Inhibitors for Glyoxylate cycle ICL1 Enzyme Activity in Candida albicans

1. Inhibitors for Glyoxylate cycle ICL1 Enzyme Activity inCandida albicans DOBLIN SANDAI Ph.D Cheah Hong Leong Infectomics Cluster, Advance Medical and Dental Institute (AMDI)/InstitutPerubatandanPergigianTermaju, UniversitiSains Malaysia

2. Introduction C. albicans & other medical relevant Candida species are commensal yeast in human oral cavity, Gastrointestinal & genitourinary tracts They are usually benign but can establish infections when host immune system become impaired or host niche become available, causing candidiasis

3. Candidiasis Superficial candidiasis: - non-fatal e.g. Oral thrush, GI candidiasis & vaginal candidiasis. • Systemic candidiasis: • Among the most common • nosocomial infections • Potential fatal with high mortality • rate (50%). • (Pfaller & Diekema, 2007; Mayer et al., 2013)

4. Candidiasis *Major fungal pathogen of humans; cause of invasive fungal infections. Factors predisposing people to candidiasis include; AIDS/HIV Patients Oral birth control Antibiotic therapy Burn patients Heart surgery Immunosuppressant High fruit diets Pregnancy period Dirty needle Young individual Use of catheters Endocrine deficiency diabetes Steroids Genetic defiency Cancer treatment

5. Candidiasis Virulence factors Clinical Manifestation Morphogenesis Transition between unicellular yeast cells and filamentous growth forms. Invade/escape. Biofilm formation Biofilms allow colonization of host surfaces where yeast or filamentous cells could not proliferate on their own. Episodic Adhesins A collection of biomolecules that are expressed at fungal surface and promote pathogenesis by helping cells to attach to host cells. Phenotypic switching Spontaneous, reversible phenomenon that generates a variety of phenotypes, helping to evade immunological detection and adapt to varying host environments. Chronic Sub-Acute Acute Candidiasis are extremely varied, ranging from Secreted hydrolytic enzymes Proteinase, phopolipase, lipase- degradation of host proteins and lipid, tissues invation and the destruction of host immune factors. Fitness attributes Survive and cause infections, involve both stress responses and metabolic adaptation, leads to physiological fitness in the host.

6. signals pathways metabolic Plasticity to assimilate the available nutrients in niches. fitness attributes fitness attributes virulence factors pathogenicity Growth & Pathogenicity Mixtures of carbon sources Carbon assimilation and growth In many microenvironments, C. albicans will enjoy complex mixtures of carbon sources

7. PCK1 PCK1 Slow decay Rapid decay Pck1p ICL1 ICL1 Growth and pathogenesis Growth Icl1p Pck1p Icl1p Growth & Pathogenicity C. albicans S. cerevisiae Alternative carbon sources + Alternative carbon sources + Glucose Glucose C. albicans and S. cerevisiae display similar responses to glucose at the transcriptional level, their responses at post-transcriptional and metabolite level diverge significantly. As a result, C. albicans can assimilate both glucose and alternative carbon sources at the same time.Sandai et al., 2012

8. Glucose Growth & Pathogenicity HXK2 G1P G6P UGP1 INO1 PGI1 Mannose-6P Inositol-1P Fructose-6P PMI40 PFK1 FBP1 Fructose-1,6P2 Glucose – gut, tissues, bloodstream FBA1 Glyceraldehyde-3P DAK2 TPI1 Glycerone-P GAP1 Fatty acids (oleic acid) – gut, tissues, macrophages, neutrophils Glycerate-1,3P2 PGK1 Glycerate-3P GPM1 Carboxylic acids (lactic acid) – gut, tissues, bloodstream Glycerate-2P ENO1 glycolysis glycolysis Phosphoenolpyruvate gluconeogenesis gluconeogenesis CDC19 Acetaldehyde Pyruvate Ethanol ADH2 PDC11 ICL1 : Isocitrate Lyse, PCK1 ; Phosphoenolpyruvate carboxykinase. Contribute to C. albicans virulence for the establishment of systemic infections, required for growth on non-fermentable carbon sources such as lactate & oleic acid. PCK1 PDA1 PDB1 LAT1 PDX1 IPF16300 PDB1 LAT1 Acetate lactate lactate ACS1 Oleic acid Ac-CoA Citrate CIT1 Oxaloacetate glyoxylate cycle glyoxylate cycle ACO1 MDH11 Malate Isocitrate ICL1 Glyoxylate IDH2 FUM12 TCA cycle TCA cycle Fumarate 2-Oxoglutarate KGD1 SDH12 Succinate Succinyl-CoA Brown, 2005 LSC2

9. ICL as antifungal drug target - ICL is involved in multiple pathogenicity mechanisms encourages the development of specific inhibitor for candidiasis treatments. - no human orthologue of this pathway has been identified, potentially making it even more suitable target for treatments.

10. Objectives 1. To screen & identify plant reference compounds that inhibit/repress ICL1 activity in C. albicans > To identify compounds that might inhibit glyoxylate cycle by screening in lactate-containing medium > To confirm the inhibition of identified compounds on ICL1 activity from C. albicans by ICL1 inhibition assay 2. To determine the possible inhibitory mechanisms of the identified compounds on ICL1 activity in C. albicans > To study the effect of compounds treatment on ICL gene transcription levels in C. albicans > To predict the binding of the identified inhibitors to ICL protein by in silico analysis

11. Methodology Primary screening for growth inhibitors in YNB-Lactate Secondary screening for growth inhibitor in YNB-Lactate & -Glucose ICL enzyme assay & ICL inhibition studies MIC determination by broth microdilution for hit compounds

12. Results & Discussions > Screening & MIC determination

13. Screening for antifungal Compounds > Primary & secondary screening identified 3 hit compounds (blue arrows), plus 1 known ICL inhibitor that showed a lactate-specific pattern in growth inhibition. > Drug control: fluconazole > Cutoff value: 60% growth percentage (blue arrow) > Abbreviations: FLC (fluconazole); ITC (itaconic acid); QCT (quercetin); CINN (cinnamic acid); GALL (gallic acid); RT (rutin); CAFF (caffeic acid); ROS (rosmarinic acid); API (apigenin)

14. Chemistry of ICL1 Enzyme Assay Glyoxylate Phenylhydrazone • can be measured by absorbance • at 324 nm with millimolar extinction • coefficient at 16.8 Phenylhydrazine Glyoxylate Phenylhydrazone

15. ICL1 Enzyme Assay Optimization U/mL = [(Slope) x (reaction volume) x Df] 16.8 x vol. of enzyme • Graph of absorbance at 324nm versus time of incubation for ICL enzyme assay. • Optimized time of incubation: 45 minutes. • ICL enzyme activity measured: 16 U/L

16. ICL1 Inhibition Study > ICL inhibition study confirmed the hit compounds (blue arrows) identified in growth inhibition screening > Cutoff value: 40% inhibitory percentage (red arrow) > Apigenin showed highest inhibitory percentage, followed by caffeic acid & rosmarinic acid

17. MIC Determination > Method: Broth microdilution method for yeasts (EUCAST EDef 7.1) > MIC definition: Lowest concentration giving rise to an inhibition of growth of > 50% of that of the drug-free control > MIC of fluconazole in lactate medium was higher than that of glucose medium - lactate-grown C. albicans had been reported to showed increased resistance to some drugs - lactate strongly influences the cell wall properties and virulence > Apigenin showed the lowest MIC among the 3 hit compounds identified

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19. Thank you