Babu L. Tekwani

8-Aminoquinolines: future role as antiprotozoal drugs.

Purpose of review 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. Recent findings The earlier efforts towards development of 8-aminoquinoline analogs have been directed to extensive derivatization programs. This has led to discovery of tafenoquine for prophylaxis against malaria infections and sitamaquine with utility for treatment of visceral leishmaniasis. Bulaquine, a primaquine pro-drug, has shown reduced methemoglobin toxicity and better malaria-transmission-blocking activity than primaquine. Stereoselective pharmacologic and toxicologic characteristics of chiral 8-aminoquinolines provided the lead for enantiomeric separation of an 8-aminoquinoline analog NPC1161B, with greatly reduced toxicity and potent antimalarial action against blood as well as tissue stages of the parasite. NPC1161B has also shown promising use as an antileishmanial agent. Better understanding of the mechanisms of toxicity and efficacy may help in development of 8-aminoquinoline analogs with superior therapeutic actions, reduced toxicity and broader utility. Summary Extensive derivatization approaches followed by better understanding of structure-activity relationships and biotransformation mechanisms of toxicity have provided 8-aminoquinoline analogs with better pharmacologic and reduced toxicologic profiles. The novel 8-aminoquinoline analogs may have broader utility in public health as future antiprotozoals.

Novel 4-aminoquinoline-pyrimidine based hybrids with improved in vitro and in vivo antimalarial activity.

A class of hybrid molecules consisting of 4-aminoquinoline and pyrimidine were synthesized and tested for antimalarial activity against both chloroquine (CQ)-sensitive (D6) and chloroquine (CQ)-resistant (W2) strains of Plasmodium falciparum through an in vitro assay. Eleven hybrids showed better antimalarial activity against both CQ-sensitive and CQ-resistant strains of P. falciparum in comparison to standard drug CQ. Four molecules were more potent (7-8-fold) than CQ in D6 strain, and eight molecules were found to be 5-25-fold more active against resistant strain (W2). Several compounds did not show any cytotoxicity up to a high concentration (60 μM), others exhibited mild toxicities, but the selective index for the antimalarial activity was very high for most of these hybrids. Two compounds selected for in vivo evaluation have shown excellent activity (po) in a mouse model of Plasmodium berghei without any apparent toxicity. The X-ray crystal structure of one of the compounds was also determined.

PROCESSING OF MAMMALIAN AND PLANT S-ADENOSYLMETHIONINE DECARBOXYLASE PROENZYMES

S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl enzyme, and the pyruvate is formed in an intramolecular reaction that cleaves a proenzyme precursor and converts a serine residue into pyruvate. The wild type potato AdoMetDC proenzyme processed much faster than the human proenzyme and did not require putrescine for an optimal rate of processing despite the presence of three acidic residues (equivalent to Glu11, Glu178, and Glu256) that were demonstrated in previous studies to be required for the putrescine activation of human AdoMetDC proenzyme processing (Stanley, B. A., Shantz, L. M., and Pegg, A. E. (1994) J. Biol. Chem. 269, 7901–7907). A fourth residue that is also needed for the putrescine stimulation of human AdoMetDC proenzyme processing was identified in the present studies, and this residue (Asp174) is not present in the potato sequence. The site of potato AdoMetDC proenzyme processing was found to be Ser73 in the conserved sequence, YVLSESS, which is the equivalent of Ser68 in the human sequence. Replacement of the serine precursor with threonine or cysteine by site-directed mutagenesis in either the potato or the human AdoMetDC proenzyme did not prevent processing but caused a significant reduction in the rate. Although the COOH-terminal regions of the known eukaryotic AdoMetDCs are not conserved, only relatively small truncations of 8 residues from the human protein and 25 residues from the potato proenzyme were compatible with processing. The maximally truncated proteins show no similarity in COOH-terminal amino acid sequence but each contained 46 amino acid residues after the last conserved sequence, suggesting that the length of this section of the protein is essential for maintaining the proenzyme conformation needed for autocatalytic processing.

Assay of β-hematin formation by malaria parasite

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

Formation of haemozoin/β-haematin under physiological conditions is not spontaneous

Malaria parasite detoxifies free haem, released as a result of haemoglobin digestion, by converting it into an stable, crystalline, black brown pigment known as ‘malaria pigment’ or ‘haemozoin’. Earlier studies have demonstrated the involvement of a parasite‐specific enzyme ‘haem polymerase’ in the formation of haemozoin. However, recently it has been proposed that the polymerization of haem may be a spontaneous process that could take place by incubation of haematin with carboxylic acids (pH 4.2–5.0) even without presence of any parasitic or biological component (FEBS Letters, 352, 54–57 (1994)). Here we report that no spontaneous haem polymerization occurs at physiological conditions and the product described in the study mentioned above is not haemozoin/β‐haematin (haem polymer) as characterized by us on the basis of solubility characteristics and thin layer chromatography. The infra‐red spectroscopic analysis of the product formed though exhibits the bands corresponding to formation of iron‐carboxylate bond, similar to that in haemozoin/β‐haematin, but was identified as haem‐acid adduct. Thus polymerization of haem may not occur spontaneously under the reaction conditions corresponding to food vacuoles of the malarial parasite, the physiological site of haemozoin formation.

Kinetics and molecular characteristics of arginine transport by Leishmania donovani promastigotes

Characteristics of transport of L-arginine were studied in Leishmania donovani promastigotes grown in vitro in a defined medium. The promastigotes exhibited a time-dependent, temperature-sensitive, pH-dependent and saturable uptake of arginine. Metabolic inhibitors caused 81-92% inhibition, indicating that arginine influx in promastigotes is an energy requiring process. The presence of Na+ ions was necessary for full activity. Considerable inhibition was also noticed with valinomycin, gramicidin and amiloride. The transporter seems to involve an -SH group at the active site. The most distinctive feature of the leishmanial transporter was that lysine and ornithine did not show significant competition with arginine transport. Other neutral and acidic amino acids, as well as polyamines were also ineffective. The arginine analogues, viz., nitro-L-arginine methyl ester, N-nitro-L-arginine, aminoguanidine, agmatine and D-arginine were not recognised by the transporter, while N-methyl-L-arginine acetate and phospho-L-arginine showed competition, indicating stereo-specificity of the transporter and recognition of both the guanidino group, as well as the arginine side chain by the transporter. No exchange of intracellular [14C]arginine taken up by the promastigotes was noticed during incubation with 2 or 5 mM arginine in the extracellular medium. Eighty percent of the arginine taken up remained in the trichloroacetic acid-soluble fraction. Pentamidine caused competitive inhibition of arginine transport, exhibiting an IC50 value of 40 microM. Results indicate the presence of a novel distinct arginine transporter in Leishmania promastigotes.

A parasite rescue and transformation assay for antileishmanial screening against intracellular Leishmania donovani amastigotes in THP1 human acute monocytic leukemia cell line.

Leishmaniasis is one of the worlds most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models. In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes. Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

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.

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.

POLYAMINE TRANSPORT SYSTEMS OF LEISHMANIA DONOVANI PROMASTIGOTES

The following observations are conjointly indicative of the presence of distinct energy-dependent, saturable and multiple polyamine transport systems in Leishmania donovani promastigotes, the causative agent for visceral leishmaniasis. Spermidine was influxed with as much as seven times higher rate than putrescine, while both spermidine and putrescine transporters exhibited equally high affinity for the respective polyamine. N-Ethylmaleimide arrested the complete functionality of both the transporters which could be restored by reduced glutathione. Putrescine transporter did not recognize spermine but spermidine was recognized to some extent, while spermidine transporter significantly recognized spermine but putrescine was absolutely spared. A few aromatic diamines viz., diaminobiphenyl and the analogs as well as aliphatic diamines viz., cadaverine and agmatine were selectively recognized by the putrescine transporter only. L. donovani promastigotes grown in presence of alpha-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, registered marked upregulation of putrescine transport while spermidine transport was only marginally induced. PA transport systems provide the alternative pool of polyamines in L. donovani promastigotes in the absence of an adequate intracellular PA repertoire.