Cinnamic Acid Analogs as Intervention Catalysts for Overcoming Antifungal Tolerance

Abstract

Disruption of fungal cell wall should be an effective intervention strategy. However, the cell wall-disrupting echinocandin drugs, such as caspofungin (CAS), cannot exterminate filamentous fungal pathogens during treatment. For potency improvement of cell wall-disrupting agents (CAS, octyl gallate (OG)), antifungal efficacy of thirty-three cinnamic acid derivatives was investigated against Saccharomyces cerevisiaeslt2Δ, bck1Δ, mutants of the mitogen-activated protein kinase (MAPK), and MAPK kinase kinase, respectively, in cell wall integrity system, and glr1Δ, mutant of CAS-responsive glutathione reductase. Cell wall mutants were highly susceptible to four cinnamic acids (4-chloro-α-methyl-, 4-methoxy-, 4-methyl-, 3-methylcinnamic acids), where 4-chloro-α-methyl- and 4-methylcinnamic acids possessed the highest activity. Structure-activity relationship revealed that 4-methylcinnamic acid, the deoxygenated structure of 4-methoxycinnamic acid, overcame tolerance of glr1Δ to 4-methoxycinnamic acid, indicating the significance of para substitution of methyl moiety for effective fungal control. The potential of compounds as chemosensitizers (intervention catalysts) to cell wall disruptants (viz., 4-chloro-α-methyl- or 4-methylcinnamic acids + CAS or OG) was assessed according to Clinical Laboratory Standards Institute M38-A. Synergistic chemosensitization greatly lowers minimum inhibitory concentrations of the co-administered drug/agents. 4-Chloro-α-methylcinnamic acid further overcame fludioxonil tolerance of Aspergillus fumigatus antioxidant MAPK mutants (sakAΔ, mpkCΔ). Collectively, 4-chloro-α-methyl- and 4-methylcinnamic acids possess chemosensitizing capability to augment antifungal efficacy of conventional drug/agents, thus could be developed as target-based (i.e., cell wall disruption) intervention catalysts.

Keywords: antifungal; antioxidant system; caspofungin; cell wall integrity; chemosensitization; cinnamic acids; intervention catalysts; small molecules; synergism.

Figure 1

Structures of antifungal agents used in this study. (a) Cinnamic acid (Basic structure), (b) 4-Chloro-α-methylcinnamic acid, (c) 4-Methoxycinnamic acid, (d) 4-Methylcinnamic acid, (e) Caspofungin (CAS), (f) Octyl gallate (OG).

Figure 2

Yeast dilution bioassay showing differential susceptibility of S. cerevisiae slt2Δ, bck1Δ, and glr1Δ mutants to cinnamic acid analogs (0.5 mM).

Figure 3

Glutathione supplementation test. The tolerance of S. cerevisiae glutathione reductase mutant (glr1Δ) to 4-methoxycinnamic acid was not abolished by supplementation of reduced (GSH) or oxidized (GSSG) glutathione, indicating glutathione limitation was not the determinant of glr1Δ hyper-tolerance to 4-methoxycinnamic acid.

Figure 4

Overcoming fludioxonil (50 μM) tolerance of A. fumigatus mitogen-activated protein kinase (MAPK) mutants (sakAΔ, mpkCΔ) by 4-chloro-α-methylcinnamic acid (0.5 mM).

Figure 5

Scheme showing overcoming fungal tolerance to antifungal agents. (a) Chemosensitization to overcome fludioxonil tolerance of A. fumigatus MAPK mutants by small molecule chemosensitizer 4-chloro-α-methylcinnamic acid; (b) Deoxygenation of 4-methoxyl group to overcome 4-methoxycinnamic acid tolerance of S. cerevisiae glr1Δ mutant.