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Articles by Babu L. Tekwani in JoVE

 JoVE Immunology and Infection

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

1National Center for Natural Products Research, School of Pharmacy, University of Mississippi, 2Department of Pharmacology, University of Mississippi

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A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

Other articles by Babu L. Tekwani on PubMed

A Physiochemical Mechanism of Hemozoin (beta-hematin) Synthesis by Malaria Parasite

Malaria parasite homogenate, the lipid extracts, and an unsaturated fatty acid, linoleic acid, which have been shown to promote beta-hematin formation in vitro, were used to investigate the mechanism of hemozoin biosynthesis, a distinct metabolic function of the malaria parasite. In vitro beta-hematin formation promoted by Plasmodium yoelii homogenate, the lipid extracts, and linoleic acid were blocked by ascorbic acid, reduced glutathione, sodium dithionite, beta-mercaptoethanol, dithiothreitol, and superoxide dismutase. Oxidized glutathione did not show any effect. Preoxidized preparations of the lipids extracts or the P. yoelii homogenate failed to catalyze beta-hematin formation. Depletion of oxygen in the reaction mixtures also inhibited the lipid-catalyzed beta-hematin formation. Under the reaction conditions similar to those used for the in vitro beta-hematin formation assay, the antioxidants and reducing agents mentioned above, except the DTT and beta-mercaptoethanol, did not cause degradation of heme. beta-Hematin formation was also inhibited by p-aminophenol, a free radical chain reaction breaker. Hemozoin biosynthesis within the digestive vacuoles of the malaria parasite may be a lipid-catalyzed physiochemical reaction. An oxidative mechanism may be proposed for lipid-mediated beta-hematin formation, which may be mediated by generation of some free radical intermediates of heme.

Peripheral Blood Leucocytes Ornithine Decarboxylase Activity in Chronic Myeloid Leukemia Patients: Prognostic and Therapeutic Implications

Leukocytes ornithine decarboxylase (ODC) activity was measured in normal individuals and in patients with chronic myeloid leukemia (CML) in chronic phase (CML-CP) as well as in accelerated phase (CML-AP), with an aim to examine the role of ODC activity in prognostic evaluation of CML patients. Our results showed that ODC activity was significantly higher in CML-CP (41.02+/-25.57nmol/h per 10(7) cells, P<0.005) and CML-AP (67.71+/-44.42nmol/h per 10(7) cells, P<0.001) patients than in normal subjects (3.12+/-1.34nmol/h per 10(7) cells). Furthermore, patients with CML-AP showed higher ODC activity than CML-CP patients (P<0.005). Patients with CML-CP who converted to accelerated phase within 24 months had higher ODC activity (84.58+/-12.81nmol/h per 10(7) cells) than patients who did not convert to accelerated phase (31.13+/-18.24nmol/h per 10(7) cells). The high value of ODC activity was also associated with less clinico-hematological response. We suggest that ODC activity reflects the neoplastic proliferative activity in CML patients and may serve as an additional prognostic marker.

Haem Polymerase As a Novel Target of Antimalarial Action of Cyproheptadine

An antihistaminic drug, cyproheptadine (20-25mg/kg x 4 days), showed significant schizontocidal activity in the blood against a lethal multidrug-resistant (MDR) strain of Plasmodium yoelii nigeriensis (highly resistant to chloroquine, mefloquine, and quinine); the protection of mice ranged between 75 and 100%. A combination of cyproheptadine (15 mg/kg) and chloroquine improved antimalarial activity compared to treatment with either drug alone, whereas a combination of cyproheptadine with quinine or mefloquine did not improve its antimalarial activity. Chloroquine and cyproheptadine inhibited haem polymerization activity in cell-free extracts and in in vivo experiments with MDR P. yoelii, but the combination did not cause a more significant inhibition than found with either drug alone. Cyproheptadine has been shown to produce dose-dependent inhibition of haem polymerization activity both in vitro and in vivo. The mechanism of the antimalarial action of cyproheptadine and its enhanced antimalarial activity with chloroquine could be due, in part, to their inhibitory effect on haem polymerization.

Synthesis and Biological Evaluation of New Imidazo[1,2-a]pyridine Derivatives Designed As Mefloquine Analogues

This paper describes the synthesis and the in vitro antimalarial profile of two new imidazo[1,2-a]pyridine derivatives 4HCl and 13HCl, structurally proposed as mefloquine (1) analogues, by exploring bioisosterism and molecular simplification tools. The synthetic route employed to access the title compounds used, as starting material, the previously described ethyl 2-methylimidazo[1,2-aJpyridine-3-carboxylate derivative (5). These novel heterocyclic derivatives 4HCl and 13HCl presented modest antimalarial activity against the W-2 and D-6 clones of Plasmodium falciparum as well as inhibitors of in vitro heme polymerization compared to mefloquine.

A Novel Active DNA Topoisomerase I in Leishmania Donovani

A common feature shared by type I DNA topoisomerases is the presence of a "serine, lysine, X, X, tyrosine" motif as conventional enzyme active site. Preliminary data have shown that Leishmania donovani DNA topoisomerase I gene (LdTOP1A) lacked this conserved motif, giving rise to different theories about the reconstitution of an active DNA topoisomerase I in this parasite. We, herein, describe the molecular cloning of a new DNA topoisomerase I gene from L. donovani (LdTOP1B) containing the highly conserved serine, lysine, X, X, tyrosine motif. DNA topoisomerase I activity was detected only when both genes (LdTOP1A and LdTOP1B) were co-expressed in a yeast expression system, suggesting the existence of a dimeric DNA topoisomerase I in Leishmania parasites.

Antimicrobial and Antiparasitic (+)-trans-hexahydrodibenzopyrans and Analogues from Machaerium Multiflorum

Machaerium multiflorum yielded two additional new (+)-trans-hexahydrodibenzopyrans (HHDBP's), machaeriol C (1) and machaeriol D (2), and three new 5,6-seco-HHDBP's, machaeridiol A (3), machaeridiol B (4), and machaeridiol C (5). Their structures and stereochemistries were determined by 1D and 2D NMR data, including HMBC, NOESY, and circular dichroism experiments. Machaeriol C (1) demonstrated in vitro antibacterial activity against Staphylococcus aureus (IC(50) 0.65 microg/mL) and methicillin-resistant S. aureus (MRSA) (IC(50) 0.70 microg/mL), while its corresponding 5,6-seco-analogues machaeridiol A (3) and machaeridiol B (4) showed antibacterial activity against S. aureus and MRSA (IC(50) 1.0-2.6 microg/mL) and antifungal activity against Candida albicans (IC(50), 2.0-3.5 microg/mL). In addition, machaeridiol B (4) demonstrated antiparasitic activities against Plasmodium falciparum D6 and W2 clones and Leishmania donavani with IC(50) values of 0.64, 0.22, and 0.9 microg/mL, respectively.

New Manzamine Alkaloids with Activity Against Infectious and Tropical Parasitic Diseases from an Indonesian Sponge

Eleven manzamine type alkaloids, two beta-carbolines, and five nucleosides have been isolated from an Indonesian sponge. Among these are the previously characterized 12,34-oxamanzamine A, 12,34-oxamanzamine E, manzamine A (1), 8-hydroxymanzamine A, 6-deoxymanzamine X, manzamine E (2), manzamine X, manzamine F (4), norharman, thymine, 2',3'-didehydro-2',3'-dideoxyuridine, uracil, thymidine, and 2'-deoxyuridine. The structures for the five new compounds have been assigned as 32,33-dihydro-31-hydroxymanzamine A (3), 32,33-dihydro-6-hydroxymanzamine A-35-one (5), des-N-methylxestomanzamine A (6), 32,33-dihydro-6,31-dihydroxymanzamine A (7), and 1,2,3,4-tetrahydronorharman-1-one (8), on the basis of NMR and X-ray data. The bioactivity and SAR of the manzamines against malaria, TB, and leishmania are also presented. The structural revision of two previously reported pyrazoles as uracil and thymine is also discussed.

Flow Cytometric Analysis of Aneuploidy and S-phase Fraction in Chronic Myeloid Leukemia Patients: Role in Early Detection of Accelerated Phase

We studied S-phase fraction (SPF) and aneuploidy in peripheral blood leucocytes of patients with chronic myeloid leukemia (CML) in chronic phase (CML-CP, n=41), accelerated phase (CML-AP, n=6), and control subjects (n=12) with an aim to find out their role in early detection of accelerated phase. The SPF and aneuploidy were studied through flow-cytometry using LT-Mod. Fit software. Mean SPF value in CML-AP (9.28+/-3.46%) and in CML-CP (4.76+/-2.30%) were significantly higher than in normal controls (0.28+/-0.21%), (P<0.005, P<0.001). CML-CP patients having higher SPF (>7%) converted to accelerated phase within 18 months of follow-up while those with lower SPF (<7%) did not. Aneuploidy was present in 34.14% of CML-CP and all patients of CML-AP whereas no control subjects showed aneuploidy. Among CML-CP patients having SPF >7%, 86% developed aneuploidy during follow-up as compared to 18.50% of CML-CP with less than 7% SPF. We conclude that peripheral blood SPF and aneuploidy could be important parameters for prediction of evolution to accelerated phase in CML patients.

Antiparasitic Alkaloids from Psychotria Klugii

Psychotria klugii yielded two new benzoquinolizidine alkaloids, klugine (1) and 7'-O-demethylisocephaeline (2), together with the previously known cephaeline (3), isocephaeline (4), and 7-O-methylipecoside (5). The structures and stereochemistry of 1 and 2 were determined by 1D and 2D NMR data and circular dichroism experiments. Cephaeline (3) demonstrated potent in vitro antileishmanial activity against Leishmania donavani (IC(50) 0.03 microg/mL) and was >20- and >5-fold more potent than pentamidine and amphotericin B, respectively, while klugine (1) (IC(50) 0.40 microg/mL) and isocephaeline (4) (IC(50) 0.45 microg/mL) were <13- and <15-fold less potent than 3. In addition, emetine (6) (IC(50) 0.03 microg/mL) was found to be as equally potent as 3, but was >12-fold more toxic than 3 against VERO cells (IC(50) 0.42 vs 5.3 microg/mL). Alkaloids 1 and 3 exhibited potent antimalarial activity against Plasmodium falciparum clones W2 and D6 (IC(50) 27.7-46.3 ng/mL). Compound 3 was cytotoxic to SK-MEL, KB, BT-549, and SK-OV-3 human cancer cells, while 1 was inactive.

Structure-activity Relationships of the Antimalarial Agent Artemisinin. 8. Design, Synthesis, and CoMFA Studies Toward the Development of Artemisinin-based Drugs Against Leishmaniasis and Malaria

Artemisinin (1) and its analogues have been well studied for their antimalarial activity. Here we present the antimalarial activity of some novel C-9-modified artemisinin analogues synthesized using artemisitene as the key intermediate. Further, antileishmanial activity of more than 70 artemisinin derivatives against Leishmania donovani promastigotes is described for the first time. A comprehensive structure-activity relationship study using CoMFA is discussed. These analogues exhibited leishmanicidal activity in micromolar concentrations, and the overall activity profile appears to be similar to that against malaria. Substitution at the C-9beta position was shown to improve the activity in both cases. The 10-deoxo derivatives showed better activity compared to the corresponding lactones. In general, compounds with C-9alpha substitution exhibited lower antimalarial as well as antileishmanial activities compared to the corresponding C-9beta analogues. The importance of the peroxide group for the observed activity of these analogues against leishmania was evident from the fact that 1-deoxyartemisinin analogues did not exhibit antileishmanial activity. The study suggests the possibility of developing artemisinin analogues as potential drug candidates against both malaria and leishmaniasis.

Molecular and Functional Characterization of Adenylate Kinase 2 Gene from Leishmania Donovani

ATP-regenerating enzymes may have an important role in maintaining ATP levels in mitochondria-like kinetoplast organelle and glycosomes in parasitic protozoa. Adenylate kinase (AK) (ATP:AMP phosphotransferase) catalyses the reversible transfer of the gamma-phosphate group from ATP to AMP, releasing two molecules of ADP. This study describes cloning and functional characterization of the gene encoding AK2 from a genomic library of Leishmania donovani and also its expression in leishmania promastigote cultures. AK2 was localized on an approximately 1.9-Mb chromosomal band as a single copy gene. L. donovani AK2 gene is expressed as a single 1.9-kb mRNA transcript that is developmentally regulated and accumulated during the early log phase. The overexpression of L. donovani AKgene in Escherichia coli yielded a 26-kDa polypeptide that could be refolded to a functional protein with AK activity. The recombinant protein was purified to apparent homogeneity. Kinetic analysis of purified L. donovani AK showed hyperbolic behaviour for both ATP and AMP, with Km values of 104 and 74 microM, respectively. The maximum enzyme activity (Vmax) was 0.18 micromol.min(-1).mg(-1) protein. P1,P5-(bis adenosine)-5'-pentaphosphate (Ap5A), the specific inhibitor of AK, competitively inhibited activity of the recombinant enzymes with estimated Ki values of 190 nM and 160 nM for ATP and AMP, respectively. Ap5A also inhibited the growth of L. donovani promastigotes in vitro which could be only partially reversed by the addition of ADP. Thus, presence of a highly regulated AK2, which may have role in maintenance of ADP/ATP levels in L. donovani, has been demonstrated.

Spectrophotometric Determination of De Novo Hemozoin/beta-hematin Formation in an in Vitro Assay

Formation of hemozoin in the malaria parasite, due to its unique nature, is an attractive molecular target. Several laboratories have been trying to unravel the molecular mechanism of hemozoin biosynthesis within the parasite digestive vacuoles. Use of different assay protocols for in vitro beta-hematin (synthetic identical to hemozoin) formation by these laboratories has led to inconsistent and often contradictory findings. Much of the difficulty may be attributed to oligomeric heme aggregates, which may be indistinguishable in some detection approaches if adequate separation of beta-hemtin is not achieved. Therefore, there is an urgent need for a widely accepted protocol for in vitro beta-hematin formation. We describe here a spectrophotometric assay for in vitro beta-hematin formation. The assay has been validated with the Plasmodium falciparum lysate, the parasite lipid extracts, and some commercially available fatty acids, which are known to initiate/catalyze beta-hematin formation in vitro. The necessity for multiple wash steps for accurate quantification of de novo hemozoin/beta-hematin formation was verified experimentally. It was necessary to wash the pellet, which contains beta-hematin and heme aggregates, sequentially with Tris/SDS buffer and alkaline bicarbonate solution for complete removal of monomeric heme and heme aggregates and accurate quantification of beta-hematin formed during the assay. The pellets and side products in the supernatant were characterized by infrared spectroscopy. No beta-hematin formation occurred in the absence of a catalytic/initiating factor. Based on these findings, a filtration-based assay that uses 96-well microplates, and which has important application in in vitro screening and identification of novel inhibitors of hemozoin formation as potential blood schizontocidal antimalarials, has been developed.

Antimalarial and Antileishmanial Activities of Aroyl-pyrrolyl-hydroxyamides, a New Class of Histone Deacetylase Inhibitors

A New Antimalarial Quassinoid from Simaba Orinocensis

A new antimalarial quassinoid, namely, orinocinolide (1), was isolated from the root bark of Simaba orinocensis, together with the previously reported simalikalactone D (2). The structure of 1 was determined primarily from 1D and 2D NMR analysis, as well as by chemical derivatization. Compound 1 was found to be as equally potent as 2 against Plasmodium falciparum clones D6 and W2 (IC(50) 3.27 and 8.53 ng/mL vs 3.0 and 3.67 ng/mL, respectively), but was 4- and 28-fold less toxic than 2 against VERO cells (IC(50) 10 vs 2.3 microg/mL) and HL-60 (IC(50) 0.7 vs 0.025 microg/mL), respectively. In addition, 2 was >46- and >31-fold more potent than pentamidine and amphotericin B (IC(50) 0.035 vs 1.6 and 1.1 microg/mL) against Leishmania donovani, while 1 was inactive. Orinocinolide (1) inhibited growth of human cancer cells SK-MEL, KB, BT-549, and SK-OV-3, but was less potent than 2 (IC(50) 0.8-1.9 vs 0.3-1.0 microg/mL) against these cells.

Piperazine-linked Bisbenzamidines: a Novel Class of Antileishmanial Agents

A series of 13 1,4-diarylpiperazines has been prepared, evaluated for antileishmanial activity and their binding affinity to DNA was measured. Among these compounds, 1,4-bis[4-(1H-benzimidazol-2-yl)phenyl]piperazine (14) emerged as the most active compound with an IC(50) value of 0.41 microM which is about sevenfold more potent than pentamidine.

An Alpha-proteobacterial Type Malate Dehydrogenase May Complement LDH Function in Plasmodium Falciparum. Cloning and Biochemical Characterization of the Enzyme

Malate dehydrogenase (MDH) may be important in carbohydrate and energy metabolism in malarial parasites. The cDNA corresponding to the MDH gene, identified on chromosome 6 of the Plasmodium falciparum genome, was amplified by RT-PCR, cloned and overexpressed in Escherichia coli. The recombinant Pf MDH was purified to homogeneity and biochemically characterized as an NAD(+)(H)-specific MDH, which catalysed reversible interconversion of malate to oxaloacetate. Pf MDH could not use NADP/NADPH as a cofactor, but used acetylpyridine adenine dinucleoide, an analogue of NAD. The enzyme exhibited strict substrate and cofactor specificity. The highest levels of Pf MDH transcripts were detected in trophozoites while the Pf MDH protein level remained high in trophozoites as well as schizonts. A highly refined model of Pf MDH revealed distinct structural characteristics of substrate and cofactor binding sites and important amino acid residues lining these pockets. The active site amino acid residues involved in substrate binding were conserved in Pf MDH but the N-terminal glycine motif, which is involved in nucleotide binding, was similar to the GXGXXG signature sequence found in Pf LDH and also in alpha-proteobacterial MDHs. Oxamic acid did not inhibit Pf MDH, while gossypol, which interacts at the nucleotide binding site of oxidoreductases and shows antimalarial activity, inhibited Pf MDH also. Treatment of a synchronized culture of P. falciparum trophozoites with gossypol caused induction in expression of Pf MDH, while expression of Pf LDH was reduced and expression of malate:quinone oxidoreductase remained unchanged. Pf MDH may complement Pf LDH function of NAD/NADH coupling in malaria parasites. Thus, dual inhibitors of Pf MDH and Pf LDH may be required to target this pathway and to develop potential new antimalarial drugs.

Three New Manzamine Alkaloids from a Common Indonesian Sponge and Their Activity Against Infectious and Tropical Parasitic Diseases

Three new manzamine-type alkaloids, 12,34-oxamanzamine E (3), 8-hydroxymanzamine J (4), and 6-hydroxymanzamine E (8), as well as 12 previously characterized manzamine alkaloids have been isolated from a common Indonesian sponge of the genus Acanthostrongylophora. The structures of the new compounds have been established on the basis of 1D and 2D NMR spectroscopic analysis and comparison of the data to literature values of related compounds. The biological activities and structure-activity relationship of the manzamines against malaria, Mycobacterium tuberculosis, Leishmania, HIV-1, and AIDS opportunistic infections are discussed. A plausible pathway for the formation of the 12,34-oxaether bridge in compound 3 is also provided.

Antimicrobial and Antileishmanial Activities of Hypocrellins A and B

Hypocrellins A and B were evaluated for in vitro antimicrobial and antileishmanial activities. Hypocrellin A exhibited promising activity against Candida albicans and moderate activity against Staphylococcus aureus, methicillin-resistant S. aureus, Pseudomonas aeruginosa, and Mycobacterium intracellulare. Hypocrellin B showed weak antimicrobial activities. Hypocrellin A exhibited potent antileishmanial activity, while hypocrellin B was only moderately active. These results of promising antifungal and antileishmanial activity of hypocrellin A may be useful for further structure-activity relationship and in vivo studies.

Targeting the Hemozoin Synthesis Pathway for New Antimalarial Drug Discovery: Technologies for in Vitro Beta-hematin Formation Assay

Clinical manifestations of malaria primarily result from proliferation of the parasite within the hosts' erythrocytes. During this process, hemoglobin is utilized as the predominant source of nutrition. The malaria parasite digests hemoglobin within the digestive vacuole through a sequential metabolic process involving multiple proteases. Massive degradation of hemoglobin generates large amount of toxic heme. Malaria parasite, however, has evolved a distinct mechanism for detoxification of heme through its conversion into an insoluble crystalline pigment, known as hemozoin. Hemozoin is identical to beta-hematin, which is constituted of cyclic heme dimers arranged in an ordered crystalline structure through intermolecular hydrogen bonding. The exact mechanism of biogenesis of hemozoin in malaria is still obscure and is the subject of intense debate. Hemozoin synthesis is an indispensable process for the parasite and is the target for action of several known antimalarials. The pathway has therefore attracted significant interest for new antimalarial drug discovery research. Formation of beta-hematin may be achieved in vitro under specific chemical and physiochemical conditions through a biocrystallization process. Based on these methods several experimental approaches have been described for the assay of formation of beta-hematin in vitro and screening of compounds as inhibitors of hemozoin synthesis. These assays are primarily based on differential solubility and spectral characteristics of monomeric heme and beta-hematin. Different factors viz., the malaria parasite lysate, lipids extracts, preformed beta-hematin, malarial histidine rich protein II and some unsaturated lipids have been employed for promoting beta-hematin formation in these assays. The assays based on spectrophotometric quantification of beta-hematin or incorporation of (14)C-heme yield reproducible results and have been applied to high throughput screening. Several novel antimalarial pharmacophores have been discovered through these assays.

Bisnortriterpenes from Salacia Madagascariensis

A new bisnortriterpene quinone methide, 20-epi-isoiguesterinol (2), and a new 6-oxophenolic triterpene, 6-oxoisoiguesterin (5), as well as two known compounds, isoiguesterin (1) and isoiguesterinol (4), were isolated from the petroleum ether extract of the roots of Salacia madagascariensis. Isoiguesterin (1) and 20-epi-isoiguesterinol (2) showed potent activity against Leishmania.

Synthesis, Antimalarial, Antileishmanial, and Antimicrobial Activities of Some 8-quinolinamine Analogues

In the present communication, newly synthesized 8-quinolinamines (25-27) related to previously reported 2-tert-butylprimaquine (2) were evaluated for their in vitro antimalarial activity against chloroquine sensitive and resistant Plasmodium falciparum strains, in vivo antimalarial activity against P. berghei infected mice, in vitro antileishmanial activity against Leishmania donovani, in vitro antimicrobial activity against various fungi and bacteria, and cytotoxicity in a panel of mammalian cell lines. No promising cytotoxicities were observed for compounds reported herein. Analogue 25 was found to exhibit curative antimalarial activity at a dose of 25 mg/kg/dayx4 in a P. berghei infected mice model, and produced suppressive activity at a lower dose of 10 mg/kg/dayx4. In vitro antileishmanial activities (IC50 and IC90) comparable to standard drug pentamidine were exhibited by all synthesized 8-quinolinamines 25-27. At the same time, promising antibacterial and antifungal activities were also observed for synthesized compounds against a panel consisting of several bacteria and fungi.

Polyamine Transport in Parasites: a Potential Target for New Antiparasitic Drug Development

The metabolism of the naturally occurring polyamines-putrescine, spermidine and spermine-is a highly integrated system involving biosynthesis, uptake, degradation and interconversion. Metabolic differences in polyamine metabolism have long been considered to be a potential target to arrest proliferative processes ranging from cancer to microbial and parasitic diseases. Despite the early success of polyamine inhibitors such as alpha-difluoromethylornithine (DFMO) in treating the latter stages of African sleeping sickness, in which the central nervous system is affected, they proved to be ineffective in checking other major diseases caused by parasitic protozoa, such as Chagas' disease, leishmaniasis or malaria. In the use and design of new polyamine-based inhibitors, account must be taken of the presence of up-regulated polyamine transporters in the plasma membrane of the infectious agent that are able to circumvent the effect of the drug by providing the parasite with polyamines from the host. This review contains information on the polyamine requirements and molecular, biochemical and genetic characterization of different transport mechanisms in the parasitic agents responsible for a number of the deadly diseases that afflict underdeveloped and developing countries.

Biomimetic Synthesis, Antimicrobial, Antileishmanial and Antimalarial Activities of Euglobals and Their Analogues

In the present communication, naturally occurring phloroglucinol-monoterpene adducts, euglobals G1-G4 (3b/a and 4a/b) and 16 new analogues (13a/b-18a/b and 19-22) were synthesized by biomimetic approach. These synthetic compounds differ from natural euglobals in the nature of monoterpene and acyl functionality. All of these compounds were evaluated for their antibacterial, antifungal, antileishmanial and antimalarial activities. Analogue 17b possessed good antibacterial activity against methicillin-resistant Staphylococcus aureus, while analogues 19-22 possessed potent antifungal activity against Candida glabrata with IC50s ranging from 1.5 to 2.5 microg/mL. Euglobals along with all synthesized analogues exhibited antileishmanial activity. Amongst these, euglobal G2 (3a), G3 (4a) and analogues 13a and 14a showed potent antileishmanial activity with IC50s ranging from 2.8 to 3.9 microg/mL. Analogue 16a possessed antimalarial activity against chloroquine sensitive D6 clone of Plasmodium falciparum. None of the compounds showed toxicity against mammalian kidney fibroblasts (vero cells) upto the concentration of 4.76 microg/ml.

Aromatic Constituents of Uvaria Grandiflora

Grandiuvarone A (1) and grandiuvarins A-C (2-4) were isolated from the bark of Uvaria grandiflora. The structures of these new aromatic compounds were elucidated on the basis of spectroscopic analyses, especially 2D NMR techniques. Only compound 1 exhibited antileishmanial activity, with IC(50)/IC(90) values of 0.7/1.5 microg/mL. The positive controls pentamidine and amphotericin B had IC(50)/IC(90) values of 1.6/6.6 and 0.17/0.34 microg/mL, respectively.

Manzamine B and E and Ircinal A Related Alkaloids from an Indonesian Acanthostrongylophora Sponge and Their Activity Against Infectious, Tropical Parasitic, and Alzheimer's Diseases

Four new manzamine-type alkaloids, 12,28-oxamanzamine E (2), 12,34-oxa-6-hydroxymanzamine E (3), 8-hydroxymanzamine B (5), and 12,28-oxaircinal A (11), were isolated from three collections of an Indonesian sponge of the genus Acanthostrongylophora together with 13 known manzamine alkaloids, ircinal A, ircinol A, xestomanzamine A, manzamines A, E, F, J, and Y, manadomanzamines A and B, neo-kauluamine, 8-hydroxymanzamine A, and manzamine A N-oxide. The structures of the new compounds were elucidated by means of 1D and 2D NMR spectroscopic methods. Three of these compounds (2, 3, and 11) possess a unique manzamine-type aminal ring system generated through an ether linkage between carbons 12-28 or between carbons 12-34. In the case of manzamine B and related metabolites, carbons 11 and 12 of the typical manzamine structure have an epoxide group and add to our growing understanding of manzamine structure-activity relationships (SAR) and metabolism. The bioactivity and SAR for a number of previously reported manzamine-related metabolites against malaria, leishmania, tuberculosis, and HIV-1 are also presented. Manzamine Y (9) showed significant inhibitory activity of GSK3, an enzyme implicated in Alzheimer's disease pathology. The toxicity of manzamine A and neo-kauluamine was evaluated against both medaka fry and eggs.

Anti-plasmodial and Anti-leishmanial Activity of Conformationally Restricted Pentamidine Congeners

A library of 52 pentamidine congeners in which the flexible pentyldioxy linker in pentamidine was replaced with various restricted linkers was tested for in-vitro activity against two Plasmodium falciparum strains and Leishmania donovani. The tested compounds were generally more effective against P. falciparum than L. donovani. The most active compounds against the chloroquine-sensitive (D6, Sierra Leone) and -resistant (W2, Indochina) strains of P. falciparum were bisbenzamidines linked with a 1,4-piperazinediyl or 1, 4-homopiperazinediyl moiety, with IC50 values (50% inhibitory concentration, inhibiting parasite growth by 50% in relation to drug-free control) as low as 7 nM based on the parasite lactate dehydrogenase assay. Seven piperazine-linked bisbenzamidines substituted at the amidinium nitrogens with a linear alkyl group of 3-6 carbons (22, 25, 27, 31) or cycloalkyl group of 4, 6 or 7 carbons (26, 32, 34) were more potent (IC50<40 nM) than chloroquine or pentamidine as anti-plasmodial agents. The most active anti-leishmanial agents were 4,4'-[1,4-phenylenebis(methyleneoxy)]bisbenzenecarboximidamide (2, IC50 approximately 0.290 microM) and 1,4-bis[4-(1H-benzimidazol-2-yl)phenyl] piperazine (44, IC50 approximately 0.410 microM), which were 10- and 7-fold more potent than pentamidine (IC50 approximately 2.90 microM). Several of the more active anti-plasmodial agents (e.g. 2, 31, 33, 36-38) were also potent anti-leishmanial agents, indicating broad antiprotozoal properties. However, a number of analogues that showed potent anti-plasmodial activity (1, 18, 21, 22, 25-28, 32, 43, 45) were not significantly active against the Leishmania parasite. This indicates differential modes of anti-plasmodial and anti-leishmanial actions for this class of compounds. These compounds provide important structure-activity relationship data for the design of improved chemotherapeutic agents against parasitic infections.

8-Aminoquinolines: Future Role As Antiprotozoal Drugs

This review focuses on recent developments on evaluation of 8-aminoquinoline analogs with broader efficacy and reduced toxicity, which would provide better drugs for treatment of protozoal infections.

A New Library of C-16 Modified Artemisinin Analogs and Evaluation of Their Anti-parasitic Activities

A library of C-16 modified artemisinin analogs was prepared and their antimalarial as well as antileishmanial activities were evaluated. Synthesis of these compounds involved the conversion of artemisinin to its phenol derivatives 7 and 12, and subsequent parallel derivatization by introducing new chemical groups through ester, carbamate, sulfate, phosphate and isourea linkages. Comparison of in vitro antimalarial activities showed that C9-beta artemisinin analogs (8a-f) are more potent than the corresponding C9-alpha diastereomers (9a-f); however, their antileishmanial activities were in the same range. Many of the 10-deoxoartemisinin analogs studied here showed promising antiparasitic activities. For example, compounds 13a-e are approximately three times more active against drug resistant W2 strain of P. falciparum, compared to artemisinin (IC(50), approximately 0.2 - 0.6 nM; cf. artemisinin = 1.6 nM). Further, a number of compounds in this series were notably leishmanicidal, with activities comparable to or better than pentamidine (e.g., 13g and 13j). Detailed in vivo studies involving these active compounds are underway to identify lead candidates for further development.

Antiprotozoal and Antimicrobial Activities of O-alkylated and Formylated Acylphloroglucinols

In the present article, we examined the antileishmanial, antimalarial, antibacterial, and antifungal activities of several newly synthesized O-alkylated phloroglucinol compounds (11-19) which are analogues of the naturally occurring antimalarial compound 1. Analogues 12 and 16 exhibited antileishmanial activity against, Leishmania donovani promastigotes with IC(50)s of 5.3 and 4.2microg/mL, respectively. Naturally occurring monomeric formylated acylphloroglucinol compounds, grandinol (2), jensenone (3), and their analogues (29-37), were also synthesized and evaluated for antileishmanial, antimalarial, antibacterial, and antifungal activities. Amongst these, both grandinol and jensenone showed mild to moderate antibacterial, antifungal, and antileishmanial activities. Jensenone (3) was effective against Candida albicans with an IC(50) of 5.5microg/mL but was ineffective against Cryptococcus neoformans and methicillin-resistant Staphylococcus aureus. Among the analogues, 34 was the most active against C. albicans and C. neoformans with IC(50)s of 2.0 and 2.5microg/mL, respectively, and was fungicidal toward Candida albicans.

Synthesis, Antimalarial, Antileishmanial, Antimicrobial, Cytotoxicity, and Methemoglobin (MetHB) Formation Activities of New 8-quinolinamines

We report the synthesis, in vitro antiprotozoal (against Plasmodium and Leishmania), antimicrobial, cytotoxicity (Vero and MetHb-producing properties), and in vivo antimalarial activities of two series of 8-quinolinamines. N1-{4-[2-(tert-Butyl)-6-methoxy-8-quinolylamino]pentyl}-(2S/2R)-2-aminosubstitutedamides (21-33) and N1-[4-(4-ethyl-6-methoxy-5-pentyloxy-8-quinolylamino)pentyl]-(2S/2R)-2-aminosubstitutedamides (51-63) were synthesized in six steps from 6-methoxy-8-nitroquinoline and 4-methoxy-2-nitro-5-pentyloxyaniline, respectively. Several analogs displayed promising antimalarial activity in vitro against Plasmodium falciparum D6 (chloroquine-sensitive) and W2 (chloroquine-resistant) clones with high selectivity indices versus mammalian cells. The most promising analogs (21-24) also displayed potent antimalarial activity in vivo in a Plasmodium berghei-infected mouse model. Most interestingly, many analogs exhibited promising in vitro antileishmanial activity against Leishmania donovani promastigotes, and antimicrobial activities against a panel of pathogenic bacteria and fungi. Several analogs, notably 21-24, 26-32, and 60, showed less MetHb formation compared to primaquine indicating the potential of these compounds in 8-quinolinamine-based antimalarial drug development.

Synthesis of Isoquinuclidine Analogs of Chloroquine: Antimalarial and Antileishmanial Activity

The isoquinuclidine (2-azabicyclo[2.2.2]octane) ring system may be viewed as a semi-rigid boat form of the piperidine ring and, when properly substituted, a scaffold for rigid analogs of biologically active ethanolamines and propanolamines. It is present in natural products (such as ibogaine and dioscorine) that display interesting pharmacological properties. In this study, we have expanded our continuing efforts to incorporate this ring system in numerous pharmacophores, by designing and synthesizing semirigid analogs of the antimalarial drug chloroquine. The analogs were tested in vitro against Plasmodium falciparum strains and Leishmania donovani promastigote cultures. Compounds 6 and 13 displayed potent antimalarial activity against both chloroquine-susceptible D6 and the -resistant W2 strains of P. falciparum. All analogs also demonstrated significant antileishmanial activity with compounds 6 and 13 again being the most potent. The fact that these compounds are active against both chloroquine-resistant and chloroquine-sensitive strains as well as leishmanial cells makes them promising candidates for drug development.

Design, Synthesis, and Biological Evaluation of Plasmodium Falciparum Lactate Dehydrogenase Inhibitors

Plasmodium falciparum lactate dehydrogenase (pfLDH) is a key enzyme for energy generation of malarial parasites and is a potential antimalarial chemotherapeutic target. It is known that the oxamate moiety, a pyruvate analog, alone shows higher inhibition against pfLDH than human LDHs, suggesting that it can be used for the development of selective inhibitors. Oxamic acid derivatives were designed and synthesized. Derivatives 5 and 7 demonstrated activities against pfLDH with IC50 values of 3.13 and 1.75 muM, respectively, and have 59- and 7-fold selectivity over mammalian LDH, respectively. They also have micromolar range activities against Plasmodium falciparum malate dehydrogenase (pfMDH), which may fill the role of pfLDH when the activity of pfLDH is reduced. Thus, certain members of these oxamic acid derivatives may have dual inhibitory activities against both pfLDH and pfMDH. It is presumed that dual LDH/MDH inhibitors would have enhanced potential as antimalarial drugs.

Growth, Drug Susceptibility, and Gene Expression Profiling of Plasmodium Falciparum Cultured in Medium Supplemented with Human Serum or Lipid-rich Bovine Serum Albumin [corrected]

In vitro cultivation of Plasmodium falciparum has been extremely useful in understanding the biology of the human malaria parasite as well as research on the discovery of new antimalarial drugs and vaccines. A chemically defined serum-free medium supplemented with lipid-rich bovine serum albumin (AlbuMAX I) offers the following advantages over human serum-supplemented media for the in vitro culture of P. falciparum: 1) improved growth profile, with more than a 2-fold higher yield of the parasites at any stage of the growth cycle; 2) suitability for in vitro antimalarial screening, as the parasites grown in AlbuMAX and human serum-supplemented media show similar sensitivity to standard and novel antimalarials as well as natural product extracts in the in vitro drug susceptibility assays; and 3) DNA microarray analysis comparing the global gene expression profile of sorbitol-synchronized P. falciparum trophozoites grown in the 2 different media, indicating minimal differences.

Antileishmanial, Antiplasmodial and Cytotoxic Activities of 12,16-dideoxy Aegyptinone B from Zhumeria Majdae Rech.f. & Wendelbo

The ethanol extract of Zhumeria majdae showed potent antileishmanial and antiplasmodial activity in vitro. Bioactivity guided fractionation of the extract led to the isolation of 12,16-dideoxy aegyptinone B. This compound exhibited potent in vitro antileishmanial activity with an IC(50) of 0.75 microg/mL and a strong antiplasmodial activity with IC(50) values of 1.3 and 1.4 microg/mL against chloroquine sensitive and resistant strains, respectively. This compound was further found to have mild cytotoxicity towards cancer cell lines.

S-Euglobals: Biomimetic Synthesis, Antileishmanial, Antimalarial, and Antimicrobial Activities

Several new euglobal analogues (named as S-euglobals) were synthesized from phloroglucinol via a biomimetic three-component reaction involving Knoevenagel condensation followed by [4+2]-Diels-Alder cycloaddition with monoterpene. Newly synthesized euglobal analogues involve monoterpenes that have not yet been encountered in natural euglobals. S-Euglobals along with previously synthesized robustadial A and B were evaluated for in vitro antileishmanial, antimalarial, antimicrobial, and cytotoxic activities. Out of 16, nine analogues were found to exhibit antileishmanial activity against Leishmania donovani promastigotes. Analogue 7 was the most potent with IC(50) of 2.4 microg/mL and IC(90) of 8 microg/mL, followed by analogues 8 and 11 (IC(50) 5.5 and 9.5 microg/mL). Antileishmanial activity of robustadial A (5) and B (6) was moderate with IC(50) of 20 and 16 microg/mL, respectively. Robustadial A and B and S-euglobal 8 exhibited weak antimalarial activity against Plasmodium falciparum (IC(50) of 2.7-4.76 microg/mL). Few of the euglobal analogues showed antibacterial activity against methicillin-resistant Staphylococcus aureus. Amongst these, analogue 11 was the most potent with IC(50) of 1.0 microg/mL and MIC of 5.0 microg/mL. Most of the compounds were not cytotoxic up to 25 microg/mL in a panel of cell lines consisting of both cancer (SK-MEL, KB, BT-549, and SK-OV-3) as well as non-cancer kidney (Vero and LLC-PK11) cells.

Structural Insights into the Plasmodium Falciparum Histone Deacetylase 1 (PfHDAC-1): A Novel Target for the Development of Antimalarial Therapy

The histone deacetylase (HDAC) enzyme from Plasmodium falciparum has been identified as a novel target for the development of antimalarial therapy. A ligand-refined homology model of PfHDAC-1 was generated from the crystal structures of human HDAC8 and HDLP using a restraint guided optimization procedure involving the OPLS/GBSA potential setup. The model was extensively validated using protein structure checking tools. A predictive docking study was carried out using a set of known human HDAC inhibitors, which were shown to have in vitro antimalarial activity against the chloroquine sensitive D6 and resistant W2 strains of P. falciparum. Pose validation and score-based active/inactive separation studies provided independent validation of the geometric accuracy and the predictive ability of the generated model. Comparative analysis was carried out with the human HDACs to identify differences in the binding site topology and interacting residues, which might be utilized to develop selective PfHDAC-1 inhibitors.

Novel Bisbenzimidazoles with Antileishmanial Effectiveness

A small library of 2,2'-[(alpha,omega-alkanediylbis(oxyphenylene)]bis-1H-benzimidazoles has been prepared and screened in vitro against Pneumocystis carinii, Trypanosoma brucei rhodesiense, and Leishmania donovani. Among the six tested compounds two derivatives emerged as promising hits characterized by IC(50) values lower than that determined for pentamidine against L. donovani.

Probing the Structures of Leishmanial Farnesyl Pyrophosphate Synthases: Homology Modeling and Docking Studies

Leishmania donovani and Leishmania major farnesyl pyrophosphate synthase ( LdFPPS and LmFPPS) are potential targets for the development of antileishmanial therapy. The protein sequence for LdFPPS was recently elucidated in our laboratory. Highly refined homology models were generated using the protein sequences of LdFPPS and the closely related LmFPPS enzyme. A ligand-refined model of LmFPPS with a bound bisphosphonate ligand was generated using restraint-guided molecular mechanics followed by quantum mechanics/molecular mechanics refinement. The ligand-refined model of LmFPPS was further validated through extensive pose validation, enrichment, and other docking studies involving known bisphosphonate inhibitors. The model was able to explain the critical binding site interactions and site-directed mutagenesis data obtained from experimental studies on related FPPS enzymes. The ligand-refined model in conjunction with the validated docking protocol could be utilized in the future for structure-based virtual screening and rational drug design studies against these targets.

Structural Analysis of Farnesyl Pyrophosphate Synthase from Parasitic Protozoa, a Potential Chemotherapeutic Target

Synthesis of farnesyl pyrophosphate (FPP), a key intermediate of the isoprenoid biosynthesis pathway, is catalyzed by FPP synthase (FPPS). Antiprotozoal properties of bisphosphonates, which target FPPS, have generated interest in FPPS as a potential antiprotozoal drug target. The genes encoding FPPS from parasitic protozoa were assessed to analyze structural and functional features of the enzyme. Comparisons of the FPPS from the parasitic protozoa and search for conserved motifs revealed that FPPS from both apicomplexan and trypanosomatid parasites show characteristic conserved regions for example first aspartate rich motif (FARM) contained within II conserved domain and the second aspartate rich motif (SARM) contained within VI conserved domain. Phylogenetic analysis of FPPS generated a tree with three distinct clusters. Overall topology of the phylogenic tree constructed with small subunit ribosomal RNA sequences was almost similar to that constructed with FPPS sequences. Comparative homology modeling and structural comparisons of FPPS from the parasitic protozoa provided significant insights into common and distinct characteristics of the enzyme. The critical interacting residues of the isopentenyl pyrophosphate binding site are conserved across the enzymes from the family except for malarial FPPS where the C-terminal residues from the BXB motif of helix J were missing. Variations noticed in aromatic residue pairs at the fourth and fifth position upstream of the FARM, which play important role in determination of chain length of the polyprenyl products, may produce functional differences among protozoan FPPSs. The structural comparison of protozoan FPPS may be useful in designing common or selective FPPS inhibitors as potential broad spectrum or selective antiprotozoal agents.

Infectious Disorders Drug Targets--an Arsenal of Knowledge on Pathogen Targets

Antileishmanial Activity Screening of 5-nitro-2-heterocyclic Benzylidene Hydrazides

A series of 53 nitro derivatives rationally designed were obtained by parallel synthesis and screened against Leishmania donovani. Six compounds exhibited IC(50) values lower than standard drugs. Brief SAR analysis revealed that substitution is important to the activity. Nitrothiophene analogues were more potent than the nitrofuran ones. This was attributed to the ability of sulfur atoms in accommodating electrons from nitro group, which facilitate its reduction and therefore the formation of free radicals lethal to parasites.

Kinetic Studies and Bioactivity of Potential Manzamine Prodrugs

The manzamines represent a class of marine natural products that show considerable promise in the control of malaria but generate GI distress in rodents when administered orally in high doses. In an effort to generate manzamine prodrugs with improved antimalarial activity and reduced GI toxicity, we prepared acetylated 8-hydroxymanzamine A analogues including 8-acetoxymanzamine A (3) and 8,12-diacetoxymanzamine A (4), and 8-methoxymanzamine A (5) beginning with 8-hydroxymanzamine A (2). The semisynthetic analogues were assayed for antimalarial and antimicrobial activities, cytotoxicity, and biological and chemical stability. Due to gradual hydrolysis of the ester group, application of monoacetate 3 as an antimalarial prodrug was investigated. The in vitro and in vivo bioassays show that acetylated analogues exhibit significant antimalarial activity (IC50( 3) 9.6-30 ng/mL), which are comparable to the parent molecule; however the monoaceate 3 was shown to actually produce higher toxicity at 30 mg/kg when administered orally.

Current Antiparasitic Drug Targets and a Paradigm Shift in Discovery of New Antiparasitic Drugs and Vaccines

Plasmodium Falciparum Serine/threonine Phoshoprotein Phosphatases (PPP): from Housekeeper to the 'holy Grail'

Availability of complete genome sequence for Plasmodium falciparum has been useful in drawing a comprehensive metabolic map of the parasite. Distinct and unique metabolic characteristics of the parasite may be exploited as potential targets for new antimalarial drug discovery research. Reversible phosphorylation of proteins is a ubiquitous process and an indispensable part of cell signaling cascades, which regulate different cellular functions. Not so long ago the role of protein phosphatases in the cell life was underestimated but now these enzymes strongly focus attention of many researches. Based on primary structure and functional characteristics protein phosphatases have been divided into number of families and subfamilies. The amino acid sequences of catalytic subunits of protein phosphatases of particular families stay highly conserved in eukaryotic organisms during evolutionary changes. Serine/threonine protein phosphatases (PPPs) constitute an important family, which are involved in mitotic and meiotic cell divisions, cell development, apoptosis and many other crucial cellular processes. Complex life cycle of the malaria parasite, which encompasses through distinct developmental stages, offers highly sophistical roles for the protein phosphatases. We have researched and analyzed characteristics of 17 putative or/and confirmed catalytic subunits of PPPs on P. falciparum genome. Evidences have been gathered that indicate functional expression of some PPP isoforms in P. falciparum. A few of them have been found to be essential or play important cellular functions in the parasite. Identification of distinct molecular and functional characteristics of these enzymes shall be useful in designing selective inhibitors of plasmodial PPPs as potential new antimalarials.

Antiparasitic, Nematicidal and Antifouling Constituents from Juniperus Berries

A bioassay-guided fractionation of Juniperus procera berries yielded antiparasitic, nematicidal and antifouling constituents, including a wide range of known abietane, pimarane and labdane diterpenes. Among these, abieta-7,13-diene (1) demonstrated in vitro antimalarial activity against Plasmodium falciparum D6 and W2 strains (IC(50) = 1.9 and 2.0 microg/mL, respectively), while totarol (6), ferruginol (7) and 7beta-hydroxyabieta-8,13-diene-11,12-dione (8) inhibited Leishmania donovani promastigotes with IC(50) values of 3.5-4.6 microg/mL. In addition, totarol demonstrated nematicidal and antifouling activities against Caenorhabditis elegans and Artemia salina at a concentration of 80 microg/mL and 1 microg/mL, respectively. The resinous exudate of J. virginiana afforded known antibacterial E-communic acid (4) and 4-epi-abietic acid (5), while the volatile oil from its trunk wood revealed large quantities of cedrol (9). Using GC/MS, the two known abietanes totarol (6) and ferruginol (7) were identified from the berries of J. procera, J. excelsa and J. phoenicea.

Indolizidine, Antiinfective and Antiparasitic Compounds from Prosopis Glandulosa Var. Glandulosa

A new potent antiinfective and antiparasitic 2,3-dihydro-1H-indolizinium chloride (1) was isolated from Prosopis glandulosa var. glandulosa. Three additional new (2-4) and one known (5) indolizidines were also isolated, and the dihydrochloride salts of 1-3 (compounds 6, 7, and 8) were prepared. Structures were determined by 1D and 2D NMR and mass spectra. Compound 1 showed potent in vitro antifungal activity against Cryptococcus neoformans and Aspergillus fumigatus (IC(50) values = 0.4 and 3.0 microg/mL, respectively) and antibacterial activity against methicillin-resistant Staphylococcus aureus and Mycobacterium intracellulare (IC(50) values of 0.35 and 0.9 microg/mL, respectively). The remarkable in vitro fungicidal activity of 1-4 against C. neoformans (MFCs = 0.63-1.25 microg/mL) and 2, 3, and 5 against A. fumigatus (MFCs = 0.63-2.5 microg/mL) were similar to amphotericin B, but >2-4-fold more potent than 6-8. Prosopilosidine (1) showed potent in vivo activity at 0.0625 mg/kg/day/ip for 5 days in a murine model of cryptococcosis by eliminating approximately 76% of C. neoformans infection from brain tissue compared to approximately 83% with amphotericin B at 1.5 mg/kg/day. Compounds 1 and 4 exhibited potent activity and high selectivity index (SI) values against chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of Plasmodium falciparum, with IC(50) values of 39 and 95 ng/mL and 42 and 120 ng/mL, respectively (chloroquine, IC(50) = 17 and 140 ng/mL). Prosopilosine (1) also showed in vivo antimalarial activity, with an ED(50) value of approximately 2 mg/kg/day/ip against Plasmodium berghei-infected mice after 3 days of treatment.

Synthesis and Antimalarial Activities of Cyclen 4-aminoquinoline Analogs

In an attempt to augment the efficacy of 7-chloro 4-aminoquinoline analogs and also to overcome resistance to antimalarial agents, we synthesized three cyclen (1,4,7,10-tetraazacyclododecane) analogs of chloroquine [a bisquinoline derivative, 7-chloro-4-(1,4,7,10-tetraaza-cyclododec-1-yl)-quinoline HBr, and a 7-chloro-4-(1,4,7,10-tetraaza-cyclododec-1-yl)-quinoline-Zn(2+) complex]. The bisquinoline displays the most potent in vitro and in vivo antimalarial activities. It displays 50% inhibitory concentrations (IC(50)s) of 7.5 nM against the D6 (chloroquine-sensitive) clone of Plasmodium falciparum and 19.2 nM against the W2 (chloroquine-resistant) clone, which are comparable to those of artemisinin (10.6 and 5.0 nM, respectively) and lower than those of chloroquine (10.7 and 87.2 nM, respectively), without any evidence of cytotoxicity to mammalian cells, indicating a high selectivity index (>1,333 against D6 clone and >521 against W2 clone). Potent antimalarial activities of the bisquinoline against chloroquine- and mefloquine-resistant strains of P. falciparum were also confirmed by in vitro [(3)H]hypoxanthine incorporation assay. The in vivo antimalarial activity of the bisquinoline, as determined in P. berghei-infected mice, is comparable to that of chloroquine (50% effective dose,

Analysis of Quaternary Structure of a [LDH-like] Malate Dehydrogenase of Plasmodium Falciparum with Oligomeric Mutants

L-Malate dehydrogenase (PfMDH) from Plasmodium falciparum, the causative agent for the most severe form of malaria, has shown remarkable similarities to L: -lactate dehydrogenase (PfLDH). PfMDH is more closely related to [LDH-like] MDHs characterized in archae and other prokaryotes. Initial sequence analysis and identification of critical amino acid residues involved in inter-subunit salt-bridge interactions predict tetrameric structure for PfMDH. The catalytically active recombinant PfMDH was characterized as a tetramer. The enzyme is localized primarily in the parasites cytosol. To gain molecular insights into PfMDH/PfLDH relationships and to understand the quaternary structure of PfMDH, dimers were generated by mutation to the potential salt-bridge interacting sites. The R183A and R214G mutations, which snapped the salt bridges between the dimers and resulted in lower dimeric state, did not affect catalytic properties of the enzyme. The mutant dimers of PfMDH were active equally as the wild-type PfMDH. The studies reveal structure of PfMDH as a dimer of dimers. The tetrameric state of PfMDH was not essential for catalytic functions of the enzyme but may be an evolutionary adaptation for cytosolic localization to support its role in NAD/NADH coupling, an important metabolic function for survival of the malaria parasite.

Bioactive (+)-manzamine A and (+)-8-hydroxymanzamine A Tertiary Bases and Salts from Acanthostrongylophora Ingens and Their Preparations

The genus Acanthostrongylophora is famous for producing a wide array of manzamine alkaloids as natural hydrochloride salts. An examination of A. ingens has now yielded two tertiary bases, (+)-8-hydroxymanzamine A (1) and (+)-manzamine A (2), by chromatography over alumina using CHCl3-MeOH-NH3.H2O as solvent. In addition, (+)-8-hydroxymanzamine A hydrochloride (3) and (+)-manzamine A hydrochloride (4) were isolated under the same conditions from the same source by silica gel chromatography. The structures of 1-4 were determined from 1D- and 2D-NMR spectra and by circular dichroism experiments, and the spectral features of the bases 1 and 2 were found to be different from those of the salts 3 and 4. Compounds 3 and 4 were deprotonated by both A12O3 and strong base to afford 1 and 2, which were converted again to their respective salts 3 and 4. Both the compounds 1 and 3 showed equally potent in vitro antimalarial activity against chloroquine-sensitive (D6) and -resistant (W2) strains of P. falciparum (IC50 = 19.5 and 22.0 ng/mL vs. 27.0 and 36.5 ng/mL, respectively), while 2 was >3-fold less potent than 4 (IC50 = 20.8 and 25.8 ng/mL vs. 6.1 and 7.3 ng/mL, respectively). Compounds 1, 3 and 4 showed good antimicrobial activities against methicillin-resistant Staphylococcus aureus and Mycobacterium intracellulare and antileishmanial activity against Leishmania donovani promastigotes. In contrast, manzamine A base (2) showed relatively weaker antimicrobial, antileishmanial and cytotoxic activities [towards cancer (HepG2: Human hepatocellular carcinoma or hepatoma), and non-cancer cells (VERO: Monkey kidney fibroblast; LLC-PK11: Pig kidney epithelial)], compared with salt 4.

Cytochrome P(450)-dependent Toxic Effects of Primaquine on Human Erythrocytes

Primaquine, an 8-aminoquinoline, is the drug of choice for radical cure of relapsing malaria. Use of primaquine is limited due to its hemotoxicity, particularly in populations with glucose-6-phosphate dehydrogenase deficiency [G6PD(-)]. Biotransformation appears to be central to the anti-infective and hematological toxicities of primaquine, but the mechanisms are still not well understood. Metabolic studies with primaquine have been hampered due to the reactive nature of potential hemotoxic metabolites. An in vitro metabolism-linked hemotoxicity assay has been developed. Co-incubation of the drug with normal or G6PD(-) erythrocytes, microsomes or recombinant cytochrome P(450) (CYP) isoforms has allowed in situ generation of potential hemotoxic metabolite(s), which interact with the erythrocytes to generate hemotoxicity. Methemoglobin formation, real-time generation of reactive oxygen intermediates (ROIs) and depletion of reactive thiols were monitored as multiple biochemical end points for hemotoxicity. Primaquine alone did not produce any hemotoxicity, while a robust increase was observed in methemoglobin formation and generation of ROIs by primaquine in the presence of human or mouse liver microsomes. Multiple CYP isoforms (CYP2E1, CYP2B6, CYP1A2, CYP2D6 and CYP3A4) variably contributed to the hemotoxicity of primaquine. This was further confirmed by significant inhibition of primaquine hemotoxicity by the selective CYP inhibitors, namely thiotepa (CYP2B6), fluoxetine (CYP2D6) and troleandomycin (CYP3A4). Primaquine caused similar methemoglobin formation in G6PD(-) and normal human erythrocytes. However, G6PD(-) erythrocytes suffered higher oxidative stress and depletion of thiols than normal erythrocytes due to primaquine toxicity. The results provide significant insights regarding CYP isoforms contributing to hemotoxicity and may be useful in controlling toxicity of primaquine to increase its therapeutic utility.

Antimicrobial, Antiparasitic and Cytotoxic Spermine Alkaloids from Albizia Schimperiana

Albizia schimperiana Oliv. (Leguminosae) is a tree distributed in the highland of Kenya, where it is used as a traditional medicine for the treatment of bacterial and parasitic infections, notably pneumonia and malaria, respectively. Bioassay guided isolation of the CH2Cl2-MeOH 1:1/ MeOH-H20 9:1 (mixed) extract of A. schimperiana afforded the new bioactive macrocyclic spermine alkaloid, namely 5,14-dimethylbudmunchiamine L1 (1) and three known budmunchiamine analogs 2-4. The structures of the compounds 1-4 were determined by 1D and 2D NMR data, including COSY, HMQC, and HMBC experiments, and ESI-HRMS. Compounds 1 and 3 exhibited significant in vitro antimicrobial activity against a panel of microorganisms, including C neoformans, methicillin-resistant S. aureus, E. coli, M. intracellulare, and A. fumigatus. In Saddition, they demonstrated strong in vitro antimalarial activities against chloroquine-susceptible (D6) and -resistant (W2) strains of Plasmodium falciparum with IC50s ranging from 120-270 ng/mL. Compounds 1-4 were also evaluated for cytotoxic activity against selected human cancer cell lines and mammalian kidney fibroblasts (VERO cells). It was observed that hydroxyl substitution of the side chain of the budmunchiamines dramatically reduced the cytotoxicity and antimicrobial activity of the alkaloids 2 and 4 without decreasing antimalarial activity.

Structure and Function of Plasmodium Falciparum Malate Dehydrogenase: Role of Critical Amino Acids in Co-substrate Binding Pocket

The malaria parasite thrives on anaerobic fermentation of glucose for energy. Earlier studies from our laboratory have demonstrated that a cytosolic malate dehydrogenase (PfMDH) with striking similarity to lactate dehydrogenase (PfLDH) might complement PfLDH function in Plasmodium falciparum. The N-terminal glycine motif, which forms a characteristic Rossman dinucleotide-binding fold in the co-substrate binding pocket, differentiates PfMDH (GlyXGlyXXGly) from other eukaryotic and prokaryotic malate dehydrogenases (GlyXXGlyXXGly). The amino acids lining the co-substrate binding pocket are completely conserved in MDHs from different species of human, primate and rodent malaria parasites. Based on this knowledge and conserved domains among prokaryotic and eukaryotic MDH, the role of critical amino acids lining the co-substrate binding pocket was analyzed in catalytic functions of PfMDH using site-directed mutagenesis. Insertion of Ala at the 9th or 10th position, which converts the N-terminal GlyXGlyXXGly motif (characteristic of malarial MDH and LDH) to GlyXXGlyXXGly (as in bacterial and eukaryotic MDH), uncoupled regulation of the enzyme through substrate inhibition. The dinucleotide fold GlyXGlyXXGly motif seems not to be responsible for the distinct affinity of PfMDH to 3-acetylpyridine-adenine dinucleotide (APAD, a synthetic analog of NAD), since Ala9 and Ala10 insertion mutants still utilized APADH. The Gln11Met mutation, which converts the signature glycine motif in PfMDH to that of PfLDH, did not change the enzyme function. However, the Gln11Gly mutant showed approximately a 5-fold increase in catalytic activity, and higher susceptibility to inhibition with gossypol. Asn119 and His174 participate in binding of both co-substrate and substrate. The Asn119Gly mutant exhibited approximately a 3-fold decrease in catalytic efficiency, while mutation of His174 to Asn or Ala resulted in an inactive enzyme. These studies provide critical insights into the co-substrate binding pocket of PfMDH, which may be important in design of selective PfMDH/PfLDH inhibitors as potential antimalarials.

Structure-activity Relationship Studies of Manzamine A: Amidation of Positions 6 and 8 of the Beta-carboline Moiety

Twenty manzamine amides were synthesized and evaluated for in vitro antimalarial and antimicrobial activities. The amides of manzamine A (1) showed significantly reduced cytotoxicity against Vero cells, although were less active than 1. The structure-activity analysis showed that linear, short alkyl groups adjacent to the amide carbonyl at position 8 are favored for antimalarial activity, while bulky and cyclic groups at position 6 provided the most active amides. Most of the amides showed potent activity against Mycobacterium intracellulare. The antimicrobial activity profile for position 8 series was similar to that for antimalarial activity profile, in which linear, slightly short alkyl groups adjacent to the amide carbonyl showed improved activity. Two amides 14 and 21, which showed potent antimalarial activity in vitro against Plasmodium falciparum were further evaluated in vivo in Plasmodium berghei infected mice. Oral administration of 14 and 21 at the dose of 30mg/kg (once daily for three days) caused parasitemia suppression of 24% and 62%, respectively, with no apparent toxicity.

Antiprotozoal, Anticancer and Antimicrobial Activities of Dihydroartemisinin Acetal Dimers and Monomers

Nine dihydroartemisinin acetal dimers (6-14) with diversely functionalized linker units were synthesized and tested for in vitro antiprotozoal, anticancer and antimicrobial activity. Compounds 6, 7 and 11 [IC(50): 3.0-6.7 nM (D6) and 4.2-5.9 nM (W2)] were appreciably more active than artemisinin (1) [IC(50): 32.9 nM (D6) and 42.5 nM (W2)] against the chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of the malaria parasite, Plasmodium falciparum. Compounds 10, 13 and 14 displayed enhanced anticancer activity in a number of cell lines compared to the control drug, doxorubicin. The antifungal activity of 7 and 12 against Cryptococcus neoformans (IC(50): 0.16 and 0.55 microM, respectively) was also higher compared to the control drug, amphotericin B. The antileishmanial and antibacterial activities were marginal. A number of dihydroartemisinin acetal monomers (15-17) and a trimer (18) were isolated as byproducts from the dimer synthesis and were also tested for biological activity.

Biologically Active Tetranorditerpenoids from the Fungus Sclerotinia Homoeocarpa Causal Agent of Dollar Spot in Turfgrass

Nine new tetranorditerpenoid dilactones (2-10), together with two previously reported norditerpenoids dilactones (1, 11), and two known putative biosynthetic intermediates, oidiolactone-E (12) and 13, were isolated from an ethyl acetate extract of a culture medium of Sclerotinia homoeocarpa. Structures and absolute configurations of these compounds were determined by spectroscopic methods and confirmed by X-ray crystallographic analysis of representative compounds. Compounds were evaluated for herbicidal, antiplasmodial, and cytotoxic activities. Compounds 1, 2, 6, 7, and 11 were more active as growth inhibitors in a duckweed bioassay (I(50) values of 0.39-0.95 microM) than more than half of 26 commercial herbicides previously evaluated using the same bioassay. Some of these compounds exhibited strong antiplasmodial activities as well, but they also had cytotoxic activity, thus precluding them as potential antimalarial agents.

Banisteriopsis Caapi, a Unique Combination of MAO Inhibitory and Antioxidative Constituents for the Activities Relevant to Neurodegenerative Disorders and Parkinson's Disease

Parkinson's disease is a neurological disorder mostly effecting the elder population of the world. Currently there is no definitive treatment or cure for this disease. Therefore, in this study the composition and constituents of the aqueous extract of Banisteriopsis caapi for monoamine oxidases (MAO) inhibitory and antioxidant activities were assessed, which are relevant to the prevention of neurological disorders, including Parkinsonism.

Antimalarial and Antileishmanial Activities of Histone Deacetylase Inhibitors with Triazole-linked Cap Group

Histone deacetylase inhibitors (HDACi) are endowed with plethora of biological functions including anti-proliferative, anti-inflammatory, anti-parasitic, and cognition-enhancing activities. Parsing the structure-activity relationship (SAR) for each disease condition is vital for long-term therapeutic applications of HDACi. We report in the present study specific cap group substitution patterns and spacer-group chain lengths that enhance the antimalarial and antileishmanial activity of aryltriazolylhydroxamates-based HDACi. We identified many compounds that are several folds selectively cytotoxic to the plasmodium parasites compared to standard HDACi. Also, a few of these compounds have antileishmanial activity that rivals that of miltefosine, the only currently available oral agent against visceral leishmaniasis. The anti-parasite properties of several of these compounds tracked well with their anti-HDAC activities. The results presented here provide further evidence on the suitability of HDAC inhibition as a viable therapeutic option to curb infections caused by apicomplexan protozoans and trypanosomatids.

Cytochrome P450-dependent Toxicity of Dapsone in Human Erythrocytes

The most prominent adverse effects seen during treatment with dapsone, an antibacterial and antiprotozoal agent, are hemolysis and methemoglobinemia. An in vitro microsomal/cytochrome P(450) (CYP)-linked assay, which allows reactive metabolites generated in situ to react with the co-incubated human erythrocytes, was employed to profile CYP isoforms responsible for hemotoxicity of dapsone. Dapsone caused a robust generation of methemoglobin in human erythrocytes in the presence of human/mouse liver microsomes, which indicates contribution of CYP-mediated metabolism for hemotoxicity. The highest methemoglobin formation with dapsone was observed with CYP2C19, with minor contributions from CYP2B6, CYP2D6 and CYP3A4. Cimetidine and chloramphenicol completely abrogated methemoglobin generation by dapsone, thus confirming a predominant contribution of CYP2C19. The results provide useful insights into CYP-dependent hemotoxicity of dapsone in human erythrocytes.

Structure-activity Relationship and Mechanism of Action Studies of Manzamine Analogues for the Control of Neuroinflammation and Cerebral Infections

Structure-activity relationship studies were carried out by chemical modification of manzamine A (1), 8-hydroxymanzamine A (2), manzamine F (14), and ircinal isolated from the sponge Acanthostrongylophora. The derived analogues were evaluated for antimalarial, antimicrobial, and antineuroinflammatory activities. Several modified products exhibited potent and improved in vitro antineuroinflammatory, antimicrobial, and antimalarial activity. 1 showed improved activity against malaria compared to chloroquine in both multi- and single-dose in vivo experiments. The significant antimalarial potential was revealed by a 100% cure rate of malaria in mice with one administration of 100 mg/kg of 1. The potent antineuroinflammatory activity of the manzamines will provide great benefit for the prevention and treatment of cerebral infections (e.g., Cryptococcus and Plasmodium). In addition, 1 was shown to permeate across the blood-brain barrier (BBB) in an in vitro model using a MDR-MDCK monolayer. Docking studies support that 2 binds to the ATP-noncompetitive pocket of glycogen synthesis kinase-3beta (GSK-3beta), which is a putative target of manzamines. On the basis of the results presented here, it will be possible to initiate rational drug design efforts around this natural product scaffold for the treatment of several different diseases.

Composition, Standardization and Chemical Profiling of Banisteriopsis Caapi, a Plant for the Treatment of Neurodegenerative Disorders Relevant to Parkinson's Disease

Banisteriopsis caapi, a woody vine from the Amazonian basin, is popularly known as an ingredient of a sacred drink ayahuasca, widely used throughout the Amazon as a medicinal tea for healing and spiritual exploration. The usefulness of Banisteriopsis caapi has been established for alleviating symptoms of neurological disorders including Parkinson's disease.

Anti-infective Discorhabdins from a Deep-water Alaskan Sponge of the Genus Latrunculia

Bioassay- and LC-MS-guided fractionation of a methanol extract from a new deep-water Alaskan sponge species of the genus Latrunculia resulted in the isolation of two new brominated pyrroloiminoquinones, dihydrodiscorhabdin B and discorhabdin Y (2), along with six known pyrroloiminoquinone alkaloids, discorhabdins A (3), C (4), E (5), and L (6), dihydrodiscorhabdin C (7), and the benzene derivative 8. Compounds 3, 4, and 7 exhibited anti-HCV activity, antimalarial activity, and selective antimicrobial activity. Although compounds 3 and 7 displayed potent and selective in vitro antiprotozoal activity, Plasmodium berghei-infected mice did not respond to these metabolites due to their toxicity in vivo.

A Structure-activity Relationship Study of the Antimalarial and Antileishmanial Activities of Nonpeptide Macrocyclic Histone Deacetylase Inhibitors

Antimicrobial and Antiparasitic Abietane Diterpenoids from the Roots of Clerodendrum Eriophyllum

Chromatographic separation of the roots of a Kenyan medicinal plant, Clerodendrum eriophyllum, led to the isolation of ten abietane diterpenoids (1-10), one of which (1) was isolated for the first time from a natural source. Using spectroscopic data, the structure of 1 was determined to be 12-hydroxy-8,12-abietadiene-3,11,14-trione. Circular dichroism (CD) spectra showed that the stereochemistry of compounds 1, 3, and 6-8 belongs to the normal series of abietane diterpenes, which confirmed the absolute stereochemistry of the isolated compounds. Compounds 1-10 were evaluated for their in vitro antiplasmodial, antileishmanial, antifungal and antibacterial activities. Compounds 3 and 7 exhibited potent antifungal activity (IC50/MIC 0.58/1.25 and 0.96/2.5 microg/mL, respectively) against C. neoformans, whereas 3, 6 and 7 showed strong antibacterial activity against Staphylococcus aureus and methicillin-resistant S. aureus with IC50/MIC values between 1.33-1.75/2.5-5 and 0.96-1.56/2.5 microg/mL, respectively. In addition, compounds 3 and 9 exhibited potent antileishmanial activity (IC50 0.08 and 0.20 microg/mL, respectively) against L. donovani, while 3 and 7 displayed weak antimalarial activity against Plasmodium falciparum, but 9 was inactive.

Non-peptide Macrocyclic Histone Deacetylase Inhibitors Derived from Tricyclic Ketolide Skeleton

Inhibition of histone deacetylase (HDAC) function is a validated therapeutic strategy for cancer treatment. Of the several structurally distinct small molecule histone deacetylase inhibitors (HDACi) reported, macrocyclic depsipeptides possess the most complex cap groups and have demonstrated excellent HDAC inhibition potency and isoform selectivity. Unfortunately, the development of macrocyclic depsipeptides has been hampered in part because of development problems characteristic of large peptides and the complex reaction schemes required for their synthesis. Herein we report that tricyclic ketolide TE-802 is an excellent mimetic for the peptide backbone of macrocyclic HDACi. Compounds derived from this template are particularly selective against HDACs 1 and 2 with nanomolar inhibitory activity. Interrogation of the association between a subset of these compounds and key HDAC isoforms, using AutoDock, enables a molecular description of the interaction between the HDAC enzyme's outer rim and the inhibitors' macrocyclic cap group that are responsible for compound affinity and presumably isoform selectivity.

The Antiplasmodial Activity of Norcantharidin Analogs

The antiplasmodial activities of sixty norcantharidin analogs were tested in vitro against a chloroquine sensitive (D6, Sierra Leone) and chloroquine resistant (W2) strains of Plasmodium falciparum. Forty analogs returned IC(50) values <500 μM against at least one of the P. falciparum strains examined. The ring open compound 24 ((1S,4R)-3-(allylcarbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid) is the most active aliphatic analog (D6 IC(50)=3.0±0.0 and W2 IC(50)=3.0±0.8 μM) with a 20-fold enhancement relative to norcantharidin. Surprisingly, seven norcantharimides also displayed good antiplasmodial activity with the most potent, 5 returning D6=8.9±0.9 and W2 IC(50)=12.5±2.2 μM, representing a fivefold enhancement over norcantharidin.

Analysis of Primaquine and Its Metabolite Carboxyprimaquine in Biological Samples: Enantiomeric Separation, Method Validation and Quantification

The clinical formulation of primaquine (PQ) is a mixture of (-)-(R)- and (+)-(S)- primaquine enantiomers which may show different pharmacokinetic and pharmacodynamic properties. To assess the efficacy and toxicity of primaquine enantiomers, a method using LC-MSD-TOF has been developed. The enantiomers were well separated using a Chiralcel OD column (250 × 4.6 mm, 10 µm) with a linear gradient of mobile phase consisting of acetonitrile (0.1% formic acid) and aqueous ammonium formate (20 mm; 0.1% formic acid) adjusted to pH 5.9 at a flow rate of 0.7 mL/min. The method was validated for linearity, precision, accuracy and limits of detection and quantification. The calibration curves were linear with all correlation coefficients being >0.999. The average recoveries of (-)-(R)- and (+)-(S)-primaquine and (-)-(R)- and (+)-(S)-carboxyprimaquine were 88 and 92%, respectively, in spiked human plasma and 89 and 93% respectively in spiked mouse plasma samples. The RSD of (-)-(R)- and (+)-(S)-primaquine and (-)-(R)- and (+)-(S)-carboxyprimaquine were 2.15, 1.74, 1.73 and 2.31, respectively, in spiked human plasma and 2.21, 1.09, 1.95 and 1.17% in spiked mouse plasma, respectively. The intra-day and inter-day precisions expressed as RSD were lower than 10% in all analyzed quality control levels. The method as reported is suitable for study of the pharmacokinetic and pharmacodynamic properties of the enantiomers of primaquine. The method was successfully applied to study plasma pharmacokinetic profile of enantiomers of primaquine and carboxyprimaquine in mice administered with primaquine in racemic form. The analytical method was found to be linear, accurate, precise and specific.

Synthesis, Antiprotozoal, Antimicrobial, β-hematin Inhibition, Cytotoxicity and Methemoglobin (MetHb) Formation Activities of Bis(8-aminoquinolines)

In continuing our search of potent antimalarials based on 8-aminoquinoline structural framework, three series of novel bis(8-aminoquinolines) using convenient one to four steps synthetic procedures were synthesized. The bisquinolines were evaluated for in vitro antimalarial (Plasmodiumfalciparum), antileishmanial (Leishmaniadonovani), antimicrobial (a panel of pathogenic bacteria and fungi), cytotoxicity, β-hematin inhibitory and methemoglobin (MetHb) formation activities. Several compounds exhibited superior antimalarial activities compared to parent drug primaquine. Selected compounds (44, 61 and 79) when tested for in vivo blood-schizontocidal antimalarial activity (Plasmodiumberghei) displayed potent blood-schizontocial activities. The bisquinolines showed negligible MetHb formation (0.2-1.2%) underlining their potential in the treatment of glucose-6-phosphate dehydrogenase deficient patients. The bisquinoline analogues (36, 73 and 79) also exhibited promising in vitro antileishmanial activity, and antimicrobial activities (43, 44 and 76) against a panel of pathogenic bacteria and fungi. The results of this study provide evidence that bis(8-aminoquinolines), like their bis(4-aminoquinolines) and artemisinin dimers counterparts, are a promising class of antimalarial agents.

Antiparasitic and Antimicrobial Indolizidines from the Leaves of Prosopis Glandulosa Var. Glandulosa

A new indolizidine alkaloid, named Δ¹,⁶-juliprosopine (1), together with previously known indolizidine analogs (2- 6), was isolated from the leaves of Prosopis glandulosa var. glandulosa, collected from Nevada, USA; while two other known indolizidines, juliprosopine (6) and juliprosine (7), were isolated from P. glandulosa leaves collected in Texas, USA. The structures of compound 1 and 7 were determined using a combination of NMR and MS techniques. Compound 7 exhibited potent antiplasmodial activity against Plasmodium falciparum D6 and W2 strains with IC (50) values of 170 and 150 ng/mL, respectively, while 1 was found to be less active (IC₅₀ values 560 and 600 ng/mL, respectively). Both compounds were devoid of VERO cells toxicity up to a concentration of 23 800 ng/mL. The antileishmanial activity of indolizidines was evaluated against Leishmania donovani promastigotes, axenic amastigotes, and amastigotes in THP1 macrophage cultures. When tested against macrophage cultures, the tertiary bases (1, 3, 6) were found to be more potent than quaternary salts (2, 5, 7), displaying IC₅₀ values between 0.8-1.7 µg/mL and 3.1-6.0 µg/mL, respectively. In addition, compound 7 showed potent antifungal activity against Cryptococcus neoformans and antibacterial activity against Mycobacterium intracellulare, while 1 was potent only against C. neoformans and weakly active against other organisms.

Identification of Novel Malarial Cysteine Protease Inhibitors Using Structure-based Virtual Screening of a Focused Cysteine Protease Inhibitor Library

Malaria, in particular that caused by Plasmodium falciparum , is prevalent across the tropics, and its medicinal control is limited by widespread drug resistance. Cysteine proteases of P. falciparum , falcipain-2 (FP-2) and falcipain-3 (FP-3), are major hemoglobinases, validated as potential antimalarial drug targets. Structure-based virtual screening of a focused cysteine protease inhibitor library built with soft rather than hard electrophiles was performed against an X-ray crystal structure of FP-2 using the Glide docking program. An enrichment study was performed to select a suitable scoring function and to retrieve potential candidates against FP-2 from a large chemical database. Biological evaluation of 50 selected compounds identified 21 diverse nonpeptidic inhibitors of FP-2 with a hit rate of 42%. Atomic Fukui indices were used to predict the most electrophilic center and its electrophilicity in the identified hits. Comparison of predicted electrophilicity of electrophiles in identified hits with those in known irreversible inhibitors suggested the soft-nature of electrophiles in the selected target compounds. The present study highlights the importance of focused libraries and enrichment studies in structure-based virtual screening. In addition, few compounds were screened against homologous human cysteine proteases for selectivity analysis. Further evaluation of structure-activity relationships around these nonpeptidic scaffolds could help in the development of selective leads for antimalarial chemotherapy.

An Entry to Curcuphenol/elvirol Core Structures Via a Retro-Aldol Reaction

Analogs of curcuphenol/elvirol, naturally occurring bisabolane sesquiterpenes, were prepared in six steps from alkyl-α-tetralones employing an aromatization reaction of cyclic dienone precursors and olefination of the key aldehyde intermediates. The in vitro antifungal activities of 6a, 6b, 6d, and 6g are also reported.

Antiparasitic and Antimicrobial Isoflavanquinones from Abrus Schimperi

The EtOH extract of Abrus schimperi (Fabaceae), collected in Kenya, demonstrated significant activity against Leishmania donovani promastigotes with IC50 value of 3.6 microg/mL. Bioassay-guided fractionation of CHCl3 fraction using Centrifugal Preparative TLC afforded two antiparasitic isoflavanquinones, namely amorphaquinone (1) and pendulone (2). They displayed IC50 values of 0.63 microg/mL and 0.43 microg/mL, respectively, against L. donovani promastigotes. Both the compounds were also evaluated against L. donovani axenic amastigotes and amastigotes in THPI macrophage cultures. In addition, compounds 1 and 2 showed antiplasmodial activity against Plasmodium falciparum D6 and W2 strains, while 2 displayed antibacterial activity against Staphylococcus aureus and methicillin-resistant S. aureus (each IC50 1.44 microg/mL). The 1H and 13C data of 1, not fully assigned previously, were unambiguously assigned using 1D and 2D NMR HMBC and HMQC experiments. In addition, the absolute stereochemistry of the isolated compounds 1 and 2 was revised as C-(3S) based on Circular Dichroism experiments. This appears to be the first report of amorphaquinone (1) and pendulone (2) from the genus Abrus.

Synthesis and Antikinetoplastid Activity of a Series of N,N'-substituted Diamines

A series of 25 N,N'-substituted diamines were prepared by controlled reductive amination of free aliphatic diamines with different substituted benzaldehydes. The library was screened in vitro for antiparasitic activity on the causative agents of human African trypanosomiasis, Chagas' disease and visceral leishmaniasis. The most potent compounds were derived from a subset of diamines that contained a 4-OBn substitution, having a 50% parasite growth inhibition in the submicromolar (against Trypanosoma cruzi) or nanomolar (against Trypanosoma brucei and Leishmania donovani) range. We conclude that members of this series of N,N'-substituted diamines provide new lead structures that have potential to treat trypanosomal and leishmanial infections.

Inhibition of Human Monoamine Oxidase A and B by 5-phenoxy 8-aminoquinoline Analogs

8-Aminoquinolines (8-AQs) are important class of anti-infective therapeutics. 5-Phenoxy 8-aminoquinoline analogs have shown improved metabolic stability compared to primaquine. In view or predictive role of monoamine oxidases (MAO) in metabolism of 8-aminoquinolines the 5-phenoxy analogs were evaluated in vitro for the inhibition of recombinant human MAO-A and MAO-B. The analogs were several folds more potent inhibitors of MAO-A and MAO-B compared to primaquine, the parent drug, with selectivity for MAO-B. 5-(4-Trifluoromethylphenoxy)-4-methylprimaquine (6) Inhibited MAO-B with IC(50) value of 150 nM (626-fold more potent than primaquine). These results will have important implications in optimizing metabolic stability of 8-AQs to improve therapeutic value and also indicate scope for development of 8-AQs as selective MAO inhibitors.

Understanding the Mechanisms for Metabolism-linked Hemolytic Toxicity of Primaquine Against Glucose 6-phosphate Dehydrogenase Deficient Human Erythrocytes: Evaluation of Eryptotic Pathway

Therapeutic utility of primaquine, an 8-aminoquinoline antimalarial drug, has been limited due to its hemolytic toxicity in population with glucose 6-phosphate dehydrogenase deficiency. Recent investigations at our lab have shown that the metabolites generated through cytochrome P(450)-dependent metabolic reactions are responsible for hemotoxic effects of primaquine, which could be monitored with accumulation of methemoglobin and increased oxidative stress. The molecular markers for succeeding cascade of events associated with early clearance of the erythrocytes from the circulation were evaluated for understanding the mechanism for hemolytic toxicity of primaquine. Primaquine alone though did not induce noticeable methemoglobin accumulation, but produced significant oxidative stress, which was higher in G6PD-deficient than in normal erythrocytes. Primaquine, presumably through redox active hemotoxic metabolites generated in situ in human liver microsomal metabolism-linked assay, induced a dose-dependent methemoglobin accumulation and oxidative stress, which were almost similar in normal and G6PD-deficient erythrocytes. Primaquine alone or in presence of pooled human liver microsomes neither produced significant effect on intraerythrocytic calcium levels nor affected the phosphatidyl serine asymmetry of the normal and G6PD-deficient human erythrocytes as monitored flowcytometrically with Annexin V binding assay. The studies suggest that eryptosis mechanisms are not involved in accelerated removal of erythrocytes due to hemolytic toxicity of primaquine.

Amino Acid, Dipeptide and Pseudodipeptide Conjugates of Ring-substituted 8-aminoquinolines: Synthesis and Evaluation of Anti-infective, β-haematin Inhibition and Cytotoxic Activities

Three new series of 8-aminoquinolines with modifications in the side-chain by conjugation with amino acids, dipeptides and pseudodipeptides have been synthesized. The synthesized compounds were tested for in vitro antimalarial activity against chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum strains, in vitro cytotoxicity in mammalian kidney cells (Vero), in vitro antileishmanial activity against Leishmania donovani, in vitro antimicrobial activity and in vitro inhibition of β-haematin formation. The promising compounds were also evaluated for in vivo blood-schizontocidal antimalarial activity against Plasmodium berghei infected mice. The analogues 55 and 101 produced highest antimalarial activities, in vitro. Analogues 52 and 59 exhibited promising antileishmanial and broad spectrum of antifungal activities, respectively.

Antiprotozoal and Antimicrobial Compounds from the Plant Pathogen Septoria Pistaciarum

Four new 1,4-dihydroxy-5-phenyl-2-pyridinone alkaloids, 17-hydroxy-N-(O-methyl)septoriamycin A (1), 17-acetoxy-N-(O-methyl)septoriamycin A (2), 13-(S)-hydroxy-N-(O-methyl)septoriamycin A (3), and 13-(R)-hydroxy-N-(O-methyl)septoriamycin A (4), together with the known compounds (+)-cercosporin (5), (+)-14-O-acetylcercosporin (6), (+)-di-O-acetylcercosporin (7), lumichrome, and brassicasterol, were isolated from an ethyl acetate extract of a culture medium of Septoria pistaciarum. Methylation of septoriamycin A (8) with diazomethane yielded three di-O-methyl analogues, two of which existed as mixtures of rotamers. We previously reported antimalarial activity of septoriamycin A. This compound also exhibited significant activity against Leishmania donovani promastigotes. Compounds 5-7 showed moderate in vitro activity against L. donovani promastigotes and chloroquine-sensitive (D6) and -resistant (W2) strains of Plasmodium falciparum, whereas compound 5 was fairly active against methicillin-sensitive and methicillin-resistant strains of Staphylococcus aureus. Compounds 5-7 also displayed moderate phytotoxic activity against both a dicot (lettuce, Lactuca sativa) and a monocot (bentgrass, Agrostis stolonifera) and cytotoxicity against a panel of cell lines.

Synthesis and Biological Evaluation of Tricyclic Guanidine Analogues of Batzelladine K for Antimalarial, Antileishmanial, Antibacterial, Antifungal and Anti-HIV Activities

Fifty analogues of batzelladine K were synthesized and evaluated for in vitro antimalarial (Plasmodium falciparum), antileishmanial (Leishmania donovani), antimicrobial (panel of bacteria and fungi), antiviral (HIV-1) activities. Analogues 14h and 20l exhibited potential antimalarial activity against chloroquine-sensitive D6 strain with IC(50) 1.25 and 0.88 μM and chloroquine-resistant W2 strain with IC(50) 1.64 and 1.07 μM, respectively. Analogues 12c and 14c having nonyl substitution showed the most potent antileishmanial activity with IC(50) 2.39 and 2.78 μM and IC(90) 11.27 and 12.76 μM respectively. Three analogues 12c, 14c and 14i were the most active against various pathogenic bacteria and fungi with IC(50) <3.02 μM and MIC/MBC/MFC <6 μM. Analogue 20l having pentyl and methyl substituents on tricycle showed promising activities against all pathogens. However, none was found active against HIV-1. Our study demonstrated that the tricyclic guanidine compounds provide new structral class for broad spectrum activity. © 2012 John Wiley & Sons A/S.

Synthesis and Evaluation of Aplysinopsin Analogs As Inhibitors of Human Monoamine Oxidase A and B

Aplysinopsins are tryptophan-derived natural products that have been isolated from a variety of marine organisms. Previous studies have shown aplysinopsin analogs to possess a variety of biological activities, including modulation of neurotransmissions. A series of fifty aplysinopsin analogs was synthesized and assayed for monoamine oxidase A and B inhibitory activity. Three compounds displayed significant MAO inhibitory activity and selectivity. The compound (E)-5-[(6-bromo-1H-indol-3-yl)methylene]-2-imino-1,3-dimethylimidazolidin-4-one (3x) possessed an IC(50) of 5.6 nM at MAO-A and had a selectivity index of 80.24. An SAR study revealed that multiple N-methylations, one of which should be at position N-2', and bromination at C-5 or C-6 are important factors for MAO-A potency and selectivity.

Pentacyclic Ingamine Alkaloids, a New Antiplasmodial Pharmacophore from the Marine Sponge Petrosid Ng5 Sp5

Two new pentacyclic ingamine alkaloids, namely 22(S)-hydroxyingamine A (2) and dihydroingenamine D (3), together with the known ingamine A (1), have been isolated from marine sponge Petrosid Ng5 Sp5 (family Petrosiidae) obtained from the open repository of the National Cancer Institute, USA. The structures of compounds 1-3 were determined using 1D and 2D NMR, and HRESIMS techniques. The absolute configuration of both the C9 and C22 of 2 was determined as (S) using a modified Mosher esterification method. Compounds 1 and 3 showed strong antiplasmodial activity against chloroquine-sensitive (D6) and -resistant (W2) strains of Plasmodium falciparum with IC₅₀ values of 90 and 78 ng/mL and 72 and 57 ng/mL, respectively, while 2 was found to be less active (IC₅₀ values of 200 and 140 ng/mL, respectively). Compounds 1-3 were found to be devoid of in vitro cytotoxicity against human solid tumor cells of breast (BT-549), ovary (SK-OV-3), and epidermoid (KB) carcinomas and skin melanoma (SK-MEL), as well as against noncancerous monkey kidney fibroblasts (VERO) and pig kidney epithelial (LLC-PK₁₁) cells, up to a maximum concentration of 10 µg/mL. Compounds 1-3 also displayed weak antimicrobial and moderate antileishmanial activities against Leishmania donovani promastigotes. These polycyclic ingamine alkaloids represent the first example of antiplasmodial leads without a β-carboline ring, which is known to be responsible for the cytotoxicity of the well-known manzamine class of marine alkaloids related to 1-3.

Synthesis and Structure-Activity Relationships of Lansine Analogues As Antileishmanial Agents

Clear and rational thinking: A series of rationally designed, lansine-derived carbazoles was synthesized and evaluated for activity against promastigotes and amastigotes of Leishmania donovani, the causative agent of leishmaniasis. Some structural modifications gave rise to compounds with enhanced activity and selectivity over lansine, allowing structure-activity relationships to be elucidated and providing a foundation for the further development of this pharmacophore.

Antimicrobial and Antiprotozoal Activities of Secondary Metabolites from the Fungus Eurotium Repens

In this study, we examined in vitro antibacterial, antifungal, antimalarial, and antileishmanial activities of secondary metabolites (1-8) isolated from the fungus Eurotium repens. All compounds showed mild to moderate antibacterial or antifungal or both activities except 7. The activity of compound 6 was the best of the group tested. The in vitro antimalarial evaluation of these compounds revealed that compounds 1-3, 5, and 6 showed antimalarial activities against both chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum with IC(50) values in the range of 1.1-3.0 μg/ml without showing any cytotoxicity to the mammalian cells. Compound 5 displayed the highest antimalarial activity. Antileishmanial activity against Leishmania donovani promastigotes was observed for compounds 1-6 with IC(50) values ranging from 6.2 to 23 μg/ml. Antileishmanial activity of compounds 5 and 6 (IC(50) values of 7.5 and 6.2 μg/ml, respectively) was more potent than 1-4 (IC(50) values ranging from 19-23 μg/ml). Compounds 7 and 8 did not show any antiprotozoal effect. Preliminary structure and activity relationship studies indicated that antibacterial, antifungal, antimalarial, and antileishmanial activities associated with phenol derivates (1-6) seem to be dependent on the number of double bonds in the side chain, which would be important for lead optimization in the future.

Antiparasitic and Anticancer Carvotacetone Derivatives of Sphaeranthus Bullatus

The CH2Cl2-MeOH (1:1) extract of the aerial parts of Sphaeranthus bullatus, an annual herb native to tropical East Africa, showed activity against chloroquine sensitive D6 (IC50 9.7 microg/mL) and chloroquine resistant W2 (IC50 15.0 microg/mL) strains of Plasmodium falciparum. Seventeen secondary metabolites were isolated from the extract through conventional chromatographic techniques and identified using various spectroscopic methods. The compounds were evaluated for their in vitro antiplasmodial, antileishmanial and anticancer activities revealing activity of four carvotacetone derivatives, namely 3-acetoxy-7-hydroxy-5-tigloyloxycarvotacetone (1), 3,7-dihydroxy-5-tigloyloxycarvotacetone (2), 3-acetoxy-5,7-dihydroxycarvotacetone (3) and 3,5,7-trihydroxy-carvotacetone (4); with antiplasmodial IC50 values of 1.40, 0.79, 0.60 and 3.40 microg/mL, respectively, against chloroquine sensitive D6 strains of P. falciparum; antiplasmodial activity of IC50 2.00, 0.90, 0.68 and 2.80 microg/mL, respectively, against chloroquine resistant W2 strains of P. falciparum; antileishmanial IC50 values of 0.70, 3.00, 0.70 and 17.00 microg/mL, respectively, against the parasite L. donovanii promastigotes, and anticancer activity against human SK-MEL, KB, BT-549 and SK-OV-3 tumor cells, with IC50 values between <1.1 - 5.3 microg/mL for 1-3. In addition, cytotoxic effects of the active compounds were evaluated against monkey kidney fibroblasts (VERO) and pig kidney epithelial cells (LLC-PK11). The structures of carvotacetone derivatives were determined by 1D and 2D NMR spectroscopy; the absolute stereochemical configuration of 3-acetoxy-7-hydroxy-5-tigloyloxycarvotacetone (1) was determined as 3R, 4R, 5S by circular dichroism, specific rotation, 1H NMR and 2D NMR ROESY and NOESY experiments.

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