Rho-Rho kinase pathway in the actomyosin contraction and cell-matrix adhesion in immortalized human trabecular meshwork cells.

PURPOSE: The outflow facility for aqueous humor across the trabecular meshwork (TM) is enhanced by agents that oppose the actomyosin contraction of its resident cells. Phosphorylation of MYPT1 (myosin light chain [MLC] phosphatase complex of Type 1) at Thr853 and Thr696 inhibits dephosphorylation of MLC, leading to an increase in actomyosin contraction. In this study, we examined the effects of Rho kinase (ROCK) inhibitors on the relative dephosphorylation of the two sites of MYPT1 using human TM cells (GTM3). METHODS: Dephosphorylation of MYPT1 at Thr853 and Thr696 was determined by western blot analysis following exposure to selective inhibitors of ROCK, namely Y-27632 and Y-39983. Consequent dephosphorylation of MLC and decreases in actomyosin contraction were assessed by western blot analysis and collagen gel contraction assay, respectively. Changes in the cell-matrix adhesion were measured in real time by electric cell-substrate impedance sensing and also assessed by staining for paxillin, vinculin, and focal adhesion kinase (FAK). RESULTS: Both ROCK inhibitors produced a concentration-dependent dephosphorylation at Thr853 and Thr696 of MYPT1 in adherent GTM3 cells. IC50 values for Y-39983 were 15 nM and 177 nM for dephosphorylation at Thr853 and Thr696, respectively. Corresponding values for Y-27632 were 658 nM and 2270 nM. Analysis of the same samples showed a decrease in MLC phosphorylation with IC50 values of 14 nM and 1065 nM for Y-39983 and Y-27632, respectively. Consistent with these changes, both inhibitors opposed contraction of collagen gels induced by TM cells. Exposure of cells to the inhibitors led to a decrease in the electrical cell-substrate resistance, with the effect of Y-39983 being more pronounced than Y-27632. Treatment with these ROCK inhibitors also showed a loss of stress fibers and a concomitant decrease in tyrosine phosphorylation of paxillin and FAK. CONCLUSIONS: Y-39983 and Y-27632 oppose ROCK-dependent phosphorylation of MYPT1 predominantly at Thr853 with a corresponding decrease in MLC phosphorylation. A relatively low effect of both ROCK inhibitors at Thr696 suggests a role for other Ser/Thr kinases at this site. Y-39983 was several-fold more potent when compared with Y-27632 at inhibiting the phosphorylation of MYPT1 at either Thr853 or Thr696 commensurate with its greater potency at inhibiting the activity of human ROCK-I and ROCK-II enzymes.

Salicylamide inhibitors of influenza virus fusion.

Structural variation of the quinolizidine heterocycle of the influenza fusion inhibitor BMY-27709 was examined by several topological dissections in order to illuminate the critical features of the ring system. This exercise resulted in the identification of a series of synthetically more accessible decahydroquinolines that retained the structural elements of BMY-27709 important for antiviral activity. The 2-methyl-cis-decahydroquinoline 6f was the most potent influenza inhibitor identified that demonstrated an EC50 of 90 ng/mL in a plaque reduction assay.

1,2-Benzisothiazol-3-one 1,1-dioxide inhibitors of human mast cell tryptase.

A library of compounds were prepared by reacting 2-(bromomethyl)-1, 2-benzisothiazol-3(2H)-one 1,1-dioxide (5) with commercially available carboxylic acids in the presence of potassium carbonate or a tertiary amine base. From this library, (1,1-dioxido-3-oxo-1, 2-benzisothiazol-2(3H)-yl)methyl N-[(phenylmethoxy)carbonyl]-beta-alanate (7b) emerged as a potent inhibitor of human mast cell tryptase (IC50 = 0.85 microM). Extension of the side chain of 7b by two carbons gave (1, 1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)methyl 5-[[(phenylmethoxy)carbonyl]amino]pentanoate (7d) which was an 8-fold more potent inhibitor (IC50 = 0.1 microM). Further modification of this series produced benzoic acid derivative (1, 1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)methyl 4-[[(phenylmethoxy)carbonyl]amino]benzoate (7n) which is the most potent inhibitor identified in this series (IC50 = 0.064 microM). These compounds exhibit time-dependent inhibition consistent with mechanism-based inhibition. For 7b, the initial enzyme velocity is not a saturable function of the inhibitor concentration and the initial Ki could not be determined (Ki > 10 microM). The steady-state rate constant, Ki, was determined to be 396 nM. On the other hand, compounds 7d and 7n are time-dependent inhibitors with a saturable initial complex. From these studies, an initial rate constant, Ki, for 7d and 7n was found to be 345 and 465 nM, respectively. The steady-state inhibition constants, Ki, for 7d and 7n were calculated to be 60 and 52 nM, respectively. Compound 7n is a 13-fold more potent inhibitor than 7b, and these kinetic studies indicate that the increase in inhibitory activity is due to an increase in initial affinity toward the enzyme and not an increase in chemical reactivity. These inhibitors generally show high selectivity for tryptase, being 40-fold weaker inhibitors of elastase, being 100-fold weaker against trypsin, and showing no inhibition against thrombin. These compounds are not inhibitors of thrombin, plasmin t-PA, urokinase, and factor Xa (IC50 > 33 microM). In the delayed-type hypersensitivity (DTH) mouse model, a model of skin inflammation, a 5% solution of 7d reduced edema by 69% compared to control animals.