Libuše Pavlíčková

Simple Method for Screening Candida Species Isolates for the Presence of Secreted Proteinases: a Tool for the Prediction of Successful Inhibitory Treatment

ABSTRACT The yeasts of the genus Candida are opportunistic pathogens associated with the rising incidence of life-threatening infections in immunocompromised individuals. Secretion of aspartic proteinases has been determined to be one of the virulence factors of the pathogenic Candida species. To analyze the extracellular proteolytic activities of a large number of Candida clinical isolates, we developed a screening system based on a solid medium containing hemoglobin as the sole nitrogen source. The cleavage of hemoglobin by the secreted proteinases results in formation of clearance zones. The visibility of such zones was enhanced by addition of an acid-base indicator. Using this system, we assessed 245 clinical isolates of Candida from patients in the hospital of the Faculty of Medicine, Palacky University, Olomouc, Czech Republic, for the presence of secreted aspartic proteases (Saps). We also used the test plates for rapid semiquantitative testing of Sap inhibitors. Most of the pepstatin analogs affected the formation of the zones of clearance as well as the growth of Candida albicans, C. tropicalis, and C. parapsilosis colonies. By contrast, the human immunodeficiency virus proteinase inhibitors saquinavir, ritonavir, nelfinavir, and indinavir had no effect on the Candida strains tested. These results are in agreement with the inhibition constants obtained for the individual inhibitors with purified Saps. Thus, the plates containing hemoglobin proved to be an appropriate tool for the rapid and reliable assessment of Sap production and inhibition.

Structure-based specificity mapping of secreted aspartic proteases of Candida parapsilosis, Candida albicans, and Candida tropicalis using peptidomimetic inhibitors and homology modeling

Abstract Secreted aspartic proteases (Saps) of pathogenic Candida spp. represent a specific target for antifungal drug development. We synthesized a series of peptidomimetic inhibitors with different isosteric groups and modifications at individual positions and tested them with purified Saps from C. albicans (Sap2p), C. tropicalis (Sapt1p), and C. parapsilosis (Sapp1p). The kinetic parameters indicated that all three proteases prefer binding of inhibitors containing bulky hydrophobic residues between positions P3 and P3′. The most divergent specificity was found for Sapp1p. The sequence alignment of Sap2p, Sapt1p, and Sapp1p, and homology modeling of Sapp1p with the crystal structure of Sapt1p and the complex of Sap2p with a peptidomimetic inhibitor showed that the overall folds of Sap2p, Sapt1p, and Sapp1p are similar. However, the N- and C-terminal loops formed by disulfide bonds between residues 47–53 and 258–292 are significantly shorter in Sapp1p, and a unique insertion following Tyr 129 in Sapp1p results in the formation of a loop that can interact with inhibitor residues. These Sapp1p structural differences might lead to its altered susceptibility to inhibition.

A Convenient Preparation of 4-Alkylidene-2,5-cyclohexadien-1-ones

Abstract 4-Alkylidene-2,5-cyclohexadien-1-ones (p-qui-nonemethides) have been prepared either by the oxidation of 4-alkylphenols with inorganic reagents (potassium ferricyanide1, manganese dioxide2, silver oxide3), or by the dehydrohalogenation of substituted 4-hydroxybenzyl chlorides (or bromides) with tertiary aliphatic amines4 or alkali carbonates5. The oxidation method is limited by the accessibility and poor stability of the starting phenols: whereas a serious disadvantage of the dehydrohalogenation methods is the necessity of preparing benzyl halides in a rather pure form.

15gag proteinase of myeloblastosis-associated virus: specificity studies with substrate-based inhibitors.

The key role of retrovirus-encoded proteinases in limited proteolysis of polyprotein precursors, a prerequisite of the formation of mature infectious virions, makes these enzymes attractive targets of specific inhibitors. A rational basis for the design of such inhibitors requires detailed knowledge of the proteinase-inhibitor interactions at the level of three-dimensional structures. Such data are available so far only for the HIV-1 proteinase whose crystal complexes with some inhibitors were subject of X-ray studies.1 The absence of such information on the myeloblastosis associated virus (MAV) proteinase, another retroviral aspartate proteinase, together with the need of finding a tight-binding yet hydrophilic inhibitor for both X-ray studies and titration of its active site, led us to investigate the interactions of the proteinase with various classes of custom-synthesized inhibitors.