We have also tested the effect of celecoxib on daf 2 mutants and found no significant lifespan extension. Similar to what we have observed with celecoxib, exposure to OSU 03012 further extends the lifespan of eat 2 and cyc 1 mutants, but not the lifespan of daf 16 and daf 2 mutants. Since OSU 03012 exhibits no detectable COX 2 inhibitory activity, our findings strongly suggest that celecoxib and its derivative OSU 03012 act on a target other than COX 2 to modulate longevity in C. elegans. It should be noted that we couldnt rule out the possibility that different mutants may exhibit varied sensitivity to the drugs. However, this is unlikely to be the case, as daf 16 mutants failed to respond to all three different concentrations of OSU 03012 we have examined.

Among all the potential secondary targets reported to date ), inhibition of PDK 1, a known IIS pathway component upstream of DAF 16, by celecoxib is particularly VEGF intriguing. It has been reported that celecoxib and a number of its derivatives exhibit different degrees of inhibitory activity against human PDK 1. Given the strong antagonistic activity of OSU 03012 on human PDK 1 both in vitro and in vivo, we have also tested the effect of OSU 03012 on pdk 1 mutants lifespan. Treatment with OSU 03012 failed to extend the lifespan of either the long lived loss offunction pdk 1 mutants, or the short lived gain of function pdk 1 mutants. To determine whether the activity of C.

elegans PDK 1 could indeed be inhibited by celecoxib and OSU 03012 in vivo, we analyzed the phosphorylation status mGluR of SGK 1, a known substrate of PDK 1, in animals exposed to both drugs. It has been reported that the Thr256 residue in the activation loop of human SGK1 is phosphorylated by PDK1, whereas the Ser422 residue in the hydrophobic motif might be phosphorylated by mTOR. The phosphorylation status of SGK 1 is assessed by immunoprecipitating SGK 1::GFP fusion proteins from drug treated BR2773 animals and blotting with anti phospho Thr, anti phospho Ser or anti phospho PDK 1 docking motif antibodies. We found that treatments with both drugs significantly reduce Threonine phosphorylation of SGK 1 by PDK 1, while Serine phosphorylation of SGK 1 remains basically unaltered. Thus, our findings strongly suggest that celecoxib and OSU 03012 might act directly on PDK 1 or a component upstream of PDK 1 in the IIS pathway to increase longevity in worms.

Previous studies have shown that DAF 16 accumulates in the nucleus when the activity of its upstream kinases is reduced. To further examine the mGluR idea that celecoxib and OSU 03012 might act on a component of the IIS pathway upstream of DAF 16, likely PDK 1, to influence longevity, we examined the nuclear localization of DAF 16 using a GFP reporter strain. In agreement with our model, we found an increased level of nuclear localized DAF 16::GFP fusions after 72 hr of treatment with celecoxib or OSU 03012, indicating that celecoxib and OSU 03012 treatments might promote DAF 16 activation.