Article pubs.acs.org/JPCB
Modulation of Non Steroidal Anti-Inflammatory Drug Induced Membrane Fusion by Copper Coordination of These Drugs: Anchoring Effect Anupa Majumdar,† Sreeja Chakraborty,† and Munna Sarkar* Chemical Sciences Division, Saha Institute of Nuclear Physics, 1/AF, Bidhannagar, Kolkata-700064, India S Supporting Information *
ABSTRACT: 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. small molecules, high concentration is required15,16 to induce fusion in vitro. Non steroidal anti-inflammatory drugs (NSAIDs) constitute the most common class of anti-inflammatory and analgesic agents. Besides their primary functions, this class of painkillers show several new and alternate functions,17,18 such as anticancer properties18−21 as well as beneficiary effects against neurodegenerative diseases, viz. Alzheimer’s Disease.22,23 They have been shown to perturb the membrane structure on incorporation.24−26 Our group has shown that only three NSAIDs, viz. Piroxicam (Px), Meloxicam (Mx), and Tenoxicam (Tx), belonging to the oxicam group, can induce membrane fusion at physiologically relevant low concentration without the aid of other fusogens.27 This property is not shared by NSAIDs belonging to other chemical groups.27 In order to use these painkillers to induce fusion in a controlled manner, as is necessary in many biotechnological and biomedical procedures, it is essential to understand the mechanism and the way to modulate the process. Modulation of the fusion process can be achieved by different strategies, of which two relevant strategies for small molecule induced fusion are (a) by changing the physicochemical properties of the participating drugs or the membranes and (b) by using designed membrane anchors.
1. INTRODUCTION Membrane fusion is an integral step in important biological processes, such as fertilization,1,2 neurotransmission,3,4 trafficking,5,6 viral infection,7,8 etc. Specific energy barriers guide the three major steps of membrane fusion,9 as shown in Figure 1. The first step involves close apposition of membranes belonging to two vesicles/cells/cell organelles to initiate membrane contact by expunging the in-between aqueous phase. This is followed by lipid mixing or rearrangements of the outer leaflet of the bilayers to form the stalk intermediate (Figure 1a). The inner leaflets then rearrange to open a pore between the two vesicles/cells/cell organelles that leads to the mixing of their contents.9 These energy barriers should be overcome for the successful completion of the fusion process. To do so, external fusogenic agents are required for inducing fusion both in vivo and in vitro.10,11 For proteins12 and peptides13 that constitute the major class of fusogenic agents, conformational reorganization provides the driving force to overcome the energy barriers of the intermediate steps of membrane fusion.14 Small molecules lack this advantage, since the energy associated with their conformational reorganization will not be adequate to provide the required driving force. However, if we can use small molecules to induce membrane fusion both in vivo and in vitro, not only will it open up a new avenue but it will also help us to have better control over the fusion process. Modulating membrane fusion is necessary in many biotechnological and biomedical procedures. Usually, for © 2014 American Chemical Society
Received: August 26, 2014 Revised: November 7, 2014 Published: November 7, 2014 13785
dx.doi.org/10.1021/jp5086087 | J. Phys. Chem. B 2014, 118, 13785−13799
The Journal of Physical Chemistry B
Article
Figure 1. Graphical presentation of (a) the different steps of membrane fusion, (b) fluorescence based lipid mixing assay and (c) fluorescence based content mixing assay.
lipidated coiled-coil peptides have been used as membrane anchors to induce fusion.37 Peptides that emote targetrecognition domains of fusogenic proteins, coupled with a phenylboronic acid derivative as a recognition domain for a sugar-like target, also serve as membrane anchors.38,39A common feature of the above strategies is the need to functionalize the interacting vesicles by incorporating suitable molecular partners. These molecular partners, on interacting with each other, bridge apposing vesicles, thereby bringing them in close contact to initiate fusion. For these kinds of membrane anchors, as mentioned above, the molecular recognition and the strength of interaction between the partners play a critical role in modulating the fusion process. Designing preformed small molecules that can act as membrane anchors to modulate and/or increase the efficacy of membrane fusion is of special importance. For these new classes of designed anchors, neither molecular recognition nor the strength of interaction between the partners will have any role in initiating and controlling the fusion process. This will pave the way for the development of relatively simpler molecular systems that can act as membrane anchors, which we aim to do in this work. Modifying the marketed NSAIDs to modulate and/or enhance the efficacy of the drugs towards a preferred function has also been the strategy of different research groups.40,41 In this work we aim to modulate or increase the efficacy of the drug induced membrane fusion process by using preformed metal complexes of two fusion inducing NSAIDs, viz. Piroxicam (Px) and Meloxicam (Mx). The metal chosen is copper, which is not only a bioactive metal but is also known to have many beneficiary effects.42 Copper complexes of these NSAIDs have been shown to have better anticancer effects than the bare drugs.43,44 In this study, addition of these complexes to model membranes such as small unilamellar vesicles (SUVs) formed with dimyristoylphosphatidylcholine (DMPC) not only induced fusion, as for the bare drugs, but also increased the overall fusion efficacy. Enhanced efficacy as compared to the bare drugs is expressed either by the increase in rates and/or by
Modulation of the fusion process by changing several physicochemical properties of the participating drugs or the membranes has already been achieved by us. The effects of drug concentration as well as temperature have been studied. It has been shown that the rates of the final step of content mixing increased with increasing drug to lipid ratio (D/L) up to a certain threshold value characteristic of each of the drugs. Beyond this threshold D/L ratio, the rates decreased due to the predominance of leakage that caused rupture of the vesicles.28 It should be mentioned that spontaneous and/or induced leakage, leading to the release of the inner aqueous content of the vesicles to the bulk water, can occur independently irrespective of whether fusion is occurring or not. The effects of changing the orientational order of the lipid chains and the headgroup spacing upon incorporation of a low concentration of cholesterol (
