Tutor: Dottoranda:

Prof. Stefano Piotto Piotto Federica Campana

Co-Tutor:

Prof. Pablo V. EscribáDepartment of Biology, University of the Balearic IslandsSpain

Università degli Studi di SalernoDottorato di Ricerca in Scienza e Tecnologie per l’Industria Chimica, Farmaceutica e Alimentare

XI CICLO

Molecular dynamics investigations of drug-cell membrane interactions

Structure and function of lipid membranes

Membrane fluidizers alter membrane physical state

Membrane physical state modulates the activity of embedded proteins

CHOL content influences the effect of membrane fluidizers

Effect of fatty acids inside membranes

Overview

Membrane properties depend on:

temperaturepressureelectrical field pHsalt concentrationpresence of proteinsprotein conformation

The physical state of a biological membrane depends on all thermodynamic variables.

Membrane physical state

It is involved in regulating the activity of all proteins that are embedded and, consequently, the expression of genes involved in stress responses.

Objectives

Escribá, P. V. (2006) Trends in Molecular Medicine. 12:34-43

Membrane Lipid Therapy (MLT)

Gα monomer Gβγ dimer

Biogenic aminesAmino acids and ionsLipidsPeptides and proteinsOthers

GαGβGγ

GPCRs and G proteins

GαGβGγ

G protein lipid moieties

Geranylgeranyol (GG) Myristic alcohol (MOH) Palmitic alcohol (POH)Myristic acid (MA) Palmitic acid (PA)

GG MOH POH

-418 2517

-3 -13

POPCPOPC-POPE

Free

ene

rgy

of b

indi

ng (k

cal/

mol

)

Lipid moieties affinity for different membrane compositions

Effect of lipid moieties on membranes

An increase in the proportion of PE gradually decreases Gα monomer binding to model membranes.

Heterotrimeric Gαβγ subunits have a greater affinity for non-lamellar phases.

Effect of hydroxylamine derivatives in modulating membrane physical state

Vigh, L., Maresca, B., Harwood, J. L. (1998) TIBS. 23:369-74

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.

N

N

O N

Cl OH

N

N

O N

NH2 OH

N

NH

N

OH

OH

N

Bimoclomol BGP-15 NG-094

HSP co-inducers

BGP-15 affinity for different CHOL concentrations

The permeation of BGP-15 is mildly influenced by the composition.

Docking of BGP-15 is enhanced by high cholesterol level.

BGP-15 affects both the level and the size distribution of CHOL-rich membrane microdomains.

BGP-15 activation of HSP involves the Rac1 signaling cascade.Membrane CHOL profoundly affects the targeting of Rac1 to membranes.

BGP-15 inhibit the rapid HSF1 acetylation observed in the early phase of heat stress, thereby promoting a prolonged duration of HSF1 binding to HSE on hsp genes.

Ability of HSP co-inducers to modify the physical state of membranes

SM/CHOL SM/CHOL/BGP-15 SM/CHOL/NG-094 SM/CHOL/BMC

46.63 46.16

43.23

45.94

Thickness

SM/CHOL SM/CHOL/BGP-15 SM/CHOL/NG-094 SM/CHOL/BMC

-974566

-1018570

-981763

-1011496

Total energy

SM/CHOL SM/CHOL/BGP-15

SM/CHOL/NG-094

SM/CHOL/BMC

0.920.89

0.84

0.92

CHOL Alignment

Effect of HSP co-inducers on membrane spatial distribution

CHOL content in lipid rafts influences the effect of HSP co-inducers

Affinity for CHOL concentration in membranes

Transparent atoms = more staticOpaque atoms = more mobile

Membrane fluidity

Pure membrane Doped membrane

NG-094 +SM/CHOL 60:40

BGP-15 +SM/CHOL 80:20

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 functions as a membrane-associated homodimer, catalyzing the committed step in the conversion of AA to prostaglandin H2 (PGH2), following AA's release from membrane phospholipds.

The COX enzyme

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.

COX-1

COX-2

Docking on COX isoforms

AA AArOH AAsOH AA AArOH AAsOH

8.29 7.94 8.52 10.25 11.09 10.93

COX-1 COX-2

Bind

ing

ener

gy (k

cal/

mol

)

Affinity for COX isoforms

AA AA-OH Fukui Indices for Radical Attack

atom Mulliken Hirshfeld atom Mulliken HirshfeldC ( 1) 0.076 0.073 C ( 1) 0.121 0.110 C ( 2) -0.023 0.014 C ( 2) -0.027 0.015 H ( 47) 0.000 0.000 H ( 47) -0.005 -0.002 H ( 48) 0.002 0.001 H ( 48) 0.007 0.003 H ( 49) 0.014 0.007 H ( 49) 0.011 0.005 O ( 50) 0.087 0.085 O ( 50) 0.108 0.111 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

The presence of αOH reduces the

probability of extraction of the

hydrogen on C13 of almost 60%

The Fukui function explains the inibitor capabilities of AAxOH

Prof. Stefano Piotto Piotto

Prof.ssa Simona Concilio

Prof. Pio Iannelli

Dott.ssa Lucia Sessa

Lab. 12

Acknowledgement