molecules Article Licofelone-DPPC Interactions: Putting Membrane Lipids on the Radar of Drug Development Catarina Pereira-Leite 1,2 , Daniela Lopes-de-Campos 1, Philippe Fontaine 3 , Iolanda M. Cuccovia 2, Cláudia Nunes 1 and Salette Reis 1,* 1 LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal; [email protected] (C.P.-L.); [email protected] (D.L.-d.-C.); [email protected] (C.N.) 2 Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes, 748, 05508-000 São Paulo, Brazil; [email protected] 3 Synchrotron SOLEIL, L’Orme des Merisiers, Saint Aubin, BP48, 91192 Gif-sur-Yvette, France; [email protected] * Correspondence: [email protected]; Tel.: +351-220-428-672 Academic Editors: Maria Emília De Sousa, Honorina Cidade and Carlos Manuel Afonso Received: 19 December 2018; Accepted: 30 January 2019; Published: 31 January 2019 Abstract: (1) Background: Membrane lipids have been disregarded in drug development throughout the years. Recently, they gained attention in drug design as targets, but they are still disregarded in the latter stages. Thus, this study aims to highlight the relevance of considering membrane lipids in the preclinical phase of drug development. (2) Methods: The interactions of a drug candidate for clinical use (licofelone) with a membrane model system made of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were evaluated by combining Langmuir isotherms, Brewster angle microscopy (BAM), polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS), and grazing-incidence X-ray diffraction (GIXD) measurements. (3) Results: Licofelone caused the expansion of the DPPC isotherm without changing the lipid phase transition profile. Moreover, licofelone induced the reduction of DPPC packing density, while increasing the local order of the DPPC acyl chains. (4) Conclusions: The licofelone-induced alterations in the structural organization of phosphatidylcholine monolayers may be related to its pharmacological actions. Thus, the combination of studying drug-membrane interactions with the pharmacological characterization that occurs in the preclinical stage may gather additional information about the mechanisms of action and toxicity of drug candidates. Ultimately, the addition of this innovative step shall improve the success rate of drug development. Keywords: lipid monolayers; DPPC; licofelone; langmuir isotherms; BAM; PM-IRRAS; GIXD 1. Introduction Drug discovery is primarily based on finding or designing new molecules to target a biomolecule in order to achieve a therapeutic benefit [1]. Proteins are the main biological targets considered in the rational design of novel drugs. However, other biomolecules have been gaining attention in the last decades, with the emergence of DNA and RNA-based gene therapy [2,3] and membrane lipid therapy [4]. Chronologically, lipids were the last biomolecules to be considered as biological targets, for two main reasons: (a) the huge amount of different lipids found in the human body; and (b) powerful lipid profiling techniques, such as high-resolution mass spectrometry, were recently developed [5]. Cell membranes are nowadays considered as a complex, dynamic, and heterogeneous conjunction of lipids, proteins, and carbohydrates, functioning as platforms to control signal transduction. Molecules 2019, 24, 516; doi:10.3390/molecules24030516 www.mdpi.com/journal/molecules Molecules 2019, 24, 516 2 of 14 In particular, membrane lipids act either as messengers or as regulators of signaling pathways [4]. The adventMolecules 2019 of functional, 24, x lipidomics has been unveiling the specific roles of membrane2 lipidsof 13 on diverse pathophysiological processes. Indeed, alterations in lipid composition and structure are currentlytransduction. related to In diverse particular, pathological membrane conditions, lipids act either including as messengers inflammation, or as regulators cancer, neurodegenerative of signaling disorders,pathways and [4]. metabolic The advent diseases of functional [4,5]. In lipidomics this context, has been membrane unveiling lipid the therapyspecific roles has emergedof membrane with the lipids on diverse pathophysiological processes. Indeed, alterations in lipid composition and structure goal of attaining a therapeutic benefit by designing new compounds able to modify membrane lipid are currently related to diverse pathological conditions, including inflammation, cancer, structures or domains [4]. neurodegenerative disorders, and metabolic diseases [4,5]. In this context, membrane lipid therapy Beyondhas emerged being with considered the goal of for attaining the rational a therapeutic design benefit of novel by designing drugs, membrane new compounds lipids able seem to also to bemodify valuable membrane for the lipid preclinical structures phaseor domains of drug[4]. development, in which the pharmacokinetic, pharmacodynamic,Beyond being and considered toxicity for profiles the rational of the design selected of novel candidate drugs, membrane are fully addressedlipids seem [also1]. Into fact, membranebe valuable model for systems the preclinical are important phase of tools drug todevelopment, predict the in lipophilicity which the pharmacokinetic, of drug candidates, withpharmacodynamic, recognized advantages and toxicity over theprofiles octanol-water of the selected system candidate [6–8], are which fully is addressed one of the [1]. main In fact, aspects influencingmembrane the model pharmacokinetic systems are important properties tools of pharmaceuticalsto predict the lipophilicity [9]. Regarding of drug pharmacodynamics,candidates, with membranerecognized lipids advantages can either over be the the therapeutic octanol-water target system of drugs [6–8], or which may indirectly is one of regulatethe main the aspects activity of influencing the pharmacokinetic properties of pharmaceuticals [9]. Regarding pharmacodynamics, membrane proteins [7,8]. Thus, drug-induced alterations in membrane lipids may result in changes membrane lipids can either be the therapeutic target of drugs or may indirectly regulate the activity in membrane-proteins activity, as already described, for instance, for phospholipase A2 [10]. Finally, of membrane proteins [7,8]. Thus, drug-induced alterations in membrane lipids may result in changes the toxicityin membrane-proteins mechanisms activity, of drugs as already may also described, include for drug-induced instance, for phospholipase modifications A2 [10]. in the Finally, structure of membranethe toxicity lipids, mechanisms as reported of drugs for may commercial also include nonsteroidal drug-induced anti-inflammatory modifications in the drugs structure [7,11 of] and antibioticsmembrane [12]. lipids, as reported for commercial nonsteroidal anti-inflammatory drugs [7,11] and Inantibiotics this context, [12]. this work aims to show the relevance of considering membrane lipids in the preclinical phase of drugIn this development. context, this Forwork that, aims a candidateto show the for relevance clinical use, of considering namely licofelone, membrane was lipids selected, in the and its interactionspreclinical with phase a membrane of drug development. model system, For viz. that, lipid a candidate monolayers, for clinical were evaluated use, namely using licofelone, complementary was experimentalselected, techniques.and its interactions with a membrane model system, viz. lipid monolayers, were evaluated using complementary experimental techniques. Licofelone (Figure1) is a dual cyclooxygenase (COX)/5-lipoxygenase (5-LOX) inhibitor designed Licofelone (Figure 1) is a dual cyclooxygenase (COX)/5-lipoxygenase (5-LOX) inhibitor designed to circumvent the toxic actions of nonsteroidal anti-inflammatory drugs, due to the additional inhibition to circumvent the toxic actions of nonsteroidal anti-inflammatory drugs, due to the additional of 5-LOXinhibition [13]. of Although 5-LOX [13]. the Although released the data released of preclinical data of preclinical and clinical and studiesclinical studies indicate indicate that licofelone that has promisinglicofelone has efficacy promising and tolerabilityefficacy and profilestolerability [13 profiles], this drug [13], this was drug not was marketed not marketed yet, raising yet, raising questions aboutquestions the underlying about the reasons. underlying In this reasons. sense, licofeloneIn this sense, was licofelone chosen for was this chosen study tofor eventually this study unravelto someeventually unknown unravel membrane some unknown lipid-related membrane mechanisms lipid-related of action mechanisms and toxicity, of action which and toxicity, may eventuallywhich contributemay eventually to clarify contribute the delayed to clarify commercialization. the delayed commercialization. FigureFigure 1. Structural 1. Structural formula formula of of licofelone licofelone andand 1,2-dipalmitoyl-1,2-dipalmitoyl-snsn-glycero-3-phosphocholine-glycero-3-phosphocholine (DPPC). (DPPC). LipidLipid monolayers monolayers made ofmade 1,2-dipalmitoyl- of 1,2-dipalmitoyl-sn-glycero-3-phosphocholinesn-glycero-3-phosphocholine (DPPC) (DPPC) at physiological at pH (7.4)physiological were chosen pH (7.4) as membrane were chosen model as membrane system. model Lipid system. monolayers Lipid monolayers are simple are and simple easy-to-make and membrane model systems that mimic the lateral interactions occurring in one leaflet of the