title: Rationally designed transmembrane peptide mimics of the multidrug transporter protein Cdr1 act as antagonists to selectively block drug efflux and chemosensitize azole-resistant clinical isolates of Candida albicans.
abstract: 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 expressing the Cdr1p homologue Cdr2p or from cells expressing a non-ABC transporter Mdr1p. Notably, the time correlation of single photon counting fluorescence measurements confirmed the specific interaction of FITC-tagged TMPMs with their respective TMH. By using mutant variants of Cdr1p, we show that these TMPM antagonists contain the structural information necessary to target their respective TMHs of Cdr1p and specific binding sites that mediate the interactions between the mimics and its respective helix. Additionally, TMPMs that were devoid of any demonstrable hemolytic, cytotoxic, and antifungal activities chemosensitize AR clinical isolates and demonstrate synergy with drugs that further improved the therapeutic potential of fluconazole in vivo.
authors.affiliation: Membrane Biology Laboratory, School of Life Sciences, New Delhi 110067, India.
authors.forename: Indresh Kumar
authors.initials: IK
authors.lastname: Maurya
authors.affiliation: International Centre for Genetic Engineering and Biotechnology, New Delhi 110067, India.
authors.forename: Chaitanya Kumar
authors.initials: CK
authors.lastname: Thota
authors.affiliation: School of Physical Sciences, New Delhi 110067, India.
authors.forename: Sachin Dev
authors.initials: SD
authors.lastname: Verma
authors.affiliation: International Centre for Genetic Engineering and Biotechnology, New Delhi 110067, India.
authors.forename: Jyotsna
authors.initials: J
authors.lastname: Sharma
authors.affiliation: Membrane Biology Laboratory, School of Life Sciences, New Delhi 110067, India.
authors.forename: Manpreet Kaur
authors.initials: MK
authors.lastname: Rawal
authors.affiliation: School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
authors.forename: Balaguru
authors.initials: B
authors.lastname: Ravikumar
authors.affiliation: School of Physical Sciences, New Delhi 110067, India.
authors.forename: Sobhan
authors.initials: S
authors.lastname: Sen
authors.affiliation: Public Health Research Institute, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103.
authors.forename: Neeraj
authors.initials: N
authors.lastname: Chauhan
authors.affiliation: School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Lynn
authors.affiliation: International Centre for Genetic Engineering and Biotechnology, New Delhi 110067, India. Electronic address: virander@icgeb.res.in.
authors.forename: Virander Singh
authors.initials: VS
authors.lastname: Chauhan
authors.affiliation: Membrane Biology Laboratory, School of Life Sciences, New Delhi 110067, India. Electronic address: rp47@mail.jnu.ac.in.
authors.forename: Rajendra
authors.initials: R
authors.lastname: Prasad
mesh_terms: D000935:Antifungal Agents / Q000737:chemistry / Q000494:pharmacology*; D001393:Azoles; D040761:Biomimetic Materials / Q000737:chemistry / Q000494:pharmacology*; D002176:Candida albicans / Q000235:genetics / Q000378:metabolism*; D025141:Drug Resistance, Fungal / Q000187:drug effects*; D005656:Fungal Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D017433:Protein Structure, Secondary
chemical_list: D000935:Antifungal Agents; D001393:Azoles; C094693:CDR1 protein, Candida albicans; D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D010455:Peptides
keywords: ABC Transporter; Antagonist; Candida albicans; Cdr1 Protein; Drug Design; Efflux Pump; Membrane Proteins; Multidrug Transporters; Time Correlation Single Photon Counting (TCSPC); Transmembrane Peptide Mimics