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Targeting the Endocannabinoid System for Neuroprotection: A (19)F-NMR Study of a Selective FAAH Inhibitor Binding with an Anandamide Carrier Protein, HSA.

Zhuang J, Yang DP, Tian X, Nikas SP, Sharma R, Guo JJ, Makriyannis A - J Pharm Pharmacol (Los Angel) (2013)

Bottom Line: We have developed a brain permeable FAAH inhibitor, AM5206, which has served as a valuable pharmacological tool to explore neuroprotective effects of this class of compounds.Our results may provide insight into the mechanism of HSA-AM5206 interactions.The findings should also help in the development of suitable formulations of the lipophilic AM5206 and its congeners for their effective delivery to specific target sites in the brain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Drug Discovery, Department of Pharmaceutical Sciences, and Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA ; Department of Chemistry, The College of Staten Island, City University of New York, 2800 Victory Boulevard, Staten Island, NY 10314, USA.

ABSTRACT
Fatty acid amide hydrolase (FAAH), the enzyme involved in the inactivation of the endocannabinoid anandamide (AEA), is being considered as a therapeutic target for analgesia and neuroprotection. We have developed a brain permeable FAAH inhibitor, AM5206, which has served as a valuable pharmacological tool to explore neuroprotective effects of this class of compounds. In the present work, we characterized the interactions of AM5206 with a representative AEA carrier protein, human serum albumin (HSA), using (19)F nuclear magnetic resonance (NMR) spectroscopy. Our data showed that as a drug carrier, albumin can significantly enhance the solubility of AM5206 in aqueous environment. Through a series of titration and competitive binding experiments, we also identified that AM5206 primarily binds to two distinct sites within HSA. Our results may provide insight into the mechanism of HSA-AM5206 interactions. The findings should also help in the development of suitable formulations of the lipophilic AM5206 and its congeners for their effective delivery to specific target sites in the brain.

No MeSH data available.


Proposed binding scenarios and the mechanism for the solubility enhancement by HSA: (A) In the aqueous buffer solution, AM5206 has a very low solubility (~70 μM), and most of it forms aggregates or even precipitates. (B) With HSA present, a fast exchange allows AM5206 to rapidly bind to the fatty acid binding site(s) and to dissolve the aggregated/precipitated AM5206. (C) More binding sites (namely, drug site 1 and non-specific binding sites) on HSA become available at higher concentrations of AM5206. The exchange rate constants among these binding sites are indicated in the diagram.
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Figure 7: Proposed binding scenarios and the mechanism for the solubility enhancement by HSA: (A) In the aqueous buffer solution, AM5206 has a very low solubility (~70 μM), and most of it forms aggregates or even precipitates. (B) With HSA present, a fast exchange allows AM5206 to rapidly bind to the fatty acid binding site(s) and to dissolve the aggregated/precipitated AM5206. (C) More binding sites (namely, drug site 1 and non-specific binding sites) on HSA become available at higher concentrations of AM5206. The exchange rate constants among these binding sites are indicated in the diagram.

Mentions: In the aqueous buffer solution, AM5206 has very low solubility (~70 μM) and it aggregates rapidly into various forms or even precipitates out of solution (Figure 7A). The addition of serum albumin can drastically increase the solubility of AM5206 and potentially dissolve all the aggregates. We now propose the following two different binding scenarios of AM5206 due to the multiple binding sites on human serum albumin. With low content of AM5206 (HSA:AM5206 ratio up to 1:0.5), it almost binds exclusively at the fatty acid binding site(s) on albumin (Figure 7B). This is probably because of the easy accessibility of some of the FA sites, particularly the FA site on subdomain IB which is the most open of all the presumed high-affinity pockets on HSA [18]. When higher amount of AM5206 is present in HSA solution, it starts to interact with many other different binding sites on albumin while AM5206 undergoes slow exchange among these binding sites (Figure 7C). Specifically, the exchange rates are 115 s-1 and 47s-1 for AM5206 to dissociate from the non-specific sites to the fatty acid site(s) or drug site 1, respectively. However, the exchange rate of AM5206 between the two specific binding sites (fatty acid site(s) and drug site 1) is ~1 s-1, almost two orders of magnitude smaller than those involving the non-specific binding sites. This observed spectroscopic exchange can be explained through a direct transfer of AM5206 between fatty acid site(s) and drug site 1. This very slow process is also compatible with an alternative explanation according to which AM5206 undergoes exchanges indirectly through the intermediacy of the non-specific sites. As for AM5206 at the non-specific sites, there exists a fast exchange with the free AM5206 present in the HSA solution. The more AM5206 added into the HSA solution, the more it associates directly to the non-specific binding sites. In the present study, the non-specific binding sites are not saturable over the entire titration experiments with HSA:AM5206 ratios up to 1:5. The complexity of the binding scenarios of serum albumin, in conjunction with the poor aqueous solubility of ligands, underscores the difficulty of obtaining accurate dissociation constants for each of the binding sites. This helps explain the frequent discrepancies of reported affinity values of drug molecules with albumin in the literature.


Targeting the Endocannabinoid System for Neuroprotection: A (19)F-NMR Study of a Selective FAAH Inhibitor Binding with an Anandamide Carrier Protein, HSA.

Zhuang J, Yang DP, Tian X, Nikas SP, Sharma R, Guo JJ, Makriyannis A - J Pharm Pharmacol (Los Angel) (2013)

Proposed binding scenarios and the mechanism for the solubility enhancement by HSA: (A) In the aqueous buffer solution, AM5206 has a very low solubility (~70 μM), and most of it forms aggregates or even precipitates. (B) With HSA present, a fast exchange allows AM5206 to rapidly bind to the fatty acid binding site(s) and to dissolve the aggregated/precipitated AM5206. (C) More binding sites (namely, drug site 1 and non-specific binding sites) on HSA become available at higher concentrations of AM5206. The exchange rate constants among these binding sites are indicated in the diagram.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3921897&req=5

Figure 7: Proposed binding scenarios and the mechanism for the solubility enhancement by HSA: (A) In the aqueous buffer solution, AM5206 has a very low solubility (~70 μM), and most of it forms aggregates or even precipitates. (B) With HSA present, a fast exchange allows AM5206 to rapidly bind to the fatty acid binding site(s) and to dissolve the aggregated/precipitated AM5206. (C) More binding sites (namely, drug site 1 and non-specific binding sites) on HSA become available at higher concentrations of AM5206. The exchange rate constants among these binding sites are indicated in the diagram.
Mentions: In the aqueous buffer solution, AM5206 has very low solubility (~70 μM) and it aggregates rapidly into various forms or even precipitates out of solution (Figure 7A). The addition of serum albumin can drastically increase the solubility of AM5206 and potentially dissolve all the aggregates. We now propose the following two different binding scenarios of AM5206 due to the multiple binding sites on human serum albumin. With low content of AM5206 (HSA:AM5206 ratio up to 1:0.5), it almost binds exclusively at the fatty acid binding site(s) on albumin (Figure 7B). This is probably because of the easy accessibility of some of the FA sites, particularly the FA site on subdomain IB which is the most open of all the presumed high-affinity pockets on HSA [18]. When higher amount of AM5206 is present in HSA solution, it starts to interact with many other different binding sites on albumin while AM5206 undergoes slow exchange among these binding sites (Figure 7C). Specifically, the exchange rates are 115 s-1 and 47s-1 for AM5206 to dissociate from the non-specific sites to the fatty acid site(s) or drug site 1, respectively. However, the exchange rate of AM5206 between the two specific binding sites (fatty acid site(s) and drug site 1) is ~1 s-1, almost two orders of magnitude smaller than those involving the non-specific binding sites. This observed spectroscopic exchange can be explained through a direct transfer of AM5206 between fatty acid site(s) and drug site 1. This very slow process is also compatible with an alternative explanation according to which AM5206 undergoes exchanges indirectly through the intermediacy of the non-specific sites. As for AM5206 at the non-specific sites, there exists a fast exchange with the free AM5206 present in the HSA solution. The more AM5206 added into the HSA solution, the more it associates directly to the non-specific binding sites. In the present study, the non-specific binding sites are not saturable over the entire titration experiments with HSA:AM5206 ratios up to 1:5. The complexity of the binding scenarios of serum albumin, in conjunction with the poor aqueous solubility of ligands, underscores the difficulty of obtaining accurate dissociation constants for each of the binding sites. This helps explain the frequent discrepancies of reported affinity values of drug molecules with albumin in the literature.

Bottom Line: We have developed a brain permeable FAAH inhibitor, AM5206, which has served as a valuable pharmacological tool to explore neuroprotective effects of this class of compounds.Our results may provide insight into the mechanism of HSA-AM5206 interactions.The findings should also help in the development of suitable formulations of the lipophilic AM5206 and its congeners for their effective delivery to specific target sites in the brain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Drug Discovery, Department of Pharmaceutical Sciences, and Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA ; Department of Chemistry, The College of Staten Island, City University of New York, 2800 Victory Boulevard, Staten Island, NY 10314, USA.

ABSTRACT
Fatty acid amide hydrolase (FAAH), the enzyme involved in the inactivation of the endocannabinoid anandamide (AEA), is being considered as a therapeutic target for analgesia and neuroprotection. We have developed a brain permeable FAAH inhibitor, AM5206, which has served as a valuable pharmacological tool to explore neuroprotective effects of this class of compounds. In the present work, we characterized the interactions of AM5206 with a representative AEA carrier protein, human serum albumin (HSA), using (19)F nuclear magnetic resonance (NMR) spectroscopy. Our data showed that as a drug carrier, albumin can significantly enhance the solubility of AM5206 in aqueous environment. Through a series of titration and competitive binding experiments, we also identified that AM5206 primarily binds to two distinct sites within HSA. Our results may provide insight into the mechanism of HSA-AM5206 interactions. The findings should also help in the development of suitable formulations of the lipophilic AM5206 and its congeners for their effective delivery to specific target sites in the brain.

No MeSH data available.