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Title: Synthesis and evaluation of novel antifungal agents targeting the fungal plasma membrane H+-ATPase
Author: Patel, Dhruvnesh Vijaykumar
ISNI:       0000 0004 9349 6331
Awarding Body: University of Hertfordshire
Current Institution: University of Hertfordshire
Date of Award: 2020
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Fungal infections now contribute significantly to microbe-related morbidity and mortality due to there being a limited number of available antifungal agents and limited modes of delivery. Over the past two decades, many pathogenic fungi have developed various modes of resistance to commonly used antifungals. Thus there is a need for novel antifungal agents with novel mechanisms of action. Inhibition of the essential plasma membrane (PM) H+-ATPase of fungi is a potentially effective therapeutic approach in antifungal drug discovery. In order to investigate this, three series (A-C) consisting of a total of thirty-three symmetrical 1,4-diene-3-one (22a-v, 23a-h and 24a-c) compounds have been synthesized. In vitro macro-broth susceptibility testing of 1,4-diene-3-ones showed wide range of inhibition against Saccharomyces cerevisiae (0.2 - 99%) and Candida albicans (0 - 99%). Compounds 22f, 22m, 22n and 22s exhibited highest potency then other compounds from the library against S. cerevisiae (IC50 = 1.21, 2.22, 0.62 and 1.87 μM), however these compounds demonstrated limited activity against C. albicans (IC50 = 114, 525, 474 and 666 μM). In contrast, compounds 23f, 23g and 23h exhibited a higher degree of antifungal activity against C. albicans (IC50 = 68.5, 57.6 and 50.7 μM) and these compounds also showed good potency against S. cerevisiae (IC50 = 8.46, 5.52 and 6.25 μM). To gain an understanding about the mechanism of action of 1,4-diene-3-ones, the H+-ATPase mediated proton pumping by S. cerevisiae was investigated by measuring the pH of the glucose-induced acidification of the external medium. The bis-pyridylidene derivatives of N-methylpiperidin-4-one (23a-c, 60 μM) were determined to be the most potent inhibitors of H+ efflux from S. cerevisiae and the steady state of proton flux from S. cerevisiae was achieved within 10 minutes of medium acidification. A similar result was observed with N-ethylmaleimide (NEM, 60 μM, positive control). Additionally, 23a, 23b and 23c have shown good potency in the macro broth susceptibility assay of S. cerevisiae (IC50 = 12.6, 8.84 and 9.45 μM). Moreover, the most potent compounds 22n and 23h in macro broth susceptibility assay against S. cerevisiae and C. albicans exhibited limited activity to inhibit the proton efflux from S. cerevisiae. To further elucidate the mechanism of action, preliminary structure-activity relationship (SAR) studies was performed. SAR of bis-benzylidene derivatives of N-methylpiperidin-4-one displayed reasonable correlation coefficient (R2 = 0.6746) between the inhibitory activity expressed as log (1/IC50) and an electronic parameter, the 1H-NMR δ-values of proton on the β-carbon. Conversely, SAR of log (1/IC50) and a lipophilic parameter, calculated logP (clogP) of bis-benzylidene derivatives of N-methylpiperidin-4-one showed R2 of 0.4138. This indicates that the inhibitory activity of compounds is due to the influence of electronic property rather than the lipophilic property. In conclusion, the inhibitory action of 1,4-diene-3-ones on yeast suggests a membrane-bound enzyme target for its action. It is hypothesized that these compounds form a covalent C-S thio-ether bond with cysteine residues of proteins of the plasma membrane and eventually inhibit the H+-ATPase.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: 1,4-diene-3-ones ; a,ß-unsaturated carbonyls ; Antifungal ; Area under the curve ; bis-(benzylidene)-1-methylpiperidin-4-one ; bis-(pyridine-3-ylmethylene)-1-methylpiperidin-4-one ; C. albicans ; Macro broth susceptibility ; Michael addition reaction ; Plasma membrane H+-ATPase ; Proton efflux ; Proton on the ß-carbon ; S. cerevisiae ; Thiol reactive compounds