CHEMICAL BIOLOGY

Chemistry based research in our group is focused on the understanding of how proteins work and how the structural features of receptor ligands influence protein – small molecule interacting systems. One major area of our activity is to develop new methods for the preparation of special polypeptides and proteins of biomedical interest; furthermore, we have intensive programs in small molecule receptor ligand development, radioactive labeling, molecule spectroscopy and molecule modeling. These combined efforts result in protein derivatives enriched in chemical information, small ligand libraries with systematic structural changes and radiolabeled receptor ligands that are used as probes for protein function and receptor signaling. Beyond the biological studies, theoretical chemical techniques support the in-depth analysis of structure – activity/function relationships.

Peptide and protein synthesis

The therapeutic value of opioid type drugs is high, but their side-effects and their misuse cause serious medical and social problems. The diverse physiological effects of the opiates and opioid peptides are mediated by the heterogeneous G-protein coupled opioid receptors. However, the lack of their high-resolution structure restricts the design of opioids free of side effects. As an alternative, structure-activity studies of ligand sets with well defined three dimensional structures made it feasible to determine the spatial arrangement of pharmacophore elements, and in an iterative way to find more selective ligands with high affinity. Such structure-activity studies were performed on unnatural amino acid containing endomorphin, deltorphin and TIPP analogues. It turned out that the gauche(–) orientation of the Phe4 side chain is important for binding to the μ-opioid receptor. Furthermore, bending of the tetrapeptide backbone was achieved by incorporating a spirocyclic benzazepinone into the endomorphin-2 sequence, and this forced secondary structure was preferred by the μ-opioid receptor. The incorporation of alicyclic β-amino acids into the endomorphin sequences resulted in another ligand set with variable opioid receptor affinities and selectivities that depended on the absolute configuration of the substituent amino acid.

Very recent research efforts target the incorporation of site-specific covalent modifications into proteins to support their physicochemical and functional characterization, because the genome-sequencing projects have revealed hundreds of thousands of new proteins without isolating and characterizing them. Chemical synthesis alone or in combination with recombinant techniques is a powerful approach for this purpose, as it allows the covalent structure to be varied and the incorporation of non-native elements. The model system is the membrane-associated prion protein (PrP), where the chemical synthesis of fluorescent-labeled membrane anchor molecules and the total chemical synthesis of the full length PrP are explored. Different anchor mimetics are conjugated to the recombinant PrP, and the resulting semisynthetic proteins are used for in vitro and in vivo studies of PrP misfolding, the basic phenomenon during prion pathogenesis (collaboration with E. Welker, BRC, Szeged). This research will provide a new methodology for protein production and novel proteins presumably with designed properties.

Molecule structure investigation

Structural investigations with various spectroscopic and computational methods are focused on the opioid receptor – opioid ligand interactions, on the membrane embedding of semisynthetic PrP, and on the development of novel strategies for conformational analysis. Structure-activity studies of the opioid receptor ligands help refine the theory on the proper three-dimensional arrangement of pharmacophore elements. For µ-opioid ligands the simultaneous fulfillment of four structural requirements were shown to result in high affinity binding. Membrane environment studies are set up to give information in atomic details about the distribution and orientation of the anchor mimetic conjugated and lipid bilayer embedded PrP derivatives.

Our strategy development involves the theoretical reproduction of IR and VCD spectra using molecular dynamics simulations and quantum chemical calculations. The advantage of this approach is that it provides atomic resolution information about all conformational states present in the equilibrium, while other high resolution experimental techniques, such as NMR provide only time-averaged results when fast conformational transitions occur.

Radioactive labeling

In vitro or in vivo investigation of radioactively labeled high affinity and selectivity ligands is an important step in the characterization of the ligand – receptor interactions and of the pharmacological properties of an administered drug. Preferred radioligands or drugs for this purpose are H-3, C-14 and I-125 labeled compounds. The tritium and carbon isotopes are incorporated by total chemical synthesis, or in the case of tritium by reduction of halogenated or unsaturated precursor compounds. For peptides not containing either aromatic amino acids or Pro or Leu, a new tritium labeling method has been developed that allows the incorporation of tritium into Val or Ala residues of peptides by the reduction of Pen or Cys residues, respectively. The most important novel tritiated μ and δ opioid receptor selective ligands are: endomorphin 1, (Tyr-Pro-Trp-Phe-NH2), endomorphin 2, (Tyr-Pro-Phe-Phe-NH2), TAPP (Tyr-D-Ala-Phe-Phe-NH2), TIPP [Ψ] (Tyr-TicΨ[CH2-NH]Phe-Phe-NH2), Ile5,6-deltorphin II (Tyr-D-Ala-Phe-Glu-Ile-Ile-Gly-NH2), nociceptin (Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln-OH), nociceptin (1-13) amide and N,N-dimethyl-Dmt-Tic-OH. [3H]β-amyloid fragments and [3H]GnRH-III are further examples of compounds prepared for receptor research. The description of the in vitro proteolytic degradation of endomorphins was achieved with the digestion of different H-3 isotopomers of endomorphins.

Being useful research tools, some of these radioligands have become commercial products, and our radiolabeling expertise is applied in numerous contract research tasks to prepare deuterium, tritium or carbon labeled compounds.