Research - Institute of Biochemistry - Neurobiology Unit - Laboratory of Chemical Biology

Csaba TÖMBÖLY
senior research associate

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Attila BORICS senior research associate
Szabolcs DVORÁCSKÓ junior research associate
Gabriella FISER junior research associate
Argha MITRA Ph.D. student
Arijit SARKAR Ph.D. student
Judit DARUSI Ph.D. student
Éva TÓTHNÉ PAPP laboratory assistant

LABORATORY OF CHEMICAL BIOLOGY

Chemistry based research in our group is focused on the understanding of protein – small molecule interactions. The potential drug target opioid, vasopressin and cannabinoid receptors are investigated by spectroscopic, theoretical and in vitro methods to obtain structure – activity/function relatioships. Beyond the preparation of small molecules, peptides and proteins enriched in chemical information, i.e. stable isotopes, unnatural building blocks, fluorophores, radiactive labels, protein modification strategies are also developed for the preparation of membrane associated proteins.


Membrane associated proteins (PI: Cs. Tömböly)

Numerous proteins are anchored to the cell membrane via co- or post-translationally linked lipid moieties. The amidation of the protein C-terminus with a glycosylphosphatidylinositol (GPI) glycolipid is a specific protein modification resulting in the protein tethered to the extracellular leaflet of the cell membrane. It is widespread throughout eukaryotes, more than 150 human GPI-anchored proteins are known, and beyond the normal physiological functions they are associated with a range of diseases including paroxysmal nocturnal hemoglobinuria, prion diseases, carcinogenesis and sleeping sickness. The GPI-anchored proteins are able to associate temporarily with sphingolipid- and cholesterol-rich lipid rafts. Clustering of the GPI-anchored proteins may serve as platforms for diverse cellular functions, e.g. for signal transduction. The complexity of the GPIs’ structure restricts the preparation of GPI-anchored protein derivatives for structural and functional studies. An important feature of the GPI-anchored proteins is that they spontaneously reintegrate into cell membranes with the retention of the biological function. This property can be exploited for structural and functional studies, because semisynthetic lipoproteins containing GPI anchor mimetic lipids can also be exogenously introduced into cell membranes. Since cholesterol favours the interaction with sphingolipids, it has the potential to target the attached protein to lipid rafts. A series of novel cholesterol based GPI mimetics was designed and prepared, and we developed a method for the cell membrane anchoring of proteins with these cholesterol anchors. Its usefulness was demonstrated in a proof-of-concept study where the red fluorescent protein mCherry and the full length prion protein were anchored to SHSY-5Y neuroblastoma cell membranes. Atomic details of the distribution and orientation of the GPI anchor mimetic cholesterol derivatives in a model lipid bilayer were obtained by theoretical methods.


Vasopressin receptors (PI: Cs. Tömböly)

The pituitary peptide hormone arginine vasopressin is responsible for various physiological effects including the regulation of the blood pressure, diuresis and the ACTH secretion. Besides, AVP has multiple effects on the regulation of memory, synaptic transmission, body temperature, anxiety and depression. The physiological effects of AVP are connected with the activation of the G-protein coupled receptors V1a, V1b, V2 and OT. In order to decipher the distribution of the vasopressin receptors, especially the V1b receptors in the brain, desamino-[Leu4, Lys8]vasopressin, the only V1b selective agonist was prepared in tritium labelled form. Then our French collaborators evidenced the presence of V1b receptors in the rat pituitary, hippocampus and cortex by binding on membrane preparations, and they also applied the V1b-selective radioligand for autoradiography studies. The corresponding fluorescent V1B selective peptide ligands were also prepared and it gave us outstanding results on cell lines expressing V1B receptors and on rat pituitary primary cultures.


Cannabinoid receptors (PI: A. Keresztes)

Our current cannabinoid receptor-related research primarily focuses on the development and in vitro/in vivo pharmacological characterization of opioid-CB1 bivalent ligands and other synthetic cannabinoids. The pharmacology and signaling properties of opioid-CB1 bivalent ligands and their in vivo efficacies in animal models of inflammation and neuropathic pain are investigated by using multidisciplinary approaches. We also synthesize and pharmacologically characterize a heterocyclic cannabinoid ligand library, predominantly with high affinity and selectivity towards the CB2 receptor. We believe that our results will contribute to a better understanding of the pharmacology of opioid-CB1 receptor dimers and may help clarifying the role of CB2 receptors in the CNS. We also believe that our results may help the development of better analgesics with moderate to low side-effect profile for chronic pain incidences.


Opioid receptors (PI: A. Borics)

Structural investigations with spectroscopic and computational methods are primarily focused on opioid receptor – opioid ligand interactions, and the mechanism of activation. Additionally, the membrane embedding of lipoproteins, and the membrane transport of steroid hormones are also modelled. Ligand-based investigation of the structural requirements of µ-opioid receptor binding has shown that the proper three-dimensional arrangement of pharmacophore elements is simultaneously controlled by four structural parameters. Based on the high resolution structure of the opioid receptors, molecular dynamics studies in membrane environment are performed for the proper representation of the receptor protein. Molecular dynamics simulations of the ligand-receptor complex are executed in order to reveal structural changes caused by ligand binding, leading to signal transduction events. Furthermore, theoretical investigations of interactions between the receptor and the different types of ligands at the atomic level are aimed the exact determination of the binding modes and epitopes. This information is then used for the design of new, potent peptide ligands, relevant in the research field of novel potential analgesics.


Projects for students interested in joining our lab:

The general applicability of our fluorescent cholesterol derivatives as GPI mimetics is restricted, because the conjugation requires a C-terminal Cys residue of the target protein. Furthermore, if the target protein contains additional Cys residues, then the specific C-terminal conjugation of the anchor molecule is doubtful. To overcome these limitations and to achieve the widespread application of cholesteryl lipoproteins in cell membrane anchoring and in lipid raft targeting as GPI-anchored protein mimetics, the strategy will be improved by introducing bioorthogonal functional group pairs into the proteins’ C- or N-terminus and into the probed cholesterol anchors.

Tritium labeling of cholesterol anchors: preparation of tools to quantitate the cell membrane concentration of the cholesteryl anchors and that of the cholesteryl lipoproteins after exogenous addition.

Introduction of novel fluorogenic probes to the cholesterol anchors, e.g. probes with fluorescence depending on pH or on metal ion concentration. These turn-on fluorescent probes are extremely useful, because cellular processes, such as internalization pathways or endocytosis of the membrane anchored protein can be visualized.

The total synthesis of the prion protein is performed in order to enrich the chemical information content (fluorophores, stable isotopic labels, unnatural amino acids, posttranslational modifications or conformational constraints) of the protein to support the spectroscopic and functional studies. Native chemical ligation and different non-Cys ligation strategies are applied and developed for this purpose.

Given the lack of selective labeled V1a receptor ligands so far, new fluorescent V1a selective vasopressin analogues will be produced and characterized on rat, mouse and human receptors expressed in model cells.

Glioblastomas, highly malignant tumors of the CNS, express cannabinoid receptors at high level making them susceptible to cannabinoid-based anti-cancer therapy. Though, cannabinoids are currently used to alleviate chemotherapy- and radiotherapy-induced side-effects and cancer-associated pain, several studies have pointed to that cannabinoid ligands display anti-tumor activity in several types of cancers. However, the underlying molecular mechanisms of the cannabinoid-driven cell death remained elusive. The role of death-receptors and their possible synergistic effects with cannabinoids are not well understood. Consequently, a deeper molecular investigation should be necessary for future use since these effects proved to be somewhat selective for tumor cells and these drugs showed low toxicity in experimental animal models. The preparation of a cannabinoid ligand library is in progress, and it will be followed by the investigation of the pharmacological and signaling properties.

The recently published high-resolution three-dimensional structure of the opioid receptors gives scope for the generation of an active receptor-ligand complex model, which could give an insight into the structural mechanism of G-protein coupled receptor binding and activation.

The possibility and circumstances of passive cell membrane transport of steroid hormones depending on the lipid composition of the membrane is studied utilizing molecular dynamics simulations.

Selected publications

Schäfer B, Orbán E, Borics A, Huszár K, Nyeste A, Welker E, Tömböly Cs (2013) “Preparation of Semisynthetic Lipoproteins with Fluorescent Cholesterol Anchor and Their Introduction to the Cell Membrane with Minimal Disruption of the Membrane.” Bioconj. Chem. 24, 1684−1697.

Borics A, Mallareddy JR, Tímári I, Kövér KE, Keresztes A, Tóth G (2012) “The Effect of Pro(2) Modifications on the Structural and Pharmacological Properties of Endomorphin-2.” J. Med. Chem. 55, 8418-8428.

Borics A, Gach K, Fichna J, Sobolewski D, Tóth G, Janecka A (2012) “Structural comparison of endomorphin-2 and its conformationally restricted analog Tyr-Pro-Phe-Phe-NH(2).” Central Eur. J. Chem. 10, 172-179.

Tumati S, Largent-Milnes TM, Keresztes A, Ren JY, Roeske WR, Vanderah TW, Varga EV (2012) “Repeated morphine treatment-mediated hyperalgesia, allodynia and spinal glial activation are blocked by co-administration of a selective cannabinoid receptor type-2 agonist.” J. Neuroimmunol. 244, 23-31.

Tumati S, Largent-Milnes TM, Keresztes AI, Yamamoto T, Vanderah TW, Roeske WR, Hruby VJ, Varga EV (2012) “Tachykinin NK1 receptor antagonist co-administration attenuates opioid withdrawal-mediated spinal microglia and astrocyte activation.” Eur. J. Pharmacol. 684, 64-70.

Petrovszki Z, Kovács G, Tömböly Cs, Benedek G, Horváth G (2012) “The Effects of Peptide and Lipid Endocannabinoids on Arthritic Pain at the Spinal Level.” Anesthesia and Analgesia 114, 1346-1352.

Ballet S, Feytens D, Buysse K, Chung NN, Lemieux C, Tumati S, Keresztes A, Van Duppen J, Lai J, Varga E, Porreca F, Schiller PW, Broeck JV, Tourwe D (2011) “Design of Novel Neurokinin 1 Receptor Antagonists Based on Conformationally Constrained Aromatic Amino Acids and Discovery of a Potent Chimeric Opioid Agonist-Neurokinin 1 Receptor Antagonist.” J. Med. Chem. 54, 2467-2476.

De Wachter R, de Graaf C, Keresztes A, Vandormael B, Ballet S, Tóth G, Rognan D, Tourwe D (2011) “Synthesis, Biological Evaluation, and Automated Docking of Constrained Analogues of the Opioid Peptide H-Dmt-D-Ala-Phe-Gly-NH(2) Using the 4- or 5-Methyl Substituted 4-Amino-1,2,4,5-tetrahydro-2-benzazepin-3-one Scaffold.” J. Med. Chem. 54, 6538-6547.

Keresztes A, Birkás E, Páhi A, Tóth G, Bakota L, Gulya K, Szűcs M (2011) “Pharmacology of a new tritiated endomorphin-2 analog containing the proline mimetic cis-2-aminocyclohexanecarboxylic acid.” Peptides 32, 722-728.

Mallareddy JR, Borics A, Keresztes A, Kövér KE, Tourwe D, Tóth G (2011) “Design, Synthesis, Pharmacological Evaluation, and Structure-Activity Study of Novel Endomorphin Analogues with Multiple Structural Modifications.” J. Med. Chem. 54, 1462-1472.

Vandormael B, De Wachter R, Martins JC, Hendrickx PMS, Keresztes A, Ballet S, Mallareddy JR, Tóth F, Tóth G, Tourwe D (2011) “Asymmetric Synthesis and Conformational Analysis by NMR Spectroscopy and MD of Aba- and alpha-MeAba-Containing Dermorphin Analogues.” CHEMMEDCHEM 6, 2035-2047.

Keresztes A, Borics A, Tóth G (2010) “Recent advances in endomorphin engineering.” CHEMMEDCHEM 5, 1176-1196.

Borics A, Tóth G (2010) “Structural comparison of mu-opioid receptor selective peptides confirmed four parameters of bioactivity.” J. Mol. Graph. Model. 28, 495-505.

De Wachter R, Brans L, Ballet S, Van den Eynde I, Feytens D, Keresztes A, Tóth G, Urbanczyk-Lipkowska Z, Tourwe D (2009) “Influence of ring substitution on the conformation and beta-turn mimicry of 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one peptide mimetics.” Tetrahedron 65, 2266-2278.

Bojnik E, Farkas J, Magyar A, Tömböly Cs, Gündüz O, Borsodi A, Corbani M, Benyhe S (2009) “Selective and high affinity labeling of neuronal and recombinant nociceptin receptors with the hexapeptide radioprobe [(3)H]Ac-RYYRIK-ol.” Neurochem. Int. 55, 458-466.

Tömböly Cs, Ballet S, Feytens D, Kövér KE, Borics A, Lovas S, Al-Khrasani M, Fürst Z, Tóth G, Benyhe S, Tourwe D (2008) “Endomorphin-2 with a beta-turn backbone constraint retains the potent mu-opioid receptor agonist properties.” J. Med. Chem. 51, 173-177.

Keresztes A, Szűcs M, Borics A, Kövér KE, Forró E, Fülöp F, Tömböly Cs, Péter A, Páhi A, Fábian G, Murányi M, Tóth G (2008) “New endomorphin analogues containing alicyclic beta-amino acids: Influence on bioactive conformation and pharmacological profile.” J. Med. Chem. 51, 4270-4279.

Szemenyei E, Barna I, Mergl Z, Keresztes A, Darula Z, Kató E, Tóth G, Rónai AZ (2008) “Detection of a novel immunoreactive endomorphin 2-like peptide in rat brain extracts.” Regulatory Peptides 148, 54-61.

Botros M, Johansson T, Zhou Q, Lindeberg G, Tömböly Cs, Tóth G, Le Greves P, Nyberg F, Hallberg M (2008) “Endomorphins interact with the substance P (SP) aminoterminal SP1-7 binding in the ventral tegmental area of the rat brain.” Peptides 29, 1820-1824.

Knutter I, Hartrodt B, Tóth G, Keresztes A, Kottra G, Mrestani-Klaus C, Born I, Daniel H, Neubert K, Brandsch M (2007) “Synthesis and characterization of a new and radiolabeled high-affinity substrate for H+/peptide cotransporters.” FEBS J. 274, 5905-5914.

Tóth G, Ioja E, Tömböly Cs, Ballet S, Tourwe D, Péter A, Martinek T, Chung NN, Schiller PW, Benyhe S, Borsodi A (2007) “Beta-methyl substitution of cyclohexylalanine in Dmt-Tic-Cha-Phe peptides results in highly potent delta opioid antagonists.” J. Med. Chem. 50, 328-333.

Ballet S, de Wachter R, van Rompaey K, Tömböly Cs, Feytens D, Tóth G, Quartara L, Cucchi P, Meini S, Tourwe D (2007) “Bradykinin analogs containing 4-amino-2-benzazepin-3-one the scaffold at the C-terminus.” J. Pept. Sci. 13, 164-170.