Investigation of the effect of the farnesyl protein transferase inhibitor R115777 on isoprenylation and intracellular signalling by the prostacyclin receptor.

The human (h) and mouse (m) prostacyclin receptors (IPs) undergo isoprenylation through attachment of a C-15 farnesyl moiety within their conserved carboxyl terminal -CSLC sequences. Herein, the effects of a novel farnesyl transferase inhibitor R115777 on signalling by the hIP and mIP, overexpressed in human embryonic kidney 293 cells, and by the hIP endogenously expressed in human erythroleukaemia cells were investigated. R115777 significantly impaired IP-mediated cyclic AMP generation (IC(50) 0.37-0.60 nm) and intracellular calcium ([Ca(2+)](i)) mobilization (IC(50) 37-65 nm), but had no effect on signalling by the control nonisoprenylated beta(2) adrenergic receptor or the alpha or beta isoforms of the human thromboxane A(2) receptor (TP). Additionally, R115777 significantly reduced IP-mediated cross-desensitization of signalling by the TP alpha, but not by the TP beta, isoform of the human TP and impaired the farnesylation-dependent processing of the chaperone HDJ-2 protein (IC(50) 4.5 nm). Furthermore, R115777 fully impaired isoprenylation of both the Ha-Ras(WT) and Ha-Ras(CSLC) in vitro and in whole cells confirming that, unlike N-Ras and Ki-Ras, the -CSLC motif associated with the IP cannot support alternative geranylgeranylation in the presence of R115777 and does not act as a substrate for geranylgeranyl transferase 1 in vitro or in whole cells. In conclusion, these data confirm that R115777 potently impairs IP isoprenylation and signalling, and suggest that clinically it may not only target Ras proteins but may also disrupt IP isoprenylation, events which could impact on physiologic processes in which prostacyclin and its receptor are implicated.

Ocular hypotensive FP prostaglandin (PG) analogs: PG receptor subtype binding affinities and selectivities, and agonist potencies at FP and other PG receptors in cultured cells.

Natural prostaglandins (PGs) such as PGD2, PGE2, PGF2(2alpha), and PGI2 exhibited the highest affinity for their respective cognate receptors, but were the least selective agents when tested in receptor binding assays. Travoprost acid ([+]-fluprostenol) was the most FP-receptor-selective compound, exhibiting a high affinity (Ki = 35 +/- 5 nM) for the FP receptor, and minimal affinity for DP (Ki = 52,000 nM), EP1 (Ki = 9540 nM), EP3 (Ki = 3501 nM), EP4 (Ki = 41,000 nM), IP (Ki > 90,000 nM), and TP (Ki = 121,000 nM) receptors. Travoprost acid was the most potent PG analog tested in FP receptor functional phosphoinositide turnover assays in the following cell types: human ciliary muscle (EC50 = 1.4 nM), human trabecular meshwork (EC50 = 3.6 nM), and mouse fibroblasts and rat aortic smooth muscle cells (EC50 = 2.6 nM). Although latanoprost acid exhibited a relatively high affinity for the FP receptor (Ki = 98 nM), it had significant functional activity at FP (EC50 = 32-124 nM) and EP1 (EC50 = 119 nM) receptors. Bimatoprost acid was less selective, exhibiting a relatively high affinity for the FP (Ki = 83 nM), EP1 (Ki = 95 nM), and EP3 (Ki = 387 nM) receptors. Bimatoprost acid exhibited functional activity at the EP1 (EC50 = 2.7 nM) and FP (EC50 = 2.8-3.8 nM in most cells) receptors. Bimatoprost (nonhydrolyzed amide) also behaved as an FP agonist at the cloned human FP receptor (EC50 = 681 nM), in h-TM (EC50 = 3245 nM) and other cell types. Unoprostone and S-1033 bound with low affinity (Ki = 5.9 microM to > 22 microM) to the FP receptor, were not selective, but activated the FP receptor. In conclusion, travoprost acid has the highest affinity, the highest FP-receptor-selectivity, and the highest potency at the FP receptor as compared to the other ocular hypotensive PG analogs known so far, including free acids of latanoprost, bimatoprost, and unoprostone isopropyl ester.