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. 2012 Aug;85(4):747-67.
doi: 10.1111/j.1365-2958.2012.08140.x. Epub 2012 Jul 13.

Specific interactions between the Candida albicans ABC transporter Cdr1p ectodomain and a D-octapeptide derivative inhibitor

Kyoko Niimi  1 , David R K Harding, Ann R Holmes, Erwin Lamping, Masakazu Niimi, Joel D A Tyndall, Richard D Cannon, Brian C Monk
Affiliations

Specific interactions between the Candida albicans ABC transporter Cdr1p ectodomain and a D-octapeptide derivative inhibitor

Kyoko Niimi et al. Mol Microbiol. 2012 Aug.

Abstract

Overexpression of the Candida albicans ATP-binding cassette transporter CaCdr1p causes clinically significant resistance to azole drugs including fluconazole (FLC). Screening of a ~1.89 × 10(6) member D-octapeptide combinatorial library that concentrates library members at the yeast cell surface identified RC21v3, a 4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative of the D-octapeptide D-NH(2) -FFKWQRRR-CONH(2) , as a potent and stereospecific inhibitor of CaCdr1p. RC21v3 chemosensitized Saccharomyces cerevisiae strains overexpressing CaCdr1p but not other fungal ABC transporters, the C. albicans MFS transporter CaMdr1p or the azole target enzyme CaErg11p, to FLC. RC21v3 also chemosensitized clinical C. albicans isolates overexpressing CaCDR1 to FLC, even when CaCDR2 was overexpressed. Specific targeting of CaCdr1p by RC21v3 was confirmed by spontaneous RC21v3 chemosensitization-resistant suppressor mutants of S. cerevisiae expressing CaCdr1p. The suppressor mutations introduced a positive charge beside, or within, extracellular loops 1, 3, 4 and 6 of CaCdr1p or an aromatic residue near the extracytoplasmic end of transmembrane segment 5. The mutations did not affect CaCdr1p localization or CaCdr1p ATPase activity but some increased susceptibility to the CaCdr1p substrates FLC, rhodamine 6G, rhodamine 123 and cycloheximide. The suppressor mutations showed that the drug-like CaCdr1p inhibitors FK506, enniatin, milbemycin α11 and milbemycin β9 have modes of action similar to RC21v3.

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Figures

Fig. 1
Fig. 1
Screens and counterscreens for ABC transporter chemosensitizer discovery in a D-octapeptide combinatorial library. Strain AD/CaCDR1 was used in cell-based primary screens (ia, growth inhibition screen in the absence of FLC; iia, chemosensitization screen in the presence of 40 μg ml−1 FLC) and to provide an enriched plasma membrane fraction containing CaCdr1p for the in vitro secondary screen (iii, oligomycin sensitive CaCdr1p ATPase activity screen). Strain AD/CaMDR1 was used in cell-based counterscreens (ib, a growth inhibition screen in the absence of FLC; iib, a chemosensitization screen in the presence of 10 μg ml−1 FLC). Screening was carried out using the following steps. 1) The combinatorial library pool d-NH2-FFX3X2X1RRR-CONH2 was identified as giving strong chemosensitization of AD/CDR1 and the lowest CaMDR1/CaCDR1 chemosensitization ratio based on the relative sizes of the growth inhibition zones in agarose diffusion assays for chemosensitization of AD/CaCDR1 to FLC (40 μg ml−1) and AD/CaMDR1 to FLC (10 μg ml−1). 2) This pool also gave strong inhibition the in vitro oligomycin sensitive ATPase activity of CaCdr1p. 3) The process was repeated (iv) with resynthesized subpools (stages 2-4). HPLC purified peptides, quality assured using mass spectrometry analysis, were used to ensure that corrected lead had been identified in stage 4.
Fig. 2
Fig. 2
Discovery of RC21 by deconvolution of a d-octapeptide combinatorial library. A, Agarose diffusion assays used to screen for chemosensitization of AD/CaCDR1B to FLC. B, Agarose diffusion assay used as a counterscreen by comparing chemosensitization to FLC of AD/CaMDR1 with AD/CaCDR1B.
Fig. 3
Fig. 3
Specificity of RC21 as a chemosensitizer. The drug efflux pump substrate FLC was used at concentrations that were 4x lower than the FLC MIC for each strain. A, Agarose diffusion assays showing RC21 chemosensitization to FLC of S. cerevisiae cells expressing drug efflux pumps or CaErg11p. Crude RC21 (not HPLC purified) was applied at 6 nmole/disk in the presence or absence of the indicated concentration of FLC. B, Agarose diffusion assays showing RC21 chemosensitization to FLC of C. albicans clinical isolates that overexpress CaCdr1p. Crude RC21 was applied at 48 nmole/disk in the presence or absence of the indicated concentration of FLC for all strains except CIA4 and MML33, for which 7.5 nmole of RC21 was applied. Strains ATC10261, IFO1060, MML610 and CIA4 are FLC susceptible strains that express CaCdr1p at low levels (Holmes et al., 2008). MML31 is derived from CIA4 by deletion of both alleles of CaCdr1p.
Fig. 4
Fig. 4
Stereospecificity of RC21 and localization of the Mtr-group in RC21 required for chemosensitization. Crude D-RC21 was compared with the HPLC purified peptides d- and l-RC21v1 and v2 quantitated spectrophotometrically. A, each peptide was used at 6 nmole/disk in the presence and absence of the indicated concentrations of FLC. The Mtr group required for AD/CaCDR1B chemosensitization is associated with an arginine residue (R6, R7 or R8) and not the tryptophan residue (W4). B, d-RC21 was used at 6 nmol per disk and the HPLC purified L-peptides were used at 28 nmole/disk. D-RC21 but not L-RC21 chemosensitized AD/CaCDR1B to FLC.
Fig. 5
Fig. 5
Localization of Mtr group on RC21 required for inhibition of R6G pumping by CaCdr1p. R6G efflux in the presence and absence of glucose was used to identify the glucose-dependent efflux of R6G from AD/CDR1 cells exposed to the indicated concentrations of: A, the mixed Mtr derivative crude RC21; B, the HPLC-purified peptide RC1; C, the HPLC-purified derivative RC21v1 (W4-Mtr); D, the HPLC-purified derivatives RC21v2 (R6-R8-Mtr). The efflux of R6G from preloaded cells into the supernatant fraction was measured as described under Experimental Procedures in the presence formula image or absence formula image of glucose. The glucose-sensitive efflux remaining after RC21 treatment is shown formula image.
Fig. 6
Fig. 6
Chemosensitization of AD/CaCDR1 requires an Mtr group on the R6 sidechain of RC21. Agarose diffusion assays were used to measure the potency of HPLC-purified congeners of RC21 and their derivatives. Chemosensitization in agarose diffusion assays was carried out as described under Experimental Procedures using strain AD/CaCDR1. Crude RC21 or HPLC purified peptide was applied at 6 nmole/disk in the presence or absence of FLC (40 μg ml−1).
Fig. 7
Fig. 7
Toxicity of RC21. A, Crude RC21, and its HPLC purified peptide backbone RC1 and HPLC-purified derivatives RC21v2 (≤100 μM) did not hemolyse human red blood cells unlike amphotericin B (AMB) which caused complete hemolysis at 3 μM. B, RC21v2 (≤100 μM) did not kill cultured HEp2 cells.
Fig. 8
Fig. 8
Molecular genetic analysis of RC21-resistant variants. A, Selection and stability of RC21 chemosensitization-resistant variants. Examples of RC21 variants (strongly resistant C1 and C2 and weakly resistant E1 and E2) selected from within the zone of inhibition and retested in an agarose diffusion assays are shown. B, Checkerboard assay showing suppression of synergy between RC21 chemosensitization of AD/CaCDR1B to FLC by the RC21 chemosensitization-resistant variant AD/CDR1B-C2. C, Mutations in RC21 chemosensitization-resistant variants occur within the CaCDR1B-URA3 transformation cassette. The CaCDR1B-Ura3 transformation cassette was obtained by PCR of the genomic DNA from strain AD/CDR1-C2 and used to transform the null parental strain AD1-8u− to Ura+. Selection on CSM-ura medium gave the strain AD/CDR1-C2.1 which showed RC21 chemosensitization-resistance identical to AD/CDR1-C2.
Fig. 9
Fig. 9
A map of RC21 suppressor mutations using a CaCdr1p homology model. The open (no ATP bound) CaCdr1p model, viewed from the side (A) and from the periplasm (B), was generated in Modeller 9v7 (Eswar et al., 2006) using the S. cerevisiae Pdr5p model of Rutledge and colleagues (Rutledge et al., 2010). A sequence alignment was generated for CaCdr1p and ScPdr5p using clustalW. Coordinates for the Pdr5p model were kindly provided by Robert Rutledge. Conserved amino acid sidechains of the PDR motif A are shown in red, the connecting sequence in blue and the PDR motif B in yellow. Conserved amino acid sidechains of the EL6 hydrophobic motif are shown in orange. Amino acid side chains of 5 of the suppressor mutants are shown as spheres, with carbons in grey and nitrogens in blue.
Fig. 10
Fig. 10
CaCdr1ps in RC21 chemosensitization-resistant variants have functional ATPase activities localized to the plasma membrane. A, SDS-PAGE of plasma membrane preparations obtained from AD1-8u−, AD/CaCDR1 and representative RC21 and RC21v2 suppressor mutants. B, CaCdr1p ATPase activity of plasma membrane preparations described in A. ATPase activity was measured as described under Experimental Procedures. The CaCdr1p ATPase activity measurements shown are for a representative of two independent experiments, with duplicate measurements of ATPase activity sensitive to vanadate (100 μM), oligomycin (20 μM) and FK506 (10 μg/ml) which did not vary by more than 5%.
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