Group leader: Csaba Tömböly

Email: tomboly.csaba[at]brc.hu

Group website:

Group members

Name

Title

 

 

Csaba TÖMBÖLY

senior research associate

publications

CV

Attila BORICS

senior research associate

publications

CV

Szabolcs DVORÁCSKÓ

research associate

publications

CV

Argha MITRA

Ph. D. student

publications

CV

Arijit SARKAR

Ph. D. student

publications

CV

Judit DARUSI

Ph. D. student

 

CV

Ádám KÖDMÖN

Scientific administrator

 

 

Éva TÓTHNÉ PAPP

laboratory assistant

 

 

Research                                  

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

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 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.

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 that are dangerous, occasionally lethal compounds worldwide 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.

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.

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. 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. 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. 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 ligands
- Radioactive and fluorescent labeling of bioactive compounds

- Radioligand receptor binding techniques (kinetic, saturation and competition binding assays, [35S]GTPgS functional binding assays)
- In vivo distribution studies of radiolabelled compounds

- In vivo analgesic methods (von-Frey test)

- Molecular simulations of complex biological systems

- In silico screening and characterization of potential drug candidates, prediction of binding

  affinities and pharmacophore interactions
- Optical spectroscopic analysis of peptide and protein secondary structure, stability and interactions
- Experience in forensic science of illicit 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 Galgóczi1,2, Florentine Marx2, Gyula Batta3, José F. Marcos4

1University of Szeged, Faculty of Sciences, Department of Microbiology (Hungary)

2Innsbruck Medical University, Division of Molecular Biology (Austria)

3University of Debrecen, Department of Organic Chemistry (Hungary)

4Department 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.

Preparation of Semisynthetic Lipoproteins with Fluorescent Cholesterol Anchor and Their Introduction to the Cell Membrane with Minimal Disruption of the Membrane.

BIOCONJ. CHEM. 2013, 24, 1684−1697.

 

Borics A, Mallareddy JR, Tímári I, Kövér KE, Keresztes A, Tóth G.

The Effect of Pro(2) Modifications on the Structural and Pharmacological Properties of Endomorphin-2.

J. MED. CHEM. 2012, 55, 8418-8428.

 

Borics, A.; Tóth, G.

Structural comparison of mu-opioid receptor selective peptides confirmed four parameters of bioactivity.

J. MOL. GRAPH. MODEL. 2010, 28, 495-505.

 

Tömböly, Cs.; Ballet, S.; Feytens, D.; Kövér, K. E.; Borics, A.; Lovas, S.; Al-Khrasani, M.; Fürst, Z.; Tóth, G.; Benyhe, S.; Tourwe, D.

Endomorphin-2 with a beta-turn backbone constraint retains the potent mu-opioid receptor agonist properties.

J. MED. CHEM. 2008, 51, 173-177.

 

Borics, A.; Murphy, R. F.; Lovas, S. Optical spectroscopic elucidation of beta-turns in disulfide bridged cyclic tetrapeptides.

BIOPOLYMERS 2007, 85, 1-11.

 

Tóth, G.; Borics, A.

Closing of the flaps of HIV-1 protease induced by substrate binding: A model of a flap closing mechanism in retroviral aspartic proteases.

BIOCHEMISTRY 2006, 45, 6606-6614.