The inhibitory profile of Ibudilast against the human phosphodiesterase enzyme family.

Ibudilast is widely used in Japan to treat ischemic stroke and bronchial asthma. Its mode of action is through the inhibition of cyclic nucleotide phosphodiesterases (PDEs). Growing evidence suggests this compound has utility in a range of neurological conditions linked to its ability to elevate cellular cyclic nucleotide concentrations, however limited data exists on Ibudilast's action on individual PDE families. We therefore used an extensive panel of human PDE enzymes to define the PDE inhibitory profile of this compound. Ibudilast preferentially inhibits PDE3A, PDE4, PDE10 and PDE11 with lesser inhibition of a number of other families. The significance of these findings is discussed in relation to Ibudilast's observed effects on certain disease states.

Measurement of agonist-dependent and -independent signal initiation of alpha(1b)-adrenoceptor mutants by direct analysis of guanine nucleotide exchange on the G protein galpha(11).

Immunoprecipitation of a fusion protein between the alpha(1b)-adrenoceptor and Galpha(11) following a [(35)S]GTPgammaS [guanosine-5'-O-(3-thio)triphosphate] binding assay resulted in incorporation of low levels of nucleotide. The agonist phenylephrine increased incorporation some 30-fold. Agonist-induced binding represented 1.0 mol of [(35)S]GTPgammaS/mol of fusion protein. This was to the G protein linked to the receptor rather than endogenous Galpha(q)/Galpha(11) as a fusion protein containing the alpha(1b)-adrenoceptor and a form of Galpha(11) (G(208)A) unable to exchange guanine nucleotides effectively, bound [(35)S]GTPgammaS very poorly. Fusion proteins between A(293)E, D(142)A, and 3CAM mutants of the alpha(1b)-adrenoceptor and Galpha(11) bound substantially greater levels of [(35)S]GTPgammaS in the absence of agonist than the fusion incorporating the wild-type receptor. Constitutive binding of the nucleotide induced by these mutants was only 20% of the level achieved by phenylephrine. These mutant receptors thus do not provide an accurate mimic of the agonist-occupied state. Phentolamine reduced the binding of [(35)S]GTPgammaS and acted as a partial inverse agonist for each of the constitutively active mutants. [(35)S]GTPgammaS binding to Galpha(11) was elevated by phenylephrine in both wild-type and constitutively active mutant forms of the fusion proteins, but agonist potency and binding affinity were 50 times higher for the fusions containing the mutated receptors. These studies provide the first direct demonstration of the capacity of constitutively active mutants of a receptor to stimulate guanine nucleotide exchange on the alpha subunit of a G(q) family G protein and defines a strategy potentially suitable for any receptor that couples to these G proteins.

Ligand rescue of constitutively active mutant receptors.

Constitutively active mutants (CAMs) of G protein-coupled receptors are found naturally in disease states and they can be generated by point mutation. As these mutants are able to activate G proteins in the absence of a ligand, they are useful tools in the study of conformational changes leading to receptor activation and in the drug discovery process. Early studies on CAMs noted that they are often expressed at lower levels than their wild-type forms. In this review we discuss the mechanisms responsible for this reduced expression and also provide an explanation for the observation that challenging cells with receptor ligands can increase the CAM expression level. The application of these observations to the development of a high-throughput reporter assay suitable for ligand identification is also discussed.