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Structure-based drug design targeting the cell membrane receptor GPBAR1: exploiting the bile acid scaffold towards selective agonism.

Di Leva FS, Festa C, Renga B, Sepe V, Novellino E, Fiorucci S, Zampella A, Limongelli V - Sci Rep (2015)

Bottom Line: A number of dual GPBAR1/FXR agonists are known, however their therapeutic use is limited by multiple unwanted effects due to activation of the diverse downstream signals controlled by the two receptors.On the other hand, designing selective GPBAR1 and FXR agonists is challenging since the two proteins share similar structural requisites for ligand binding.The presence of the 3α-NH2 group on the steroidal scaffold is responsible for the selectivity over FXR unveiling unprecedented structural insights into bile acid receptors activity modulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, University of Naples "Federico II", Via D. Montesano 49, I-80131 Naples, Italy.

ABSTRACT
Bile acids can regulate nutrient metabolism through the activation of the cell membrane receptor GPBAR1 and the nuclear receptor FXR. Developing an exogenous control over these receptors represents an attractive strategy for the treatment of enterohepatic and metabolic disorders. A number of dual GPBAR1/FXR agonists are known, however their therapeutic use is limited by multiple unwanted effects due to activation of the diverse downstream signals controlled by the two receptors. On the other hand, designing selective GPBAR1 and FXR agonists is challenging since the two proteins share similar structural requisites for ligand binding. Here, taking advantage of our knowledge of the two targets, we have identified through a rational drug design study a series of amine lithocholic acid derivatives as selective GPBAR1 agonists. The presence of the 3α-NH2 group on the steroidal scaffold is responsible for the selectivity over FXR unveiling unprecedented structural insights into bile acid receptors activity modulation.

No MeSH data available.


Related in: MedlinePlus

Concentration-response curve on GPBAR1 and RT-PCR experiments on GLUTAg cells.(A) Concentration-response curve on GPBAR1 activation by compound 5. (B) Effect of 5 on relative mRNA expression of pro-glucagon in GLUTAg cells left untreated (NT) or stimulated with 10 μM compound 5. Values are normalized relative to GAPDH mRNA and are expressed relative to those of nontreated cells (NT), which are arbitrarily set to 1: *p < 0.05 vs NT.
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f7: Concentration-response curve on GPBAR1 and RT-PCR experiments on GLUTAg cells.(A) Concentration-response curve on GPBAR1 activation by compound 5. (B) Effect of 5 on relative mRNA expression of pro-glucagon in GLUTAg cells left untreated (NT) or stimulated with 10 μM compound 5. Values are normalized relative to GAPDH mRNA and are expressed relative to those of nontreated cells (NT), which are arbitrarily set to 1: *p < 0.05 vs NT.

Mentions: Derivatives 3–8 were tested for their activity on FXR and GPBAR1. For this purpose, we performed a luciferase reporter assay using HepG2 and HEK-293T cells transfected with FXR and GPBAR1, respectively (Fig. 6). Unfortunately compounds 6–8, featuring the amino group at C-3 in β configuration were proved to be cytotoxic on HEK-293T cells at 10 μM (data not shown). Of interest, the results shown in Fig. 6 demonstrate that all 3α-amino LCA derivatives generated in this study are agonists of GPBAR1 (Fig. 6A). Indeed, we found that the substitution of the hydroxyl group at C-3 on LCA scaffold with a α-amino group produces selective GPBAR1 agonists, devoid of FXR agonist properties (Fig. 6C). On the other hand, none of the tested compounds turned out to be GPBAR1 antagonist (Fig. 6B). However, when compound 3 was tested at 50 μM in the presence of CDCA (Fig. 6D), the relative luciferase/renilla units ratio RLU/RRU was higher than that of CDCA, indicating that 3 might be also an FXR modulator. Overall, LCA 3α-amino derivatives thus represent promising templates for generating selective GPBAR1 modulators. The potency of compound 5, a selected member of our set of LCA derivatives, was further investigated by a detailed measurement of concentration-response curve on GPBAR1. As shown in Fig. 7A, compound 5 effectively induced GPBAR1 transactivation in a concentration-dependent manner, with a relative EC50 of 6.8 μM. Moreover, the agonism of 5 on GPBAR1 was also substantiated by RT-PCR experiments where 5 was effective in inducing the expression of pro-glucagon mRNA in GLUTAg cells, an intestinal endocrine cell line (Fig. 7B).


Structure-based drug design targeting the cell membrane receptor GPBAR1: exploiting the bile acid scaffold towards selective agonism.

Di Leva FS, Festa C, Renga B, Sepe V, Novellino E, Fiorucci S, Zampella A, Limongelli V - Sci Rep (2015)

Concentration-response curve on GPBAR1 and RT-PCR experiments on GLUTAg cells.(A) Concentration-response curve on GPBAR1 activation by compound 5. (B) Effect of 5 on relative mRNA expression of pro-glucagon in GLUTAg cells left untreated (NT) or stimulated with 10 μM compound 5. Values are normalized relative to GAPDH mRNA and are expressed relative to those of nontreated cells (NT), which are arbitrarily set to 1: *p < 0.05 vs NT.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4645117&req=5

f7: Concentration-response curve on GPBAR1 and RT-PCR experiments on GLUTAg cells.(A) Concentration-response curve on GPBAR1 activation by compound 5. (B) Effect of 5 on relative mRNA expression of pro-glucagon in GLUTAg cells left untreated (NT) or stimulated with 10 μM compound 5. Values are normalized relative to GAPDH mRNA and are expressed relative to those of nontreated cells (NT), which are arbitrarily set to 1: *p < 0.05 vs NT.
Mentions: Derivatives 3–8 were tested for their activity on FXR and GPBAR1. For this purpose, we performed a luciferase reporter assay using HepG2 and HEK-293T cells transfected with FXR and GPBAR1, respectively (Fig. 6). Unfortunately compounds 6–8, featuring the amino group at C-3 in β configuration were proved to be cytotoxic on HEK-293T cells at 10 μM (data not shown). Of interest, the results shown in Fig. 6 demonstrate that all 3α-amino LCA derivatives generated in this study are agonists of GPBAR1 (Fig. 6A). Indeed, we found that the substitution of the hydroxyl group at C-3 on LCA scaffold with a α-amino group produces selective GPBAR1 agonists, devoid of FXR agonist properties (Fig. 6C). On the other hand, none of the tested compounds turned out to be GPBAR1 antagonist (Fig. 6B). However, when compound 3 was tested at 50 μM in the presence of CDCA (Fig. 6D), the relative luciferase/renilla units ratio RLU/RRU was higher than that of CDCA, indicating that 3 might be also an FXR modulator. Overall, LCA 3α-amino derivatives thus represent promising templates for generating selective GPBAR1 modulators. The potency of compound 5, a selected member of our set of LCA derivatives, was further investigated by a detailed measurement of concentration-response curve on GPBAR1. As shown in Fig. 7A, compound 5 effectively induced GPBAR1 transactivation in a concentration-dependent manner, with a relative EC50 of 6.8 μM. Moreover, the agonism of 5 on GPBAR1 was also substantiated by RT-PCR experiments where 5 was effective in inducing the expression of pro-glucagon mRNA in GLUTAg cells, an intestinal endocrine cell line (Fig. 7B).

Bottom Line: A number of dual GPBAR1/FXR agonists are known, however their therapeutic use is limited by multiple unwanted effects due to activation of the diverse downstream signals controlled by the two receptors.On the other hand, designing selective GPBAR1 and FXR agonists is challenging since the two proteins share similar structural requisites for ligand binding.The presence of the 3α-NH2 group on the steroidal scaffold is responsible for the selectivity over FXR unveiling unprecedented structural insights into bile acid receptors activity modulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, University of Naples "Federico II", Via D. Montesano 49, I-80131 Naples, Italy.

ABSTRACT
Bile acids can regulate nutrient metabolism through the activation of the cell membrane receptor GPBAR1 and the nuclear receptor FXR. Developing an exogenous control over these receptors represents an attractive strategy for the treatment of enterohepatic and metabolic disorders. A number of dual GPBAR1/FXR agonists are known, however their therapeutic use is limited by multiple unwanted effects due to activation of the diverse downstream signals controlled by the two receptors. On the other hand, designing selective GPBAR1 and FXR agonists is challenging since the two proteins share similar structural requisites for ligand binding. Here, taking advantage of our knowledge of the two targets, we have identified through a rational drug design study a series of amine lithocholic acid derivatives as selective GPBAR1 agonists. The presence of the 3α-NH2 group on the steroidal scaffold is responsible for the selectivity over FXR unveiling unprecedented structural insights into bile acid receptors activity modulation.

No MeSH data available.


Related in: MedlinePlus