MANAVATHU EK, DIMMOCK JR, CUTRIGHT JL, CHANDRASEKAR PH; Interscience Conference on Antimicrobial Agents and Chemotherapy.

Abstr Intersci Conf Antimicrob Agents Chemother Intersci Conf Antimicrob Agents Chemother. 2000 Sep 17-20; 40: 201.

Wayne State Univ., Detroit, MI

NC1175 is a novel thiol-blocking conjugated styryl ketone that exhibits activity against a broad spectrum of pathogenic fungi by inhibiting the plasma membrane H[+]-ATPase of fungi. To further investigate the mechanism of action of NC1175 against H[+]-ATPase of fungi, we examined the kinetics of inhibition of Candida albicans H[+]-ATPase and compared the results with those obtained for two known thiol-blocking agents, N-ethylmaleimide (NEM) and p-chloromercuriphenylsulfonate (PCMPS). Plasma membrane fractions were prepared from C. albicans 90028 using a previously described procedure and the enzyme activity was assayed by ATP hydrolysis. The optimum temperature and pH for the H[+]-ATPase of C. albicans were 25 degrees C and 7.0, respectively. The apparent K[m] and the V[max] of the enzyme for the hydrolysis of ATP were 1.8 mM and 3.85 micro-mol Pi/min/mg protein. The inhibitory effect of NC1175 on the enzyme was examined in the presence of 0 micro-M to 100 micro-M of the inhibitor with excess substrate. Double reciprocal plot (1/s vs. 1/v) revealed that both the K[m] and the V[max] were affected suggesting that the inhibition of C. albicans H[+]-ATPase by NC1175 is caused by mixed inhibition. Analysis of the inhibition data by Dixon plot provided an apparent inhibition constant (K[i]) of 8.5 micro-M for NC1175 whereas the K[i]s of NEM and PCMPS were 380 micro-M and 150 micro-M, respectively. The K[i] of NC1175 is in the same range as the MIC (6.25 micro-M) of the drug for C. albicans and is approximately 18 to 45 fold lower than those of other known thiol-blocking agents. These data suggest that the antifungal activity of this novel conjugated styryl ketone is at least partly due to the inhibition H[+]-ATPase, an enzyme essential for the regulation of intracellular pH as well as the uptake of nutrients in fungi.KEYWORDS: Candida albicans; H+ATPase inhibition; Styryl ketone

Publication Types:

• Meeting Abstracts

Keywords:

• Antifungal Agents
• Antigens, Fungal
• Candida albicans
• Cell Membrane
• Fungi
• Hydrogen-Ion Concentration
• Hydrolysis
• Ketones
• Kinetics
• Miconazole
• Microbial Sensitivity Tests
• Motor Activity
• Proton-Translocating ATPases
• immunology
• metabolism
• pharmacokinetics

Other ID:

• GWAIDS0010628

UI: 102248126

From Meeting Abstracts