Insights on Rigidity and Flexibility at the Global and Local Levels of Protein Structures and Their Roles in Homologous Psychrophilic, Mesophilic, and Thermophilic Proteins: A Computational Study.

The rigidity and flexibility of homologous psychrophilic (P), mesophilic (M), and thermophilic (T) proteins have been investigated at the global and local levels in terms of "packing factors" and "atomic fluctuations" obtained from B-factors. For comparison of atomic fluctuations, correction of errors by considering errors in B-factors from all sources in a consolidated manner and conversion of the fluctuations to the same temperature have been suggested and validated. The results indicate no differences in the global values like the average packing factor among the three classes of protein homologues, but at local levels there are differences. A comparison of homologous protein triplets show that the average atomic fluctuations at a given temperature mainly obey the order P > M > T. Packing factors and the atomic fluctuations are anti-correlated, suggesting that altering the rigidity of the active site might be a potential strategy to make tailor-made psychrophilic or thermophilic proteins from their mesophilic homologues. The computer codes developed and used in this work are available at the link https://github.com/Munna-Sarkar/proteins-rigidity-flexibility.git.

Painkiller Isoxicam and Its Copper Complex Can Form Inclusion Complexes with Different Cyclodextrins: A Fluorescence, Fourier Transform Infrared Spectroscopy, and Nuclear Magnetic Resonance Study.

The interaction of a painkiller Isoxicam, belonging to the oxicam group of nonsteroidal anti-inflammatory drugs (NSAIDs) and its copper complex with different cyclodextrins (ß-CD, γ-CD, HPßCD, and HPγCD), has been investigated in both solution and the solid state. Steady state and time-resolved fluorescence spectroscopy, fluorescence anisotropy, 1H NMR, and FTIR spectroscopy are used. Both the drug and its copper complex form a host-guest inclusion complex with all CDs. Fluorescence spectroscopy is used to determine binding constants and stoichiometries of the host-guest complex. The strongest binding is seen for γ-CD. 1H NMR study showed that Isoxicam penetrates into the CD cavity from the more accessible wider side. For ß- and γ-CD, Isoxicam showed one type of binding, i.e., formation of an inclusion complex, whereas, for HPßCD and HPγCD, it showed two types of binding, i.e., inclusion in the CD cavities and interaction with the outer surface of the CD molecules mainly near the hydroxy propyl group. Deeper penetration occurred into the larger diameter cavity of γ-CD and HPγCD compared to ß-CD and HPßCD. From FTIR and 1H NMR study, it is seen that predominantly the π-electron-rich benzene part of the drug and its complex penetrate into the host cavity.

Interaction of a common painkiller piroxicam and copper-piroxicam with chromatin causes structural alterations accompanied by modulation at the epigenomic/genomic level.

BACKGROUND: NSAIDs are the most common class of painkillers and anti-inflammatory agents. They also show other functions like chemoprevention and chemosuppression for which they act at the protein but not at the genome level since they are mostly anions at physiological pH, which prohibit their approach to the poly-anionic DNA. Complexing the drugs with bioactive metal obliterate their negative charge and allow them to bind to the DNA, thereby, opening the possibility of genome level interaction. To test this hypothesis, we present the interaction of a traditional NSAID, Piroxicam and its copper complex with core histone and chromatin. METHODS: Spectroscopy, DLS, and SEM studies were applied to see the effect of the interaction on the structure of histone/chromatin. This was coupled with MTT assay, immunoblot analysis, confocal microscopy, micro array analysis and qRT-PCR. RESULTS: The interaction of Piroxicam and its copper complex with histone/chromatin results in structural alterations. Such structural alterations can have different biological manifestations, but to test our hypothesis, we have focused only on the accompanied modulations at the epigenomic/genomic level. The complex, showed alteration of key epigenetic signatures implicated in transcription in the global context, although Piroxicam caused no significant changes. We have correlated such alterations caused by the complex with the changes in global gene expression and validated the candidate gene expression alterations. CONCLUSION AND GENERAL SIGNIFICANCE: Our results provide the proof of concept that DNA binding ability of the copper complexes of a traditional NSAID, opens up the possibility of modulations at the epigenomic/genomic level.

Spectroscopic studies on the interaction of DNA with the copper complexes of NSAIDs lornoxicam and isoxicam.

Non Steroidal Anti-inflammatory Drugs (NSAIDs) form the most common class of anti-inflammatory and analgesic agents. They also show anticancer properties for which they exert their effects by interacting at the protein but not at the genomic level. This is because most NSAIDs are anions at physiological pH, which prohibit their approach to the polyanionic DNA backbone. Complexing NSAIDs with bioactive metal like copper obliterates this disadvantage. Here, copper complexes of two oxicam NSAIDs, Lornoxicam (Lx) and Isoxicam (Isx) have been chosen to study their interaction with calf thymus (ct) DNA and have been synthesized as per reported protocols. UV-vis absorption showed that DNA binding to Cu(II)-Lx complex alters the absorption spectra indicating changes in the electronic environment of the complex, whereas, for Cu(II)-Isx there was only small changes. Hence, UV-vis absorption was used to determine the binding constant, stoichiometry and thermodynamic parameters of Cu(II)-Lx. However, UV-melting studies and CD difference spectra showed that both Cu(II)-Lx and Cu(II)-Isx can interact with the DNA backbone albeit with different binding modes. The probable binding mode was determined by kinetics of EtBr displacement and viscosity measurements. Our results point to an intercalative mode of binding for Cu(II)-Lx and external groove binding for Cu(II)-Isx.

Small Mismatches in Fatty Acyl Tail Lengths Can Effect Non Steroidal Anti-Inflammatory Drug Induced Membrane Fusion.

Biological membranes are made up of a variety of lipids with diverse physicochemical properties. The lipid composition modulates different lipidic parameters, such as hydration, dynamics, lipid packing, curvature strain, etc. Changes in these parameters affect various membrane-mediated processes, such as membrane fusion which is an integral step in many biological processes. Packing defects, which originate either from mismatch in the headgroup region or in the hydrophobic acyl tail region, play a major role in modulating membrane dynamics. In this study, we demonstrate how even a small mismatch in the fatty acyl chain length, achieved by incorporation of low concentrations (up to 30 mol %) of dipalmitoylphosphatidylcholine (DPPC) into dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicles (SUVs), alters several lipidic parameters like packing, dynamics, and headgroup hydration. This in turn affects non steroidal anti-inflammatory drug (NSAID) induced membrane fusion. Dynamic light scattering, differential scanning calorimetry, second-derivative absorption spectrophotometry, and steady-state and time-resolved fluorescence have been used to elucidate the effect of small mismatch in the tails in DMPC/DPPC mixed vesicles and how it modulates membrane fusion induced by the oxicam NSAIDs, meloxicam (Mx), piroxicam (Px), and tenoxicam (Tx). Fusion kinetics was monitored using fluorescence based fusion assays. At low DPPC concentration of 10 mol %, additional fluidization promotes lipid mixing to some extent for Mx, but at higher mol % of DPPC, subsequent increase in rigidity of membrane interior along with increase in headgroup hydration, synergistically inhibits fusion to various extents for the three different drugs, Mx, Px, and Tx.

Counterion effects on nano-confined metal-drug-DNA complexes.

We have explored morphology of DNA molecules bound with Cu complexes of piroxicam (a non-steroidal anti-inflammatory drug) molecules under one-dimensional confinement of thin films and have studied the effect of counterions present in a buffer. X-ray reflectivity at and away from the Cu K absorption edge and atomic force microscopy studies reveal that confinement segregates the drug molecules preferentially in a top layer of the DNA film, and counterions enhance this segregation.

Piroxicam, a traditional non-steroidal anti-inflammatory drug (NSAID) causes apoptosis by ROS mediated Akt activation.

BACKGROUND: Piroxicam (Px) belongs to the oxicam group of the non-steroidal anti-inflammatory drugs (NSAIDs) and have been shown to exert chemopreventive and chemotherapeutic effects in animal models and cultured animal cells. However, little is known about the mode of action of Px and its cellular targets. METHODS: We explored the role of Px, in triggering apoptosis and examined the involvement of upstream cellular mechanisms in apoptosis induction by Px. RESULTS AND DISCUSSION: Our studies with human breast cancer cells MCF-7 show that Px induces reactive oxygen species (ROS) generation along with apoptotic cell death. ROS release lead to Akt activation. On evaluation it became evident that ROS mediated apoptosis induction was due to Akt activation (hyper phosphorylation). Silencing the expression of Akt using siRNA and a specific Akt inhibitor, triciribine further confirmed the findings. However Px failed to cause ROS generation, cell death or Akt phosphorylation in another human breast cancer cells MDA-MB-231 which is estrogen receptor negative and more aggressive compared to MCF-7 cells. This suggests that Px has cell type specific effects. Thus we revealed for the first time that Px can induce apoptosis by ROS mediated Akt hyperphosphorylation/activation.

Differential Effect of Oxicam Non-Steroidal Anti-Inflammatory Drugs on Membranes and Their Consequence on Membrane Fusion.

Non-steroidal anti-inflammatory drugs (NSAIDs) are the most commonly used analgesics and antipyretics, which form an interesting drug group because of their new and alternate functions. The ability of the NSAIDs belonging to the oxicam chemical group to induce membrane fusion at low physiologically relevant concentrations is a new function that has drawn considerable attention. Membrane fusion is dependent on the interplay of physicochemical properties of both drugs and membranes. Here, we have elucidated the effects of different oxicam drugs, Meloxicam, Piroxicam, Tenoxicam, Lornoxicam, and Isoxicam, on an identical membrane-mimetic system. This highlights only the differential effects of the drugs on drug-membrane interactions, which in turn modulate their role as membrane fusogens. The partitioning behavior and the location of the drugs in dimyristoylphosphatidylcholine vesicles have been studied using second-derivative absorption spectroscopy, fluorescence quenching, steady-state fluorescence anisotropy, and time-resolved fluorescence lifetime measurements. Fusion kinetics has been monitored by fluorescence assays and dynamic light scattering was used to provide a snapshot of the vesicle diameter distribution at different time points. The differential perturbing effect of the drugs on the membrane is dependent both on their partitioning and location. Although partitioning governs the extent of fusion, the location modulates the rates of each step.

Lipid composition is an important determinant of antimicrobial activity of alpha-melanocyte stimulating hormone.

We have reported strong antimicrobial activity of cationic neuropeptide α-MSH against Staphylococcus aureus. Clinical S. aureus isolates non-susceptible to the peptide had higher amount of cationic phospholipid. To elucidate the molecular basis of lipid selectivity and antimicrobial activity of α-MSH, studies were carried out on SUVs having different combinations of neutral DMPC and anionic lipids DMPG to mimic mammalian and bacterial membrane. The peptide interacted with the DMPG containing vesicles only, as evident from the changes in Trp fluorescence. CD spectroscopy revealed that despite interaction, the peptide retained its native random coil structure. The perturbation of the vesicles caused by peptide interaction is strongly dependent on peptide concentration as seen both by DLS and Tb(3+)/DPA based fluorescence leakage assay. Our data clearly demonstrate the preference of α-MSH to interact with anionic DMPG containing vesicles leading to significant permeabilization which is the molecular basis behind the selectivity of α-MSH for bacterial systems.

Modulation of non steroidal anti-inflammatory drug induced membrane fusion by copper coordination of these drugs: anchoring effect.

Membrane fusion, an integral event in several biological processes, is characterized by several intermediate steps guided by specific energy barriers. Hence, it requires the aid of fusogens to complete the process. Common fusogens, such as proteins/peptides, have the ability to overcome theses barriers by their conformational reorganization, an advantage not shared by small drug molecules. Hence, drug induced fusion at physiologically relevant drug concentrations is rare and occurs only in the case of the oxicam group of non steroidal anti-inflammatory drugs (NSAIDs). To use drugs to induce and control membrane fusion in various biochemical processes requires the understanding of how different parameters modulate fusion. Also, fusion efficacy needs to be enhanced. Here we have synthesized and used Cu(II) complexes of fusogenic oxicam NSAIDs, Meloxicam and Piroxicam, to induce fusion in model membranes monitored by using DSC, TEM, steady-state, and time-resolved spectroscopy. The ability of the complexes to anchor apposing model membranes to initiate/facilitate fusion has been demonstrated. This results in better fusion efficacy compared to the bare drugs. These complexes can take the fusion to its final step. Unlike other designed membrane anchors, the role of molecular recognition and strength of interaction between molecular partners is obliterated for these preformed Cu(II)-NSAIDs.

Spectroscopic studies of the binding of Cu(II) complexes of oxicam NSAIDs to alternating G-C and homopolymeric G-C sequences.

Drugs belonging to the Non-steroidal anti-inflammatory (NSAID) group are not only used as anti-inflammatory, analgesic and anti-pyretic agents, but also show anti-cancer effects. Complexing them with a bioactive metal like copper, show an enhancement in their anti-cancer effects compared to the bare drugs, whose exact mechanism of action is not yet fully understood. For the first time, it was shown by our group that Cu(II)-NSAIDs can directly bind to the DNA backbone. The ability of the copper complexes of NSAIDs namely meloxicam and piroxicam to bind to the DNA backbone could be a possible molecular mechanism behind their enhanced anticancer effects. Elucidating base sequence specific interaction of Cu(II)-NSAIDs to the DNA will provide information on their possible binding sites in the genome sequence. In this work, we present how these complexes respond to differences in structure and hydration pattern of GC rich sequences. For this, binding studies of Cu(II) complexes of piroxicam [Cu(II)-(Px)2 (L)2] and meloxicam [Cu(II)-(Mx)2 (L)] with alternating GC (polydG-dC) and homopolymeric GC (polydG-polydC) sequences were carried out using a combination of spectroscopic techniques that include UV-Vis absorption, fluorescence and circular dichroism (CD) spectroscopy. The Cu(II)-NSAIDs show strong binding affinity to both polydG-dC and polydG-polydC. The role reversal of Cu(II)-meloxicam from a strong binder of polydG-dC (Kb=11.5×10(3) M(-1)) to a weak binder of polydG-polydC (Kb=5.02×10(3) M(-1)), while Cu(II)-piroxicam changes from a strong binder of polydG-polydC (Kb=8.18×10(3) M(-1)) to a weak one of polydG-dC (Kb=2.18×10(3) M(-1)), point to the sensitivity of these complexes to changes in the backbone structures/hydration. Changes in the profiles of UV absorption band and CD difference spectra, upon complex binding to polynucleotides and the results of competitive binding assay using ethidium bromide (EtBr) fluorescence indicate different binding modes in each case.

Binding of Cu(II) complexes of oxicam NSAIDs to alternating AT and homopolymeric AT sequences: differential response to variation in backbone structure.

Besides their principal functions as painkillers and anti-inflammatory agents, drugs belonging to the nonsteroidal anti-inflammatory drug (NSAID) group also have anticancer properties. Cu(II) complexes of these drugs enhance the anticancer effect. How they exert this effect is not clear. As a possible molecular mechanism, our group has already shown that the Cu(II) complexes of two oxicam NSAIDs with anticancer properties, viz. piroxicam and meloxicam, can directly bind to the DNA backbone. AT stretches are abundant in the eukaryotic genome. These stretches are more accessible to binding of different ligands, resulting in expression of different functions. AT stretches containing both alternating base pairs and homopolymeric bases in individual strands show subtle differences in backbone structures. It is therefore of interest to see how the Cu(II)-NSAID complexes respond to such differences in backbone structure. Binding studies of these complexes with alternating polydA-dT and homopolymeric polydA-polydT have been conducted using UV-vis absorption titration studies, UV melting studies and circular dichroism spectroscopy. Competitive binding with the standard intercalator ethidium bromide and the minor groove binder 4',6-diamidino-2-phenylindole was monitored using fluorescence to identify the possible binding mode. Our results show that Cu(II)-NSAID complexes are highly sensitive to the subtle differences in backbone structures of polydA-dT and polydA-polydT and respond to them by exhibiting different binding properties, such as binding constants, effect on duplex stability and binding modes. Both complexes have a similar binding mode with polydA-dT, which is intercalative, but for polydA-polydT, the results point to a mixed mode of binding.

Effect of lipid molecule headgroup mismatch on non steroidal anti-inflammatory drugs induced membrane fusion.

Membrane fusion is an essential process guiding many important biological events, which most commonly requires the aid of proteins and peptides as fusogenic agents. Small drug induced fusion at low drug concentration is a rare event. Only three drugs, namely, meloxicam (Mx), piroxicam (Px), and tenoxicam (Tx), belonging to the oxicam group of non steroidal anti-inflammatory drugs (NSAIDs) have been shown by us to induce membrane fusion successfully at low drug concentration. A better elucidation of the mechanism and the effect of different parameters in modulating the fusion process will allow the use of these common drugs to induce and control membrane fusion in various biochemical processes. In this study, we monitor the effect of lipid headgroup size mismatch in the bilayer on oxicam NSAIDs induced membrane fusion, by introducing dimyristoylphosphatidylethanolamine (DMPE) in dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicles (SUVs). Such headgroup mismatch affects various lipid parameters which includes inhibition of trans-bilayer motion, domain formation, decrease in curvature, etc. Changes in various lipidic parameters introduce defects in the membrane bilayer and thereby modulate membrane fusion. SUVs formed by DMPC with increasing DMPE content (10, 20, and 30 mol %) were used as simple model membranes. Transmission electron microscopy (TEM) and differential scanning calorimetry (DSC) were used to characterize the DMPC-DMPE mixed vesicles. Fluorescence assays were used to probe the time dependence of lipid mixing, content mixing, and leakage and also used to determine the partitioning of the drugs in the membrane bilayer. How the inhibition of trans-bilayer motion, heterogeneous distribution of lipids, decrease in vesicle curvature, etc., arising due to headgroup mismatch affect the fusion process has been isolated and identified here. Mx amplifies these effects maximally followed by Px and Tx. This has been correlated to the enhanced partitioning of the hydrophobic Mx compared to the more hydrophilic Px and Tx in the mixed bilayer.

Membrane fusion induced by small molecules and ions.

Membrane fusion is a key event in many biological processes. These processes are controlled by various fusogenic agents of which proteins and peptides from the principal group. The fusion process is characterized by three major steps, namely, inter membrane contact, lipid mixing forming the intermediate step, pore opening and finally mixing of inner contents of the cells/vesicles. These steps are governed by energy barriers, which need to be overcome to complete fusion. Structural reorganization of big molecules like proteins/peptides, supplies the required driving force to overcome the energy barrier of the different intermediate steps. Small molecules/ions do not share this advantage. Hence fusion induced by small molecules/ions is expected to be different from that induced by proteins/peptides. Although several reviews exist on membrane fusion, no recent review is devoted solely to small moleculs/ions induced membrane fusion. Here we intend to present, how a variety of small molecules/ions act as independent fusogens. The detailed mechanism of some are well understood but for many it is still an unanswered question. Clearer understanding of how a particular small molecule can control fusion will open up a vista to use these moleucles instead of proteins/peptides to induce fusion both in vivo and in vitro fusion processes.

Effect of increase in orientational order of lipid chains and head group spacing on non steroidal anti-inflammatory drug induced membrane fusion.

Membrane fusion is a key event in many biological processes. The fusion process, both in vivo and in vitro, is induced by different agents which include mainly proteins and peptides. For protein- and peptide-mediated membrane fusion, conformational reorganization serves as a driving force. Small drug molecules do not share this advantage; hence, drug induced membrane fusion occurring in absence of any other fusogenic agent and at physiologically relevant concentration of the drugs is a very rare event. To date, only three drugs, namely, meloxicam (Mx), piroxicam (Px), and tenoxicam (Tx), belonging to the oxicam group of non steroidal anti-inflammatory drugs (NSAIDs), have been shown by us to induce fusion at very low drug to lipid ratio without the aid of any other fusogenic agent. In our continued effort to understand the interplay of different physical and chemical parameters of both the participating drugs and the membrane on the mechanism of this drug induced membrane fusion, we present here the effect of increase in orientational order of the lipid chains and increase in head group spacing. This is achieved by studying the effect of low concentration cholesterol (<10 mol %) at temperatures above the chain-melting transition. Low concentration cholesterol (<10 mol %), above the gel to fluid transition temperature, is mainly known to increase orientational order of the lipid chains and increase head group spacing. To isolate the effect of these parameters, small unilameller vesicles (SUVs) formed by dimyristoylphosphatidylcholine (DMPC) with an average diameter of 50-60 nm were used as simple model membranes. Fluorescence assays were used to probe the time dependence of lipid mixing, content mixing, and leakage and also used to determine the partitioning of the drugs in the membrane bilayer. Differential scanning calorimetry (DSC) was used to study the effect of drugs in the presence of cholesterol on the chain-melting temperature which reflects the fluidization effect of the hydrophobic tail region of the bilayer. Our results show contradictory effect of low concentration cholesterol on the fusion induced by the three drugs, which has been explained by parsing the effect of orientational order and increase in head group spacing on the fusion process.

Non-steroidal anti-inflammatory drug induced membrane fusion: concentration and temperature effects.

Membrane fusion is a critical step in many biological events. The fusion process is always induced by different fusogenic agents of which proteins and peptides form the largest group. The mechanistic details of the fusion process vary depending on the nature of the fusogenic agents. However, membrane fusion induced by small drug molecules at physiologically relevant concentration has not been observed. Only recently our group has shown that three painkillers, namely, meloxicam, piroxicam, and tenoxicam, belonging to the oxicam group of non-steroidal anti-inflammatory drugs (NSAIDs) share this property. In this work, we present the effect of drug concentration and temperature on the kinetics of the fusion process. Small unilameller vesicles (SUVs) formed by dimyristoylphosphatidylcholine (DMPC) with an average diameter of 50-60 nm were used as model membranes. Fluorescence assays were used to probe the time dependence of lipid mixing, content mixing, and leakage whereas transmission electron microscopy (TEM) was used to image the fusion process and to calculate the average diameter of the vesicles. The results show that, in this fusion process, lipid mixing and content mixing are two sequential events and can occur even at a very low drug to lipid ratio (D/L) of 0.018. For a D/L ratio greater than 0.045, leakage of the vesicles leading to rupture compete with the fusion thereby inhibiting it. Temperature variation in the presence of drugs gives linear Arrhenius plots and is used to calculate the activation energies for the lipid mixing and content mixing, which are less compared to that seen in SUVs with a smaller diameter of 45 nm. Thermodynamic parameters of the transition state are calculated. The fusogenic property of the drugs has been interpreted in terms of the ability of the drugs to introduce membrane perturbation even at such low D/L ratios as studied here.

Interaction of Oxicam NSAIDs with lipid monolayer: anomalous dependence on drug concentration.

Surface pressure (pi) versus specific molecular area (A) isotherms of Langmuir monolayers of dimyristoylphosphatidylcholine (DMPC) lipid on pure water were studied in pristine form and in presence of three non-steroidal anti-inflammatory drugs, meloxicam (MX), piroxicam (PX) and tenoxicam (TX) in the subphase. Data were taken at three drug/lipid (D/L) ratios of 0.026, 0.05, and 0.1. Integration of drug to the lipid monolayer was measured by the increase in A (Delta A) of DMPC monolayer due to the presence of drugs. All three drugs could be integrated in the monolayer resulting in a positive value of Delta A for D/L ratio of 0.026. Above this D/L value, there is an anomalous, monotonic decrease in Delta A for MX and TX resulting, finally, in negative Delta A values. For PX, however, decrease in Delta A values at D/L of 0.05 is partially compensated at D/L of 0.1. We have tentatively explained these observations by invoking two competing forces in the overall drug-lipid interaction. One of these is an 'in-plane' force that tends to integrate the drug molecule to the plane formed by the lipid monolayer and the other is an 'out-of-plane' force that perturbs the drug and the lipid molecules such that the monolayer plane is no longer well defined.

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.

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.

Interaction of piroxicam with mitochondrial membrane and cytochrome c.

Modulation of surface properties of biomembranes by any ligand leading to permeabilization, fusion, rupture, etc. is a fundamental requirement for many biological processes. In this work, we present the interaction of piroxicam, a long acting Non-Steroidal Anti-Inflammatory Drug (NSAID) with isolated mitochondria, membrane mimetic systems, intact cells and a mitochondrial protein cytochrome c. Dye permeabilization study on isolated mitochondria indicates that piroxicam can permeabilize mitochondrial membrane. Direct imaging by Scanning Electron Microscope (SEM) shows that piroxicam induces changes in mitochondrial membrane morphology leading to fusion and rupture. Transmission Electron Microscope (TEM) imaging of piroxicam treated DMPC vesicles and mixed micelles formed from CTAB and SDS show that causing membrane fusion is a general property of piroxicam at physiological pH. In intact cells viz., V79 Chinese Hamster lung fibroblast, piroxicam is capable of releasing cytochrome c from mitochondria into the cytosol in a dose dependent manner along with the enhancement of downstream proapoptotic event viz., increase in caspase-3 activity. We have also shown that piroxicam can reduce cytochrome c within a time frame relevant to its lifetime in blood plasma. UV-visible spectroscopy has been used to study the reaction mechanism and kinetics in detail, allowing us to propose and validate a Michaelis-Menten like reaction scheme. CD spectroscopy shows that small but significant changes occur in the structure of cytochrome c when reduced by piroxicam.