Functional insight into the promiscuous role of multidrug transporter of Candida albicans

1. Functional insight into the promiscuous role of multidrug transporter of Candida albicans SHWETA NIM

2. Candida Drug Resistance1 (CDR1) protein is a major multidrug transporter K G I S D L M V P V F S Y G N S Y N C A P G P R E Q S T E N A G F A V V F D F A A T G F G V C P H T L V Out L E Y Y Y C Y Q G T G C A T G F K E Q S C G F N R V E R S E C N M V E A G Q L E L Y S S I A Q TMS M V L P N M A K I Y N Y E V S S F L V Y Q TMS R S N N G S S N S S I G T Y T Y T V W N V T N T F G TMS C R Y M Q K P T D F P G P N T S T Y N S W N P L S R N V Q Y A G L M H S L Q N N S R Y S W K 619 K 1229 1466 Y N R N G N P L P R E 1217 548 622 A Q 1302 1378 R S 535 764 1314 T R 676 K N A P R N Y N R W M Lipid bilayer P F G M Y A G I V L L V F I F F F F N G S F M L M C F F A V G L W G I TMD R TMS TMS S V I V G V F M V F F N A Y W I G G F L M P G F F V F M L W L F W T F S F L F T I M P Y L V D I F L G T I F V A Q S C F M S A V Y F Y P S F N I L F Y F G F S I I A L N F F W Y G L V A Q V A F I A V L L V V Y I Q V I C F A Y I V K A L M V M I S I E P A M I K F A P Q F A T L V T L S L G F M L S T T A V N L F F A V T Y L S Y P T I A H M V L T G F T N A C F I V M T P P L TMS P M S A L L E I Y I W L F Y G 597 1358 I L 786 I 654 F S M G F L N L F Y C Q F L 1280 S E 644 E 513 70 1251 N 1195 G V E 1336 A 1488 R S C M S L R P A S K T R D P K S I D G I L TMS K S M S D C D T L D S A I D G Y I G P R I F V P Q G S Q R R S A Y Q T S T I F G K L K G E F A D Q L S V R R T A Y T Y F Q P R I S D P L G K N F G D V S M L M T A V G L A G E A K W A L Q T L H G I N V A A G L V G A V I G ATP Q S V A H Y F I T M A A S R L D E L N A R F P R H A T V A K V L K F A A R L Q G T S L E A P L Q F Q R A Q L T D F E N E W E E E W S S V E A F R T A K M G T I G Q V L G D H L M K L ATP L L P V G W I A F R T S L I E D S T S G P K S S F E Q A K R F N D A K S R W I M C M I S K P E C F D G L M L Y F I G G E A T A H A A F G L T D N S T Q L D C W K A E S E D S P D S V K F G A P S A V F A E V K S R W K D S TMS P K R K I F E TMS K K L H D Q E A G V L T R N E T L S T I K P K W R K L K P K W K N A E I N R I P F F T L L T V F S Y N Y L T Y R D A D M L M K S R A V K M R G L S K L L A M S L A D D P A Y Y A P L P L S E V N W L D K A Q S G E W L K Y R L D S T A N G P E N E K Y NBD2 S Y D A K D L S N G N P F P G TMS H L S L D T L D K Y T S V A V Q I L G Y S P L L T N D C K A G L T S T T P A Q Y V N A K Q F D L E V F D A A P K K V V L E K E L Y E F N S E D I G E R K E V A S E R P K A H S L S TMS E W P K I W R T Q N T K K K E I T V N I S C C E H E D E E G L S I V T I P F TMS P Y A N V V G W L Y T H Y R Q\ P A A L R R N C E R H D A V K A S K I N E T R V M F R G R M A T K Y Q P G E I V S Q E M T K C P A L A S E K K K S A P E R Y V K P S Q S D Q H E F G R L Y R R N D V T R N L R A D E N E Y I L I L Q D I T G L F E Q Q D R K Y T H D A V T V Q R T S L Q Y G M F N E G S N N H P L Y A K L L I M A Y I V A S G T L Y D G I D E K R G Y K V E F V H S A E P I Q S E G A S Y S D I D Q G Y F A S T S D K E I G I V E N I E E I E N S N H K T S K H A K A Y T N F S I K S E M V A C L I Y T V N Y I G G N Y L R T A G P S K L N E E A L H G Y D F L E E T V V Y L E H D Y G T G R K N F E V L E S E F P K R Q R D Q R D G E N G S R Q N R D E N T V H Q G K S Q T H H D A V I G Q E V E A V R D S Y D A F Y G T A L A L D V A G N V D T V I T D V L G Y F F E R Q D Q N T P D G I N K V H L S V T D F G D Y L T A N E R Y D R E V G R N A DRUG TMD1 TMD2 NBD1 IN ATP ADP+Pi ADP+Pi ATP DRUG Eukaryotic Cell 2 (2003)1361 Annual reviews of Microbiology, 2012., Rawal et al JBC (2013)

3. White et al, 2012

4. Mechanisms of fungal drug resistance Alteration in drug import Alteration in Intracellular drug processing Mutation in drug target Enzymes. ERG Mutation in enzymes of ergosterol pathway. Change in sterol composition of PM Modification/ degradation

5. 1 Mechanisms of fungal drug resistance Over-expression of efflux pumps HUGO ABC transport protein superfamily Nine families Fungal pdr family Alteration in drug import Alteration in Intracellular drug processing Mutation in drug target Enzymes. ERG Mutation in enzymes of ergosterol pathway. Change in sterol composition of PM Modification/ degradation Cdr1p (C. albicans) Pdr5p (S.cerevisiae) 10 phylogenetic clusters Cluster A

6. TMDs are the most variable part of Cdr1 protein B A C D Rawal et al JBC (2013)

7. Promiscuous Cdr1 protein Antiarrhythmics Quinidine Anticancer Drugs Antibiotics Tamoxifen Chloramphenicol Daunorubicin Doxorubicin Vinblastin sulfate Anisomycin Nimodipine Cycloheximide Topotecan Novobiocin Etoposide CyclosporineA Crystal Violet Fumitremorgin C Niguldipine Mitoxantrone Calcium Channel Blocker Gefitinib Verapamil Nefidipine Miscellaneous Compounds Doxorubicin Yohimbine 4-NQO Azoles Daunorubicin Berberine TK inhibitor Terbinafine Miconazole Fluconazole Gefitinib Dexamethazone Voriconazole Verapamil Itraconazole Ketoconazole Fluorescent compounds Benzimidazole Rhodamine 6G Benomyl Rhodamine 123 Aromatic imidazole Tritylimidazole Herbicides Clotrimazole Linuron Propanil Linuron Lysomotropic amino esters Diuron Triazoles Chlorbromuron Steroid DE-11 DM-11 AT-12 DM-11 DE-11 Progesterone Anisomycin

8. Poly-specificity of Cdr1 protein comes from its overlapping multiple binding sites within a large drug binding pocket

9. Same size, different charges Rhodamine6G Rhodamine B Rhodamine 123

11. Same size, same charges Tetrazolium chloride Tetrazolium bromide

14. These residues proved to be exciting as till no role of the TMS12 has been reported in fungal ABC transporter to play the role in drug binding or efflux.

15. Nim et al, Commumnicated in JBC, September 2015

16. Strategies to combat antifungal resistance Cell Surface Level Designing of modulators/inhibitors/peptide mimics to block efflux pumps X X Signal Transduction Level Identification of TFs and their inhibitors to block the expression of CDR genes X

17. Modulators of ABC drug transporters of Candida Curcumin (a natural product of turmeric, from rhizomes of Curcuma longa) Farnesol (Quorum sensing molecule) Disulfiram (Alcohol deterrent) Shukla et al BBRC 2005 Sharma et al AAC,20010;2012

18. FK520, is a homologue of immunosuppressive and antifungal FK506 • inhibits fungicidal synergism with azoles • competitively inhibits drug efflux • mediated by ABC multidrug transporter. FK520 FK506

19. Extracellular R6G Fluconazole accumulation (pmoles/mg dry wt) FLU KTC MCZ ctrl CDR1 ctrl CDR1 ctrl CDR1 Control 86 % % of mutants FK520 Drug 14 % Drug+FK520

20. DRUG Control DRUG + FK520 FK520 KTC FLC MCZ

21. Selective distribution of amino acids which exclusively interact with FK 520

22. Drug Strain Fold change in MIC Nim et al, FEMS Yeast Research, 2014 Editor’s Choice “Paper of the Month”

23. Macrocyclicditerpenes from Euphorbia species as potent inhibitors of drug-efflux pumps Nim et al, Communicated in bioorg. med.Chem, September 2015

24. ACKNOWLEDGEMENT Prof. Rajendra Prasad Attilio Di Pietro CNRS-University of Lyon France Maria Jose Umbelino Ferreira University of Lisbon, Portugal Pierre Falson Institut de Biologie et de Chimie des Protéines France

25. THANK YOU