Modulation of cell death and pathogenicity by eugenol tosylate congeners in Candida Albicans

Abstract

The global prevalence of serious fungal diseases is increasing rapidly, which affects more than a billion people every year with a significant mortality rate. On the other hand, the development of new drugs to treat these fungal infections is slow, while the current antifungal therapy is insufficient and bound with limitations. Such circumstances are contributing to the development of drug resistance and also to the provision of a platform for new emerging multi-drug resistant species such as Candida auris. Thus, development of novel antifungal drugs with minimum or no toxicity and multi-target mechanisms of action can resolve these issues. In this regard, we synthesized seven novel eugenol tosylate congeners (ETC-1 – ETC 7) from a natural compound eugenol, which is well known and has been extensively studied for its antimicrobial and other pharmacological properties. These ETCs were tested for their antifungal activity against various fluconazole susceptible and resistant strains of Candida albicans. In this study, we selected C. albicans as a target organism due to the fact that this opportunistic pathogen is the most predominant species of genus Candida that can cause infections in humans and is widely used as a model organism in fungal research for its various unique characteristics. Antifungal susceptibility testing was done by determining Minimum Inhibitory Concentrations (MIC) and Minimum Fungicidal Concentrations (MFC) and the results revealed the enhanced and potent antifungal effect of these compounds in comparison to their parent compound eugenol against both FLC susceptible and resistant isolates of C. albicans. The order of antifungal potency based on susceptibility results was ETC-5>ETC-6>ETC 7>ETC-1>ETC-4>ETC-2>ETC-3. To determine the multi-target mechanisms of antifungal action of these compounds, effect on both established and emerging drug targets was studied. The effect on the ergosterol biosynthesis pathway was evaluated by applying both in silico and in vitro approaches. The in silico studies, which includes molecular docking, confirmed vii

that these ETCs target sterol 14α-demethylase (CYP51), an essential enzyme in the ergosterol biosynthesis pathway, and thereby impaired ergosterol biosynthesis in both susceptible and resistant isolates. Test compound ETC-5 showed best docking score and is efficiently involved in its high affinity with the active site of CYP51 protein, followed by ETC-6 and ETC-7 respectively. Therefore, based on antifungal susceptibility and molecular docking results, only three most active compounds (ETC-5, ETC-6 and ETC-7) were selected for further studies. In silico findings were further corroborated by in vitro studies, where the test compounds significantly reduced ergosterol biosynthesis and downregulated the expression of sterol 14 α–demethylase encoding gene ‘ERG11’. The concentration dependent fungicidal effect was detected in a cell viability assay by using MUSE Cell Analyzer, where complete cell death was observed when cells were treated with higher concentrations of the test compounds. To study the effect of the most active ETCs (ETC-5, ETC-6 and ETC-7) on major virulence factors of C. albicans, the effect of these compounds on adherence (alamarBlue-based assay), morphogenesis (microscopy), secretion of hydrolytic enzymes (plate assay methods) and biofilm formation (XTT reduction assay) was studied. Furthermore, to study the in depth effect at molecular level, expression of genes related to pathogenicity of C. albicans such as ALS1, ALS2, ALS3, ALS9, CPH1, HWP1, SAP1, SAP2, SAP3 and PLB1 was tested by using quantitative reverse transcription PCR (RT-qPCR). From these results, it was evident that all the tested ETCs significantly reduced the virulence factors of C. albicans and down regulated the expression of their related genes. To study the in depth mechanism of antifungal action of the most active ETCs (ETC-5, ETC 6 and ETC-7), the mode of cell death was determined. External induction of apoptosis in yeast cells is considered as an ideal model for the development of novel antifungal drugs. Therefore, we studied apoptotic effect of ETC-5, ETC-6 and ETC-7 in C. albicans cells by analysing major markers of yeast apoptosis, which include phosphatidylserine externalization, DNA damage, mitochondrial depolarization and decrease in cytochrome c oxidase activity. viii

FITC annexin V/PI double staining and TUNEL assay results revealed significant apoptotic effect through phosphatidylserine externalization and DNA damage, however necrotic effects were also observed at higher concentrations of these ETCs. Mitochondrial membrane potential and cytochrome c oxidase activity was also markedly decreased in C. albicans cells after being exposed to these test entities, which suggested the possible involved apoptotic pathway activated by these ETCs could reside in the metacaspase dependent pathway. Based on the above results it is evident that these test compounds have potent antifungal activity, however it is very important to determine their cytotoxic effect. Therefore, cytotoxicity of these test compounds (ETC-5, ETC-6 and ETC-7) was determined by in vitro haemolytic assay using horse red blood cells (RBCs). All these compounds were observed to have very low toxicity on RBCs, with only 1.98% to 15.18% cell haemolysis at varying concentrations ranging from sub-MIC to MFC values. These in vitro results advocate that ETC-5, ETC-6 and ETC-7 are safe to use for in vivo studies using animal models. Collectively, the findings of this study indicated that newly synthesized ETCs possessed potent antifungal activity and had multi-target mechanisms of action. The antifungal nature of these compounds is related to their ability to inhibit ergosterol biosynthesis and to subsequently result in apoptosis and necrosis. At sub-inhibitory concentrations, these test entities significantly inhibit the virulence factors and drastically reduced the biofilm formation. The overall results indicated that ETC-5 was the most active compound followed by ETC-6 and ETC-7, against both fluconazole susceptible and resistant isolates of C. albicans. Thus, these eugenol derivatives have the ability to be developed as new antifungal agents with multi-target mechanisms of action.