[Albumin] (uM)
Graduate Category: Health Sciences Degree Level:Master Abstract ID#: 441
Interactions of the Endocannabinoid Anandamide with Its Carrier Proteins Xin Sun, Fera Soedarsono, Yingpeng Liu, Han Zhou, Richard W. Mercier, Mark K. Williams, De-Ping Yang, Jason Guo, Alexandros Makriyannis Center for Drug Discovery, Northeastern University, Boston, MA 02115 The endocannabinoid anandamide (arachidonoylethanolamine, AEA) was demonstrated to be a neuroprotective agent against excitotoxicity and neuronal injury in vivo. Recently, a number of endogenous proteins including the fatty acid binding proteins (FABPs), albumin, and heat shock protein 70 (HSP70), were identified as potential carriers that facilitate the transport of AEA across cellular membranes. Due to the intermediate binding affinity range and the highly lipophilic nature of the anandamide molecule, it is challenging to obtain accurate Kd values for anandamide with its carrier proteins.
Binding of anandamide to its carrier proteins
INTRODUCTION Anandamide is an endogenous cannabinoid neurotransmitter that exhibits neuroprotective properties. The biosynthesis and physiology of AEA are well understood, but its mechanism of uptake has been elusive. Despite intensive studies on the AEA transport process, the question remains on how the uncharged lipophilic AEA can cross cellular membranes and transverse in the hydrophilic cytosol. Recently, it was reported that the cellular uptake of AEA can be significantly potentiated by a class of carrier proteins. By inhibiting these anandamide carrier proteins, the uptake of AEA inside the cell was found to be drastically reduced. These findings provide a potential new therapeutic modality for neuroprotection through dual inhibition of FAAH and anandamide carrier proteins. Here we selected three different fatty acid binding proteins as well as serum albumin (HSA) as representative anandamide carrier proteins and studied their interactions with AEA. Schematics of carrier protein mediated transport of anandamide across plasma membrane3. The carrier protein (orange) can also facilitate the intracellular diffusion of anandamide to its catabolic enzyme (red) for degradation.
Kd = 0.98uM
15
20
0
5
15
20
Bound AEA
Kd = 0.11uM
Kd = 0.39uM
0 0
5
10
15
20
0
5
[FABP4] (uM)
10
15
20
[Albumin] (uM)
Anandamide (AEA) binds to four different carrier proteins with sub-micromolar affinity.
Different microscopic binding affinity of AEA to albumin. The highest affinity site for AEA is Drug Site I, followed by Drug Site II and possibly some other fatty acid binding sites.
Kd = 0.39uM
0
5
10
15
CONCLUSIONS
20
Comparison of the saturation binding curves of AEA to human serum albumin when titrating with protein (left) or titrating with ligand (right) 37ºC HSA : AEA
HSA : AEA = 1:7
1:7
1:5
5ºC
1:3
12ºC
1:1
22ºC
1:0
37ºC
13.0 12.5 12.0 11.5 11.0 10.5 10.0 ppm
13.0 12.5 12.0 11.5 11.0 10.5 10.0 ppm
Downfield region of 1H-NMR spectra of human serum albumin (HSA) Left: titrated with anandamide (AEA) at 37ºC; Right: variable temperature experiments with a protein: ligand ratio of 1:7. HSA : Ligand
1:10 w/ Warfarin 1:5
1:10
Free Albumin
1:5
1:0
13.0 12.5 12.0 11.5 11.0 10.5 10.0 ppm
Downfield region of 1H-NMR spectra of human serum albumin (HSA) titrated with warfarin (Site I marker) or Ltryptophan (Site II marker).
10
15
20
0
5
10
[Albumin] (uM) HSA + L-Tryptophan
15
20
HSA + Warfarin
Kd = 0.57uM
5
10
[Albumin] (uM)
HSA only
0
[Albumin] (uM)
w/ L-Tryptophan
5
Kd = 7.74uM
[Albumin] (uM)
Kd=0.39uM
0
Bound AEA
[FABP5] (uM)
[FABP 7] (uM)
Bound AEA
10
Bound AEA
10
Kd = 0.71uM
Bound AEA
5
H-bonding between the His288 Hd1 and the side chain carboxyl group of Glu153 in Drug Site I of HSA (pdb id: 1AO6).
Bound AEA
0
Bound AEA
In this study, we developed a novel radioactivity-based assay with saturated protein concentrations and have determined the affinities of anandamide with four of its carrier proteins. Our approach also has the advantage of being able to distinguish different microscopic binding affinities when more than one binding site is present on the protein. Further NMR experiments provided detailed residue-specific information of these binding sites upon on anandamide binding. The new assay will allow us to pursue the role of these proteins as modulators of AEA levels and use the information to design selective ligands as potential medications which produce their effects through modulation of the endocannabinoid system.
Bound AEA
RESULTS
Bound AEA
ABSTRACT
15
20
Kd = 9.60uM
0
5
10
15
20
[Albumin] (uM) HSA + Warfarin + L-Tryptophan
• The endocannabinoid anandamide (AEA) interacts with a range of carrier proteins with sub-micromolar affinity. • For the carrier protein HSA, there are multiple binding sites that are accessible for anandamide. We can determine not only the average binding affinity for anandamide to HSA, but also the microscopic affinity for different binding sites. • Modulation of endocannabinoid transport may provide novel therapeutic opportunities for treating inflammation, neuropathic pain, drug addiction and other disorders.
REFERENCES 1. Kaczocha, M.; Glaser, S. T.; Deutsch, D. G. Identification of intracellular carriers for the endocannabinoid anandamide. Proc Natl Acad Sci USA 106 (2009), 6375-6380. 2. David, B. et al. A combined spectroscopic and crystallographic approach probing drug-human serum albumin interactions, Bioorganic & Medicinal Chemistry 18 (2010), 7486-7496. 3. Felder, C. C., Dickason-Chesterfield, A. K., Moore, S. A. Cannabinoids Biology: The search for new therapeutic targets, Molecular Interventions (2006), 149-161.
ACKNOWLEDGEMENT We would like to acknowledge the financial support for this research by NIH grants DA032020 (J.G.), DA003801 (A.M.) and DA009158 (A.M.) from the National Institute on Drug Abuse.
Interactions of the Endocannabinoid Anandamide with Its Carrier Proteins Xin Sun, Fera Soedarsono, Yingpeng Liu, Han Zhou, Richard W. Mercier, Mark K. Williams, De-Ping Yang, Jason Guo, Alexandros Makriyannis Center for Drug Discovery, Northeastern University, Boston, MA 02115 The endocannabinoid anandamide (arachidonoylethanolamine, AEA) was demonstrated to be a neuroprotective agent against excitotoxicity and neuronal injury in vivo. Recently, a number of endogenous proteins including the fatty acid binding proteins (FABPs), albumin, and heat shock protein 70 (HSP70), were identified as potential carriers that facilitate the transport of AEA across cellular membranes. Due to the intermediate binding affinity range and the highly lipophilic nature of the anandamide molecule, it is challenging to obtain accurate Kd values for anandamide with its carrier proteins.
Binding of anandamide to its carrier proteins
INTRODUCTION Anandamide is an endogenous cannabinoid neurotransmitter that exhibits neuroprotective properties. The biosynthesis and physiology of AEA are well understood, but its mechanism of uptake has been elusive. Despite intensive studies on the AEA transport process, the question remains on how the uncharged lipophilic AEA can cross cellular membranes and transverse in the hydrophilic cytosol. Recently, it was reported that the cellular uptake of AEA can be significantly potentiated by a class of carrier proteins. By inhibiting these anandamide carrier proteins, the uptake of AEA inside the cell was found to be drastically reduced. These findings provide a potential new therapeutic modality for neuroprotection through dual inhibition of FAAH and anandamide carrier proteins. Here we selected three different fatty acid binding proteins as well as serum albumin (HSA) as representative anandamide carrier proteins and studied their interactions with AEA. Schematics of carrier protein mediated transport of anandamide across plasma membrane3. The carrier protein (orange) can also facilitate the intracellular diffusion of anandamide to its catabolic enzyme (red) for degradation.
Kd = 0.98uM
15
20
0
5
15
20
Bound AEA
Kd = 0.11uM
Kd = 0.39uM
0 0
5
10
15
20
0
5
[FABP4] (uM)
10
15
20
[Albumin] (uM)
Anandamide (AEA) binds to four different carrier proteins with sub-micromolar affinity.
Different microscopic binding affinity of AEA to albumin. The highest affinity site for AEA is Drug Site I, followed by Drug Site II and possibly some other fatty acid binding sites.
Kd = 0.39uM
0
5
10
15
CONCLUSIONS
20
Comparison of the saturation binding curves of AEA to human serum albumin when titrating with protein (left) or titrating with ligand (right) 37ºC HSA : AEA
HSA : AEA = 1:7
1:7
1:5
5ºC
1:3
12ºC
1:1
22ºC
1:0
37ºC
13.0 12.5 12.0 11.5 11.0 10.5 10.0 ppm
13.0 12.5 12.0 11.5 11.0 10.5 10.0 ppm
Downfield region of 1H-NMR spectra of human serum albumin (HSA) Left: titrated with anandamide (AEA) at 37ºC; Right: variable temperature experiments with a protein: ligand ratio of 1:7. HSA : Ligand
1:10 w/ Warfarin 1:5
1:10
Free Albumin
1:5
1:0
13.0 12.5 12.0 11.5 11.0 10.5 10.0 ppm
Downfield region of 1H-NMR spectra of human serum albumin (HSA) titrated with warfarin (Site I marker) or Ltryptophan (Site II marker).
10
15
20
0
5
10
[Albumin] (uM) HSA + L-Tryptophan
15
20
HSA + Warfarin
Kd = 0.57uM
5
10
[Albumin] (uM)
HSA only
0
[Albumin] (uM)
w/ L-Tryptophan
5
Kd = 7.74uM
[Albumin] (uM)
Kd=0.39uM
0
Bound AEA
[FABP5] (uM)
[FABP 7] (uM)
Bound AEA
10
Bound AEA
10
Kd = 0.71uM
Bound AEA
5
H-bonding between the His288 Hd1 and the side chain carboxyl group of Glu153 in Drug Site I of HSA (pdb id: 1AO6).
Bound AEA
0
Bound AEA
In this study, we developed a novel radioactivity-based assay with saturated protein concentrations and have determined the affinities of anandamide with four of its carrier proteins. Our approach also has the advantage of being able to distinguish different microscopic binding affinities when more than one binding site is present on the protein. Further NMR experiments provided detailed residue-specific information of these binding sites upon on anandamide binding. The new assay will allow us to pursue the role of these proteins as modulators of AEA levels and use the information to design selective ligands as potential medications which produce their effects through modulation of the endocannabinoid system.
Bound AEA
RESULTS
Bound AEA
ABSTRACT
15
20
Kd = 9.60uM
0
5
10
15
20
[Albumin] (uM) HSA + Warfarin + L-Tryptophan
• The endocannabinoid anandamide (AEA) interacts with a range of carrier proteins with sub-micromolar affinity. • For the carrier protein HSA, there are multiple binding sites that are accessible for anandamide. We can determine not only the average binding affinity for anandamide to HSA, but also the microscopic affinity for different binding sites. • Modulation of endocannabinoid transport may provide novel therapeutic opportunities for treating inflammation, neuropathic pain, drug addiction and other disorders.
REFERENCES 1. Kaczocha, M.; Glaser, S. T.; Deutsch, D. G. Identification of intracellular carriers for the endocannabinoid anandamide. Proc Natl Acad Sci USA 106 (2009), 6375-6380. 2. David, B. et al. A combined spectroscopic and crystallographic approach probing drug-human serum albumin interactions, Bioorganic & Medicinal Chemistry 18 (2010), 7486-7496. 3. Felder, C. C., Dickason-Chesterfield, A. K., Moore, S. A. Cannabinoids Biology: The search for new therapeutic targets, Molecular Interventions (2006), 149-161.
ACKNOWLEDGEMENT We would like to acknowledge the financial support for this research by NIH grants DA032020 (J.G.), DA003801 (A.M.) and DA009158 (A.M.) from the National Institute on Drug Abuse.
