all the compounds exhibited moderate to high cytotoxic activity. The most potent compound in the dataset was docked with hemoglobin and its pharmacophore features were generated. These features were used as input to the Pharmit server for screening of six databases. The compound with the best score from ChEMBL was 2016904, previously reported as a VEGFR-2 inhibitor. The 11 compounds selected presented the best Gold scores with drug-like properties and can be used for drug development.

Molecular Targets for Malarial Chemotherapy: A Review.

The malaria parasite resistance to the existing drugs is a serious problem to the currently used antimalarials and, thus, highlights the urgent need to develop new and effective anti-malarial molecules. This could be achieved either by the identification of the new drugs for the validated targets or by further refining/improving the existing antimalarials; or by combining previously effective agents with new/existing drugs to have a synergistic effect that counters parasite resistance; or by identifying novel targets for the malarial chemotherapy. In this review article, a comprehensive collection of some of the novel molecular targets has been enlisted for the antimalarial drugs. The targets which could be deliberated for developing new anti-malarial drugs could be: membrane biosynthesis, mitochondrial system, apicoplasts, parasite transporters, shikimate pathway, hematin crystals, parasite proteases, glycolysis, isoprenoid synthesis, cell cycle control/cycline dependent kinase, redox system, nucleic acid metabolism, methionine cycle and the polyamines, folate metabolism, the helicases, erythrocyte G-protein, and farnesyl transferases. Modern genomic tools approaches such as structural biology and combinatorial chemistry, novel targets could be identified followed by drug development for drug resistant strains providing wide ranges of novel targets in the development of new therapy. The new approaches and targets mentioned in the manuscript provide a basis for the development of new unique strategies for antimalarial therapy with limited off-target effects in the near future.

Structuring of Organic Solvents at Solid Interfaces and Ramifications for Antimalarial Adsorption on β-Hematin Crystals.

A critical aspect of material synthesis is solvent structuring at solid-liquid interfaces, which can impact the adsorption of solute and growth modifiers on an underlying substrate. In general, the impact of solvent structuring on molecular sorbate interactions with solid sorbents is poorly understood. This is particularly true for processes that occur in organic media, such as hematin crystallization, which is crucial to the survival of malaria parasites. Here, we use chemical force microscopy and molecular modeling to analyze the interactions between functional moieties of known antimalarials and the interface between β-hematin crystals and a mixed organic (octanol)-aqueous solvent. We show that the β-hematin surface, patterned in parallel hydrophobic and hydrophilic stripes, engenders the assembly of up to five layers of octanol molecules aligned parallel to the crystal surface. In contrast, studies of solvent structuring on a disordered glass surface reveal that octanol molecules align perpendicular to the interface. The distinct octanol arrays direct molecule adsorption at the respective interfaces. At both substrates, we also find stabilized pockets of aqueous nanophase lining the surfaces. A combination of experimental analyses and modeling of solvent structuring provides crucial insights into the association of hematin molecules with growing crystals as well as the adsorption and mobility of antimalarial drugs. Moreover, our findings offer a general perspective on the collective behaviors of complex organic solvents that may apply to a broad range of interactions at solid-liquid interfaces.

Heme crystallization in a Chagas disease vector acts as a redox-protective mechanism to allow insect reproduction and parasite infection

Heme crystallization as hemozoin represents the dominant mechanism of heme disposal in blood feeding triatomine insect vectors of the Chagas disease. The absence of drugs or vaccine for the Chagas disease causative agent, the parasite Trypanosoma cruzi, makes the control of vector population the best available strategy to limit disease spread. Although heme and redox homeostasis regulation is critical for both triatomine insects and T. cruzi, the physiological relevance of hemozoin for these organisms remains unknown. Here, we demonstrate that selective blockage of heme crystallization in vivo by the antimalarial drug quinidine, caused systemic heme overload and redox imbalance in distinct insect tissues, assessed by spectrophotometry and fluorescence microscopy. Quinidine treatment activated compensatory defensive heme-scavenging mechanisms to cope with excessive heme, as revealed by biochemical hemolymph analyses, and fat body gene expression. Importantly, egg production, oviposition, and total T. cruzi parasite counts in R. prolixus were significantly reduced by quinidine treatment. These effects were reverted by oral supplementation with the major insect antioxidant urate. Altogether, these data underscore the importance of heme crystallization as the main redox regulator for triatomine vectors, indicating the dual role of hemozoin as a protective mechanism to allow insect fertility, and T. cruzi life-cycle. Thus, targeting heme crystallization in insect vectors represents an innovative way for Chagas disease control, by reducing simultaneously triatomine reproduction and T. cruzi transmission.

Early Onset of Kinetic Roughening due to a Finite Step Width in Hematin Crystallization.

The structure of the interface of a growing crystal with its nutrient phase largely determines the growth dynamics. We demonstrate that hematin crystals, crucial for the survival of malaria parasites, transition from faceted to rough growth interfaces at increasing thermodynamic supersaturation Δμ. Contrary to theoretical predictions and previous observations, this transition occurs at moderate values of Δμ. Moreover, surface roughness varies nonmonotonically with Δμ, and the rate constant for rough growth is slower than that resulting from nucleation and spreading of layers. We attribute these unexpected behaviors to the dynamics of step growth dominated by surface diffusion and the loss of identity of nuclei separated by less than the step width w. We put forth a general criterion for the onset of kinetic roughening using w as a critical length scale.

Deconstructing Quinoline-Class Antimalarials to Identify Fundamental Physicochemical Properties of Beta-Hematin Crystal Growth Inhibitors.

A versatile approach to control crystallization involves the use of modifiers, which are additives that interact with crystal surfaces and alter their growth rates. Elucidating a modifier's binding specificity to anisotropic crystal surfaces is a ubiquitous challenge that is critical to their design. In this study, we select hematin, a byproduct of malaria parasites, as a model system to examine the complementarity of modifiers (i.e., antimalarial drugs) to β-hematin crystal surfaces. We divide two antimalarials, chloroquine and amodiaquine, into segments consisting of a quinoline base, common to both drugs, and side chains that differentiate their modes of action. Using a combination of scanning probe microscopy, bulk crystallization, and analytical techniques, we show that the base and side chain work synergistically to reduce the rate of hematin crystallization. In contrast to general observations that modifiers retain their function upon segmentation, we show that the constituents do not act as modifiers. A systematic study of quinoline isomers and analogues shows how subtle rearrangement and removal of functional moieties can create effective constituents from previously ineffective modifiers, along with tuning their inhibitory modes of action. These findings highlight the importance of specific functional moieties in drug compounds, leading to an improved understanding of modifier-crystal interactions that could prove to be applicable to the design of new antimalarials.

Unraveling heme detoxification in the malaria parasite by in situ correlative X-ray fluorescence microscopy and soft X-ray tomography

A key drug target for malaria has been the detoxification pathway of the iron-containing molecule heme, which is the toxic byproduct of hemoglobin digestion. The cornerstone of heme detoxification is its sequestration into hemozoin crystals, but how this occurs remains uncertain. We report new results of in vivo rate of heme crystallization in the malaria parasite, based on a new technique to measure element-specific concentrations at defined locations in cell ultrastructure. Specifically, a high resolution correlative combination of cryo soft X-ray tomography has been developed to obtain 3D parasite ultrastructure with cryo X-ray fluorescence microscopy to measure heme concentrations. Our results are consistent with a model for crystallization via the heme detoxification protein. Our measurements also demonstrate the presence of considerable amounts of non-crystalline heme in the digestive vacuole, which we show is most likely contained in hemoglobin. These results suggest a tight coupling between hemoglobin digestion and heme crystallization, highlighting a new link in the crystallization pathway for drug development.

Antimalarials inhibit hematin crystallization by unique drug–surface site interactions

In malaria pathophysiology, divergent hypotheses on the inhibition of hematin crystallization posit that drugs act either by the sequestration of soluble hematin or their interaction with crystal surfaces. We use physiologically relevant, time-resolved in situ surface observations and show that quinoline antimalarials inhibit β-hematin crystal surfaces by three distinct modes of action: step pinning, kink blocking, and step bunch induction. Detailed experimental evidence of kink blocking validates classical theory and demonstrates that this mechanism is not the most effective inhibition pathway. Quinolines also form various complexes with soluble hematin, but complexation is insufficient to suppress heme detoxification and is a poor indicator of drug specificity. Collectively, our findings reveal the significance of drug-crystal interactions and open avenues for rationally designing antimalarial compounds.

Quantification of labile heme in live malaria parasites using a genetically encoded biosensor

Heme is ubiquitous, yet relatively little is known about the maintenance of labile pools of this cofactor, which likely ensures its timely bioavailability for proper cellular function. Quantitative analysis of labile heme is of fundamental importance to understanding how nature preserves access to the diverse chemistry heme enables, while minimizing cellular damage caused by its redox activity. Here, we have developed and characterized a protein-based sensor that undergoes fluorescence quenching upon heme binding. By genetically encoding this sensor in the human malarial parasite, Plasmodium falciparum, we have quantified cytosolic labile heme levels in intact, blood-stage parasites. Our findings indicate that a labile heme pool (∼1.6 µM) is stably maintained throughout parasite development within red blood cells, even during a period coincident with extensive hemoglobin degradation by the parasite. We also find that the heme-binding antimalarial drug chloroquine specifically increases labile cytosolic heme, indicative of dysregulation of this homeostatic pool that may be a relevant component of the antimalarial activity of this compound class. We propose that use of this technology under various environmental perturbations in P. falciparum can yield quantitative insights into fundamental heme biology.

Synthesis of 1,3,5-trisubstituted pyrazolines as potential antimalarial and antimicrobial agents

A series of novel 1,3,5-trisubstituted pyrazolines derivatives have been synthesized from chalcones and nicotinic acid hydrazide in two steps. In first step, chalcones were prepared by treatment of 4-hydroxy acetophenone with different substituted benzaldehyde by Claisen-Schimidt Condensation. In second step, various pyrazoline derivatives were prepared by reflux reaction of chalcones with nicotinic acid hydrazide in ethanolic solution. Compounds were confirmed by elemental analyses, IR, 1H NMR and 13C NMR spectral data and were evaluated for antimalarial and antibacterial activity. Compounds 5n (IC50=0.022μM for MRC-2 and IC50=0.192μM for RKL-9) displayed better antiplasmodial activity than the chloroquine (CQ) against chloroquine-sensitive (MRC-2) and chloroquine-resistant (RKL-9) P. falciparum strains. The in vitro cytotoxicity study conducted on the human HepG2 cell line (>30μM) and selectivity index (100–220) indicate that this series presents an interesting selective antiplasmodial profile. Further, in vitro heme crystallization inhibition assay showed compound 5e inhibited formation of β-hematin more efficiently than CQ. In addition, antibacterial and antifungal evaluations were conducted, compounds 5c, 5i and 5j displayed better antibacterial activity against S. aureus, B. subtilis, E. coli and P. aeruginosa than ciproafloxacin. Antifungal activity of compound 5l against A. niger (MIC-3.25μg/ml) and C. albicans (MIC-6.5μg/ml) was found to be better than the standard drug fluconazole.

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

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.