LMA addresses issues on rational design of novel drugs by applying:

conformational analysis,
biophysical studies and
theoretical calculations.

Members

Research Staff
Dr. Thomas M. Mavromoustakos, Research Director

Dr. George Bonas, Special Functional Scientist B' (currently occupied in GSRT)
Dr. Maria Zervou, Special Functional Scientist C'
Eleni Siapi, MSc., Special Technical Scientist
Dr. Nikolaos-Ploutarxos Benetis (GSRT ENTER, 2003-2005)
Dr. Ioanna Kyrikou (GSRT EPAN YB/76, 2004-2007)
Dr. Panagiotis Zoumpoulakis (GSRT EPAN YB/76, 2004-2007)
Agnes Kapou, MSc., Ph.D. student
Efthimia Mantzourani, MSc , Ph.D. student (GSRT EPAN YB/60 2003-2006)
Michael Smyrniotakis, MSc., Ph.D. student (GSRT 02 PRAXE 173)
Eirini Chalkefs, graduate student
Kostas Potamitis, graduate student
Aggeliki Politi, graduate student

Collaborating Staff
Dr. Anastasia Zoga
Styliani Geka, MSc.
Ioannis Touris, MSc.

Conformational Analysis and Molecular Modelling of Novel Bioactive Molecules
The application of rational drug design is approved to be more efficient compared to the traditional way of drug discovery since it aims to understand the molecular basis of a disease. The relationship between the conformational properties of bioactive molecules and their pharmacological profile has been well documented in the Medicinal Chemistry. NMR spectroscopy and molecular modelling consist indispensable tools in conformational studies. LMA explores the molecular basis of Hypertension and Multiple Sclerosis aiming to provide the stereoelectronic requirements for novel drugs.

High resolution NMR. The best method for determining experimentally the conformation of molecules in solution or in formations simulating more complex biological environment (micelles, vesicles, bicelles).

Molecular Modelling. The information obtained by HR-NMR in combination with molecular graphics algorithms offer a powerful approach for obtaining reliable conformations of drugs in solution or in membrane environment. This approach leads to putative bioactive conformations of drug molecules at the binding site of the receptor.

Molecular Basis of Hypertension. Superimposition of the bioactive conformation of AT1 antagonist Losartan (commercially available antihypertensive drug) with [Sar1 Tyr(OMe)4 ]AII, a peptide antagonist of the peptidormone AII which is implicated in the hypertension process. The models have derived with the combination of 2D HR-NMR spectroscopy and Molecular Modelling.

Synthesis of novel anti-hypertensive drugs
Several studies of AT1 antagonists, already approved for the treatment of hypertension as well as synthetic ones, led to the design of a new series of drugs. A patent application is already submitted and their synthesis is in progress in collaboration with University of Athens.

Running projects:

• EPAN GSRT YB/76 (2004-2007). Production of new double action (angiotensin II inhibitors with adrenergic inhibitors) anti-hypertensive drugs and overlapping of intra-intentions with angiotensin II inhibitors for the reduction of reconstruction. (Proj. coordinator: Dr. T. Mavromoustakos, NHRF)
• EPAN GSRT YB/60 (2003-2006). New directions in immunotherapy of Multiple Sclerosis with the use of novel drastic cyclic analogues of epitope 87-99 of the basic protein of myelin, alone or conjugated with mannan (Proj. coordinator: Prof. I. Matsoukas, Univ. of Patras, School of Chemistry)
• Bilateral Scientific and Technological collaboration between Greece-Slovenia ΕPΑΝ-Μ.4.3.6.1 (2003-2005). Conformational Analysis and Drug Membrane Interactions of Bioactive Compounds
• GSRT 02 PRAXE 173 (2003-2005). Production of novel anti-hypertensive drugs (Proj. coordinator: Dr. T. Mavromoustakos, NHRF)

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Drug:Membrane Interactions
Membranes, are based mainly on a bilayer arrangement of phospholipids and play an important role for the formation of cells and hence life. These membranes serve not only as a barrier, but also as a location of essential biological processes. Different types of protein receptors are located inside membranes thus an interaction between the drug and membrane has to take place.

Because of their complexity and inherent instability it is necessary to combine several experimental and computational methods in order to study the interactions of drugs with membranes. Differential Scanning Calorimetry and solid state NMR studies are performed by LMA while X-ray diffraction and Raman Spectroscopy are also used in the means of collaboration with other institutes.

Drug:membrane interactions: to comprehend on the molecular requirements that drive drug activity.
The combined use of DSC, small angle x-ray diffraction and solid state NMR spectroscopy have shown that Δ8-THC, an active compound of marihuana, anchors to the interface of lipid bilayers in order to maximize (left) the amphipathic interactions while the almost inactive analog Me-Δ8-THC(right) is located deeper in the membrane bilayer since it is governed mainly by lipophobic interactions.

Differential Scanning Calorimetry (DSC) is a thermodynamic technique suitable for studying phase transitions of various materials. DSC has been used extensively to investigate the thermotropic properties of membranes and to obtain information on the effects of drugs on membranes.

Solid State NMR Spectroscopy is the method of choice for a detailed molecular study of the drug-membrane interactions. The solid state NMR experiment can be carried out by observing the wide-line spectrum (stationary experiment) where the nucleus under observation is either introduced by isotropic labelling (e.g. 2H), or is already present in natural abundance in the system (e.g. 31P, 13C). Information from the spectra is then obtained by line-shape analysis including spectral simulation which is based on specific conformational and motional models.

Simulation of solid state spectra of membrane:drug system. Dedicated software has been developed by LMA for the simulation of experimental CP P-31 NMR broadlines. This aims to identify the interaction between certain pharmaceutical molecules with model membranes. From such a study the structural and dynamical parameters of the drug:membrane system are determined with the phospholipid lamellae in the gel or the liquid crystalline phase. The program was developed from scratch by application of basic principles.

Experimental (left) CP P-31 NMR of water dispersion of DPPC/Glu in molar ratio 80/20 and simulated (right)

Enter the Simulation Software page here

X-ray diffraction. X-ray diffraction is a very useful and direct method for characterizing materials having periodicity in their structures. Lipid bilayers can be packed into a stack of lamellae which give coherent Bragg-like reflection orders. Thus, information can be obtained about their structures (e.g., d-spacing) using Bragg's Law from small angle x-ray diffraction and about the effects of drugs on the bilayer structure (e.g., topography of the drug) from the electron density profiles analysis. Wide-angle x-ray experiments give information on the change of the polar headgroups from the gel to the liquid-crystalline phase.

Small X-ray diffraction orders of DMSO containing 1% concentration of different AT1 antagonists. Loss of intense diffraction orders, prove the incorporation of the drug in the membrane bilayers.

Raman Spectroscopy. The transition in lipid bilayers is accompanied by several structural changes in the lipids as well as systematic alterations in the bilayer geometry, but the most prominent feature is the trans-gauche isomerization taking place in the acyl chain conformation. The average number of gauche conformers indicates the effective fluidity which depends not only on the temperature, but also on perturbations due to the presence of a drug molecule intercalating between the lipids. Raman spectroscopy is a suitable technique to measure this ratio quantitatively and therefore give direct information about the effects of drugs on membrane fluidity.

Running projects:

• Bilateral Scientific and Technological Collaboration between Greece - Romania EPAN - Μ.4.3.6.1 (2003-2005). Correlation between the morphological structure and ionic transport efficiency of the bioactive components in polymeric collageneous and phospholipid membranes.
• ENTER 01ΕR-88 (2003-2005). Use of theoretical calculations for the design and synthesis of novel antihypertensive drugs. (Proj. coordinator: Dr. T. Mavromoustakos, NHRF)
  

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Docking and 3D QSAR Studies
Rational design must take into account drug-receptor interactions. This analysis comprehends on the physicochemical parameters which determine interactions between the ligand and the active site. This aids to the design of novel analogs which will possess the pharmacophoric segments suitable for specific interactions with the receptor. The theoretical activity of the designed analogue can be checked using 3D QSAR studies.

Docking of losartan in the transmembrane region of AT1 receptor. The lipid bilayer is influencing the topographical location of the ligand.

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