DEVELOPMENT OF SPECIFIC INHIBITORS OF TRANSGLUTAMINASE: PHENYLTHIOUREA DERIVATIVES

A series of phenylthiourea derivatives of (alpha),(omega)-diaminoalkanes was designed to retain three structural domains which might have structure-function potentials: (a) an aminoalkyl side chain for inhibition of transglutaminase-catalyzed reactions; (b) a hydrophobic phenyl group for ready uptake by cells; and (c) a thiourea domain for potential inhibition of cell proliferation. The compounds were synthesized and evaluated as inhibitors of transglutaminase, and their cellular effects were also examined. Five compounds of phenylthiourea-(CH(,2))(,n)-NH(,2) with n = 2, 3, 4, 5, and 6 were tested as inhibitors of both cellular transglutaminase and Factor XIIIa-catalyzed reactions, i.e., (a) amine incorporation into various glutamine-containing substrates, and (b) fibrin clot stabilization. In all the assays the inhibitory activity of the compounds increases with increasing number of methylene groups in the side chain up to a maximum for n = 5. A further increase to n = 6 results in decreased activity. The K(,i) value (4.9 x 10('-5) M) of 1-(5-aminopentyl)-3-phenylthiourea (PPTU). with n = 5, for the inhibition of guinea pig liver transglutaminase-catalyzed amine incorporation into B chain of oxidized insulin is in close agreement to its K(,m(app)) value (7.1 x 10('-5) M) directly obtained using ('14)C-labeled PPTU. The cellular uptake and distribution of PPTU was examined using Chinese hamster ovary (CHO) cells. The labeled PPTU is rapidly taken up, within 1 hour, by the cells and distributed evenly to nuclear and supernatant fractions. Uptake appears to reach a plateau at 2 hours of incubation. The effect of PPTU on the proliferation of CHO normal cells and B16 melanoma cells was also evaluated. PPTU at concentrations of 4.2 x 10('-3) M and 2.4 x 10('-3) M caused 50% inhibition of CHO and B16 cell proliferation, respectively. A similar dose-dependent effect was observed with 1-(2-aminoethyl)-3-phenylthiourea, with n = 2, indicating that side chain length is not a factor but that the phenylthiourea group may be important for cellular uptake and inhibition of cell proliferation. These results suggest that 1-(5-aminopentyl)-3-phenylthiourea (PPTU) is an inhibitor which may be useful for preventing both cellular transglutaminase and Factor XIIIa-catalyzed reactions, and cell proliferation.