This document is a thesis submitted by Federica Campana for a PhD in molecular dynamics investigations of drug-cell membrane interactions. It summarizes research on how drug molecules interact with and influence cell membranes. Key findings include that membrane cholesterol content influences the effects of membrane fluidizers and heat shock protein co-inducers. Hydroxylamine derivatives were found to modify membrane physical state and induce heat shock proteins. Hydroxy arachidonic acid was identified as a potential new non-steroidal anti-inflammatory drug that targets cyclooxygenase enzymes with less affinity than arachidonic acid.

1. Tutor: Dottoranda:
Prof. Stefano Piotto Piotto Federica Campana
Co-Tutor:
Prof. Pablo V. Escribá
Department of Biology, University of the Balearic Islands
Spain
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 interactions

2. 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

4. Membrane properties depend on:
temperature
pressure
electrical field
pH
salt concentration
presence of proteins
protein 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.

5. Objectives

6. Escribá, P. V. (2006) Trends in Molecular Medicine. 12:34-43
Membrane Lipid Therapy (MLT)

7. Gα monomer Gβγ dimer
Biogenic amines
Amino acids and ions
Lipids
Peptides and proteins
Others
Gα
Gβ
Gγ
GPCRs and G proteins

8. Gα
Gβ
Gγ

9. G protein lipid moieties
Geranylgeranyol (GG) Myristic alcohol (MOH) Palmitic alcohol (POH)Myristic acid (MA) Palmitic acid (PA)

10. -4
18 2517
-3 -13
GG MOH POH
Freeenergyofbinding(kcal/mol)
POPC
POPC-POPE
Lipid moieties affinity for different membrane compositions

11. 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.

12. Effect of hydroxylamine derivatives in
modulating membrane physical state

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

14. 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
Bimoclomol BGP-15 NG-094
HSP co-inducers

15. 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.

16. Ability of HSP co-inducers to modify the physical state of
membranes
46.63 46.16
43.23
45.94
SM/CHOL SM/CHOL/BGP-15 SM/CHOL/NG-094 SM/CHOL/BMC
Thickness
-974566
-1018570
-981763
-1011496
SM/CHOL SM/CHOL/BGP-15 SM/CHOL/NG-094 SM/CHOL/BMC
Total energy
0.92
0.89
0.84
0.92
SM/CHOL SM/CHOL/BGP-15 SM/CHOL/NG-094 SM/CHOL/BMC
CHOL Alignment

17. Effect of HSP co-inducers on membrane spatial distribution

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

19. Affinity for CHOL concentration in membranes

20. Transparent atoms = more static
Opaque atoms = more mobile
Membrane fluidity
Pure membrane Doped membrane
NG-094 +
SM/CHOL 60:40
BGP-15 +
SM/CHOL 80:20

21. 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)

22. Hydroxy arachidonic acid, a new
potential non steroidal anti-
inflammatory molecule

23. 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.

24. COX-1
COX-2
Docking on COX isoforms

25. 8.29 7.94 8.52 10.25 11.09 10.93
AA AArOH AAsOH AA AArOH AAsOH
Bindingenergy(kcal/mol)
COX-1 COX-2
Affinity for COX isoforms

26. 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 0.015
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

27. Prof. Stefano Piotto Piotto
Prof.ssa Simona Concilio
Prof. Pio Iannelli
Dott.ssa Lucia Sessa
Lab. 12
Acknowledgement