Università degli Studi di Salerno Dottorato di Ricerca in Scienza e Tecnologie per l’Industria Chimica, Farmaceutica e Alimentare XI CICLO Molecular dynamics investigations of drug-cell membrane interactionsTutor: Dottoranda:Prof. Stefano Piotto Piotto Federica CampanaCo-Tutor:Prof. Pablo V. EscribáDepartment of Biology, University of the Balearic IslandsSpain
Overview Structure and function of lipid membranesMembrane fluidizers alter membrane Membrane physical state modulates physical state the activity of embedded proteinsCHOL content influences the effect of Effect of fatty acids inside membrane fluidizers membranes
Membrane physical stateMembrane properties depend on:temperaturepressureelectrical fieldpHsalt concentrationpresence of proteinsprotein conformationThe physical state of a biologicalmembrane depends on allthermodynamic variables.It is involved in regulating theactivity of all proteins that areembedded and, consequently, theexpression of genes involved instress responses.
G protein lipid moietiesGeranylgeranyol (GG) Myristic alcohol (MOH) Myristic acid (MA) Palmitic alcohol (POH) Palmitic acid (PA)
Lipid moieties affinity for different membrane compositions GG MOH POH Free energy of binding (kcal/mol) 17 18 25 -4 -3 -13 POPC POPC-POPE
Effect of lipid moieties on membranesAn increase in the proportion of PE gradually decreases Gαmonomer binding to model membranes.Heterotrimeric Gαβγ subunits have a greater affinity for non-lamellar phases.
Effect of hydroxylamine derivatives inmodulating membrane physical state
Vigh, L., Maresca, B., Harwood, J. L. (1998) TIBS. 23:369-74
HSP co-inducers Cl OH NH2 OH O N O N N NN N Bimoclomol BGP-15 NG-094 Preservation of the chemical architecture of a cell or of an organism under stressful conditions is termed homeostasis. One of the best known mechanisms protecting cells from various stresses is the heat-shock response, which results in the induction of the synthesis of heat-shock proteins (HSPs or stress proteins). Hydroxylamine derivatives, interacting with lipid bilayers, promote the formation of chaperone molecules in eukaryotic cells and induce the expression of heat-shock genes.
BGP-15 affinity for different CHOL concentrationsBGP-15 affects both the level and the sizedistribution of CHOL-rich membranemicrodomains.BGP-15 activation of HSP involves theRac1 signaling cascade.Membrane CHOL profoundly affects thetargeting of Rac1 to membranes.BGP-15 inhibit the rapid HSF1 acetylationobserved in the early phase of heatstress, thereby promoting a prolongedduration of HSF1 binding to HSE on hspgenes.The permeation of BGP-15 is mildlyinfluenced by the composition.Docking of BGP-15 is enhanced by highcholesterol level.
Ability of HSP co-inducers to modify the physical state of membranes Thickness 46.63 46.16 45.94 43.23SM/CHOL SM/CHOL/BGP-15 SM/CHOL/NG-094 SM/CHOL/BMC Total energySM/CHOL SM/CHOL/BGP-15 SM/CHOL/NG-094 SM/CHOL/BMC CHOL Alignment-974566 -981763 -1011496 -1018570 0.92 0.92 0.89 0.84 SM/CHOL SM/CHOL/BGP-15 SM/CHOL/NG-094 SM/CHOL/BMC
Effect of HSP co-inducers on membrane spatial distribution
CHOL content in lipid rafts influences the effect of HSP co-inducers
Membrane fluidity Pure membrane Doped membrane BGP-15 + SM/CHOL 80:20 NG-094 + SM/CHOL 60:40Transparent atoms = more staticOpaque atoms = more mobile
BGP-15 and MβCD work together to induce HSP70 HSP70 without BGP-15 HSP70 with BGP-15 Effect of cholesterol removal in HEK293 lines (Crul et al, unpublished results)
Hydroxy arachidonic acid, a new potential non steroidal anti- inflammatory molecule
The COX enzyme The COX functions as a membrane-associated homodimer, catalyzing the committed step in the conversion of AA to prostaglandin H2 (PGH2), following AAs release from membrane phospholipds.Lopez, D. H., Fiol-de Roque, M. A., Noguera-Salva, M. A., Teres, S., Campana, F., Piotto, S., Castro, J. A., Mohaibes, R. J., Escribá P.V., Busquets. X. 2-Hydroxy Arachidonic Acid: A New Non-Steroidal Anti-Inflammatory Drug. British Journal of Pharmacology. Submitted.
Affinity for COX isoformsBinding energy (kcal/mol) 8.29 7.94 8.52 10.25 11.09 10.93 AA AArOH AAsOH AA AArOH AAsOH COX-1 COX-2
The Fukui function explains the inibitor capabilities of AAxOH AA AA-OH Fukui Indices for Radical Attack atom Mulliken Hirshfeld atom Mulliken Hirshfeld C ( 1) 0.076 0.073 C ( 1) 0.121 0.110 C ( 2) -0.023 0.014 C ( 2) -0.027 0.015The presence of αOH reduces the H ( 47) 0.000 0.000 H ( 47) -0.005 -0.002 H ( 48) 0.002 0.001 H ( 48) 0.007 0.003probability of extraction of the H ( 49) 0.014 0.007 H ( 49) 0.011 0.005 O ( 50) 0.087 0.085 O ( 50) 0.108 0.111hydrogen on C13 of almost 60% O ( 51) 0.027 0.038 O ( 51) 0.056 0.065 H ( 52) 0.028 0.018 H ( 52) 0.013 0.008 H ( 53) 0.034 0.022 H ( 53) 0.033 0.020 H ( 54) 0.032 0.023 H ( 54) 0.042 0.032 H ( 55) 0.019 0.014
AcknowledgementProf. Stefano Piotto PiottoProf.ssa Simona Concilio Prof. Pio Iannelli Dott.ssa Lucia Sessa Lab. 12