Optimization of an

FABP Ligand Binding Assay

and Determination of
Xenobiotic Affinities
Wendy Ni, King Yabut, Nina Isoherranen
Outline
• Background – King’s Project
• Function
• My Project
• Optimizing Fluorescence Assay
• Changing Variables
• Drug Displacement
• Testing Controls
• Drug Screening
• Moving Forward
What are Fatty Acid Binding Proteins (FABPs)?
• FABPs bind lipophilic and poorly water-soluble compounds
• helps endogenous molecules travel through cytosol to nucleus
• FABPs are found everywhere in the body
• highly conserved tertiary structure
Gene Common Name Localization
FABP1 Liver FABP Liver, intestine, pancreas, kidney, lung, stomach
FABP2 Intestinal FABP Intestine, liver
FABP3 Heart FABP Cardiac and skeletal muscle, brain, kidney, lung, stomach, testis, adrenal gland,
mammary gland, placenta, ovary, brown adipose tissue
FABP4 Adipocyte FABP Adipocyte, macrophages, dendritic cells, skeletal muscle fibers
FABP5 Epidermal FABP Skin, tongue, adipocyte, macrophage, dendritic cells, mammary gland, brain,
stomach, intestine, kidney, liver, lung, heart, skeletal muscle, testis, retina,
lens, spleen, placenta
Smathers and Petersen, 2011
FABP5 Regulates Catabolism of Endogenous Compounds

• FABP5 is expressed in the brain and liver

• FABP5 binds endocannabinoid
anandamide (AEA) to be degraded by
fatty acid amide hydroxylase (FAAH)
• AEA is important for regulating cognition,
learning, and memory

FABP5 AEA
Yu et. al., 2014
 Evidence of FABP5 Binding to Xenobiotics
THC
• THC competitively inhibit AEA binding with
FABP5

Compound I-FABP Ki (μM) L-FABP Ki (μM) L-FABP (site 2) Ki (μM)

Ibuprofen 58±8 47.6±9.8 448±39
Progesterone 20±4.6 0.027±0.0011 No binding detected
Midazolam 12±3 7.9±0.2 No binding detected
20±(4-OH) 9.5±1.1(4-OH)
Diclofenac 86.3 -- --
Tolfenamic acid 6.1 -- --
Fenofibric acid 6.1 -- --

Trevaskis et. al., 2011 Elmes et. al., JBC 2015

Hypothesis
• Because many drugs are also lipophilic and poorly water soluble,
FABP5 may have a role in xenobiotic metabolism

King’s Spring Rotation Talk, 2018

Aims of the Project
Can FABP5 help move xenobiotics to metabolizing sites on the
membrane of ER? If so how?
1. Does FABP5 bind with xenobiotics?
2. Does binding with FABP5 affect xenobiotic metabolism?
a) turnover rate of the substrate
b) formation rate of product or metabolite
3. How does FABP5 binding affect deliver xenobiotics to the ER?
a) mechanism
b) models of substrate channeling
My Project
Aim 1: Determine if FABP5 binds with drugs of interest
• Fluorescent Assay
• Method – Titration to calculate Kd of ANS
• Changing variables
• NBD-stearate vs. ANS
• Waiting time
• Expanding [ANS]
• Curve Fit

• Drug Displacement
• Drug Screening
• Method – Qualitative Decrease in Fluorescence in the Presence of Drugs
 Fluorescence Assay

8-anilino-1-naphthalenesulfonic acid (ANS)

• Fluorescent probe that binds to hydrophobic surfaces (Van der Waals)
and cation groups (form ion pairs) on proteins
• blue shift in emission spectra when ANS is in polar environment
• red shift in emission spectra when ANS is bound to protein

Hawe et. al., 2007

 Fluorescence Assay

Method
• Purpose: determine Kd of ANS for
calculating Ki of the drug
• Well plate to set up titration
• FABP5 = 1μM (1:200)
• ANS = 1μL at varying concentrations
• EtOH = 1μL (no ANS control)
• ANS dissolved in EtOH
• add buffer to final volume of 200μL
• Set up on ice
• Between scans, plate at room
temperature
• Automix on fluorimeter
• mixing with pipet caused values to
greatly deviate
 Fluorescence Assay

NBD-Stearate vs. ANS

• Emission Test Scan 200
ANS Emission Spectra with FABP5 (λem = 470nm)

• No background 180

fluorescence with ANS 160

while NBD increased in 140

fluorescence with 120

Fluroescence
1uM FABP5 + 1.8uM ANS
increasing concentration 100 1uM FABP5 + 1 uM ANS
1uM FABP5 + 1uL EtOH

• More tests with NBD- 80

60
0uM FABP5 + 1.8uM ANS
0uM FABP5 + 1 uM ANS

stearate in the future 40

20

0
430 450 470 490 510 530 550
Wavelength (nm)
 Fluorescence Assay

Waiting Time
• Scan every 5 mins for a total of 20 mins
• Fluorescence values have the least variability around 15-20 mins
• More time for FABP5 and ANS to equilibrate? Mixing? Temperature?
 Fluorescence Assay

Expanding [ANS] Range

• Removed lower points that have had high variability
• Added higher points to capture asymptotic behavior
 Fluorescence Assay

Fit of the Curve

• Morrison vs. One Site Specific
• Morrison is a better fit and Kd value is closer to literature value
• Morrison is sensitive to variation and gives different Kd’s with the same plate

𝐸𝑡 +𝑋+𝐾𝑑 − 𝐸𝑡 +𝑋+𝐾𝑑 2 − 4𝐸𝑡 𝑋

𝑋
𝑌 = 𝐹𝑚𝑎𝑥 𝑌 = 𝐵𝑚𝑎𝑥
2𝐸𝑡 𝐾𝑑 + 𝑋
Fluorescence Assay
 Fluorescence Assay

Optimizing the Curve

• Kd value comparable but lots of variability
• Same general method as Lee’s paper
• Literature value Morrison Kd = 1.32 ± 0.07 μM (n = 13)
• More trials until Kd is consistent -> Drug Displacement
 Drug Fluorescence Displacement Assay

Method
Purpose: to calculate Ki for different
drugs
• Add varying concentrations of a
lipophilic drug to outcompete FABP5
binding with ANS
• Test Controls
• Positive Control – Arachidonic Acid
• Vehicle Control – EtOH
• Drug Screening
• Calculate Ki

Lee et. al., 2015

 Drug Fluorescence Displacement Assay

Drug Screening
• Method
• Purpose: qualitative assessment of FABP5-
drug binding
• Positive control: Arachidonic Acid
• fluorescence decreased by 48.28%
• Drugs have been shown to bind FABP5:
• Ibuprofen
• Rosiglitazone
• Pioglitazone
• Fenofibrate
• THC
• Retinoic Acid
• Drugs have been shown to bind other FABPs:
• Progesterone
• Midazolam
• Diclofenac

Lee et. al., 2015; Trevaskis et. al., 2011

 Drug Fluorescence Displacement Assay

Vehicle Control
• Purpose: Test how solvent can change fluorescence EtOH vs. DMSO
• EtOH decreases fluorescence
• What concentration of ANS good for drug screening (dynamic range)
• still 1μM of FABP5
 Fluorescence Displacement Assay

Redo ANS Titration

• Modify fluorescence displacement assay with DMSO as solvent
Drug Screening – 2/28/19
• Tested for FABP5-drug binding by
measuring displacement of ANS
(decrease fluorescence)
• 5μM ANS and 10μM of each drug
• Showed little or no decrease in
fluorescence
• Next trial -> decrease ANS concentration
Drug Screening – 3/4/19
• Repeat drug
screening with
1μM ANS and
10μM of each
drug
• Too high of
[DMSO]?
Moving Forward
• Reproduce ANS titration Kd values
• test other variables (mixing and temperature)
• Drug Fluorescence Displacement Assay
• Test NBD-Stearate as a fluorescence probe
• Repeat Fluorescence Displacement Assay with FABP1
Thank you!
Questions?
FABP-Drug Binding Affinities
THC data
• Addition of THC increased fluorescence
• Two trials present contradicting results – inconclusive