Professor Ioannis P. Gerothanassis PhD, DSc

H.F.R.I. 2019

H.F.R.I. 2019

GENERAL PROJECT INFORMATION

CATEGORY II
SCIENTIFIC AREA Natural Sciences
PROJECT TITLE Investigation of interactions of unsaturated fatty acids with albumin and the anti-apoptotic protein Bcl-2 with emphasis in the use of NMR methodologies at a cellular level (in-cell NMR)
PRINCIPAL INVESTIGATOR Ioannis P. Gerothanassis
HOST INSTITUTION University of Ioannina
– PROJECT STARTING DATE 10/6/2020
PROJECT DURATION 36 months
APPROVED BUDGET 180.000 €

WP Number: 1

WP Objectives: Fast screening of bioactive unsaturated lipids interactions with bovine/human serum albumin and the anti-apoptotic Bcl-2 proteins in vitro.

Tasks: (i) Characterization of the epitopes and the binding constants of FFAs as well as their binding sites to the BSA protein using STD-NMR. Determination of the preferred conformational changes of FFAs upon binding to BSA with the use of Tr-NOESY NMR.

(ii) Characterization of the epitopes and the binding constants of FFAs as well as their binding sites to the Bcl-2 protein using STD-NMR. Determination of the preferred conformational changes of FFAs upon binding to Bcl-2 with the use of Tr-NOESY NMR.

Task (i): Characterization of the epitopes and the binding constants of FFAs as well as their binding sites to the BSA protein using STD-NMR. Determination of the preferred conformational changes of FFAs upon binding to BSA with the use of Tr-NOESY NMR.

(a) Conformational investigation of polyunsaturated FFAs in solution.

  1. A DFT study of the 1H- and 13C-NMR chemical shifts of geometric isomers of 18:2 ω-7 conjugated linoleic acid (CLA) and nine model compounds is presented, using five functionals and two basis sets. The results are compared with available experimental data from solution NMR. The experimental 1H chemical shifts exhibit highly diagnostic resonances due to the olefinic protons of the conjugated double bonds. The “inside” olefnic protons of the conjugated double bonds are deshielded than those of the “outside” protons. Furthermore, in the cis/trans isomers the signals of the cis bonds are more deshielded than those of the trans bonds. These regularities of the experimental 1H chemical shifts of the olefinic protons of the conjugated double bonds are reproduced very accurately for all functionals and basis sets used. We conclude that proton NMR chemical shifts are more discriminating than carbon and DFT calculations can provide a valuable tool: (i) for the accurate prediction of 1H-NMR chemical shifts even with less demanding functionals and basis sets; (ii) for the unequivocal identification of geometric isomerism of CLAs that occur in nature, and (iii) to derive high resolution structures in solution. The research results were published: “DFT Calculations of 1H- and 13C-NMR Chemical Shifts of Geometric Isomers of Conjugated Linoleic Acid (18:2 ω-7) and Model Compounds in Solution”. T. Venianakis, C. Oikonomaki, M.G. Siskos, P.C. Varras, A. Primikyri, E. Alexandri, I.P. Gerothanassis, Molecules, 25(16), 3660, https://doi.org/10.3390/molecules25163660 (2020).

ADMINISTRATION EXPERIENCE

  • Academic Administration
    1. Chairman of the Greek Rectors Conference 2010.
    2. Rector of the University of Ioannina (UoI): 2006-2010.
    3. Vice Rector of the UoI: 2003-2006.
    4. Chairman of the Dept. of Architecture Engineering, UoI: 2009-2010.
    5. Chairman of the Dept. of Plastic Arts and Art Sciences, UoI: 2007-2010.
    6. Chairman of the Dept. of Biological Applications & Technologies, UoI: 2004-2005.
    7. Chairman of the Dept. of Chemistry, UoI: 2000-2002 and 2002-2004.
    8. Vice Chairman of the Dept. of Chemistry, UoI: 1999-2000.
    9. Chairman of the Section of Organic Chemistry and Biochemistry, Dept. of Chemistry, UoI: 1997-1998, 1998-1999 and 1999-2000.
DFT structure
DFT structure of the low energy conformer C of the (9Z.11E)-CLA in the gas-phase, with a discrete solvation molecule of DMSO on the basis of 1H-NMR
  1. DFT study of the 1H NMR chemical shifts, δ(1H), of geometric isomers of 18:3 conjugated linolenic acids (CLnAs), hexadecatrienyl pheromones, and model triene-containing compounds is presented, using standard functionals (B3LYP and PBE0) as well as corrections for dispersion interactions (B3LYP-D3, APFD, M06–2X and ωB97XD). The results are compared with literature experimental δ(1H) data in solution. The closely spaced “inside” olefinic protons are significantly more deshielded due to short-range through-space H…H steric interactions and appear close to or even beyond δ-values of aromatic systems. Several regularities of the computational δ(1H) of the olefinic protons of the conjugated double bonds are reproduced very accurately for the lowest-energy DFT-optimized single conformer for all functionals used and are in very good agreement with experimental δ(1H) in solution. Examples are provided of literature studies in which experimental resonance assignments deviate significantly from DFT predictions and, thus, should be revised. We conclude that DFT calculations of 1H chemical shifts of trienyl compounds are powerful tools (i) for the accurate prediction of δ(1H) even with less demanding functionals and basis sets; (ii) for the unequivocal identification of geometric isomerism of conjugated trienyl systems that occur in nature; (iii) for tackling complex problems of experimental resonance assignments due to extensive signal overlap; and (iv) for structure elucidation in solution. The research results were published: “DFT Calculations of 1H NMR Chemical Shifts of Geometric Isomers of Conjugated Linolenic Acids, Hexadecatrienyl Pheromones and Model Triene-Containing Compounds: Structures in Solution and Revision of NMR Assignments”. T. Venianakis, Ch. Oikonomaki, M.G. Siskos, A. Primikyri, I.P. Gerothanassis, Molecules, 26, 3477. https://doi.org/10.3390/molecules26113477 (2021).
DFT calculations of 1H NMR
DFT calculations of 1H NMR chemical shifts of geometric isomers of trienyl compounds result in 3D structures in solution and revision of literature NMR assignements
  1. Structures of low-energy conformers of α-linolenic acid (ALA;18:3-cis-9,12,15 ω-3), eicosapentaenoic acid (EPA;20:5-cis 5,8,11,14,17 ω-3) and docosahexaenoic acid (DHA;22:6-cis 4,7,10,13,16,19 ω-3) in the liquid state, based on NMR and density functional theory calculations of 1H NMR chemical shifts, are provided. A critical discussion of the J. Mol. Liq. 314 (2020) 113376 article is also presented on a number of issues related to: (i) the method of assignment of 1H and 13C chemical shifts of ALA, EPA and DHA; (ii) attribution of NMR signals of impurities to those of EPA and (iii) the conclusion of an unusually strong electron 13C and 1H shielding of =HC–CH2–CH= protons and carbons, due to an asymmetry in a twist between the two ends, which results in torque at the methylene site near the geometric center. The present data demonstrate that the folding effects of the chain as a function of the fragment number (CH=CH–CH2)n, were n=3, 5 and 6 for ALA, EPA and DHA, do not result in unusually strong electron 1H and 13C shielding. The research results were published: “Molecular models of three ω-3 fatty acids based on NMR and DFT calculations of 1H NMR chemical shifts”. T. Venianakis, A. Primikyri, E. Alexandri, G. Papamokos, I.P. Gerothanassis, J. Mol. Liq.342, 117460 (2021); “NMR and DFT studies of monounsaturated and ω-3 polyunsaturated free fatty acids in the liquid state reveal a novel atomistic structural model of DHA”. T. Venianakis, M. Siskos, G. Papamokos* and I.P.  Gerothanassis*, J. Mol. Liq., 376, 121459 https://doi.org/10.1016/j.molliq.2023.121459 (2023).
structure of DHA 1
structure of DHA 2

Figure: Perspectives (a) and (b) of the energy minimized helical structure of DHA in the monomeric state. In (b), the hydrogen atoms are not shown.

Structural folding parameters of the DFT

Figure: Structural folding parameters of the DFT minimum energy conformers of ALA (a), EPA (b) and DHA (c).

(b) Determination of preferred conformational changes of mono-unsaturated and poly-unsaturated FFAs upon binding to serum albumin.

  1. (i) An approach based on the combined use of saturation transfer difference (STD), Tr-NOESY and Interligand NOEs for PHArmacophore Mapping (INPHARMA) NMR techniques and docking calculations is reported, for the first time, for mapping interactions and specific binding sites of caproleic acid (10:1 cis-9), oleic acid (18:1 cis-9), linoleic acid (18:2 cis-9,12) and linolenic (18:3, cis-9,12,15) free fatty acids (FFAs) with non-labelled serum albumin (BSA/HSA). Significant negative interligand NOEs between the FFAs and the drugs ibuprofen and warfarin, of known X-ray binding sites, through competition experiments were observed. This demonstrates efficient spin-diffusion process mediated by the protons of the receptor (BSA/HSA) binding pocket and, thus, atomic level structural recognition of the unsaturated fatty acids irrespective of the length of the carbon chain and the number of the cis allylic double bonds. The interligand NOEs and docking calculations were interpreted in terms of significant folding of the bis allylic region and the presence of two orientations of the FFAs in the warfarin binding site, due to two anchoring groups of polar amino acids. This conformational flexibility is the reason that the location and conformational states of the FFAs in the binding site of warfarin could not be determined accurately, despite numerous X-ray structural studies. Furthermore, the computational strategy of the present work was shown to be an excellent tool for accurate description of electrostatic and hydrogen bonding interactions between polyunsaturated FFAs and serum albumin. The combined use, therefore, of STD, Tr-NOESY and INPHARMA NMR and docking calculations allows investigation of albumin – FFAs interactions without the need of enriched albumin with 15N and 13C isotopes or FFAs selectively enriched with 13C and, thus, may find promising applications in the field of protein-lipid recognition research. The research results were published in: “NMR and computational studies reveal novel aspects in molecular recognition of unsaturated fatty acids with non-labeled serum albumin”. E. Alexandri, A. Primikyri, G. Papamokos, T. Venianakis, V.G. Gkalpinos, A.G. Tzakos, A. Karydis-Messinis, D. Moschovas, A. Avgeropoulos and I.P. Gerothanassis, FEBS Journal,289, 5617-5636 https://doi.org/10.1111/febs.16453 (2022).
Selected regions of inter-ligand 2D Tr-NOESY NMR spectra of: (A) α-linolenic acid

Figure: Selected regions of inter-ligand 2D Tr-NOESY NMR spectra of: (A) α-linolenic acid (ALA) (2.5 mM) with HSA (25 μΜ) in 50 mM PBS buffer in D2O with 10% DMSO-d6 after the addition of warfarin (W) (2.5 mM). (B) warfarin (W) (2.5 mM) with HSA (20 μΜ) in 50 mM PBS buffer in D2O with 10% DMSO-d6 after the addition of α-linolenic acid (ALA) (2.5 mM) (mixing time = 300 ms, T = 310 K, number of scans = 112, experimental time = 17h 19 min). The red cross-peaks denote inter-ligand NOE connectivities.

Poses with best scores for binding site 7 of HSA with: a. caproleic acid

Figure:Poses with best scores for binding site 7 of HSA with: a. caproleic acid, b. oleic acid, c. linoleic acid, d.α-linolenic acid, e. warfarin; f. Superposition of the four FFAs; for α-linolenic acid, spatial deviation is observed with respect to the rest of the FFAs.

  1. The interaction of 9/10-nitro-oleic acid (NO2-OA) with human serum albumin (HSA) was investigated. The molecular mechanism of the biological action of NO2-OA is to our knowledge based on a reversible covalent reaction–Michael addition of nucleophilic amino acid residues of proteins. Since HSA is an important fatty acid transporter, a key question is whether NO2-OA can bind covalently or non-covalently to HSA, similarly to oleic acid (OA), which can interact with the FA1-FA7 binding sites of the HSA molecule. 1H NMR studies and competition analysis with OA and the drugs ibuprofen and warfarin were used to investigate a potential non-covalent binding mode. NO2-OA/HSA binding was confirmed to compete with warfarin for FA-7 with significantly higher affinity. NO2-OA competes with ibuprofen for FA-3 and FA-6, however in contrast to the situation with warfarin, the binding affinities are not significantly different. The described interactions are based exclusively on non-covalent binding. No covalent binding of NO2-OA to HSA was detected by MS/MS. More detailed studies based on MALDI-TOF-MS and Ellman’s assay indicated that HSA can be covalently modified in the presence of NO2-OA to a very limited extent. It was also shown that NO2-OA has higher affinity to HSA than that of OA. The research results were published:“Serum Albumin as a Primary Non-covalent Binding Protein for Nitro-oleic Acid”. LenkaHernychová; Eleni Alexandri, Andreas G. Tzakos; M. Zatloukalová, A, Primikyri, I.P. Gerothanassis, L. Uhrik, M. Šebela, D. Kopečný, L. Jedinak, J. Vacekc, Int. J. Biomol. Macromol., 203, 116-129 https://doi.org/10.1016/j.ijbiomac.2022.01.050 (2022).
gerothanassis ioannis
  1. Medium- and long-chain saturated and unsaturated free fatty acids (FFAs) are known to bind to human serum albumin (HSA), the main plasma carrier protein. Atomic-level structural data regarding the binding mode in Sudlow’s sites I (FA7) and II (FA4, FA3) of the polyunsaturated ω-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), however, are largely unknown. Herein, we report the combined use of saturation transfer difference (STD) and Interligand NOEs for Pharmacophore Mapping (INPHARMA) NMR techniques and molecular docking calculations to investigate the binding mode of DHA and EPA in Sudlow’s sites I and II of HSA. The docking calculations and the significant number of interligand NOEs between DHA and EPA and the drugs warfarin and ibuprofen, which are stereotypical ligands for Sudlow’s sites I and II, respectively, were interpreted in terms of competitive binding modes and the presence of two orientations of DHA and EPA at the binding sites FA7 and FA4. The exceptional flexibility of the long-chain DHA and EPA and the formation of strongly folded structural motives are the key properties of HSA–PUFA complexes. The research results were publiched:“Molecular Basis for the Selectivity of DHA and EPA in Sudlow’s Drug Binding Sites in Human Serum Albumin with the Combined Use of NMR and Docking Calculations”. E. Alexandri, T. Venianakis, A. Primikyri*, G. Papamokos* and I.P. Gerothanassis*, Molecules, 28, 3724 https://doi.org/10.3390/molecules28093724 (2023).
Poses for binding site FA7 of HSA with DHA (a) and EPA
Figure. Poses for binding site FA7 of HSA with DHA (a) and EPA (b) for the two anchoring sites(up and down) and their superpositions (c).

Task (ii): Characterization of the epitopes and the binding constants of FFAs as well as their binding sites to the Bcl-2 protein using STD-NMR. Determination of the preferred conformational changes of FFAs upon binding to Bcl-2 with the use of Tr-NOESY NMR.

We investigated in detail the in vitro interaction profile of the FFA DHA with the recombinant Bcl-2 protein. To determine the binding interface of DHA we employed STD NMR methodology. In the STD spectrum we observed that DHA demonstrated a direct interaction with the protein with all the protons participating in this interaction. Competition experiments were performed with the selective inhibitor of Bcl-2 protein, ABT-263, that mimics the BH3 domain of the Bcl-2 family and binds with high affinity (nM). ABT-263 was added in the mixture of DHA and Bcl-2 in a molar ratio 1:1 with the protein and 1:40 with DHA. Interestingly, we observed competition as expected for a such high affinity inhibitor. The experimental results are under evaluation with docking calculations which will be published in the international literature.

WP Number: 2

Objectives – Description of Work: Study of structural and conformational properties of lipid interactions with the target protein, Bcl-2, at the cellular level using in-cell NMR.

Tasks: (i) Cell culture and cell preparation for NMR analysis.

(ii) Western blot analysis of Bcl-2.

(iii) STD in-cell NMR experiment.

(iv) Tr-NOESY in-cell NMR experiment.

The research progress will be updated soon.

WP Number: 3

Objectives – Description of Work: Investigation of in vitro cytotoxicity in cancer cell lines.

Tasks: Cytotoxic activity of FFAs will be tested against: (i) Human T-cell leukemia Jurkat Bcl-2 cell line overexpressing Bcl-2 and (ii) the corresponding Jurkat Puro control cell line.

The research progress will be updated soon.

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Biogranic chemistry Ioannis P. Gerothanassis

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