Structure-function analysis of signal and growth inhibition by carboxyamido-triazole, CAI

Evidence is accumulating that calcium homeostasis and calcium-regulated events may be selectively important in generation and maintenance of the malignant phenotype. CAI, a carboxyamido-triazole with a halogenated benzophenone tail, is a novel inhibitor of receptor-operated calcium influx and arachidonic acid release which inhibits malignant proliferation, invasion, and metastasis. The focus of this investigation was structural analysis of CAI and to determine if the inhibition of calcium influx and arachidonic acid release by CAI and its antiproliferative activity were mediated through the same chemical domains. Four families of molecular modifications of the CAI parent were synthesized: (I) modification or substitution of the triazole ring; (II) removal of the substituted benzophenone tail; (III) dehalogenation or partial truncation of the benzophenone moiety; and (IV) removal of the triazole and altered substitutions of the benzophenone tail. Compounds were tested for the inhibition of calcium influx and arachidonic acid release and inhibition of proliferation and colony formation in soft agar using the malignant CHO line transfected with the m5 muscarinic receptor and the A2058 human melanoma cell line. Only CAI and Group I compounds inhibited stimulated calcium influx, arachidonic acid release, and proliferation. Linear regression analysis of the relationship of the 50% inhibitory concentration values for all compounds in inhibition of calcium influx and arachidonate release was statistically significant (r2 = 0.993). Similarly, a linear relationship was demonstrated between inhibition of calcium influx and inhibition of tumor cell proliferation (r2 = 0.971). Groups II-IV had minimal or no signal or growth inhibitory activity. This investigation provides the first evidence for a coordinate link between calcium influx, calcium-mediated arachidonic acid release, and malignant proliferation and metastasis and constitutes the initial analysis of structurally important domains of the CAI molecule.