Bonded His93 As a Sensitive Probe for Identifying Inhibitors of the Endocannabinoid Transport Protein FABP7

Chem Biol Drug Des 2015; 85: 534–540 Research Article Hydrogen-Bonded His93 As a Sensitive Probe for Identifying Inhibitors of the Endocannabinoid Transport Protein FABP7 Sergiy Tyukhtenko1, Karrie Chan1, Rubin Jiang1, receptor-mediated signal transduction (2). The biosynthesis Han Zhou1, Richard W. Mercier1, De-Ping Yang2, and physiology of AEA are well understood (3,4), but its Alexandros Makriyannis1 and Jason J. Guo1,* mechanism of uptake has been elusive. Despite intensive studies on the AEA transport process (5–9), the question 1Center for Drug Discovery, Department of Pharmaceutical remains on how the uncharged lipophilic endocannabinoid Sciences and Department of Chemistry and Chemical ligand AEA crosses the cellular membrane to access the Biology, Northeastern University, 360 Huntington Avenue, hydrophilic cytosol. Recently, it was reported (10,11) that a Boston, MA 02115, USA 2 group of carrier proteins, particularly fatty acid binding pro- Physics Department, College of the Holy Cross, 1 College teins (FABPs), can significantly enhance the cellular uptake Street, Worcester, MA 01610, USA and subsequent inactivation of AEA. The findings provide *Corresponding author: Jason J. Guo, [email protected] a potential new therapeutic modality to the treatment of pain, inflammation, and drug abuse through dual inhibition The human brain FABP (FABP7) has been shown to be an intracellular carrier protein that can significantly of the deactivating enzymes such as fatty acid amide potentiate the uptake of the endocannabinoid ananda- hydrolase (FAAH) (12) and the endocannabinoid transport mide. For this reason, there is a great interest in the proteins. discovery and development of FABP7 inhibitors for treating stress, pain, inflammation, and drug abuse. FABPs are intracellular lipid chaperones that exhibit unique We found that in the 1H-NMR spectrum of the protein, patterns of tissue expression and are expressed most abun- a well-separated downfield resonance arising from the dantly in tissues involved in active lipid metabolism (13). hydrogen-bonded His93 side chain is very sensitive to Human brain FABP (FABP7), which has been shown to sig- ligand binding. Using this characteristic spectral mar- nificantly potentiate AEA uptake (10), is expressed in various ker together with another well-resolved upfield reso- regions in the brain and is distinguished from other FABPs by nance from the side chain of Val84, we have identified its strong affinity for polyunsaturated fatty acids (14,15). Cur- that an adipocyte FABP (FABP4) inhibitor BMS309403 rently only a few specific FABP inhibitors have been also binds tightly to FABP7. Our data demonstrated described (16,17), and there are no selective ligands avail- that this unique His93 downfield resonance can be used as a sensitive probe for rapidly and unambigu- able for FABP7. In this work, we sought to obtain structural ously identifying novel high-affinity FABP7 ligands. The insights on the FABP7 binding pocket due to a growing inter- findings should help accelerate the discovery of poten- est in the discovery and development of selective FABP7 1 tial drug leads for the modulation of endocannabinoid inhibitors (18). We found that in the 1D H-NMR spectrum, a transport. well-separated downfield resonance arising from the hydrogen-bonded His93 side chain is very sensitive to ligand bind- Key words: anandamide transport, endocannabinoids, fatty ing. Using this characteristic spectral marker, we have acid binding protein, NMR successfully demonstrated that a selective adipocyte FABP (FABP4) inhibitor (16) BMS309403 also binds tightly to Abbreviations: AA, arachidonic acid; AEA, anandamide, FABP7. Thus, this unique His93 can be exploited as a sensi- N-arachidonoylethanolamide; DHA, docosahexaenoic acid; tive probe for NMR-based high-throughput screening to rap- FABP7, fatty acid binding protein 7, brain fatty acid binding idly identify novel FABP7 ligands that can subsequently be protein; HSQC, heteronuclear single quantum coherence; OA, developed into selective probes or drug leads. oleic acid. Methods and Materials Received 20 August 2014, revised 18 September 2014 and accepted for publication 19 September 2014 Chemicals Docosahexaenoic acid (DHA), arachidonic acid (AA), oleic Anandamide (N-arachidonoylethanolamide, AEA) is the first acid (OA), and all other common chemicals were endocannabinoid isolated from porcine brain (1) that binds purchased from Sigma-Aldrich (St. Louis, MO, USA). Three to the human cannabinoid receptor and stimulates of the four representative cannabinoid ligands, D9-tetrahy- 534 ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12440 His93 As a Sensitive Probe for Identifying FABP7 Inhibitors drocannabinol (D9-THC), CP55940 (2-[(1R,2R,5R)-5- Lipidex competition binding assay hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2- Ligand binding to purified human FABP7 was analyzed by yl)phenol), and SR141716A (rimonabant; 5-(4-Chlor the Lipidex assay (14,15,19). Briefly, 2.5 lM of purified and ophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)- delipidated recombinant FABP7 protein was incubated 1H-pyrazole-3-carboxamide), were obtained from the with the same concentration of oleic acid at a constant National Institute on Drug Abuse (NIDA). The cannabinoid hot to cold ratio of 1:50 at 37 °C for 15 min. Solutions ligand WIN55212-2 ((R)-(+)-[2,3-Dihydro-5-methyl-3-(4- with different competitor concentrations were subsequently morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]- added followed by an additional 30 min incubation at 1-napthalenylmethanone) was purchased from Tocris 37 °C. The mixtures were cooled on ice for 15 min, and Bioscience (R&D Systems, Minneapolis, MN, USA). The 50 lL of a 50% (v/v) precooled Lipidex 1000 suspension selective adipocyte FABP (FABP4) inhibitor, BMS309403 was added with subsequent vortexing for 15 min while (2-[20-(5-Ethyl-3,4-diphenyl-1H-pyrazol-1-yl)biphenyl-3-yl- maintaining the temperature below 4 °C to remove any oxy]-acetic acid), was purchased from EMD Chemicals free oleic acid from the mixture. Raw data for constructing (San Diego, CA, USA). The [9, 10-3H] oleic acid and the competition binding curve were obtained by separation Lipidex 1000 resin used for the radioactive competition of protein-bound and free fatty acid (Lipidex bound) by binding assay were from Perkin Elmer (Waltham, MA, USA). vacuum filtration using Pall 96-well filter plates (0.45 lm pore size) and scintillation counting of the protein-bound fractions. Expression, purification, and refolding of human brain FABP Human FABP7 encoding cDNA in pCMV6-XL5 was pur- Results and Discussion chased from OriGene (Rockville, MD, USA). The Champion pET directional TOPO expression kit from Invitrogen was The 1D 1H-NMR spectrum of human FABP7 (Figure 1A) is employed for subcloning and expression in BL21 E. coli substantially crowded with severe overlapping in the entire cells. The recombinant FABP7 contained a polyhistidine tag amide, aromatic, and aliphatic regions. However, two dis- at the N-terminus (12 additional amino acids) that allowed tinctive peaks can be readily identified: one at the far single-step purification by immobilized cobalt affinity chro- downfield region (> 11 ppm) and the other at the high matography. Extraction from inclusion bodies and His-tag purification were performed under strong denaturing condi- tion using 8 M urea. The purified FABP7 was first refolded in A a cold refolding buffer (1 M arginine, 5 mM DTT, 150 mM NaCl, 20 mM PBS, pH 7.4) followed by dialysis against NMR buffer (10 mM PBS, 100 mM NaCl, 1 mM DTT, and 0.05% sodium azide, pH 7.4) using Spectra/Por dialysis membrane (MWCO 6–8 K) and further delipidated over a Lipidex 1000 column at 37 °C according to a reported pro- cedure (19). Uniformly 15N-enriched FABP7 protein was obtained by first growing cells in an M9 minimal medium 15 containing 1 g/L of NH4Cl (Cambridge Isotope Laborato- ries, Andover, MA, USA) followed by the same purification and refolding protocol as above. B High-resolution NMR experiments All one- and two-dimensional high-resolution NMR experiments were conducted at 298 K on a Bruker AVANCEII 700 MHz NMR spectrometer. The 1D 1H-NMR spectra of the human recombinant FABP7 were acquired using a WATERGATE 3-9-19 scheme with a water flipback (Bruker pulse sequence: p3919fpgp) pulse to minimize solvent sat- uration transfer. For 2D 1H-15N correlation, a fast hetero- 1 nuclear single quantum coherence (Fast-HSQC) detection Figure 1: (A) One-dimensional H-NMR spectrum of human scheme (20,21) was employed. To study ligand binding, recombinant FABP7. Two very distinctive peaks, one located at the far downfield region (> 11 ppm) and the other at the upfield NMR titration experiments were performed to monitor region (< 0 ppm), are highlighted. (B) Strong H-bond between the complex formation. All ligands were first dissolved in His93 He2 and the side chain carboxyl group of Glu72 in the dimethyl sulfoxide-d6 (DMSO-d6) to create concentrated binding pocket of FABP7. The Val84 Hc2 protons experience stock solutions. The final concentration of DMSO-d6 in the strong shielding by the ring current from the His93 imidazole side protein–ligand solution did not exceed 2% (v/v). chain. Chem Biol Drug Des 2015; 85: 534–540 535 Tyukhtenko et al. upfield region (< 0 ppm). These two peaks can be somewhat different result with the His93 He2 peak gradu- assigned unambiguously based on earlier NMR structural ally shifting from 11.96 to 11.92 ppm but with no new studies of FABP7 (PDB ID: 1JJX) (22,23). The peak at peaks appearing in the spectrum. This suggests a fast 11.96 ppm is assigned to He2 of the His93 residue, while exchange process on the NMR time scale and reflects a the peak at À0.38 ppm is assigned to the Hc2 of Val84. relatively lower binding affinity of oleic acid. The strong downfield shift of the His93 He2 proton can be attributed to a hydrogen bonding interaction with the side The robustness of this characteristic His93 as a spectral chain carboxyl group of Glu72 (22) (Figure 1B).

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