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Luciferin Amides Enable in Vivo Bioluminescence Detection of Endogenous Fatty Acid Amide Hydrolase Activity.

Mofford DM, Adams ST, Reddy GS, Reddy GR, Miller SC - J. Am. Chem. Soc. (2015)

Bottom Line: Here, we synthesized luciferin amides and found that these molecules are hydrolyzed to substrates for firefly luciferase by the enzyme fatty acid amide hydrolase (FAAH).In the presence of luciferase, these molecules enable highly sensitive and selective bioluminescent detection of FAAH activity in vitro, in live cells, and in vivo.The potency and tissue distribution of FAAH inhibitors can be imaged in live mice, and luciferin amides serve as exemplary reagents for greatly improved bioluminescence imaging in FAAH-expressing tissues such as the brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, Massachusetts 01605, United States.

ABSTRACT
Firefly luciferase is homologous to fatty acyl-CoA synthetases. We hypothesized that the firefly luciferase substrate d-luciferin and its analogs are fatty acid mimics that are ideally suited to probe the chemistry of enzymes that release fatty acid products. Here, we synthesized luciferin amides and found that these molecules are hydrolyzed to substrates for firefly luciferase by the enzyme fatty acid amide hydrolase (FAAH). In the presence of luciferase, these molecules enable highly sensitive and selective bioluminescent detection of FAAH activity in vitro, in live cells, and in vivo. The potency and tissue distribution of FAAH inhibitors can be imaged in live mice, and luciferin amides serve as exemplary reagents for greatly improved bioluminescence imaging in FAAH-expressing tissues such as the brain.

No MeSH data available.


Related in: MedlinePlus

(A) Bioluminescence imaging with CycLuc1or CycLuc1-amide in ubiquitouslyexpressing transgenic luciferase mice treated with vehicle only orthe indicated FAAH inhibitor. (B) Total flux from the brain and kidneysquantitated as a function of inhibitor concentration and normalizedto the vehicle-only signal, represented as the mean ± SEM for n = 3 mice. Data were fit by nonlinear regression to determinerelative IC50 values in the brain (PF3845, 0.14 mg/kg;URB597, 0.40 mg/kg; URB937, ND) and kidneys (PF3845, 0.03 mg/kg; URB597,0.07 mg/kg; URB937, 0.33 mg/kg).
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fig5: (A) Bioluminescence imaging with CycLuc1or CycLuc1-amide in ubiquitouslyexpressing transgenic luciferase mice treated with vehicle only orthe indicated FAAH inhibitor. (B) Total flux from the brain and kidneysquantitated as a function of inhibitor concentration and normalizedto the vehicle-only signal, represented as the mean ± SEM for n = 3 mice. Data were fit by nonlinear regression to determinerelative IC50 values in the brain (PF3845, 0.14 mg/kg;URB597, 0.40 mg/kg; URB937, ND) and kidneys (PF3845, 0.03 mg/kg; URB597,0.07 mg/kg; URB937, 0.33 mg/kg).

Mentions: To visualize FAAH activity throughout themouse, we next turnedto transgenic mice that express luciferase in all tissues.20 When d-luciferin or CycLuc1 is introducedinto these mice, the weakest light emission is from the head, andbioluminescence is dominated by superficial tissues (Figures 5 and S9). In marked contrast, injection of CycLuc1-amide revealed the strongestbioluminescence signals from the brain and kidneys (Figure 5), tissues known to have highFAAH activity.9 Ventral bioluminescencewas less well-defined, which may reflect rapid transit of releasedluciferin out of FAAH-expressing tissues such as the liver (Figure S9). Pretreatment of mice with PF3845completely blocked bioluminescence from luciferin amides in the brainand in all peripheral tissues (Figures 5, S10, and S11) but hadno effect on bioluminescence from the parent luciferins (Figure S12). The aminoluciferin amides (Figure 1) readily sense FAAHactivity in vivo (Figure S9), and can be imaged at extremely low doses (as low as 8 nmol/kg; Figure S13). Although d-luciferin amidesenses FAAH activity in vitro and in live cells,it works poorly in live mice and cannot sense FAAH activity in thebrain (Figure S9). This is consistent withour contention that the improved biodistribution properties of aminoluciferinsand low Km values render them superiorfor use as luminogenic sensors in vivo.4 Interestingly, CycLuc1-methyl amide did not exhibitan advantage over CycLuc1-amide in the mouse (Figure S9). Presumably, inhibition of luciferase by uncleavedluciferin primary amides is not an issue at the substrate concentrationsachieved in vivo.


Luciferin Amides Enable in Vivo Bioluminescence Detection of Endogenous Fatty Acid Amide Hydrolase Activity.

Mofford DM, Adams ST, Reddy GS, Reddy GR, Miller SC - J. Am. Chem. Soc. (2015)

(A) Bioluminescence imaging with CycLuc1or CycLuc1-amide in ubiquitouslyexpressing transgenic luciferase mice treated with vehicle only orthe indicated FAAH inhibitor. (B) Total flux from the brain and kidneysquantitated as a function of inhibitor concentration and normalizedto the vehicle-only signal, represented as the mean ± SEM for n = 3 mice. Data were fit by nonlinear regression to determinerelative IC50 values in the brain (PF3845, 0.14 mg/kg;URB597, 0.40 mg/kg; URB937, ND) and kidneys (PF3845, 0.03 mg/kg; URB597,0.07 mg/kg; URB937, 0.33 mg/kg).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4507478&req=5

fig5: (A) Bioluminescence imaging with CycLuc1or CycLuc1-amide in ubiquitouslyexpressing transgenic luciferase mice treated with vehicle only orthe indicated FAAH inhibitor. (B) Total flux from the brain and kidneysquantitated as a function of inhibitor concentration and normalizedto the vehicle-only signal, represented as the mean ± SEM for n = 3 mice. Data were fit by nonlinear regression to determinerelative IC50 values in the brain (PF3845, 0.14 mg/kg;URB597, 0.40 mg/kg; URB937, ND) and kidneys (PF3845, 0.03 mg/kg; URB597,0.07 mg/kg; URB937, 0.33 mg/kg).
Mentions: To visualize FAAH activity throughout themouse, we next turnedto transgenic mice that express luciferase in all tissues.20 When d-luciferin or CycLuc1 is introducedinto these mice, the weakest light emission is from the head, andbioluminescence is dominated by superficial tissues (Figures 5 and S9). In marked contrast, injection of CycLuc1-amide revealed the strongestbioluminescence signals from the brain and kidneys (Figure 5), tissues known to have highFAAH activity.9 Ventral bioluminescencewas less well-defined, which may reflect rapid transit of releasedluciferin out of FAAH-expressing tissues such as the liver (Figure S9). Pretreatment of mice with PF3845completely blocked bioluminescence from luciferin amides in the brainand in all peripheral tissues (Figures 5, S10, and S11) but hadno effect on bioluminescence from the parent luciferins (Figure S12). The aminoluciferin amides (Figure 1) readily sense FAAHactivity in vivo (Figure S9), and can be imaged at extremely low doses (as low as 8 nmol/kg; Figure S13). Although d-luciferin amidesenses FAAH activity in vitro and in live cells,it works poorly in live mice and cannot sense FAAH activity in thebrain (Figure S9). This is consistent withour contention that the improved biodistribution properties of aminoluciferinsand low Km values render them superiorfor use as luminogenic sensors in vivo.4 Interestingly, CycLuc1-methyl amide did not exhibitan advantage over CycLuc1-amide in the mouse (Figure S9). Presumably, inhibition of luciferase by uncleavedluciferin primary amides is not an issue at the substrate concentrationsachieved in vivo.

Bottom Line: Here, we synthesized luciferin amides and found that these molecules are hydrolyzed to substrates for firefly luciferase by the enzyme fatty acid amide hydrolase (FAAH).In the presence of luciferase, these molecules enable highly sensitive and selective bioluminescent detection of FAAH activity in vitro, in live cells, and in vivo.The potency and tissue distribution of FAAH inhibitors can be imaged in live mice, and luciferin amides serve as exemplary reagents for greatly improved bioluminescence imaging in FAAH-expressing tissues such as the brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, Massachusetts 01605, United States.

ABSTRACT
Firefly luciferase is homologous to fatty acyl-CoA synthetases. We hypothesized that the firefly luciferase substrate d-luciferin and its analogs are fatty acid mimics that are ideally suited to probe the chemistry of enzymes that release fatty acid products. Here, we synthesized luciferin amides and found that these molecules are hydrolyzed to substrates for firefly luciferase by the enzyme fatty acid amide hydrolase (FAAH). In the presence of luciferase, these molecules enable highly sensitive and selective bioluminescent detection of FAAH activity in vitro, in live cells, and in vivo. The potency and tissue distribution of FAAH inhibitors can be imaged in live mice, and luciferin amides serve as exemplary reagents for greatly improved bioluminescence imaging in FAAH-expressing tissues such as the brain.

No MeSH data available.


Related in: MedlinePlus