THC shows activity against cultured Plasmodium falciparum.

The FDA approved drug Dronabinol was identified in a previous study applying virtual screening using the haemozoin crystal as a target against malaria parasites. The active ingredient of dronabinol is synthetic tetrahydrocannabinol (THC), which is one of the major cannabinoids from Cannabis sativa. Traditional use of cannabis for malaria fever was reported in the world's oldest pharmacopoeia, dating to around 5000 years ago. In this research we report that THC inhibits ß-haematin (synthetic haemozoin) and malaria parasite growth. Due the psychoactivity of THC, CBD, the other major naturally occurring cannabinoid that lacks the off-target psychoactive effects of THC, was also tested and inhibited ß-haematin but showed only a mild antimalarial activity. To evaluate whether THC inhibit haemozoin formation, we performed a cellular haem fractionation assay that indicated that is not the likely mechanism of action. For the first time, the cannabinoid chemical structure is raised as a new chemical class to be further studied for malaria treatment, aiming to overcome the undesirable psychoactive effects of THC and optimize the antimalarial effects.

Novel Antimalarial Tetrazoles and Amides Active against the Hemoglobin Degradation Pathway in Plasmodium falciparum.

Malaria control programs continue to be threatened by drug resistance. To identify new antimalarials, we conducted a phenotypic screen and identified a novel tetrazole-based series that shows fast-kill kinetics and a relatively low propensity to develop high-level resistance. Preliminary structure-activity relationships were established including identification of a subseries of related amides with antiplasmodial activity. Assaying parasites with resistance to antimalarials led us to test whether the series had a similar mechanism of action to chloroquine (CQ). Treatment of synchronized Plasmodium falciparum parasites with active analogues revealed a pattern of intracellular inhibition of hemozoin (Hz) formation reminiscent of CQ's action. Drug selections yielded only modest resistance that was associated with amplification of the multidrug resistance gene 1 (pfmdr1). Thus, we have identified a novel chemical series that targets the historically druggable heme polymerization pathway and that can form the basis of future optimization efforts to develop a new malaria treatment.

Identification of 2,4-Disubstituted Imidazopyridines as Hemozoin Formation Inhibitors with Fast-Killing Kinetics and In Vivo Efficacy in the Plasmodium falciparum NSG Mouse Model.

A series of 2,4-disubstituted imidazopyridines, originating from a SoftFocus Kinase library, was identified from a high throughput phenotypic screen against the human malaria parasite Plasmodium falciparum. Hit compounds showed moderate asexual blood stage activity. During lead optimization, several issues were flagged such as cross-resistance against the multidrug-resistant K1 strain, in vitro cytotoxicity, and cardiotoxicity and were addressed through structure-activity and structure-property relationship studies. Pharmacokinetic properties were assessed in mice for compounds showing desirable in vitro activity, a selectivity window over cytotoxicity, and microsomal metabolic stability. Frontrunner compound 37 showed good exposure in mice combined with good in vitro activity against the malaria parasite, which translated into in vivo efficacy in the P. falciparum NOD-scid IL-2Rγnull (NSG) mouse model. Preliminary mechanistic studies suggest inhibition of hemozoin formation as a contributing mode of action.

Correlation between anti-malarial and anti-haemozoin activities of anti-malarial compounds.

BACKGROUND: Despite noticeable improvement in anti-malarial treatment, rapid growth of resistant malaria strains points out the need for continuous development of novel anti-malarials to fight the disastrous infection. Haemozoin is considered as a novel inhibitory pathway for new anti-malarial drugs, therefore, this study aimed to systematically review all articles investigating the correlation between anti-malarial and anti-haemozoin activities of anti-malarial compounds. METHODS: A literature search was conducted on 22 October 2017 in eight databases for relevant in vitro articles reporting the correlation between anti-malarial and anti-haemozoin of anti-malarial compounds, based on the constructed search terms and inclusion criteria. ToxRtool was used to assess quality of each study. RESULTS: A total of ten articles were included in the review. In vitro anti-malarial and anti-haemozoin activity had a good correlation for quinolines for sensitive strains (R2 ranging from 0.66 to 0.95) and xanthones (Spearman ρ = 0.886). However, these correlations were reached after removing some compounds which had non-detectable anti-malarial or anti-haemozoin effects. Other structures (acridines, pyrolidines) showed negligible correlation with Spearman ρ ranging from 0.095 to 0.381 for acridines, and r varying from 0.54 to 0.62 for pyrolidines. Some good correlations were only shown in a logarithmic manner or when the anti-malarial activity was normalized. CONCLUSION: The results raised a relative relationship between anti-haemozoin and in vitro anti-malarial activities. Some studies reported compounds that were effective in the inhibition of haemozoin formation, but failed to inhibit the parasite survival and vice versa. The correlation results in these studies were calculated after these compounds were removed from their analysis. The ability of anti-malarial compounds to accumulate inside the reaction site might strengthen their anti-malarial activity.

Lapatinib, Nilotinib and Lomitapide Inhibit Haemozoin Formation in Malaria Parasites.

With the continued loss of antimalarials to resistance, drug repositioning may have a role in maximising efficiency and accelerating the discovery of new antimalarial drugs. Bayesian statistics was previously used as a tool to virtually screen USFDA approved drugs for predicted ß-haematin (synthetic haemozoin) inhibition and in vitro antimalarial activity. Here, we report the experimental evaluation of nine of the highest ranked drugs, confirming the accuracy of the model by showing an overall 93% hit rate. Lapatinib, nilotinib, and lomitapide showed the best activity for inhibition of ß-haematin formation and parasite growth and were found to inhibit haemozoin formation in the parasite, providing mechanistic insights into their mode of antimalarial action. We then screened the USFDA approved drugs for binding to the ß-haematin crystal, applying a docking method in order to evaluate its performance. The docking method correctly identified imatinib, lapatinib, nilotinib, and lomitapide. Experimental evaluation of 22 of the highest ranked purchasable drugs showed a 24% hit rate. Lapatinib and nilotinib were chosen as templates for shape and electrostatic similarity screening for lead hopping using the in-stock ChemDiv compound catalogue. The actives were novel structures worthy of future investigation. This study presents a comparison of different in silico methods to identify new haemozoin-inhibiting chemotherapeutic alternatives for malaria that proved to be useful in different ways when taking into consideration their strengths and limitations.

The in Vitro Antiplasmodial and Antiproliferative Activity of New Ferrocene-Based α-Aminocresols Targeting Hemozoin Inhibition and DNA Interaction.

The conjugation of organometallic complexes to known bioactive organic frameworks is a proven strategy revered for devising new drug molecules with novel modes of action. This approach holds great promise for the generation of potent drug leads in the quest for therapeutic chemotypes with the potential to overcome the development of clinical resistance. Herein, we present the in vitro antiplasmodial and antiproliferative investigation of ferrocenyl α-aminocresol conjugates assembled by amalgamation of the organometallic ferrocene unit and an α-aminocresol scaffold possessing antimalarial activity. The compounds pursued in the study exhibited higher toxicity towards the chemosensitive (3D7) and -resistant (Dd2) strains of the Plasmodium falciparum parasite than to the human HCC70 triple-negative breast cancer cell line. Indication of cross-resistance was absent for the compounds evaluated against the multi-resistant Dd2 strain. Structure-activity analysis revealed that the phenolic hydroxy group and rotatable σ bond between the α-carbon and NH group of the α-amino-o-cresol skeleton are crucial for the biological activity of the compounds. Spectrophotometric techniques and in silico docking simulations performed on selected derivatives suggest that the compounds show a dual mode of action involving hemozoin inhibition and DNA interaction via minor-groove binding. Lastly, compound 9 a, identified as a possible lead, exhibited preferential binding for the plasmodial DNA isolated from 3D7 P. falciparum trophozoites over the mammalian calf thymus DNA, thereby substantiating the enhanced antiplasmodial activity of the compounds. The presented research demonstrates the strategy of incorporating organometallic complexes into known biologically active organic scaffolds as a viable avenue to fashion novel multimodal compounds with potential to counter the development drug resistance.

Acute toxicity of cyanide in aerobic respiration: Theoretical and experimental support for murburn explanation.

The inefficiency of cyanide/HCN (CN) binding with heme proteins (under physiological regimes) is demonstrated with an assessment of thermodynamics, kinetics, and inhibition constants. The acute onset of toxicity and CN's mg/Kg LD50 (µM lethal concentration) suggests that the classical hemeFe binding-based inhibition rationale is untenable to account for the toxicity of CN. In vitro mechanistic probing of CN-mediated inhibition of hemeFe reductionist systems was explored as a murburn model for mitochondrial oxidative phosphorylation (mOxPhos). The effect of CN in haloperoxidase catalyzed chlorine moiety transfer to small organics was considered as an analogous probe for phosphate group transfer in mOxPhos. Similarly, inclusion of CN in peroxidase-catalase mediated one-electron oxidation of small organics was used to explore electron transfer outcomes in mOxPhos, leading to water formation. The free energy correlations from a Hammett study and IC50/Hill slopes analyses and comparison with ligands ( CO/ H 2 S/ N 3 - ) $\left( {\text{CO}}/{{{{\text{H}}_{2}}\text{S}}/{\text{N}_{3}^{\text{-}}}\;}\; \right)$ provide insights into the involvement of diffusible radicals and proton-equilibriums, explaining analogous outcomes in mOxPhos chemistry. Further, we demonstrate that superoxide (diffusible reactive oxygen species, DROS) enables in vitro ATP synthesis from ADP+phosphate, and show that this reaction is inhibited by CN. Therefore, practically instantaneous CN ion-radical interactions with DROS in matrix catalytically disrupt mOxPhos, explaining the acute lethal effect of CN.

Preclinical Antimalarial Combination Study of M5717, a Plasmodium falciparum Elongation Factor 2 Inhibitor, and Pyronaridine, a Hemozoin Formation Inhibitor.

Antimalarial drug resistance in the Plasmodium falciparum parasite poses a constant challenge for drug development. To mitigate this risk, new antimalarial medicines should be developed as fixed-dose combinations. Assessing the pharmacodynamic interactions of potential antimalarial drug combination partners during early phases of development is essential in developing the targeted parasitological and clinical profile of the final drug product. Here, we have studied the combination of M5717, a P. falciparum translation elongation factor 2 inhibitor, and pyronaridine, an inhibitor of hemozoin formation. Our test cascade consisted of in vitro isobolograms as well as in vivo studies in the P. falciparum severe combined immunodeficient (SCID) mouse model. We also analyzed pharmacokinetic and pharmacodynamic parameters, including genomic sequencing of recrudescent parasites. We observed no pharmacokinetic interactions with the combination of M5717 and pyronaridine. M5717 did not negatively impact the rate of kill of the faster-acting pyronaridine, and the latter was able to suppress the selection of M5717-resistant mutants, as well as significantly delay the recrudescence of parasites both with suboptimal and optimal dosing regimens.

Virtual screening as a tool to discover new ß-haematin inhibitors with activity against malaria parasites.

Malaria remains a major public health problem. With the loss of antimalarials to resistance, the malaria burden will likely continue for decades. New antimalarial scaffolds are crucial to avoid cross-resistance. Here, we present the first structure based virtual screening using the ß-haematin crystal as a target for new inhibitor scaffolds by applying a docking method. The ZINC15 database was searched for compounds with high binding affinity with the surface of the ß-haematin crystal using the PyRx Virtual Screening Tool. Top-ranked compounds predicted to interact with ß-haematin were submitted to a second screen applying in silico toxicity and drug-likeness predictions using Osiris DataWarrior. Fifteen compounds were purchased for experimental testing. An NP-40 mediated ß-haematin inhibition assay and parasite growth inhibition activity assay were performed. The benzoxazole moiety was found to be a promising scaffold for further development, showing intraparasitic haemozoin inhibition using a cellular haem fractionation assay causing a decrease in haemozoin in a dose dependent manner with a corresponding increase in exchangeable haem. A ß-haematin inhibition hit rate of 73% was found, a large enrichment over random screening, demonstrating that virtual screening can be a useful and cost-effective approach in the search for new haemozoin inhibiting antimalarials.

Silymarin, a polyphenolic flavonoid impede Plasmodium falciparum growth through interaction with heme.

A polyphenolic flavonoid, Silymarin isolated from Silybum marianum is widely known for its hepatoprotective action. In the present study anti-plasmodial activity of Silymarin has been demonstrated for the first time having IC50 of 14 ± 0.33 µM against the NF-54 strain of P. falciparum with high selectivity index (>100). The parasitostatic action is exerted through inhibition of ß-hematin/hemozoin formation which is due to the interaction (Kd = 3.63 ± 0.9µM) of silymarin with free heme in a Stoichiometry of 1:1 Silymarin: heme complex resulting into heme-induced membrane damage in the parasite. Silymarin could hinder the glutathione and hydrogen peroxide-induced heme detoxification. Silymarin also induces apoptosis in the parasite through the elevation of caspase-3 level in a dose-dependent manner. Results from the docking studies suggest that Silymarin interacts with heme.

In vitro anti-malarial efficacy of chalcones: cytotoxicity profile, mechanism of action and their effect on erythrocytes.

BACKGROUND: Malaria extensively leads to mortality and morbidity in endemic regions, and the emergence of drug resistant parasites is alarming. Plant derived synthetic pharmaceutical compounds are found to be a foremost research to obtain diverse range of potent leads. Amongst them, the chalcone scaffold is a functional template for drug discovery. The present study involves synthesis of ten chalcones with various substitution pattern in rings A and B and assessment of their anti-malarial efficacy against chloroquine sensitive and chloroquine resistant strains as well as of their cytotoxicity and effect on haemozoin production. METHODS: The chalcones were synthesized by Claisen-Schmidt condensation between equimolar quantities of substituted acetophenones and aryl benzaldehydes (or indole-3-carboxaldehyde) and were screened for anti-malarial activity by WHO Mark III schizont maturation inhibition assay. The cytotoxicity profile of a HeLa cell line was evaluated through MTT viability assay and the selectivity index (SI) was calculated. Haemozoin inhibition assay was performed to illustrate mode of action on a Plasmodium falciparum strain. RESULTS: The IC50 values of all compounds were in the range 0.10-0.40 µg/mL for MRC-2 (a chloroquine sensitive strain) and 0.14-0.55 µg/mL for RKL-9 (a chloroquine resistant strain) of P. falciparum. All the chalcones showed low cellular toxicity with minimal haemolysis. The statistically significant reduction (p < 0.05) in the haemozoin production suggests a similar mechanism than that of chloroquine. CONCLUSIONS: Out of ten chalcones, number 7 was found to be a lead compound with the highest potency (IC50 = 0.11 µg/mL), as compared to licochalcone (IC50 = 1.43 µg/mL) and with high selectivity index of 85.05.

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

Backgound: Exploring potent compounds is critical to generating multi-target drug discovery. Hematin crystallization is an important mechanism of malaria. METHODS: 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 ß-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 ß-hematin inhibition. RESULTS: The target compounds exhibited high ß-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. CONCLUSION: 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.

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

Malaria caused by Plasmodium affects millions people worldwide. Plasmodium consumes hemoglobin during its intraerythrocytic stage leaving toxic heme. Parasite detoxifies free heme through formation of hemozoin (ß-hematin) pigment. Proteolysis of hemoglobin and formation of hemozoin are two main targets for antimalarial drugs. Quinoline antimarial drugs and analogs (ß-carbolines or nitroindazoles) were studied as inhibitors of ß-hematin formation. The most potent inhibitors were quinacrine, chloroquine, and amodiaquine followed by quinidine, mefloquine and quinine whereas 8-hydroxyquinoline and ß-carbolines had no effect. Compounds that inhibited ß-hematin increased free hemin that promoted peroxidative reactions as determined with TMB and ABTS substrates. Hemin-catalyzed peroxidative reactions were potentiated in presence of proteins (i.e. globin or BSA) while antioxidants and peroxidase inhibitors decreased peroxidation. Free hemin increased by chloroquine action promoted oxidative reactions resulting in inhibition of proteolysis by three cysteine proteases: papain, ficin and cathepsin B. Glutathione reversed inhibition of proteolysis. These results show that active quinolines inhibit hemozoin and increase free hemin which in presence of H2O2 that abounds in parasite digestive vacuole catalyzes peroxidative reactions and inhibition of cysteine proteases. This work suggests a link between the action of quinoline drugs with biochemical processes of peroxidation and inhibition of proteolysis.

Magnesium Lithospermate B Derived from Salvia miltiorrhiza Ameliorates Right Ventricle Remodeling in Pulmonary Hypertensive Rats via Inhibition of NOX/VPO1 Pathway.

Right ventricle (RV) remodeling is a major pathological feature in pulmonary arterial hypertension (PAH). Magnesium lithospermate B (MLB) is a compound isolated from the roots of Salvia miltiorrhiza and it possesses multiple pharmacological activities such as anti-inflammation and antioxidation. This study aims to investigate whether MLB is able to prevent RV remodeling in PAH and the underlying mechanisms. In vivo, SD rats were exposed to 10% O2 for 21 d to induce RV remodeling, which showed hypertrophic features (increases in the ratio of RV weight to tibia length, cellular size, and hypertrophic marker expression), accompanied by upregulation in expression of NADPH oxidases (NOX2 and NOX4) and vascular peroxidase 1 (VPO1), increases in hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) production and elevation in phosphorylation levels of ERK; these changes were attenuated by treating rats with MLB. In vitro, the cultured H9c2 cells were exposed to 3% O2 for 24 h to induce hypertrophy, which showed hypertrophic features (increases in cellular size and hypertrophic marker expression). Administration of MLB or VAS2870 (a positive control for NOX inhibitor) could prevent cardiomyocyte hypertrophy concomitant with decreases in NOX (NOX2 and NOX4) and VPO1 expression, H2O2 and HOCl production, and ERK phosphorylation. Based on these observations, we conclude that MLB is able to prevent RV remodeling in hypoxic PAH rats through a mechanism involving a suppression of NOX/VPO1 pathway as well as ERK signaling pathway. MLB may possess the potential clinical value for PAH therapy.

Evaluation of ferrocenyl phosphines as potent antimalarials targeting the digestive vacuole function of Plasmodium falciparum.

Owing to their lipophilic nature and chemical stability, ferrocene and its derivatives have been widely explored as antimicrobial agents, in combination with other active chemical 'war heads'. A prime example is ferroquine, an analogue of chloroquine obtained by covalently bonding ferrocene to 4-aminoquinoline, which possesses superior efficacy against multi-drug resistant malaria parasites. Herein, we explored the possibility of combining the ferrocenyl moiety with a phosphine unit and the subsequent inclusion of gold(i) to derive a molecular framework with demonstrated potential in inhibiting parasitic diseases. A library of 24 compounds consisting of 5 non-functionalized ferrocenyl enones and 19 ferrocenyl phosphine derivatives were synthesized, verified and tested against Plasmodium (P.) falciparum, which allowed us to identify compounds with low micromolar potency against both normal and chloroquine-resistant strains. Through flow cytometry combined with microscopic examination of Giemsa-stained thin smears, we observed that most of the active compounds interfered with trophozoite development as well as schizont maturation. The gold complex, namely G3, derived from the hydrophosphination of the terminal furan bearing an enone substrate showed the highest inhibitory potential. We demonstrate that G3 is affecting the parasite's metabolic processes as evident from the swollen digestive vacuole. Furthermore, G3 significantly affected heme de-toxification as determined through the ß-hematin assay, which caused apparent oxidative stress on parasites leading to death. Collectively, these results point out the potential of gold-conjugated ferrocenyl phosphine derivatives as antimalarials targeting the digestive vacuole function and metabolism of parasites.

Hydrazonophenol, a Food Vacuole-Targeted and Ferriprotoporphyrin IX-Interacting Chemotype Prevents Drug-Resistant Malaria.

The rapid emergence of resistance against frontline antimalarial drugs essentially warrants the identification of new-generation antimalarials. Here, we describe the synthesis of ( E)-2-isopropyl-5-methyl-4-((2-(pyridin-4-yl)hydrazono)methyl)phenol (18), which binds ferriprotoporphyrin-IX (FeIII-PPIX) ( Kd = 33 nM) and offers antimalarial activity against chloroquine-resistant and sensitive strains of Plasmodium falciparum in vitro. Structure-function analysis reveals that compound 18 binds FeIII-PPIX through the -C═N-NH- moiety and 2-pyridyl substitution at the hydrazine counterpart plays a critical role in antimalarial efficacy. Live cell confocal imaging using a fluorophore-tagged compound confirms its accumulation inside the acidic food vacuole (FV) of P. falciparum. Furthermore, this compound concentration-dependently elevates the pH in FV, implicating a plausible interference with FeIII-PPIX crystallization (hemozoin formation) by a dual function: increasing the pH and binding free FeIII-PPIX. Different off-target bioassays reduce the possibility of the promiscuous nature of compound 18. Compound 18 also exhibits potent in vivo antimalarial activity against chloroquine-resistant P. yoelii and P. berghei ANKA (causing cerebral malaria) in mice with negligible toxicity.

Structure-Activity Relationship Studies and Plasmodium Life Cycle Profiling Identifies Pan-Active N-Aryl-3-trifluoromethyl Pyrido[1,2- a]benzimidazoles Which Are Efficacious in an in Vivo Mouse Model of Malaria.

Structure-activity relationship studies involving N-aryl-3-trifluoromethyl pyrido[1,2- a]benzimidazoles (PBI) identified several compounds possessing potent in vitro activities against the asexual blood, liver, and gametocyte stages of the Plasmodium parasite with no cross-resistance to chloroquine. Frontrunner lead compounds with good in vitro absorption, distribution, metabolism, and excretion (ADME) profiles were subjected to in vivo proof-of-concept studies in NMRI mice harboring the rodent P. berghei infection. This led to the identification of compounds 10 and 49, effecting 98% and 99.93% reduction in parasitemia with mean survival days of 12 and 14, respectively, at an oral dose of 4 × 50 mg/kg. In vivo pharmacokinetics studies on 10 revealed slow absorption, low volume of distribution, and low clearance profiles. Furthermore, this series displayed a low propensity to inhibit the human ether-a-go-go-related gene (hERG) potassium ion channel whose inhibition is associated with cardiotoxicity.

Multistage Antiplasmodium Activity of Astemizole Analogues and Inhibition of Hemozoin Formation as a Contributor to Their Mode of Action.

A drug repositioning approach was leveraged to derivatize astemizole (AST), an antihistamine drug whose antimalarial activity was previously identified in a high-throughput screen. The multistage activity potential against the Plasmodium parasite's life cycle of the subsequent analogues was examined by evaluating against the parasite asexual blood, liver, and sexual gametocytic stages. In addition, the previously reported contribution of heme detoxification to the compound's mode of action was interrogated. Ten of the 17 derivatives showed half-maximal inhibitory concentrations (IC50s) of <0.1 µM against the chloroquine (CQ)-sensitive Plasmodium falciparum NF54 ( PfNF54) strain while maintaining submicromolar potency against the multidrug-resistant strain, PfK1, with most showing low likelihood of cross-resistance with CQ. Selected analogues ( PfNF54-IC50 < 0.1 µM) were tested for cytotoxicity on Chinese hamster ovarian (CHO) cells and found to be highly selective (selectivity index > 100). Screening of AST and its analogues against gametocytes revealed their moderate activity (IC50: 1-5 µM) against late stage P. falciparum gametocytes, while the evaluation of activity against P. berghei liver stages identified one compound (3) with 3-fold greater activity than the parent AST compound. Mechanistic studies showed a strong correlation between in vitro inhibition of ß-hematin formation by the AST derivatives and their antiplasmodium IC50s. Analyses of intracellular inhibition of hemozoin formation within the parasite further yielded signatures attributable to a possible perturbation of the heme detoxification machinery.

Hemozoin inhibiting 2-phenylbenzimidazoles active against malaria parasites.

The 2-phenylbenzimidazole scaffold has recently been discovered to inhibit ß-hematin (synthetic hemozoin) formation by high throughput screening. Here, a library of 325,728 N-4-(1H-benzo[d]imidazol-2-yl)aryl)benzamides was enumerated, and Bayesian statistics used to predict ß-hematin and Plasmodium falciparum growth inhibition. Filtering predicted inactives and compounds with negligible aqueous solubility reduced the library to 35,124. Further narrowing to compounds with terminal aryl ring substituents only, reduced the library to 18, 83% of which were found to inhibit ß-hematin formation <100 µM and 50% parasite growth <2 µM. Four compounds showed nanomolar parasite growth inhibition activities, no cross-resistance in a chloroquine resistant strain and low cytotoxicity. QSAR analysis showed a strong association of parasite growth inhibition with inhibition of ß-hematin formation and the most active compound inhibited hemozoin formation in P. falciparum, with consequent increasing exchangeable heme. Pioneering use of molecular docking for this system demonstrated predictive ability and could rationalize observed structure activity trends.

Synthesis, Biological Evaluation, and Molecular Modeling Studies of Chiral Chloroquine Analogues as Antimalarial Agents.

In a focused exploration, we designed, synthesized, and biologically evaluated chiral conjugated new chloroquine (CQ) analogues with substituted piperazines as antimalarial agents. In vitro as well as in vivo studies revealed that compound 7c showed potent activity (in vitro 50% inhibitory concentration, 56.98 nM for strain 3D7 and 97.76 nM for strain K1; selectivity index in vivo [up to at a dose of 12.5 mg/kg of body weight], 3,510) as a new lead antimalarial agent. Other compounds (compounds 6b, 6d, 7d, 7h, 8c, 8d, 9a, and 9c) also showed moderate activity against a CQ-sensitive strain (3D7) and superior activity against a CQ-resistant strain (K1) of Plasmodium falciparum Furthermore, we carried out docking and three-dimensional quantitative structure-activity relationship (3D-QSAR) studies of all in-house data sets (168 molecules) of chiral CQ analogues to explain the structure-activity relationships (SAR). Our new findings specify the significance of the H-bond interaction with the side chain of heme for biological activity. In addition, the 3D-QSAR study against the 3D7 strain indicated the favorable and unfavorable sites of CQ analogues for incorporating steric, hydrophobic, and electropositive groups to improve the antimalarial activity.