Anticociceptive 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 via agonism of cannabinoid (CB) receptors.  Endocannabinoids may also diffuse into the cell, but because of their hydrophobicity, require the help of fatty acid binding protein (FABP) to be transported throughout cells.  There are several isoforms of FABPs that are expressed in different tissues, including FABP5 (epidermal), and FABP7 (brain). FABPs transport anandamide for inactivation by fatty acid amide hydrolase (FAAH).  Inhibition of FABP5 and FABP7 arrests the inactivation of the endocannabinoid, leading to higher extracellular anandamide levels, which results in anti-inflammatory and anti-nociceptive effects (Figure 1).

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.

Virtual Screening

This project started with the in silico screening of one million commercially available compounds in FABP7 using footprint similarity scoring (FPS) with oleic acid as the reference compound. 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).  Based on these results, 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 advanced forward.

Figure 4. Predicted binding pose and structures of the four most potent test compounds compared to oleic acid (red).

Structure Activity Relationship Studies

Several analogues of SB-FI-26 were synthesized in SAR studies.  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. 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 SB-FI-26 bound to FABP5, FABP7

The FABP5-bound SB-FI-26 crystal structure was resolved at 2.20 Å resolution and confirmed the binding pose prediction made in previous in silico studies. The FABP7-bound structure was also resolved.

Since then, we have generated a library of truxillic acid derivatives that target FABP5.