Comparative study of interactions of aliskiren and AT1 receptor antagonists with lipid bilayers.

The renin-angiotensin-aldosterone system (RAAS) plays a key role in the regulation of blood pressure. Renin is the rate limiting enzyme of the RAAS and aliskiren is a highly potent and selective inhibitor of the human renin. Renin is known to be active both in the circulating blood stream as well as locally, when bound to the (pro)-renin receptor ((P)RR). In this study we have investigated a possible mechanism of action of aliskiren, in which its accumulation in the plasma membrane is considered as an essential step for effective inhibition. Aliskiren's interactions with model membranes (cholesterol rich and poor) have been investigated by applying different complementary techniques: differential scanning calorimetry (DSC), Raman spectroscopy, magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy and small- and wide-angle X-ray scattering (SAXS and WAXS). In addition, in silico molecular dynamics (MD) calculations were applied for further confirmation of the experimental data. Aliskiren's thermal effects on the pre- and main transition of dipalmitoyl-phosphatidylcholine (DPPC) membranes as well as its topographical position in the bilayer show striking similarities to those of angiotensin II type 1 receptor (AT1R) antagonists. Moreover, at higher cholesterol concentrations aliskiren gets expelled from the membrane just as it has been recently demonstrated for the angiotensin receptor blocker (ARB) losartan. Thus, we propose that both the AT1R and the (P)RR-bound renin active sites can be efficiently blocked by membrane-bound ARBs and aliskiren when cholesterol rich membrane rafts/caveolae are formed in the vicinity of the receptors.

Interactions of the AT1 antagonist valsartan with dipalmitoyl-phosphatidylcholine bilayers.

Valsartan is a marketed drug with high affinity to the type 1 angiotensin (AT1) receptor. It has been reported that AT1 antagonists may reach the receptor site by diffusion through the plasma membrane. For this reason we have applied a combination of differential scanning calorimetry (DSC), Raman spectroscopy and small and wide angle X-ray scattering (SAXS and WAXS) to investigate the interactions of valsartan with the model membrane of dipalmitoyl-phosphatidylcholine (DPPC). Hence, the thermal, dynamic and structural effects in bulk as well as local dynamic properties in the bilayers were studied with different valsartan concentrations ranging from 0 to 20 mol%. The DSC experimental results showed that valsartan causes a lowering and broadening of the phase transition. A splitting of the main transition is observed at high drug concentrations. In addition, valsartan causes an increase in enthalpy change of the main transition, which can be related to the induction of interdigitation of the lipid bilayers in the gel phase. Raman spectroscopy revealed distinct interactions between valsartan with the lipid interface localizing it in the polar head group region and in the upper part of the hydrophobic core. This localization of the drug molecule in the lipid bilayers supports the interdigitation view. SAXS measurements confirm a monotonous bilayer thinning in the fluid phase, associated with a steady increase of the root mean square fluctuation of the bilayers as the valsartan concentration is increased. At high drug concentrations these fluctuations are mainly governed by the electrostatic repulsion of neighboring membranes. Finally, valsartans' complex thermal and structural effects on DPPC bilayers are illustrated and discussed on a molecular level.

Interactions at the bilayer interface and receptor site induced by the novel synthetic pyrrolidinone analog MMK3.

This work presents a thorough investigation of the interaction of the novel synthetic pyrrolidinone analog MMK3 with the model membrane system of dipalmitoylphosphatidylcholine (DPPC) and the receptor active site. MMK3 has been designed to exert antihypertensive activity by functioning as an antagonist of the angiotensin II receptor of subtype 1 (AT(1)). Its low energy conformers were characterized by 2D rotating-frame Overhauser effect spectroscopy (ROESY) in combination with molecular dynamics (MD) simulations. Docking study of MMK3 shows that it fits to the AT(1) receptor as SARTANs, however, its biological activity appears to be lower. Thus, differential scanning calorimetry (DSC), Raman spectroscopy and small angle X-ray scattering (SAXS) experiments on the interaction of MMK3 with DPPC bilayers were carried out and results demonstrate that the drug is well incorporated into the membrane leaflets and furthermore causes partial bilayer interdigitation, although less effective than SARTANs. Thus, it appears that the nature of the bilayer matrix and the stereoelectronic active site requirements of the receptor are responsible for the low bioactivity of MMK3.

Efforts to understand the molecular basis of hypertension through drug:membrane interactions.

Biological membranes play an essential role in the drug action. They constitute the first barrier for drugs to exert their biological action. AT1 antagonists are amphiphilic molecules and are hypothesized to act on AT1 receptor through incorporation (first step) and lateral diffusion through membrane bilayers (second step). Various biophysical methods along with Molecular Modelling were applied in order to explore the plausible two step proposed mechanism of action for this class of antihypertensive drugs.

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.