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Cite This: J. Chem. Inf. Model. 2019, 59, 4018−4033 pubs.acs.org/jcim

A Comparative Linear Interaction Energy and MM/PBSA Study on

SIRT1−Ligand Binding Free Energy Calculation
Eko Aditya Rifai,† Marc van Dijk,† Nico P. E. Vermeulen,† Arry Yanuar,‡ and Daan P. Geerke*,†
†
AIMMS Division of Molecular and Computational Toxicology, Department of Chemistry and Pharmaceutical Sciences, Vrije
Universiteit Amsterdam, De Boelelaan 1108, 1081 HZ Amsterdam, The Netherlands
‡
Faculty of Pharmacy, Universitas Indonesia, Depok 16424, Indonesia
*
S Supporting Information
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ABSTRACT: Binding free energy (ΔGbind) computation can play an important

role in prioritizing compounds to be evaluated experimentally on their affinity for
target proteins, yet fast and accurate ΔGbind calculation remains an elusive task. In
this study, we compare the performance of two popular end-point methods, i.e.,
linear interaction energy (LIE) and molecular mechanics/Poisson−Boltzmann
surface area (MM/PBSA), with respect to their ability to correlate calculated
binding affinities of 27 thieno[3,2-d]pyrimidine-6-carboxamide-derived sirtuin 1
(SIRT1) inhibitors with experimental data. Compared with the standard single-
trajectory setup of MM/PBSA, our study elucidates that LIE allows to obtain
direct (“absolute”) values for SIRT1 binding free energies with lower compute
requirements, while the accuracy in calculating relative values for ΔGbind is comparable (Pearson’s r = 0.72 and 0.64 for LIE and
MM/PBSA, respectively). We also investigate the potential of combining multiple docking poses in iterative LIE models and
find that Boltzmann-like weighting of outcomes of simulations starting from different poses can retrieve appropriate binding
orientations. In addition, we find that in this particular case study the LIE and MM/PBSA models can be optimized by
neglecting the contributions from electrostatic and polar interactions to the ΔGbind calculations.

■ INTRODUCTION
A quantitative knowledge of protein−ligand binding affinities is
essential in understanding molecular recognition; hence,
efficient and accurate binding free energy (ΔGbind) calculations
are a central goal in computer-aided drug design.1 An arsenal
of ΔGbind calculation approaches is available, ranging from
rigorous alchemical methods such as free energy perturbation
(FEP)2 and thermodynamic integration (TI)3 to fast empirical
scoring functions featured in molecular docking.4 The latter are
prominent in predicting protein−ligand binding poses and in
discriminating binders and nonbinders within large chemical
databases,5 but they typically lack accuracy in quantitatively
ranking and predicting ΔGbind values.6 In contrast, rigorous
alchemical methods may provide reliable estimates for ΔGbind
but require extensive sampling of multiple intermediate
nonphysical states and are thus computationally more
expensive and still impractical for use in high-throughput
scenarios.7
Compared to these counterparts, the alternate end-point
methods offer an intermediate in terms of effectiveness and
efficiency in ΔGbind computation by allowing one to explicitly
explore ligand, protein−ligand, and solvent configurational
space in the protein−ligand bound and unbound states only.8
This provides advantages both over rigorous free energy
calculations (in terms of efficiency) and empirical scoring
functions (in terms of potential accuracy). Frequently applied
end-point methods make use of linear interaction energy (LIE)
theory9 and the molecular mechanics/Poisson−Boltzmann
surface area (MM/PBSA) approach.10
In LIE, ΔGbind is assumed to be linearly proportional with
the differences in van der Waals and electrostatic interactions
involving the ligand and its environment, as obtained from
simulations of its protein-bound and unbound states in solvent.
Differences in these interactions (modeled using Lennard-
Jones (LJ) and Coulomb potential-energy functions) are scaled
by LIE parameters α and β, respectively.9 Originally, β was set
to several fixed values according to a series of studied
systems.9,11−15 Later, it turned out that fixed values for β are
usually only suitable for particular systems of interest and not
generally transferable between different systems.16 To mitigate
this, a proposal was made to treat both α and β as freely
adjustable parameters that can be fitted based on a set of
experimentally determined ΔGbind values.17,18 The fitted values
of α and β (and optionally offset parameter γ) determine the
LIE scoring function to be used for predicting ΔGbind for
ligands with unknown affinity, which is given by9
ΔGbind = αΔV vdw + β ΔV ele + γ (1)
with γ set to zero in this work (unless noted otherwise), and
ΔV vdw = ⟨Vligvdw vdw
− surr ⟩bound − ⟨Vlig − surr ⟩unbound (2)
Received: July 24, 2019
Published: August 28, 2019

© 2019 American Chemical Society 4018 DOI: 10.1021/acs.jcim.9b00609

J. Chem. Inf. Model. 2019, 59, 4018−4033
Journal of Chemical Information and Modeling Article

Table 1. Compound ID, Molecular Structure, and Experimental Values for Binding Free Energies (ΔGbind) of SIRT1 Inhibitors
Used in the Current Study, with ΔGbind Values Derived from Experimental Data Obtained by Disch et al.37,a

4019 DOI: 10.1021/acs.jcim.9b00609

J. Chem. Inf. Model. 2019, 59, 4018−4033
Journal of Chemical Information and Modeling Article

Table 1. continued

4020 DOI: 10.1021/acs.jcim.9b00609

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Journal of Chemical Information and Modeling
Table 1. continued
a
The compound numbering (IDs) as listed below is the same as used by Disch et al.37
and
ΔV ele = ⟨V ligele− surr ⟩bound − ⟨V ligele− surr ⟩unbound
The terms on the right-hand side in eqs 2 and 3 are the MD-
averaged van der Waals (vdw) and electrostatic (ele)
interaction energies, respectively, between the ligand (lig)
and its surroundings (surr) as obtained in complex with the
protein bound or unbound in solvent.
Another popular end-point method is molecular mechanics
combined with Poisson−Boltzmann and surface area (MM/
PBSA). MM/PBSA calculations can be performed using either
results from protein−ligand complex simulations in a single-
trajectory (one-average) setup or from three separate
simulations per compound (i.e., of the complex, the protein,
and the unbound ligand) in a multi-trajectory or three-average
Article
setup.19 Use of the single-trajectory approach is more
widespread owing to its simplicity, efficiency, precision, and
(3) accuracy compared to the multi-trajectory setup.6,20,21 This
single-trajectory approach of MM/PBSA resembles LIE in a
way that they both do not account explicitly for changes in
internal energy and configurational entropy of the ligand and
protein upon binding. A difference is that single-trajectory
MM/PBSA assumes the conformational distribution for the
bound and unbound ligand to be the same, while LIE does
not.22 Molecular dynamics (MD) trajectories of the protein−
ligand complex as obtained in the one-average strategy can be
used to evaluate each free energy term on the right-hand side
of ΔGbind within the following equation10,23
ΔGbind = Gcomplex − (Gprotein + Gligand) (4)
4021 DOI: 10.1021/acs.jcim.9b00609
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Journal of Chemical Information and Modeling
In eq 4, Gcomplex represents the free energy of the complex,
and Gprotein and Gligand represent the free energies of the
unbound protein and ligand, respectively. The separate free
energy terms Gx for the protein, ligand, or protein−ligand
complex are each quantified as23
Gx = V bonded + V vdw + V ele + G polar + Gnonpolar − TS
where V bonded
(5)
comprises bond-stretch, angle-bend, torsion, and
improper-dihedral energies, and Vvdw and Vele are the van der
Waals and electrostatic nonbonded interaction energies,
respectively. Together, the sum of these terms make up the
vacuum MM energy terms, while Gpolar and Gnonpolar constitute
the solvation free energies calculated using a continuum
(implicit) solvation model, representing the free energy change
due to converting a solute in vacuum into its solvated state. In
MM/PBSA, Gpolar is obtained by solving the Poisson−
Boltzmann equation, while the Gnonpolar term is often
approximated by a solvent accessible surface area (SASA)
term.24 Replacing Poisson−Boltzmann with Generalized
Born25 for calculating Gpolar is on the basis of the alternative
MM/GBSA approach.10 Here, T and S denote the temperature
of the system and the entropy of the solute in vacuum,
respectively. The TS terms entering eq 4 via Gcomplex, Gprotein,
and Gligand account for the change in conformational entropy of
the protein−ligand complex upon ligand binding. This can be
approximated, for example, via normal-mode analysis of the
protein−ligand coordinates obtained from the simulation of
the complex. Often this term is neglected,26 which is justified,
for example, by the typical interest in relative binding free
energies of similar compounds.
As previously reported by Genheden and Ryde27 among
others, LIE can be more compute efficient up to almost 1 order
of magnitude when compared to MM/GBSA (which is due to
the entropy27 and polar term28 in eq 5), while the relative
accuracy of both methods in predicting experimental data can
be system dependent, and this conclusion also applies to MM/
PBSA.27 In the current work, we evaluate if LIE can achieve
similar (or higher) accuracy compared to MM/PBSA in
calculating binding affinities of 27 thieno[3,2-d]pyrimidine-6-
carboxamide analogs for the sirtuin 1 (SIRT1) receptor. SIRT1
is a member of the sirtuin family which is responsible for
regulating and/or deacetylating stress resistance and metabo-
lism processes,29 and inhibition of SIRT1 has been proposed
for treating cancer, HIV-1 infections, mental retardation
syndrome, and parasitic diseases.30 Results will also be
compared to a MM/GBSA model of Karaman and Sippl31
for these ligands binding to SIRT1.
Although LIE needs experimental data to train the model
parameters in eq 1, their calibration gives access to direct
estimates of ΔGbind for individual (query) compounds. This
makes it straightforward to use a Boltzmann-like weighting
scheme to combine results of multiple MD simulations starting
from different protein conformations and/or ligand binding
modes into a single calculation per compound.32,33 Previously,
we successfully used this strategy to compute binding affinities
for flexible proteins such as Cytochrome P450s that can bind
substrates and/or inhibitors in multiple ways.33−36 In the
current work, we evaluate the potential of this strategy to
compute SIRT1 binding free energies by concatenating results
of MD simulations starting with binding poses obtained from
unsupervised docking and clustering. In addition, we
investigate if the Boltzmann-like weighting scheme can identify
4022
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binding poses as those manually selected by Karaman and
Sippl31 in their MM/GBSA study as the most probable ones.
In a final step, we monitor the contributions from the different
terms in the central LIE and MM/PBSA equations to the
calculations in (trends in) binding affinity, in order to evaluate
the possibility to further increase efficiency and accuracy in
computing SIRT1 affinities.
■ METHODS
Reference Data and Input File Preparation. Reference
data for the SIRT1 ligand set (i.e., experimentally determined
values ΔGexp for protein−ligand binding free energies) were
taken from Disch et al.37 (Table 1). All 27 compounds have a
thieno[3,2-d]pyrimidine-6-carboxamide scaffold, and ΔGexp
ranges between −29.3 and −50.3 kJ mol−1. Values for ΔGexp
were consistently obtained from the set of IC50 inhibition
constants reported by Disch et al., using the Cheng−Prusoff
relation38,39
IC
ΔGexp = RT ln 50
2 (6)
where R is the gas constant, T is the temperature, and the
concentration of agonist used to measure the IC50 data is
assumed to be equal to its EC50 value.39
Analogous to previous MD studies,31,40 the SIRT1 crystal
structure with PDB ID 4I5I41 was used as the protein template
structure for docking and subsequent MD. Along with the
protein atom coordinates, the positions of the zinc ion and one
of the water molecules in the binding pocket31 were retained to
obtain the template. Protein structure preparation was
performed using the QuickPrep function of Molecular
Operating Environment (MOE) version 2015.1042 to add
hydrogens, assign protonation states and partial charges, and
perform energy minimization prior to docking. During
minimization, the protein and zinc ion were described using
the Amber14SB force field.43
To obtain starting structures for ligand binding poses, two
alternative strategies were used: ligand−binding poses were
either taken directly from Karaman and Sippl31 or obtained
from our docking. An advantage of using the Karaman poses
can be that these were carefully selected for all ligands such
that their scaffold showed maximal structural overlap with the
scaffold of ligands 11c, 28, and 31, which were co-crystallized
in PDB structures 4JSR, 4JT8, and 4JT9 of the homologous
SIRT3 protein, respectively,37 after fitting these onto the 4I5I
SIRT1 structure. In this way, the superimposed poses of all
ligands resemble the poses of the co-crystallized inhibitors,
which suggests that they occupy both the acetyl lysine
substrate channel and the nicotinamide C-pocket of SIRT1
that overlaps with the NAD+ binding channel.31,37
Ligand-binding poses were also generated using the
PLANTS (Protein−Ligand Ant System) docking software
version 1.244 with the speed 1 setting and evaluated using the
ChemPLP scoring function.45 Coordinates for the docking
center were defined as the average of the center-of-mass of
ligands in the Karaman binding poses, and a docking radius of
0.8 nm was used. For each ligand, 100 docking poses were
generated and subjected to hierarchical clustering with the
maximum number of clusters set to 3. For each cluster, a
representative pose with the smallest deviation to the average
cluster coordinate set was selected as an initial structure for
MD simulations. Ligand topologies for MD were generated
according to the general Amber force field (GAFF)46 at the
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Journal of Chemical Information and Modeling
AM1-BCC47 level of semi-empirical theory by using the
antechamber48 and sqm packages of AmberTools15.49 This
topology generation was performed and converted to
GROMACS format by using ACPYPE (Rev: 7268).50
MD Simulations. Prior to MD, the protein−ligand
complex was immersed in a dodecahedral periodic box and
solvated in approximately 15,300 TIP3P51 water molecules.
Nine Na+ counterions were added to neutralize the net charge
of the system, and the system was energy minimized using a
steepest descent algorithm. Thermal pre-equilibration and
(grid-based) pair-list updates during MD were performed as
described previously.34 The equations of motion were
integrated using a leap-frog algorithm.52 Particle mesh Ewald
(PME)53 was used to treat the long-range electrostatic
interactions during MD. The cutoff for nonbonded inter-
actions was set to 0.9 nm. Hydrogen atoms were assigned a
mass of 4.032 amu,54 and all bonds were constrained using the
LINCS algorithm,55 allowing a time-step of 4 fs. Coordinative
bonds with a length of approximately 0.2 nm were added
between the Zn2+ ion and sulfur atoms of its coordinating
residues (Cys131, Cys134, Cys155, Cys158) to ensure
coordination during simulation and kept constant using the
LINCS algorithm as well. A Berendsen thermostat and
barostat56 were used to maintain the system temperature and
pressure close to their reference values of 300 K and 1.01325
bar, using coupling constants of 0.1 and 0.5 ps, respectively. All
MD stages, including 20 ns production runs in explicit solvent
were carried out using GROMACS 4.5.5,57 in which energy
and coordinate trajectories were written out to disk every 10
ps, and center-of-mass motion was removed every 10 ps. Every
ligand pose was used twice as the starting structure of
replicated simulations33 starting from different Maxwell−
Boltzmann distributions of randomly assigned atomic veloc-
ities. Separate duplicated 20 ns production simulations were
performed of the unbound ligands in water in periodic
dodecahedral boxes with minimal solute atom-box edge
distances of 1.8 nm, using identical settings as in the
protein−ligand complex simulations described above. Energy
minimization and MD simulations were run in an automated
fashion as implemented in eTOX ALLIES,58 and the
trajectories obtained from the production runs were used for
subsequent LIE and MM/PBSA calculations as described
below.
LIE Calculations. Average electrostatic and van der Waals
ligand−environment interaction energies were obtained by
averaging over concatenated duplicated simulations performed
per protein−ligand binding pose or per ligand free in solvent.
LIE model parameters were calibrated based on experimental
ΔGbind values (Table 1) in an ordinary least squares (OLS)
fitting procedure using the Python scikit-learn 0.17 pack-
age.35,59
Inspired by the ability to deal with flexible proteins and/or
possible multiple ligand-binding modes in LIE binding affinity
computation for, for example, Cytochrome P450s
(CYPs)32,34,35 or nuclear receptors,60,61 we also report here
SIRT1 LIE models in which results from MD simulations
starting from different binding poses are combined into a
single ΔGbind calculation per ligand. For this purpose, the
weight Wi of the contribution from simulations starting from
an individual binding pose i to the binding free energy is
calculated as32,62
4023
Article
e−Δ Gcalc ,i / kBT
Wi =
∑i e−Δ Gcalc ,i / kBT (7)
where kB is Boltzmann’s constant, T is the temperature of the
simulated system, and ΔGcalc,i is the calculated binding free
energy for a given binding pose according to eq 1. The Wi’s can
be used to combine results from simulations starting from N
different binding poses by using the following equation
N N
ΔGbind = α ∑ Wi ΔVivdw + β ∑ Wi ΔV iele + γ
(8)
i i
where α and β (and the optional offset parameter γ) are again
parametrized based on curated experimental values for ΔGbind,
but they need to be trained in an iterative fashion (in which
simulation data for multiple binding modes are combined into
individual computations) when N > 132 because the Wi’s
depend on the LIE model parameter values.
MM/PBSA Calculations. MM/PBSA models were gen-
erated using g_mmpbsa24,63 by first PBC correcting the MD
trajectories. The Zn2+ ion was included as part of the protein in
the MM/PBSA calculations. We used a single-trajectory MM/
PBSA protocol which makes use of protein−ligand complex
simulations only and assumes the protein and ligand
conformation in the bound and unbound states to be identical.
Thus, in eq 5, the Vbonded terms as well as the intramolecular
nonbonded interaction energies cancel out,64 resulting in the
final equation
vdW ele
ΔGbind = Vcomplex + V complex + ΔG polar + ΔGnonpolar (9)
with VvdW ele
complex and Vcomplex being the protein−ligand van der
Waals and electrostatic interaction energies, respectively, while
polar polar
ΔG polar = Gcomplex polar
− (Gprotein + Gligand ) (10)
and
nonpolar nonpolar
ΔGnonpolar = Gcomplex nonpolar
− (Gprotein + Gligand ) (11)
The Poisson−Boltzmann equation for the polar term is
evaluated using the following equation10,63,65
4πρ f (r )
∇·[ε(r )∇·φ(r )] − ε(r )κ(r )2 sinh[φ(r )] + =0
kBT
(12)
where φ(r) is the solute’s electrostatic potential, ε(r) is the
dielectric constant, and ρf(r) is the fixed charge density. As the
calculation of the polar solvation energy is known to depend
on the chosen value for the dielectric constant εsolute of the
complex,66 its value was varied in order to inspect its effect on
predicted ΔGbind values. By default, εsolute was set to 2 and
tuned to 1, 4, or 8 to examine the sensitivity of calculated
binding free energies to the solute dielectric constant. Other
default settings in g_mmpbsa were left unaltered (i.e., use of
atomic radii as proposed by Bondi,67 linear PB equation solver,
0.05 nm grid resolution, and smoothed van der Waals
surface).24
To estimate nonpolar solvation energies, g_mmpbsa assumes
a linear dependence of Gnonpolar on the SASA, expressed as
follows68
Gnonpolar = γsurf SASA + b
(13)
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Table 2. Model Parameters α and β of LIE Models for SIRT1 and Their Respective Root-Mean-Square Error (RMSE),
Standard Deviation of Error in Prediction (SDEP, based on leave-one-out cross validation), and Correlation Metrics
Represented by r2, q2, Pearson’s r (rP), and Spearman’s ρ (ρS) for the Data Set of 27 Compoundsa
α β RMSE SDEP r2 q2 rP ρS
LIEsingle 0.40 −0.04 4.21 4.66 0.52 0.35 0.72 0.72
LIEγ=−10.24 0.30 −0.05 4.00 4.54 0.52 0.38 0.72 0.73
LIEmult 0.42 −0.19 6.16 6.59 0.16 −0.30 0.40 0.43
LIEcomb 0.40 −0.05 4.30 4.88 0.49 0.29 0.70 0.70
LIEBoltzmann4 0.40 −0.02 4.21 4.67 0.49 0.35 0.70 0.69
LIEfirst1ns 0.41 −0.04 3.96 4.39 0.54 0.42 0.74 0.70
LIEfirst5ns 0.41 −0.02 4.41 4.91 0.46 0.28 0.68 0.68
LIEfirst10ns 0.40 −0.04 4.27 4.73 0.51 0.33 0.71 0.72
LIEfirst15ns 0.40 −0.04 4.27 4.71 0.51 0.34 0.71 0.71
LIElast1ns 0.41 0.03 4.58 5.01 0.48 0.25 0.69 0.65
LIElast5ns 0.40 −0.03 4.28 4.75 0.52 0.32 0.72 0.69
LIElast10ns 0.40 −0.05 4.41 4.83 0.50 0.30 0.70 0.70
LIElast15ns 0.40 −0.05 4.30 4.74 0.52 0.33 0.72 0.72
LIEequil 0.41 −0.02 4.50 5.30 0.45 0.16 0.67 0.66
LIEβ=0 0.41 − 4.24 4.40 0.51 0.42 0.71 0.67
LIEα=0 − −1.67 31.3 32.1 0.05 −29.9 0.23 0.18
vdwbound 0.19 − 3.79 3.94 0.58 0.54 0.76 0.76
vdwunbound 0.35 − 4.39 4.56 0.49 0.38 0.70 0.67
a
RMSEs and SDEPs are given in kJ mol−1.

where γsurf is related to the surface tension of the solvent and is
set to its default value of 2.26778 kJ mol−1 nm−2, while a
default value (3.84982 kJ mol−1) was also used for fitting
parameter b. A solvent probe radius of 0.14 nm was employed
to determine the SASA.24 Due to its high computational
demand and large standard error compared to other
contributing terms,6,20,31,66,69−72 the entropic term was not
included in the MM/PBSA models presented in this work.
Protein−ligand Interaction Profiling. To analyze
protein−ligand interactions that occurred during the MD
simulations, we built a dedicated Python-based biomolecular
analysis library MDInteract, which we made freely available at
https://github.com/MD-Studio/MDInteract. MDInteract adds
structure analysis methods to the API of the popular pandas
data analysis toolkit.73 The MD trajectory and structure file
formats are imported using the MDTraj library.74 MD
trajectories were analyzed for the following biomolecular
interaction types at a 100 ps resolution similar to previous
studies performed by us:35,61,75
• Hydrophobic interactions: identified as all contacts
between carbon atoms within 0.4 nm having only C or
H atoms as neighbors. Interacting pairs from the same
source atom to multiple target atoms in the same
residue, in both directions, were filtered to retain only
the pair with the smallest distance. Note that atoms
involved in π- or T-stacking were separately considered
(see below).
• π- and T-stacking: identified as the geometric centers of
two aromatic rings within 0.55 nm, with an offset of no
more than 0.2 nm after projection of the geometric
center of one ring onto the other and an angle between
the normals of both rings of 180° ± 30° for π-stacking or
90° ± 30° for T-stacking.76 Aromatic rings were
detected using a SSSR algorithm (Smallest Set of
Smallest Rings) filtering for aromatic planar rings.
• Hydrogen bonds (H-bond): identified as all interactions
between H-bond donor−acceptor pairs within 0.41 nm
where the donor−H−acceptor angle was within 50°
4024
from the ideal in-plane 180° orientation and the heavy
atom acceptor neighbor−acceptor−H angle did not
deviate more than 90°.77 Donor atom SYBYL types: N.3,
N.2, N.acid, N.ar, N.am, N.4, N.pl3, N.plc, and O.3 with
at least one attached hydrogen. Acceptor atom SYBYL
types: N.3, N.2, N.1, N.acid, N.ar, O.3, O.co2, O.2, S.m,
and S.a not of a donor character. H-bonds part of a salt
bridge were labeled as such by the salt-bridge detection
method.
• Salt bridges: identified as all interactions between the
heavy atoms representing the geometric centers of two
charged groups of opposite signs within 0.55 nm
distance.78 The H-bond component of the salt bridge
was added by the H-bond detection method.
• Cation−π interactions: identified as all interactions
between the center of an aromatic ring and a center of
positive charge if the distance was within 0.6 nm and the
offset between charge center and ring center after
projection on the same plane was less than 0.2 nm. In
case the charge center was a tertiary amine, the angle
between charge center and ring plane should be within
90° ± 30°.79
• Halogen bonds: identified as pairs of halogen bond donor
(I, Br, Cl, F) and acceptor atoms (O, N, and S having a
single atom neighbor of type P, C, or S) within 0.41 nm
where the donor angle (C−X−O) was within 165° ±
30° and the acceptor angle (Y−O−X) was within 120°
± 30°.80
The frequency of interactions per ligand pose to interacting
amino acid residues was normalized by the number of
replicates (i.e., by dividing interaction counts by two) and
was represented as heat maps using the seaborn Python
package version 0.8.81 Ligand−residue interaction counts that
are less than 20 were discarded for further analysis.
■ RESULTS AND DISCUSSION
LIE and MM/PBSA Models. Tables 2 and 3 summarize the
predictive ability of our LIE and MM/PBSA models, which
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Table 3. Performance of MM/PBSA Models for SIRT1 in When calibrating a LIE model using per ligand the binding
Terms of Their Correlation Metrics, Represented by r2, q2, pose proposed by Karaman and Sippl31 as a (single) starting
Pearson’s r (rP), and Spearman’s ρ (ρS) for the Data Set of pose for MD, we obtain a model (LIEsingle in Table 2) with
27 Compounds RMSE = 4.21 kJ mol−1, which is within the typical
experimental uncertainty of 4−5 kJ mol−1.87,88 Overall, this
r2 rP ρS
suggests satisfactory performance of the model parameters
MM/PBSAε=2 0.41 0.64 0.61 used. Including the optional offset γ parameter did not improve
MM/PBSAε=1 0.39 0.63 0.60 model performance further: The resulting LIEγ=−10.24 model
MM/PBSAε=4 0.22 0.47 0.45 (with γ = −10.24 kJ mol−1) showed a slight reduction in
MM/PBSAε=8 0.11 0.33 0.31 RMSE and SDEP of 0.21 and 0.12 kJ mol−1, respectively, but it
MM/PBSAf irst1ns 0.28 0.53 0.46 did not show an increase in correlation coefficients compared
MM/PBSAf irst5ns 0.41 0.64 0.64 to LIEsingle (Table 2). Figure 1 illustrates that LIEsingle shows a
MM/PBSAf irst10ns 0.42 0.65 0.63
slightly better correlation between calculated and experimental
MM/PBSAf irst15ns 0.41 0.64 0.63
ΔGbind values than the MM/PBSA model in which εsolute is set
MM/PBSAlast1ns 0.36 0.60 0.56
to its default g_mmpbsa value of 2 (MM/PBSAε=2 in Table 3).
MM/PBSAlast5ns 0.35 0.59 0.56
This is confirmed by the slightly higher r2 (0.52 vs 0.41) and
MM/PBSAlast10ns 0.36 0.60 0.58
Pearson’s and Spearman’s coefficients (0.72 vs 0.64 or 0.61) of
MM/PBSAlast15ns 0.39 0.62 0.60
LIEsingle compared to MM/PBSAε=2 (Tables 2 and 3). It should
MM/PBSAele 0.19 0.43 0.08
be kept in mind however that α and β in the LIE equations are
MM/PBSAvdW 0.60 0.77 0.76
fitted parameters. Here, q2 of LIEsingle (0.35) is in fact slightly
MM/PBSApolar 0.36 −0.60 −0.57
lower than r2 for MM/PBSAε=2 (0.41), and the value of −0.04
MM/PBSASASA 0.57 0.76 0.75
for β hints at possible overfitting. As discussed in more detail
MM/PBSAnoele 0.04 0.20 0.14
below, this could be resolved by constraining β to 0 in a
MM/PBSAnopolar 0.49 0.70 0.71
separate LIEβ=0 model, which has a q2 value of 0.42 (Table 2).
In accordance with previous findings from MM/PBSA
was evaluated in terms of obtained deviations from and studies on other protein systems,66 we found a clear effect of
correlation with experimental data. The latter was assessed by εsolute on obtained correlations in the MM/PBSA models.
calculating r2, Pearson’s r (rP), and Spearman’s ρ (ρS) Setting its value to 1 or 4 led to slightly and significantly lower
coefficients.82 For the calibrated LIE models, we also report correlation coefficients, respectively, while using εsolute = 8
correlation coefficients q2 obtained from leave-one-out cross further decreased the quality of the model when compared to
validation (LOO-CV). Deviations of computed ΔGbind values MM/PBSAε=2 (Table 3). Even though, values of 6−7 or higher
from experiments were analyzed in terms of root-mean-square have been reported as average estimates for dielectric
errors (RMSEs) and standard deviations of errors in permittivities of protein binding pockets,89 εsolute is often set
predictions based on LOO-CV (SDEPs). In Tables 2 and 3, to 1 or 2.24,90 Our finding that the most successful models of
RMSEs and SDEPs are only reported for our LIE models MM/PBSA in predicting SIRT1−ligand binding use a low
because the MM/PBSA calculations do not allow one to value of εsolute is in line with the hydrophobic acetyl−lysine
directly compare predicted and experimental ΔGbind values for binding interface in SIRT1;91 as discussed by Karaman and
individual ligands.66,83−86 This is exemplified by the range of Sippl,31 the binding site of all inhibitors is characterized by six
predicted values obtained from one of the MM/PBSA models hydrophobic (Ala22, Ile30, Phe33, Phe57, Ile107, Phe174) and
discussed below, as presented in Figure 1 (right panel). two charged side chains (Asp108, His123).

Figure 1. Scatter plots for experimental (exp) and calculated (calc) free energies ΔGbind of ligand−SIRT1 binding. Calculated values are obtained
using the LIEsingle (left panel) or MM/PBSAε=2 model (right panel). The solid blue lines represent linear fits to the data, while the distributions of
data are represented by kernel density plots.

4025 DOI: 10.1021/acs.jcim.9b00609

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Figure 2. Ligand−residue interaction profiles for MD simulations used in the LIEsingle and MM/PBSA models (upper panel) and in the LIEmult
model (lower panel). Interaction counts are presented for individual SIRT1 residues (x-axis labels) and averaged over duplicated 20-ns runs per
ligand (y-axis labels in upper panel) or per combination of ligand and starting pose used for MD (y-axis labels in lower panel).

Our MM/PBSAε=2 model performs similarly as one of the
MM/GBSA models presented by Karaman and Sippl,31 who
used a εsolute value of 1 and reported r2 values ranging between
0.4 and 0.6 in their MM/GBSA study on SIRT binding. In
addition, the similar performance of LIEsingle and MM/PBSAε=2
is in line with the earlier work of Uciechowska et al., who
found comparable performance of LIE and MM/PBSA in
reproducing experimental data for a smaller set of SIRT2
binders.72
4026
Inclusion of Multiple Binding Poses. In a next step, we
investigated if we could use the iterative LIE scheme to
improve calculations and to develop a binding affinity model in
an unsupervised manner. For that purpose, multiple binding
poses were directly obtained from docking and clustering.
These were used as starting structures in MD simulations i in
eq 8. The resulting LIEmult model showed significantly higher
RMSE and lower correlation coefficients than the LIEsingle
model (Table 2). We analyzed if this decrease in predictive
quality was due to either a lack of correct MD starting poses in
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Journal of Chemical Information and Modeling
the LIEmult model or the fact that the protein does not bind its
ligands in multiple orientations (or due to a combination of
both).
We first explored the interactions between the ligands and
SIRT1 residues during simulations used in the LIEsingle and
LIEmult models. The obtained interaction heat maps are
depicted in Figure 2. They show high frequencies in three
ligand−SIRT1 hot-spot interactions that were present in the
starting poses31 and persistent during the simulations used in
the LIEsingle and MM/PBSA models (i.e., with Phe33, Ile107,
and Asp108; Figure 3). Figure 2 shows that these frequencies
Figure 3. Residues of SIRT1 (Phe33, Ile107, Asp108) that show hot-
spot interactions with the ligands during the simulations used, for
example, in the LIEsingle model. All ligands included in the model are
depicted in stick representation using different colors (in their starting
structure used for MD), while the interacting residues are labeled and
shown in ball-and-stick representation.
are significantly lower for most of the LIEmult simulations,
especially those involving interactions with Asp108. This
finding indicates that the unsupervised selection of starting
poses for MD leads to a larger spread in protein−ligand
interactions during simulation than in the LIEsingle model. For
most ligands, we found a relatively large root-mean-square
deviation (RMSD) in atomic positions between the MD
starting poses used in LIEmult and the LIEsingle pose; for only
eight of the ligands, at least one of the three docked starting
poses showed an RMSD of less than 0.1 nm (Table S1). Only
when combining results of the MD simulations initiated from
the starting poses of LIEsingle with those from LIEmult, we could
train a combined model LIEcomb with similar accuracy and
correlation coefficients (and α and β) as LIEsingle (Table 2).
These findings indicate that docking and clustering for the
purpose of LIEmult training did not lead to sufficiently
appropriate binding poses, while the retained predictive quality
of the LIEcomb model (compared to LIEsingle) implies that
incorporating too many binding poses does not necessarily
lead to lower predictive quality. Thus, when being able to
generate and select appropriate binding poses from docking
and/or experimental information on protein−ligand inter-
4027
Article
actions, iterative LIE training can subsequently be performed
in an unsupervised manner.
Here, we evaluated if Boltzmann-like weighting (eq 7)
within the LIEcomb model predicted the manually prepared
binding poses of Karaman and Sippl as the most probable ones
by inspecting per ligand the starting pose used in the MD
simulation with the highest weight Wi (Figure 4). We found
that for 22 of the 27 ligands, the simulation starting from the
binding pose used in the LIEsingle model shows the largest
weight Wi. For the five ligands for which the largest weight
comes from a simulation starting from one of the docked poses
used in LIEmult (19, 20, 21, 27, 38), we inspected the
corresponding pose (Figure 5) and discovered that the
scaffolds of three out of five ligands (19, 20, 21) show clear
structural overlap with the ligand pose used for LIEsingle (cf.
Figure 5 and the relatively low RMSDs in Table S1).
Interestingly, docked starting poses of ligands 27 (pose 1)
and 38 (pose 1) for the simulations with the highest weight Wi
in the LIEcomb model show less overlap (Figure 5) and higher
RMSDs (Table S1), but they still share similar interaction
profiles with the simulations used for these ligands in LIEsingle
(Figure 2). This highlights that similarity in protein−ligand
interactions may well be a better measure for comparable
modes and/or affinities of binding than structural overlap
alone, in agreement with results from previous studies in which
we used iterative LIE to evaluate binding to flexible proteins
such as CYPs or nuclear receptor FXR.34,35,61 This is further
exemplified by MD simulations starting from poses which
show relatively high contributions to LIEcomb calculations such
as in the case of pose 1 of compound 26, which does not have
the lowest RMSD of poses 1−3 when compared to the LIEsingle
starting pose (Table S1), but shows a similar pattern of
protein−ligand interactions (Figure 2). Subsequently, we
trained a LIE model using per ligand the simulation with the
highest weight Wi from LIEcomb, and we found that this
LIEBoltzmann4 model performs similarly as LIEsingle and LIEcomb
(Table 2), confirming that the latter does not utilize too many
different MD starting poses.
Impact of Simulation Length. We also explored the
effect of simulation length on ΔGbind computations by the
LIEsingle and MM/PBSAε=2 models. For that purpose, models
were developed based on averages for the interaction energy
and solvation terms over either the first or the last 1, 5, 10, or
15 ns of the production simulations starting from the Karaman
and Sippl poses (and using ε = 2 in the MM/PBSA models).
The performance of these models is listed in Tables 2 and 3
(using subscripts f irstXns or lastXns) and was compared to the
models based on 20-ns averages as described above.
MM/PBSA studies in the literature often report simulations
of tens of nanoseconds from which the last few nanoseconds
are used to compute averages for the different terms in eq 5 or
9.64 Our analysis shows that for the current system of interest a
simulation length of 5 ns (in the MM/PBSAf irst5ns model) is
sufficient to obtain comparable correlation coefficients for
MM/PBSAε=2. However, by running longer simulation times
and taking the last 1 or 5 ns to average over gives a slight
deterioration in model performance (cf. MM/PBSAlast1ns and
MM/PBSAlast5ns in Table 3, respectively). It is also worth
mentioning that 1-ns LIE simulations are sufficient to achieve a
model (LIEf irst1ns) that shows even slightly better performance
compared to LIEsingle (Table 2). The finding that shorter
simulation lengths can improve LIE calculations is in line with
previous results in iterative models that showed improved
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Figure 4. Boltzmann weights Wi for the simulations (x-axis) used per ligand (y-axis) in the combined LIEcomb model. Starting poses 0 and 1−3 were
starting poses for the simulations used in the LIEsingle and LIEmult model, respectively, and the intensity of the color indicates the size of Wi per
simulation.

Figure 5. Comparison of ligand starting poses for the MD simulations that have the highest weights Wi in the combined LIEcomb model (solid cyan)
with the LIEsingle pose (transparent green).

performance when restricting sampling to local parts of
conformational space,62,92 and it can be seen as a clear
advantage of LIE in terms of its efficiency. It prompted us to
generate a LIE model (LIEequil) using the output of our thermal
equilibration simulation at 300 K only, but this in turn showed
a slightly higher RMSE (4.50 kJ mol−1) and lower rP (0.67)
than those for LIEsingle and LIEf irst1ns (Table 2). The reason that
longer equilibration may not necessarily lead to improved
correlation for the MM/PBSA model (which is in line with
previous findings in literatures66,93) and that longer simulations
are more required for MM/PBSA than for LIE is due to the
relatively slow convergence of the MM/PBSA polar term.90,94
This is illustrated in Figure 6, which shows an example of a
4028
typically observed time series of the different MM/PBSA and
LIE terms. In conclusion, longer equilibration times do not
necessarily lead to an improved agreement with experimental
data which makes the selection of the optimal simulation time
to be an effective model parameter.
Contributions of Different LIE and MM/PBSA Terms
to ΔGbind Calculations. In the LIE models discussed above, β
is close or practically equal to zero (Table 2). This indicates
that electrostatic interactions do not significantly contribute to
trends in binding affinity, which can be in line with the
hydrophobic character of the SIRT1 binding pocket.91 In a
previous LIE study on a protein with a hydrophobic active site
(i.e., Cytochrome P450 1A2 or CYP1A2), fitted values for β
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Figure 6. Time series of different ligand−environment interaction
energies and solvation free energy terms used in the LIE and/or MM/
PBSA models, as obtained in one of the MD simulations of ligand 11a
either bound to SIRT1 or free in water (red, ΔGpolar; orange,
ΔGnonpolar; light green, ⟨Vele lig−surr⟩f ree; green, ⟨Vlig−surr⟩bound; dark green,
ele
Vcomplex; light blue, ⟨Vlig−surr⟩f ree; blue, ⟨Vlig−surr⟩bound; dark blue, Vvdw
ele vdw vdw
complex).
Plotted values are averages over 10 consecutive data points.
were similarly small and constrained to zero in the final
presented model.95 As for CYP1A2, a SIRT1 LIEβ=0 model
with β set to zero shows nearly identical performance as
LIEsingle when considering RMSEs and correlation metrics and
a slightly lower SDEP (and higher q2) (Table 2). The low
contribution of electrostatic interactions to predicted trends in
binding affinity can be understood from the relatively low
absolute values for ΔVele obtained from MD (Table S2),
suggesting that hot-spot and other hydrogen-bonding inter-
actions with SIRT1 residues compensate for the ligand−
solvent interactions in the unbound state. As a result, training a
LIE model with α set to zero (and using only β as fitting
parameter) is of no meaning at all (cf. LIEα=0 in Table 2).
Similarly as for LIE, we tried to simplify the MM/PBSA
models by only taking van der Waals interactions or the
nonpolar/SASA term into account. Table 3 shows that models
that only include the van der Waals VvdW complex interactions (MM/
PBSAvdW, rP = 0.77) or the SASA ΔGnonpolar term in eq 9 (MM/
PBSASASA, rP = 0.76) show higher predictivity than models
based on electrostatic Vele
complex interactions (rP = 0.43) or the
polar ΔGpolar term only (rP = −0.60) and even than the overall
MM/PBSAε=2 model. Our finding that LIE and MM/PBSA
models with a reduced number of model terms can give similar
or slightly improved performance would make it of interest to
explore the use of extended LIE approaches such as the one
recently proposed by He and co-workers,96 in which MM/
PBSA-like terms are included together with six fitting
parameters. Based on our current findings, this number may
also be effectively reduced in a model for ligand binding to
SIRT1. In addition to the small electrostatic contribution to
trends in binding to the hydrophobic SIRT1 pocket (Table
S2), the reason that neglecting electrostatics and polar
contributions can improve our MM/PBSA predictions can
be explained by the relatively large uncertainty in determining
complex and especially ΔG
Vele polar
(cf. Figure 6). The difficulty to
reach convergence in the latter can be due to fluctuations in
contributions not only from the protein binding site but also
from distant protein residues that may be less relevant for
ligand binding.22 The uncertainty in determining ΔGpolar was
4029
Article
also highlighted in a previous benchmarking study on
g_mmpbsa,24 in which it was observed that ΔGpolar is more
prone to be different (by more than 20 kJ mol−1) between
results of g_mmpbsa and AMBER simulation tools97,98 than
the other MM/PBSA terms. This was attributed to the
different algorithms implemented in APBS63 (used in
g_mmpbsa) and the PBSA package of AMBER and/or subtle
differences in the implementation of the AMBER force field
used in AMBER and GROMACS.
As the MM/PBSA method only considers protein−ligand
interactions and MM/PBSAvdw was found to be the best
performing MM/PBSA model in Table 3, we conducted
further investigation on a LIE model with β set to zero and in
which ΔVvdw in eq 1 is replaced by ⟨Vvdw lig−surr⟩bound. This LIE
model is annotated as vdwbound in Table 2 and thus considers
van der Waals interaction energies in the protein−ligand
bound state only. It shows the highest accuracy of the LIE
models presented in Table 2, with RMSE = 3.79 kJ mol−1 and
rP = 0.76. Interestingly, when calibrating a LIE model
vdwunbound with β = 0 and ΔVvdw in eq 1 replaced by
lig−surr⟩unbound, comparable performance was obtained as for,
⟨Vvdw
for example, LIEsingle (Table 2). Comparable correlation
coefficients could even be obtained by considering ligand
SASAs only (for which r2 = 0.43 and rP = 0.66). In this
particular case, binding prediction can apparently be made
highly efficient by considering ligand simulations or properties
only. This is probably again due to the dominance of nonpolar
protein−ligand contacts in determining trends in binding
affinity, but we note that the most predictive LIE and MM/
PBSA models are the ones based on protein−ligand van der
Waals interactions (Tables 2 and 3), as exemplified, for
example, by the high q2 value for the vdwbound LIE model.
■
CONCLUSIONS
By comparing fitted LIE models for a set of 27 compounds
binding to SIRT1 with single-trajectory-based MM/PBSA
models, we find calculated ΔGbind values from our LIE models
with a RMSE of 4.2 kJ mol−1, while the obtained correlations
are comparable (Pearson’s r = 0.72 and 0.64 for LIEsingle and
MM/PBSAε=2, respectively). This indicates that if agreement
with experimental affinities in absolute terms is not of interest,
both methods have a comparable predictive quality. We also
found that longer equilibration may not necessarily lead to
improved correlation with experiments and that longer
simulations are more required for MM/PBSA than for LIE
due to the relatively slow convergence of the MM/PBSA polar
term. In addition to the higher cost in computing this term
when compared to the terms in the central LIE equation, this
makes LIE more efficient.
For the studied protein and ligands, calibration of an
iterative LIE model (that uses weighted contributions from
multiple simulations starting from different ligand-binding
poses) did not lead to satisfactory computations when
generating starting poses for MD simulations in unsupervised
docking and clustering. During these simulations, we found
relatively low frequencies in ligand interactions with hot-spot
protein residues, as analyzed using our Python library
MDInteract. In other words, careful selection of proper MD
starting poses can be crucial in generating iterative LIE models.
Encouragingly, when combining results from simulations
starting from the docked poses and from poses based on
homologous crystal structure data (in the LIEcomb model),
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Boltzmann-like weighting of the multiple simulations was able
to identify appropriate starting configurations.
For our simulations with starting structures obtained in a
supervised way, we found relatively small values for the
difference ΔVele in electrostatic interactions in the bound and
unbound state. Apparently, ligand interactions with hot-spot
residues can largely compensate for the loss in polar
interactions with the solvent upon binding. As a result, LIE
parameter β was fitted to values close to zero. By constraining
it to zero, we could obtain a model with reduced overfitting
compared to models in which β was used as a free parameter.
This was illustrated by a lower SDEP and higher q2 for the
former model in leave-one-out cross validation. We found that
the correlation between calculated and experimental ΔGbind
values could be maximized by including protein−ligand van
der Waals interactions only (i.e., either in a MM/PBSA model
complex in eq 9 as the only term or in a LIE model with α
with VvdW
as the only free parameter and which only includes results from
the bound-complex simulations). On the other hand, our
models could be made most efficient by only including ligand
properties, e.g., in a SASA-only-based model or in a LIE model
that only includes van der Waals interaction energies involving
the unbound ligand. The finding that predictive models could
be obtained in terms of van der Waals interactions only for this
specific protein is in line with the hydrophobic character of its
binding site and with the uncertainty in determining the
ΔGpolar MM/PBSA term.
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jcim.9b00609.
Root-mean-square deviations (RMSDs) of ligand−atom
positions between starting poses used in the three
simulations per ligand in the LIEmult model and the
starting pose of the LIEsingle simulations (Table S1).
ΔVvdw and ΔVele values in kJ mol−1 per ligand as used in
the LIEsingle model (Table S2) (PDF)
Ligand force field parameters: GROMACS include
topology (.itp) files per ligand (using ligand IDs from
Table 1 for file nomenclature) and a separate atom type
file (attype.itp) to specify van der Waals parameters used
in the GROMACS simulations (ZIP)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: d.p.geerke@vu.nl.
ORCID
Daan P. Geerke: 0000-0002-5262-6166
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
Dr. Berin Karaman (Biruni University) and Prof. Wolfgang
Sippl (Martin-Luther-University of Halle-Wittenberg) are
gratefully acknowledged for sharing their SIRT1−ligand
complex structures with us. We thank The Netherlands
Organisation for Scientific Research (NWO, VIDI Grant
723.012.105) for financial support, and E.A.R. gratefully
acknowledges financial support from the Indonesia Endow-
4030
Article
ment Fund for Education (LPDP), Ministry of Finance,
Republic of Indonesia.
■
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4033 DOI: 10.1021/acs.jcim.9b00609

J. Chem. Inf. Model. 2019, 59, 4018−4033