Fatty Acid Binding Protein Inhibitors

Fatty acids play many important roles in the body.  One such fatty acid is anandamide, an endocannabinoid that is linked to the regulation of stress, pain, and inflammation. Due to their hydrophobicity, fatty acids require the help of fatty acid binding proteins (FABP) to be transported throughout the cytosol. Anandamide agonizes cannabinoid receptors (CB receptors) on cell surface, resulting in an analgesic effect. Anandamide also readily diffuses across the membrane, where FABP transports anandamide for hydrolysis by FAAH (fatty acid amide hydrolase). After hydrolysis, anandamide is unable to agonize the CB receptor and loses its analgesic effect. Accordingly, inhibitition of FABP would arrest the hydrolysis of the endocannabinoid anandamide, leading to higher extracellular concentrations, yielding anti-inflammatory and anti-nociceptive effects (Figure 1). While there are many isoforms of FABP, FABP3, FABP5 and FABP7 have been of specific interest for drug design.

fabp reasoning

Figure 1. Anandamide (AEA) can either bind to the CB receptor or enter the cell through diffusion. Binding to the CB receptor causes anti-inflammatory and anti-nociceptive effects. Diffusion into the cell leads transportation of AEA by fatty acid binding proteins (FABPs) and hydrolysis of AEA by fatty acid amide hydrolase (FAAH). Inhibition of FABPs will hamper AEA breakdown, resulting in pain relief.


Computer-aided Drug Design

This project started with the in-silico screening of one million commercially available compounds, from ChemDiv, using utilizing molecular footprints was conducted on FABP7 with oleic acid as the reference compound.  Footprint similarity scoring (FPS) is a type of DOCK scoring function that divides a molecule into residues and scores it based on the contributions of each residue.  Each compound screened had its own footprint signature, and it was compared to the footprint signature of oleic acid to determine which compounds would bind the best to FABP7 (Figure 2).  From the virtual screening, 48 compounds were purchased and assayed in-vitro against FABP5.

Figure 2 

Figure 2. Footprint signature of oleic acid (red) is compared to the footprint signature of a candidate molecule (blue).

Fluorescence Displacement Assay

The 48 purchased compounds were tested in a fluorescence displacement assay to determine the degree to which the compounds displaced NBD-stearate from FABP5, with arachidonic acid as the control.  About 1/3 of the compounds displaced NBD-stearate, causing a decrease in fluorescence (Figure 3).  The four most potent compounds were selected for further evaluation (Figure 4).

Figure 3 

Figure 3. Fluorescence displacement assay of 48 compounds.  Aracdonic acid (black), a compound that binds strongly with FABP5, was used as a control.  The four most potent compounds (red) (SB-FI-19, SB-FI-26, SB-FI-27, SB-FI-31) were take forward.

 Figure 4

Figure 4. Left – Predicted binding pose of test compounds compared to oleic acid (red). Right – Structural formulae of four most potent compounds from the fluorescence displacement assay.

Berger, W.T.; Ralph, B.P.; Kaczocha, M.; Sun, J.; Balius, T.E.; Rizzo, R.C.; Haj-Dahmane, S.; Ojima, I.; Deutsch, D.G. Targeting Fatty Acid Binding Protein (FABP) Anandamide Transporters – A Novel Strategy for Development of Anti-Inflammatory and Anti-Nociceptive Drugs. PLoS One. 2012, 7(12), e50968.


In-vivo Studies of SB-FI-26 and other α-Truxillic Acid Derivatives

Analofgues of SB-FI-26 were synthesized as part of an SAR study to determine why functional groups are needed to optimize the activity of the molecule.  Three α-truxillic acid derivatives, SB-FI-50, SB-FI-60, and SB-FI-62, were synthesized (Figure 5).  The compounds were tested in various pain models in mice.  The experiments were formalin model of inflammatory pain, carrageenan model of inflammatory pain, and acetic acid writhing model of visceral pain (Figure 6).

Figure 5 

Figure 5. α-Truxillic acid derivatives. 

Figure 6a 

Figure 6b

Figure 6. a) The effect of the four α-truxillic acid derivatives on carrageenan induced hyperalgesia (left) and paw edema (right) in mice. b) The effect of α-truxillic acid derivatives on the first phase (left) and second phase (right) of formalin induced inflammatory pain model in mice. c) The effect of α-truxillic acid derivatives on the acetic acid induced visceral pain model in mice.  d) Dose dependent effect of SB-FI-26 on acetic acid induced visceral pain model in mice.

Kaczocha, M.;Rebecchi, M.J.; Ralph, B.P.; Teng, Y.G.; Berger, W.T.; Galbavy, W.; Elmes, M.W.; Glaser, S.T.; Wang, L.; Rizzo, R.C.; Deutsch, D.G.; Ojima, I. Inhibition of Fatty Acid Binding Proteins Elevates Brain Anandamide Levels and Produces Analgesia. PLoS One. 2014, 9(4), e94200.

Crystal Structure of Mouse FABP5

Mouse FABP5 was overexpressed in e. coli and purified.  The protein was crystalized and the structure was solved at 2.33 Å resolution.  The crystals were then soaked in saturated solutions of anandamide, giving the FABP5-anandamide complex.  The structure of this complex was solved at 2.1 Å resolution.  The mouse FABP5 crystals were soaked in a second endocannabinoid call 2-AG, 2-acrachidonoylglycerol.  The structure of the mouse FABP5-2-AG complex was solved at 2.0 Å resolution (Figure 7).

figure 7a Figure 7b

Figure 7. (Left) Ribbon diagram of mouse FABP5-anandamide (AEA) complex.  (Right) Ribbon diagram of mouse FABP5-2-AG complex.


Crystal Structure of Human FABP5

The structures of FABP5-anandamide complex and FABP5-BMS-309403 complex were solved.  It was discovered that human FABP5 formed domain-swapped dimers when ligand molecules bind (Figure 8).

Figure 8 

Figure 8. Ribbon diagram of human FABP5 domain-swapped dimer.

Sanson, B.; Wang, T.; Sun, J.; Wang, L.; Kaczocha, M.; Ojima, I.; Deutsch, D.; Li, H. Crystallographic Study of FABP5 as an Intracellular Endocannabinoid Transporter. Acta Cryst. 2014, D70, 290-298.


Associated Publications:

1. “SAR Studies on Truxillic Acid Mono Esters as a New Class of Antinociceptive Agents, Targeting Fatty Acid Binding Proteins”,  Su Yan, Matthew W. Elmes, Simon Tong, Kongzhen Hu, Monaf Awwa, Gary Y. H. Teng, Yunrong Jing, Matthew Freitag, Qianwen Gan, Timothy Clement, Longfei Wei, Joseph M. Sweeney,  Olivia M. Joseph, Gregory S. Carbonetti, Liqun Wang, Jerome Falcone, Norbert Smietalo, Yuchen Zhou, Brian Ralph, Hao-Chi Hsu, Huilin Li, Robert C. Rizzo, Dale G. Deutsch, Martin Kaczocha and Iwao Ojima, Eur. J. Med. Chem., 154, 233-252 (2018). PMC5999033

2. “The anti-nociceptive agent SBFI-26 binds to anandamide transporters FABP5 and FABP7 at two different sites”, H.-C. Hsu, S. Tong, Y. Zhou, M. Elmes, S. Yan, M. Kaczocha, D. G Deutsch, R. C Rizzo, I. Ojima, and H. Li, Biochemistry 56(27) 3454-3462 (2017). PMC5884075

3. “Incarvillateine produces antinociceptive and motor suppressive effects via adenosine receptor activation”, Jinwoo Kim, Diane M. Bogdan, Matthew W. Elmes, Monaf Awwa, Su Yan, Joyce Che, Garam Lee, Dale G. Deutsch, Robert C. Rizzo, Martin Kaczocha and Iwao Ojima, PLoS ONE 14(6): e0218619 (2019). PMID: 31237895

4. “Docetaxel/cabazitaxel and fatty acid binding protein 5 inhibitors produce synergistic inhibition of prostate cancer growth”, Gregory Carbonetti, Cynthia Converso, Timothy Clement, Changwei Wang, Lloyd Trotman, Iwao Ojima, and Martin Kaczocha, The Prostate 80(1), 88-98 (2020), PMC7063589




Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400 Phone: (631) 632-7890