A detailed analysis of the interactions of the cyclic pep-tides QZ59-RRR and QZ59-SSS is presented in both the X-ray structures of mouse P-glycoprotein and the human P-glycoprotein model
Trang 1models and ligand binding of human P-glycoprotein
Ilza K Pajeva1,2,*, Christoph Globisch1,* and Michael Wiese1
1 Pharmaceutical Institute, University of Bonn, Germany
2 Center of Biomedical Engineering, Bulgarian Academy of Science, Sofia, Bulgaria
Introduction
Since its discovery in 1976 [1], P-glycoprotein (P-gp)
continues to be the main focus of research interest In
addition to its involvement in cancer multidrug
resis-tance (MDR) [2], the protein acts as a protector of
nor-mal tissues against xenobiotics, and is a significant
factor for the absorption, distribution, metabolism and
excretion of drugs [3] These observations explain the
strong research interest in P-gp, currently making it the
most studied ABC transporter A number of
experimen-tal studies have been reported to date attempting to
explain its structure–function relationships, broad sub-strate specificity and interactions of its ligands [4–15] Investigations of P-gp by computational methods have developed over the years as a function of the data available for modeling In recent years, a number of three-dimensional structures of related MDR trans-porters have become available and these data have stimulated P-gp homology modeling Several models of the protein have been published based on the struc-tures of bacterial MDR transporters [16–19]
Keywords
binding sites; homology model; ligand
interactions; multidrug resistance;
P-glycoprotein
Correspondence
M Wiese, Pharmaceutical Institute,
University of Bonn, An der Immenburg 4,
53121 Bonn, Germany
Fax: +49 228 737929
Tel: +49 228 735213
E-mail: mwiese@uni-bonn.de
*These authors contributed equally to this
work
(Received 7 August 2009, revised 21
September 2009, accepted 29 September
2009)
doi:10.1111/j.1742-4658.2009.07415.x
An homology model of human P-glycoprotein, based on the X-ray struc-ture of the recently resolved mouse P-glycoprotein, is presented The model corresponds to the inward-facing conformation competent for drug binding From the model, the residues involved in the protein-binding cav-ity are identified and compared with those in the outward-facing confor-mation of human P-glycoprotein developed previously based on the Sav1866 structure A detailed analysis of the interactions of the cyclic pep-tides QZ59-RRR and QZ59-SSS is presented in both the X-ray structures
of mouse P-glycoprotein and the human P-glycoprotein model generated
by ligand docking The results confirm the functional role of transmem-brane domains TM4, TM6, TM10 and TM12 as entrance gates to the protein cavity, and also imply differences in their functions The analysis
of the cavities in both models suggests that the ligands remain bound to the same residues during the transition from the inward- to the outward-facing conformations The analysis of the ligand–protein interactions in the X-ray complexes shows differences in the residues involved, as well as
in the specific interactions performed by the same ligand within the same protein This observation is supported by docking of the QZ59 ligands into human P-glycoprotein, thus aiding in the understanding of the com-plex behavior of P-glycoprotein substrates and inhibitors The results con-firm the possibility for multispecific drug interactions of the protein, and are important for elucidating the P-glycoprotein function and ligand interactions
Abbreviations
HB, hydrogen bond; MDR, multidrug resistance; MOE, Molecular Operating Environment; P-gp, P-glycoprotein; TM, transmembrane.
Trang 2Very recently, the X-ray structure of mouse P-gp,
with 87% sequence identity to human P-gp, has been
refined to 3.8 A˚ resolution [20] The apo and
drug-bound structures have been obtained that are open to
the cytoplasm, thus corresponding to the inward-facing
conformation of P-gp This conformation is considered
to represent the initial stage of the transport cycle
competent for drug binding The large internal cavity,
formed by the bundles of the transmembrane (TM)
helices, can accommodate more than one compound
simultaneously, and implies a common mechanism of
polyspecific drug recognition The experimental data
obtained so far on the similarities [21,22] and
differ-ences [23] in substrate specificity between mouse and
human P-gp, despite the strong similarity of their
primary structures, raise questions regarding the way
in which the co-crystallized ligands may interact with
human P-gp [24]
In this article, we describe a homology model of
human P-gp for the inward-facing conformation of the
protein using the reported three-dimensional structure
of mouse P-gp [20] We further compare our model
with the previously published homology model of P-gp
[18] for the open to the extracellular space or
outward-facing conformation based on the Sav1866 structure
[25,26] The comparison has been performed in
rela-tion to the residues exposed to the binding cavity of
the protein in both conformations The same algorithm
for the identification of the binding site residues has
been applied The analysis confirms the functional role
of TM4, TM6, TM10 and TM12 for the entrance gates
(portals) to the cavity, in agreement with the X-ray
data, and also suggests differences in their functions
Next, the interactions of the QZ59 compounds in the
X-ray structures of mouse P-gp were analyzed in
detail The analysis reveals differences in the specific
interactions of each ligand with the protein The
dock-ing of the QZ59 stereoisomers into the human P-gp
model, and the subsequent analysis of the ligand
interactions, confirms the possibility for multispecific
interactions of the ligands with the protein
Results
Homology modeling of human P-gp
In the template structure of mouse P-gp (PDB ID
code: 3G61 chain A), the missing residues (982–1000)
in the disrupted helix TM12 were replaced with the
homologous part of TM6 (amino acids 339–357) by
superposing the backbone atoms of the three terminal
amino acids (Fig S1) In Fig 1, the distribution of the
residues, identified as outliers in the Ramachandran
plot (Fig S2), are shown In total, 95 outliers were identified, depicted in a space-filled rendering mode in dark yellow (Fig 1)
The homology model of the template structure was minimized using the Amber 99 force field with the ligands as environment One hundred models were generated using the best intermediate option with medium minimization, including the prevention of clashes, to stay as close as possible to the initial structure The final template model was selected according to the best score of the Molecular Operat-ing Environment (MOE) scorOperat-ing function A multise-quence alignment was performed by the ‘Align’ tool
in MOE (see Materials and methods), including the template (PDB ID: 3G61 chain A), Swissprot data-base human MDR1 (code P08183) and closest relative
to the human P-gp [hamster MDR1 (code P21448)] sequences
To obtain the final homology model, 100 structures were generated using the best intermediate option and
Fig 1 X-Ray structure of mouse P-gp (PDB ID code 3G61): distri-bution of the amino acid outliers (95 residues) identified from the Ramachandran plot (Fig S2); the outliers are rendered in dark-yellow, space-filled mode.
Trang 3medium minimization with the Amber 99 force field
for each model to remove the bad contacts The best
model was selected according to the MOE scoring
function and was investigated by the protein report
function in MOE No outliers in the TM domains of
the protein were found that were important in
estimat-ing the drug-bindestimat-ing competency The deviatestimat-ing amino
acids, located mostly within the loop regions of the
nucleotide-binding domains (31 outliers, data not
shown), were then minimized, together with the
adja-cent residues, keeping the remaining protein fixed
After minimization, the outliers in the Ramachandran
plot (Fig S3) were reduced to 20 In Fig 2, the
loca-tions of the outliers within the final model of human
P-gp are shown in a space-filled rendering mode in
dark yellow The rmsd of all a-carbon atoms between
the homology model after minimization of the
Ramachandran plot outliers and the template with the
modified TM12 was 0.188 A˚
The protonation state of the model was assigned by
the protonate 3D module in MOE, which considers
the solvent accessibility and regional neighboring of
the amino acids
Comparison of the cavities in the inward- and outward-facing conformations of the human P-gp models
In Table 1, the residues involved in the binding cavity in the inward- and outward-facing conforma-tions of the homology model of human P-gp are shown The residues reported for the outward-facing conformation have been identified in a previous study in which a homology model of P-gp was developed on the basis of the crystal structure of the multidrug transporter Sav1866 (see table 9 in ref [18]) To have an equal basis for comparison, the same approach was applied for the identification of the binding pockets in the cavity of the inward-facing conformation of P-gp (the module ‘Site Finder’ in MOE, see Materials and methods) In Table 1, the residues related to the binding of the substrates dibromobimane (d), verapamil (v) and rhodamine (r) are marked, according to the experi-mental findings of Loo and coworkers [4,5,7,8,11] Figure 3 illustrates the cavity and binding pockets identified In Fig 3A, the general view of the cavity and, in Fig 3B, a closer view (from the inside) are shown Clearly outlined are the large hydrophobic spheres occupying the entrances (portals) from the inner leaflet of the membrane to the protein cavity: the first formed by TM4 (light green) and TM6 (light magenta), and the second by TM10 (dark green) and TM12 (dark magenta)
Comparing the residues reported in Table 1 for the inward- and outward-facing models, differences can be seen in the residues of the TMs exposed to the cavity
in both conformations
Amino acid residues involved in the interactions with the QZ59 compounds in mouse P-gp The binding sites of the cyclic peptides QZ59 were analyzed in the X-ray structures using the ‘Ligand Interactions’ module in MOE (see Materials and meth-ods) In Table 1, for each TM, the residues involved in the interactions of the QZ59 ligands are denoted by ‘q’
in the table row ‘sub’
For the same ligand, different amino acids involved
in the interactions were identified as a function of the protein molecule in the asymmetric cell unit of the crystal For the stereoisomer QZ59-RRR (PDB ID 3G60), 11 common residues were found for both P-gp molecules (A and B) in the unit cell; mouse Tyr303 (human Tyr307), Leu335 (human L339) and Ser725 (human Ala729) were also involved in the case of molecule A (Fig 4A), and Met68 (human Met69),
Fig 2 Homology model of human P-gp: distribution of the amino
acid outliers (20 residues) identified from the Ramachandran plot
(Fig S3); outliers are rendered in dark-yellow, space-filled mode.
Trang 4Table 1 Amino acids involved in the binding cavity of the inward (‘in’) and outward (‘out’)-facing conformations of human P-gp; the numbers and letters of the residues correspond to human P-gp; sub, residues related to substrate binding; grey color, light (in), dark (out).
TM1
54: G T L A A I I H G A G L P L M M L V F G E M
in a
x x x x out
subb
TM2
117: Y Y S G I G A G V L V A A Y I Q V S F W
in x out
sub
TM3
190: I G M F F Q S M A T F F T G F I V
in out
sub
TM4
219: L A I S P V L G L S A A V W A K I L S
in out c
d
TM5
296: N I S I G A A F L L I Y A S Y A L A F W
out
TM6
330: Q V L T V F F S V L I G A F S V G Q A S P
out
v d
q
d
q r
TM7
718: A I I N G G L Q P A F A d
I I F S K I I G V F
out
TM8
762: L G I I S F I T F F L Q G F T F
in x out
sub q
TM9
831: S R L A V I T Q N I A N L G T G I I I S F I Y
in out
sub r
TM10
857: L T L L L L A I V P I I A I A G V V E
in
sub v
d
d
TM11
938: F G I T F S F T Q A M M Y F S Y A G C F
out
sub v
d
v d
TM12
974: V L L V F S A V d
V F G A M A V G Q V S S F
out
r d
r q r d v q d
a
x, residues involved in the binding pocket of the QZ59 ligands (identified by docking, see Materials and methods).bd, dibromobimane [4,5,7,8]; q, QZ59 (RRR and SSS); r, rhodamine [7,11]; v, verapamil [7,8] c no residue involved in the outward-facing conformation d A729 (mouse S725); V981 (mouse A977).
Trang 5Phe71 (human Phe72) and Leu971 (human Leu975) in
molecule B (Fig 4B) Arene–arene interactions were
identified with Phe332 (human Phe336) and Phe974
(human Phe978) for molecules A and B, respectively
Compared with the residues reported for QZ59-RRR
in [20, fig 3], mouse Ser725 (human Ala729) and Ala981 (human Ala985) for the ligand in molecule A, and Phe71 (human Phe72), Leu971 (human Leu975) and Ala981 (human Ala985) for the ligand in mole-cule B, were also identified, with Ala981 (human
A
B
Fig 3 Binding cavity in the inward-facing conformation of the
homology model of human P-gp: (A) general view (face); (B) closer
view (from inside) The pockets are filled with alpha spheres; grey
spheres indicate hydrophobic atoms and red spheres hydrophilic
atoms The protein backbone is colored by the secondary structure;
colors used for the portal TMs: light green (TM4), magenta (TM6),
dark green (TM10), purple (TM12).
A
B
Fig 4 The interaction panel of the ligand QZ59-RRR in the X-ray structure of mouse P-gp (PDB ID code 3G60): (A) ligand 1, mole-cule A; (B) ligand 2, molemole-cule B The receptor exposure is shown
by the size and intensity (the darker the color, the more exposed the residue) of the disks drawn behind some of the residues to denote the difference in their solvent exposure as a result of the presence of the ligand; the ligand solvent exposure is shown by smudges drawn behind some of the ligand atoms to denote the extent of solvent exposure Symbols: , arene–arene interactions; , proximity contour; , ligand exposure; , receptor exposure.
Trang 6Ala985) being well exposed according to the size and
intensity of the disk drawn around the residue
(Fig 4A,B)
Figure 5 illustrates the interactions for the
QZ59-SSS stereoisomer (PDB ID 3G61) Similar to
QZ59-RRR, differences have been recorded in the
interactions of the ligand in the different molecules
in the unit cell For ligand 1 in molecule A, only
nonspecific van der Waals’ interactions were
exhib-ited (Fig 5A), whereas, for ligand 3 in molecule B
(Fig 5C), a hydrogen bond (HB) was formed with
residue Tyr303 (human Tyr307) Ligand 2 (mole-cule A) and ligand 4 (mole(mole-cule B) were not fully resolved and the analyses of the ligand interactions thus involved only parts of the structures For ligand 2 (Fig 5B), arene–arene interactions occurred with Phe974 (human Phe978) No specific interac-tions were recorded for ligand 4 (Fig 5D) Obvi-ously, the same ligand can occupy different positions within the protein-binding pocket
Compared with the residues reported in ref [20,
fig 3] for QZ59-SSS, mouse Phe71, Phe728, Leu971
Fig 5 The interaction panel of the ligand QZ59-SSS in the X-ray structure of mouse P-gp (PDB ID code 3G61): (A) ligand 1, molecule A; (B) ligand 2, molecule A; (C) ligand 3, molecule B; (D) ligand 4, molecule B The receptor exposure is shown by the size and intensity (the darker the color, the more exposed the residue) of the disks drawn behind some of the residues to denote the difference in their solvent exposure
as a result of the presence of the ligand; the ligand solvent exposure is shown by smudges drawn behind some of the ligand atoms
to denote the extent of solvent exposure Symbols: , arene–arene interactions; , proximity contour; , ligand exposure; , receptor exposure.
Trang 7and Ile977 (Fig 5B) were also identified, with Tyr303
performing a specific HB interaction (Fig 5C)
Amino acid residues involved in the interactions
with QZ59 compounds in the homology model of
human P-gp
The analysis of the residues involved in the
interac-tions was performed in two ways: (a) using the
QZ59-SSS ligands as incorporated from the X-ray sources;
and (b) using docking (by GOLD, see Materials and
methods) to better explore the binding possibilities of
the ligands In the homology model of human P-gp,
the same amino acids as in the X-ray structures
inter-acted with the QZ59-SSS ligand, and two additional
residues were identified: human Gln725 and Met986
(data not shown)
The docking of the QZ59 stereoisomers yielded
sta-ble and similar solutions Several runs were performed
using either one or two ligands simultaneously to
define the binding pocket The poses were fully
repro-ducible with close GoldScore values The top score
poses were used for further analysis In Table 1, the
residues involved in the binding sites of the QZ59
ligands identified in the best docking poses in the
human P-gp inward-open model are marked by ‘x’
Figure 6 visualizes the interaction of QZ59-RRR
and QZ59-SSS with the residues in the human P-gp
model Figure 6A shows the general view of the best
docked poses of both stereoisomers in the two binding
sites superimposed on the P-gp backbone Similar to
the SSS enantiomer, the RRR enantiomer could
poten-tially occupy the two binding sites Figure 6B shows a
closer view of the interactions of the two peptides in
the lower located binding site, and Fig 6C in the
upper site Comparing the residues comprising the
binding sites of the ligands in human and mouse P-gp
(Table 1: ‘q’ with ‘x’; Figs 4 and 5 with Fig 6),
simi-larities and differences in relation to the particular amino acids involved and their specific interactions can
be outlined The residues in the ligand-binding sites in
Fig 6 (A) A front overview of the P-gp binding cavity with the
inhibitors QZ59-RRR and QZ59-SSS (in space-filling form) docked
into the two binding sites and superimposed on the P-gp backbone
(grey line) (B) Closer view (from the bottom) of the protein–ligand
interactions and the surface of the binding site in the lower binding
site (C) Closer view (from the bottom) of the protein–ligand
interac-tions and the surface of the binding site in the upper binding site.
The Gaussian contact surface is presented with the following color
coding: green, hydrophobic; magenta, HB; blue, mildly polar The
HB distance and score are colored in magenta; the residues are
col-ored in green; the structures of the ligands are rendered in stick
form and colored according to the atom types; the HB atoms are
shown as balls and the distance between (=O) of QZ59-RRR and
(–N<) of Gln725 is 2.84 A ˚ (64% score, see Materials and methods).
A
B
C
Trang 8the X-ray structures of mouse P-gp partially overlap
with those in the docked poses in the human P-gp
model Although Tyr303 (human Tyr307) performs an
HB interaction with QZ59-SSS in the X-ray structure
(Fig 5C), human Gln725 is involved in an HB–donor
interaction in human P-gp, and Phe343 can perform
arene–arene interactions with QZ59-RRR (Fig 6B)
Thus, it can be proposed that the QZ59 ligands will
bind to human P-gp in a similar, but not identical,
manner as to mouse P-gp, interacting specifically with
different residues from the same environment Such a
result is not surprising considering the observed
differ-ences mentioned above in the interactions of the same
ligand with two different molecules of the same protein
in the X-ray structures (Figs 4 and 5) Notably, the
hydrophobic residues with the human P-gp codes
Phe336, Phe343, Phe728, Phe978 and Val982 are the
most involved amino acids in both the X-ray and
docked poses of the ligands The same residues have
been proven experimentally to relate to other P-gp
substrates [4,7,8,10,11,14]
Discussion
For the different TMs, differences between the residues
exposed to the cavity in both the open and closed
con-formations have been observed The most striking
dif-ference is the absence of amino acids of TM4 and
TM10 facing the cavity Although residues of TM4
and TM10 are broadly accessible in the inward-open
conformation, they are fully buried in the
outward-open form (Table 1) At the same time, the same
residues of TM6 and TM12 face the cavity in both
conformations and, in addition, these domains possess
the highest number of experimentally proven amino
acids involved in drug binding Among them are a
number of hydrophobic residues, such as human
Phe336, Phe339, Phe340, Phe343, Phe978, Val981,
Val982, Ala985, identified in the binding sites of the
QZ59 ligands; moreover, some have been found to
per-form specific (arene–arene) interactions, such as
Phe336 (mouse Phe332, Fig 4A), Phe978 (mouse
Phe974, Figs 4B and 5B) and Phe343 (Fig 6B) TM6
and TM12 are also the most involved domains in
interactions with other P-gp substrates, such as
verapa-mil, rhodamine and dibromobimane, proven by
drug-binding experiments (Table 1, row ‘sub’) In the
most recent study, Loo et al [5] also found the largest
number of mutations in TM6 and TM12 in
arginine-scanning mutagenesis experiments Considering that
these TMs form the two portals (TM4–TM6 and
TM10–TM12) for entering the cavity from the inner
leaflet of the membrane, the differences observed
above also suggest differences in their role for the function of protein and ligand binding TM4 and TM10, being parts of the portals, could be involved in weaker interactions with the entering ligands that are lost during the transport cycle In Table 1, three resi-dues only are reported as belonging to these domains, related to interactions with P-gp substrates: human Ser222 (TM4), Ile868 and Gly872 (TM10) (Table 1) Figuratively, TM4 and TM10 could function as ‘portal keepers’, preventing the substances that enter the cav-ity from the inner leaflet of the membrane escaping back In contrast, TM6 and TM12 could be regarded
as the ‘portal carriers’, being mainly responsible for ligand interactions Interestingly, the TM6 and T12 residues mostly perform hydrophobic interactions, whereas the more specific HB-type interactions could
be related to other domains, such as, for example, Tyr307 (mouse Tyr303) of TM5 (Fig 5C) and Gln725
of TM7 (Fig 6A) Studying the interactions of specific P-gp inhibitors, representatives of the third-generation MDR modulators, we found specific HB interactions with Tyr117 (TM2), Tyr307 (TM5) and an arene– arene-type interaction with Tyr953 (TM11) [27], thus outlining, in addition, the role of TM2, TM5, TM7 and TM11 in ligand interactions It is worth noting that these residues also face the cavity in both confor-mations of the protein (Table 1)
From the above analysis, it is most likely that the ligands remain bound to the same residues during the transition from the inward- to the outward-facing con-formation of the protein, suggesting that the ligand is not flipped
Next, the detailed analyses of the ligand interactions with the protein, as recorded here for the QZ59 com-pounds (Figs 4 and 5), show differences in the residues involved, as well as in the specific interactions of the same ligand with the same protein For QZ59-RRR, Phe332 (human Phe336) is in a favorable position for arene–arene interactions in molecule A, whereas Phe974 (human Phe978) is involved in such interac-tions in molecule B (Fig 4A,B); Tyr303 (human Tyr307) forms an HB interaction with ligand 3 in mol-ecule B (Fig 5C); at the same time, no such interac-tion is recorded for ligand 1 in molecule A (Fig 5A) Whether these differences can be related to the experi-mental conditions under which the ligands were co-crystallized, or reflect the possibility for different binding locations and orientations of the same ligand
in the same protein environment, remains to be proven The docking of QZ59 ligands into the human P-gp binding cavity supports the latter suggestion The results confirm the possibility for binding of the same ligand in two different binding sites, as shown for
Trang 9QZ59-RRR in Fig 6B,C This observation, illustrated
here by the analysis of the X-ray complexes and
sup-ported by binding simulation, could help, for example,
to better understand the complex behavior of P-gp
substrates and inhibitors in functional assays
In conclusion, the results of this study confirm the
possibility for multispecific interactions of the protein
with its ligands, and aid in the elucidation of P-gp
function and drug interactions
Materials and methods
Homology modeling
Chain A from the crystal structure of mouse P-gp (PDB ID:
3G61 [20]) was used as template for the homology model
The Swissprot database sequences of human P-gp (P08183)
and hamster P-gp (P21448) were used for alignment [28]
The alignment was obtained with the ‘Align’ tool in
MOE [29], using the default tree-based approach with the
blosum62 substitution matrix and increased values for the
gap penalty of 15 and gap extension penalty of 2
The homology model was calculated by the ‘Homology
Model’ method in MOE using a rotamer library and loop
dictionary derived from the X-ray structures to predict the
coordinates of deviating residues The best intermediate
approach with the medium minimization option and the
Amber 99 force field was used to derive the model The
co-crystallized ligand structures were defined as environment
and kept fixed The best model (out of 100) was selected
according to the GB⁄ VI scoring function (Coulomb and
generalized Born interaction energies of the model and
environment) The stereochemical quality of the model was
inspected by the protein report of MOE The
MOE-ProSu-perpose module was used to calculate the rmsd values
Identification of the binding pockets
The ‘Site Finder’ tool in MOE [29] was employed for the
identification of the binding sites in the inward-open
con-formation of the P-gp homology model The program is
based on the methodology of convex hulls which produces
pockets invariant to rotation of the atomic coordinates It
treats the set of three-dimensional points by triangulation
and associates each resulting simplex with a sphere, coded
as ‘alpha sphere’ The radius of the sphere is proportional
to the convex hull of the point set Each sphere is classified
as either ‘hydrophobic’ or ‘hydrophilic’ depending on
whether the sphere is a good HB point in the receptor
Hydrophilic spheres not close to hydrophobic ones
are eliminated as they generally correspond to water sites
The generated pockets consist of one or more alpha
spheres, and at least one is hydrophobic The following
settings were used: radius of a hydrophilic HB sphere,
1.4 A˚; radius of a hydrophobic sphere, 1.8 A˚; isolate donor–acceptor distance, 3 A˚; connection distance between clusters of alpha spheres, 2.5 A˚; minimum site size, 3; minimum site radius, 2 A˚
Docking of the QZ59 ligands into human P-gp models
The QZ59 ligands were prepared in Sybyl [30] QZ59-RRR was extracted from its complex 3G60 and QZ59-SSS from 3G61 The chirality and atom types of the compounds were checked; the missing hydrogen atoms were added, and the geometries were optimized with the Tripos force field and Gasteiger–Hueckel charges The minimized structures were subsequently exported as mol2 files for docking with the GOLD Suite [31,32], which applied a genetic optimization algorithm for docking flexible ligands into protein-binding sites The binding pocket was defined on the basis of the co-crystallized ligands in the X-ray structure Considering the substantial volume of the binding cavity (around
6000 A˚3 [20]), the pocket was extended by 10 A˚ and out-ward-facing amino acids were deselected The default setting for the genetic algorithm and the original GoldScore function were used to rank the ligand poses Docking was performed by the ‘slow (most accurate)’ option for balance between speed and accuracy
Analysis of the ligand interactions The X-ray complexes (mouse P-gp) and docked poses (human P-gp) of the QZ59 compounds were analyzed by the ‘Ligand Interactions’ tool in MOE The method imple-mented is fully described in [33] The information content displayed in the ligand interactions panel consists of the selected ligand and the receptor-interacting entities, namely
HB residues, close but non-bonded residues (approaching the ligand but not having any strong interactions, i.e HBs), solvent molecules and ions The solvent-accessible surface area and the ligand proximity outline were also estimated HBs were assigned to each pair of heavy atoms from the ligand and receptor according to probability criteria derived from a large training set [34,35] The HB scores were expressed as percentages and the HB directionality was noted The ligand and residue solvent accessibility metrics were estimated by measuring the exposed surface area once each of the atoms had been assigned a van der Waals’-like radius of +1.4 A˚ (water solvent) The solvent exposure of receptor residues was calculated by examining the difference between the solvent-exposed surface area of the receptor with and without the presence of the ligand For the ligands, the surface accessibility calculation was carried out
on the ligand + receptor complex The default settings were applied for the definition of hydrogen-bonded and proximity interactions
Trang 10I.P and M.W gratefully acknowledge the generous
support by the Alexander von Humboldt Foundation,
Germany I.P also thanks the National Science
Foun-dation of Bulgaria
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