No abstract

Preclinical pharmacological characterization of a novel inhibitor (UM8190) of prolylcarboxypeptidase (PRCP) was investigated. We synthesized and evaluated a library of proline-based analogs as prospective recombinant PRCP (rPRCP) inhibitors and inhibitors of PRCP-dependent prekallikrein (PK) activation on human pulmonary artery endothelial cells (HPAEC). Among the newly synthesized compounds, UM8190 was further characterized in vivo using methods that encompassed a mouse carotid artery thrombosis model and animal model of food consumption. (S)-N-dodecyl-1-((S)-pyrrolidine-2-carbonyl) pyrrolidine-2-carboxamide [Compound 3 (UM8190)] was selected for further evaluation from the initial assessment of its PRCP inhibitory action (Ki= 43 µM) coupled with its ability to block PRCP-dependent PK activation on HPAEC (Ki= 34 µM). UM8190 demonstrated excellent selectivity against a panel of carboxypeptidases and serine proteases and blocked bradykinin (BK) generation and BK-induced permeability by 100%, suggesting that it may be useful in preventing the local production of large amounts of BK. Furthermore, UM8190 showed an anorexigenic effect when systemically administered to fasted mice, reducing food intake in a dose- and time-dependent manner. In a mouse carotid artery thrombosis model, it also demonstrated an antithrombotic effect. UM8190 is a selective PRCP inhibitor and it may represent a new anorexigenic, and antithrombotic drug, that works by inhibiting PRCP–mediated mechanisms.

A novel binding site for angiotensins II and III that is unmasked by parachloromercuribenzoate has been reported in rat, mouse and human brains. Initial studies of this binding site indicate that it is not expressed in the adrenal, liver or kidney of the rat and mouse. To determine if this binding site occurs in other mouse tissues, 8 tissues were assayed for expression of this binding site by radioligand binding assay and compared with the expression of this binding site in the forebrain. Particulate fractions of homogenates of testis, epididymis, seminal vesicles, heart, spleen, pancreas, lung, skeletal muscle, and forebrain were incubated with (125)I-sarcosine(1), isoleucine(8) angiotensin II in the presence or absence of 0.3mM parachloromercuribenzoate plus 10microM losartan and 10microM PD123319 (to saturate AT(1) and AT(2) receptors). Specific (3microM angiotensin II displaceable) high affinity binding occurred in the testis>forebrain>epididymis>spleen>pancreas>lung when parachloromercuribenzoate was present. Binding could not be reliably observed in heart, skeletal muscle and seminal vesicles. High affinity binding of (125)I-sarcosine(1), isoleucine(8) angiotensin II was observed in the absence of parachloromercuribenzoate in the pancreas on occasion. This suggests that this novel angiotensin binding site may have a functional role in these tissues.

We recently reported the existence of a novel, non‐AT1, non‐AT2 binding site for angiotensins II (Ang II) and III (Ang III) in the rat, mouse and human brain. While the absence of this binding site in the liver, adrenal, and kidney suggested that this binding site was brain‐specific, we now report its presence in other mouse tissues. 125I‐Sar1,Ile8 Ang II binding was determined as described previously (Karamyan et al., Eur J. Pharmacol., 2008, 590: 87) in tissue homogenates with or without 0.3 mM parachloromercuribenzoate (PCMB), in the presence of 10 μM losartan and 10 μM PD123319 to saturate AT1 and AT2 receptors, respectively. In the absence of 0.3 mM PCMB specific (3 μM Ang II displaceable) binding of 125I‐ Sar1,Ile8 Ang II could not be measured reliably. In the presence of 0.3 mM PCMB, saturable, high affinity binding was observed in the testis (4.1±2.2 pmol/g, KD = 5.0±2.7 nM) with comparable density to the brain (4.2±2.0 pmol/g), but slightly lower affinity (KD = 2.7±1.2 nM, p<0.05). Measurable binding was present in 5 of 6 pancreas and 4 of 7 spleen samples with KD values similar to the brain, but at lower (23 and 30%, respectively) binding density. Binding was generally undetectable in the lung and heart. The presence of this binding site in testis, pancreas and spleen may indicate additional functions for angiotensins in these tissues. Supported by the Peptide Radioiodination Service Center of the University of Mississippi.

To further characterize the non‐AT1, non‐AT2 binding site in mouse brain, differential centrifugation was used to assess its subcellular distribution. The fraction enriched in plasma membranes (P2) from both cerebral cortex (CTX) and hypothalamus (HT) showed a higher density of 125I‐Ang II binding relative to the nuclear/mitochondrial/debris (P1) fraction: CTX P1 Bmax= 21.9±3.6 fmol/mg protein, CTX P2 Bmax = 63.1±11.1 fmol/mg protein; HT P1 Bmax = 24.5±7.6 fmol/mg protein, HT P2 Bmax = 38.3±9.8 fmol/mg protein. The enriched binding in CTX and HT plasma membranes (71 and 66% of total binding, respectively) suggests that the non‐AT1, non‐AT2 angiotensin binding site is primarily localized to plasma membrane. Since p‐chloromercuribenzoate (PCMB), the agent that unmasks this binding site, may simulate pathophysiological conditions in the brain associated with an altered redox state, we examined the effect of reduced glutathione (GSH) on the ability of PCMB to unmask the binding site in P2 membranes. GSH at concentrations ranging from 0.3 to 10 mM inhibited 125I‐Ang II binding to the non‐AT1, non‐AT2 binding site from 78 to 87% in a concentration‐related manner. A small residual amount of 125I‐Ang II binding to the non‐AT1, non‐AT2 binding site in the absence of PCMB was also inhibited by 0.3 to 10 mM GSH by 51 to 70%. Functionality of the non‐AT1, non‐AT2 binding site may thus depend on redox state of the brain.