Although Cys39 and Cys80 are positioned away from the Cofilin1 actin interface, their relatively selleck inhibitor buried positions could result in disruption of protein folding if conjugated to the bulky cucurbitacin molecules, similar to the cucurbitacin induced structural rearrangements observed for human serum albumin, which could also inhibit F actin sev ering activity. In addition to cucurbitacin E binding to thiols on F actin, Cofilin1 and DTT, cucurbitacin B was reported to form adducts with thiol containing N acetylcysteine and glutathione, but not thiol free vitamin C or as corbic acid. Although NAC blocked the cytotoxicity of cucurbitacin B, vitamin C and ascorbic acid did not.

These results suggest Inhibitors,Modulators,Libraries that rather than inhibiting the effect of cucurbitacin B through anti oxidant properties, which NAC, vitamin C and ascorbic acid all share, the in hibitory effect of NAC is more likely to be a consequence of its direct conjugation with cucurbitacin Inhibitors,Modulators,Libraries B. In fact, the ability of the reactive oxygen scavenging NAC to reverse the effects of cucurbitacin has widely been interpreted as evidence that generation of reactive oxygen species is part of its mechanism of action. Our alternative interpret ation is that the neutralization of the biological effects of cucurbitacin compounds is via direct conjugation with NAC in tissue culture medium or in cells, meaning that all experiments in which NAC and cucurbitacins have been combined should be interpreted with caution.

Conclusions Although cucurbitacin compounds have been proposed to be potential anti cancer drugs and are used to inhibit spe cific signal transduction pathways, the results of this study and others support the conclusion that cucur bitacins non specifically bind protein targets by forming thioether Inhibitors,Modulators,Libraries bonds via a Michaels Inhibitors,Modulators,Libraries type addition. This would allow cucurbitacins to be conjugated with a broad array of potential protein targets, many of which would be inhi bited or disrupted as a consequence. As a result, their value as chemical biology probes is limited and must be confirmed by independent means. For example, although cucurbitacin I was reported to be a selective inhibitor of Jak STAT3 signalling and has been used to test the in volvement of this pathway in various processes, the ability of cucurbitacin I to activate Rac1 was not replicated by Jak2 or Stat3 knockdown by siRNA.

The findings Inhibitors,Modulators,Libraries in this study also indicate that the binding mode of cucurbitacin compounds to protein targets means that optimization for selectivity would be unlikely to work, which would make it very difficult useful site to minimize toxicities or improve the therapeutic window for future clinical de velopment. Optimistically, alternative ways that their po tential therapeutic utility could be improved in the future would be through targeted delivery to tumour cells, for ex ample through antibody conjugation or incorporation in liposome microparticles.