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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.

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(a) Selected region ofa 350 ms NOESY spectrum of [Y]6-AII (90% H2O/10%D2O). The red and green linesdenote the NOE connectivities for the trans and cis isomers, respectively. Solution structures of the distinctive cis (b) and trans (c) conformers of theengineered AII analogue.
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fig2: (a) Selected region ofa 350 ms NOESY spectrum of [Y]6-AII (90% H2O/10%D2O). The red and green linesdenote the NOE connectivities for the trans and cis isomers, respectively. Solution structures of the distinctive cis (b) and trans (c) conformers of theengineered AII analogue.

Mentions: To further probethe [Y]6-AII analogue structure in solution we used NMR.A selected region of the 1H–1H 2D NOESYspectrum of the analogue is shown in Figure 2. Interestingly, in aqueous solution [Y]6-AII showed twodistinct sets of proton resonances that correspond to discrete cis and trans conformational populations.This was in contrast to the native AII where a single set of peakswas observed, representing the single conformer (trans) (Supplementary Figure S2–S4).This structural plasticity (coexistence of nearly equal populationsin solution of two different discrete conformations) could be favorablefor recognition selectivity. Due to excellent dispersion of the resonancesof the cis and trans conformers,deconvolution and complete resonance assignment was achieved (Supplementary Tables S1 and S2). Structure calculationsfor the distinctive cis and trans isomers were performed, and the structural origin of the stabilizationof the relevant conformational potencies was mapped (Figure 2). As was expected, for the [Y]6-AII cis isomer the calculations produced a family of structureswith the aromatic rings of Tyr6 and Phe8 stackedaround the Pro7 ring, thus leading to a compact hydrophobicmotif (Figure 2b). The structural architectureof this motif was found to mimic closely the conformation adoptedby Tyr-Pro-Phe minicores recorded in the crystallographic proteindatabase (Supplementary Figure S5). Thecompactness of the cis over the trans form was also probed through a more reduced accessibility of thepeptide bonds as determined from both the amide proton temperaturecoefficients and translational diffusion values (i.e., for Tyr4 we determined a diffusion coefficient of 1.9 × 10–10 m2 s–1 for the cis and 2.3 × 10–10 m2 s–1 for the trans, Supplementary Figure S6).


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) Selected region ofa 350 ms NOESY spectrum of [Y]6-AII (90% H2O/10%D2O). The red and green linesdenote the NOE connectivities for the trans and cis isomers, respectively. Solution structures of the distinctive cis (b) and trans (c) conformers of theengineered AII analogue.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: (a) Selected region ofa 350 ms NOESY spectrum of [Y]6-AII (90% H2O/10%D2O). The red and green linesdenote the NOE connectivities for the trans and cis isomers, respectively. Solution structures of the distinctive cis (b) and trans (c) conformers of theengineered AII analogue.
Mentions: To further probethe [Y]6-AII analogue structure in solution we used NMR.A selected region of the 1H–1H 2D NOESYspectrum of the analogue is shown in Figure 2. Interestingly, in aqueous solution [Y]6-AII showed twodistinct sets of proton resonances that correspond to discrete cis and trans conformational populations.This was in contrast to the native AII where a single set of peakswas observed, representing the single conformer (trans) (Supplementary Figure S2–S4).This structural plasticity (coexistence of nearly equal populationsin solution of two different discrete conformations) could be favorablefor recognition selectivity. Due to excellent dispersion of the resonancesof the cis and trans conformers,deconvolution and complete resonance assignment was achieved (Supplementary Tables S1 and S2). Structure calculationsfor the distinctive cis and trans isomers were performed, and the structural origin of the stabilizationof the relevant conformational potencies was mapped (Figure 2). As was expected, for the [Y]6-AII cis isomer the calculations produced a family of structureswith the aromatic rings of Tyr6 and Phe8 stackedaround the Pro7 ring, thus leading to a compact hydrophobicmotif (Figure 2b). The structural architectureof this motif was found to mimic closely the conformation adoptedby Tyr-Pro-Phe minicores recorded in the crystallographic proteindatabase (Supplementary Figure S5). Thecompactness of the cis over the trans form was also probed through a more reduced accessibility of thepeptide bonds as determined from both the amide proton temperaturecoefficients and translational diffusion values (i.e., for Tyr4 we determined a diffusion coefficient of 1.9 × 10–10 m2 s–1 for the cis and 2.3 × 10–10 m2 s–1 for the trans, Supplementary Figure S6).

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