Synthetic arylquinuclidine derivatives exhibit antifungal activity against <it>Candida albicans</it>, <it>Candida tropicalis </it>and <it>Candida parapsilopsis</it>

<p>Abstract</p> <p>Background</p> <p>Sterol biosynthesis is an essential pathway for fungal survival, and is the biochemical target of many antifungal agents. The antifungal drugs most widely used to treated fungal infections are compounds that inhibit cytochrome P450-dependent C14α-demethylase (CYP51), but other enzymes of this pathway, such as squalene synthase (SQS) which catalyses the first committed step in sterol biosynthesis, could be viable targets. The aim of this study was to evaluate the antifungal activity of SQS inhibitors on <it>Candida albicans</it>, <it>Candida tropicalis </it>and <it>Candida parapsilopsis </it>strains.</p> <p>Methods</p> <p>Ten arylquinuclidines that act as SQS inhibitors were tested as antiproliferative agents against three ATCC strains and 54 clinical isolates of <it>Candida albicans</it>, <it>Candida tropicalis </it>and <it>Candida parapsilopsis</it>. Also, the morphological alterations induced in the yeasts by the experimental compounds were evaluated by fluorescence and transmission electron microscopy.</p> <p>Results</p> <p>The most potent arylquinuclidine derivative (3-[1'-{4'-(benzyloxy)-phenyl}]-quinuclidine-2-ene) (WSP1267) had a MIC₅₀of 2 μg/ml for all species tested and MIC₉₀varying from 4 μg/ml to 8 μg/ml. Ultrathin sections of <it>C. albicans </it>treated with 1 μg/ml of WSP1267 showed several ultrastructural alterations, including (a) loss of cell wall integrity, (b) detachment of the plasma membrane from the fungal cell wall, (c) accumulation of small vesicles in the periplasmic region, (d) presence of large electron-dense vacuoles and (e) significantly increased cell size and cell wall thickness. In addition, fluorescence microscopy of cells labelled with Nile Red showed an accumulation of lipid droplets in the cytoplasm of treated yeasts. Nuclear staining with DAPI revealed the appearance of uncommon yeast buds without a nucleus or with two nuclei.</p> <p>Conclusion</p> <p>Taken together, our data demonstrate that arylquinuclidine derivatives could be useful as lead compounds for the rational synthesis of new antifungal drugs.</p

Kinetic Characterization of Squalene Synthase from Trypanosoma cruzi: Selective Inhibition by Quinuclidine Derivatives▿

The biosynthesis of sterols is a major route for the development of antitrypanosomals. Squalene synthase (SQS) catalyzes the first step committed to the biosynthesis of sterols within the isoprenoid pathway, and several inhibitors of the enzyme have selective antitrypanosomal activity both in vivo and in vitro. The enzyme from Trypanosoma cruzi is a 404-amino-acid protein with a clearly identifiable membrane-spanning region. In an effort to generate soluble recombinant enzyme, we have expressed in Escherichia coli several truncated versions of T. cruzi SQS with a His tag attached to the amino terminus. Deletions of both the amino- and carboxyl-terminal regions generated active and soluble forms of the enzyme. The highest levels of soluble protein were achieved when 24 and 36 amino acids were eliminated from the amino and carboxyl regions, respectively, yielding a protein of 41.67 kDa. The Michaelis-Menten constants of the purified enzyme for farnesyl diphosphate and NAD (NADPH) were 5.25 and 23.34 μM, respectively, whereas the Vmax was 1,428.56 nmol min−1mg−1. Several quinuclidine derivatives with antiprotozoal activity in vitro were found to be selective inhibitors of recombinant T. cruzi SQS in comparative assays with the human enzyme, with 50% inhibitory concentration values in the nanomolar range. These data suggest that selective inhibition of T. cruzi SQS may be an efficient strategy for the development of new antitrypanosomal agents

Bleeding risk assessment using multiple electrode aggregometry in patients following coronary artery bypass surgery

Individual variability in the response to antiplatelet therapy (APT), frequently administered preoperatively, has been established by various platelet function assays and could reflect bleeding tendency after coronary artery bypass surgery (CABG). Our hypothesis is that multiple electrode whole-blood aggregometry (MEA) can identify patients at risk for excessive bleeding. We enrolled 211 patients (155 male and 56 female) undergoing isolated CABG in a prospective observational study. Patients were divided into four groups with respect to their preoperative APT management. MEA, using the ASPI and the ADP test, was performed prior to surgery. The primary endpoint was chest tube output (CTO) and the secondary endpoint was perioperative packed red blood cell concentrate (PRBC) administration. Patients were characterized as bleeders if their 24 h CTO exceeded the 75th percentile of distribution. 24 h CTO value of 11.33 ml/kg presented 75th percentile of distribution, thus cut-off value for "bleeder category". The proportion of patients characterized as bleeders was significantly different among the groups in regard to preoperative APT (p = 0.039). Significant differences in both ASPI (p < 0.001) and ADP (p = 0.038) tests were observed between different preoperative APT groups. Significant correlations between the ASPI test (r = -0.170, p = 0.014) and ADP test (r = -0.206, p = 0.003) with 24 h CTO were found. The receiver operating curve revealed an ASPI test value of <20 area under curve (AUC) units (AUC 0.603, p = 0.023) and an ADP test <73 AUC (AUC 0.611, p = 0.009) as a "bleeder" determinant. The proportion of patients transfused with PRBC did not significantly differ among the groups in regard to preoperative APT (p = 0.636). Comparison of the ASPI test values between patients with respect to PRBC administration revealed lower values in the ASPI test in a group of patients transfused with PRBC (mean, 27.88 vs. 40.32 AUC, p = 0.002). Our study showed that MEA is a useful method of predicting CABG patients with excessive postoperative bleeding

Synthetic arylquinuclidine derivatives exhibit antifungal activity against Candida albicans, Candida tropicalis and Candida parapsilopsis

10 p. : il.Background: Sterol biosynthesis is an essential pathway for fungal survival, and is the biochemical target of many antifungal agents. The antifungal drugs most widely used to treated fungal infections are compounds that inhibit cytochrome P450-dependent C14a-demethylase (CYP51), but other enzymes of this pathway, such as squalene synthase (SQS) which catalyses the first committed step in sterol biosynthesis, could be viable targets. The aim of this study was to evaluate the antifungal activity of SQS inhibitors on Candida albicans, Candida tropicalis and Candida parapsilopsis strains. Methods: Ten arylquinuclidines that act as SQS inhibitors were tested as antiproliferative agents against three ATCC strains and 54 clinical isolates of Candida albicans, Candida tropicalis and Candida parapsilopsis. Also, the morphological alterations induced in the yeasts by the experimental compounds were evaluated by fluorescence and transmission electron microscopy. Results: The most potent arylquinuclidine derivative (3-[1’-{4’-(benzyloxy)-phenyl}]-quinuclidine-2-ene) (WSP1267) had a MIC50 of 2 μg/ml for all species tested and MIC90 varying from 4 μg/ml to 8 μg/ml. Ultrathin sections of C. albicans treated with 1 μg/ml of WSP1267 showed several ultrastructural alterations, including (a) loss of cell wall integrity, (b) detachment of the plasma membrane from the fungal cell wall, (c) accumulation of small vesicles in the periplasmic region, (d) presence of large electron-dense vacuoles and (e) significantly increased cell size and cell wall thickness. In addition, fluorescence microscopy of cells labelled with Nile Red showed an accumulation of lipid droplets in the cytoplasm of treated yeasts. Nuclear staining with DAPI revealed the appearance of uncommon yeast buds without a nucleus or with two nuclei. Conclusion: Taken together, our data demonstrate that arylquinuclidine derivatives could be useful as lead compounds for the rational synthesis of new antifungal drugs

Biphenylquinuclidines as inhibitors of squalene synthase and growth of parasitic protozoa

In this paper we describe the preparation of some biphenylquinuclidine derivatives and their evaluation as inhibitors of squalene synthase in order to explore their potential in the treatment of the parasitic diseases leishmaniasis and Chagas disease. The compounds were screened against recombinant Leishmania major squalene synthase and against Leishmania mexicana promastigotes, Leishmania donovani intracellular amastigotes and Trypanosoma cruzi intracellular amastigotes. Compounds that inhibited the enzyme, also reduced the levels of steroids and caused growth inhibition of L. mexicana promastigotes. However there was a lower correlation between inhibition of the enzyme and growth inhibition of the intracellular parasites, possibly due to delivery problems. Some compounds also showed growth inhibition of T. brucei rhodesiense trypomastigotes, although in this case alternative modes of action other than inhibition of SQS are probably involved

Alterations on the growth and ultrastructure of <em>Leishmania chagasi </em>induced by squalene synthase inhibitors

Leishmaniasis is an important disease in widely dispersed regions of the world. In South America, visceral leishmaniasis (VL) is mainly caused by Leishmania chagasi. The morbidity associated with the infection is high, and death may occur in some untreated patients. Treatment has been based upon pentavalent antimonial drugs for more than half a century and problems, including development of resistance to antimonials and lack of efficacy against VL/HIV co-infections, have emphasized the need for new drugs. Squalene synthase (SQS) is an essential enzyme for the biosynthesis of protozoal sterol molecules. In this work, nineteen synthetic quinuclidines, potentially inhibitors of SQS, were tested against promastigote forms of L. chagasi and the IC50 values of the compounds were determined. The most active compounds had IC50 values of around 30 nM and induced complete growth arrest and cell lysis at sub-micromolar concentrations. We analyzed the morphological structure of the parasites treated with these compounds by transmission electron microscopy of thin sections. Treated parasites showed significant ultrastructural changes, which varied from discrete alterations to total destruction of the cells, depending on the drug concentration and the time of incubation. One important change observed was a typical swelling of the unique and highly branched mitochondrion, where the inner membrane lost its organization. There was an increase in the number of autophagosomal structures. Changes in the organization of the nuclear chromatin and alterations in the flagellar pocket and flagellar membrane were also observed. Crown Copyright (c) 2007 Published by Elsevier B.V. All rights reserved.</p

Quinuclidine Derivatives as Potential Antiparasitics▿

There is an urgent need for the development of new drugs for the treatment of tropical parasitic diseases such as Chagas' disease and leishmaniasis. One potential drug target in the organisms that cause these diseases is sterol biosynthesis. This paper describes the design and synthesis of quinuclidine derivatives as potential inhibitors of a key enzyme in sterol biosynthesis, squalene synthase (SQS). A number of compounds that were inhibitors of the recombinant Leishmania major SQS at submicromolar concentrations were discovered. Some of these compounds were also selective for the parasite enzyme rather than the homologous human enzyme. The compounds inhibited the growth of and sterol biosynthesis in Leishmania parasites. In addition, we identified other quinuclidine derivatives that inhibit the growth of Trypanosoma brucei (the causative organism of human African trypanosomiasis) and Plasmodium falciparum (a causative agent of malaria), but through an unknown mode(s) of action