Losartan and metabolite EXP3179 activate endothelial function without lowering blood pressure in AT2 receptor KO mice.

BACKGROUND: We have documented profound release of nitric oxide (NO) and endothelium-derived hyperpolarization factor (EDHF) by angiotensin II (ANGII) receptor 1 (AT1) blocker (ARB) losartan and its unique metabolite EXP3179, a pleiotropic effect that may help rationalize the protective properties of ARBs. Since blood pressure (BP) lowering by ARBs likely require an ANGII-dependent switch from AT1 to ANGII receptor 2 (AT2) signaling, a receptor known to stimulate endothelial NO release, we investigated the contribution of AT1 and AT2 to losartan and EXP3179's endothelial function-activating properties. EXPERIMENTAL APPROACH: Two AT1 ligands were used in an attempt to block the AT1-dependent endothelium-enhancing effects of EXP3179. AT2-null mice were used to evaluate the acute ex vivo and chronic in vivo effects of EXP3179 (20µM) and losartan (0.6 g/l), respectively, on endothelial function, BP and aortic stiffness. KEY RESULTS: Ex vivo blockade of AT1 receptors did not attenuate EXP3179's effects on NO and EDHF-dependent endothelial function activation. We observed significant reductions in PE-induced contractility with EXP3179 in both WT and AT2 knockout (KO) aortic rings. In vivo, a 1-month chronic treatment with losartan did not affect pulse wave velocity (PWV) but decreased PE-induced contraction by 74.9 % in WT (p < 0.0001) and 47.3 % in AT2 KO (p < 0.05). Presence of AT2 was critical to losartan's BP lowering activity. CONCLUSION: In contrast to BP lowering, the endothelial function-enhancing effects of losartan and EXP3179 are mostly independent of the classic ANGII/AT1/AT2 pathway, which sheds light on ARB pleiotropism.

Losartan metabolite EXP3179 is a unique blood pressure-lowering AT1R antagonist with direct, rapid endothelium-dependent vasoactive properties.

BACKGROUND AND PURPOSE: Losartan is an anti-hypertensive angiotensin II (ANGII) type 1 receptor (AT1R) blocker (ARB) with many unexpected therapeutic properties, even in non-blood pressure (BP)-related diseases. Administered as a prodrug, losartan undergoes serial metabolism into EXP3179, a metabolite alleged to lack AT1R-blocking properties, and EXP3174, the dominant AT1R antagonist. Having observed that losartan can decrease vascular tone in mice with low AT1R expression and inhibit Marfan aortic widening at very high doses, we investigated whether EXP3179 may have unique, AT1R-independent effects on vascular tone and endothelial function. EXPERIMENTAL APPROACH: We compared the AT1R blocking capabilities of EXP3179 and EXP3174 using AT1R-expressing cell lines. Their BP lowering and vasoactive properties were studied in normal, hypertensive and transgenic rodents, and ex vivo wire myography. KEY RESULTS: We observed that both EXP3179 and EXP3174 can fully block (100%) AT1R signaling in vitro and significantly decrease BP in normotensive and spontaneously hypertensive rats. Only EXP3179 prevented PE-induced contraction by up to 65% (p < 0.01) in L-NAME and endothelium removal-sensitive fashion. Use of transgenic mice revealed that these effects involve the eNOS/caveolin-1 axis and the endothelium-dependent hyperpolarization factor (EDHF). CONCLUSION AND IMPLICATIONS: We provide direct structure-activity evidence that EXP3179 is a BP-lowering AT1R blocker with unique endothelial function-enhancing properties not shared with losartan or EXP3174. The major pharmacological effects of losartan in patients are therefore likely more complex than simple blockade of AT1R by EXP3174, which helps rationalize its therapeutic and prophylactic properties, especially at very high doses. Reports relying on EXP3179 as an AT1R-independent losartan analogue may require careful re-evaluation.

Prostaglandin F2α and angiotensin II type 1 receptors exhibit differential cognate G protein coupling regulation.

Promiscuous G protein-coupled receptors (GPCRs) engage multiple Gα subtypes with different efficacies to propagate signals in cells. A mechanistic understanding of Gα selectivity by GPCRs is critical for therapeutic design, since signaling can be restrained by ligand-receptor complexes to preferentially engage specific G proteins. However, details of GPCR selectivity are unresolved. Here, we investigated cognate G protein selectivity using the prototypical promiscuous Gαq/11 and Gα12/13 coupling receptors, angiotensin II type I receptor (AT1R) and prostaglandin F2α receptor (FP), bioluminescence resonance energy transfer-based G protein and pathway-selective sensors, and G protein knockout cells. We determined that competition between G proteins for receptor binding occurred in a receptor- and G protein-specific manner for AT1R and FP but not for other receptors tested. In addition, we show that while Gα12/13 competes with Gαq/11 for AT1R coupling, the opposite occurs for FP, and Gαq-mediated signaling regulated G protein coupling only at AT1R. In cells, the functional modulation of biased ligands at FP and AT1R was contingent upon cognate Gα availability. The efficacy of AT1R-biased ligands, which poorly signal through Gαq/11, increased in the absence of Gα12/13. Finally, we show that a positive allosteric modulator of Gαq/11 signaling that also allosterically decreases FP-Gα12/13 coupling, lost its negative modulation in the absence of Gαq/11 coupling to FP. Together, our findings suggest that despite preferential binding of similar subsets of G proteins, GPCRs follow distinct selectivity rules, which may contribute to the regulation of ligand-mediated G protein bias of AT1R and FP.

Stereo- and Regiochemical Transannular Cyclization of a Common Hexahydro-1H-azonine to Afford Three Different Indolizidinone Dipeptide Mimetics.

Sets of azabicyclo[X.Y.0]alkanone amino acids have been effectively used to identify active conformers in peptide-based drug discovery, but they usually require multiple routes to synthesize. Employing a common method from the same nine-membered unsaturated lactam precursor, we developed conditions for stereo- and regiochemical transannular cyclizations to synthesize three different indolizidin-2- and 9-one amino acid (I2aa and I9aa) analogues. For example, (3S,5R,6R,9S)- and (3S,5S,6S,9S)-I2aa diastereomers were prepared from hexahydro-1H-azonines by using iodine in THF and in MeCN with DIB as an additive. The regioselectivity of the transannular cyclization was influenced by amine protection to favor the synthesis of the I9aa isomer. Moreover, side chains were added onto the I2aa and I9aa ring systems by way of olefin intermediates that underwent Pd-catalyzed C-H bond activation and allylic oxidation.

Paired Utility of Aza-Amino Acyl Proline and Indolizidinone Amino Acid Residues for Peptide Mimicry: Conception of Prostaglandin F2α Receptor Allosteric Modulators That Delay Preterm Birth.

Peptide mimicry employing a combination of aza-amino acyl proline and indolizidinone residues has been used to develop allosteric modulators of the prostaglandin F2α receptor. The systematic study of the N-terminal phenylacetyl moiety and the conformation and side chain functions of the central turn dipeptide residue has demonstrated the sensitive relationships between modulator activity and topology. Examination of aza-Gly-Pro and aza-Phe-Pro analogs 2a and 2b in a murine preterm labor model featuring treatment with lipopolysaccharide demonstrated their capacity to extend significantly (>20 h) the average time of delivery offering new prototypes for delaying premature birth.

4-Vinylproline.

Enantiomerically pure 4-vinylproline (Vyp) was synthesized by a five-step approach from N-(Boc)iodo-alanine (2) featuring copper-catalyzed SN2' substitution of the corresponding zincate onto ( Z)-1,4-dichlorobut-2-ene to prepare methyl 2- N-(Boc)amino-4-(chloromethyl)hexenoate (3). Intra- and intermolecular displacement of the chloride provided respectively Vyp and methyl 2- N-(Boc)amino-4-(azidomethyl)hexenoate (7) suitable for the synthesis of constrained peptide analogs.

Peptidomimetic Synthesis by Way of Diastereoselective Iodoacetoxylation and Transannular Amidation of 7-9-Membered Lactams.

Azacyclo- and azabicycloalkanone peptidomimetics were synthesized regio- and diastereoselectively by iodoacetoxylation and transannular amidation reactions on unsaturated lactam precursors contingent on ring size, olefin position, solvent, and hypervalent iodine(III) reagent. 4-Iodopyrrolizidinone 1, 7-iodoindolizidinone 2, and 4-iodo-5-acetoxylactams (e.g., 6 and 7) were made stereospecifically from 7-9-membered olefins 16, iodine, and hypervalent iodine(III) in acetonitrile or toluene, respectively. X-ray crystallography demonstrated potential for mimicry of natural peptide turn side chain and backbone conformations.

Insight into Transannular Cyclization Reactions To Synthesize Azabicyclo[X.Y.Z]alkanone Amino Acid Derivatives from 8-, 9-, and 10-Membered Macrocyclic Dipeptide Lactams.

An efficient method for synthesizing different functionalized azabicyclo[X.Y.0]alkanone amino acid derivatives has been developed employing electrophilic transannular cyclizations of 8-, 9-, and 10-membered unsaturated macrocycles to form 5,5-, 6,5-, 7,5-, and 6,6-fused bicylic amino acids, respectively. Macrocycles were obtained by a sequence featuring peptide coupling of vinyl-, allyl-, homoallyl-, and homohomoallylglycine building blocks followed by ring-closing metathesis. X-ray crystallographic analyses of the 8-, 9-, and 10-membered macrocyclic lactam starting materials as well as certain bicyclic amino acid products provided insight into their conformational preferences as well as the mechanism for the diastereoselective formation of specific azabicycloalkanone amino acids by way of transannular iodolactamization reactions.