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Predicting a Drug’s Membrane Permeability: A Computational Model

Validated With in Vitro Permeability Assay Data
Brian J. Bennion,†,# Nicholas A. Be,†,# M. Windy McNerney,†,‡ Victoria Lao,† Emma M. Carlson,§
Carlos A. Valdez,∥ Michael A. Malfatti,† Heather A. Enright,† Tuan H. Nguyen,⊥ Felice C. Lightstone,†
and Timothy S. Carpenter*,†
†
Biosciences and Biotechnology Division, Lawrence Livermore National Laboratory, Livermore, California 94550, United States
‡
War Related Illness and Injury Study Center, Veterans Affairs, Palo Alto, California 94304, United States
§
U.S. Naval Academy, Annapolis, Maryland 21402, United States
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

∥
Nuclear and Chemical Sciences Division, Lawrence Livermore National Laboratory, Livermore, California 94550, United States
⊥
Global Security Directorate, Lawrence Livermore National Laboratory, Livermore, California 94550, United States
*
S Supporting Information
Downloaded via 157.47.108.80 on December 1, 2022 at 04:48:53 (UTC).

ABSTRACT: Membrane permeability is a key property to consider

during the drug design process, and particularly vital when dealing with
small molecules that have intracellular targets as their efficacy highly
depends on their ability to cross the membrane. In this work, we
describe the use of umbrella sampling molecular dynamics (MD)
computational modeling to comprehensively assess the passive
permeability profile of a range of compounds through a lipid bilayer.
The model was initially calibrated through in vitro validation studies
employing a parallel artificial membrane permeability assay (PAMPA).
The model was subsequently evaluated for its quantitative prediction of
permeability profiles for a series of custom synthesized and closely
related compounds. The results exhibited substantially improved
agreement with the PAMPA data, relative to alternative existing
methods. Our work introduces a computational model that underwent progressive molding and fine-tuning as a result of its
synergistic collaboration with numerous in vitro PAMPA permeability assays. The presented computational model introduces
itself as a useful, predictive tool for permeability prediction.

■ INTRODUCTION
Most drugs need to pass through at least one cellular
membrane to reach their intended target. Although tight
binding of a drug molecule to its intended target is important
for potency, poor membrane permeability often translates into
poor or nonexistent in vivo efficacy. Thus, a detailed
understanding of the partitioning of a given species in the
membrane is vitally important from a pharmacokinetics and
rational drug design standpoint. In eukaryotic systems, two
possible transport modes are available for a molecule to pass
through a membrane: active and passive.1 Active transport
involves a transport protein that uses energy (e.g., ATP
hydrolysis) to shuttle a molecule across a membrane. In
contrast, passive transport, which is the most common mode of
drug passage through membranes, involves diffusion of a
molecule through the membrane with no outside assistance or
energy input. The rate of passive diffusion across a membrane is
proportional to the partition coefficient of the compound
between the membrane (lipophilic environment) and the
external medium (aqueous milieu), the diffusion coefficient of
the compound through the membrane, and the compound’s
concentration gradient across the membrane.2 Important
chemical properties of small molecules for the process of
membrane binding and diffusion are lipophilicity, molecular
weight, and measures of molecular polarity.3 Thus, the
development of a successful drug involves a fine balance
among all these properties in a molecular scaffold, which is no
trivial matter.
Membrane permeability is a key metric in the drug design
pipeline. A drug intended for an intracellular target, but with
poor permeability will have low efficacy. As a result of these
characteristics several well-characterized in vitro and in silico
permeability prediction methods have been developed. These
methods are relatively high throughput, and are important in
the early stages of the drug discovery process with some of the
most common and relatively simple in vitro methods being the
parallel artificial membrane permeability assay (PAMPA), the
immobilized artificial membrane (IAM) technique, and
immobilized liposome chromatography (ILC). The PAMPA
in vitro technique was developed in 1998 by Kansy et al.4 and
Received: March 27, 2017
Revised: April 28, 2017
Published: April 28, 2017

© 2017 American Chemical Society 5228 DOI: 10.1021/acs.jpcb.7b02914

J. Phys. Chem. B 2017, 121, 5228−5237
The Journal of Physical Chemistry B
Figure 1. Control compounds used in the calibration data set.
originally established to rapidly predict passive permeability
through the gastrointestinal tract, but has since been adapted
for use in other systems such as the blood brain barrier (BBB),5
for which it demonstrated a good prediction of BBB
penetration.5,6 Briefly, the technique involves a donor and an
acceptor compartment separated by a filter supporting a liquid
artificial membrane. The artificial membrane can be composed
of a variety of phospholipid mixtures. The compound to be
tested is placed in the donor compartment and is allowed to
permeate between the donor and the acceptor compartments
through the artificial membrane. As the assay can be performed
in 96-well plates, high throughput screening of drug candidates
is possible. Alternatively, IAMs mimic the phospholipid
environment of the cellular plasma membrane by anchoring
synthetic lipid analogues to silica particles in monolayer density.
These particles are then used as the packing material for a high-
performance liquid chromatography column.7−10 The IAM
chromatographic retention factors are used to generate
predictions of membrane permeability. These systems have
shown reasonable results for prediction of permeability, despite
the retention time in the column not reflecting actual passage
across the membrane.11,12 The ILC method is used to study
solute-membrane interactions, where liposomes are non-
covalently immobilized to gel beads. The liposomes can have
varied compositions, including numerous different phospholi-
pids. Lipids extracted from human red cells have also been used
in ILC assays.13
An alternative approach to assess membrane permeability is
to employ computational methods. Most in silico prediction
approaches use quantitative structure-permeability relationship
(QSPR) models. These predictive, quantitative models applied
in drug design are methods that utilize statistical relationships
extracted from experimental permeability measurements
combined with the physiochemical properties of a set of
training compounds. Thus, in QSPR studies, the permeability
of the compound is an outcome that is the result of the various
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interactions that a compound experiences during passage
through the membrane. The interactions between the
compound and the membrane are assumed to be governed
by the chemical structure and properties of the compound,
which are termed descriptors. As such, a mathematical model of
biological permeability is optimized based on a combination of
a variety of descriptors for the small molecule compound. Thus,
a change in structure can result in a change in permeability.
QSPR models have been developed to model and predict,
among others, oral bioavailability,14 intestinal absorption,15
Caco-2 permeability,16 and blood-brain barrier penetration.17
The degree of success of these models is extremely dependent
on the compounds in the training set. Thus, the applicability of
the approach can be limited, and transferability can be a major
issue.18 Even successful QSPR models can be limited because
they do not provide any insight into the mechanisms that
govern the permeability.19
For prediction of passive membrane permeability, the most
critical parameter in QSPR studies is lipophilicity.20 Lip-
ophilicity is a crucial factor governing passive membrane
partitioning,21 and calculated lipophilicity metrics are often
utilized to predict drug absorption.22 The parameter that
determines the lipophilicity of a molecule is LogP (the partition
coefficient of the molecule between an aqueous and lipophilic
phase, usually water and octanol). LogP is a crucial factor
governing passive membrane partitioning; an increase in LogP
enhances permeability.21 While the partition coefficient is used
to calculate properties such as membrane permeability and
water solubility, it also has importance in the prediction of
biological activities, ADME, and toxicological end points. Thus,
reliable prediction of LogP is of massive importance in the drug
design process, as it is important to know lipophilic properties
of a compound before synthesis.23 There are several methods
used to calculate the LogP parameter for a initial, rapid
prediction of lipophilicity and, thus, permeability. Atomic-based
LogP predictions take each atom’s contribution to the LogP of
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the compound and combine them additively.24 This method is
in essence a look-up table per atom and suited to smaller, less
complex molecules. More advanced “hybrid” LogP methods use
the LogP contribution of each atom, as well as a contribution
from the neighboring atoms, and combine them with correction
factors. In a fragment-based approach, data determined
experimentally from different chemical moieties, or chemical
“fragments” are modeled, and these contributions are again
added up with correction factors. The motivation is that
atomic-based approaches do not always properly capture
certain molecular subtleties. Thus, fragment-based methods
tend to be better for complex, larger molecules. However, the
assumption is the molecule contains fragments that are similar
to those from which the model was constructed.
One of the most powerful (though computer intensive) in
silico techniques to simulate the molecular process of diffusion
at the atomic level is molecular dynamics (MD).25 Coupling
MD with free energy techniques provides a powerful tool to
study membrane permeability in detail.26 While computational
models can often correlate well with experiment, knowledge-
based QSPR methods are often less accurate (but inexpensive)
compared to MD methods.27 Although human membranes are
a complicated mixture of lipids and proteins, with a judicious
choice of lipid, a single bilayer can provide a good first
approximation of the physicochemical properties of a cellular
membrane. Indeed, phosphatidylcholine lipids are the major
phospholipid within cellular membranes.28 Previous studies
have investigated the permeation of small compounds (and a
limited number of drug-like molecules) through homogeneous
lipid bilayers.29−35 Thus, with increasing computing power,
these methods will become an increasingly valuable tool for
parameter prediction.
The work described herein demonstrates robust consistency
between the in vitro PAMPA approach and the predictive
capacity of MD calculations. Our predictive MD approach is
calibrated using the equivalent PAMPA experimental measure-
ments. The semiquantitative predictive power of the method-
ology is then rigorously tested using a custom synthesized set of
structurally related compounds (compounds LLNL1−
LLNL18), demonstrating the potential utility of these methods
for permeability assessment of novel chemical entities.
■
EXPERIMENTAL AND THEORETICAL METHODS
In this study, two sets of compounds were characterized, the
calibration data set and the prediction data set. The calibration
data set was selected to cover a wide range of experimentally
known permeability values and is comprised of the following
compounds: progesterone, chlorpromazine, promazine, atro-
pine, diazepam, theophylline, 2-PAM, Hi-6, and MMB4 (Figure
1). The calibration compounds were commercially obtained
(Sigma-Aldrich). The prediction data set (compounds
LLNL1−LLNL18) is a structurally related set of compounds
that were designed and synthesized in order to provide a set of
compounds with a narrow range of permeabilities with which to
rigorously test our methodology and predictive capabilities.
General Synthetic Procedure. The prediction set of
compounds used in this study was synthesized via an amide
forming reaction between an amine and an ethyl ester species.
The library of generated materials can be assembled from an
ethyl ester and a series of amines that serve as the point
structural diversity origin. Since the carboxylic acid component
(ethyl ester) is not an activated species, the amine was used in
slight excess to it (1.2 equiv) and the mixture was heated to 70
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°C in an overnight basis in ethanol. In some instances, the
products precipitate out of the solution (purity >99% after
ethanol washes during filtration) while in some cases flash
column chromatography (3:7 → 7:3 EtOAc:hexanes) is needed
for their purification. Purity of the compounds was assessed by
1
H NMR (DMSO-d6) and in all cases found to be >98%.
Molecular Dynamics Setup. The simulation setup and
protocol was the same as our previous published inves-
tigation.36 In brief, each simulation contained a single copy of
the compound harmonically restrained at specified locations
along the z-axis in a solvated DOPC bilayer system. The
complete system contained 5124 water molecules, 72 DOPC
molecules, and one small compound. A typical system
contained a total of ∼19 300 atoms. This system is comparable
to similar studies. We acknowledge that larger membrane
systems may be required to investigate the permeability of
larger molecules. The simulations were run using GROMACS
4.5.5,37 with the Berger et al. force field used for the DOPC
molecules38 as adapted by the Tieleman group (http://wcm.
ucalgary.ca/tieleman/downloads), the GROMOS 53A6 force
field used for the small compounds,39 and the SPC model used
to represent the water,40 with the simulation parameters the
same as Carpenter et al.36 The topologies for the small
molecule compounds were generated using the Automated
Topology Builder server (https://atb.uq.edu.au).41−43 The
topology builder combines a knowledge-based approach with
quantum mechanical calculations to produce parameters that
are compatible and consistent with a given force field. During
the initial stage, the molecule was optimized at the HF/STO-
3G level of theory. The molecule was then reoptimized at the
B3LYP/6-31G* level of theory44−46 in implicit water. The
charges were initially estimated by fitting the electrostatic
potential using a Kollman−Singh scheme.47 The Hessian
matrix was then calculated and used to estimate the force
constants for both the bonds and angles. All compounds were
modeled in their neutral forms and other physiologically
relevant charged states. The ChemAxon/Chemicalize server48
was used to calculate the pKa values for the ionizable atoms of
each compound, and thus determine the most prevalent charge
species at physiological pH of 7.4. The parameters for the
calibration data set are freely available from the Automated
Topology Builder server.
Free Energy Calculations. The potential of mean force
(PMF) free-energy profiles for the partitioning of compounds
was calculated using umbrella sampling simulations. A single
harmonic restraint with a force constant of 1000 kJ mol−1 nm−2
was applied to the z-axis (normal to the bilayer) distance
between the center of mass of the compound and the center of
mass of the DOPC bilayer. One hundred separate simulations
were performed, with the compound harmonically restrained in
increments of 0.1 nm along the z-axis direction. These 100
simulations completely span the 10 nm distance from bulk
water, through the entire membrane and out into bulk water
again. Each simulation window was initially run for ∼50 ns, for
a total of ∼5 μs of simulation time for a complete set. As the
position of a molecule within the membrane can shift its pKa
value, most compounds were simulated in both their neutral
forms and most physiologically relevant charge species,
resulting in ∼10 μs of simulation data for each compound. A
total of 27 different compounds were simulated for a combined
∼250 μs of simulation time. On the basis of our previous
experience, the initial 20 ns of each simulation was discarded as
equilibration time and as such only the final ∼30 ns of each
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simulation was used for all subsequent analyses. The weighted
histogram analysis method,49 as implemented within GRO-
MACS, was used to calculate the PMF for each compound, and
all free-energy profiles were normalized to zero in bulk water.
Diffusion and Permeability. The membrane permeability
of a small molecule was calculated using the same methodology
as previously published.36 Briefly, information from the PMF
1D energy landscape was combined with diffusion coefficients
within the membrane to calculate the membrane permeability
rates. The position-specific diffusion coefficients were calculated
from the MD simulation data using the method of Hummer.50
For each umbrella sampling simulation, the compound
positional variance and autocovariance as a function of lag
time were calculated. These calculations were repeated for a
number of subsamples of each trajectory. The resulting
autocovariance curves decay roughly exponentially with
increasing lag time. The characteristic time, τ, of each
autocovariance decay was estimated by making a least-squares
fit to the log of the autocovariance data. The diffusion
coefficient for each subsample was then calculated as:
var(Z)
D(⟨Z⟩) =
τZ (1)
where ⟨Z⟩ is the average of the z-axis position of the compound
center of mass during the simulation and var. (z) is the variance
of the compound center of mass (the autocovariance at “lag
zero”), and the average over the subsamples was calculated. The
standard deviation of the diffusion coefficient over the
subsamples was also calculated for use in a sensitivity analysis,
described below. The effective membrane permeability, Peff, was
calculated from the effective resistivity, Reff, (as derived by
Marrink and Berendsen51),
1 Z
Peff
= R eff = ∫0 R (z ) d z
(2)
where R(z) is the resistivity of every “slice” of the membrane at
position “z”,
e β(ΔG(z))
R (z ) =
D(z) (3)
where β is the inverse of the Boltzmann constant times the
temperature, and ΔG(z) is the free energy from the PMF
calculations. The integral is over the width of the membrane.
Error Estimation. As a metric for confidence in our results,
the error of Peff was determined. In order to calculate this
overall error of Peff, we first had to separately resample both the
PMF and diffusion profiles. The sensitivity analysis used a
random resampling of the position-specific diffusion coef-
ficients. As described previously, for each compound, position-
specific diffusion coefficients were calculated as well as the
standard deviation of each position-specific coefficient. These
calculations were based on independent subsamples of the
trajectory from each umbrella-sampling simulation. Since the
mean and variation of each calculated coefficient is an
independent calculation, the correlation of the variations is
zero. The resampled diffusion coefficients were thus based on
independent draws from a set of normal distributions, where
each such distribution has a mean and standard deviation
corresponding to the calculated mean and standard deviation of
each position-specific diffusion coefficient. We used 50 random
draws for each coefficient to produce a set of 50 diffusion
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profiles that were distributed around the calculated data
according to the normal distribution of that data.
Resampling of the PMF curves was calculated using the
bootstrapping method as implemented within the g_wham52
analysis program of GROMACS. Again, 50 separate PMF
profiles were generated, from which the mean and standard
deviations calculated. These standard deviations of the PMF
were used as an additional quality control metric. Any PMF
profile that exhibited a standard deviation of >0.5 kcal mol−1
was subjected to extended simulation and supplementary
sampling in order to reduce this deviation. The PMF histogram
overlap was also checked to ensure correct sampling.
In order to determine the error of the Peff, the data in the 50
diffusion profiles were combined with the data in the 50 PMF
profiles using eq 3 to produce 50 similar, but different, Peffs.
The standard deviation for this set of 50 Peffs was taken as the
error.
The Parallel Artificial Membrane Permeability Assay
(PAMPA). PAMPA was applied to screen compounds for
passive diffusion. This study employed the Gentest Precoated
PAMPA Plate System (Corning Discovery Labware). The
system is composed of a 96-well plate/insert system. Two fluid-
filled compartments (donor well and receiver well) are
separated by a polyvinylidene fluoride (PVDF) filter plate
precoated with a phospholipid-oil-phospholipid trilayer primar-
ily consisting of DOPC phospholipids.53 Compounds of
interest were dissolved in Hanks Balanced Salt Solution
(HBSS) at a concentration of 100 μM. Solutions were added
to donor wells at a volume of 0.3 mL. The filter plate,
containing acceptor wells filled with 0.2 mL blank HBSS, was
placed on top of the donor plate. The entire system was
incubated for 5 h at 25 °C. Following incubation, solution was
removed from donor and acceptor wells. Compounds were
then reserved for analysis via ultra performance liquid
chromatography (UPLC). Certain compounds exhibited very
low traversal to the acceptor well, such that material could not
be detected via UPLC upon initial analysis. Such compounds
were concentrated from donor and acceptor solution fractions
by extraction into methanol, followed by vacuum concentration
and resuspension in the minimal amount of appropriate UPLC
solvent.
UPLC Conditions. Compounds in solution were quantified
using the Acquity ultra performance liquid chromatography
(UPLC) system (Waters) with the Acquity BEH C18 column
(1.7 μm × 2.1 mm × 50 mm) at a pressure of 7500 PSI. Here,
10 μL were injected for each sample. Detection was based on
retention time and UV absorbance. Specific detection protocols
were determined, where necessary for each compound
(Supporting Information).
Permeability Calculations. The concentration data was
used to calculate effective permeability (cm/s) as follows:
−ln[1 − CA(t )/Ceq]
Pe =
A × (1/VD + 1/VA) × t (4)
where A is the filter insert area (0.3 cm2), VD is the volume of
the donor well (0.3 mL), VA is the volume of the acceptor/
receiver well (0.2 mL), CA(t) is the concentration in the
acceptor well at time t, and t = incubation time (18 000 s). Ceq
was calculated according to the following equation:
Ceq = [CD(t ) × VD + CA(t ) × VA]/(VD + VA) (5)
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where CD(t) is the concentration in donor well at time t, and

the other values are the same as previously defined.
LogP Calculations. CLogP. CLogP is a proprietary method
that is owned by BioByte/Pomona College. This method is a
fragment-based approach, whereby experimentally determined
fragments are modeled using QSPR (rather than per atom).
The CLogP program54−56 has become the benchmark, or gold-
standard, method for LogP prediction in the last 35 years and is
the most extensively tested fragment-based LogP calculator
used in drug design.
miLogP. The Molinspiration method for LogP prediction
(miLogP) is also based on group/fragment contributions. The
fragment contributions were obtained by fitting a training set of
>12 000 mostly drug-like molecules with experimental LogP
data. The authors state that miLogP is calculated using the Figure 2. PMF free energy curves for the passage of the control
methodology developed by Molinspiration as a sum of compounds from water (x-axis >3 nm) to the center of the bilayer (x-
fragment-based contributions and correction factors.57,58 axis <1 nm). As the membranes are symmetrical, the absolute
Chemicalize LogP. The LogP calculations (termed “(c)- distances of the compound from the bilayer center are used generate
LogP”) implemented from the Chemaxon/Chemicalize serv- these curves. Generally, a negative free energy at the bilayer center
er48 are based on a pool of predefined fragments. This pool of correlates with a more permeable compound.
fragments is based on the data in Viswanadhan et al.,59 with a
few minor adaptations (such as redefining atomic types to
accommodate electron delocalization and contributions of ionic
forms, or considering the potential for hydrogen bonding to
form a six membered ring between suitable donor/acceptor
atoms).
MOE SLogP. Chemical Computing Group’s MOE60 uses a
hybrid LogP prediction method termed “SLogP”. This method
is based on an atomic contribution model.61 The SLogP
training set to calibrate the model was ∼7000 structures.

■ RESULTS
Our computational methodology is first calibrated against the
well-established PAMPA protocol using a set of diverse, highly
studied compounds. The predictive power of the approach is
then tested with a synthesized set of structurally similar
compounds and compared against the equivalent predictive
power of readily available LogP calculators.
Calibration. We are focused on improving the predictive
capability of our membrane permeability model based on our
previous work.36 In order to do this, we continue to run
simulations and carry out experimental assay validation on our
data set of “calibration” compounds (Figure 1). This set of
compounds is comprised of molecules that fit a specific set of
criteria: (1) a significant amount of previous experimental work
is available to compare against our results, and (2) the
compounds must also cover a wide range of permeability values
to evaluate the performance of our model across this range.
The potential of mean force (PMF) free energy curves for
these compounds is generated from the umbrella sampling MD
simulations (Figure 2), and in keeping with previous results,36 it
shows that (as a general rule) if a compound has a higher free
energy in the middle of the bilayer, it will be less permeable.
The PMF values for the calibration compounds (along with
diffusion data also extracted from the MD simulations) are used
to determine Peff (effective permeability) of the compounds
(termed “PeffPMF”). These PeffPMF values are then compared to
the equivalent P eff measured from running the same
compounds through in vitro PAMPA assays (termed
“PeffPAMPA”), which also measure passive permeability (Figure
3).
The linear correlation between our predicted P effPMF
permeability rates and those experimentally measured PeffPAMPA
5232
Figure 3. Comparison between the experimentally measured Peff
(using PAMPA, x-axis) and the computationally predicted Peff (using
the PMF, y-axis). The plots are divided into regions of differing
permeability. A red region is defined as impermeable, an orange region
is defined as having a low permeability, a yellow region is defined as
having a medium permeability, and a green region is defined as having
a high permeability.
is extremely good (R2 = 0.97). Using these measured
experimental permeability rates, as well as permeability data
from literature, we are able to divide our data into four groups;
high permeability (green), medium permeability (yellow), low
permeability (orange), and impermeable (red). The boundaries
between these regions are defined by using the PeffPAMPA
midpoints between different compounds of known permeability
categories (identified from literature). The boundary between
the “impermeable” and “low permeability” groups is chosen
such that the size of the “low permeability” region is uniformly
distributed about the PeffPAMPA value for the “low permeability”
control compound (theophylline). These cutoff values of the
experimentally measured PeffPAMPA are determined such that
impermeable compounds have a log PeffPAMPA of ←6.14, low
permeability compounds have a log PeffPAMPA of > −6.14 and <
−5.66, medium permeability compounds have a log PeffPAMPA of
> −5.66 and < −5.33, and high permeability compounds have a
log PeffPAMPA of > −5.33. By fitting against the linear regression
line, we can use these in vitro cutoff values to define the
equivalent cutoff values for our predicted log PeffPMF (Figure 3
and Table 1). Thus, the cutoff values for our predictions are
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Table 1. Experimentally Measured and Computationally
Predicted Permeation Rates of the Calibration Compounds
impermeable compounds, log PeffPMF < −2.05; low permeabilty
compounds, log PeffPMF > −2.05 and <0.15; medium
permeabilty compounds, log PeffPMF > 0.15 and <1.62; high
permeability compounds, log PeffPMF > 1.62. These values are in
agreement with our previous studies, where the boundary
between impermeable/poorly permeable and highly permeable
is defined as a log PeffPMF of ∼0.36 By defining what the PeffPMF
cutoffs and thresholds are for these different permeability
categories, we are able to compute the PeffPMFs for novel
compounds and use those values to predict the permeability
categories for those compounds.
Prediction. Using these cutoffs, we attempt to semi-
quantitatively predict the permeability of 18 of our synthesized
set of structurally related compounds. This prediction is
significantly more difficult than correlating the control
compounds, as the range of permeabilities of these compounds
is much smaller. Despite this, however, we were able to
correctly predict the permeability category of 78% (14 of 18) of
the novel compounds (Figure 4). The four compounds that are
Figure 4. Prediction of the permeability category of our set of 18
structurally related compounds. Almost 80% of the compounds had
their permeability category correctly predicted.
assigned into the wrong permeability category are computa-
tionally predicted to have a “low permeability”, while
experimentally they are classified as impermeable (Table 2).
Thus, we have obtained a few “false positive” results. However,
we have successfully identified (and categorized) all eight of the
compounds with some level of permeability (low, medium, or
high). From a drug design point of view, a false positive is a
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better outcome than obtaining false negatives that actually do
possess permeability, but are predicted to be impermeable.
Comparison to LogP Predictions. The semiquantitative
permeability prediction results generated from our PMF-based
methodology are compared with the equivalent calibration and
predictions using several commonly used LogP calculation
tools (Table 3). LogP prediction tools are used in lieu of QSPR
models, which are primarily built upon LogP values. These
LogP calculation tools are the atomic/hybrid methodology of
MOE’s SLogP, and the fragment-based methods of miLogP
(from Molinspiriation), (c)LogP (from Chemicalize), and
CLogP (from Biobyte/Pomona College). The details of these
techniques are described in the Methods section. The LogP
calculation tools are subjected to the same analytic approach as
for the PeffPMF results. The various LogP calculations are first
calibrated against the PeffPAMPA measurements for the control
compounds to define LogP cutoffs between the four different
permeability categories. These defined thresholds for each of
the LogP tools are used to categorize our 18 custom
compounds (SI, Figures S1 and S2).
The LogP prediction tools performed more poorly than our
PMF-based methodology in both the correlation against the
control compounds and the semiquantitative prediction. While
the correlation coefficient for our PMF-based methodology is
0.97, the range for the LogP tools is only 0.44−0.75. The LogP
tools have difficulty with the unusual structures of our negative
controls that are known to be impermeable. Furthermore, all of
the LogP tools have a significant number of false negative
permeability predictions, with no method able to correctly
identify more than two (of the eight) compounds that exhibit
any form of experimentally categorized permeability. In fact,
two of the methods (SLogP and miLogP) fail to identify any of
these compounds that have categorized permeability. Indeed,
regardless of calibration, each of the LogP calculation tools
assign a higher LogP (greater permeability) to some
compounds measured to be impermeable than those measured
to have a medium (or even high) permeability. In comparison,
our PMF-based methodology has no such occurrences and
correctly identifies all of the compounds with experimentally
measured categorized permeability. Indeed, even the four
impermeable compounds that our method incorrectly catego-
rize as low permeability compounds still all have lower
predicted permeability that the four compounds correctly
identified to have low permeability. Thus, a “manual
recalibration” of the impermeable/low permeability threshold
cutoffs would allow our data to be categorized such that all 18
compounds are predicted correctly. This is impossible to
achieve with any of the LogP data sets. Additionally, the
compounds miscategorized by the PMF method are only a
single cutoff (predicts to be “low permeability” but measures as
“impermeable”) removed from their correct region, while many
of the LogP predictions are misplaced by two, or even three
regions (predicts to be “impermeable” but measures as “high
permeability”). As can be expected, the fragment-based
approaches perform marginally better than the atomic/hybrid
method. CLogP is the top performing LogP tool (albeit only
slightly, with only a ∼ 60% success rate and only predicting 2/8
permeable compounds). This is again anticipated as CLogP is
the current gold-standard tool for LogP prediction.
■
DISCUSSION
Assessing permeability is of critical importance to under-
standing and predicting in vivo efficacy and bioavailability of
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The Journal of Physical Chemistry B Article

Table 2. Measured and Predicted Permeabilities for the Set of Structurally Related LLNL Compounds
permeability category
compound logPeffPMF log PeffPAMPA measured predicted
LLNL1 −6.52 ± 0.39 −1.17 ± 0.33 impermeable low
LLNL2 −6.53 ± 0.23 −1.69 ± 0.21 impermeable low
LLNL3 −6.12 ± 0.29 −0.16 ± 0.24 low low
LLNL4 −6.52 ± 0.21 −0.90 ± 0.29 impermeable low
LLNL5 −6.57 ± 0.17 −2.27 ± 0.30 impermeable impermeable
LLNL6 −6.73 ± 0.74 −3.90 ± 0.19 impermeable impermeable
LLNL7 −6.45 ± 0.26 −3.28 ± 0.27 impermeable impermeable
LLNL8 −6.84 ± 0.12 −0.93 ± 0.26 impermeable low
LLNL9 −6.46 ± 0.41 −3.57 ± 0.30 impermeable impermeable
LLNL10 −5.50 ± 0.20 0.27 ± 0.42 medium medium
LLNL11 −5.91 ± 0.15 −0.10 ± 0.51 low low
LLNL12 −5.80 ± 0.14 −0.73 ± 0.25 low low
LLNL13 −6.86 ± 0.41 −2.59 ± 0.65 impermeable impermeable
LLNL14 −7.05 ± 0.17 −3.82 ± 0.18 impermeable impermeable
LLNL15 −6.10 ± 0.37 −0.64 ± 1.75 low low
LLNL16 −5.83 ± 0.24 −0.64 ± 0.30 low low
LLNL17 −5.36 ± 0.16 1.46 ± 0.19 medium medium
LLNL18 −5.23 ± 0.28 1.82 ± 0.12 high high

Table 3. Comparison between the Characterized Permeability of Novel Synthesized Compounds LLNL1-18 as Predicted Using
PMF Methods versus Various LogP Prediction Tools
method calibration compound correlation (R2) LLNL1−LLNL18 compounds correct (%) false positives false negatives “permeable” compounds correct
PMF 0.97 78 4 0 8/8
SLogP 0.53 56 0 8 0/8
miLogP 0.75 56 0 8 0/8
(c)LogP 0.45 56 1 7 1/8
CLogP 0.44 61 1 6 2/8

candidate drug compounds. A range of physiological mem-
branes are relevant to such concerns. Permeability across
gastrointestinal membranes is critical to bioavailability62
following oral administration. Transdermal permeation must
be evaluated for topical administration and management of skin
pathologies.63 Traversal across the BBB represents a particularly
confounding challenge, and is critical to delivery of compounds
requiring central nervous system access for treatment of
infection, neurologic malignancies, and neurodegenerative
disease,64,65 among other maladies. Computational prediction
of permeability represents a need that has been historically
challenging and is often limited by the requirement for a robust
training set, which ultimately restricts applicability of the
resultant model. This study sought to address this need by
refining and prospectively validating an MD-based model,
demonstrating effective prediction of permeability for candidate
compounds across a physiological lipid membrane.
The results of our study show that the developed
computational model can predict the PAMPA-defined perme-
ability category of a compound with greater accuracy than
compared LogP-based methods. This is demonstrated both
when examining well-characterized calibration compounds
exhibiting a wide range of permeation capacity, as well when
prospectively studying a set of novel compounds with a more
narrow dynamic range. The methodology is extremely
successful in characterizing the nine selected calibration
compounds (R2 = 0.97), and is able to correctly categorize
∼80% of our internal “prediction set” of 18 compounds.
As expected, the correlation with the calibration compounds
is higher than with the prediction set of compounds because
5234
the calibration compounds are structurally diverse and their
experimental permeabilities are spread over ∼5 log units, while
the prediction set are structurally similar and only spread over
<2 log units. Calculations based on this prediction set are thus a
challenging task, especially as the properties of many of the
compounds are so similar. Furthermore, several of the
experimental permeabilities of the prediction set are measured
at the lower end of the sensitivity threshold of the PAMPA
methodology, resulting in higher relative uncertainty in this
region due to the inherent challenge of experimentally
measuring very low levels of diffusion precisely (Figure 4).
Amplified relative uncertainty at the low end of the
permeability spectrum is correspondingly observed in computa-
tional predictions; we have previously shown that equivalent
errors in the PMF profile have a larger effect on the calculated
Peff when the compound has a lower permeability versus a
higher permeability (if there is an energy barrier at the bilayer
center rather than an energy well).36 Thus, any error in
calculating the PMF for these poorly permeable compounds
will be reflected in larger error in the Peff.
These results are a significant improvement in accuracy over
predictions made using current high throughput LogP
calculation techniques. Indeed, some of the LogP-based
methods fail completely when attempting to categorize the
permeability of the compounds. Depending on which LogP
tool is used, the same compound could be characterized into
three different regions. Furthermore, our PMF-based predicted
permeabilities are not dependent on training sets, but only the
physical properties recapitulated in the MD simulations. Our
first-principles method is especially useful for a novel set of
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J. Phys. Chem. B 2017, 121, 5228−5237
The Journal of Physical Chemistry B
compounds for which there is no QSPR or experimental data
available, and we do not run the risk of overfitting our model to
a narrow spectrum of physicochemical parameters.
Limitations in permeability and, ultimately, bioavailability, of
candidate drug compounds can substantially slow and derail the
development process. Ideally, computational model systems
would serve as a first pass screen for permeation. Existing
methods such as QSPR and LogP are not computationally
intensive, but are restrictive in their input capacity, and were
shown in our study to produce a large proportion of false
negatives, thereby running the risk of ruling out potentially
promising leads. Although MD-based simulations are more
computationally intensive than LogP calculations (a few days
compared to a few minutes), the result is a more accurate,
actionable model that does not discard valuable compounds.
Further, the computational time required for the method
described in our study is still less than the time required to
synthesize, purify, and experimentally characterize the same
novel compound.
From a drug design perspective, this predictive capability
would facilitate compound evaluation by ruling out imperme-
able candidates with a demonstrably low false-negative rate.
The resultant data would provide a more in depth character-
ization of the permeability of hits identified though high
throughput virtual screening or analogues of existing lead
compounds. Application of the described predictive methods
would facilitate faster iterative improvement, allowing for
selective retention of compounds with access to the intended
physiological target. Such a model could represent an important
component of a more comprehensive design pipeline, capable
of evaluating leads independent of structure or novelty of the
given compound.
■
ASSOCIATED CONTENT
* Supporting Information
S Immobilized artificial membrane liquid chromatography: proposed
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jpcb.7b02914.
Figures S1 and S2, the calibration plots and permeability
prediction plots for the LogP-based methods (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*(T.S.C.) Biosciences and Biotechnology Division, L-367,
Physical and Life Sciences Directorate, Lawrence Livermore
National Laboratory 7000 East Ave., Livermore, CA 94550. E-
mail: carpenter36@llnl.gov.
ORCID
Timothy S. Carpenter: 0000-0001-7848-9983
Author Contributions
# (13) Beigi, F.; Gottschalk, I.; Lagerquist Hagglund, C. L.; Haneskog,
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript. The authors denoted with the # label
contributed equally.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We thank the Defense Threat Reduction Agency (DTRA) for
funding (CB14-CBS-03-1-0127). We thank D. A. Kirshner for a
helpful discussion and the Livermore Computing Grand
Challenge for the computer time. This work was performed
5235
Article
under the auspices of the U.S. Department of Energy by
Lawrence Livermore National Laboratory under Contract DE-
AC52−07NA27344. LLNL-JRNL-725792.
■ ABBREVIATIONS
MD, molecular dynamics; PAMPA, parallel artificial membrane
permeability assay; IAM, immobilized artificial membrane; ILC,
immobilized liposome chromatography; BBB, blood brain
barrier; QSPR, quantitative structure-permeability relationship;
PMF, potential of mean force
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J. Phys. Chem. B 2017, 121, 5228−5237