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

Csaba TÖMBÖLY
senior research associate

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Attila BORICS
senior research associate

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Szabolcs DVORÁCSKÓ 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 and structural mechanisms of protein function. The potential drug target opioid, cannabinoid, vasopressin and sigma receptors are investigated by spectroscopic, theoretical and in vitro methods to obtain structure – activity/function relationships. Beyond the preparation of small molecules, peptides and proteins enriched in chemical information, i.e. stable isotopes, unnatural building blocks, fluorophores, radioactive labels, protein modification strategies are also developed for the preparation of membrane associated proteins.

Research directions

Membrane associated proteins, cannabinoid, vasopressin and sigma receptors (PI: Cs. Tömböly)

1. The exogenous introduction of semisynthetic lipoproteins into the plasma membrane is a cell surface modification method that offers strict control over the composition of the engineered cell membrane. Therefore, it is an accurate approach for the investigation of membrane protein dynamics, for visualizing the cellular traffic of membrane proteins, and for improving the efficacy of biologically active proteins and cell based therapeutics. In order to deliver proteins into the plasma membrane with synthetic anchor molecules, we have developed a mild method and demonstrated that a cholesterol anchor and the attached protein had been incorporated into the membrane of live cells in stoichiometric ratio. Our cholesterol anchor is water soluble, non-toxic and appropriate for live cell cultures without membrane perturbing detergents. Additional advanced feature of our anchor is a fluorescent or radioactive reporter in the headgroup that facilitates the direct imaging of the attached protein. Since cholesterol favours the interaction with sphingolipids, it has the potential to target the attached protein to lipid rafts. The usefulness of our anchors was demonstrated in a proof-of-concept study where the red fluorescent protein mCherry and the full length prion protein were anchored to SH-SY5Y 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.

2. Synthetic compounds that interact with the cannabinoid receptors (CBR) or modulate the endocannabinoid signaling have therapeutic potential in the treatment of neurological diseases, psychiatric disorders, neuropathic pain and obesity. On one hand we focus on the development of opioid-CB1 bivalent ligands and other synthetic cannabinoids with the aim to develop better analgesics with moderate to low side-effect profile for chronic pain incidences. The pharmacology and signaling properties of the novel ligands and their in vivo efficacies in animal models of inflammation and neuropathic pain are investigated.On the other hand, in our ongoing collaboration with the Hungarian Institute for Forensic Sciences, several current illicit synthetic cannabinoids and their novel derivatives were structurally and pharmacologically characterized. We investigated their interactions with the CBRs and possible off-target receptors in vitro, and also their in vivo effects on the cardiovascular system. There are highly potent synthetic CB1R agonists among illicit drugs worldwide that are dangerous, occasionally lethal compounds and there is no specific antidote for the intoxication of such synthetic cannabinoids. In this context an endogenous peptide family (hemopressins or pepcans) is investigated to develop CBR-targeting peptide therapeutics without psychoactive side effects.

3. 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 contribute to the understanding of the physiological roles of this receptor family, different synthetic peptides with tritium or fluorescent reporters are prepared for autoradiography, binding and fluorescent microscopy studies.

4. The sigma-1 receptor is an intracellular receptor and chaperone which is not coupled to G-proteins and which binds ligands of diverse structure. The main physiological functions of the sigma-1 receptor include the modulation of Ca2+ signaling, the inhibition of voltage-dependent K+-channels and the release of various neurotransmitters. Since sigma-1 agonists exhibit neuroprotective effect in cellular and animal models, they represent drug candidates for the treatment of neurodegenerative diseases. We have optimized an in vitro radioligand binding method to screen diverse compound libraries for sigma-1 receptor binding and to find novel structures for pharmacological development.

The structural mechanism of G protein-coupled receptor activation (PI: A. Borics)

G protein-coupled receptors (GPCRs) represent one of the largest protein superfamily of the human genome and participate in crucial physiological functions. GPCRs are involved in many diseases, hence approximately 34% of all prescription pharmaceuticals target members of this receptor family. High-resolution structures of active and inactive state GPCRs are available but the process of transition between these states is poorly understood. We intend to identify the role of ligand-receptor interactions with respect to the electrostatic equilibrium in order to reveal general aspects of the function of this receptor family. Our investigations are based on the introduction of a new perspective, focusing on the electrostatic charge distribution within the receptor. Our most recent. unbiased, atomistic molecular dynamics simulations of the mu-opioid receptor in a physiological environment revealed that external stimulus is propagated to the intracellular surface of the receptor through subtle, concerted movements of highly conserved polar amino acid side chains along the 7th transmembrane helix. We suggest that the initiation event of GPCR activation is the shift of macroscopic polarization between the ortho- and allosteric binding pockets and the intracellular G protein-binding interface. Our future directions involve the extension of this study to further class A GPCRs and experimental verification of the above hypothesis utilizing newly designed, interaction specific ligands, mutant receptors, in vitro functional assays and STDD NMR spectroscopic measurements.

Specific projects for prospective M. Sc. and Ph. D. Students Cs. Tömböly

1. Semisynthetic protein lipidation:The general applicability of our fluorescent cholesterol derivatives as membrane anchor moieties 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 modifications and in lipid raft targeting, bioorthogonal functional group pairs that can be introduced into the C- or N-terminus of proteins and into the probed cholesterol anchors are investigated.

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

3. Receptor research: Synthetic derivatives of an endogenous peptide family (hemopressins or pepcans) are investigated in vitro and in vivo to develop cannabinoid receptor targeting compounds without psychoactive side effects.

A. Borics

1. Investigation of the activation mechanism of G protein-coupled receptors based on recently published high-resolution three-dimensional structures, using state-of-the-art molecular modeling tools and procedures.

2. Investigation of the effect of cell membrane composition and specific lipid-protein interactions on the activation mechanism of G protein-coupled receptors.

3. Design, synthesis and characterization of peptide ligand probes targeting specific pharmacophore interactions with G protein-coupled receptors.

Expertise

Semisynthesis and chemical modification of proteins

Design and synthesis of various GPCR pgands

Radioactive and fluorescent labepng of bioactive compounds

Radiopgand receptor binding techniques (kinetic, saturation and competition binding assays)

In vivo distribution studies of radiolabelled compounds

In vivo analgesic methods (von-Frey test)

Molecular simulations of complex biological systems

In sipco screening and characterization of potential drug candidates, prediction of binding affinities and pharmacophore interactions

Optical spectroscopic analysis of peptide and protein secondary structure, stabipty and interactions

Experience in forensic science of ilpcit synthetic cannabinoids

Current collaborations

A. Borics

Design, synthesis and structural and functional characterization of novel opioid receptor ligands
Anna Janecka
Medical University of Łódź, Department of Biomolecular Chemistry (Poland)

Structural characterization of small, cysteine-rich antimicrobial proteins of fungal origin
László Galgóczi, Florentine Marx, Gyula Batta, José F. Marcos
University of Szeged, Faculty of Sciences, Department of Microbiology (Hungary)
Innsbruck Medical University, Division of Molecular Biology (Austria)
University of Debrecen, Department of Organic Chemistry (Hungary)
Department of Biotechnology, Instituto de Agroquímica y Tecnología de Alimentos (Spain)

Selected publications

Mitra, A.; Sarkar, A.; Szabó, M. R.; Borics, A. Correlated motions of conserved polar motifs lay out a plausible mechanism of G protein-coupled receptor activation. https://doi.org/10.1101/2020.01.27.920769

Dvorácskó, Sz; Keresztes, A.; Mollica, A.; Stefanucci, A.; Macedonio, G.; Pieretti, S.; Zádor, F.; Walter, F.R.; Deli, M.A.; Kékesi, G.; Bánki, L.; Tuboly, G.; Horváth, Gy.; Tömböly, Cs. Preparation of bivalent agonists for targeting the mu opioid and cannabinoid receptors. EUR J. MED. CHEM. 2019, 178, 571-588.

M.P. Dimmito, A. Stefanucci, S. Pieretti, P. Minosi, Sz. Dvorácskó, Cs. Tömböly, G. Zengin, A. Mollica “Discovery of Orexant and Anorexant Agents with Indazole Scaffold Endowed with Peripheral Antiedema Activity.” Biomolecules 9(9), 492 (2019).

Corbani, M., Marir, R., Trueba, M., Chafai, M., Vincent, A., Borie, A.M., Desarménien, M.G., Ueta, Y., Tömböly, Cs., Olma, A., Manning, M., Guillon, G. “Neuroanatomical distribution and function of the vasopressin V1B receptor in the rat brain deciphered using specific fluorescent ligands.” Gen. Comp. Endocrinol. 258, 15-32 (2018).

Adamska-Bartlomiejczyk, A.; Janecka, A.; Szabó, M. R.; Cerlesi, M. C.; Calo, G.; Kluczyk, A.; Tömböly, Cs.; Borics, A. Cyclic mu-opioid receptor ligands containing multiple N-methylated amino acid residues BIOORG. MED. CHEM. LETT. 2017, 1644-1648.

Schäfer B.; Orbán, E.; Fiser, G.; Marton, A.; Vizler, C.; Tömböly, Cs. Semisynthesis of membrane-anchored cholesteryl lipoproteins on live cell surface by azide–alkyne click reaction. TETRAHEDRON LETT. 2016, 57, 868-873.

Váradi, A.; Marrone, G. F.; Palmer, T. C.; Narayan, A.; Szabó, M. R.; Le Rouzic, V.; Grinnell, S. G.; Subrath, J. J.; Warner, E.; Kalra, S.; Hunkele, A.; Pagirsky, J.; Eans, S. O.; Medina, J. M.; Xu, J.; Pan, Y. X.; Borics, A.; Pasternak, G. W.; McLaughlin, J. P.; Majumdar, S. Mitragynine/Corynantheidine Pseudoindoxyls As Opioid Analgesics with Mu Agonism and Delta Antagonism, Which Do Not Recruit beta-Arrestin-2. J. MED. CHEM. 2016, 59, 8381-8397.

Dvorácskó, Sz.; Tömböly, Cs.; Berkecz, R.; Keresztes, A. Investigation of receptor binding and functional characteristics of hemopressin(1-7) NEUROPEPTIDES 2016, 58, 15-22.

Dvorácskó, Sz.; Stefanucci, A.; Novellino, E.; Mollica, A. The design of multitarget ligands for chronic and neuropathic pain FUTURE MED. CHEM. 2015, 7, 2469-2483.

Orbán, E.; Kele, Z.; Tömböly, C. Tritium labelling of a cholesterol amphiphile designed for cell membrane anchoring of proteins. J. LABELLED COMPD. RAD. 2015, 58, 7-13.

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.