The Antinociceptive Agent SBFI-26 Binds to Anandamide Transporters FABP5 and FABP7 at Two Different Sites

Article abstract of DOI:10.1021/acs.biochem.7b00194

Human FABP5 and FABP7 are intracellular endocannabinoid transporters. SBFI-26 is an α-truxillic acid 1-naphthyl monoester that competitively inhibits the activities of FABP5 and FABP7 and produces antinociceptive and anti-inflammatory effects in mice. The

Full text of DOI:10.1021/acs.biochem.7b00194

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Article
The antinociceptive agent SBFI-26 binds to anandamide
transporters FABP5 and FABP7 at two different sites
Hao-Chi Hsu, Simon Tong, Yuchen Zhou, Matthew W. Elmes, Su Yan, Martin
Kaczocha, Dale G. Deutsch, Robert C Rizzo, Iwao Ojima, and Huilin Li
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00194 • Publication Date (Web): 20 Jun 2017
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Biochemistry
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The antinociceptive agent SBFI-26 binds to anandamide transporters FABP5
and FABP7 at two different sites
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HaoꢀChi Hsu1, Simon Tong2, Yuchen Zhou3, Matthew W. Elmes4, Su Yan2, Martin Kaczocha4,5, Dale G.
Deutsch4,6, Robert C. Rizzo3,6,7, *, Iwao Ojima2,6, *, and Huilin Li1,6,*
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1 CryoꢀEM Structural Biology Laboratory, Van Andel Research Institute, Grand Rapids, MI 49503
2Department of Chemistry, Stony Brook University, Stony Brook, NY 11794
3Department of Applied Mathematics and Statistics, Stony Brook University, Stony Brook, NY 11794
4Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794
5Department of Anesthesiology, Stony Brook University, Stony Brook, NY, 11794
6Institute of Chemical Biology and Drug Discovery, Stony Brook University, Stony Brook, NY 11794
7Laufer Center for Physical & Quantitative Biology, Stony Brook University, Stony Brook, NY 11794
*Corresponding author (H.L.)
Address: Van Andel Research Institute,
333 Bostwick Ave., NE, Grand Rapids, MI 49503. Tel: 616ꢀ234ꢀ5306. Eꢀmail: Huilin.Li@vai.org.
*Corresponding author (R.R.)
Address: Department of Applied Mathematics & Statistics, Stony Brook University, Stony Brook NY,
11794ꢀ3600. Eꢀmail: rizzorc@gmail.com
*Corresponding author (I.O.)
Address: Institute of Chemical Biology & Drug Discovery, Department of Chemistry, Stony Brook
University, Stony Brook NY 11794ꢀ3400. Eꢀmail: iwao.ojima@stonybrook.edu.
Keywords
Anandamide transporter, fatty acid binding proteins (FABP), antiꢀnociceptive agents, truxillic acids,
structural biology, DOCK, footprint similarity, docking, SBFIꢀ26.
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ABSTRACT
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Human FABP5 and FABP7 are intracellular endocannabinoid transporters. SBFIꢀ26 is an αꢀtruxillic acid
1ꢀnaphthyl monoester that competitively inhibits the activities of FABP5 and FABP7 and produces
antinociceptive and antiꢀinflammatory effects in mice. The synthesis of SBFIꢀ26 yields several
stereoisomers, and it is not known how the inhibitor binds the transporters. Here we report coꢀcrystal
structures of SBFIꢀ26 in complex with human FABP5 and FABP7 at a resolution of 2.2 Å and 1.9 Å,
respectively. We found that only (S)ꢀSBFIꢀ26 was present in the crystal structures. The inhibitor largely
mimics the fatty acid binding pattern, but it also has several unique interactions. Notably, the FABP7
complex corroborates key aspects of the ligand binding pose at the canonical site previously predicted by
virtual screening. In FABP5, SBFIꢀ26 was unexpectedly found to bind at the substrate entry portal region
in addition to binding at the canonical ligandꢀbinding pocket. Our structural and binding energy analyses
indicate that both (R) and (S) forms appear to bind the transporter equally well. We suggest that the (S)
enantiomer observed in the crystal structures may be a result of the crystallization process selectively
incorporating the (S)ꢀSBFIꢀ26ꢀFABP complexes into the growing lattice, or that the (S)ꢀenantiomer may
bind to the portal site more rapidly than to the canonical site, leading to an increased local concentration
of the (S) enantiomer for binding to the canonical site. Our work reveals two binding poses of SBFIꢀ26 in
its target transporters. This knowledge will guide the development of more potent FABP inhibitors based
upon the SBFIꢀ26 scaffold.
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INTRODUCTION
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The endocannabinoids such as anandamide (AEA), 2ꢀarachidonoylglyerol (2ꢀAG), and the related Nꢀ
acylethanolamines (NAEs) are fatty acid neurotransmitters that activate cannabinoid receptors (CBs) in
the brain and the immune system 1. Tissue AEA levels are regulated by fatty acid binding protein
(FABP)ꢀmediated intracellular transport to the catalytic enzyme, fatty acid amide hydrolase (FAAH) 2, 3
FABP5 and FABP7 are the intracellular transporters of AEA 4, 5. Inhibition of FABPs reduces AEA
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inactivation in vitro and elevates AEA levels in vivo 4, 6-8. Thus, FABP5 and FABP7 are novel targets for
modulating AEA levels and signaling pathways 9, 10
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FABPs are a family of intracellular lipidꢀbinding proteins. FABPs reversibly bind hydrophobic ligands
such as fatty acids and their acyl derivatives from the cell membrane and traffic them throughout various
intracellular compartments. There are ten known FABP isoforms in humans that are expressed in many
tissues and regulate many cellular processes, including lipid metabolism, inflammation, sleep, and
neuronal signaling 11-16. For example, FABP5 was first found in epidermal cells (and therefore is also
called EꢀFABP), but the protein is found in many cell types including the brain. We recently found that
FABP5 is also expressed in nociceptive dorsal root ganglia and the spinal cord, and inhibition of FABP5
exerts peripheral and supraꢀspinal analgesic effects 17. FABP7 was first identified in brain and therefore is
also called BꢀFABP. Although the sequence identity of FABP family members varies from 20% to 70%,
they share a similar tertiary structure that is essentially a waterꢀfilled ligandꢀbinding pocket. This pocket
comprises a βꢀbarrel formed by ten antiparallel βꢀstrands and an Nꢀterminal helixꢀturnꢀhelix (HTH) cap
(Fig. 1A) 5, 18-21
.
Interestingly, the structures of FABPs in apo form and in their ligandꢀbound complexes are usually
similar 9. Because the ligands are large and the space between the HTH cap and the βꢀbarrel is too small
for the ligands to freely diffuse through, a “portal hypothesis” has been proposed in which the portal,
composed of αꢀhelix H2 and two loops of the S3ꢀS4 and S5ꢀS6 strands, may open for the ligand’s
entrance into the deep substrateꢀbinding pocket (Fig. 1A) 18, 22. However, how and the extent to which the
portal region opens have not been well characterized 23. It has also been unclear whether the portal region
plays a role in ligand selection.
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Figure 1. FABP5 is an intracellular anandamide transporter and SBFI-26 is an antinociceptive
targeting FABP5. (A) Crystal structure of the mouse FABP5 in complex with the endocannabinoid
anandamide (AEA) (PDB 4AZP). FABP has a βꢀbarrel fold with a helixꢀturnꢀhelix (HTH) cap. It is
hypothesized that H2 of the HTH cap and the loops between S3ꢀS4 and S5ꢀS6 form the substrate entry
port, which is highlighted in red. (B) The chemical structure of (S)ꢀSBFIꢀ26.
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SBFIꢀ26 is an αꢀtruxillic acid 1ꢀnaphthyl monoester (Fig. 1B). As we reported earlier 7, 24, SBFIꢀ26 was
originally identified using a computational docking protocol (DOCK6 program) 25 in which a previously
published crystal structure 26 of FABP7 in complex with a fatty acid was employed for a largeꢀscale
virtual screen. The screening protocol docked over 1 million purchasable compounds (ZINC database) 27
to the fattyꢀacidꢀremoved FABP7 structure 24. Candidate ligands were prioritized for purchase and
experimental testing was based on several criteria, including the use of footprint similarity scoring to
identify compounds having similar interaction patterns as the cognate ligand 28, 29. SBFIꢀ26 was
synthesized as a mixture of both the (S) and (R) enantiomers 24. It produced antinociceptive and antiꢀ
inflammatory effects in mice and competitively inhibited the activities of FABP5 and FABP7 with Ki
values of 0.9 ꢀM and 0.4 ꢀM, respectively 7, 24. The previous computational docking has suggested the (R)
enantiomer is a better ligand for FABP7, but the active form of SBFIꢀ26 (and how it interacts with these
FABPs) has yet to be experimentally determined. In this work, we found that only the (S) enantiomer of
SBFIꢀ26 was present in the coꢀcrystal structures with FABP5 and FABP7. Serendipitously, we captured
SBFIꢀ26 at the canonical substrateꢀbinding site as well as at the substrateꢀentry portal site in FABP5. By a
detailed computational energetic analysis, we found that the canonical sites of FABP5 and FABP7 could
accommodate both enantiomers of SBFIꢀ26 equally well.
MATERIALS AND EXPERIMENTAL DETAILS
Chemical Synthesis of 1-Naphthyl α-Truxillate (SBFI-26)
The synthesis of SBFIꢀ26 followed the procedures reported previously 7. Briefly, αꢀtruxillic acid was
prepared in 93ꢀ95% yield by the modified literature method 24 through irradiation of transꢀcinnamic acid
at 350 nm with a 280 mW/cm2 light. Then, αꢀtruxillic acid (595 mg, 2.0 mmol) was reacted with thionyl
chloride (3 mL) and one drop of dimethyl formamide (DMF) at reflux for 3 h, followed by the removal of
excess thionyl chloride and DMF in vacuo to give αꢀtruxillic acid dichloride. The acid chloride thus
obtained was reacted with 1ꢀnaphthol (240 mg, 1.68 mmol) and pyridine (0.5 mL) in tetrahydrofuran
(THF; 15 mL) at reflux for 3 h. The reaction was quenched with water (2 mL) and extracted with ethyl
acetate (15 mL). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The
crude product was purified by flashed column chromatography on silica gel (ethyl acetate/hexanes) to
give SBFIꢀ26 as white solid (390 mg, 55%): m.p.: 195–196 °C; 1H NMR (500 MHz, CDCl3) δ 4.16 (dd, J
= 10.6, 7.4 Hz, 1 H), 4.40 (dd, J = 10.6, 7.4 Hz, 1 H), 4.63ꢀ4.69 (m, 2 H), 6.28 (d, J = 8.2 Hz, 1 H), 7.12
(d, J = 8.2 Hz, 1 H), 7.24 (d, J = 8.2 Hz, 1 H), 7.33ꢀ7.27 (m, 2 H), 7.38ꢀ7.46 (m, 8 H), 7.49ꢀ7.51 (m, 2 H),
7.63 (d, J = 8.2 Hz, 1 H), 7.77 (d, J = 8.2 Hz, 1 H); Mass (ESꢀAPI, negative mode) m/z calculated for
C28H21O4 [Mꢀ1]ꢀ: 421.1, found 421.1 (negative mode); HRMS (TOF) m/e calculated for C28H22O4H+:
423.1589. Found: 423.1596 (ꢁ =1.7 ppm).
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Protein Expression and Purification
Cloning and expression of FABP5 and FABP7 were described elsewhere 24. Briefly, a pET28a vector
consisting of human FABP5 or FABP7 gene was expressed in the BL21(DE3) E. coli strain. Cells were
grown at 37 °C to OD600 = 0.5ꢀ0.6 before being induced with 0.5 mM Isopropyl βꢀDꢀ1ꢀ
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thiogalactopyranoside. Both human FABP5 and FABP7 were expressed at 18 °C for 16 h. Cells were
harvested by centrifugation and were lysed by passing through a Microfluidizer cell disruptor in 10 mM
potassium phosphate (pH 8.0), 10 mM imidazole, and 0.25 M NaCl. The homogenates were clarified by
spinning at 27,000 × g, and the supernatant was applied to a HiTrapꢀNi column (GE Healthcare) preꢀ
equilibrated with the lysis buffer. Hisꢀtagged proteins were eluted with a 10–300 mM imidazole gradient
in 10 mM potassium phosphate (pH 8.0) and 0.25 M NaCl. To remove the Nꢀterminal Hisꢀtag, FABPs
were incubated with thrombin overnight at 4 °C in 50 mM Tris (pH 8.5) and 0.15 M NaCl. After clearing
the undigested HisꢀFABPs by passing through HiTrapꢀNi column, FABPs were applied to a Superdex 75
column (16 x 1000 mm, GE Healthcare) equilibrated with 10 mM Tris (pH 8.5), 1 mM dithiothreitol, and
0.15 M NaCl. Delipidation was performed by incubating FABPs with hydroxyalkoxypropylꢀdextran
(Sigma) for 1 h at 37 °C. The purified FABP5 and FABP7 were concentrated to 25 mg/ml and 50 mg/ml,
respectively.
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Co-crystallization and structure determination
Before crystallization, FABP5 was incubated with SBFIꢀ26 at room temperature for 30 min at a molar
ratio of 1: 3 (protein: ligand). FABP5ꢀSBFIꢀ26 complex was crystallized at 293 K by the hangingꢀdrop
vapor diffusion method using 0.1 M HEPES, pH 7.5, 2% polyethylene glycol 400, and 2.1 M ammonium
sulfate as precipitant. Diffraction data of FABP5ꢀSBFIꢀ26 were collected at the X6A beamline of
National Synchrotron Light Source (NSLS), Brookhaven National Laboratory, and were processed with
Mosflm software. The space group of FABP5ꢀSBFIꢀ26 crystal was P1 and the structure was solved by
molecular replacement program PHASER using the apoꢀFABP5 structure (PDB ID 4LKP) as the initial
search model. The FABP7ꢀSBFIꢀ26 complex was obtained by mixing FABP7 and SBFIꢀ26 at a 1:2 molar
ratio for 30 min at room temperature. The FABP7ꢀSBFIꢀ26 coꢀcrystals were grown at 293 K by the
sittingꢀdrop vapor diffusion method using 0.1 M Tris, pH 8.5, 0.1 M lithium sulfate, and 33%
polyethylene glycol 4000 as the mother liquor. Diffraction data of FABP7ꢀSBFIꢀ26 to resolution of 1.9 Å
were collected at Lilly Research Laboratories Collaborative Access Team (LRLꢀCAT) beamline of
Advanced Photon Source (APS), Argonne National Laboratory, and were processed with Mosflm
software. The space group of FABP7ꢀSBFIꢀ26 crystal was P21 and the structure was solved by molecular
replacement program PHASER using previously solved FABP7 structure (PDB ID 1FDQ) as the initial
search model. After building the corresponding inhibitor models in Coot 30, the refinements were
performed using Phenixꢀrefine 31; the statistics are provided in Table 1.
Computational analysis
Ligand RMSD values between predicted and experimental binding geometries were computed using the
Hungarian algorithm 32, as implemented into the program DOCK 25, following alignment of the predicted
FABP7ꢀ(R)-SBFIꢀ26 structure (based on pdb code 1FE3) with the new FABP7ꢀ(S)-SBFIꢀ26 crystal
structure, and the new FABP5ꢀ(S)-SBFIꢀ26 crystal structure. The Chimera program 33 MatchMaker
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feature was employed for the alignments (Cꢀalpha backbone atoms). Perꢀresidue interaction energy
profiles (molecular footprints) and reꢀdocking calculations were computed using DOCK.
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Molecular dynamics (MD) simulation protocols, analysis, and free energy calculations followed that
recently reported by Zhou et al. 34 using the AMBER 16 suite of programs 35. The module tleap was
employed to prepare and assemble each system. Briefly, setups employed the ff99SB 36 force field for the
protein and GAFF 37 force field for the SBFIꢀ26 ligand which was augmented with AM1ꢀBCC 38 partial
atomic charges. The TIP3P 39 explicit water model was used to solvate the system. The MD simulations
(pmemd module) were carried out at 298.15 K under NPT conditions, with 120 ns of data collection after
a nineꢀstep minimization/equilibration schedule, during which positional restraints imposed on the ligand
and protein were gradually decreased to relax the complex in an orderly manner. The data collection
phase employed a weak restraint (0.1 kcal mol–1Å–2) on the protein backbone atoms and no restraints on
SBFIꢀ26. The MD trajectories were processed with the cpptraj module to gauge geometric stability
through root mean square deviation (RMSD) analysis. Free energies of binding were estimated using the
“single trajectory” MMꢀGBSA 40, 41 implicit solvent model using periodically saved snapshots taken from
the explicit solvent simulations. Although 120 ns is not necessarily a long MD simulation, under
conditions that employ a weak protein backbone restraint, as little as 2ꢀ20 ns appears to be sufficient to
establish whether a predicted docked pose can be categorized as geometrically or energetically stable as
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previously discussed 34, 42, 43
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RESULTS
1. Structure determination of SBFI-26 in complex with human FABP5 or FABP7
We expressed the human FABP5 and FABP7 in E. coli and purified the proteins to homogeneity. A
standard delipidation protocol was used to remove the endogenous fatty acids in these proteins. We coꢀ
crystallized FABP5 or FABP7 in an excess amount of a racemic mixture of SBFIꢀ26. The coꢀcrystals of
FABP5ꢀSBFIꢀ26 and FABP7ꢀSBFIꢀ26 diffracted to a resolution of 2.2 Å and 1.9 Å, respectively. The
statistics of the diffraction data and the structure refinement are listed in Table 1. We solved these
structures by the molecular replacement method.
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Fig. 2. Overall structures of (S)-SBFI-26 in complex with FABP5 and FABP7. (A) SBFIꢀ26 bound at
the canonical site in human FABP5. (B) SBFIꢀ26 bound at the portal site of the human FABP5. (C) SBFIꢀ
26 bound at the canonical site in human FABP7. In all panels, the 2mFoꢀDFc electron density map of
SBFIꢀ26 is rendered at 1.0σ and shown as grey mesh, the protein structure is shown as ribbons, and SBFIꢀ
26 is modeled in yellow stick form.
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By examining the FoꢀFc and 2FoꢀFc maps (Fig. 2, Supplementary Fig.1), we found that SBFIꢀ26
was clearly present in the coꢀcrystallized structures with FABP5 and with FABP7. The FABP5ꢀSBFIꢀ26
complex was crystallized in the P1 space group. There were eight FABP5 molecules in the asymmetric
unit: four proteins were found to each bind to an SBFIꢀ26 at the canonical substrate binding pocket (Fig.
2A), and the remaining four proteins each bound to the inhibitor at a previous unobserved site at the
substrate entry portal region (Fig. 2B). The four FABP5ꢀSBFIꢀ26 portalꢀsite structures and the four
canonicalꢀsite structures are very similar among themselves, with the average RMSD of 0.36 Å and 0.55
Å, respectively. In FABP7, SBFIꢀ26 resides in the canonical substrateꢀbinding pocket (Fig. 2C). The
electron density was detailed enough that we were able to unambiguously assign the SBFIꢀ26 as the (S)
enantiomer in all structures (Fig. 2A-C). We therefore conclude that only the (S) form, but not the (R)
form, of SBFIꢀ26 is able to bind FABP5 or FABP7 under our crystallization conditions.
2. Conformational changes of FABP5 and FABP7 upon binding to SBFI-26
The crystal structure of the human apo FABP5 was reported previously; its portal area is nearly
closed 44 (Fig. 3A, left). In our structure of SBFIꢀ26 bound at the portal site of human FABP5, the S3ꢀS4
loop moves outward by 5 Å, and the H2 and S3ꢀS4 strands also move outward slightly (Fig. 3A, middle
and right). These movements create enough space to accommodate the bulky naphthalene ring and the
phenyl ring, enabling SBFIꢀ26 to partially insert into the slightly open portal site. In this pose, the SBFIꢀ
26 carboxylate is still outside facing the solvent. To allow SBFIꢀ26 to fully enter the ligand binding
pocket and bind to the canonical site, the H2 and S3ꢀS4 strands move further out, and the S5ꢀS6 loop
moves outward by as much as 8.0 Å (Fig. 3B, left and middle). Interestingly, the side chains of Leu32 of
helix H2, and Leu60 and Lys61 of S3ꢀS4 also rotate outward, further increasing the portal size (Fig. 3B,
right). Therefore, in FABP5, even when SBFIꢀ26 has reached its canonical site, the portal remains wide
open. This is very different from FABP7, in which the portal region is essentially closed when the SBFIꢀ
26 is bound to the canonical site (Fig. 3C, left). The structure of the human FABP7 in the apo form is not
known, but the structure of human FABP7 in complex with a fatty acid has been reported 26. Compared
with the FAꢀbound FABP7, the H2 helix and the S3ꢀS4 and S5ꢀS6 loops move slightly outwards in the
FABP7ꢀSBFIꢀ26 structure, creating a small gap for the side chain of Phe58 to swing inward and close the
portal (Fig. 3C, middle and right).
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Fig. 3. The SBFI-26 induced conformational changes in FABP5 and FABP7. (A) Left: The closed
state of apoꢀFABP5 shown in surface view (PDB ID 4LKT). Only a shallow pocket is seen in the portal
area (red arrow). Middle: The portal area is partially open after insertion of naphthalene group of SBFIꢀ26.
The red dashed curve marks the opened portal area. Right: Insertion of the naphthyl group in portalꢀsite
FABP5ꢀSBFIꢀ26 (green ribbon) disrupts the weak hydrophobic interactions found in apoꢀFABP5 (wheat
ribbon). The helix H2 and the S3ꢀS4 loop move outwards and the portal area of the portal position SBFIꢀ
26ꢀFABP5 complex starts to open (green). The side chains of Leu60 and Lys 61 are also changed from an
inward position to an outward position. (B) Left: The portal area is wide open in the canonicalꢀsite
FABP5ꢀSBFIꢀ26 structure. Middle: Conformational changes in canonical site FABP5ꢀSBFIꢀ26 (cyan
ribbon) relative to the apoꢀFABP (wheat ribbon). SBFIꢀ26 is shown in yellow stick form. Right:
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Superposition of the portalꢀsite (green ribbon) and the canonical site SBFIꢀ26ꢀFABP5 structures (cyan
ribbon). Only the canonical site inhibitor is shown for clarity (yellow stick form). (C) Left: The portal
area is in a closed state in FABP7ꢀSBFIꢀ26 structure. Middle, superposition of the fatty acidꢀbound
FABP7 structure (light blue ribbon, PDB ID 1FDQ) and the FABP7ꢀSBFIꢀ26 structure (magenta ribbon).
Right: The side chain of Phe58 in FABP7ꢀSBFIꢀ26 complex (magenta) rotates from an outside position in
fatty acidꢀbound FABP7 (light blue) to close the portal in the FABP7ꢀSBFIꢀ26 structure.
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It is unclear whether the closed portal configuration of FABP7 or the open port configuration of
FABP5 represents the end state of substrate binding, because in both structures, SBFIꢀ26 has fully entered
the substrate pocket and is bound at the canonical site. It is possible that the portal region fluctuates in
solution between the open and the closed forms while the ligand is bound at the canonical site. This
scenario is supported by a previous NMR and deuterium exchange studies revealing the highly dynamic
nature of these human FABP proteins at the portal and helical lid regions 45. Interestingly, a previous
work suggested that the interaction between Leu60 in the S3ꢀS4 loop and Met35 in H2 specifies the
ligandꢀbound active state of FABP5 44. Another work indicated that the inwardꢀpointing orientation of the
Phe57 side chain in the S3ꢀS4 loop (equivalent of Phe58 in FABP7) is required for FABP4 to undergo a
conformational change that results in the exposure of its nuclear localization signal to the solution 46.
These observations appear to reflect the fact that the portal region is highly dynamic and actively
sampling multiple conformations.
3. Interaction between SBFI-26 and the canonical sites of FABP5 and FABP7
In FABPs, the native substrate fatty acids bind to and form salt bridges and hydrogen bonds (Hꢀ
bonds) with two arginines and a tyrosine inside the canonical ligand binding sites. These residues are
Arg109, Arg129, and Tyr 131 in FABP5, and Arg107, Arg127, and Tyr 129 in FABP7. In the canonicalꢀ
site FABP5ꢀSBFIꢀ26 complex, SBFIꢀ26 appears to mimic the native substrates, with its carboxylate
forming a salt bridge with Arg129, an Hꢀbond with Tyr131, and four Hꢀbonds with Arg109 via an ordered
water molecule (Fig. 4A). Similar interactions were also observed in the FABP7ꢀSBFIꢀ26 complex, in
which the carboxyl group of SBFIꢀ26 formed a salt bridge with Arg127 and a waterꢀmediated network of
hydrogen bonds was formed among the carboxyl group, Arg127, Tyr129, Arg107, and Thr54 (Fig. 4B).
Additionally, a water mediated four Hꢀbonds among the backbone oxygens of Gly34 and Thr37, the NH
group of Arg127, and the carbonyl oxygen of SBFIꢀ26. These Hꢀbonds are ligandꢀspecific and are not
observed in the fatty acid–bound FABPs. In FABP7, the hydrophobic residues Phe17, Met21, Leu24,
Val26, Thr30, Pro 39, Phe58, Ala76, Phe105, Met116, and Leu118 formed hydrophobic interactions with
the two phenyl rings and the naphthalene ring of SBFIꢀ26 (Fig 4C). These hydrophobic portal residues
also contribute to the binding of the native substrate fatty acids 26.
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Fig. 4. The binding profiles of SBFI-26 in the canonical sites of FABP5 and FABP7. (A) The FABP5ꢀ
SBFIꢀ26 canonical position (cyan). The carboxyl group of SBFIꢀ26 forms a salt bridge with Arg129 and
hydrogen bonds with Tyr131 and Arg109. Water molecules are shown as red spheres. (B) In the FABP7ꢀ
SBFIꢀ26ꢀ complex, the carboxyl group of SBFIꢀ26 forms a salt bridge with Arg127 and hydrogen bonds
with Tyr129, Arg107, and Thr54. SBFIꢀ26 specific interactions between the carbonyl group and Gly34
and Thr37 are mediated by a water molecule. (C) Hydrophobic interactions between SBFIꢀ26 and FABP7.
4. Structural comparison between the DOCK-predicted pose for SBFI-26 and the crystallographic
pose
Determination of the crystal structures of SBFIꢀ26 in complex with either FABP7 or FABP5 afforded the
opportunity to compare the original DOCK binding geometry (pose) reported previously 7, 24 with those
observed in the present work (Fig. 5). The theoretical and experimental poses of SBFIꢀ26 with FABP7
show remarkable overlap and have a low heavyꢀatom rootꢀmeanꢀsquareꢀdeviation (RMSD) of 2.7 Å,
despite the fact the DOCK prediction originally led to the identification of the (R) enantiomer but the
FABP7 Xꢀray structure coꢀcrystallized in the (S) enantiomer (Fig. 5A). Although the differences in
stereochemistry preclude perfect overlap and the docking calculations employed a rigid protein, the
position of the key carboxylic group that interacts with Arg127 (see discussion below) was wellꢀoverlaid,
and there was overall good correspondence in the positions of the phenyl and naphthalene rings (Fig. 5A).
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Figure 5. Comparison of the original DOCK pose in orange 7, 24 for (R)ꢀSBFIꢀ26 made using FABP7 with
oleic acid removed (PDB 1FE3) and the current crystallographic poses of (S)ꢀSBFIꢀ26 determined with
FABP7 (A) and FABP5 (B) in green and cyan, respectively. Protein residues are hidden for clarity.
Overlay of the current FABP7 (green) and FABP5 (cyan) crystal structures with the previously published
FABP7 (orange) structure (C). Structural comparisons made after alignment of the Cꢀalpha backbone
atoms in common. For FABP5, only the structure with a canonical site ligand is shown.
Geometrically, after aligning the two FABP7 structures on their protein backbones (Cα RMSD =
0.73 Å, Fig. 5C), the docked (R)-SBFIꢀ26 showed only minor steric clashes in the new FABP7 structure,
which could be easily alleviated by local energy minimization, resulting in even better overlap (2.4 Å).
Interestingly, the reverse experiment — placing the (S)ꢀSBFIꢀ26 crystal into the FABP7 site that
originally accommodated oleic acid — yielded some steric clashes that could not be ameliorated by a
simple energy minimization, thereby explaining why the (R) form was selected from the virtual screen.
Thus, although there is substantial similarity between the two different FABP7 crystal structures (i.e., Cꢀ
alpha RMSD = 0.73 Å), there does appear to be enough difference to preclude (S)ꢀSBFIꢀ26 from adopting
the same pose without at least some conformational changes to the FABP7 structure originally containing
oleic acid. Given that a rigid protein approximation was employed, this was not allowable. Notably, reꢀ
docking the (S) form back into FABP7 or FABP5 from the current work yielded very low ligand RMSD
values of 0.69 Å and 0.88 Å relative to their respective crystallographic poses.
For FABP5, however, there were greater structural differences (Fig. 5B, RMSD = 4.2 Å) between
the (R) predicted pose made in FABP7 and the experimental pose. These were likely due to the fact that
FABP5 with (S)-SBFIꢀ26 was not yet in an intermediateꢀlike state, i.e., its portal region was still open
relative to the closed portal in FABP7 (Cα RMSD = 2.90 Å, Fig. 5C). Rotations of ~ 180° about the
cyclobutane and naphthalene ester bonds in (S)-SBFIꢀ26 showed there were differences even between the
two experimental geometries in FABP5 or FABP7 (Fig. 5A-B, green vs cyan poses). Despite these
differences, the DOCK pose still showed good overlay with the FABP5 experimental pose in terms of the
carboxylic acid, although the phenyl and naphthalene rings were more offset. As before, energy
minimization of the docked structure in the xꢀray structure (in this case FABP5) yielded a slightly better
RMSD (3.8 Å).
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5. Energetic comparison, ensemble-based geometric stability, and binding free energies for SBFI-26
with FABP7
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From an energetic perspective, the perꢀresidue interaction footprints of SBFIꢀ26 computed from the
original prediction made in FABP7 (PDB 1FE3) yielded striking overlap with that computed using the
current Xꢀray pose (Fig. 5). Specifically, similarly favorable van der Waals (VDW) energies (Fig. 6A)
included, among others, those at positions Phe17, Met21, Pro39, Thr54, Asp77, and Arg127, and the good
geometric overlap noted above for the carboxylic acid of SBFIꢀ26 yielded nearly identical electrostatic
(ES) footprints patterns (Fig. 6B), in particular the strong peaks at positions Arg107 and Arg127. Given
the differences in stereochemistry and the fact that the calculations were performed using two different
crystal structures, such good correspondence is notable.
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Figure 6. Perꢀresidue van der Waals (A) and electrostatic (B) interaction energy profiles (termed
footprints) computed for SBFIꢀ26 from the current FABP7 crystal structure (green, S form) versus that
from the previously reported DOCK prediction (orange, R form) made using FABP7 with oleic acid
removed (PDB ID 1FE3). Plots explicitly show only the top 30 protein residues based on interaction
strength, with the remaining interactions summed into the residue labeled REMAIN.
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Figure 7. (A) Molecular dynamics simulation results for (S)ꢀSBFIꢀ26 and (R)ꢀSBFIꢀ26 with FABP7
showing 100 evenly spaced ligand snapshots from 20ꢀns production runs. Protein residues omitted for
clarity. (B) Estimated free energies of binding in kcal/mol (MMꢀGBSA method) plotted as running
averages versus time (100ꢀps blocks). (C) Ligand heavy atom RMSD in angstroms plotted as running
averages versus time (100ꢀps blocks).
To further characterize the SBFIꢀ26 binding poses with FABP7, we employed allꢀatom molecular
dynamics (MD) simulations to assess geometric stability and estimate free energies of binding (MMꢀ
GBSA method) using protocols recently reported to target botulinum neurotoxin serotype E 34. As
highlighted in Fig. 7A, which shows 100 evenly spaced snapshots taken over the course of 20ꢀns MD
simulations, both enantiomers preserved their initial starting geometries. Interestingly, the simulation of
(S)ꢀSBFIꢀ26 starting from the xꢀray pose showed larger upꢀandꢀdown movement at one phenyl group
position, as well as leftꢀtoꢀright movement at the naphthalene position (Fig. 7A left). This observation is
consistent with the FABP7 binding site having sufficient wiggle room to accommodate both enantiomers,
as illustrated by an overlay of the MD snapshot ensembles (Fig. 7A, right). This could be advantageous
when designing new analogs. Quantitatively, the MD simulations yielded very similar and stable free
energies of binding (Fig. 7B). Accompanying ligand RMSD values were similarly stable, with the xꢀray
pose (green) yielding slightly lower values than the DOCK pose (orange). Taken together, the Xꢀray,
docking, footprintꢀsimilarity, and MDꢀsimulation results strongly suggest that both the S and R forms of
SBFIꢀ26 are geometrically and energetically compatible with the FABP7 binding site.
DISCUSSION
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Over the past several years, SBFIꢀ26 and related compounds have been actively developed as
potential analgesics targeting the intracellular anandamide transporters FABP5 and FABP7 7. Highꢀ
resolution structures of SBFIꢀ26 in complex with these proteins would aid our medicinal chemistry efforts
to design more potent inhibitors based on the SBFIꢀ26 scaffold. In this report, we describe the coꢀcrystal
structures of SBFIꢀ26 in complex with FABP5 and FABP7. We found that SBFIꢀ26 has an overall similar
binding profile as the native substrate fatty acids: the carboxylate — a key moiety of the inhibitor —
forms a salt bridge and hydrogenꢀbond interactions just as the fatty acids do, and there are extensive
hydrophobic interactions between the phenyl rings and naphthalene ring and the substrate pocket of the
FABPs, similar to the hydrophobic interaction of the aliphatic chain of the fatty acid. A comparison of the
binding geometry of SBFIꢀ26 to FABP7 with the computational prediction we reported earlier showed
remarkable agreement 7, 24. Unique to SBFIꢀ26 are several waterꢀmediated hydrogen bonds between the
carbonyl oxygen of the inhibitor and the backbone oxygens of Gly34 and Thr37, indicating a crucial role
of water molecules in SBFIꢀ26 binding to FABPs.
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An interesting finding from our structures is that with FABP5, SBFIꢀ26 binds at a portal site as
well as the canonical site in the substrate pocket. Both binding poses were captured in coꢀcrystal
structures hence are stable. However, their binding characteristics with the transporter resembles that of
the binding intermediates, because the portal regions are either partially open (portal site) or wide open
(canonical site), which contrasts with the portalꢀclosed structure of FABP7 in complex with SBFIꢀ26 or
with a fatty acid. The unique binding profile of SBFIꢀ26 in FABP5 may afford an opportunity for the
development of FABP5ꢀspecific inhibitors in the future.
It is currently unclear why only the S form binds to both FABP5 and FABP7 in our coꢀcrystal
structures. It is possible that the crystallization process may have selectively incorporated the S form
complex into the crystal lattice, leaving the R form in solution. Alternatively, we suggest that the S form
may bind to the portal site more rapidly than to the canonical site, leading to an increased local
concentration of the S enantiomer for binding to the canonical site. Importantly, our computational
analysis showed that both the S form and the R form are geometrically and energetically compatible in the
binding pocket of FABP7ꢀSBFIꢀ26 complex. Future work is needed to understand and to further improve
the interaction between SBFIꢀ26 and the human anandamide transporters.
ACKNOWLEDGEMENTS
This work was supported by NIH (DA035923 to D.G.D., I.O., R.R., M.K. and H.L., DA035949 to M.K.,
and GM083669 to R.R.), and the Van Andel Research Institute (to H.L.). We thank Brian Ralph and
William J. Allen for computational assistance, and the Institute for Advanced Computational Science at
Stony Brook University for providing computational resources. Diffraction data for this study were
collected at X6A beamline of National Synchrotron Light Source (NSLS), Brookhaven National
Laboratory and the Lilly Research Laboratories Collaborative Access Team (LRLꢀCAT) beamline at
Advanced Photon Source (APS), Argonne National Laboratory. We thank Laura Morisco, Jordi Benach,
and Stephen Wasserman for help with data collection. APS was supported by the U.S. Department of
Energy, Office of Science, Office of Basic Energy Sciences, under contract no. DEꢀAC02ꢀ06CH11357.
Use of the LRLꢀCAT beamline at Sector 31 of the APS was provided by Eli Lilly Company, which
operates the facility.
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Table 1. Data collection and refinement statistics
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FABP5ꢀSBFIꢀ26
FABP7ꢀSBFIꢀ26
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Data collection
Wavelength
Space group
Cell dimensions
a, b, c (Å)
α, β, γ (°)
Resolution (Å)
Rmerge (%)
1.00000
P 1
0.97931
P 1 21 1
68.99, 78.11, 78.83
61.66, 69.61, 78.01
54.94 – 2.20 (2.32 – 2.20)*
5.6 (38.6)
11.2 (2.3)
164362
53.84, 73.33, 70.53
90, 92.98, 90
53.77 – 1.85 (1.95 – 1.85)
10.7 (55.1)
8.0 (2.3)
174183
I / σI
Total reflections
Completeness (%)
Redundancy
92.5 (89)
2.6 (2.5)
99.9 (100)
3.7 (3.7)
Refinement
Resolution (Å)
No. reflections
Rwork / Rfree
No. of nonꢀhydrogen 9367
atoms
40.76 – 2.20
63854
0.1967 / 0.2369
53.77 – 1.85
46755
0.1688 / 0.1982
4998
Protein
Ligand
Water
8466
557
344
41.22
40.95
46.48
39.19
4248
168
582
30.60
29.34
44.21
35.82
Bꢀfactors
Protein
Ligand/ion
Water
R.m.s. deviations
Bond lengths (Å)
Bond angles (°)
Ramachandran
statistics (%)
Favored
0.008
0.97
0.005
0.73
97.9
2.1
0
99
1
0
Allowed
Outliers
REFERENCES
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