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. Fatty acids, because of their hydrophobicity, require the help of fatty acid binding protein (FABP) to be transported throughout cells. Anandamide targets cannabinoid receptors (CB receptors) on cell surface. Activation of the CB receptor, leads to pain relief and stress relief. Through diffusion, anandamide also enters the cell, where FABP5, mostly expressed in epidermal tissue, and FABP7, mostly expressed in the brain, transport the anandamide for inactivation in FAAH (fatty acid amide hydrolase). Inhibition of FABP5 and FABP7 would arrest the inactivation of the endocannabinoid, leading to higher extracellular anandamide levels, which will result in anti-inflammatory and anti-nociceptive effects (Figure 1). Inhibitors of FABP5 and FABP7 can be anti-inflammatory and anti-nociceptive drugs.
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 inactivation of AEA by fatty acid amide hydrolase (FAAH). Inhibition of FABPs will hamper AEA breakdown, resulting in pain relief.
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. 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. 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. 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. α-Truxillic acid derivatives.
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 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. 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.