We therefore next examined the effects of existing and novel small molecule inhibitors of PKC. Rottlerin, a na tural product, has been identified as a PKC inhibitor for many years, with an in vitro IC50 of approximately 5 uM in our kinase assays, in good agreement with the literature. We and others have scientific research shown that rottlerin, at the concentrations employed herein, is not cytostatic or cytotoxic to normal primary cells Inhibitors,Modulators,Libraries or cell lines, and is well tolerated when administered orally or intraperitoneally to mice. Exposure of PCSC and PrCSC cultures to rottlerin produced a significant dose dependent inhibition of proliferation as early as 24 hr after exposure. Similarly, rottlerin induced cytoto xicity in both CSC cultures in a dose dependent fashion, as assessed by LDH release.
The duration of PKC inhibition required to irreversibly prevent CSC proliferation was next assessed. Exposure to rottlerin efficiently decreased the clonogenic capacity of PCSC. Eighteen hr of exposure to rottlerin, followed by washout, was sufficient to decrease the clonogenic capacity of PCSC by 40%, and increasing the duration of the exposure to 48 hr reduced the clonogenic Inhibitors,Modulators,Libraries potential by more than 90%. As previously reported, we have sought to develop novel PKC inhibitory molecules with greater specificity for PKC compared to essential PKC isozymes, such as PKC, using pharmacophore modeling and structure activity relationships. We designed and syn thesized a set of analogs based on this strategy.
In this 2nd generation of PKC inhibitors, the head group was made to resemble that of stauros porine, a potent general PKC inhibitor, and other bisindoyl Inhibitors,Modulators,Libraries maleimide kinase inhibitors, with two other domains conserved from the rottlerin scaffold to preserve isozyme specificity. The first such chimeric molecule reported, KAM1, was indeed active, like staurosporine, but was also more PKC specific, and showed potent activity against Ras mutant human cancer cells in culture and in vivo animal models, while not producing cytotoxicity in non transformed cell lines. KAM1 induced cytotoxicity as assessed Inhibitors,Modulators,Libraries by LDH release in a dose dependent fashion in both PCSC and PrCSC cultures at concentrations as low as 2. 5 uM and 5 uM. On the basis of SAR analyses of KAM1, we then de Inhibitors,Modulators,Libraries signed thirty six new 3rd generation analogs.
The syn thetic chemistry platform that was used to prepare KAM1 was modified to synthesize these additional analogs, which were then tested for biochemical and cellular activity. The PKC inhibitory activity and isozyme specificity of this 3rd generation was quantitated in vitro. A number of these Bicalutamide Casodex 3rd generation analogs demonstrated significant increases in potency and isozyme specificity over rottlerin and KAM1. The new compound selected for study in this report, BJE6 106, is much more potent than rottlerin. BJE6 106 has an PKC IC50 in the range of 0.