Inhibitors of Phosphatidylinositol-specific Phospholipase C with Myo-inositol Scaffold

Background: Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes catalyze the conversion of phosphatidylinositol-4,5-bisphosphate into the second messengers diacylglycerol and inositol- 1,4,5-trisphosphate, both of which play crucial roles in regulating biochemical processes. Despite the wellestablished link between elevated PI-PLC activity and pathophysiological conditions, no PI-PLC inhibitors are currently in clinical development. Moreover, existing inhibitors demonstrate only limited potency. Objective: Due to the structural similarity with known inhibitors with a myo-inositol backbone, DL-1-Ododecylsulfonyl- myo-inositol-3,5-bisphosphate, designated as acid sphingomyelinase inhibitor, and derivatives thereof should be tested for inhibition of PI-PLC activity. Methods: The newly synthesized compounds were evaluated for their ability to inhibit PI-PLC activity in porcine platelet lysate and porcine brain homogenate, as well as their inhibitory potency against the recombinant isoenzymes PLCγ1 and PLCγ2. The assay measured the release of diacylglycerol from L-α- phosphatidylinositol using HPLC coupled with MS detection. Furthermore, the specificity of selected compounds was assessed by determining their inhibitory potency against other surface-active enzymes through HPLC-based assays. Results: It was found that DL-1-O-dodecylsulfonyl-myo-inositol-3,5-bisphosphate inhibits PI-PLC activity at micromolar concentrations. However, its maximum achievable inhibitory effect was limited to approximately 70%. Through structural modifications, inhibitors were developed that led to near complete inhibition of PIPLC activity. The study also revealed that the alleged PI-PLC inhibitor U73122, still frequently cited in the literature to demonstrate PI-PLC involvement in biochemical processes, is unsuitable for this purpose. Consistent with observations by others, its inhibitory activity in bionucleophile-containing cell or tissue preparations was found to be significantly lower than its activity against purified PI-PLC enzymes. Additionally, U73122 was shown to inhibit other enzymes, such as cytosolic phospholipase A2α, fatty acid amide hydrolase, and monoacylglycerol lipase, which, like PI-PLC, metabolize lipophilic substrates. In contrast, the newly developed myo-inositol derivatives exhibited reduced sensitivity to bionucleophiles and significantly improved selectivity against the tested surface-active enzymes compared to U73122. Conclusion: New compounds exhibiting significant inhibitory activity against PI-PLC have been identified. The findings could prove valuable in the development of clinically applicable PI-PLC inhibitors, particularly for the treatment of cancer. Additionally, the myo-inositol derivatives developed demonstrated greater suitability for studying PI-PLC's role in physiological processes in tissue homogenates compared to the maleimide derivative U73122, which is commonly used for this purpose in scientific research. This advantage arises from the fact that U73122 is a non-specific 'pan-assay interference compound' (PAIN).