Inhibition Of Hemozoin Formation Research Articles

Antiplasmodial activity of novel keto-enamine chalcone-chloroquine based hybrid pharmacophores

A series of novel keto-enamine chalcone-chloroquine based hybrids were synthesized following new methodology developed in our laboratory. The synthesized compounds were screened against chloroquine sensitive strain (3D7) of Plasmodium falciparum in an in vitro model. Some of the compounds were showing comparable antimalarial activity at par with chloroquine. Compounds with significant in vitro antimalarial activity were then evaluated for their in vivo efficacy in Swiss mice against Plasmodium yoelii (chloroquine resistant N-67 strain), wherein compounds 25 and 27 each showed an in vivo suppression of 99.9% parasitaemia on day 4. Biochemical studies reveal that inhibition of hemozoin formation is the primary mechanism of action of these analogues.

Synthesis and Antimalarial Activities of a Diverse Set of Triazole‐Containing Furamidine Analogues

Four different series of triazole diamidines have been prepared by the Pinner method from the corresponding triazole dinitriles. Copper-catalyzed "click chemistry" was used for the synthesis of 1,4- and 4,5-substituted triazoles, aryl magnesium acetylide reagents for the 1,5-substituted triazoles, with a thermal dipolar addition reaction employed for the 2,4-substituted triazoles. In vitro antimalarial activity against two different PfCRT-modified parasite lines (Science 2002, 298, 210-213) of Plasmodium falciparum and inhibition of hemozoin formation were determined for each compound. Several diamidines with potent nanomolar antimalarial activities were identified, and selected molecules were resynthesized as their diamidoxime triazole prodrugs. One of these prodrugs, OB216, proved to be highly potent in vivo with an ED50 value of 5 mg kg(-1) (po) and an observed 100 % cure rate (CD100) of just 10 mg kg(-1) by oral (po) administration in mice infected with P. vinckei.

Enone- and chalcone-chloroquinoline hybrid analogues: in silico guided design, synthesis, antiplasmodial activity, in vitro metabolism, and mechanistic studies.

Analogues of the previously reported antimalarial hybrid compounds 8b and 12 were proposed with the aim of identifying compounds with improved solubility and retained antimalarial potency. In silico characterization predicted improved solubilities of the analogues, particularly at low pH; they retained acceptable predicted permeability properties but were predicted to be susceptible to hepatic metabolism. These analogues were synthesized and found to exhibit notable in vitro antimalarial activity. Compounds 25 and 27 were the most active of the analogues. In vitro metabolism studies indicated susceptibility of the analogues to hepatic metabolism. There was also evidence of primary glucuronidation for analogues 24-27. Presumed cis-trans isomerism of 12, 22, and 23 under in vitro metabolism assay conditions was also observed, with differences in the nature and rates of metabolism observed between isomers. Biochemical studies strongly suggested that inhibition of hemozoin formation is the primary mechanism of action of these analogues.

Synthesis, Structure−Activity Relationship, and Mode-of-Action Studies of Antimalarial Reversed Chloroquine Compounds

We have previously shown that a "reversed chloroquine (RCQ)" molecule, composed of a chloroquine-like moiety and a resistance reversal-like moiety, can overcome chloroquine resistance in P. falciparum ( Burgess , S. J. ; Selzer , A. ; Kelly , J. X. ; Smilkstein , M. J. ; Riscoe , M. K. ; Peyton , D. H. J. Med. Chem. 2006 , 49 , 5623 . Andrews , S. ; Burgess , S. J. ; Skaalrud , D. ; Kelly , J. X. ; Peyton , D. H. J. Med. Chem. 2010 , 53 , 916 ). Here, we present an investigation into the structure-activity relationship of the RCQ structures, resulting in an orally active molecule with good in vitro and in vivo antimalarial activity. We also present evidence of the mode of action, indicating that the RCQ molecules inhibit hemozoin formation in the parasite's digestive vacuole in a manner similar to that of chloroquine.

Additive In Vitro Antiplasmodial Effect of N-Alkyl and N-Benzyl-1,10-Phenanthroline Derivatives and Cysteine Protease Inhibitor E64

Potential new targets for antimalarial chemotherapy include parasite proteases, which are required for several cellular functions during the Plasmodium falciparum life cycle. Four new derivatives of N-alkyl and N-benzyl-1,10-phenanthroline have been synthesized. Those are (1)-N-methyl-1,10-phenanthrolinium sulfate, (1)-N-ethyl-1,10-phenanthrolinium sulfate, (1)-N-benzyl-1,10-phenanthrolinium chloride, and (1)-N-benzyl-1,10-phenanthrolinium iodide. Those compounds had potential antiplasmodial activity with IC50 values from 260.42 to 465.38 nM. Cysteine proteinase inhibitor E64 was used to investigate the mechanism of action of N-alkyl and N-benzyl-1,10-phenanthroline derivatives. A modified fixed-ratio isobologram method was used to study the in vitro interactions between the new compounds with either E64 or chloroquine. The interaction between N-alkyl and N-benzyl-1,10-phenanthroline derivatives and E64 was additive as well as their interactions with chloroquine were also additive. Antimalarial mechanism of chloroquine is mainly on the inhibition of hemozoin formation. As the interaction of chloroquine and E64 was additive, the results indicated that these new compounds had a mechanism of action by inhibiting Plasmodium proteases.

X-ray Crystallographic Structure of the Potent Antiplasmodial Compound 2,7-Dibromocryptolepine Acetic Acid Solvate

AbstractThe structure of 2,7-dibromocryptolepine acetic acid solvate, C 16 H 11 N 2 Br 2 [1.5(C 2 H 4 O 2 )][C 2 H 3 O 2 − ][0.5H 2 O], M r = 460.17, has been determined from X-ray diffraction data. The crystals are monoclinic, space group P2 1 /c with Z = 4 molecules per unit cell and a = 7.3243(3), b = 18.7804(6), c = 15.8306(7) A, β = 94.279(1)°, V c = 2171.5(2) A, crystal density D c = 1.667 g/cm 3 . The structure was determined using direct methods and refined by full-matrix least-squares to a conventional R-index of 0.0496 for 4,908 reflections and 258 parameters. The cryptolepine nucleus of the 2,7-dibromocryptolepine molecule is highly planar and the two Br atoms are in this plane within 0.06 and 0.01 A, respectively. The crystal structure is maintained via hydrogen bonding between N(10) in the cryptolepine nucleus and the oxygen of one of the three solvated acetic acid molecules. The acetic acid molecules also form hydrogen bonded chains. Acetic acid B is deprotonated and its two C-O bond lengths are equivalent, unlike those in A and C. Acetic acid C lies very close to a crystallographic centre of symmetry. To avoid overlap the two repeats cannot exist together and are subject to 50% statistical disorder. O(1C) of this methanol is furthest from the two-fold axis and its occupancy refines to a value of 1.0 and is assumed to exist alternately as a water oxygen hydrogen bonding to methanol O(1C) across the two-fold axis at a distance of 2.775 A.Index AbstractThe antiplasmodial activity of the analogue 2,7-dibromocryptolepine is nine times greater than that of cryptolepine itself against chloroquine-resistant Plasmodium falciparum (multi-drug resistant strain K1) with IC 50 values = 0.44 and 0.049 μM, respectively. This analogue also showed promising in vivo activity against P. berghei in mice. Parasitaemia was suppressed by 91.4% compared to untreated infected control animals at a dose of 25 mg/kg given daily by i.p. injection with no apparent toxicity to the mice, in contrast to cryptolepine which was toxic to mice when given i.p. at 20 mg/kg. Further experiments showed a dose-dependent relationship with ED 50 and ED 90 values of 6.92 and 21.46 mg/kg/day, respectively. Although 2,7-dibromocryptolepine was not toxic to the mice its cytotoxic activity is similar to that of cryptolepine, but unlike cryptolepine it does not appear to intercalate into DNA as assessed using thermodenaturation techniques (ΔTm values = 3 and 9 °C, respectively). Like cryptolepine, 2,7-dibromocryptolepine inhibits the formation of haemozoin, but its increased antiplasmodial potency does not appear to be due to more potent inhibition of haemozoin formation, nor to increased accumulation into the acidic parasite food vacuole suggesting that 2,7-dibromocryptolepine has additional modes of action compared to cryptolepine. The X-ray structure of this compound will help to determine whether or not 2,7-dibromocryptolepine is able to intercalate into DNA and facilitate the design of new cryptolepine analogues with DNA binding properties appropriate for antimalarial (with no DNA intercalation) or anticancer (sequence-specific binding) applications. [IMAGE]

Antiplasmodial Activity of [(Aryl)arylsulfanylmethyl]Pyridine

A series of [(aryl)arylsufanylmethyl]pyridines (AASMP) have been synthesized. These compounds inhibited hemozoin formation, formed complexes (K(D) = 12 to 20 muM) with free heme (ferriprotoporphyrin IX) at a pH close to the pH of the parasite food vacuole, and exhibited antimalarial activity in vitro. The inhibition of hemozoin formation may develop oxidative stress in Plasmodium falciparum due to the accumulation of free heme. Interestingly, AASMP developed oxidative stress in the parasite, as evident from the decreased level of glutathione and increased formation of lipid peroxide, H(2)O(2), and hydroxyl radical (.OH) in P. falciparum. AASMP also caused mitochondrial dysfunction by decreasing mitochondrial potential (DeltaPsim) in malaria parasite, as measured by both flow cytometry and fluorescence microscopy. Furthermore, the generation of .OH may be mainly responsible for the antimalarial effect of AASMP since .OH scavengers such as mannitol, as well as spin trap alpha-phenyl-n-tertbutylnitrone, significantly protected P. falciparum from AASMP-mediated growth inhibition. Cytotoxicity testing of the active compounds showed selective activity against malaria parasite with selectivity indices greater than 100. AASMP also exhibited profound antimalarial activity in vivo against chloroquine resistant P. yoelii. Thus, AASMP represents a novel class of antimalarial.

Bilirubin inhibits Plasmodium falciparum growth through the generation of reactive oxygen species

Free heme is very toxic because it generates highly reactive hydroxyl radicals ( OH) to cause oxidative damage. Detoxification of free heme by the heme oxygenase (HO) system is a very common phenomenon by which free heme is catabolized to form bilirubin as an end product. Interestingly, the malaria parasite, Plasmodium falciparum, lacks an HO system, but it forms hemozoin, mainly to detoxify free heme. Here, we report that bilirubin significantly induces oxidative stress in the parasite as evident from the increased formation of lipid peroxide, decrease in glutathione content, and increased formation of H 2O 2 and OH. Bilirubin can effectively inhibit hemozoin formation also. Furthermore, results indicate that bilirubin inhibits parasite growth and induces caspase-like protease activity, up-regulates the expression of apoptosis-related protein (Gene ID PFI0450c), and reduces the mitochondrial membrane potential. OH scavengers such as mannitol, as well as the spin trap α-phenyl- n- tert-butylnitrone, effectively protect the parasite from bilirubin-induced oxidative stress and growth inhibition. These findings suggest that bilirubin, through the development of oxidative stress, induces P. falciparum cell death and that the malaria parasite lacks an HO system probably to protect itself from bilirubin-induced cell death as a second line of defense.

Insights into the Mechanism of Action of Ferroquine. Relationship between Physicochemical Properties and Antiplasmodial Activity

Ferroquine (FQ) is a 4-aminoquinoline antimalarial which contains a quinoline nucleus similar to chloroquine, but a novel ferrocenic group in its side chain. Previous work has demonstrated that this compound has excellent activity against malaria parasites, both in vitro and in vivo, with especially good activity against chloroquine-resistant parasites, but details of its mechanism of action have not previously been reported. In this study, we have investigated the physicochemical properties of FQ for comparison with chloroquine (CQ). Like CQ, FQ forms complexes with hematin in solution (log K = 4.95 +/- 0.05). FQ is an even stronger inhibitor of beta-hematin formation than CQ (IC(50) = 0.78 equiv relative to hematin for FQ vs 1.9 for CQ). These data suggest that the mechanism of action of FQ is likely to be similar to that of CQ and probably involves hematin as the drug target and inhibition of hemozoin formation. However, both the basicity and lipophilicity of FQ are significantly different from those of CQ. The lipophilicity of FQ and CQ are similar when protonated at the putative food vacuole pH of 5.2 (log D = -0.77 and -1.2 respectively), but differ markedly at pH 7.4 (log D = 2.95 and 0.85 respectively). In addition, the pK(a) values of FQ are lower (pK(a1) = 8.19 and pK(a2) = 6.99) than those of CQ (10.03 and 7.94, respectively). This suggests that there will be somewhat less vacuolar accumulation of FQ compared with CQ. Single crystal structure determination of FQ shows the presence of a strong internal hydrogen bond between the 4-amino group and the terminal N atom. This, together with the electron donating properties of the ferrocene moiety, probably explains the decreased pK(a). Interestingly, the decreased accumulation arising from the less basic behavior of this compound is partly compensated for by its stronger beta-hematin inhibition. Increased lipophilicity, differences in geometric and electronic structure, and changes in the N-N distances in FQ compared to CQ probably explain its activity against CQ-resistant parasites.

A colorimetric high-throughput β-hematin inhibition screening assay for use in the search for antimalarial compounds

Antimalarial drugs such as chloroquine are believed to act by inhibiting hemozoin formation in the food vacuole of the malaria parasite. We have developed a new assay for measuring and detecting inhibition of synthetic hemozoin (β-hematin) formation. Aqueous pyridine (5% v/v, pH 7.5) forms a low-spin complex with hematin but not with β-hematin. Its absorbance obeys Beer’s law, making it useful for quantitating hematin concentration in hematin/β-hematin mixtures, allowing compounds to be investigated for inhibition of β-hematin formation. The assay is rapid (60 min incubation) and requires no centrifugation. The β-hematin inhibition data show good agreement with alternative assay methods reported by four laboratories. The assay was adapted for high-throughput colorimetric screening, allowing visual identification of β-hematin inhibitors. In this mode, the assay successfully detected all 18 β-hematin inhibitors in a set of 47 compounds tested, with no false positive results. The quantitative in vitro antimalarial activities of a set of 13 aminoquinolines and quinoline methanols were found to correlate significantly with β-hematin inhibition values determined using the assay.

Haemozoin Formation as a Target for the Rational Design of New Antimalarials

Owing to the widespread occurrence of drug resistance, there is now an urgent need for new antimalarial drugs. There is compelling evidence that the target of the historically most important group of antimalarials, the quinolines, remains useful for the design of new drugs. Currently available data suggest that these drugs inhibit the disposal of haemoglobin derived haematin during the blood-stage of the malaria parasite. This article reviews recent findings concerning the disposal of haematin in the food vacuole of the malaria parasite P. falciparum, the interaction of quinoline antimalarials with haematin and their inhibition of haemozoin formation. Emphasis is also placed on recently reported structure-activity relationships and possible ways forward in the discovery and design of new drugs of this type. Keywords: malaria, antimalarials, haemozoin, haematin, quinolines, haem

Carbon isosteres of the 4-aminopyridine substructure of chloroquine: effects on pK(a), hematin binding, inhibition of hemozoin formation, and parasite growth.

Unlike diprotic chloroquine (CQ), its two 4-aminoquinoline carbon isosteres (1, 2) are monoprotic at physiological pH. Compared to CQ, hematin binding affinity of 1 decreased 6.4-fold, and there was no measurable binding for 2. Although 1 was a weak inhibitor of hemozoin formation, neither isostere inhibited P. falciparum in vitro. Evidently, the CQ-hematin interaction is largely a function of its pyridine substructure, but inhibition of hemozoin formation and parasite growth depends on its 4-aminopyridine substructure.

Artemisinin Antimalarials Do Not Inhibit Hemozoin Formation

The mechanism of action of artemisinin antimalarials may be ascribed to C-centered radicals that alkylate biomolecules or to the peroxide moiety, which inhibits a specific, as yet undefined, target (4). It is also proposed that artemisinins kill the parasite through inhibition of hemozoin formation, thereby allowing buildup of toxic heme monomer (1, 2, 5). Artemisinin and dihydroartemisinin may be unstable under aqueous conditions used for hemozoin studies; ring-opened products (3) may therefore bind to the heme and inhibit hemozoin formation. 10-Deoxodihydroartemisin (Fig. ​(Fig.1)1) has no oxygen at C-10 and is less able to undergo ring opening under aqueous conditions. Therefore, even though it is an active antimalarial it may not interfere with hemozoin formation. To test this, dihydroartemisinin and 10-deoxodihydroartemisinin were screened for inhibition of β-hematin (hemozoin) formation by using both the HPIA (heme polymerization inhibitory activity) assay (2) (hematin in acetic acid at pH 2.7, 37°C, 18 h) and the BHIA (β-hematin inhibitory activity) assay (hemin in dimethyl sulfoxide-acetate buffer at pH 5.0, 37°C, 18 h). The first identifies ligands that bind axially with the protoporphyrin iron, and the second identifies ligands undergoing π-π interactions with hematin (6). FIG. 1. Structures of artemisinin, dihydroartemisinin, and 10-deoxodihydroartemisinin. Dihydroartemisinin showed a dose-dependent inhibition in the HPIA assay but not in the BHIA assay. The 10-deoxy compound was inactive in both assays (Fig. ​(Fig.2).2). Thus, peroxidic antimalarials do not interfere with hemozoin formation in the parasite and are differentiated from quinoline antimalarials in that they cannot bind via π-π interactions with the heme molecule. This is also evident in their failure to inhibit β-hematin formation in the BHIA assay (6). It is uncertain if dihydroartemisinin undergoes ring opening under the HPIA assay conditions. In this case, it cannot be excluded that in the HPIA assay (pH 2.7), the ring-opened form of dihydroartemisinin will form an axial ligand with the porphyrin iron, thus inhibiting β-hematin formation. 10-Deoxodihydroartemisinin cannot easily undergo ring opening and therefore cannot bind porphyrin—it has no effect on β-hematin formation, yet it displays potent antimalarial activity. FIG. 2. Results of in vitro assays of inhibition of β-hematin formation. DHA, dihydroartemisinin. Thus, inhibition of β-hematin formation in the HPIA assay by artemisinin (2) and dihydroartemisinin (this letter), but not by 10-deoxodihydroartemisinin, reflects a reactivity that is not related to their antimalarial action. Furthermore, binding of artemisinins with Fe(III)PPIX is not necessary for antimalarial activity. These data also confirm that the HPIA and BHIA assays are useful for distinguishing compounds forming π-π interactions with heme from those forming axial ligands.

Optimization of xanthones for antimalarial activity: the 3,6-bis-omega-diethylaminoalkoxyxanthone series.

Hydroxyxanthones have been identified as novel antimalarial agents. The compounds are believed to exert their activity by complexation to heme and inhibition of hemozoin formation. Modification of the xanthone structure was pursued to improve their antimalarial activity. Attachment of R-groups bearing protonatable nitrogen atoms was conducted to enhance heme affinity through ionic interactions with the propionate side chains of the metalloporphyrin and to facilitate drug accumulation in the parasite food vacuole. A series of 3,6-bis-omega-diethylaminoalkoxyxanthones with side chains ranging from 2 to 8 carbon atoms were prepared and evaluated. Measurement of heme affinity for each of the derivatives revealed a strong correlation (R(2) = 0.97) between affinity and antimalarial potency. The two most active compounds in the series contained 5- and 6-carbon side chains and exhibited low nanomolar 50% inhibitory concentration (IC(50)) values against strains of chloroquine-susceptible and multidrug-resistant Plasmodium falciparum in vitro. Both of these xanthones exhibit stronger heme affinity (8.26 x 10(5) and 9.02 x 10(5) M(-1), respectively) than either chloroquine or quinine under similar conditions and appear to complex heme in a unique manner.

The propionate of heme binds N4O2 Schiff base antimalarial drug complexes.

Schiff base N4O2 complexes offer a flexible template on which to develop novel antimalarial drug complexes that inhibit the aggregation of hemozoin, a detoxification product of the malaria parasite Plasmodium falciparum. The efficacies of these complexes are dependent on the charges of the complexes. Further evidence suggests that these complexes inhibit hemozoin formation via a specific drug/heme propionate salt that prevents the formation of the requisite axial propionato linkage in the repeating dimeric unit of the hemozoin aggregate.

Inhibition of Hemozoin Formation in Plasmodium falciparum Trophozoite Extracts by Heme Analogs: Possible Implication in the Resistance to Malaria Conferred by the β-Thalassemia Trait

Human falciparum malaria, caused by the intracellular protozoa Plasmodium falciparum, results in 1-2 million deaths per year. P. falciparum digests host erythrocyte hemoglobin within its food vacuole, resulting in the release of potentially toxic free heme. A parasite-specific heme polymerization activity detoxifies the free heme by cross-linking the heme monomers to form hemozoin or malaria pigment. This biochemical process is the target of the widely successful antimalarial drug chloroquine, which is rapidly losing its effectiveness due to the spread of chloroquine resistance. We have shown that chloroquine resistance is not due to changes in the overall catalytic activity of heme polymerization or its chloroquine sensitivity. Therefore, the heme polymerization activity remains a potential target for novel antimalarials. In this study, we investigated the ability of heme analogs to inhibit heme polymerization and parasite growth in erythrocytes. Incorporation of radioactive hemin substrate into an insoluble hemozoin pellet was used to determine heme polymerization. Incorporation of radioactive hypoxanthine into the nucleic acid of dividing parasites was used to determine the effects of heme analogs on parasite growth. Microscopic and biochemical measurements were made to determine the extent of heme analog entry into infected erythrocytes. The heme analogs tin protoporphyrin IX (SnPP), zinc protoporphyrin IX (ZnPP), and zinc deuteroporphyrin IX, 2,4 bisglycol (ZnBG) inhibited polymerization at micromolar concentrations (ZnPP << SnPP < ZnBG). However, they did not inhibit parasite growth since they failed to gain access to the site of polymerization, the parasite's food vacuole. Finally, we observed high ZnPP levels in erythrocytes from two patients with beta-thalassemia trait, which may inhibit heme polymerization. The heme analogs tested were able to inhibit hemozoin formation in Plasmodium falciparum trophozite extracts. The increased ZnPP levels found in thalassemic erythrocytes suggest that these may contribute, at least in part, to the observed antimalarial protection conferred by the beta-thalassemia trait. This finding may lead to the development of new forms of antimalarial therapy.