Hirak Chakraborty

Incorporation of NSAIDs in micelles: implication of structural switchover in drug–membrane interaction

Non-steroidal anti-inflammatory drugs (NSAIDs) of oxicam group are not only effective as anti-inflammatory agents but also show diverse functions. Their principal targets are cyclooxygenases, which are membrane-associated enzymes. To bind with the targets these drugs have to pass through the membrane and hence their interactions with biomembranes should play a major role in guiding their interactions with cyclooxygenases. Here we have studied the interactions of three NSAIDs of oxicam group viz. piroxicam, meloxicam and tenoxicam with micelles having different headgroup charges, as simple membrane mimetic systems. Spectroscopic methods have been used to understand the interaction of these drugs with Cetyl N,N,N-trimethyl ammonium bromide (cationic), Sodium dodecyl sulphonate (anionic) and Triton X-100 (neutral) micelles. Our results demonstrate that the environment of the drugs i.e. the nature of the micelles plays a decisive role in choosing a specific prototropic form of the drugs for incorporation. Additionally it induces a switch over or change between different prototropic forms of piroxicam, which is correlated with the change in their reactivities in presence of different surface charges, given by the change in pK(a) values. These results together, indicate that in vivo, the diverse nature of biomembranes might play a significant role in choosing the particular form of oxicam NSAIDs that would be presented to their targets.

Photophysical studies of oxicam group of NSAIDs: piroxicam, meloxicam and tenoxicam

Oxicam group of non steroidal anti-inflammatory drugs has been chosen as a prototype molecular group that shows diverse biological functions and dynamic structural features. Photophysical studies of three drugs from this group viz., piroxicam, meloxicam and tenoxicam have been carried out in different solvents with varying polarity, H-bond character and viscosity. The spectral responses of different prototropic forms of these drugs towards varying solvent parameters have been studied, with the aim to characterize their interaction in biomimetic environment non-invasively. The nature of the lowest transition has been identified. The extinction coefficient, quantum yield and viscosity dependence on the nature of the solvents, all indicate the extreme sensitivity of these drugs to their microenvironment.

Membrane fusion: a new function of non steroidal anti-inflammatory drugs.

Membrane fusion is an important event in many biological processes and is characterized by several intermediate steps of which content mixing between the two fusing vesicles signals the completion of the process. Fusion induced solely by small drug molecules is not a common event. Non Steroidal Anti-Inflammatory Drugs (NSAIDs), that control pain and inflammation, are also capable of exhibiting diverse functions. In this study we present a new function of NSAIDs belonging to the oxicam group, as membrane fusogenic agents. Small Unilamellar Vesicles (SUVs) formed by the phospholipid, dimyristoylphosphatidylcholine (DMPC), were used as model membranes. Fluorescence assays using terbium/dipicolinic acid (Tb/DPA) were used to monitor content mixing and corresponding leakage in presence of the drugs. Transmission Electron Microscope (TEM) was also used to image fusion bodies in drug treated vesicles as compared to the untreated ones. The results show that the three oxicam NSAIDs viz. Meloxicam, Piroxicam and Tenoxicam can induce fusion of DMPC vesicles and lead the fusion process to completion at a very low drug to lipid ratio (D/L) of 0.045. The oxicam drugs exhibit differential fusogenic behavior as reflected in the kinetics of content mixing and leakage, both of which can be described by a single exponential rate equation. Moreover, not all NSAIDs can induce membrane fusion. Indomethacin, an acetic acid group NSAID and ibuprofen, a propionic acid group NSAID, did not induce fusion of vesicles. This new property of NSAIDs has important applications in biochemical processes.

Membrane dipole potential is sensitive to cholesterol stereospecificity: Implications for receptor function

Dipole potential is the potential difference within the membrane bilayer, which originates due to the nonrandom arrangement of lipid dipoles and water molecules at the membrane interface. Cholesterol, an essential lipid in higher eukaryotic membranes, has previously been shown to increase membrane dipole potential. In this work, we explored the effect of stereoisomers of cholesterol, ent-cholesterol and epi-cholesterol, on membrane dipole potential, monitored by the dual wavelength ratiometric approach utilizing the probe di-8-ANEPPS. Our results show that cholesterol and ent-cholesterol share comparable ability in increasing membrane dipole potential. In contrast, epi-cholesterol displays a slight reduction in membrane dipole potential. Our results constitute the first report on the effect of stereoisomers of cholesterol on membrane dipole potential, and imply that an extremely subtle change in sterol structure can significantly alter the dipolar field at the membrane interface. These results assume relevance in the context of differential abilities of these stereoisomers of cholesterol in supporting the activity of the serotonin1A receptor, a representative G protein-coupled receptor. The close correlation between membrane dipole potential and receptor activity provides new insight in receptor-cholesterol interaction in terms of stereospecificity. We envision that membrane dipole potential could prove to be a sensitive indicator of lipid-protein interactions in biological membranes.

Multiple Functions of Generic Drugs: Future Perspectives of Aureolic Acid Group of Anti-Cancer Antibiotics and Non-Steroidal Anti-Inflammatory Drugs

Non-steroidal anti-inflammatory drugs and aureolic acid group of anti-cancer drugs belong to the class of generic drugs. Research with some members of these two groups of drugs in different laboratories has unveiled functions other than those for which they were primarily developed as drugs. Here we have reviewed the molecular mechanism behind the multiple functions of these drugs that might lead to employ them for treatment of diseases in addition to those they are presently employed.

Excitements and challenges in GPCR oligomerization: molecular insight from FRET.

G protein-coupled receptors (GPCRs) are the largest family of proteins involved in signal transduction across cell membranes, and they represent major drug targets in all clinical areas. Oligomerization of GPCRs and its implications in drug discovery constitute an exciting area in contemporary biology. In this Review, we have highlighted the application of fluorescence resonance energy transfer (FRET) in exploring GPCR oligomerization, with special emphasis on possible pitfalls and experimental complications involved. Based on FRET photophysics, we discuss some of the possible complications, and recommend that FRET results in complex cellular environments should be interpreted with caution. Although both hetero- and homo-FRET are used in measurements of GPCR oligomerization, we suggest that homo-FRET enjoys certain advantages over hetero-FRET. Given the seminal role of GPCRs as current drug targets, we envision that methodological progress in studying GPCR oligomerization would result in better therapeutic strategies.

Molecular rheology of neuronal membranes explored using a molecular rotor: Implications for receptor function

The role of membrane cholesterol as a crucial regulator in the structure and function of membrane proteins and receptors is well documented. However, there is a lack of consensus on the mechanism for such regulation. We have previously shown that the function of an important neuronal receptor, the serotonin1A receptor, is modulated by cholesterol in hippocampal membranes. With an overall objective of addressing the role of membrane physical properties in receptor function, we measured the viscosity of hippocampal membranes of varying cholesterol content using a meso-substituted fluorophore (BODIPY-C12) based on the BODIPY probe. BODIPY-C12 acts as a fluorescent molecular rotor and allows measurement of hippocampal membrane viscosity through its characteristic viscosity-sensitive fluorescence depolarization. A striking feature of our results is that specific agonist binding by the serotonin1A receptor exhibits close correlation with hippocampal membrane viscosity, implying the importance of global membrane properties in receptor function. We envision that our results are important in understanding GPCR regulation by the membrane environment, and is relevant in the context of diseases in which GPCR signaling plays a major role and are characterized by altered membrane fluidity.

Depth-Dependent Organization and Dynamics of Archaeal and Eukaryotic Membranes: Development of Membrane Anisotropy Gradient with Natural Evolution

The lipid composition of archaea is unique and has been correlated with increased stability under extreme environmental conditions. In this article, we have focused on the evolution of membrane organization and dynamics with natural evolution. Dynamic anisotropy along the membrane normal (i.e., gradients of mobility, polarity, and heterogeneity) is a hallmark of fluid phase diester or diether phospholipid membranes. We monitored gradients of mobility, polarity, and heterogeneity along the membrane normal in membranes made of a representative archaeal lipid using a series of membrane depth-dependent fluorescent probes, and compared them to membranes prepared from a typical diether lipid from higher organisms (eukaryotes). Our results show that the representative dynamic anisotropy gradient along the membrane normal is absent in membranes made from archaeal lipids. We hypothesize that the dynamic gradient observed in membranes of diester and diether phospholipids is a consequence of natural evolution of membrane lipids in response to the requirement of carrying out complex cellular functions by membrane proteins.

Differential Membrane Dipolar Orientation Induced by Acute and Chronic Cholesterol Depletion

Cholesterol plays a crucial role in cell membrane organization, dynamics and function. Depletion of cholesterol represents a popular approach to explore cholesterol-sensitivity of membrane proteins. An emerging body of literature shows that the consequence of membrane cholesterol depletion often depends on the actual process (acute or chronic), although the molecular mechanism underlying the difference is not clear. Acute depletion, using cyclodextrin-type carriers, is faster relative to chronic depletion, in which inhibitors of cholesterol biosynthesis are used. With the overall goal of addressing molecular differences underlying these processes, we monitored membrane dipole potential under conditions of acute and chronic cholesterol depletion in CHO-K1 cells, using a voltage-sensitive fluorescent dye in dual wavelength ratiometric mode. Our results show that the observed membrane dipole potential exhibits difference under acute and chronic cholesterol depletion conditions, even when cholesterol content was identical. To the best of our knowledge, these results provide, for the first time, molecular insight highlighting differences in dipolar reorganization in these processes. A comprehensive understanding of processes in which membrane cholesterol gets modulated would provide novel insight in its interaction with membrane proteins and receptors, thereby allowing us to understand the role of cholesterol in cellular physiology associated with health and disease.

Protein-dependent membrane interaction of a partially disordered protein complex with oleic acid : Implications for cancer lipidomics

Bovine α-lactalbumin (BLA) forms cytotoxic complexes with oleic acid (OA) that perturbs tumor cell membranes, but molecular determinants of these membrane-interactions remain poorly understood. Here, we aim to obtain molecular insights into the interaction of BLA/BLA-OA complex with model membranes. We characterized the folding state of BLA-OA complex using tryptophan fluorescence and resolved residue-specific interactions of BLA with OA using molecular dynamics simulation. We integrated membrane-binding data using a voltage-sensitive probe and molecular dynamics (MD) to demonstrate the preferential interaction of the BLA-OA complex with negatively charged membranes. We identified amino acid residues of BLA and BLA-OA complex as determinants of these membrane interactions using MD, functionally corroborated by uptake of the corresponding α-LA peptides across tumor cell membranes. The results suggest that the α-LA component of these cytotoxic complexes confers specificity for tumor cell membranes through protein interactions that are maintained even in the lipid complex, in the presence of OA.