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.

[1]  A. Edison,et al.  Comparison of the extracellular proteinase activity produced by a low-virulence mutant of Candida albicans and its wild-type parent , 1988, Infection and immunity.

[2]  R. Johnson.  Renin inhibitors. Substitution of the leucyl residues of Leu-Leu-Val-Phe-OCH3 with 3-amino-2-hydroxy-5-methylhexanoic acid. , 1982, Journal of medicinal chemistry.

[3]  F. Salituro,et al.  Inhibition of aspartic proteases by pepstatin and 3-methylstatine derivatives of pepstatin. Evidence for collected-substrate enzyme inhibition. , 1985, Biochemistry.

[4]  T. L. Blundell,et al.  High resolution X-ray analyses of renin inhibitor-aspartic proteinase complexes , 1987, Nature.

[5]  D. Nisato,et al.  Stereospecific synthesis of N-protected statine and its analogues via chiral tetramic acid , 1987 .

[6]  I. Pichová,et al.  Enzymological Characterization of Secreted Proteinases from Candida parapsilosis and Candida lusitaniae , 2001 .

[7]  J. Collins,et al.  Structure‐based subsite specificity mapping of human cathepsin D using statine‐based inhibitors , 1997, Protein science : a publication of the Protein Society.

[8]  K. Haynes.  Virulence in Candida species. , 2001, Trends in microbiology.

[9]  P. Tamburini,et al.  Role of aspartic proteases in disseminated Candida albicans infection in mice , 1997, Infection and immunity.

[10]  T. Bertsch,et al.  Germ Tubes and Proteinase Activity Contribute to Virulence of Candida albicans in Murine Peritonitis , 1999, Infection and Immunity.

[11]  D. Coy,et al.  Solid phase synthesis of peptides containing the CH2NH peptide bond isostere , 1987, Peptides.

[12]  D. Rich,et al.  Inhibition of cathepsin D by substrate analogues containing statine and by analogues of pepstatin. , 1986, Journal of medicinal chemistry.

[13]  T L Blundell,et al.  X-ray studies of aspartic proteinase-statine inhibitor complexes. , 1991, Biochemistry.

[14]  S. C. Smith,et al.  Synthesis of all the stereoisomers of statine (4-amino-3-hydroxy-6-methylheptanoic acid). Inhibition of pepsin activity by N-carbobenzoxy-L-valyl-L-valyl-statine derived from the four stereoisomers. , 1979, Journal of medicinal chemistry.

[15]  B. Dunn,et al.  Extracellular aspartic proteinases from Candida albicans, Candida tropicalis, and Candida parapsilosis yeasts differ substantially in their specificities. , 1994, Biochemistry.

[16]  I. Pichová,et al.  Secreted aspartic proteases of Candida albicans, Candida tropicalis, Candida parapsilosis and Candida lusitaniae. Inhibition with peptidomimetic inhibitors. , 2001, European journal of biochemistry.

[17]  J. Symerský,et al.  High-resolution structure of the extracellular aspartic proteinase from Candida tropicalis yeast. , 1997, Biochemistry.

[18]  A. Cassone,et al.  Biotyping and Virulence Properties of Skin Isolates ofCandida parapsilosis , 1999, Journal of Clinical Microbiology.

[19]  I. Pichová,et al.  Simple Method for Screening Candida Species Isolates for the Presence of Secreted Proteinases: a Tool for the Prediction of Successful Inhibitory Treatment , 2003, Journal of Clinical Microbiology.

[20]  B. Hube,et al.  Candida albicans proteinases: resolving the mystery of a gene family. , 2001, Microbiology.

[21]  B. Dunn.  Structure and mechanism of the pepsin-like family of aspartic peptidases. , 2002, Chemical reviews.

[22]  S. Foundling,et al.  Enzymic characteristics of secreted aspartic proteases of Candida albicans. , 2000, Biochimica et biophysica acta.

[23]  Professor Dr. Miklos Bodanszky,et al.  The Practice of Peptide Synthesis , 1994, Springer Lab Manual.

[24]  C. Abad-Zapatero,et al.  Candida proteases and their inhibition: prospects for antifungal therapy. , 2001, Current medicinal chemistry.

[25]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[26]  J. Navaza,et al.  Structure of a secreted aspartic protease from C. albicans complexed with a potent inhibitor: Implications for the design of antifungal agents , 1996, Protein science : a publication of the Protein Society.

[27]  J. F. Höfling,et al.  Candida albicans proteinases , 2006 .

[28]  R. Falchetto,et al.  Cloning and sequencing of two Candida parapsilosis genes encoding acid proteases. , 1993, Journal of general microbiology.

[29]  C. Abad-Zapatero,et al.  Antifungal drug targets: Candida secreted aspartyl protease and fungal wall beta-glucan synthesis. , 1995, Infectious agents and disease.

[30]  G. Gilfillan,et al.  Candida dubliniensis: phylogeny and putative virulence factors. , 1998, Microbiology.

[31]  Dan Backman,et al.  Kinetic and mechanistic analysis of the association and dissociation of inhibitors interacting with secreted aspartic acid proteases 1 and 2 from Candida albicans. , 2003, Biochimica et biophysica acta.

[32]  G. Santoni,et al.  De Bernardis, F. et al. Protective role of anti-mannan and anti-aspartyl proteinase antibodies in an experimental model of Candida albicans vaginitis in rats. Infect. Immun. 65, 3399-3405 , 1997 .

[33]  T. Aoyagi,et al.  Pepstatin, a new pepsin inhibitor produced by Actinomycetes. , 1970, The Journal of antibiotics.

[34]  B. Anderson,et al.  The crystal structure of a major secreted aspartic proteinase from Candida albicans in complexes with two inhibitors. , 1995, Structure.

[35]  S. Challacombe,et al.  Candida albicans Secreted Aspartyl Proteinases in Virulence and Pathogenesis , 2003, Microbiology and Molecular Biology Reviews.

[36]  B. Hube,et al.  Multiplicity of genes encoding secreted aspartic proteinases in Candida species , 1994, Molecular microbiology.

[37]  U. Reichard,et al.  Secreted Aspartic Proteinase Family ofCandida tropicalis , 2001, Infection and Immunity.

[38]  J. Bobbitt,et al.  Organic oxoammonium salts. 3. A new convenient method for the oxidation of alcohols to aldehydes and ketones , 1991 .

[39]  P. Štrop,et al.  Cloning, Bacterial Expression, and Characterization of the Mason-Pfizer Monkey Virus Proteinase (*) , 1995, The Journal of Biological Chemistry.