Mimics of the binding sites of opioid receptors obtained by molecular imprinting of enkephalin and morphine ( molecular recognition / synthetic polymers / ligand - receptor interactions / molecularly imprinted sorbent assay )

Molecular imprinting of morphine and the endogenous neuropeptide [Leu5]enkephalin (Leu-enkephalin) in methacrylic acid-ethylene glycol dimethacrylate copolymers is described. Such molecular imprints possess the capacity to mimic the binding activity of opioid receptors. The recognition properties ofthe resultant imprints were analyzed by radioactive ligand binding analysis. We demonstrate that imprinted polymers also show high binding affinity and selectivity in aqueous buffers. This is a major breakthrough for molecular imprinting technology, since the binding reaction occurs under conditions relevant to biological systems. The antimorphine imprints showed high binding affinity for morphine, with Kd values as low as 10-7 M, and levels of selectivity similar to those of antibodies. Preparation of imprints against Leu-enkephalin was greatly facilitated by the use of the anilide derivative rather than the free peptide as the print molecule, due to improved solubility in the polymerization mixture. Free Leu-enkephalin was efficiently recognized by this polymer (Kd values as low as 10-7 M were observed). Four tetraand pentapeptides, with unrelated amino acid sequences, were not bound. The imprints showed only weak affi'nity for two D-amino acid-containing analogues of Leuenkephalin. Enantioselective recognition of the L-enantiomer of phenylalanylglycine anilide, a truncated analogue of the N-terminal end of enkephalin, was observed. The study of molecular recognition, with implications for intermolecular chemistry, chemical selectivity, and ultimately in drug design, is at present a rapidly developing field of research (1, 2). Numerous model systems have evolved that mimic the interaction between a substrate (the guest) and a receptor (the host). Molecular imprinting (3) has, particularly during the last few years, developed into a powerful technique for the preparation of highly substrateand enantioselective polymers (4-7). The technique entails polymerization around a print species using monomers with chemical functionalities complementary to those of the print molecule (4-7). The interactions developed between complementary functionalities present in the imprint molecule and the monomer(s) prior to the initiation of polymerization are conserved in the resultant polymer. Subsequent removal of the print species exposes recognition sites within the polymer possessing a "memory" for this compound in terms of complementarity of both shape and chemical functionality. Antitheophylline and antidiazepam molecularly imprinted polymers (MIPs) have been applied as antibody mimics in an immunoassay-like technique, molecularly imprinted sorbent assay (MIA), for drug determinations in human serum (8). The imprinted antibody mimics showed high binding affinities and selectivities comparable to those demonstrated by the corresponding antigen-antibody systems. Assay results showed excellent correlation with those obtained by using a traditional immunoassay technique. AnThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. other important application of MIPs is their use in HPLC separations. In particular, when the print molecule is one of the optical antipodes of a chiral compound, the resultant MIP shows a very high enantioseparative capability. Chiral stationary phases have, to date, been developed for resolution of amino acid derivatives (9-13), sugar derivatives (14, 15), and drug compounds, such as ,B-blocking agents (16). Opioid receptors are a family of receptors involved in pain perception (for comprehensive state-of-the-art reviews of opioid research, see ref. 17). At least three classes of opioid receptors, ,u, 8, and K, are present in brain that differ in their ligand selectivity and in their pharmacological effect. Structural and functional characterization of the receptors is still preliminary. This is due to experimental difficulties in purification of the receptors, reconstitution of them in a membrane environment, and isolation and cloning of their genes (18). The advancement of opioid receptor knowledge has, to a large extent, relied on the identification and characterization of selective opioid agonists and antagonists. Several studies of ligand-receptor interactions have used antibodies against enkephalins (19-23) or morphine (24-29) as mimics of the binding site of opioid receptors. Here we report the imprinting of the endogenous neuropeptide [Leu5]enkephalin (Leuenkephalin), and of morphine, and the characterization of the binding properties of the resultant imprints. MIPs have several distinct advantages that make them attractive for this kind of study, such as simple and rapid preparation, the stability of imprinted structures, and the wide variety of substances amenable to imprinting. By the choice of the functional monomer(s) employed, it may be possible to undertake systematic studies of particular binding interactions and contributions to binding. In this study, radioligand displacement (MIA) was used as the diagnostic tool to evaluate the selective binding abilities of the imprints. Strong ligand-imprint interaction requires the use of apolar organic solvents, which is a disadvantage for the object of this present study. Hence, this particular issue was addressed and some basic protocols for the optimization of ligand binding to MIPs in both organic and aqueous media were developed. A comparison is made among the binding properties of imprints, natural opioid receptors (17), and several anti-enkephalin and anti-morphine monoclonal antibodies. MATERIALS AND METHODS Polymer Preparation. Morphine MIPs were made by carefully heating 428 mg (1.5 mmol) of morphine free base and 516 Abbreviations: DADLE, [D-Ala2,D-Leu5]enkephalin; MIP, molecularly imprinted polymer; Leu-enkephalin, [Leu5]enkephalin; MIA, molecularly imprinted sorbent assay; DALE, [D-Ala2,Leu5]enkephalin; Met-enkephalin, [Met5]enkephalin. *Present address: Astra Pain Control, S-151 85 Sodertalje, Sweden. tPresent address: Johnson & Johnson Medical, Oststrasse 1, 22844 Norderstedt, Germany. tPresent address: Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology, 711 10 Heraklion, Crete, Greece. §To whom reprint requests should be addressed.