Luc Koymans

Contribution of mutations in the cytochrome P450 14α-demethylase (Erg11p, Cyp51p) to azole resistance in Candida albicans

The cytochrome P450 14alpha-demethylase, encoded by the ERG11 (CYP51) gene, is the primary target for the azole class of antifungals. Changes in the azole affinity of this enzyme caused by amino acid substitutions have been reported as a resistance mechanism. Nine Candida albicans strains were used in this study. The ERG11 base sequence of seven isolates, of which only two were azole-sensitive, were determined. The ERG11 base sequences of the other two strains have been published previously. In these seven isolates, 12 different amino acid substitutions were identified, of which six have not been described previously (A149V, D153E, E165Y, S279F, V452A and G4655). In addition, 16 silent mutations were found. Two different biochemical assays, subcellular sterol biosynthesis and CO binding to reduced microsomal fractions, were used to evaluate the sensitivity of the cytochromes for fluconazole and itraconazole. Enzyme preparations from four isolates showed reduced itraconazole susceptibility, whereas more pronounced resistance to fluconazole was observed in five isolates. A three-dimensional model of C. albicans Cyp51p was used to position all 29 reported substitutions, 98 in total identified in 53 sequences. These 29 substitutions were not randomly distributed over the sequence but clustered in three regions from amino acids 105 to 165, from 266 to 287 and from 405 to 488, suggesting the existence of hotspot regions. Of the mutations found in the two N-terminal regions only Y132H was demonstrated to be of importance for azole resistance. In the C-terminal region three mutations are associated with resistance, suggesting that the non-characterized substitutions found in this region should be prioritized for further analysis.

P450 inhibitors of use in medical treatment: focus on mechanisms of action.

A number of cytochrome P450s are targets for compounds that are clinically used or under clinical evaluation for treatment of patients with mycotic infections, such as dermatophytosis, superficial and systemic candidiasis, cryptococcosis and aspergillosis, with skin diseases, such as psoriasis or ichthyosis, and other retinoid-sensitive malignancies, e.g., neuro-ectodermal glioma. Some of the P450 inhibitors are candidates for the treatment of hirsutism or prostate cancer, others are potent inhibitors of the P450 isomerase involved in the synthesis of thromboxane A2, a potent platelet aggregation inducer and vasoconstrictor.

Aromatase inhibitors — mechanisms for non-steroidal inhibitors

SummaryThe conversion of androgens to estrogens occurs in a variety of cells and tissues, such as ovarian granulosa and testicular cells, placenta, adipose tissue, and various sites of the brain. The extragonadal synthesis of estrogens has great pathophysiological importance. Estrogens produced by, for example, adipose tissue have a role in the pathogenesis of certain forms of breast cancer and endometrial adenocarcinoma. The biosynthesis of estrogens is catalyzed by the aromatase, an enzyme localized in the endoplasmic reticulum that consists of two components: a cytochrome P450 (P450 Arom, P450 19 product of theCYP 19 gene) and the NADPH cytochrome P450 reductase. The alignment of the amino acid sequences of human P450 19 with other mammalian P450s shows little sequence similarity, which indicates not only that P450 19 is a unique form of the P450 superfamily but also that the aromatase may be a good target for the development of selective P450 inhibitors.Aminoglutethimide (AG) is the pioneer drug of the reversible competitive nonsteroidal aromatase inhibitors. Since AG is a nonspecific aromatase inhibitor and presents some problems with tolerability, a number of structural analogues have been synthesized. For example, rogletimide is slightly less potent than AG but has the advantage of not inhibiting the cholesterol side-chain cleavage and is devoid of sedative action. Elongation of the ethyl substituent of AG and rogletimide leads to an increase in aromatase inhibition. Further studies led to the discovery of a new generation of much more potent aromatase inhibitors. An example is fadrozole. However, although fadrozole is a poor inhibitor of the cholesterol side-chain cleavage, it suppresses aldosterone release by ACTH-stimulated human adrenocortical cells. More selective aromatase inhibitors are the triazole derivatives. Examples are CGS 20267, CGS 47645, R 76 713, and ICI D1033.R 76 713s aromatase inhibitory effect is largely due to its (+)-S-enantiomer, vorozole. Computer modeling studies of the interaction of vorozole with part of the “I-helix” of P450 19 suggest that the chlorine-substituted phenyl ring of vorozole interacts with the gamma-carbonyl group of Glu-302. Thr-310, which corresponds to the highly conserved Thr-252 in P450 101, interacts with vorozoles triazole ring, and the 1-methyl-benzotriazole moiety binds near Asp-309.

Review Article Cytochromes P450 in fungi

Summary. The article gives an overview on the history of the discovery of P450 cytochromes and on their occurrence in nature, especially on their interactions with metabolic pathways in fungi. The significance of the P450 cytochromes in the ergosterol synthesis as well as in the inhibitory mechanisms caused by imidazole and triazole antimycotics is described is detail.

On the detection of multiple-binding modes of ligands to proteins, from biological, structural, and modeling data

There are several indications that a given compound or a set of related compounds can bind in different modes to a specific binding site of a protein. This is especially evident from X-ray crystallographic structures of ligand-protein complexes. The availability of multiple binding modes of a ligand in a binding site may present an advantage in drug design when simultaneously optimizing several criteria. In the case of the design of anti-HIV compounds we observed that the more active compounds that are also resilient against mutation of the non-nucleoside binding site of HIV1-reverse transcriptase make use of more binding modes than the less active and resilient compounds.

Correlations between Factors Determining the Pharmacokinetics and Antiviral Activity of HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors of the Diaryltriazine and Diarylpyrimidine Classes of Compounds

AbstractObjective: To investigate the important factors that determine the bioavailability and the antiviral activity of the diaryltriazine (DATA) and diarylpyrimidine (DAPY) non-nucleoside reverse transcriptase inhibitors (NNRTIs) of HIV-1 in animal species and humans using cell-based assays, physicochemical and comput ed parameters. Methods: This naturalistic study included 15 parameters ranging from molecular mechanics calculations to phase I clinical trials. The calculated parameters were solvent-accessible surface area (SASA), polar surface area and Gibbs free energy of solvation. Physicochemical parameters comprised lipophilicity (octanol/water partition coefficient [cLogP]), ionisation constant (pKa), solubility and aggregate radius. Cell-based assays included human colonic adenocarcinoma cell (Caco-2) permeability (transepithelial transport), drug metabolism and antiviral activity (negative logarithm of the molar effective concentration inhibiting viral replica tion by 50% [pEC50]). Exposure was tested in rats, dogs and human volunteers. Results: Of the 15 parameters, eight correlated consistently among one another. Exposure (area under the plasma concentration-time curve [AUC]) in humans correlated positively with that in rats (r = 1.00), with transepithelial transport (r = 0.83), lipophilicity (r = 0.60), ionisability (r = 0.89), hydrodynamic radius of aggregates (r = 0.66) and with antiviral activity (r = 0.61). Exposure in humans was also seen to correlate negatively with SASA (r = −0.89). No consistent correlation was found between exposure in dogs and the eight parameters. Of the 14 DATA/DAPY molecules, 11 form aggregates with radii between 34 and 100nm. Conclusions: We observed correlations between exposure in humans with expo sure in rats, transepithelial transport (Caco-2 cells), ionisability, lipophilicity, aggregate radius and SASA in the class of DATA/DAPY NNRTI compounds. The lipophilic DATA/DAPY compounds form aggregates. It can be assumed that absorption in the intestinal tract and endocytosis in infected cells of these lipophilic compounds are governed by the common phenomenon of aggregate formation. As the lymphatic system offers a pathway for intestinal uptake of aggregates, this may offer a therapeutic advantage in the treatment of HIV-1 infection. Although it was not the objective of the study, we found that the rat was a better in vivo model than the dog for the prediction of systemic exposure in this particular set of compounds.