Pharmacological characterisation of new hU-II analogs modified at Tyr9

Madura Batuwangala1, Valeria Camarda2, Erika Marzola3, David Lambert1, Leong Ng1, Remo Guerrini3, Severo Salvadori3, Domenico Regoli2, Girolamo Calo’2
1University of Leicester, Department of Cardiovascular Sciences, Leicester, United Kingdom, 2University of Ferrara, Department of Pharmacology, Ferrara, Italy, 3University of Ferrara, Department of Pharmaceutical Sciences, Ferrara, Italy

Urotensin-II (U-II) is a cyclic peptide that selectively binds and activates the previously orphan UT receptor (Ames et al., 1999). In this study we have characterised nine new Tyr9 containing U-II(4-11) analogs in vitro and ex vivo using the calcium mobilization and rat aorta bioassays, respectively

U-II analogs were screened using the Flexstation II fluorometric imaging plate reader with HEK293 cells expressing recombinant rat UT (HEK293rUT). Thoracic aortae from Sprague-Dawley rats were isolated and cut into helical strips. The strips were suspended in organ baths, equilibrated for 1 hour with one gram preload and washed every 20 mins. Experiments were carried out after this initial equilibration period. Tissue responsiveness was assessed by the addition of 1 micromolar noradrenaline. Protocols were carried out as described previously (Camarda et al., 2006). All experimental data are from n > 5 separate experiments

Using the calcium mobilization assay and with respect to U-II(4-11) we demonstrated that i) Ortho and Meta Tyr9 modifications do not alter pharmacological activity, ii) chirality of Tyr9 is an important factor, iii) N-methylation as well as cyclization of the Tyr9 side chain on the nitrogen or C-alpha chiral carbon is detrimental for biological activity. Moreover 3,5-diiodination of the Tyr9 aromatic ring slightly increased peptide potency 7.74(7.19-8.29) vs 7.60(7.34-7.87) (95% confidence interval) with reference to the control peptide and produced a clear reduction of peptide efficacy (alpha = 0.55) thus generating the partial agonist [(3,5-diiodo)Tyr9]U-II(4-11). This pharmacological behaviour was confirmed in rat aorta bioassay experiments where [(3,5-diiodo)Tyr9]U-II(4-11) displayed reduced efficacy (alpha = 0.54) compared to U-II and a similar potency when evaluated as an agonist (pEC50 7.70(7.41-7.98)) or as an antagonist (pA2 7.75 (6.91-8.59))

These data indicate that Tyr9 diiodonation serves as a means of generating new U-II analogs. Further studies in cell lines expressing the native human and recombinant UT receptor are warranted in order to characterise this new partial agonist further

Ames RS et al., (1999) Nature 401: 282-286
Camarda V et al., (2006). Br J Pharmacol 147: 92-100

MB holds an International PhD studentship for joint degree from University of Leicester and University of Ferrara