A series of structurally related jatrophane diterpenoids (1-6), including the new euphosquamosins... more A series of structurally related jatrophane diterpenoids (1-6), including the new euphosquamosins A-C (4-6), was purified from the Iranian spurge Euphorbia squamosa and evaluated for its capacity to inhibit drug efflux by multidrug transporters of Candida albicans. Three of these compounds showed an interesting profile of activity. In particular, deacetylserrulatin B (2) and euphosquamosin C (6) strongly inhibited the drug-efflux activity of the primary ABC-transporter CaCdr1p, an effect that translated, in a yeast strain overexpressing this transporter, into an increased sensitivity to fluconazole. These compounds were transported by CaCdr1p, as shown by the observation of an 11-14-fold cross-resistance of yeast growth, and could also inhibit the secondary MFS-transporter CaMdr1p. In contrast, euphosquamosin A (4) was selective for CaCdr1p, possibly as a result of a different binding mode. Taken together, these observations suggest jatrophane diterpenes to be a new class of potent inhibitors of multidrug transporters critical for drug resistance in pathogenic yeasts.
Thirteen macrocyclic diterpenes (1-13) of the jatrophane and lathyrane types, either isolated fro... more 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 an...
Reduced intracellular accumulation of drugs (due to rapid efflux) mediated by the efflux pump pro... more Reduced intracellular accumulation of drugs (due to rapid efflux) mediated by the efflux pump proteins belonging to ABC (ATP Binding Cassette) and MFS (Major Facilitators) superfamily is one of the most common strategies adopted by multidrug resistance (MDR) pathogenic yeasts. To combat MDR, it is essential to understand the structure and function of these transporters so that inhibitors/modulators to these can be developed. The sequence alignments of the ABC transporters reveal selective divergence within much conserved domains of Nucleotide-Binding Domains (NBDs) which is unique to all fungal transporters. Recently, the role of conserved but divergent residues of Candida Drug Resistance 1 (CDR1), an ABC drug transporter of human pathogenic Candida albicans, has been examined with regard to ATP binding and hydrolysis. In this paper, we focus on some of the recent advances on the relevance of divergent and conserved amino acids of CaCdr1p and also discuss as to how drug interacts wi...
The ABC transporter Cdr1 protein of Candida albicans, which plays a major role in antifungal resi... more The ABC transporter Cdr1 protein of Candida albicans, which plays a major role in antifungal resistance, has two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). The 12 transmembrane helices of TMDs that are interconnected by extracellular and intracellular loops (ICLs) mainly harbor substrate recognition sites where drugs bind while cytoplasmic NBDs hydrolyze ATP which powers drug efflux. The coupling of ATP hydrolysis to drug transport requires proper communication between NBDs and TMDs typically accomplished by ICLs. This study examines the role of cytoplasmic ICLs of Cdr1p by rationally predicting the critical residues on the basis of their interatomic distances. Among nine pairs that fall within a proximity of <4 Å, an ion pair between K577 of ICL1 and E315 of NBD1 was found to be critical. The substitution, swapping and changing of the length or charge of K577 or E315 by directed mutagenesis led to a misfolded, non-rescuable protein entrapped in intra...
There are currently few antifungals in use which show efficacy against fungal diseases. These ant... more There are currently few antifungals in use which show efficacy against fungal diseases. These antifungals mostly target specific components of fungal plasma membrane or its biosynthetic pathways. However, more recent class of antifungals in use is echinocandins which target the fungal cell wall components. The availability of mostly fungistatic antifungals in clinical use, often led to the development of tolerance to these very drugs by the pathogenic fungal species. Thus, the development of clinical multidrug resistance (MDR) leads to higher tolerance to drugs and its emergence is helped by multiple mechanisms. MDR is indeed a multifactorial phenomenon wherein a resistant organism possesses several mechanisms which contribute to display reduced susceptibility to not only single drug in use but also show collateral resistance to several drugs. Considering the limited availability of antifungals in use and the emergence of MDR in fungal infections, there is a continuous need for the ...
Drug-resistant pathogenic fungi use several families of membrane-embedded transporters to efflux ... more Drug-resistant pathogenic fungi use several families of membrane-embedded transporters to efflux antifungal drugs from the cells. The efflux pump Cdr1 (Candida drug resistance 1) belongs to the ATP-binding cassette (ABC) superfamily of transporters. Cdr1 is one of the most predominant mechanisms of multidrug resistance in azole-resistant (AR) clinical isolates of Candida albicans. Blocking drug efflux represents an attractive approach to combat the multidrug resistance of this opportunistic human pathogen. In this study, we rationally designed and synthesized transmembrane peptide mimics (TMPMs) of Cdr1 protein (Cdr1p) that correspond to each of the 12 transmembrane helices (TMHs) of the two transmembrane domains of the protein to target the primary structure of the Cdr1p. Several FITC-tagged TMPMs specifically bound to Cdr1p and blocked the efflux of entrapped fluorescent dyes from the AR (Gu5) isolate. These TMPMs did not affect the efflux of entrapped fluorescent dye from cells e...
The molecular basis of polyspecificity of Mdr1p, a major drug/H+ antiporter of Candida albicans, ... more The molecular basis of polyspecificity of Mdr1p, a major drug/H+ antiporter of Candida albicans, is not elucidated. We have probed the nature of the drug-binding pocket by performing systematic mutagenesis of the 12 transmembrane segments. Replacement of the 252 amino acid residues with alanine or glycine yielded 2/3 neutral mutations while 1/3 led to the complete or selective lost of resistance to drugs or substrates transported by the pump. Using the GlpT-based 3D-model of Mdr1p, we roughly categorized these critical residues depending on their type and localization 1°/ main structural impact ("S" group), 2°/ exposure to the lipid interface ("L" group), 3°/ buried but not facing the main central pocket, inferred as critical for the overall H+/drug antiport mechanism ("M" group) and finally 4°/ buried and facing the main central pocket ("B" group). Among "B" category, 13 residues were essential for the large majority of drugs/substr...
The present study examines the kinetics of steroids efflux mediated by the Candida drug resistanc... more The present study examines the kinetics of steroids efflux mediated by the Candida drug resistance protein 1 (Cdr1p) and evaluates their interaction with the protein. We exploited our in-house mutant library for targeting the 252 residues forming the twelve transmembrane helices (TMHs) of Cdr1p. The screening revealed 65 and 58 residues critical for β-estradiol and corticosterone transport, respectively. Notably, up to 83% critical residues for corticosterone face the lipid interface compared to 54% for β-estradiol. Molecular docking identified a possible peripheral corticosterone-binding site made of 8/14 critical/non-critical residues between TMHs 3, 4 and 6. β-estradiol transport was severely hampered by alanine replacements of Cdr1p core residues involving TMHs 2, 5 and 8, in a binding site made of 10/14 critical residues mainly shared with rhodamine 6G with which it competes. By contrast, TMH11 was poorly impacted, although being part of the core domain. Finally, we observed th...
Macrocyclic diterpenes were previously found to be able to modulate the efflux pump activity of C... more 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 transform...
Twenty-nine jatrophane esters (1-10, 12-30) and one lathyrane (11) diterpenoid ester isolated fro... more 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. Howe...
The ABC (ATP-Binding Cassette) transporter Cdr1 (Candida drug resistance 1) protein (Cdr1p) of Ca... more 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 ...
An analysis of Candida albicans ABC transporters identified conserved related α-helical sequence ... more An analysis of Candida albicans ABC transporters identified conserved related α-helical sequence motifs immediately C-terminal of each Walker A sequence. Despite the occurrence of these motifs in ABC subfamilies of other yeasts and higher eukaryotes, their roles in protein function remained unexplored. In this study we have examined the functional significance of these motifs in the C. albicans PDR transporter Cdr1p. The motifs present in NBD1 and NBD2 were subjected to alanine scanning mutagenesis, deletion, or replacement of an entire motif. Systematic replacement of individual motif residues with alanine did not affect the function of Cdr1p but deletion of the M1-motif in NBD1 (M1-Del) resulted in Cdr1p being trapped within the endoplasmic reticulum. In contrast, deletion of the M2-motif in NBD2 (M2-Del) yielded a non-functional protein with normal plasma membrane localization. Replacement of the motif in M1-Del with six alanines (M1-Ala) significantly improved localization of th...
A series of structurally related jatrophane diterpenoids (1-6), including the new euphosquamosins... more A series of structurally related jatrophane diterpenoids (1-6), including the new euphosquamosins A-C (4-6), was purified from the Iranian spurge Euphorbia squamosa and evaluated for its capacity to inhibit drug efflux by multidrug transporters of Candida albicans. Three of these compounds showed an interesting profile of activity. In particular, deacetylserrulatin B (2) and euphosquamosin C (6) strongly inhibited the drug-efflux activity of the primary ABC-transporter CaCdr1p, an effect that translated, in a yeast strain overexpressing this transporter, into an increased sensitivity to fluconazole. These compounds were transported by CaCdr1p, as shown by the observation of an 11-14-fold cross-resistance of yeast growth, and could also inhibit the secondary MFS-transporter CaMdr1p. In contrast, euphosquamosin A (4) was selective for CaCdr1p, possibly as a result of a different binding mode. Taken together, these observations suggest jatrophane diterpenes to be a new class of potent inhibitors of multidrug transporters critical for drug resistance in pathogenic yeasts.
Thirteen macrocyclic diterpenes (1-13) of the jatrophane and lathyrane types, either isolated fro... more 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 an...
Reduced intracellular accumulation of drugs (due to rapid efflux) mediated by the efflux pump pro... more Reduced intracellular accumulation of drugs (due to rapid efflux) mediated by the efflux pump proteins belonging to ABC (ATP Binding Cassette) and MFS (Major Facilitators) superfamily is one of the most common strategies adopted by multidrug resistance (MDR) pathogenic yeasts. To combat MDR, it is essential to understand the structure and function of these transporters so that inhibitors/modulators to these can be developed. The sequence alignments of the ABC transporters reveal selective divergence within much conserved domains of Nucleotide-Binding Domains (NBDs) which is unique to all fungal transporters. Recently, the role of conserved but divergent residues of Candida Drug Resistance 1 (CDR1), an ABC drug transporter of human pathogenic Candida albicans, has been examined with regard to ATP binding and hydrolysis. In this paper, we focus on some of the recent advances on the relevance of divergent and conserved amino acids of CaCdr1p and also discuss as to how drug interacts wi...
The ABC transporter Cdr1 protein of Candida albicans, which plays a major role in antifungal resi... more The ABC transporter Cdr1 protein of Candida albicans, which plays a major role in antifungal resistance, has two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). The 12 transmembrane helices of TMDs that are interconnected by extracellular and intracellular loops (ICLs) mainly harbor substrate recognition sites where drugs bind while cytoplasmic NBDs hydrolyze ATP which powers drug efflux. The coupling of ATP hydrolysis to drug transport requires proper communication between NBDs and TMDs typically accomplished by ICLs. This study examines the role of cytoplasmic ICLs of Cdr1p by rationally predicting the critical residues on the basis of their interatomic distances. Among nine pairs that fall within a proximity of <4 Å, an ion pair between K577 of ICL1 and E315 of NBD1 was found to be critical. The substitution, swapping and changing of the length or charge of K577 or E315 by directed mutagenesis led to a misfolded, non-rescuable protein entrapped in intra...
There are currently few antifungals in use which show efficacy against fungal diseases. These ant... more There are currently few antifungals in use which show efficacy against fungal diseases. These antifungals mostly target specific components of fungal plasma membrane or its biosynthetic pathways. However, more recent class of antifungals in use is echinocandins which target the fungal cell wall components. The availability of mostly fungistatic antifungals in clinical use, often led to the development of tolerance to these very drugs by the pathogenic fungal species. Thus, the development of clinical multidrug resistance (MDR) leads to higher tolerance to drugs and its emergence is helped by multiple mechanisms. MDR is indeed a multifactorial phenomenon wherein a resistant organism possesses several mechanisms which contribute to display reduced susceptibility to not only single drug in use but also show collateral resistance to several drugs. Considering the limited availability of antifungals in use and the emergence of MDR in fungal infections, there is a continuous need for the ...
Drug-resistant pathogenic fungi use several families of membrane-embedded transporters to efflux ... more Drug-resistant pathogenic fungi use several families of membrane-embedded transporters to efflux antifungal drugs from the cells. The efflux pump Cdr1 (Candida drug resistance 1) belongs to the ATP-binding cassette (ABC) superfamily of transporters. Cdr1 is one of the most predominant mechanisms of multidrug resistance in azole-resistant (AR) clinical isolates of Candida albicans. Blocking drug efflux represents an attractive approach to combat the multidrug resistance of this opportunistic human pathogen. In this study, we rationally designed and synthesized transmembrane peptide mimics (TMPMs) of Cdr1 protein (Cdr1p) that correspond to each of the 12 transmembrane helices (TMHs) of the two transmembrane domains of the protein to target the primary structure of the Cdr1p. Several FITC-tagged TMPMs specifically bound to Cdr1p and blocked the efflux of entrapped fluorescent dyes from the AR (Gu5) isolate. These TMPMs did not affect the efflux of entrapped fluorescent dye from cells e...
The molecular basis of polyspecificity of Mdr1p, a major drug/H+ antiporter of Candida albicans, ... more The molecular basis of polyspecificity of Mdr1p, a major drug/H+ antiporter of Candida albicans, is not elucidated. We have probed the nature of the drug-binding pocket by performing systematic mutagenesis of the 12 transmembrane segments. Replacement of the 252 amino acid residues with alanine or glycine yielded 2/3 neutral mutations while 1/3 led to the complete or selective lost of resistance to drugs or substrates transported by the pump. Using the GlpT-based 3D-model of Mdr1p, we roughly categorized these critical residues depending on their type and localization 1°/ main structural impact ("S" group), 2°/ exposure to the lipid interface ("L" group), 3°/ buried but not facing the main central pocket, inferred as critical for the overall H+/drug antiport mechanism ("M" group) and finally 4°/ buried and facing the main central pocket ("B" group). Among "B" category, 13 residues were essential for the large majority of drugs/substr...
The present study examines the kinetics of steroids efflux mediated by the Candida drug resistanc... more The present study examines the kinetics of steroids efflux mediated by the Candida drug resistance protein 1 (Cdr1p) and evaluates their interaction with the protein. We exploited our in-house mutant library for targeting the 252 residues forming the twelve transmembrane helices (TMHs) of Cdr1p. The screening revealed 65 and 58 residues critical for β-estradiol and corticosterone transport, respectively. Notably, up to 83% critical residues for corticosterone face the lipid interface compared to 54% for β-estradiol. Molecular docking identified a possible peripheral corticosterone-binding site made of 8/14 critical/non-critical residues between TMHs 3, 4 and 6. β-estradiol transport was severely hampered by alanine replacements of Cdr1p core residues involving TMHs 2, 5 and 8, in a binding site made of 10/14 critical residues mainly shared with rhodamine 6G with which it competes. By contrast, TMH11 was poorly impacted, although being part of the core domain. Finally, we observed th...
Macrocyclic diterpenes were previously found to be able to modulate the efflux pump activity of C... more 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 transform...
Twenty-nine jatrophane esters (1-10, 12-30) and one lathyrane (11) diterpenoid ester isolated fro... more 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. Howe...
The ABC (ATP-Binding Cassette) transporter Cdr1 (Candida drug resistance 1) protein (Cdr1p) of Ca... more 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 ...
An analysis of Candida albicans ABC transporters identified conserved related α-helical sequence ... more An analysis of Candida albicans ABC transporters identified conserved related α-helical sequence motifs immediately C-terminal of each Walker A sequence. Despite the occurrence of these motifs in ABC subfamilies of other yeasts and higher eukaryotes, their roles in protein function remained unexplored. In this study we have examined the functional significance of these motifs in the C. albicans PDR transporter Cdr1p. The motifs present in NBD1 and NBD2 were subjected to alanine scanning mutagenesis, deletion, or replacement of an entire motif. Systematic replacement of individual motif residues with alanine did not affect the function of Cdr1p but deletion of the M1-motif in NBD1 (M1-Del) resulted in Cdr1p being trapped within the endoplasmic reticulum. In contrast, deletion of the M2-motif in NBD2 (M2-Del) yielded a non-functional protein with normal plasma membrane localization. Replacement of the motif in M1-Del with six alanines (M1-Ala) significantly improved localization of th...
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Papers by Manpreet Rawal