Targeting fatty acid binding protein (FABP) anandamide transporters - a novel strategy for development of anti-inflammatory and anti-nociceptive drugs

Abstract

Fatty acid binding proteins (FABPs), in particular FABP5 and FABP7, have recently been identified by us as intracellular transporters for the endocannabinoid anandamide (AEA). Furthermore, animal studies by others have shown that elevated levels of endocannabinoids resulted in beneficial pharmacological effects on stress, pain and inflammation and also ameliorate the effects of drug withdrawal. Based on these observations, we hypothesized that FABP5 and FABP7 would provide excellent pharmacological targets. Thus, we performed a virtual screening of over one million compounds using DOCK and employed a novel footprint similarity scoring function to identify lead compounds with binding profiles similar to oleic acid, a natural FABP substrate. Forty-eight compounds were purchased based on their footprint similarity scores (FPS) and assayed for biological activity against purified human FABP5 employing a fluorescent displacement-binding assay. Four compounds were found to exhibit approximately 50% inhibition or greater at 10 µM, as good as or better inhibitors of FABP5 than BMS309403, a commercially available inhibitor. The most potent inhibitor, γ-truxillic acid 1-naphthyl ester (ChemDiv 8009-2334), was determined to have K(i) value of 1.19±0.01 µM. Accordingly a novel α-truxillic acid 1-naphthyl mono-ester (SB-FI-26) was synthesized and assayed for its inhibitory activity against FABP5, wherein SB-FI-26 exhibited strong binding (K(i) 0.93±0.08 µM). Additionally, we found SB-FI-26 to act as a potent anti-nociceptive agent with mild anti-inflammatory activity in mice, which strongly supports our hypothesis that the inhibition of FABPs and subsequent elevation of anandamide is a promising new approach to drug discovery. Truxillic acids and their derivatives were also shown by others to have anti-inflammatory and anti-nociceptive effects in mice and to be the active component of Chinese a herbal medicine (Incarvillea sinensis) used to treat rheumatism and pain in humans. Our results provide a likely mechanism by which these compounds exert their effects.

Figure 1. Scheme demonstrating anandamide inactivation and FABP drug target.

Anandamide crosses the membrane by diffusion but requires FABPs for transport through the cytoplasm to the endoplasmic reticulum for breakdown by FAAH. FABP inhibitors prevent AEA from being delivered to FAAH for breakdown resulting in increased AEA levels at the receptor.

Figure 2. Use of molecular footprint comparisons to prioritize compounds from virtual screening.

In this example the van der Waals footprint made by the reference ligand oleic acid (red) is compared with that of a candidate molecule (blue) from the virtual screen.

Figure 3. Sequence and structural alignment of FABP7 (grey) with FABP5 (green).

Key binding residues consisting of ARG106, ARG126, and TYR128 are identical (blue) however MET115 in FABP7 is substituted for VAL115 in FABP5 (purple). The native substrate oleic acid is shown in cyan.

Figure 4. A flow chart describing the discovery of ligands through virtual screening, biological assay, and medicinal chemistry.

Figure 5. Four compounds from the FABP7 computational virtual screen which show experimental activity in an FABP5 fluorescence displacement assay.

The predicted binding pose for each ligand (blue stick) is shown in relationship to the reference oleic acid (red stick, gray surface).

Figure 6. VDW (top) and ES (bottom) footprints for active compounds (blue) compared with the native substrate oleic acid (red).

The VDW clash between oleic acid and MET115 is offset by the strong favorable ES interactions at ARG106, ARG126, and TYR 128.

Figure 7. Highlighted in red is the α-truxillic acid core structure of both (-)-Incarvillateine and FABP inhibitor SB-FI-26.

Figure 8. High throughput fluorescence displacement assay with NBD-stearate.

Shown in blue is the NBD-stearate FABP5 complex with no inhibitor, in black is arachidonic acid a potent inhibitor of FABP5, and in red are the four lead compounds.

Figure 9. Verification of high throughput fluorescent displacement assay results.

(A) Replicate testing of the lead compounds shows that 8009-2334 (SB-FI-26) exhibits the best inhibition of FABP5. One * indicates p<0.05, two ** indicates p<0.01, three *** indicates p<0.001 (n = 3). (B) Controls show that all lead compounds exhibited no detectable fluorescence in the assay nor did they add significantly to the fluorescence of the NBD-stearate probe.

Figure 10. Binding analysis of SB-FI-26 (α-truxillic acid 1-naphthyl ester), SB-FI-49 (γ-truxillic acid 1-naphthyl ester), and BMS309403.

(A) Assay in triplicate shows that SB-FI-26 derivative attains a K_i within nanoMolar ranges. (B) Analysis of γ-truxillic acid 1-naphthyl ester implies that 8009-2334 is an impure form of the compound and that the pure, synthesized gamma derivative is as potent as BMS309403. (C) BMS309403 was found to be slightly more potent in this study (K_i, 0.75 µM) than published (K_i, 0.89 µM), but still within range of this value.

Figure 11. FABP inhibitor SB-FI-26 does not reduce amplitude of EPSCs and is a weak agonist at PPARα and PPARγ receptors.

(A) Summary graph of effect of SB-FI-26 (10 µM) on amplitude of EPSCs. (B) Superimposed average EPSC traces taken at the time point indicated by numbers in panel A. Note that application of α-1-napthol-truxillic acid did not inhibit the amplitude of EPSCs. (C) PPARα activation by SB-FI-26 and the PPARα agonist GW7647. (D) Activation of PPARγ receptors by SB-FI-26 compared to the PPARγ agonist rosiglitazone (n = 3).

Figure 12. SB-FI-26 inhibits the cellular uptake of AEA.

(A) AEA uptake in wild-type HeLa (un-shaded) or FABP5 shRNA-expressing HeLa (shaded) cells in the presence or absence of SB-FI-26. (B) AEA hydrolysis in FAAH-transfected HeLa cells in the presence or absence of SB-FI-26 or the FAAH inhibitor URB597. **, p<0.01; ***, p<0.001 (n = 3).

Figure 13. Antinociceptive effects of SB-FI-26.

(A) SB-FI-26 (20 mg/kg, i.p.) reduces pain associated with the first phase (left panel) but not the second phase (right panel) of the formalin test. Intraplantar injection of vehicle or 20 µg SB-FI-26 (checkered bars) did not evoke nocifensive behavior. *, p<0.05 (n = 6). (B) SB-FI-26 (20 mg/kg, i.p.) alleviates carrageenan-induced thermal hyperalgesia in mice. Concurrent administration of rimonabant and SR144528 (SR1/SR2) blocked the antinociceptive effects of SB-FI-26. **, p<0.01 versus carrageenan-injected animals; ##, p<0.01 versus SR1/SR2-treated animals (n = 6–9). (C) SB-FI-26 (20 mg/kg, i.p.) reduces carrageenan-induced paw edema. *, p<0.05 (n = 6–9).