Research Article pubs.acs.org/acschemicalneuroscience
Solution Structures of the Prototypical 18 kDa Translocator Protein Ligand, PK 11195, Elucidated with 1H/13C NMR Spectroscopy and Quantum Chemistry Yong-Sok Lee,† Fabrice G. Siméon,‡ Emmanuelle Briard,‡ and Victor W. Pike*,‡ †
Center for Molecular Modeling, Division of Computational Bioscience, Center for Information Technology, National Institutes of Health, Building 12a, Room 2049, Bethesda, Maryland 20892, United States ‡ PET Radiopharmaceutical Sciences Section, Molecular Imaging Branch, National Institute of Mental Health, National Institutes of Health, Building 10, Room B3 C346A, 10 Center Drive, Bethesda, Maryland 20892, United States S Supporting Information *
ABSTRACT: Eighteen kilodalton translocator protein (TSPO) is an important target for drug discovery and for clinical molecular imaging of brain and peripheral inflammatory processes. PK 11195 [1a; 1-(2-chlorophenyl)-N-methyl-(1-methylpropyl)-3-isoquinoline carboxamide] is the major prototypical high-affinity ligand for TSPO. Elucidation of the solution structure of 1a is of interest for understanding small-molecule ligand interactions with the lipophilic binding site of TSPO. Dynamic 1H/13C NMR spectroscopy of 1a revealed four quite stable but interconverting rotamers, due to amide bond and 2-chlorophenyl group rotation. These rotamers have been neglected in previous descriptions of the structure of 1a and of the binding of 1a to TSPO. Here, we used quantum chemistry at the level of B3LYP/ 6-311+G(2d,p) to calculate 13C and 1H chemical shifts for the rotamers of 1a and for the very weak TSPO ligand, N-desmethyl-PK 11195 (1b). These data, plus experimental NMR data, were then used to characterize the structures of rotamers of 1a and 1b in organic solution. Energy barriers for both the amide bond and 2′-chlorophenyl group rotation of 1a were determined from dynamic 1H NMR to be similar (ca.17 to 18 kcal/mol), and they compared well with those calculated at the level of B3LYP/6-31G*. Furthermore, the computed barrier for Z to E rotation is considerably lower in 1a (18.7 kcal/mol) than in 1b (25.4 kcal/mol). NMR (NOE) unequivocally demonstrated that the E rotamer of 1a is the more stable in solution by about 0.4 kcal/mol. These detailed structural findings will aid future TSPO ligand design and support the notion that TSPO prefers to bind ligands as amide E-rotamers. KEYWORDS: PK 11195, dynamic 1H/13C NMR, TSPO, rotamer, variable temperature, energetics, quantum chemistry, structure
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periphery.17,18 TSPO has therefore become a significant target for CNS drug development.9 Furthermore, radioligands19−21 for the imaging of TSPO in vivo are keenly pursued as potential biomarkers of inflammatory conditions.22,23 These considerations drive the efforts to discover new and selective high-affinity TSPO ligands as potential new CNS drugs or improved imaging radioligands. The interaction of 1a and other ligands with TSPO has been modeled extensively.24−27 From these studies, it is clear that the amido carbonyl group of 1a plays a critical role in its binding to TSPO, perhaps by forming a directional hydrogen bond within a generally lipophilic binding site. Hence, precise knowledge of the position and orientation of the carbonyl group of 1a in solution and when bound to TSPO can be informative about the topography of the TSPO binding site, and also aid in future ligand design. Earlier studies have not considered the possible existence of stable rotamers of 1a in solution (Figure 1), nor
K 11195 (1-(2-chlorophenyl)-N-methyl-(1-methylpropyl)3-isoquinoline carboxamide, 1a (Chart 1),1 is the major prototypical high-affinity ligand for the 18 kDa translocator protein (TSPO), formerly known as the peripheral benzodiazepine receptor. TSPO was first discovered as a result of its ability to bind diazepam2 and was later distinguished from central benzodiazepine receptors by location, function, structure, and pharmacology.3−5 Several functions have been postulated for TSPO but perhaps the most evidence-based is as a mitochondrial membrane-based transporter, channel, or exchanger for cholesterol.6 Besides isoquinoline carboxamides7 and certain benzodiazepines (e.g., 2; Ro-5-4864, 4′-chlorodiazepam; Chart 1), TSPO also binds with high affinity to ligands belonging to many other structural classes8,9 including quinoline carboxamides,10 pyrazolopyrimide acetamides,11 2-arylindole-3-acetamides,12 N,N-dialkyl-2-phenylindol-3-ylglyoxylamides,13 and aryloxyanilides14−16 (Chart 1). High-affinity ligands from these and other structural classes invariably feature a single tertiary amido group.8,9 TSPO is now implicated in various neuropsychiatric conditions, especially anxiety, and is expressed heavily in microglia in response to various inflammatory conditions in brain and This article not subject to U.S. Copyright. Published 2012 by the American Chemical Society
Received: Revised: Accepted: Published: 325
January 20, 2012 February 8, 2012 February 14, 2012 February 14, 2012 dx.doi.org/10.1021/cn3000108 | ACS Chem. Neurosci. 2012, 3, 325−335
ACS Chemical Neuroscience
Research Article
Chart 1. Some High Affinity TSPO Ligands from Different Structural Classesa
a PK 11195 (1a; an isoquinoline carboxamide); Ro 5-4864 (2; a benzodiazepine); PBR 28 (3; an aryloxyanilide); FGIN 1 (4; an indoleacetamide); DPA-713 (5; a pyrazalopyrimidine); IGA-1 (6; an N1-methyl-2-phenylindol-3-ylglyoxylamide).
of the N-methyl protons in the E and Z rotamers that was not seen in the spectra acquired in d6-DMSO (Figure S2, Supporting Information). Namely, each signal appeared not as the expected singlet but as an almost completely resolved pair of peaks with small separation (
