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CAN-296 is a heat stable, complex carbohydrate (molecular mass 4300 Da) isolated from the cell wall of the filamentous fungus Mucor rouxii. It possesses potent in-vitro fungicidal activity against a wide spectrum of pathogenic yeasts, including azole-resistant isolates of Candida albicans and Candida glabrata. As a preliminary step in the study of the mode of action of this novel antifungal agent, we investigated the effect of various cations on the antifungal activity as well as the binding of CAN-296 to intact cells and cell-wall fractions of C. albicans. The antifungal activity of CAN-296 was inhibited by low concentrations of calcium, magnesium and lithium and by high concentrations of barium, cobalt and manganese, but not by potassium and copper. The calcium-mediated inhibition of the antifungal activity of CAN-296 was readily reversible by the removal of calcium by dialysis, and the fungicidal activity of the inhibited compound was fully restored. The uptake/binding of CAN-296 to intact cells and to the cell-wall fraction of C. albicans was time and concentration dependent. Maximum uptake/binding was obtained at 5 mg/L within 60 min and was associated with the aggregation of intact cells. Washing intact cells and the cell-wall fraction preincubated with radiolabelled CAN-296 with 150-fold excess of unlabelled compound failed to remove CAN-296 associated with the intact cells and the cell-wall fraction, suggesting that the binding of CAN-296 to C. albicans is tight. The uptake/binding of CAN-296 and the drug-mediated aggregation of intact cells were inhibited by calcium in a concentration-dependent manner. The fact that CAN-296 is a fungicidal agent that binds to intact cells and the cell-wall fraction of C. albicans very tightly, together with the observation that calcium was able to inhibit the fungicidal activity as well as the uptake/binding of CAN-296, suggests that the mode of action of this novel antifungal agent may involve interaction with the cell wall of C. albicans.

Involvement of calcium inhibitable binding to the cell wall in the fungicidal activity of CAN-296