Edward Borowski

Voriconazole and multidrug resistance in Candida albicans

In vitro activity of voriconazole against fluconazole‐resistant Candida albicans clinical isolates with identified molecular basis of multidrug resistance (MDR) and recombinant Saccharomyces cerevisiae expressing C. albicans genes coding for major multidrug transporters, CaCdr1p, CaCdr2p or CaMdr1p, was compared with that of fluconazole, ketoconazole and clotrimazole. It was found that overexpression of the MDR genes made the yeast cells less susceptible to voriconazole. The voriconazole resistance indexes, defined as a ratio of minimum inhibitory concentrations (MICs) determined for MDR and sensitive cells, were comparable with those determined for fluconazole. Voriconazole effectively competed with rhodamine 6G for the active efflux mediated by CaCdr1p and CaCdr2p.

Synthesis and biological evaluation of 2,7-Dihydro-3H-dibenzo[de,h]cinnoline-3,7-dione derivatives, a novel group of anticancer agents active on a multidrug resistant cell line

A series of anthrapyridazone derivatives with one or two basic side chains at various positions in the tetracyclic chromophore have been synthesized. The key intermediates in the synthesis are 2,7-dihydro-3H-dibenzo[de,h]cinnoline-3,7-diones 1, 12 and 15 monosubstituted at position 2 (4d, 16a-e), or 6 (2a-f) or disubstituted at positions 2 and 6 (4a-c) or 2 and 8 (17a-e) with appropriate alkylaminoalkylamines. All analogues showed in vitro cytotoxic activity against murine leukemia (L1210) and human leukemia (K562) cell lines. The compounds were also active against human leukemia multidrug resistant (K562/DX) cell line with resistance index (RI) in the range 1-3 depending on the compounds structure. Two of the most active in vitro compounds 4a and 11 were tested in vivo against murine P388 leukemia and displayed antileukemic activity comparable with that of Mitoxantrone. DNA-binding assays were performed and DNA affinity data were correlated with the structures of the compounds. The cytoplasmatic membrane affinity values (log k(IAM)) have also been determined and the correlation with the resistance indexes discussed. The anthrapyridazones constitute a novel group of antitumor compounds that can overcome multidrug resistance.

Differential ability of cytostatics from anthraquinone group to generate free radicals in three enzymatic systems: NADH dehydrogenase, NADPH cytochrome P450 reductase, and xanthine oxidase.

The antitumor drugs of the anthraquinone group are widely used agents in the treatment of a variety of human neoplasms. However, their clinical effectiveness is limited by several factors, among which dose-dependent cardiotoxicity is of great importance. Numerous data indicate that the cardiac effects of these drugs are the consequence of one-electron transfer from reduced nucleotides to atmospheric oxygen. This process is catalyzed primarily by NADH dehydrogenase, NADPH cytochrome P450 reductase, and xanthine oxidase, and leads to the formation of reactive oxygen species. In our previous studies we have shown that the NADH dehydrogenase catalyzed electron transfer phenomenon is correlated with the affinity of anthraquinone drugs to the enzyme. In this work data are presented on the ability of compounds belonging to several structural types of anthraquinone cytostatics (sugar- and quinone-modified derivatives of DR and ADR, and anthracenedione compounds) to stimulate free radical formation in the above three enzymatic systems. It has been shown that the three oxidoreductases exhibit different structural requirements with respect to their substrate properties for anthraquinones. Therefore, evaluation of the structural factors determining the ability of anthraquinone compounds to generate active oxygen species cannot be limited to a single oxidoreductase system but must include all types of enzymatic systems involved in the catalysis of one-electron transfer reactions.

Molecular modeling of amphotericin B-ergosterol primary complex in water II.

The work presented is a part of our continual study on the behavior of the polyene macrolide antibiotic amphotericin B (AmB) complexes with sterols on the molecular level. In contrast to the previously researched AmB-ergosterol binary complex, the AmB-ergosterol-AmB aggregates simulated of 2:1 stoichiometry retain significantly higher stability and relatively rigid, sandwich geometry. Van der Waals forces with a considerable share of the electrostatic interactions are responsible for such behavior. System of the intermolecular hydrogen bonds also seems to be of notable importance for the complexs structure preservation. The most energetically favored geometries match fairly close the geometric criteria and the network of interactions postulated in the contemporary hypothetical and computational models of antibiotic-sterol complexes. On the basis of works previously published and the present study novel hypotheses on the AmB selectivity towards sterols varying in chemical structure and on the possible mechanisms of channel structure formation were presented.

Influence of a lipid bilayer on the conformational behavior of amphotericin B derivatives — A molecular dynamics study

Amphotericin B (AmB) is an effective but very toxic antifungal antibiotic. In our laboratory a series of AmB derivatives of improved selectivity of action was synthesized and tested. To understand molecular basis of this improvement, comparative conformational studies of amphotericin B and its two more selective derivatives were carried out in an aqueous solution and in a lipid membrane. These molecular simulation studies revealed that within a membrane environment the conformational behavior of the derivatives differs significantly from the one observed for the parent molecule. Possible reasons for such a difference are analyzed. Furthermore, we hypothesize that the observed conformational transition within the polar head of AmB derivatives may lead to destabilization of antibiotic-induced transmembrane channels. Consequently, the selective toxicity of the derivatives should increase as ergosterol-rich liquid-ordered domains are more rigid and conformationally ordered than their cholesterol-containing counterparts, and as such may better support less stable channel structure.

Novel Nystatin A1 derivatives exhibiting low host cell toxicity and antifungal activity in an in vitro model of oral candidosis

Opportunistic oral infections caused by Candida albicans are frequent problems in immunocompromised patients. Management of such infections is limited due to the low number of antifungal drugs available, their relatively high toxicity and the emergence of antifungal resistance. Given these issues, our investigations have focused on novel derivatives of the antifungal antibiotic Nystatin A1, generated by modifications at the amino group of this molecule. The aims of this study were to evaluate the antifungal effectiveness and host cell toxicity of these new compounds using an in vitro model of oral candidosis based on a reconstituted human oral epithelium (RHOE). Initial studies employing broth microdilution, revealed that against planktonic C. albicans, Nystatin A1 had lower minimal inhibitory concentration than novel derivatives. However, Nystatin A1 was also markedly more toxic against human keratinocyte cells. Interestingly, using live/dead staining to assess C.albicans and tissue cell viability after RHOE infection, Nystatin A1 derivatives were more active against Candida with lower toxicity to epithelial cells than the parent drug. Lactate dehydrogenase activity released by the RHOE indicated a fourfold reduction in tissue damage when certain Nystatin derivatives were used compared with Nystatin A1. Furthermore, compared with Nystatin A1, colonisation of the oral epithelium by C. albicans was notably reduced by the new polyenes. In the absence of antifungal agents, confocal laser scanning microscopy showed that C. albicans extensively invaded the RHOE. However, the presence of the novel derivatives greatly reduced or totally prevented this fungal invasion.

The structure, including stereochemistry, of levorin A1

The constitution and stereostructure of levorin A1 1, an aromatic heptaeneantifungal antibiotic, was established on the basis of NMR studies, which contained DQFCOSY,ROESY, HSQC and HMBC experiments. Mycosamine moiety was used as an internalchiral probe to determine the absolute configuration of levorin A1 stereogenic centers: 13S,15R, 17S, 18R, 19S, 21R. The relative configuration of the remaining stereogenic centers wasassigned as follows: 36S*, 37R*, 38S*, 40S* and 41S*. The configuration at C-5 still remainsto be established. The geometry of the heptaene chromophore was defined as 22E, 24E, 26Z,28Z, 30E, 32E and 34E.

The ability of new sugar-modified derivatives of antitumor anthracycline, daunorubicin, to stimulate NAD(P)H oxidation in different cellular oxidoreductase systems: NADH dehydrogenase, NADPH cytochrome P450 reductase, and xanthine oxidase

Numerous data indicate that cellular oxidoreductases may be responsible for the cardiotoxic effects of antitumor anthracycline drugs as a consequence of the mediation by these agents of one-electron transfer from reduced nucleotides to atmospheric oxygen. This process is catalyzed primarily by NADH dehydrogenase, NADPH cytochrome P450 reductase, and xanthine oxidase and leads to the formation of reactive oxygen species (ROS). In this work the data on the ability of new amino sugar derivatives of daunorubicin to stimulate NAD(P)H oxidation in the above oxidoreductase systems are presented. They represent analogues of daunorubicin in which the amino sugar nitrogen is bounded to an unsubsituted, or amino- or nitro-substituted benzyl group. It was found that the ability of examined sugar-modified derivatives of daunorubicin to stimulate NAD(P)H oxidation differs considerably depending on the subsituent in the phenyl ring. It was also determined that this ability was not identical in the three enzymatic systems studied, showing that these derivatives have different affinities for the enzymes examined. More similarities were observed in their interaction with NADH dehydrogenase and NADPH cytochrome P450 reductase than with xanthine oxidase.

Light-Induced Transformation of the Aromatic Heptaene Antifungal Antibiotic Candicidin D into Its All-Trans Isomer

Illumination of the aromatic heptaene macrolide antifungal antibiotic candicicin D with UV light results in an isomerization of the molecule. The product formed after irradiation of the candicidin complex with UV light (λ = 365 nm), namely, iso-candicidin D, was isolated and subjected to 2D NMR studies, consisting of DQF-COSY, ROESY, TOCSY, HSQC, and HMBC experiments. The obtained spectral data unambiguously evidenced that iso-candicidin D was the all-trans isomer of the native antibiotic, and straightening of the heptaenic chromophore was the only light-induced structural change that occurred. Hence, iso-candicidin D was proclaimed to be a prototype of a novel class of polyene macrolide antifungal antibiotics: the all-trans aromatic heptaenes, containing a macrolide ring similar to that of amphotericin B.

Molecular basis for the DNA damage induction and anticancer activity of asymmetrically substituted anthrapyridazone PDZ-7

Anthrapyridazones, imino analogues of anthraquinone, constitute a family of compounds with remarkable anti-cancer activity. To date, over 20 derivatives were studied, of which most displayed nanomolar cytotoxicity towards broad spectrum of cancer cells, including breast, prostate and leukemic ones. BS-154, the most potent derivative, had IC50 values close to 1 nM, however, it was toxic in animal studies. Here, we characterize another anthrapyridazone, PDZ-7, which retains high cytotoxicity while being well tolerated in mice. PDZ-7 is also active in vivo against anthracycline-resistant tumor in a mouse xenograft model and induces DNA damage in proliferating cells, preferentially targeting cells in S and G2 phases of the cell cycle. Activation of Mre11-Rad50-Nbs1 (MRN) complex and phosphorylation of H2AX suggest double-stranded DNA breaks as a major consequence of PDZ-7 treatment. Consistent with this, PDZ-7 treatment blocked DNA synthesis and resulted in cell cycle arrest in late S and G2 phases. Analysis of topoisomerase IIα activity and isolation of the stabilized covalent topoisomerase IIα - DNA complex in the presence of PDZ-7 suggests that this compound is a topoisomerase IIα poison. Moreover, PDZ-7 interfered with actin polymerization, thereby implying its action as a dual inhibitor of processes critical for dividing cells. Using nuclear magnetic resonance (NMR) spectroscopy we show that PDZ-7 interacts with DNA double helix and quadruplex DNA structure. Taken together, our results suggest that PDZ-7 is a unique compound targeting actin cytoskeleton and DNA.