Limits...
Electronic sculpting of ligand-GPCR subtype selectivity: the case of angiotensin II.

Magnani F, Pappas CG, Crook T, Magafa V, Cordopatis P, Ishiguro S, Ohta N, Selent J, Bosnyak S, Jones ES, Gerothanassis IP, Tamura M, Widdop RE, Tzakos AG - ACS Chem. Biol. (2014)

Bottom Line: This is especially the case for the angiotensin receptor subtypes AT1R and AT2R, where a functional negative control has been described and AT2R activation highlighted as an important cancer drug target.We describe a strategy to fine-tune ligand selectivity for the AT2R/AT1R subtypes through electronic control of ligand aromatic-prolyl interactions.Through this strategy an AT2R high affinity (Ki = 3 nM) agonist analogue that exerted 18,000-fold higher selectivity for AT2R versus AT1R was obtained.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, Medical Research Council , Cambridge CB2 0QH, United Kingdom.

ABSTRACT
GPCR subtypes possess distinct functional and pharmacological profiles, and thus development of subtype-selective ligands has immense therapeutic potential. This is especially the case for the angiotensin receptor subtypes AT1R and AT2R, where a functional negative control has been described and AT2R activation highlighted as an important cancer drug target. We describe a strategy to fine-tune ligand selectivity for the AT2R/AT1R subtypes through electronic control of ligand aromatic-prolyl interactions. Through this strategy an AT2R high affinity (Ki = 3 nM) agonist analogue that exerted 18,000-fold higher selectivity for AT2R versus AT1R was obtained. We show that this compound is a negative regulator of AT1R signaling since it is able to inhibit MCF-7 breast carcinoma cellular proliferation in the low nanomolar range.

Show MeSH

Related in: MedlinePlus

(a) Competition binding assays of [Y]6-AII analogueto AT1R, AT2R wild type, and mutants: AT1R, open circle; wild typeAT2R, black circles; AT2R-Y189A, blue diamonds; AT2R-Y189N, greentriangle; AT2R-F272(6.51)A, red square; AT2R-F272(6.51)H, orange triangle. Kd and Ki valuesare given in Supplementary Table S3. (b,c)Plots of fold selectivity values for the two AII receptor subtypes(IC50(AT1R)/IC50(AT2R)]) versus % of cis (b) and the value of Hammet substituent constants σpara (c) for the different AII analogues (see also Supplementary Table S5). (d) PC12W cells, eithertransduced with the Ad-AT2R or untransduced, were used for the evaluationof the AT2R agonistic effect of [Y]6-AII in the presenceof either 1 nM AII or [Y]6-AII. Agonist-induced neuriteoutgrowth by AII or [Y]6-AII for 24 h stimulation was quantifiedby counting neurite-positive cell numbers in five randomly selectedphotos/well. The neurite outgrowth-positive cells were defined asthe cells with neurite length longer than their cell diameters. Thisexperiment was carried out in triplicate and repeated twice.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4374176&req=5

fig3: (a) Competition binding assays of [Y]6-AII analogueto AT1R, AT2R wild type, and mutants: AT1R, open circle; wild typeAT2R, black circles; AT2R-Y189A, blue diamonds; AT2R-Y189N, greentriangle; AT2R-F272(6.51)A, red square; AT2R-F272(6.51)H, orange triangle. Kd and Ki valuesare given in Supplementary Table S3. (b,c)Plots of fold selectivity values for the two AII receptor subtypes(IC50(AT1R)/IC50(AT2R)]) versus % of cis (b) and the value of Hammet substituent constants σpara (c) for the different AII analogues (see also Supplementary Table S5). (d) PC12W cells, eithertransduced with the Ad-AT2R or untransduced, were used for the evaluationof the AT2R agonistic effect of [Y]6-AII in the presenceof either 1 nM AII or [Y]6-AII. Agonist-induced neuriteoutgrowth by AII or [Y]6-AII for 24 h stimulation was quantifiedby counting neurite-positive cell numbers in five randomly selectedphotos/well. The neurite outgrowth-positive cells were defined asthe cells with neurite length longer than their cell diameters. Thisexperiment was carried out in triplicate and repeated twice.

Mentions: Sincethe [Y]6-AII analogue experimentally fulfilled theselectivity criteria for AT2R hypothesized at the onset of this study,we measured its binding to AT1R and AT2R recombinantly expressed inmammalian cells. Interestingly, the analogue ligated AT2R with highaffinity (Ki = 3.4 ± 0.8 nM), whereassaturable binding to AT1R was in the submillimolar range (Supplementary Table S3). In order to elucidatewhether the [Y]6-AII AT2R subtype selectivity was due tothe effectiveness of the electronic tuning of the aromatic-prolylinteraction, we then performed binding experiments of the rest ofthe analogues to the AT1R and AT2R (Figure 3, Supplementary Tables S4 and S5). Notably,the AT2R/AT1R fold selectivity of all the AII analogues was directlycorrelated to the aro-pro compactness of the analogues as describedabove (Figure 3, SupplementaryTable S5): the [Y]6-AII analogue presented a 18,000-foldhigher selectivity (IC50AT1R/IC50AT2R); [4-OPO3H2-F]6-AII and [F]6-AII showedsimilar receptor subtype selectivities (4479 and 4786, respectively).In contrast and as expected, [4-NO2-F]6-AIIpresented a much lower (26-fold less) selectivity for the AT2R/AT1Rreceptor subtypes (694).


Electronic sculpting of ligand-GPCR subtype selectivity: the case of angiotensin II.

Magnani F, Pappas CG, Crook T, Magafa V, Cordopatis P, Ishiguro S, Ohta N, Selent J, Bosnyak S, Jones ES, Gerothanassis IP, Tamura M, Widdop RE, Tzakos AG - ACS Chem. Biol. (2014)

(a) Competition binding assays of [Y]6-AII analogueto AT1R, AT2R wild type, and mutants: AT1R, open circle; wild typeAT2R, black circles; AT2R-Y189A, blue diamonds; AT2R-Y189N, greentriangle; AT2R-F272(6.51)A, red square; AT2R-F272(6.51)H, orange triangle. Kd and Ki valuesare given in Supplementary Table S3. (b,c)Plots of fold selectivity values for the two AII receptor subtypes(IC50(AT1R)/IC50(AT2R)]) versus % of cis (b) and the value of Hammet substituent constants σpara (c) for the different AII analogues (see also Supplementary Table S5). (d) PC12W cells, eithertransduced with the Ad-AT2R or untransduced, were used for the evaluationof the AT2R agonistic effect of [Y]6-AII in the presenceof either 1 nM AII or [Y]6-AII. Agonist-induced neuriteoutgrowth by AII or [Y]6-AII for 24 h stimulation was quantifiedby counting neurite-positive cell numbers in five randomly selectedphotos/well. The neurite outgrowth-positive cells were defined asthe cells with neurite length longer than their cell diameters. Thisexperiment was carried out in triplicate and repeated twice.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4374176&req=5

fig3: (a) Competition binding assays of [Y]6-AII analogueto AT1R, AT2R wild type, and mutants: AT1R, open circle; wild typeAT2R, black circles; AT2R-Y189A, blue diamonds; AT2R-Y189N, greentriangle; AT2R-F272(6.51)A, red square; AT2R-F272(6.51)H, orange triangle. Kd and Ki valuesare given in Supplementary Table S3. (b,c)Plots of fold selectivity values for the two AII receptor subtypes(IC50(AT1R)/IC50(AT2R)]) versus % of cis (b) and the value of Hammet substituent constants σpara (c) for the different AII analogues (see also Supplementary Table S5). (d) PC12W cells, eithertransduced with the Ad-AT2R or untransduced, were used for the evaluationof the AT2R agonistic effect of [Y]6-AII in the presenceof either 1 nM AII or [Y]6-AII. Agonist-induced neuriteoutgrowth by AII or [Y]6-AII for 24 h stimulation was quantifiedby counting neurite-positive cell numbers in five randomly selectedphotos/well. The neurite outgrowth-positive cells were defined asthe cells with neurite length longer than their cell diameters. Thisexperiment was carried out in triplicate and repeated twice.
Mentions: Sincethe [Y]6-AII analogue experimentally fulfilled theselectivity criteria for AT2R hypothesized at the onset of this study,we measured its binding to AT1R and AT2R recombinantly expressed inmammalian cells. Interestingly, the analogue ligated AT2R with highaffinity (Ki = 3.4 ± 0.8 nM), whereassaturable binding to AT1R was in the submillimolar range (Supplementary Table S3). In order to elucidatewhether the [Y]6-AII AT2R subtype selectivity was due tothe effectiveness of the electronic tuning of the aromatic-prolylinteraction, we then performed binding experiments of the rest ofthe analogues to the AT1R and AT2R (Figure 3, Supplementary Tables S4 and S5). Notably,the AT2R/AT1R fold selectivity of all the AII analogues was directlycorrelated to the aro-pro compactness of the analogues as describedabove (Figure 3, SupplementaryTable S5): the [Y]6-AII analogue presented a 18,000-foldhigher selectivity (IC50AT1R/IC50AT2R); [4-OPO3H2-F]6-AII and [F]6-AII showedsimilar receptor subtype selectivities (4479 and 4786, respectively).In contrast and as expected, [4-NO2-F]6-AIIpresented a much lower (26-fold less) selectivity for the AT2R/AT1Rreceptor subtypes (694).

Bottom Line: This is especially the case for the angiotensin receptor subtypes AT1R and AT2R, where a functional negative control has been described and AT2R activation highlighted as an important cancer drug target.We describe a strategy to fine-tune ligand selectivity for the AT2R/AT1R subtypes through electronic control of ligand aromatic-prolyl interactions.Through this strategy an AT2R high affinity (Ki = 3 nM) agonist analogue that exerted 18,000-fold higher selectivity for AT2R versus AT1R was obtained.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, Medical Research Council , Cambridge CB2 0QH, United Kingdom.

ABSTRACT
GPCR subtypes possess distinct functional and pharmacological profiles, and thus development of subtype-selective ligands has immense therapeutic potential. This is especially the case for the angiotensin receptor subtypes AT1R and AT2R, where a functional negative control has been described and AT2R activation highlighted as an important cancer drug target. We describe a strategy to fine-tune ligand selectivity for the AT2R/AT1R subtypes through electronic control of ligand aromatic-prolyl interactions. Through this strategy an AT2R high affinity (Ki = 3 nM) agonist analogue that exerted 18,000-fold higher selectivity for AT2R versus AT1R was obtained. We show that this compound is a negative regulator of AT1R signaling since it is able to inhibit MCF-7 breast carcinoma cellular proliferation in the low nanomolar range.

Show MeSH
Related in: MedlinePlus