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Completed Research Projects
Title: Clinical and Epidemiology Leishmania Research
Synopsis: This project was designed to develop a blood test for leishmaniasis and determine the number of Gulf War veterans infected.
Overall Summary: See project objective.
Overall Project Objective: Development of serological test for viscerotropic leishmaniasis and determination of infection rate.
Results to Date: We have now documented the antileishmanial activity of xanthones and provide experimental evidence of their ability to block parasite access to heme. We have evidence that this mode of action may play a role in the antiparasitic action of pentamidine (a drug employed as a secondary line therapy for leishmaniasis and trypanosomiasis and in front-line therapy of PCP), and that our overall drug design scheme may be converging on a similar path (manuscript submitted). We recognized that the "heme problem" of Leishmania parasites may be exploitable by heme-complexing xanthones that we produced for our VA Merit Review funded malaria project. We observed the poor activity of 3,6-diethylminoxanthone and 4,5-diamidino-xanthone in the Plasmodium system and against Leishmania promastigotes (the insect form of the parasite). A molecular model was constructed on the accumulated evidence and our recently completed 1H-n.m.r. study confirming the carbonyl-iron coordination, the stacking of coplanar aromatic molecules, and hydrogen bonding between the hydroxyl sustituents of 4,5-dihydroxyxanthone and heme/-Es propionate groups. Synthesis of nitrogenated xanthones was planned with amino-and amidino-side chains in the lower half of the pharmacophore. 4,5-Diamidinoxanthone was prepared based on a computerized docking model and found to exert comparatively weak antimalarial activity in vitro. 3,6-bis-Diethylaminoxanthone was also prepared but it too exhibited only weak activity against P. falciparum. These findings indicated that the protonatable species could not be directly attached to the aromatic ring on in conjugation with the carbonyl moiety. Presumably this arrangement results in formation of bis-cations (formed once the drug enters the acidic environment or the parasite vacuole) that draw electrons away from the carbonyl and diminish the interaction with heme iron. A decision was made to retain the oxygen atom (a conjugatively electron-donating species) as a bridge for an R-group containing the protonable nitrogen. Molecular models were constructed from a ball-and-stick kit reproducing bond lengths, geometry, and angle strains demonstrated that an optimal chain length in the range of C4 to C6 would permit a relatively strain-free, close association between the ammonium ions of the xanthone and the carboxylate groups of heme. Since the strength of this ionic interaction is dependent on both bond distance and the dielectric constant of the surrounding milieu in the vacuole (which is unknown), it was decided to proceed with preparation of the straight-chain di-substituted series from C2 to C8 (i.e., excepting C7), each with a terminal diethylamino group. We developed in vitro conditions for invasion of murine (BALB/c) bone marrow derived macrophages by promastogotes of Leishmania mexicana M379. We synthesized 3,6-bis-diethylaminoalkoxyxanthones with varying chain lengths from C2 to C8 is accomplished with full analytical figures (August 99 to December 99). Drug screening indicates that the corresponding C5 and C6 derivatives (i.e., 3,6-bis-diethylamino-amyloxyxanthoneand 3,6-bis-diethylaminohexyloxyxanthone respectively) exhibit remarkable potency against the intracellular amastigotes of L. mexicana (e.g., IC50 values of below 4 x 10-9M). In a follow-up experiment we have found that C5 clears macrophages heavily infected with amastigotes of L. mexicana within 48 hrs of drug administration. We discovered that C5 and C6 are also the most potent members of the 3,6-series against drug resistant intracellular parasites of P. falciparum (e.g., IC50 values of 150 and 100nM, respectively). We synthesized and tested a stable form of an active primaquine metabolite, 5-acetoxy-6-methoxy-8-aminoquinoline. This was produced initially for synergy studies with exifone, but is now being tested as a reagent used in functional screening for inherited enzyme deficiencies in the glutathione cycle (i.e., G6PD). In addition, analogs, in which the acetoxy group have been replaced by a peptide for selective cleavage by a protease, are underway.
Project: VA-6E
Agency: Department Of Veterans Affairs
Location: VAMC Portland/Croet Portland
P.I. Name: Michael Riscoe
Status: Complete
Study Start Date: October 01, 1994
Estimated Completion Date: March 31, 2000
Specific Aims: To determine the possibility and rate of infection of PGW veterans with Leishmania spp, and to develop novel anti-leishmanial chemotherapeutics.
Methodology: We have recently identified xanthones as a novel class of antimalarial agents (Ignatushchenko et al., FEBS Letters, 409: 67-73, 1997). Selected xanthones were shown to form stable, soluble complexes with heme and to block heme polymerization, a key process in the survival of the intra-erythrocytic form of the Plasmodium parasite. Considerind the specific nutritional requirements of Leishmania, we speculated that these organisms would be exquisitely sensitive to the effects of heme- and porphyrin-complexing xanthones.
Publications:
Ignatushchenko M , Riscoe M , Winter R . Am J Trop Med Hyg.2000;62:77-81.
Kelly J X, Ignatushchenko M , Bouwer H G, Peyton D H, Hinrichs D , Winter R , Riscoe M . Mol Biochem Parasitol.2003;126:43-9.
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