Lytic reactions of drugs with lipid membranes

Article abstract of DOI:10.1039/c8sc04831b

Propranolol is shown to undergo lipidation reactions in three types of lipid membrane: (1) synthetic single-component glycerophospholipid liposomes; (2) liposomes formed from complex lipid mixtures extracted from E. coli or liver cells; and (3) in cellulo in Hep G2 cells. Fourteen different lipidated propranolol homologues were identified in extracts from Hep G2 cells cultured in a medium supplemented with propranolol. This isolation of lipidated drug molecules from liver cells demonstrates a new drug reactivity in living systems. Acyl transfer from lipids to the alcoholic group of propranolol was favoured over transfer to the secondary amine. Migration of acyl groups from the alcohol to the amine was diminished. Other drugs that were examined did not form detectable levels of lipidation products, but many of these drugs did affect the lysolipid levels in model membranes. The propensity for a compound to induce lysolipid formation in a model system was found to be a predictor for phospholipidosis activity in cellulo.

Full text of DOI:10.1039/c8sc04831b

Showcasing research from Dr John Sanderson’s laboratory,
Chemistry Department, Durham University, UK.
As featured in:
Lytic reactions of drugs with lipid membranes
Propranolol is shown to undergo transesterification reactions
with glycerophospholipids in liposomal membranes, leading to
the formation of a lipidated drug and a lysolipid. Similar lipidated
reaction products are formed in vivo. Drugs that promote
lysolipid formation by either hydrolysis or transesterification
are found to have lower EC50 values for phospholipidosis.
See John M. Sanderson et al.,
Chem. Sci., 2019, 10, 674.
rsc.li/chemical-science
Registered charity number: 207890

Chemical
Science
View Article Online
View Journal | View Issue
EDGE ARTICLE
Lytic reactions of drugs with lipid membranes†
a
Hannah M. Britt,a Clara A. Garcıa-Herrero, a Paul W. Denny, b Jackie A. Mosely
Cite this: Chem. Sci., 2019, 10, 674
´
a
*
and John M. Sanderson
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
Propranolol is shown to undergo lipidation reactions in three types of lipid membrane: (1) synthetic single-
component glycerophospholipid liposomes; (2) liposomes formed from complex lipid mixtures extracted
from E. coli or liver cells; and (3) in cellulo in Hep G2 cells. Fourteen different lipidated propranolol
homologues were identified in extracts from Hep G2 cells cultured in a medium supplemented with
propranolol. This isolation of lipidated drug molecules from liver cells demonstrates a new drug reactivity
in living systems. Acyl transfer from lipids to the alcoholic group of propranolol was favoured over
transfer to the secondary amine. Migration of acyl groups from the alcohol to the amine was diminished.
Other drugs that were examined did not form detectable levels of lipidation products, but many of these
drugs did affect the lysolipid levels in model membranes. The propensity for a compound to induce
lysolipid formation in a model system was found to be a predictor for phospholipidosis activity in cellulo.
Received 29th October 2018
Accepted 29th November 2018
DOI: 10.1039/c8sc04831b
rsc.li/chemical-science
formation of a lysolipid alongside the lipidated peptide.1 When
the nucleophile is a serine hydroxyl group, the process is
Introduction
formally a transesterication. More recently, evidence has
emerged to suggest that some membrane proteins may also be
lipidated by direct transfer from the membrane. For example,
the lipidation prole of the lens protein aquaporin-0 has been
found to be highly complex.4 Aquaporin-0 has two lipidation
sites that do not correspond to known consensus sequences for
lipidation enzymes and are partially lipidated with numerous
different acyl groups, with typically up to eight identiable. The
relative abundance of the acyl modications at each site
correlates with the acyl ester content of the lipids in the plasma
Lipids are the key component of many materials, including
liposomes used in technology applications and the membranes
of cells and organelles in biological systems. In this article we
examine whether organic molecules that partition into
membranes, such as propranolol (1), can undergo direct
chemical reactions with lipids (Scheme 1). Demonstration of
such reactivity between membrane lipids and membrane-
associated drugs in vivo would constitute a signicant addi-
tion to our understanding of membrane chemistry. Reactivity of
this nature is likely to lead to biological effects that may account
for the adverse activities of some drugs and the unusual phar-
macokinetic proles of others. By contrast, exploiting this
reactivity could offer opportunities for the purposeful design of
new membrane-active compounds. The direct transfer of acyl
groups from membrane lipids to suitably disposed molecules
embedded within the membrane has a precedent, having been
demonstrated for peptides in vitro.1–3 In these cases, peptide
lipidation results from the high affinity of the peptide for the
membrane, combined with the appropriate positioning of
reactive groups in the membrane interface. These aminolysis
reactions involve nucleophilic attack on a lipid ester group by
a suitably disposed nucleophile on the peptide, typically a lysine
3-amino group or the N-terminal amino group, and lead to the
aChemistry Department, Durham University, South Road, Durham, DH1 3LE, UK.
E-mail: j.m.sanderson@durham.ac.uk
bDepartment of Biosciences, Durham University, Stockton Road, Durham, DH1 3LE, UK
† Electronic supplementary information (ESI) available: These might include
comments relevant to but not central to the matter under discussion, limited
experimental and spectral data, and crystallographic data. See DOI:
10.1039/c8sc04831b
Scheme
headgroup.
1
Lipidation reactions involving propranolol. Key: HG,
674 | Chem. Sci., 2019, 10, 674–680
This journal is © The Royal Society of Chemistry 2019

View Article Online
Edge Article
Chemical Science
membrane leaet proximal to the site. Whilst these observa- Lipids Inc., BC, Canada). All experiments were conducted at
tions are not direct proof that this protein is lipidated by concentrations of 1.27 mM for lipids and (when present)
transfer from the membrane, they do give a strong indication 0.127 mM for membrane binding compounds. Using published
that acyl transfer from the membrane is likely to be signicant partitioning data5 these concentrations produce a bound
in vivo. Fundamentally, aquaporin-0 is a moderately high propranolol to lipid ratio of about 1 : 30. Samples at pH 7.4 were
molecular weight protein that is permanently membrane- buffered using bicarbonate at a NaCl concentration of 90 mM.
embedded in vivo, whilst peptides such as melittin that are Calibration curves for analyte concentration were generated by
lipidated in vitro are of medium molecular weight with modest tting a logistic model to data obtained using authentic stan-
membrane affinity. It is therefore of key interest to establish dards of lysolipids, propranolol and acyl propranolol derivatives
whether low molecular weight organic molecules, with at known concentrations.
concomitantly lower membrane affinity, are able to undergo the
same process. We selected the b-blocker propranolol to probe
this reactivity as it has both alcoholic and amine functionalities
alongside well-characterised membrane binding characteris-
tics.5–7 Furthermore, synthetic versions of O- and N-acyl
propranolol derivatives have been described as prodrugs,8–11
providing precedents for understanding their properties in vitro
and synthesising reference compounds for this work. In addi-
tion, propranolol is known to induce phospholipidosis,12,13 an
adverse activity associated with disorders in lysosomal phos-
pholipid storage, characterised by the formation of lamellar
bodies visible by microscopy of susceptible cells.14–20 In prin-
ciple therefore, propranolol may undergo both aminolysis and
transesterication reactions, both of which lead to the forma-
tion of lysolipids, as shown in Scheme 1 for reaction with the
acyl group at the sn-1 position of the lipid (although reaction
can occur at either the sn-1 or sn-2 position). The lysolipid
product would be formed as an equilibrium mixture of the 1-
and 2-acyl species.1 All of the products could be reasonably ex-
pected to display signicant biological activity as a consequence
of the presence of a fatty acyl chain. Lysolipids in particular, are
capable of inducing signicant deleterious biological effects at
levels as low as 1 mol%.21
Lysolipids can also be formed by lipid hydrolysis, with
concomitant formation of a fatty acid. Membrane-associated
drugs have the potential to inuence the rate of this hydro-
lysis by either changing the bulk properties of the membrane,
mediated by secondary effects on interfacial water activity, or by
direct involvement in acid or base catalysis.22,23 This article
describes the propensity of a number of membrane-active drugs
to undergo direct lipidation reactions with membrane lipids, or
promote other lytic reactions of lipids.
Hep G2 culture and extraction
Hep G2 cells from ATCC were grown to conuence at 37 ꢁC, 5%
CO2, and 95% humidity in Dulbecco's Modied Eagle Medium
(DMEM) with 10% foetal bovine serum (Gibco/ThermoFisher).
Cells (106) were incubated at 37 ꢁC, 5% CO2, and 95%
humidity overnight to adhere. The medium was removed and
replaced with either fresh medium (5 mL) for two controls, or
medium containing 30 mM propranolol (5 mL). Following
incubation for 72 h, the medium was removed from asks and
replaced with phosphate buffered saline (5 mL). Cells were
collected by centrifugation for 10 min at 1000 ꢀ g, and decanted
into a glass tube before extraction with CHCl3 : MeOH (2 : 1; 3
mL). The CHCl3 : MeOH solution was washed with H2O (0.6
mL), isolated, and the solvent removed in vacuo. Samples were
resuspended in IPA : MeCN : H2O for liquid chromatography-
mass spectrometry (LC-MS) analysis.
Mass spectrometry
LC-MS was conducted on a Synapt G2-S (Waters Corp., UK)
using electrospray ionisation. LC was conducted using an Acq-
uity UPLC with a CSH C18 1.7 mm (2.1 ꢀ 150 mm) column with
solvent A ¼ H2O : MeCN 4 : 6 and B ¼ MeCN : iPrOH, 1 : 9 (both
A and B containing 10 mM NH4HCO2 + 0.1% formic acid) and
the following A : B gradient: 60 : 40 to 57 : 43 over 2 min, 57 : 43
to 50 : 50 over 0.1 min, 50 : 50 to 46 : 54 over 9.9 min, 46 : 54 to
30 : 70 over 0.1 min, 30 : 70 to 1 : 99 over 5.9 min. MS data were
processed using the instrument manufacturers soware (Mas-
sLynx, version 4.1) and the xcms package (version 1.52.0)24 in
the R statistical computing environment (version 3.4.1).25
Full methods are in the ESI.†
Results and discussion
Propranolol lipidation in liposomes composed of single lipids
Experimental
Materials
and binary mixtures
Phospholipids and lysolipids, including E. coli Extract Polar
(catalogue number 100600P) and Liver Polar Lipid Extract
(Bovine, catalogue number 181108P) were purchased as
powders from Avanti Polar Lipids (via Instruchemie B.V., The
Netherlands). Propranolol was used as a racemic mixture.
Our initial objective was to establish that incubation of
propranolol (1) with liposomes could lead to the generation of
lipidated propranolol products. We were able to obtain unam-
biguous evidence for propranolol lipidation through the use of
liposomes with well-dened lipid compositions and compar-
ison of the lipidated products with authentic standards
prepared chemically. At 37 ꢁC and pH 7.4, using liposomes
Liposome preparation and sample set up
Liposomes were prepared by extrusion of lipid dispersions 10ꢀ composed
of
either
1-palmitoyl-2-oleoyl-sn-glycero-3-
through polycarbonate lters (Whatman) with 100 nm track- phosphocholine (POPC, Fig. 1a) or 1,2-dioleoyl-sn-glycero-3-
etched pores, using a LIPEX thermobarrel extruder (Northern phosphocholine (DOPC, Fig S1b†), lipidated propranolol (2
This journal is © The Royal Society of Chemistry 2019
Chem. Sci., 2019, 10, 674–680 | 675

View Article Online
Chemical Science
Edge Article
Fig. 2 Product formation following the addition of propranolol (0.127
mM) to DOPC liposomes (1.27 mM) at pH 7.4, 37 ꢁC. The total
concentration of oleoyl propranolol (N- plus O-oleoyl) is shown as
blue triangles. The lysolipid concentration, shown as magenta circles,
is the concentration (1- plus 2-oleoyl) after subtraction of the lysolipid
concentration in a control sample without propranolol. The inset
shows a magnified view of the first data points. Errors are mean ꢂ s,
n ¼ 3.
Fig. 1 LC-MS analysis of lipidated products formed after addition of
propranolol to POPC membranes. LC separation (A) was conducted on
a C18 column using the protocol outlined in the methods section. (B) CID
MSMS spectrum of O-oleoyl propranolol. (C) CID MSMS spectrum of N-
oleoyl propranolol. Spectra for the corresponding palmitoyl products
are in Fig. S2.† For assignments, see Tables S1–S3 and Fig. S3, S4.†
and 3, Scheme 1) was detectable in both cases, alongside
increased levels of lysolipid. Lysolipids were found in both
total content of lipidated propranolol decreases, the reasons for
which are not currently understood, although hydrolysis or
insolubility of one or both of the acyl derivatives are likely
causes. Consequently, only data obtained within the rst 48 h
have been used for comparative analyses. As indicated in
Fig. 1a, acyl transfer to propranolol from the lipid is expected to
lead to the formation of a stoichiometric equivalent of lysolipid,
which should lead to identical concentration changes for acyl
propranolol and lysolipid, assuming that there are no
competing processes. For DOPC, a linear t to the data over the
rst 48 h gave a rate of acyl propranolol formation of 8.7 (ꢂ2.8)
ꢀ 10ꢃ5 mM hꢃ1, which was 0.6 (ꢂ0.3) times the rate of lysolipid
formation in the same period. This indicates that either
propranolol binding changes bilayer properties to facilitate
hydrolysis, or that propranolol is able to catalyse lipid hydro-
lysis in addition to undergoing transesterication reactions.
The lipidated propranolol products themselves may also
promote lipid hydrolysis, for example by inducing changes to
membrane properties that modify interfacial water activity, or
by direct involvement in acid–base catalysis.
samples as
a mixture of 1- and 2-oleoyl-sn-glycero-3-
phosphocholine (OPC), in addition to 1- and 2-palmitoyl-sn-
glycero-3-phosphocholine (PPC) in the case of POPC. The
predominant products in both cases arose from a trans-
esterication reaction involving the alcoholic group of
propranolol to form the O-acyl product (2). O- and N-acyl
modications to propranolol were distinguishable by the
shorter retention times for O-acyl propranolol derivatives in
comparison to their N-acyl counterparts (Fig. 1a and S1†) and by
their fragmentation patterns in tandem mass spectra produced
by collision-induced decay (CID). The CID MSMS spectra of O-
acyl propranolol (2) yielded a fragment of m/z 260, with species
of m/z 155 and m/z 183 having the highest relative abundance
(Fig. 1b and S1–S3, Table S1†). In contrast, N-acyl propranolol
homologues (3) were particularly notable for the high relative
abundance of fragments with an intact acyl chain, at m/z 338
and m/z 380 for N-oleoyl propranolol, alongside a high relative
abundance for the fragment at m/z 260 corresponding to the
loss of the acyl chain (Fig. 1c, S4, Tables S2 and S3†).
Aer instrument calibration, using data from authentic
standards to convert raw ion intensities to concentrations
(Fig. S5, Table S4†), the concentrations of both total lipidated Propranolol lipidation in lipid extracts
propranolol and lysolipid are seen to increase steadily over the
Having demonstrated lipidation of propranolol in vitro, it was of
rst 48 h following mixing (Fig. 2). At these early time points,
the predominant lipidated product is the O-acyl ester (Fig. S6†).
The concentration of the N-acyl amide exhibits a small latency
period before concentrations begin to increase, which indicates
that the N-acyl product may be formed by O to N migration of
the acyl group rather than direct aminolysis of the lipid. Such
a migration has been described in the literature for propranolol
ester derivatives in solution.8,10
fundamental interest to probe whether this reaction also
occurred in cellulo. In order to address this question propran-
olol was rst incubated with liposomes composed of commer-
cial lipid mixtures extracted from E. coli or liver cells (Fig. S7†)
so that the likelihood of lipidation occurring in complex
membranes could be assessed and the scale of the analytical
challenge understood, given the likely complexity of the
products.
The data are reproducible at time points up to 48 h, but show
greater sample-to-sample variability over longer periods,
particularly for the concentration of lysolipid. Aer 72 h the
In both of these experiments, the ability to identify lipidation
products was conrmed, with each extract yielding a series of
acyl-modied derivatives (Fig. 3a, S8, Tables 1 and S5†) with
676 | Chem. Sci., 2019, 10, 674–680
This journal is © The Royal Society of Chemistry 2019

View Article Online
Edge Article
Chemical Science
Table 1 Fatty acid profiles of bovine liver and Hep G2 phospholipids
and the acylated propranolol derivatives observed by LCMS analysis
after either propranolol (1) incubation with bovine liver extract lipo-
somes, or the growth of Hep G2 cells for 72 h in the presence of
propranolol (1)
Fatty
acid
Abund.a
(%)
Errorc
(ppm)
Source
Observed m/zb
Liver extract
14 : 0
16 : 0
18 : 0
18 : 1
18 : 2
20 : 3
20 : 4
22 : 4
—
11
29
10
8
4
16
2
6.4
33.4
13.8
5.7
31.5d
0.7
0.7
—
470.3618
498.3947
526.4276
524.4099
522.3951
548.4109
546.3932
574.4227
470.3637
3.4
0.0
3.0
0.9
0.7
1.0
2.8
5.8
+0.6
Hep G2 cells 14 : 0
16 : 0
16 : 1
18 : 0
18 : 1
18 : 2
20 : 1
20 : 2e
20 : 3e
498.3940, 498.3937f ꢃ1.4, ꢃ2.0f
496.3762
526.4266
ꢃ5.8
+1.2
524.4106, 524.4132f +0.4, +5.4f
522.3971, 522.3934f +4.6, ꢃ2.4f
552.4423
550.4255
548.4078
546.3949
570.3966
+1.1
ꢃ0.9
ꢃ4.7
+0.4
+3.3
—
20 : 4e 1.0
22 : 6
0.8
a
Proportion (mol%) of each fatty acyl chain found in total bovine liver
phospholipids26 or in lipids isolated from Hep G2 cells cultured in the
absence of propranolol in a medium containing 10% foetal bovine
serum.27 Some fatty acids reported in this study were not found as
modications to propranolol, including C18:3 (0.09 mol%), C20:5
Fig. 3 Overlaid extracted ion chromatograms (EICs) of the molecular
ions corresponding to the lipidated propranolol species formed by
incubation of propranolol with liver lipids. (a) Liposomes formed from
bovine liver extract ([M + H]+; calculated m/z ꢂ 6 ppm). (b) Acyl
modified propranolol derivatives extracted from Hep G2 cells cultured
in a medium containing 30 mM propranolol ([M + H]+; calculated m/z ꢂ
7 ppm).
b
(0.31 mol%) and C22:5 (0.92%). Observed m/z of acyl propranolol
following fatty acyl transfer from the lipid. Data are for the O-acyl
c
species unless otherwise stated. Error between observed and
calculated m/z for [M + H]+ following transfer of this fatty acid to
d
e
propranolol. Comprises 18:1n-7 (12.5%) and 18:1n-9 (19.0%). Not
shown in Fig. 3b. Retention times: 192 s (20:4); 224 s (20:3); 247 s
(20:2). f N-Acyl (amide).
relative abundances qualitatively in line with those expected
based on the fatty acid prole of the extract, including modi-
cations with cyclopropyl fatty acids for the E. coli extract and
a high relative abundance for stearoyl-modied propranolol for
the liver extract. Details of the lipid species identied are in the
ESI (Tables S6 and S7†). Some products anticipated on the basis
of the relative fatty acid abundances were not observed,
including modication with C22:5. A number of reasons could
account for the failure to observe these products, including
their presence at concentrations below the detection limit,
differences in the fatty acid prole of the commercial mixture
from published data, peak overlap leading to ion suppression,
and their presence in a form that is not reactive.
Propranolol lipidation in cellulo
Fig. 4 Tandem mass spectrometry analysis of samples extracted from
Hep G2 cells cultured in a medium containing propranolol. (a) Extracted
With knowledge that acylated propranolol derivatives could be
identied in complex mixtures of natural lipids, propranolol ion chromatograms (EICs) of product ions formed by targeted CID frag-
mentation of the ions with m/z 498.396 (magenta) and m/z 524.411 (blue),
lipidation in cellulo was probed. The Hep G2 cell line was
selected for this work in order to build on the data described
above for liver cell extracts and because propranolol is known to
induce phospholipidosis (PLD) in Hep G2 cells with an EC50
corresponding to [M + H]+ for palmitoyl propranolol and oleoyl propran-
olol respectively. A target mass window of ꢂ4 m/z was used for CID. The
chromatograms in (a) are the sum of the monoisotopic EICs for each ion in
Fig. S3 and S4† with a mass window of ꢂ8 ppm. Panels (b) and (c) show
between 12.6 and 16 mM.13 In our hands, incubation of Hep G2 individual mass spectra corresponding to the indicated peaks in (a).
This journal is © The Royal Society of Chemistry 2019
Chem. Sci., 2019, 10, 674–680 | 677

View Article Online
Chemical Science
Edge Article
cells with 30 mM propranolol induced PLD as expected discussed above. The identities of some of the lipidated
(Fig. S9†). By performing tests with POPC liposomes beforehand propranolol species were further conrmed by LC-MSMS anal-
(Fig. S10†), the use of a chloroform/methanol mixture for the ysis of the Hep G2 extract, with the palmitoyl- and oleoyl-
chemical extraction of Hep G2 cells was veried as the best modied propranolol parent species targeted for fragmenta-
approach for recovering both O- and N-acyl propranolol deriv- tion (Fig. 4). As evidenced by the relatively early retention times
atives. When compared with the starting liposome mixture, and a high relative abundance of product ions with m/z 155 and
a
partial reduction in the relative intensity of O-oleoyl m/z 183, the predominant products were O-acyl propranolol
propranolol was found in the extracts, indicating a reduced derivatives. Only trace quantities of the N-acyl species could be
extraction efficiency for propranolol modied with unsaturated detected (Fig. 4, peaks iii and vii). The CID target mass window
fatty acyl groups. However, in the worst-case scenario all species was sufficiently wide to conrm the presence of other related
could at least be partially recovered. Extraction of Hep G2 cells species in the mixture, including O-palmitoleoyl (Fig. 4, peak i),
by this approach, followed by LC-MS analysis, identied a range O-linoleoyl (peak iv) and O-stearoyl propranolol (peak vi).
of lipids (Fig. S11, Table S8†) alongside 14 different lipidated
propranolol species (Fig. 3b, Table 1).
In contrast to the experiments with extracted lipid mixtures,
the reactivity of propranolol in Hep G2 membranes might be
The predominant fatty acyl modications to propranolol expected to produce a response that is inuenced by the pres-
observed in the chromatograms, palmitoyl and oleoyl (Table 1), ence of lipid homeostasis in the cell. Indeed signicant differ-
were those anticipated on the basis of published fatty acid ences were found in the intensities of some lipids when
proles for Hep G2 cells cultured in the same medium, comparing cells cultured with or without propranolol (Table
considered alongside the differences in extraction efficiency S9†). These included decreases in the levels of some triglycer-
ides and phosphatidylserine, and striking increases in the levels
of ether-linked lipids, which would have the effect of reducing
the ester content of the membrane that is transferrable in
intrinsic lipidation reactions.
The biological effects of drug lipidation
As we had evidence that propranolol reacts with membrane
lipids both in vitro and in vivo, leading to the generation of
lysolipids and a lipidated drug, it was of fundamental interest to
establish whether this reactivity had the potential to induce
detectable physiological effects. Previous work has established
that some classes of drug linked with phospholipidosis are able
to promote lipid hydrolysis in model systems in vitro, potentially
by acting as phase transfer catalysts.22,23 We therefore explored
whether propranolol and other compounds known to induce
phospholipidosis (Fig. 5) were subject to lipidation in a model
Fig. 5 Drug molecules studied in this work in addition to propranolol
(1).
Fig. 6 (a) Evolution of the 1/2-oleoyl-sn-glycero-3-phosphocholine (OPC) concentration in liposomes composed of DOPC (1.27 mM) following
exposure to different drugs (0.127 mM) at pH 7.4 and 37 ꢁC. Errors are mean ꢂ s, n ¼ 3. Raw data are shown as points, linear fits to the reaction rate over
the first 24 h as lines. (b) Relationship between the rate of lysolipid formation in DOPC and phospholipidosis activity12,13 for each of the drugs in part (a).
678 | Chem. Sci., 2019, 10, 674–680
This journal is © The Royal Society of Chemistry 2019

View Article Online
Edge Article
Chemical Science
DOPC system or otherwise inuence the levels of lysolipid, and
examine the correlation between these activities and phospho-
lipidosis activity. Interestingly, only propranolol underwent any
lipidation reactions in DOPC membranes. However, all of the
compounds with appreciable phospholipidosis activity yielded
detectable increases in the initial rate of lipid hydrolysis over
24 h (Fig. 6a) compared to a control in the same conditions
without compound. It may be the case that the amino group of
propranolol is positioned in such a manner that it can promote
the transesterication reactions of propranolol, but in the
absence of proximal acyl group acceptor, the amino groups of
the other drugs favour reactions involving interfacial water.
Although there is a correlation between phospholipidosis
activity and hydrolysis rates, with for example uoxetine
producing the fastest rate of lipid hydrolysis and having the
lowest EC50 (Fig. 6b), the relationship is not simple, as indicated
by the non-linearity in Fig. 6b.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
The authors thank the EPSRC (EP/M506321/1) for funding.
Notes and references
1 R. H. Dods, J. A. Mosely and J. M. Sanderson, Org. Biomol.
Chem., 2012, 10, 5371–5378.
2 R. H. Dods, B. Bechinger, J. A. Mosely and J. M. Sanderson,
J. Mol. Biol., 2013, 425, 4379–4387.
3 C. J. Pridmore, J. A. Mosely, A. Rodger and J. M. Sanderson,
Chem. Commun., 2011, 47, 1422–1424.
It might be expected that both the lipidated drug and the
lysolipid have the potential to disrupt lipid membranes as
a consequence of their shape, which favours detergent-like
activity. Both N-oleoyl and N-palmitoyl propranolol were
found to have measurable micelle-forming behavior, with CMCs
of 9 mM and 10 mM respectively (Fig. S12†). Addition of both of
these N-acyl compounds at a concentration of 1 mol% to POPC
liposomes loaded with the markers 8-aminonaphthalene-1,3,6-
trisulfonic acid (ANTS, 12.5 mM) and p-xylene-bis-pyridinium
bromide (DPX, 45 mM) resulted in the loss of membrane
integrity with concomitant increase in uorescence (Fig. S13†).
Similar results were obtained for monooleoyl and monop-
4 V. S. Ismail, J. A. Mosely, A. Tapodi, R. A. Quinlan and
J. M. Sanderson, Biochim. Biophys. Acta, 2016, 1858, 2763–
2768.
5 A. Avdeef, K. J. Box, J. E. Comer, C. Hibbert and K. Y. Tam,
Pharm. Res., 1998, 15, 209–215.
6 L. Herbette, A. M. Katz and J. M. Sturtevant, Mol. Pharmacol.,
1983, 24, 259–269.
¨
7 S. D. Kramer, A. Braun, C. Jakits-Deiser and
H. Wunderli-Allenspach, Pharm. Res., 1998, 15, 739–744.
8 C. G. Jordan, J. Pharm. Sci., 1998, 87, 880–885.
9 J. Quan, N. Wang, X.-Q. Cai, Q. Wu and X.-F. Lin, J. Mol.
Catal. B: Enzym., 2007, 44, 1–7.
almitoyl PC. Parallel analyses using O-acyl species proved to be 10 J. M. Quigley, C. G. M. Jordan and R. F. Timoney, Int. J.
problematic due to the rapid hydrolysis of these species in the
absence of membranes.
Pharm., 1994, 101, 145–163.
11 C. Udata, G. Tirucherai and A. K. Mitra, J. Pharm. Sci., 1999,
88, 544–550.
12 U. M. Joshi, P. Rao, S. Kodavanti, V. G. Lockard and
H. M. Mehendale, Biochim. Biophys. Acta, 1989, 1004, 309–
320.
Conclusions
This work has demonstrated the fundamental point that low
molecular weight organic molecules are capable of generating 13 S. A. Shahane, R. Huang, D. Gerhold, U. Baxa, C. P. Austin
lytic chemical processes in lipid membranes, including direct and M. Xia, J. Biomol. Screening, 2014, 19, 66–76.
intermolecular reactions such as transesterication, or the 14 J. M. Alakoskela, P. Vitovic and P. K. Kinnunen,
promotion of other reactions such as hydrolysis. However, as ChemMedChem, 2009, 4, 1224–1251.
evidenced by the reactivity of propranolol in comparison to 15 N. Anderson and J. Borlak, FEBS Lett., 2006, 580, 5533–5540.
drugs such as uoxetine and sertraline, the selectivity for lip- 16 U. M. Hanumegowda, G. Wenke, A. Regueiro-Ren,
idation vs. hydrolysis is complex and most likely linked to
a number of factors, including the molecular disposition in the
R. Yordanova, J. P. Corradi and S. P. Adams, Chem. Res.
Toxicol., 2010, 23, 749–755.
membrane interface, the distribution prole (log D) and the pKa 17 I. Loryan, E. Hoppe, K. Hansen, F. Held, A. Kless, K. Linz,
values of any ionisable groups. Furthermore, it is clear that the
activities observed in vitro are also likely to occur in vivo. This
has been demonstrated directly by the observation of the
products of lipidation reactions of propranolol in Hep G2 cells
V. Marossek, B. Nolte, P. Ratcliffe, D. Saunders,
R. Terlinden, A. Wegert, A. Welbers, O. Will and
M. Hammarlund-Udenaes, Mol. Pharm., 2017, 14, 4362–
4373.
and indirectly by correlations between the hydrolysis promoting 18 R. Lowe, H. Y. Mussa, F. Nigsch, R. C. Glen and J. B. Mitchell,
activity of drugs in vitro and their EC50 for phospholipidosis. In J. Cheminf., 2012, 4, 2.
the case of propranolol it is also striking that the O-acyl esters, 19 J. K. Morelli, M. Buehrle, F. Pognan, L. R. Barone, W. Fieles
which normally rapidly rearrange to the N-acyl amide counter-
and P. J. Ciaccio, Cell Biol. Toxicol., 2006, 22, 15–27.
part in vitro, exhibit increased stability in the membrane envi- 20 P. Nioi, B. K. Perry, E. J. Wang, Y. Z. Gu and R. D. Snyder,
ronment. This observation highlights the fact that there is Toxicol. Sci., 2007, 99, 162–173.
much concerning the chemistry that occurs in the membrane 21 A. Arouri and O. G. Mouritsen, Prog. Lipid Res., 2013, 52, 130–
interface that remains to be understood.
140.
This journal is © The Royal Society of Chemistry 2019
Chem. Sci., 2019, 10, 674–680 | 679

View Article Online
Chemical Science
Edge Article
22 M. Baciu, S. C. Sebai, O. Ces, X. Mulet, J. A. Clarke, 25 R Core Team, R: A Language And Environment For Statistical
G. C. Shearman, R. V. Law, R. H. Templer, C. Plisson, Computing, 2017.
C. A. Parker and A. Gee, Philos. Trans. R. Soc., A, 2006, 364, 26 P. Couture and A. J. Hulbert, J. Membr. Biol., 1995, 148, 27–
2597–2614. 39.
23 D. Casey, K. Charalambous, A. Gee, R. V. Law and O. Ces, J. R. 27 P. J. Gunn, C. J. Green, C. Pramfalk and L. Hodson, Physiol.
Soc., Interface, 2014, 11, 20131062.
Rep., 2017, 5, e13532.
24 C. A. Smith, E. J. Want, G. O'Maille, R. Abagyan and
G. Siuzdak, Anal. Chem., 2006, 78, 779–787.
680 | Chem. Sci., 2019, 10, 674–680
This journal is © The Royal Society of Chemistry 2019