Maria Zervou

Structure‐Based Design of Inhibitors of the Aspartic Protease Endothiapepsin by Exploiting Dynamic Combinatorial Chemistry

Structure-based design (SBD) can be used for the design and/or optimization of new inhibitors for a biological target. Whereas de novo SBD is rarely used, most reports on SBD are dealing with the optimization of an initial hit. Dynamic combinatorial chemistry (DCC) has emerged as a powerful strategy to identify bioactive ligands given that it enables the target to direct the synthesis of its strongest binder. We have designed a library of potential inhibitors (acylhydrazones) generated from five aldehydes and five hydrazides and used DCC to identify the best binder(s). After addition of the aspartic protease endothiapepsin, we characterized the protein-bound library member(s) by saturation-transfer difference NMR spectroscopy. Cocrystallization experiments validated the predicted binding mode of the two most potent inhibitors, thus demonstrating that the combination of de novo SBD and DCC constitutes an efficient starting point for hit identification and optimization.

Losartan's molecular basis of interaction with membranes and AT1 receptor.

Physicochemical methods were used to study the thermal and dynamic changes caused by losartan in the membrane bilayers. In addition, molecular modeling was implemented to explore its topography both in membranes and AT(1) receptor. Its incorporation resulted in the modification of thermal profile of dipalmitoyl phosphatidylcholine (DPPC) bilayers in a concentration dependent way up to 20mol% as it is depicted from the combination of differential scanning calorimetry (DSC) and MAS data. In particular, the presence of losartan caused lowering of the phase transition temperature and abolishment of the pretransition. T(1) experiments revealed the location of the drug into the membrane bilayers. The use of a combination of biophysical methods along with docking experiments brought out a possible two-step mechanism which involves incorporation of losartan at the interface of membrane bilayers and diffusion in the upper parts of AT(1) receptor helices IV-VII.

13C NMR analysis of the triacylglycerol composition of Greek virgin olive oils

High‐resolution 13C NMR spectroscopy was used to study the potential of the method for the quantitative analysis of the most abundant fatty acids in the triacylglycerols of virgin olive oil samples harvested at different geographic areas of Greece. The analysis of the results was compared with data obtained with gas chromatography. Both methods showed results that differed only by 1–2 mol% of the fatty acids. NMR data provided the α/β ratio of oleic and linoleic acids. The results showed a preference for the β‐position in these unsaturated acids.

Antihypertensive drug valsartan in solution and at the AT1 receptor: conformational analysis, dynamic NMR spectroscopy, in silico docking, and molecular dynamics simulations.

The conformational properties of AT1 antagonist valsartan have been analyzed both in solution and at the binding site of the receptor. Low energy conformations of valsartan in solution were explored by NMR spectroscopy and molecular modeling studies. The NMR results showed the existence of two distinct and almost isoenergetic conformations for valsartan (cis:trans ratio around the amide bond approximately 40:60) that coalesce at the temperature range of 55-60 degrees C in agreement with previous in solution conformational analysis study (Fang et al. Magn. Reson. Chem. 2007, 45, 929-936). Quantum mechanics and ONIOM calculations revealed that the bulky valsartan substituents actually contribute to stabilization of the transition state for interconversion. In silico docking and Molecular Dynamic studies were applied to study binding of valsartan at the AT1 receptor site models, explicitly solvated and embedded in lipid bilayers and solvent molecules. These studies revealed that the majority of docked poses adopted a trans (major) conformation. Of paramount and maybe biological importance are the MD simulations results which showed that the two acidic groups of valsartan are bridged through LYS199 enabling it for multiple hydrogen bond interactions. In a lipid bilayer environment these interactions are enhanced, designating the important role of lipid bilayers for the better binding of valsartan and its stabilization at the active site.

Comparative Biophysical Studies of Sartan Class Drug Molecules Losartan and Candesartan (CV-11974) with Membrane Bilayers

The interactions of the antihypertensive AT(1) antagonists candesartan and losartan with membrane bilayers were studied through the application of DSC, Raman, and solid state (31)P NMR spectroscopies. (1)H and (13)C NMR resonances of candesartan were assigned on the basis of 1D and 2D NMR spectroscopy. A (31)P CP NMR broadline fitting methodology in combination with ab initio computations was implemented and, in conjunction with DSC and Raman results, provided valuable information regarding the perturbation, localization, orientation, and dynamic properties of the drugs in membrane models. In particular, results indicate that losartan anchors in the mesophase region of the lipid bilayers with the tetrazole group oriented toward the polar headgroup, whereas candesartan has less definite localization spanning from water interface toward the mesophase and upper segment of the hydrophobic region. Both sartan molecules decrease the mobilization of the phospholipids alkyl chains. Losartan exerts stronger interactions compared with candesartan, as depicted by the more prominent thermal, structural, and dipolar (1)H-(31)P changes that are caused in the lipid bilayers. At higher concentrations, candesartan strengthens the polar interactions and induces increased order at the bilayer surface. At the highest concentration used (20 mol %), only losartan induces formation of microdomains attributed to the flexibility of its alkyl chain. These results in correlation to reported data with other AT(1) antagonists strengthen the hypothesis that this class of molecules may approach the active site of the receptor by insertion in the lipid core, followed by lateral diffusion toward the binding site. Further, the similarities and differences of these drugs in their interactions with lipid bilayers establish, at least in part, their pharmacological properties.

Insights into the molecular basis of action of the AT1 antagonist losartan using a combined NMR spectroscopy and computational approach

The drug:membrane interactions for the antihypertensive AT1 antagonist losartan, the prototype of the sartans class, are studied herein using an integrated approach. The pharmacophore arrangement of the drug was revealed by rotating frame nuclear Overhauser effect spectroscopy (2D ROESY) NMR spectroscopy in three different environments, namely water, dimethyl sulfoxide (DMSO), and sodium dodecyl sulfate (SDS) micellar solutions mimicking conditions of biological transport fluids and membrane lipid bilayers. Drug association with micelles was monitored by diffusion ordered spectroscopy (2D DOSY) and drug:micelle intermolecular interactions were characterized by ROESY spectroscopy. The localisation of the drug in the micellar environment was investigated by introducing 5-doxyl and 16-doxyl stearic acids. The use of spin labels confirmed that losartan resides close to the micelle:water interface with the hydroxymethyl group and the tetrazole heterocyclic aromatic ring facing the polar surface with the potential to interact with SDS charged polar head groups in order to increase amphiphilic interactions. The spontaneous insertion, the diffusion pathway and the conformational features of losartan were monitored by Molecular Dynamics (MD) simulations in a modeled SDS micellar aggregate environment and a long exploratory MD run (580ns) in a phospholipid dipalmitoylphosphatidylcholine (DPPC) bilayer with the AT1 receptor embedded. MD simulations were in excellent agreement with experimental results and further revealed the molecular basis of losartan:membrane interactions in atomic-level detail. This applied integrated approach aims to explore the role of membranes in losartans pathway towards the AT1 receptor.

Conformation and Bioactivity. Design and Discovery of Novel Antihypertensive Drugs

Peptidomimitism is applied to the medicinal chemistry in order to synthesize drugs that devoid of the disadvantages of peptides. AT1 antagonists constitute a new generation of drugs for the treatment of hypertension designed and synthesized to mimic the C-terminal segment of Angiotensin II and to block its binding action on AT1 receptor. An effort was made to understand the molecular basis of hypertension by studying the conformational analysis of Ang II and its derivatives as well as the AT1 antagonists belonging to SARTANs class of molecules. Such studies offer the possibility to reveal the stereoelectronic factors responsible for bioactivity of AT1 antagonists and to design and synthesize new analogs. An example will be given which proves that drugs with better pharmacological and financial profiles may arise based on this rational design.

NMR metabolite profiling of Greek grape marc spirits.

This (1)H NMR based study profiles metabolites in Greek grape marc distillates, tsipouro and tsikoudia. Eightysix samples of indigenous and international varieties, stemming from major vine growing regions of Greece were investigated. The monitoring protocol addressed the global metabolic profile of untreated samples and accomplished the unambiguous assignment of 35 metabolites. NMR spectra were acquired by applying the robust, sensitive and rapid WET1D NMR pulse sequence, which succeeded to unveil the presence of minor compounds in a high ethanol matrix. PCA classified the samples according to their provenance, incorporating also information related to the variety, vintage year and production process within each formed regional assembly. Metabolites such as fusel alcohols, polyols, ethyl esters, mono- and di-saccharides were associated with the classification of samples. OPLS-DA ascribed to samples of common regional entity characteristic genotypic metabolites and probed to the potential influence of the vintage effect. Finally, metabolite profiling underlined the influence of the fermentation and distillation procedures.

Leveraging NMR and X-ray Data of the Free Ligands to Build Better Drugs Targeting Angiotensin II Type 1 G-Protein Coupled Receptor.

The angiotensin II type 1 receptor (AT1R) has been recently crystallized. A new era has emerged for the structure-based rational drug design and the synthesis of novel AT1R antagonists. In this critical review, the X-ray crystallographic data of commercially available AT1R antagonists in free form are analyzed and compared with the conformational analysis results obtained using a combination of NMR spectroscopy and Molecular Modeling. The same AT1R antagonists are docked and compared in terms of their interactions in their binding site using homology models and the crystallized AT1R receptor. Various aspects derived from these comparisons regarding rational drug design are outlined.

Comparative study of the AT1 receptor prodrug antagonist candesartan cilexetil with other sartans on the interactions with membrane bilayers

Drug-membrane interactions of the candesartan cilexetil (TCV-116) have been studied on molecular basis by applying various complementary biophysical techniques namely differential scanning calorimetry (DSC), Raman spectroscopy, small and wide angle X-ray scattering (SAXS and WAXS), solution ¹H and ¹³C nuclear magnetic resonance (NMR) and solid state ¹³C and ³¹P (NMR) spectroscopies. In addition, ³¹P cross polarization (CP) NMR broadline fitting methodology in combination with ab initio computations has been applied. Finally molecular dynamics (MD) was applied to find the low energy conformation and position of candesartan cilexetil in the bilayers. Thus, the experimental results complemented with in silico MD results provided information on the localization, orientation, and dynamic properties of TCV-116 in the lipidic environment. The effects of this prodrug have been compared with other AT₁ receptor antagonists hitherto studied. The prodrug TCV-116 as other sartans has been found to be accommodated in the polar/apolar interface of the bilayer. In particular, it anchors in the mesophase region of the lipid bilayers with the tetrazole group oriented toward the polar headgroup spanning from water interface toward the mesophase and upper segment of the hydrophobic region. In spite of their localization identity, their thermal and dynamic effects are distinct pointing out that each sartan has its own fingerprint of action in the membrane bilayer, which is determined by the parameters derived from the above mentioned biophysical techniques.