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1.
Biochim Biophys Acta ; 1858(11): 2858-2870, 2016 11.
Article in English | MEDLINE | ID: mdl-27569110

ABSTRACT

The ABC (ATP-Binding Cassette) transporter Cdr1 (Candida drug resistance 1) protein (Cdr1p) of Candida albicans, shows promiscuity towards the substrate it exports and plays a major role in antifungal resistance. It has two transmembrane domains (TMDs) comprising of six transmembrane helices (TMH) that envisage and confer the substrate specificity and two nucleotide binding domains (NBDs), interconnected by extracellular loops (ECLs) and intracellular loops (ICLs) Cdr1p. This study explores the diverse substrate specificity spectrum to get a deeper insight into the structural and functional features of Cdr1p. By screening with the variety of compounds towards an in-house TMH 252 mutant library of Cdr1p, we establish new substrates of Cdr1p. The localization of substrate-susceptible mutants in an ABCG5/G8 homology model highlights the common and specific binding pockets inside the membrane domain, where rhodamines and tetrazoliums mainly engage the N-moiety of Cdr1p, binding between TMH 2, 11 and surrounded by TMH 1, 5. Whereas, tin chlorides involve both N and C moieties located at the interface of TMH 2, 11, 1 and 5. Further, screening of the in house TMH mutant library of Cdr1p displays the TMH12 interaction with tetrazolium chloride, trimethyltin chloride and a Ca2+ ionophore, A23187. In silico localization reveals a binding site at the TMH 12, 9 and 10 interface, which is widely exposed to the lipid interface. Together, for the first time, our study shows the molecular localization of Cdr1p substrates-binding sites and demonstrates the participation of TMH12 in a peripheral drug binding site.


Subject(s)
Amino Acids/chemistry , Antifungal Agents/metabolism , Drug Resistance, Multiple, Fungal/genetics , Fungal Proteins/chemistry , Membrane Transport Proteins/chemistry , Mutation , Amino Acid Substitution , Amino Acids/metabolism , Antifungal Agents/pharmacology , Binding Sites , Calcimycin/metabolism , Calcimycin/pharmacology , Candida albicans/chemistry , Fungal Proteins/antagonists & inhibitors , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Expression , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Microbial Sensitivity Tests , Molecular Docking Simulation , Mutagenesis , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Rhodamines/metabolism , Rhodamines/pharmacology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Structural Homology, Protein , Substrate Specificity , Tetrazoles/metabolism , Tetrazoles/pharmacology , Tin Compounds/metabolism , Tin Compounds/pharmacology
2.
Bioorg Med Chem ; 25(13): 3278-3284, 2017 07 01.
Article in English | MEDLINE | ID: mdl-28479022

ABSTRACT

Macrocyclic diterpenes were previously found to be able to modulate the efflux pump activity of Candida albicans multidrug transporters. Most of these compounds were jatrophanes, but only a few number of lathyrane-type diterpenes was evaluated. Therefore, the aim of this study was to evaluate the ability of nineteen structurally-related lathyrane diterpenes (1-19) to overcome the drug-efflux activity of Cdr1p and Mdr1p transporters of C. albicans, and get some insights on their structure-activity relationships. The transport assay was performed by monitoring Nile Red (NR) efflux in a Saccharomyces cerevisiae strain overexpressing the referred efflux pumps from C. albicans. Moreover, a chemosensitization assay was performed in order to evaluate the type of interaction between the inhibitory compounds and the antifungal drug fluconazole. Compounds 1-13 were previously isolated from Euphorbia boetica or obtained by derivatization, and compounds 14-19 were prepared by chemical transformations of compound 4. In the transport assays, compounds 14-19 revealed the strongest inhibitory activity of the Cdr1p efflux pump, ranging from 65 to 85%. Concerning Mdr1p efflux pump, the most active compounds were 1, 3, 6, 8, and 12 (75-85%). When used in combination with fluconazole, epoxyboetirane K (2) and euphoboetirane N (18) revealed synergistic effects in the AD-CDR1 yeast strain, overexpressing the Cdr1p transporter, through their ability to reduce the effective concentration of the antifungal drug by 23- and 52-fold, respectively.


Subject(s)
Antifungal Agents/pharmacology , Candida albicans/drug effects , Diterpenes/pharmacology , Fungal Proteins/antagonists & inhibitors , Models, Biological , Saccharomyces cerevisiae/metabolism , Transcription Factors/antagonists & inhibitors , Antifungal Agents/chemical synthesis , Antifungal Agents/chemistry , Biological Transport/drug effects , Candida albicans/metabolism , Diterpenes/chemical synthesis , Diterpenes/chemistry , Dose-Response Relationship, Drug , Fungal Proteins/metabolism , Membrane Transport Proteins/metabolism , Microbial Sensitivity Tests , Molecular Structure , Structure-Activity Relationship , Transcription Factors/metabolism
3.
J Nat Prod ; 80(2): 479-487, 2017 02 24.
Article in English | MEDLINE | ID: mdl-28106996

ABSTRACT

Twenty-nine jatrophane esters (1-10, 12-30) and one lathyrane (11) diterpenoid ester isolated from Euphorbia species were evaluated for their capacity to inhibit drug-efflux activities of the primary ABC transporter CaCdr1p and the secondary MFS transporter CaMdr1p of Candida albicans, in yeast strains overexpressing the corresponding transporter. These diterpenoid esters were obtained from Euphorbia semiperfoliata (1-10), E. insularis (11), and E. dendroides (12-30) and included five new compounds, euphodendroidins P-T (26-30). The jatrophane esters 12 and 23 were found to inhibit the efflux of Nile Red (NR) mediated by the two multidrug transporters, at 85-64% for CaCdr1p and 79-65% for CaMdr1p. In contrast, compound 21 was selective for CaCdr1p and induced a strong inhibition (92%), whereas compound 8 was selective for CaMdr1p, with a 74% inhibition. It was demonstrated further that potency and selectivity are sensitive to the substitution pattern on the jatrophane skeleton. However, these compounds were not transported and showed no synergism with fluconazole cytotoxicity.


Subject(s)
Antifungal Agents/isolation & purification , Antifungal Agents/pharmacology , Candida albicans/metabolism , Diterpenes/isolation & purification , Diterpenes/pharmacology , Euphorbia/chemistry , ATP-Binding Cassette Transporters/metabolism , Antifungal Agents/chemistry , Biological Transport/drug effects , Candida albicans/drug effects , Diterpenes/chemistry , Esters , Fluconazole/pharmacology , Membrane Transport Proteins/metabolism , Microbial Sensitivity Tests , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular
4.
Planta Med ; 82(13): 1180-5, 2016 Aug.
Article in English | MEDLINE | ID: mdl-27145238

ABSTRACT

Thirteen macrocyclic diterpenes (1-13) of the jatrophane and lathyrane types, either isolated from Euphorbia species or obtained by chemical derivatization, were evaluated for their ability to inhibit the drug efflux activity of Candida albicans CaCdr1p and CaMdr1p multidrug transporters overexpressed in a Saccharomyces cerevisiae strain. Their inhibitory potential was assessed through a functional assay of Nile Red accumulation monitored by flow cytometry. A chemosensitization assay, using the checkerboard method, was also performed with the active compounds in order to evaluate their type of interaction with fluconazole.In the transport assay, most compounds were found to inhibit both transporters, most likely as non-substrates, as shown by relative resistance indices close to unity. In contrast, the jatrophanes euphopubescenol (10) and euphomelliferene A (11) were selective for CaMdr1p and CaCdr1p, respectively. Moreover, when used in combination with fluconazole, compounds 12 and 13 displayed strong synergistic interactions (FICI = 0.071) against the yeast strain overexpressing CaMdr1p, decreasing the MIC80 of the antifungal agent 13-fold. Both compounds were also able to reduce the effective concentration of this antifungal agent by 4- to 8-fold against an azole-resistant clinical isolate of C. albicans (F5).


Subject(s)
Antifungal Agents/pharmacology , Candida albicans/drug effects , Diterpenes/pharmacology , Drug Resistance, Multiple, Fungal , Euphorbia/chemistry , Membrane Transport Proteins/drug effects , Plant Extracts/pharmacology , Antifungal Agents/isolation & purification , Diterpenes/isolation & purification , Microbial Sensitivity Tests
5.
FEMS Yeast Res ; 14(4): 624-32, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24628911

ABSTRACT

FK520, a homolog of antifungal FK506, displays fungicidal synergism with azoles in Candida albicans and inhibits drug efflux mediated by ABC multidrug transporter. This study establishes the molecular basis of interaction of FK520 with Cdr1 protein, which is one of the major ABC multidrug transporters of C. albicans. For this, we have exploited an in-house library of Cdr1 protein consisting of 252 mutant variants where the entire primary structure of the two transmembrane domains comprising of 12 transmembrane helices was subjected to alanine scanning. With these mutant variants of Cdr1 protein, we could identify the critical amino acids of the transporter protein, which if replaced with alanine, not only abrogated FK520-dependent competitive inhibition of drug efflux but simultaneously decreased susceptibility to azoles. Notably, the replacement of most of the residues with alanine was inconsequential; however, there were close to 13% mutant variants, which showed abrogation of drug efflux and reversal of fungicidal synergy with azoles. Of note, all the intrahelical residues of Cdr1 protein, which abrogated inhibitor's ability to block the efflux and reversed fungicidal synergy, were common. Taken together, our results provide evidence of cross-talk of FK520 with Cdr1 by interacting with the select intrahelical residues of the protein to chemosensitize isolates of Candida.


Subject(s)
Antifungal Agents/pharmacology , Azoles/pharmacology , Candida albicans/drug effects , Drug Resistance, Fungal/drug effects , Fungal Proteins/metabolism , Membrane Transport Proteins/metabolism , Antifungal Agents/metabolism , Azoles/metabolism , DNA Mutational Analysis , Protein Binding
6.
RSC Adv ; 10(5): 2915-2931, 2020 Jan 14.
Article in English | MEDLINE | ID: mdl-35496110

ABSTRACT

Two series of piperazinyl-pyrrolo[1,2-a]quinoxaline derivatives were prepared via a Buchwald-Hartwig cross-coupling reaction and then evaluated for their ability to inhibit the drug efflux activity of CaCdr1p and CaMdr1p transporters of Candida albicans overexpressed in a Saccharomyces cerevisiae strain. In the initial screening of twenty-nine piperazinyl-pyrrolo[1,2-a]quinoxaline derivatives, twenty-three compounds behaved as dual inhibitors of CaCdr1p and CaMdr1p. Only four compounds showed exclusive inhibition of CaCdr1p or CaMdr1p. Further biological investigations were developed and for example, their antifungal potential was evaluated by measuring the growth of control yeast cells (AD1-8u-) and efflux pump-overexpressing cells (AD-CDR1 and AD-MDR1) after exposition to variable concentrations of the tested compounds. The MIC80 values of nineteen compounds ranging from 100 to 901 µM for AD-CDR1 demonstrated that relative resistance index (RI) values were between 8 and 274. In comparison, only seven compounds had RI values superior to 4 in cells overexpressing Mdr1p. These results indicated substrate behavior for nineteen compounds for CaCdr1p and seven compounds for CaMdr1p, as these compounds were transported via MDR transporter overexpressing cells and not by the AD1-8u- cells. Finally, in a combination assay with fluconazole, two compounds (1d and 1f) have shown a synergistic effect (fractional inhibitory concentration index (FICI) values ≤ 0.5) at micromolar concentrations in the AD-MDR1 yeast strain overexpressing CaMdr1p-protein, indicating an excellent potency toward chemosensitization.

7.
Future Med Chem ; 10(18): 2177-2186, 2018 Sep 01.
Article in English | MEDLINE | ID: mdl-30043631

ABSTRACT

AIM: Resistance against antifungals used for Candida albicans (Ca) treatment is mediated by two multidrug transporters, Mdr1p and Cdr1p, which are of enormous interest to the development of modulators combined with antifungals. EXPERIMENTAL: A set of chalcones was synthesized by condensation reactions in laboratory and was then subject to biological assays to evaluate the effects on different yeast strains.  Results: The obtained chalcones were screened using the checkerboard liquid chemosensitization assays. Compounds 4, 10, 12 and 18, when combined with fluconazole, triggered strong sensitization on yeast strains overexpressing CaMdr1p and CaCdr1p, whereas displaying no cytotoxicity by themselves towards control strains and transporter-expressing yeast cells. In the Nile Red transport assay, the two most active compounds, 12 and 18 showed moderate-to-high accumulation of Nile Red with different behaviors towards the two transporters. CONCLUSION: Chalcones are promising drug candidates for further development to make azole antifungals active again.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Antifungal Agents/chemistry , Azoles/chemistry , Candida albicans/metabolism , Drug Resistance, Fungal , Fungal Proteins/metabolism , ATP-Binding Cassette Transporters/antagonists & inhibitors , Antifungal Agents/pharmacology , Azoles/pharmacology , Candida albicans/drug effects , Chalcones/chemistry , Chalcones/pharmacology , Drug Resistance, Fungal/drug effects , Fungal Proteins/antagonists & inhibitors , Oxazines/metabolism , Structure-Activity Relationship
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