title: β-lactam substituted polycyclic fused pyrrolidine/pyrrolizidine derivatives eradicate C. albicans in an ex vivo human dentinal tubule model by inhibiting sterol 14-α demethylase and cAMP pathway.
abstract: BACKGROUND
  Further quest for new anti-fungal compounds with proven mechanisms of action arises due to resistance and dose limiting toxicity of existing agents. Among the human fungal pathogens C. albicans predominate by infecting several sites in the body and in particular oral cavity and root canals of human tooth.
  
  
  METHODS
  In the present study, we screened a library of β-lactam substituted polycyclic fused pyrrolidine/pyrrolizidine compounds against Candida sp. Detailed molecular studies were carried out with the active compound 3 on C. albicans. Morphological damage and antibiofilm activity of compound 3 on C. albicans was studied using scanning electron microscopy (SEM). Biochemical evidence for membrane damage was studied using flow cytometry. In silico docking studies were carried out to elucidate the mechanism of action of compound 3. Further, the antifungal activity of compound 3 was evaluated in an ex vivo dentinal tubule infection model.
  
  
  RESULTS
  Screening data showed that several new compounds were active against Candida sp. Among them, Compound 3 was most potent and exerted time kill effect at 4h, post antifungal effect up to 6h. When used in combination with fluconazole or nystatin, compound 3 revealed an minimum inhibitory concentration (MIC) decrease by 4 fold for both drugs used. In-depth molecular studies with compound 3 on C. albicans showed that this compound inhibited yeast to hyphae (Y-H) conversion and this involved the cAMP pathway. Further, SEM images of C. albicans showed that compound 3 caused membrane damage and inhibited biofilm formation. Biochemical evidence for membrane damage was confirmed by increased propidium iodide (PI) uptake in flow cytometry. Further, in silico studies revealed that compound 3 docks with the active site of the key enzyme 14-α-demethylase and this might inhibit ergosterol synthesis. In support of this, ergosterol levels were found to be decreased by 32 fold in compound 3 treated samples as analyzed by high performance liquid chromatography (HPLC). Further, the antifungal activity of compound 3 was evaluated in an ex vivo dentinal tubule infection model, which mimics human tooth root canal infection. Confocal laser scanning microscopy studies showed 83% eradication of C. albicans and a 6 log reduction in colony forming unit (CFU) after 24h treatment in the infected tooth samples in this model.
  
  
  CONCLUSIONS
  Compound 3 was found to be very effective in eradicating C. albicans by inhibiting cAMP pathway and ergosterol biosynthesis.
  
  
  CONCLUSIONS
  The results of this study can pave the way for developing new antifungal agents with well deciphered mechanisms of action and can be a promising antifungal agent or medicament against root canal infection.
authors.affiliation: Department of Human Genetics, College of Biomedical Sciences, Technology & Research, Sri Ramachandra University, Porur, Chennai- 600 116, India.
authors.forename: Meiyazhagan
authors.initials: M
authors.lastname: Gowri
authors.affiliation: Department of Human Genetics, College of Biomedical Sciences, Technology & Research, Sri Ramachandra University, Porur, Chennai- 600 116, India.
authors.forename: Winfred
authors.initials: W
authors.lastname: Sofi Beaula
authors.affiliation: Department of Bioinformatics, Alagappa University, Karaikudi- 630003, India.
authors.forename: Jayashree
authors.initials: J
authors.lastname: Biswal
authors.affiliation: Department of Bioinformatics, Alagappa University, Karaikudi- 630003, India.
authors.forename: Prabhu
authors.initials: P
authors.lastname: Dhamodharan
authors.affiliation: Department of Medicinal Chemistry, College of Biomedical Sciences, Technology & Research,Sri Ramachandra University, Porur, Chennai- 600 116, India.
authors.forename: Raghavan
authors.initials: R
authors.lastname: Saiharish
authors.affiliation: Department of Biotechnology, Indian Institute of Technology, Madras (IIT M), Chennai- 600 025, India.
authors.forename: Surabi
authors.initials: S
authors.lastname: Rohan prasad
authors.affiliation: Department of Community Medicine, Sri Ramachandra Medical College & Research Institute, Sri Ramachandra University, Porur, Chennai-600 116, India.
authors.forename: Ravishankar
authors.initials: R
authors.lastname: Pitani
authors.affiliation: Department of Conservative Dentistry and Endodontics, College of Dental Sciences, Sri Ramachandra University, Porur, Chennai-600 116, India.
authors.forename: Deivanayagam
authors.initials: D
authors.lastname: Kandaswamy
authors.affiliation: Department of Organic Chemistry, University of Madras, Guindy Campus, Chennai- 600 025, India.
authors.forename: Ragavachary
authors.initials: R
authors.lastname: Raghunathan
authors.affiliation: Department of Bioinformatics, Alagappa University, Karaikudi- 630003, India. Electronic address: jjkanthan@gmail.com.
authors.forename: Jeyaraman
authors.initials: J
authors.lastname: Jeyakanthan
authors.affiliation: Department of Biotechnology, Indian Institute of Technology, Madras (IIT M), Chennai- 600 025, India. Electronic address: rayala@iitm.ac.in.
authors.forename: Suresh K
authors.initials: SK
authors.lastname: Rayala
authors.affiliation: Department of Human Genetics, College of Biomedical Sciences, Technology & Research, Sri Ramachandra University, Porur, Chennai- 600 116, India. Electronic address: ganeshv@sriramachandra.edu.
authors.forename: Ganesh
authors.initials: G
authors.lastname: Venkatraman
mesh_terms: D058888:14-alpha Demethylase Inhibitors / Q000494:pharmacology*; D000935:Antifungal Agents / Q000737:chemistry / Q000494:pharmacology; D002176:Candida albicans / Q000254:growth & development* / Q000648:ultrastructure; D002177:Candidiasis / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D000242:Cyclic AMP / Q000378:metabolism*; D003786:Dental Pulp Cavity / Q000378:metabolism / Q000382:microbiology* / Q000648:ultrastructure; D006801:Humans; D008954:Models, Biological; D015290:Second Messenger Systems; D058886:Sterol 14-Demethylase / Q000378:metabolism*; D047090:beta-Lactams / Q000737:chemistry / Q000494:pharmacology
chemical_list: D058888:14-alpha Demethylase Inhibitors; D000935:Antifungal Agents; D047090:beta-Lactams; D000242:Cyclic AMP; D058886:Sterol 14-Demethylase
keywords: C. albicans; Pyrrolidine; Pyrrolizidine; RT-PCR; Yeast to hyphae; β-lactam