Synthesis and Evaluation of Chirally Defined Side Chain Variants of 7-Chloro-4-Aminoquinoline To Overcome Drug Resistance in Malaria Chemotherapy.

A novel 4-aminoquinoline derivative [(S)-7-chloro-N-(4-methyl-1-(4-methylpiperazin-1-yl)pentan-2-yl)-quinolin-4-amine triphosphate] exhibiting curative activity against chloroquine-resistant malaria parasites has been identified for preclinical development as a blood schizonticidal agent. The lead molecule selected after detailed structure-activity relationship (SAR) studies has good solid-state properties and promising activity against in vitro and in vivo experimental malaria models. The in vitro absorption, distribution, metabolism, and excretion (ADME) parameters indicate a favorable drug-like profile.

Heme oxygenase-1 alleviates cigarette smoke-induced restenosis after vascular angioplasty by attenuating inflammation in rat model.

Cigarette smoke is not only a profound independent risk factor of atherosclerosis, but also aggravates restenosis after vascular angioplasty. Heme oxygenase-1 (HO-1) is an endogenous antioxidant and cytoprotective enzyme. In this study, we investigated whether HO-1 upregulating by hemin, a potent HO-1 inducer, can protect against cigarette smoke-induced restenosis in rat's carotid arteries after balloon injury. Results showed that cigarette smoke exposure aggravated stenosis of the lumen, promoted infiltration of inflammatory cells, and induced expression of inflammatory cytokines and adhesion molecules after balloon-induced carotid artery injury. HO-1 upregulating by hemin treatment reduced these effects of cigarette smoke, whereas the beneficial effects were abolished in the presence of Zincprotoporphyrin IX, an HO-1 inhibitor. To conclude, hemin has potential therapeutic applications in the restenosis prevention after the smokers' vascular angioplasty.

Targeting of hematin by the antimalarial pyronaridine.

Pyronaridine, 2-methoxy-7-chloro-10[3',5'-bis(pyrrolidinyl-1-methyl-)4'hydroxyphenyl]aminobenzyl-(b)-1,5-naphthyridine, a new Mannich base schizontocide originally developed in China and structurally related to the aminoacridine drug quinacrine, is currently undergoing clinical testing. We now show that pyronaridine targets hematin, as demonstrated by its ability to inhibit in vitro beta-hematin formation (at a concentration equal to that of chloroquine), to form a complex with hematin with a stoichiometry of 1:2, to enhance hematin-induced red blood cell lysis (but at 1/100 of the chloroquine concentration), and to inhibit glutathione-dependent degradation of hematin. Our observations that pyronaridine exerted this mechanism of action in situ, based on growth studies of Plasmodium falciparum K1 in culture showing antagonism of pyronaridine in combination with antimalarials (chloroquine, mefloquine, and quinine) that inhibit beta-hematin formation, were equivocal.

Regulation of globin gene transcription by heme in erythroleukemia cells: analysis of putative heme regulatory motifs in the p45 NF-E2 transcription factor.

The function of the NF-E2 transcription factor, a p45/small Maf heterodimer, was analyzed in the erythroleukemia cell lines MEL and CB3. In contrast to MEL cells, CB3 cells are null for p45 and thus express only extremely low levels of adult globin transcripts upon induction by agents promoting erythroid differentiation. We investigated the response of erythroleukemia cells to hemin treatment. Hemin rapidly induces beta-globin gene transcript levels in MEL cells, but not in CB3 cells. Stable expression of the large p45 NF-E2 subunit in CB3 cells restores hemin mediated beta-globin gene transcription, suggesting that the presence of a functional NF-E2 is required for strong induction of beta-globin mRNA levels by hemin in erythroleukemia cells. We performed mutagenesis of two potential heme-regulatory motifs (HRMs) in p45 NF-E2 and found that the mutated versions are expressed and can still recognize a NF-E2 DNA binding element. In addition, we showed that p45 NF-E2 HRM mutants are able to restore beta-globin gene transcription in CB3 cells upon induction by hemin. Our results suggest that globin gene activation by heme appears to be independent of the putative HRMs in the p45 subunit of the NF-E2 transcription factor.

Virulence of Staphylococcus aureus small colony variants in the Caenorhabditis elegans infection model.

Small colony variants (SCVs) of Staphylococcus aureus are slow-growing morphological variants that have been implicated in persistent, relapsing, and antibiotic-resistant infections. The altered phenotype of SCVs in most strains has been attributed to defects in electron transport due to mutations in hemin or menadione biosynthesis. The pathogenic capacity of SCVs compared to phenotypically normal strains is variable depending on the attribute examined, with some studies showing reduced virulence of SCVs and others demonstrating normal or heightened virulence. Recently, the nematode Caenorhabditis elegans has been successfully employed as an alternative host to investigate virulence mechanisms of a variety of bacterial pathogens, including S. aureus. In this study, we show that clinical SCVs as well as hemB- and menD-deficient mutants of S. aureus are greatly reduced in virulence in the C. elegans infection model.

Antimalarial 9-anilinoacridine compounds directed at hematin.

Antimalarial 9-anilinoacridines are potent inhibitors of parasite DNA topoisomerase II both in vitro and in situ. 3,6-diamino substitution on the acridine ring greatly improves parasiticidal activity against Plasmodium falciparum by targeting DNA topoisomerase II. A series of 9-anilinoacridines were investigated for their abilities to inhibit beta-hematin formation, to form drug-hematin complexes, and to enhance hematin-induced lysis of red blood cells. Inhibition of beta-hematin formation was minimal with 3,6-diamino analogs of 9-anilinoacridine and greatest with analogs with a 3,6-diCl substitution together with an electron-donating group in the 1'-anilino position. On the other hand, the presence of a 1'-N(CH3)2 group in the anilino ring produced compounds that strongly inhibited beta-hematin formation but which did not appear to be sensitive to the nature of the substitutions in the acridine nucleus. The derivatives bound hematin, and Job's plots of UV-visible absorbance changes in drug-hematin complexes at various molar ratios indicated a stoichiometric ratio of 1:2. The drugs enhanced hematin-induced red blood cell lysis at low concentrations (<4 microM). These studies open up the novel possibility of development of 9-anilinoacridine antimalarials that target not only DNA topoisomerase II but also beta-hematin formation, which should help delay the rapid onset of resistance to drugs acting at only a single site.

On the mechanism of hemozoin production in malaria parasites: activated erythrocyte membranes promote beta-hematin synthesis.

The ferriprotoporphyrin IX (FP) molecules released by intraerythrocytic malaria parasites during hemoglobin digestion are converted to beta-hematin and are stored in the parasites' food vacuoles. It has been demonstrated in cell-free medium that the incorporation of FP into beta-hematin under physiological conditions requires a catalyst from parasite lysates or pre-formed beta-hematin. In the present studies, lysates of Plasmodium falciparum-infected erythrocytes were suspended in 1 M NaOH and were washed with phosphate buffer, pH 7.6. When the cell extracts were incubated with hematin in 0.5 M sodium acetate buffer, pH 5, for 20 hr at 37 degrees C, a large quantity of beta-hematin was formed. To determine whether parasite components were necessary for the beta-hematin formation, normal erythrocyte ghosts were similarly treated with 1 M NaOH and then incubated with hematin. In repeated experiments it was found that, on the average, 70% of the hematin was converted to beta-hematin. Membranes treated with HCl or CH(3)COOH also promoted the formation of beta-hematin, while untreated membranes were ineffective. The possibility that metabolic activities in the food vacuoles of malaria parasites may activate membrane fragments, from hemoglobin vesicles, to promote beta-hematin formation is discussed in this paper.

Assay of beta-hematin formation by malaria parasite.

Novel leads are urgently required for designing antimalarials due to the reduced efficacy of presently available drugs. The malaria parasite has a unique reaction of heme polymerization, which has attracted much attention in the recent past as a target for the design of antimalarial drugs. The process is hampered by non-availability of a proper assay method. Currently available methods are cumbersome and require advanced instrumentation or radioactive substrates. Here, we are describing an assay for hemozoin formation that is simple and reproducible. This assay has routinely been used by us for the identification of potential compounds with antimalarial activity.

A site-directed Staphylococcus aureus hemB mutant is a small-colony variant which persists intracellularly.

Although small-colony variants (SCVs) of Staphylococcus aureus have been recognized for many years, this phenotype has only recently been related to persistent and recurrent infections. Clinical S. aureus SCVs are frequently auxotrophic for menadione or hemin, two compounds involved in the biosynthesis of the electron transport chain elements menaquinone and cytochromes, respectively. While this observation as well as other biochemical characteristics of SCVs suggests a link between electron-transport-defective strains and persistent infections, the strains examined thus far have been genetically undefined SCVs. Therefore, we generated a stable mutant in electron transport by interrupting one of the hemin biosynthetic genes, hemB, in S. aureus by inserting an ermB cassette into hemB. We isolated a hemB mutant, due to homologous recombination, by growth at a nonpermissive temperature and selection for erythromycin resistance. This mutant showed typical characteristics of clinical SCVs, such as slow growth, decreased pigment formation, low coagulase activity, reduced hemolytic activity, and resistance to aminoglycosides. Additionally, the mutant was able to persist within cultured endothelial cells due to decreased alpha-toxin production. Northern and Western blot analyses showed that expression of alpha-toxin and that of protein A were markedly reduced, at both the mRNA and the protein level. The SCV phenotype of the hemB mutant was reversed by growth with hemin or by complementation with intact hemB. Hence, a defect in the electron transport system allows S. aureus SCVs to resist aminoglycosides and persist intracellularly.

Persistent and relapsing infections associated with small-colony variants of Staphylococcus aureus.

Small-colony variants (SCVs) of Staphylococcus aureus were cultured from five patients with persistent and relapsing infections. All five SCV strains were nonhemolytic and nonpigmented and grew very slowly on routine culture media in an ambient atmosphere. In several instances, these phenotypic characteristics led to the initial misidentification of the organisms in the clinical microbiology laboratory. All four strains available for further analysis were shown to be auxotrophs that reverted to normal growth and morphology in the presence of menadione, hemin, and/or a CO2 supplement. Similarly, these isolates were resistant to aminoglycosides under routine conditions but susceptible in the presence of the metabolic supplements. For two patients, the large and small colony forms isolated concurrently were indistinguishable when analyzed by pulsed field gel electrophoresis and thus represented phenotypic variants within individual clones. We propose a model relating the phenotypic characteristics of S. aureus SCVs with the clinical pattern of persistent and relapsing infection.

Heme polymerase: modulation by chloroquine treatment of a rodent malaria.

The biosynthesis of the beta-hematin of malarial pigment (hemozoin) is catalyzed by a newly discovered enzyme, heme polymerase, which is described for Plasmodium berghei in this report. This novel enzyme is present in the insoluble fraction of hemolysates of infected erythrocytes but is not present in normal erythrocytes. The substrate is ferriprotoporphyrin IX (FP) released from hemoglobin. At pH 5 and 37 degrees C the enzyme is saturated by 100 microM FP. The pH optimum is between 5 and 6 and the reaction is linear for 6 hours. All heme polymerase activity is destroyed by heating at 100 degrees C for 3 minutes. Chloroquine treatment of malarious mice reduces by 80 percent the activity of this enzyme, without inhibiting release of FP from hemoglobin, and thereby causes excess nonpolymerized, nonhemozoin FP to accumulate. Since the accumulated FP is accessible to bind chloroquine, we propose that it is the mediator of the antimalarial activity of chloroquine.

Induction by hemin of proliferation and of differentiation of progenitor erythroid cells responsible for erythropoietin.

Erythroid progenitor cells were obtained from rat fetal liver by immunolysis of the whole erythroid population with an antiserum directed against adult rat erythrocytes, followed by separation on a density gradient. Immediately after their isolation, these cells contained only minute amounts of globin mRNAs and their heme synthesis was negligible. In the absence of erythropoietin (Epo), they did not proliferate or differentiate. In the presence of Epo, they proliferated, synthetized heme and globins actively, accumulated large amounts of globin mRNAs, and developed hemoglobinized colonies in methylcellulose. Hemin, in concentrations of 5-100 microM, induced, in the absence of Epo, the proliferation and differentiation of these cells (e.g., accumulation of globin mRNAs, synthesis of heme and globins, and increased density of membrane antigens characteristic of the erythrocyte). Nevertheless, Epo and hemin actions were not superimposable: in methylcellulose, Epo induced the appearance of large (greater than or equal to 32 cells) hemoglobinized colonies in 48 h, whereas hemin induced smaller and fewer colonies in only 24 h. Succinylacetone (SA, inhibitor of heme synthesis) mostly prevented the effects of Epo on cell proliferation and differentiation; SA inhibition was relieved by hemin. Thus, hemin seems to intervene in erythroid differentiation as a factor of both proliferation and maturation.

The chemical and enzymatic oxidation of hematohemin IX. Identification of hematobiliverdin IX alpha as the sole product of the enzymatic oxidation.

Oxidative cleavage of hematohemin IX in pyridine solution in the presence of ascorbic acid (coupled oxidation), followed by esterification of the products with boron trifluoride/methanol produced the four possible hematobiliverdin dimethyl esters in 11.1% overall yield. Transetherifications took place simultaneously with the esterification reaction and resulted in the formation of the dimethyl ester of hematobiliverdin IX gamma 8a,13a-dimethyl ether (1.8%), the dimethyl ester of hematobiliverdin IX beta 13a,18a-dimethyl ether (1.9%), the dimethyl ester of hematobiliverdin IX delta 8a-monomethyl ether (1.4%), and the dimethyl ester of hematobiliverdin IX alpha 18a-monomethyl ether (0.4%). The latter was the sole product obtained after the enzymatic oxidation of hematohemin with heme oxygenase, after esterification of the reaction product with boron trifluoride/methanol. When the esterification step was omitted hematobiliverdin IX alpha was obtained from the enzymatic oxidation. The structures of the hematobiliverdin derivatives were secured by their NMR and mass spectra data. Saponification of the dimethyl esters afforded the hematobiliverdin methyl ethers, which were excellent substrates of biliverdin reductase and were readily reduced to the corresponding bilirubins. Hematobiliverdin IX alpha was also a good substrate of biliverdin reductase. It is concluded that the enzymatic oxidation of hematohemin IX by heme oxygenase is alpha-selective, while biliverdin reductase shows no selectivity in the reduction of the four hematobiliverdin isomers.

Effect of Griseofulvin on 5-aminolevulinate synthase and on ferrochelatase in mouse liver neoplastic nodules.

Treatment of mice with griseofulvin for 8 months induced hepatocellular nodules in the liver which persist after discontinuation of griseofulvin feeding. We investigated the porphyrogenic effect of griseofulvin on these nodules and surrounding nonneoplastic liver after renewed short-term exposure of tumor-bearing mice to this agent. Griseofulvin treatment for 4 days led to marked elevation of the activity of 5-aminolevulinate synthase in peritumoral (3.8-fold) and control (6-fold) liver. The increase in enzyme activity was much less pronounced in the nodules (1.5-fold). Ferrochelatase activity was markedly decreased under the same experimental conditions in both peritumoral and control livers (to 18 and 13.5%, respectively, of the pretreatment values), but the effect was considerably smaller in nodules (to 40% of the pretreatment value). These results may explain the lack of porphyrin accumulation in tumor tissue.

Additive inhibition of in vitro rabbit reticulocyte heme and protein synthesis with combinations of isoniazid, chloramphenicol and ethanol.

Isoniazid, chloramphenicol and ethanol are well known hematopoietic toxins. These agents inhibit in vitro rabbit reticulocyte heme synthesis and, as a result, inhibit protein synthesis. When combinations of isoniazid (2 x 10(-3)M) and ethanol (0.1M), isoniazid and chloramphenicol (10(-3)M) or ethanol and chloramphenicol were present in the same incubation, their inhibitory effects on heme and protein synthesis were additive. These results suggest that the development of clinical hematological toxicity in man may result from additive interaction of chemically unrelated compounds which inhibit the heme synthetic pathway.

Formalin pigment (acid hematin) and related pigments.

Black to brown amorphous to microcrystalline granules are encountered in histologic sections prepared from tissues fixed in formalin having a low pH. This pigment is produced by acid acting upon hemoglobin and is known as formalin pigment or acid hematin. A similar pigment is also observed in sites of bleeding ulcers in areas of acid production such as the stomach. These pigments exhibit many physical and histochemical properties similiar to pigments produced by some animal parasites as in malaria, schistosoma and pulmonary mites. These parasites disintegrate erythrocytes in an unknown manner, and liberate an acid hematin-like pigment which is phagocytized by the reticuloendothelial system. Since formalin pigment can be considered as an artifact, confusion with other pigments can be avoided by the use of neutral buffered formalin for the fixation of tissues.

Evidence supporting a physiological role for the hemin-controlled translational repressor of globin synthesis in reticulocytes.

The synthesis of heme and globin in rabbit reticulocytes was compared at 35 and 25 degrees C. The lower temperature decreased heme synthesis significantly more than globin synthesis and resulted in a much greater accumulation of globin dimers. After 16 h of incubation in the absence of iron, globin synthesis in reticulocytes which were at 35 degrees C could not be stimulated by iron, whereas cells which were at 25 degrees C responded with nearly control levels of globin synthesis. Since the formation of the hemin-controlled translational repressor in reticulocyte lysates is also decreased much more than protein synthesis at reduced temperature the results provide evidence for a physiological role for the translational repressor in controlling globin synthesis in reticulocytes.

Incorporation of ALA-14C in circulating bilirubin and hemoglobin heme of hepatectomized dogs.

After intravenous injection of delta amino-levulinic acid-14C (ALA-14C) (50 muCi) in hepatectomized dogs, bilirubin-14C reaches a peak after 3-6 hours and then slowly falls. During this time radioactivity of hemoglobin heme is much lower and rises sluggishly. Bilirubin specific activity is several hundred times that of hemoglobin heme. Incorporation of isotope is also much greater for bilirubin. This difference between bilirubin-14C and hemoglobin heme-14C provides evidence that bilirubin is produced outside the liver by a mechanism other than breakdown of hemoglobin. In intact dogs with cannulated common duct, intravenous injection of ALA-14C produces similar results but the rise of bilirubin-14C produced in 9 hours in the hepatectomized dogs is about 12% of the total blood bilirubin-14C of non hepatectomized animals with cannulated common duct. If we include bile bilirubin-14C produced in the same time by intact animals with cannulated common duct, nonhemoglobin bile production is only 4.2% of the total.