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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

Predicted binding mode of 2 in hGPBAR1 (A) and rFXR (B).2 is shown as cyan sticks. The receptors are shown as gray and orange (helices H3, H4, and H12 in FXR) cartoons and sticks. Extracellular loops of GPBAR1 and nonpolar hydrogens are omitted for clarity.
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f2: Predicted binding mode of 2 in hGPBAR1 (A) and rFXR (B).2 is shown as cyan sticks. The receptors are shown as gray and orange (helices H3, H4, and H12 in FXR) cartoons and sticks. Extracellular loops of GPBAR1 and nonpolar hydrogens are omitted for clarity.

Mentions: The fact that activation of GPBAR1 and FXR can have similar effects acting on different downstream signals has prompted to develop dual GPBAR1/FXR agonists2131415. However, these compounds present a reduced therapeutic window, exposing patients to multiple unwanted effects due to activation of the diverse downstream signals controlled by the two receptors616. This leads to seek for selective ligands able to specifically activate only either one receptor. Indeed, selective GPBAR1 agonism allows controlling glucose metabolism through the release of glucagon-like peptide (GLP)-1 without affecting the FXR-related pathways. In order to design selective agonists, it is essential to know the BAs structural requisites to interact with GPBAR1 and FXR. Previous studies showed that the introduction of an ethyl group at C-6 on the CDCA ring B (6-ECDCA/OCA/INT-747)17, as well as the introduction on a sulfate group at position 24 or at position 23 on a shortened side chain (INT-767 and 2)1415, led to the development of potent dual agonists. Conversely, a marked selectivity toward GPBAR1 over FXR has been achieved through the methylation at C-23 position on the BA side chain (INT-777)18, and by stereochemical modification on ring B generating EUCDCOH, which is the first example of UDCA derivative substituted at C-6 with a β-oriented ethyl group19. Finally, independently from the functional group at C-24 on the side chain, the removal of the 3α-OH from the CDCA scaffold provides potent and selective FXR agonists19. In spite of these examples, a comprehensive understanding of the effects of BAs modifications on the activity and selectivity towards GPBAR1 and FXR is possible only if the binding mode of these compounds to the two receptors is elucidated. This is difficult because of the lack of the tridimensional structure of human GPBAR1 (hGPBAR1). We have lifted this limitation building the hGPBAR1 structure by homology modeling and performing a series of atomistic simulations and experiments on ligand/receptor binding using the hGPBAR1 model15 and the available rFXR crystal structure20. Our results are in fully agreement with previous mutagenesis data, reporting a decrease in the binding affinity for bile acid derivatives in the Asn93Ala, Glu169Ala and Tyr240Ala mutant forms of hGPBAR121. In particular, we found that in GPBAR1 the 3α-OH on BA scaffold forms a stable H-bond interaction with the negatively charged side chain of Glu169 on the transmembrane helix-5 (TM-5) (Fig. 2A). At variance with GPBAR1, in FXR the 3α-OH group H-bonds with a positively charged residue, His444 on helix H12 (Fig. 2B). This ligand/receptor interaction stabilizes the cation−π interaction between His444 and Trp466 in the activation function-2 (AF-2) domain, which is essential for FXR activation20. The opposite charge on the interacting partner of 3α-OH group in the two receptors provides a hint to achieve a selective binding. In fact, the replacement of the 3α-OH on the BA scaffold with a positively charged group should lead to a selective GPBAR1 activation over FXR. Therefore, we have modified the scaffold of LCA by replacing the 3α-OH with a protonable -NH2 group and developed a small set of amine LCA derivatives (Fig. 3). The compounds have been synthesized and tested on GPBAR1 and FXR through specific pharmacological assays. These experiments demonstrate that through rational drug design we have achieved selectivity identifying the first amine LCA derivatives as selective GPBAR1 agonists. The binding mode of the most potent compound of the series to GPBAR1 has been also elucidated using docking simulations. Our findings open new routes to design selective GPBAR1 ligands and towards a rational modulation of BA receptors activity.


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)

Predicted binding mode of 2 in hGPBAR1 (A) and rFXR (B).2 is shown as cyan sticks. The receptors are shown as gray and orange (helices H3, H4, and H12 in FXR) cartoons and sticks. Extracellular loops of GPBAR1 and nonpolar hydrogens are omitted for clarity.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Predicted binding mode of 2 in hGPBAR1 (A) and rFXR (B).2 is shown as cyan sticks. The receptors are shown as gray and orange (helices H3, H4, and H12 in FXR) cartoons and sticks. Extracellular loops of GPBAR1 and nonpolar hydrogens are omitted for clarity.
Mentions: The fact that activation of GPBAR1 and FXR can have similar effects acting on different downstream signals has prompted to develop dual GPBAR1/FXR agonists2131415. However, these compounds present a reduced therapeutic window, exposing patients to multiple unwanted effects due to activation of the diverse downstream signals controlled by the two receptors616. This leads to seek for selective ligands able to specifically activate only either one receptor. Indeed, selective GPBAR1 agonism allows controlling glucose metabolism through the release of glucagon-like peptide (GLP)-1 without affecting the FXR-related pathways. In order to design selective agonists, it is essential to know the BAs structural requisites to interact with GPBAR1 and FXR. Previous studies showed that the introduction of an ethyl group at C-6 on the CDCA ring B (6-ECDCA/OCA/INT-747)17, as well as the introduction on a sulfate group at position 24 or at position 23 on a shortened side chain (INT-767 and 2)1415, led to the development of potent dual agonists. Conversely, a marked selectivity toward GPBAR1 over FXR has been achieved through the methylation at C-23 position on the BA side chain (INT-777)18, and by stereochemical modification on ring B generating EUCDCOH, which is the first example of UDCA derivative substituted at C-6 with a β-oriented ethyl group19. Finally, independently from the functional group at C-24 on the side chain, the removal of the 3α-OH from the CDCA scaffold provides potent and selective FXR agonists19. In spite of these examples, a comprehensive understanding of the effects of BAs modifications on the activity and selectivity towards GPBAR1 and FXR is possible only if the binding mode of these compounds to the two receptors is elucidated. This is difficult because of the lack of the tridimensional structure of human GPBAR1 (hGPBAR1). We have lifted this limitation building the hGPBAR1 structure by homology modeling and performing a series of atomistic simulations and experiments on ligand/receptor binding using the hGPBAR1 model15 and the available rFXR crystal structure20. Our results are in fully agreement with previous mutagenesis data, reporting a decrease in the binding affinity for bile acid derivatives in the Asn93Ala, Glu169Ala and Tyr240Ala mutant forms of hGPBAR121. In particular, we found that in GPBAR1 the 3α-OH on BA scaffold forms a stable H-bond interaction with the negatively charged side chain of Glu169 on the transmembrane helix-5 (TM-5) (Fig. 2A). At variance with GPBAR1, in FXR the 3α-OH group H-bonds with a positively charged residue, His444 on helix H12 (Fig. 2B). This ligand/receptor interaction stabilizes the cation−π interaction between His444 and Trp466 in the activation function-2 (AF-2) domain, which is essential for FXR activation20. The opposite charge on the interacting partner of 3α-OH group in the two receptors provides a hint to achieve a selective binding. In fact, the replacement of the 3α-OH on the BA scaffold with a positively charged group should lead to a selective GPBAR1 activation over FXR. Therefore, we have modified the scaffold of LCA by replacing the 3α-OH with a protonable -NH2 group and developed a small set of amine LCA derivatives (Fig. 3). The compounds have been synthesized and tested on GPBAR1 and FXR through specific pharmacological assays. These experiments demonstrate that through rational drug design we have achieved selectivity identifying the first amine LCA derivatives as selective GPBAR1 agonists. The binding mode of the most potent compound of the series to GPBAR1 has been also elucidated using docking simulations. Our findings open new routes to design selective GPBAR1 ligands and towards a rational modulation of BA receptors activity.

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