This document summarizes Federica Campana's doctoral thesis on investigating drug-cell membrane interactions using molecular dynamics simulations. The thesis examines how membrane composition influences the effects of membrane fluidizers and heat shock protein co-inducers. It also analyzes the binding of anti-inflammatory molecules like hydroxyarachidonic acid to cyclooxygenase enzymes. The overall goal is to better understand how drug molecules interact with and modulate lipid bilayer properties at a molecular level.

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

2. Overview
Structure and function of lipid
membranes
Membrane fluidizers alter membrane Membrane physical state modulates
physical state the activity of embedded proteins
CHOL content influences the effect of Effect of fatty acids inside
membrane fluidizers membranes

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

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

8. Gα
Gβ
Gγ

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

10. 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

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. HSP co-inducers
Cl OH NH2 OH
O N O N
N N
N 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.

15. BGP-15 affinity for different CHOL concentrations
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.
The permeation of BGP-15 is mildly
influenced by the composition.
Docking of BGP-15 is enhanced by high
cholesterol level.

16. Ability of HSP co-inducers to modify the physical state of
membranes
Thickness
46.63 46.16 45.94
43.23
SM/CHOL SM/CHOL/BGP-15 SM/CHOL/NG-094 SM/CHOL/BMC
Total energy
SM/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

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

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

25. Affinity for COX isoforms
Binding 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

26. 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 0.015
The 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 0.003
probability 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 0.111
hydrogen 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

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