Human Serum Albumin Binding in a Vial: A Novel UV-pH Titration Method to Assist Drug Design

Article abstract of DOI:10.1021/acs.jmedchem.0c00046

The knowledge on human serum albumin (HSA) binding is of utmost importance as it affects pharmacokinetic behavior and bioavailability of drugs. In this article, we report a novel method to screen for ionizable molecules with high HSA binding affinity base

Full text of DOI:10.1021/acs.jmedchem.0c00046

This is an open access article published under a Creative Commons Attribution (CC-BY)
License, which permits unrestricted use, distribution and reproduction in any medium,
provided the author and source are cited.
pubs.acs.org/jmc
Article
Human Serum Albumin Binding in a Vial: A Novel UV-pH Titration
Method To Assist Drug Design
̋ ́ ́ ́
̈
̈
Gergo Dargo, David Bajusz, Kristof Simon, Judit Muller, and Gyorgy T. Balogh*
Cite This: J. Med. Chem. 2020, 63, 1763−1774
Read Online
ACCESS
Metrics & More
Article Recommendations
*
sı Supporting Information
ABSTRACT: The knowledge on human serum albumin (HSA)
binding is of utmost importance as it affects pharmacokinetic
behavior and bioavailability of drugs. In this article, we report a
novel method to screen for ionizable molecules with high HSA
binding affinity based on pK shifts using UV-pH titration. We
a
investigated the HSA binding of 27 drugs and compared the results
to experimental data from conventional methods. In most cases,
significant shifts (ΔpK > 0.1) were observed for drugs with high
a
HSA binding, while no change could be detected for low-affinity
binders. We showed the pivotal role of ionization centers in the
formation of strong interactions between drug and HSA using
molecular docking studies. We also verified our findings by testing
five modified analogues designed by structural considerations.
Significant decreases in their HSA binding proved that the UV-pH titration method combined with an in silico support can be used
as a medicinal chemistry tool to assist rational molecular design.
INTRODUCTION
affinity shows wide diversity for drug-like compounds; thus, it
is essential to predict the unbound fraction of APIs from the
early stages of drug discovery for the estimation of PK
behavior.
■
Active pharmaceutical ingredients (API) entering the system-
atic circulation may interact with various components of the
blood, which directly affects their pharmacokinetic and
1
−5
For the measurement of HSA-binding properties of APIs,
pharmacodynamic behaviors.
Blood plasma consists of
1
8,19,25−27
approximately 7−9% of plasma proteins (albumins, glyco-
proteins, and fibrinogen), which contribute a major part to
API-specific interactions. Among them, human serum albumin
several in vitro methods exist,
including equilibrium
28,29
30
31
dialysis (ED),
chromatographic methods,
ultrafiltration (UF), ultracentrifugation,
3
2−35
36,37
NMR spectroscopy,
X-ray
38
39
(
HSA) is the most abundant (about 54−60% of blood
crystallography, capillary electrophoresis (CE), etc. These
methods differ in throughput and time of the measurement
have different sample requirements and limitations, and each
has its advantages and disadvantages (Table 1.). Also, most of
the methods are able to provide information solely about the
APIs’ affinity to HSA, while structural information regarding
the interactions between HSA and APIs can be acquired by
only a few timely and expensive techniques (e.g., NMR and X-
ray diffraction). Besides the experimental methods, in silico
models can be used to predict HSA binding, while molecular
modeling studies can also be used to explore possible binding
modes between HSA and APIs, providing additional structural
6
−8
proteins),
which is present in the blood at particularly high
8
−11
concentrations (35−50 g/L).
It plays an important role in
maintaining the osmotic pressure of the blood, serves as a
transport protein for endogenous substances (e.g., fatty acids
and steroid hormones), and it is also the main contributor to
5
,7,8,10
the binding of drug molecules.
shaped structure is composed of three main domains (I−III),
each containing two subdomains (A and B).
The protein’s heart-
8
−10
Besides the
several recently identified low- to high-affinity binding sites of
1
2,13
HSA,
the two specific drug binding sites (site I: warfarin
site on the IIA subdomain and site II: indole-benzodiazepine
site on the IIIA subdomain) are considered to be the most
significant, regarding plasma protein binding.
molecules usually form reversible complexes with HSA by
electrostatic and hydrophobic interactions. These HSA−API
complexes cannot cross biological membranes; therefore, they
40
8
,11,14−18
information on complex formation. To give a thoroughly
detailed description of the complexes, data from orthogonal
Drug
Received: January 10, 2020
Published: January 29, 2020
5
,19
become therapeutically inactive.
Moreover, strong binding
may also have a significant effect on the pharmacokinetic (PK)
2
0−22
19−21
behavior of APIs (e.g., altered clearance,
drug−drug interactions,
distribution,
and toxicity ). The HSA binding
19,23
24
©
2020 American Chemical Society
https://dx.doi.org/10.1021/acs.jmedchem.0c00046
1
763
J. Med. Chem. 2020, 63, 1763−1774

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Table 1. Summary of Commonly Used Experimental Methods for the Investigation of HSA−API binding 8,19,25−27a
1
measurement time/
method type
throughput
pros
cons
Quantitative Binding Data
chromatography and capillary
0.5 h/API HT
accurate results
no NSB, volume shift, or membrane
leakage
nonbiological systems
calibration of immobilized HSA column is needed before
measurements
3
2−35,39
electrophoresis
high solvent consumption and waste generation
poor sensitivity for low-binding APIs
expensive chiral columns
membrane separation methods
reliable results
Gibbs−Donnan effects
NSB on filter membranes and plastic devices
possible leakage of membrane
subsequent HPLC measurements
long time to reach equilibrium
volume shifts during incubation
dilution effects
equilibrium dialysis (ED)
rapid equilibrium dialysis
3 to 24 h
2−7 h HT
1−2 h HT
reaching equilibrium faster than ED
small volumes can be measured
NSB and volume shifts minimalized
fast separation of free and protein-
bound API
possible leakage of membrane
28,29
(
RED)
plate accessories can be expensive (although inexpensive
41
devices have been developed )
30
ultrafiltration (UF)
NSB on filter membrane and device
small volumes can be measured
possibility of molecular sieving
good approximation of physiological
conditions
moderate NSB and Donnan effect
pH and temperature controls are more difficult
31,42
ultracentrifugation
10−24 h MT
time-consuming, careful pH and temperature controls are
needed
errors due to the Johnston−Ogston effect
sedimentation of unbound API may occur
sample harvesting is difficult due to the floating lipid layer
expensive instrumentation
Structural Information
38
X-ray crystallography
days to months, LT yields the most accurate structural
information
impurities of the protein may hinder crystallization
binding sites can be identified
difficulty to determine correct crystallization conditions
solubility problems of API may arise
precipitation or growth of tiny crystals may occur
Binding and Structural Information
3
6,37
NMR
hours to days, LT
noninvasive technique
no separation step or subsequent
measurement needed
APIs with low aqueous solubility cannot be measured in
physiological buffers
b
UV-pH titration
0.5 h to 1−2 h MT fast method for screening
only ionizable molecules can be measured
absorbance of HSA may interfere
may provide additional structural
information of binding
APIs with low aqueous solubility can
be measured
a
b
API, active pharmaceutical ingredient; NSB, nonspecific binding; HT, high throughput; MT, medium throughput; and LT, low throughput. If
aqueous pK values without HSA have already been determined.
a
HA F A + H+
−
methods are needed to be evaluated and compared
simultaneously.
(1)
−
+
This study aims to present a UV-pH titration method as a
novel approach to screen for high-affinity HSA-binding
ionizable APIs. By means of the method, additional structural
information might also be gathered, and the role of ionization
centers in the interaction between HSA and APIs can be
further elucidated to help medicinal chemists in their efforts
toward rational molecular design.
[
A ][H ]
K =
a
[
HA]
(2)
−
where HA and A are the neutral and dissociated forms of the
acid, respectively. After rearrangement of eq 2 and taking
logarithms, we acquire the Henderson−Hasselbalch equation
−
i
[A ]
y
Theoretical Basis of UV-pH Titration. Spectrophotom-
etry can be applied for the measurement of proton dissociation
j
z
j
z
pH = pK + logj
z
z
a
j
k
[
HA]
(3)
{
constants (pK ) provided that the compound has a
a
chromophore in proximity to the ionization center and the
absorbance changes sufficiently as a function of pH. In aqueous
solutions, the dissociation equilibrium of a monoprotic acid
If there is a pH-dependent change in absorbance, the ratio of
−
43
HA and A can be determined spectrophotometrically. At a
pH where the molecule exists entirely in its neutral form, its
absorbance at a given wavelength is given by
and the acid dissociation constant (K ) can be written as
a
1
764
https://dx.doi.org/10.1021/acs.jmedchem.0c00046
J. Med. Chem. 2020, 63, 1763−1774

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 1. pH-dependent UV−vis absorbance changes and detectable pK values of HSA in the absence (left) and in the presence (right) of blank
a
correction.
AHA = εHA × l × [HA]
(4)
equilibrium and the acid dissociation constant can be written
as
where εHA and [HA] are the molar absorption coefficient and
concentration of HA, respectively, and l is the optical path
length. At a pH where the molecule exists entirely in its
HA·G F A·G + H+
−
(9)
−
+
−
[A·G ][H ]
[HA·G]
dissociated form, the absorbance of A is given by
*
K =
a
−
(10)
AA
−
= εA
−
× l × [A ]
(5)
where G is the guest molecule, and K* is the apparent proton
−
a
where εA− and [A ] are the molar absorption coefficient and
concentration of A , respectively. At an intermediate pH where
dissociation constant of the complexed API. Due to different
absorption properties of the complexed species, a significant
−
both species are present, the absorbance is given as
44
spectral change may occur. If absorption of the guest
A = ε × l × [HA] + εA
−
× l × [A−]
(6)
molecule is negligible and addition of absorbances is assumed,
then the absorbances of the molecular species can be given as
i
HA
−
Combining eqs 4−6, the ratio of HA and A can be written
AHA = εHA × l × [HA] + εHAG × l × [HA·G]
(11)
(12)
as
−
−
× l × [A·G−]
[
A ]
A − A
AA− = εA− × l × [A ] + ε
−
i
HA
AG
=
[
HA]
AA
−
− Ai
(7)
A = ε × l × [HA] + ε
× l × [HA·G]
i
HA
HAG
Combining eqs 3 and 7 provides a modified Henderson−
Hasselbalch equation, which can be used to calculate pK
values based on absorbance changes
−
−
+ εA
−
× l × [A ] + ε
−
× l × [A·G ]
(13)
a
AG
4
3
Substituting eqs 11−13 into eq 8, the apparent pK values
a
i
y
(pK ) can be calculated with shifts from pK values that are
A − A
a,app
a
j
i
HA z
j
z
pH = pK + logj
z
z
a
j
k
proportional to the ratio of bound and unbound fractions.
AA
−
− A
i {
(8)
RESULTS AND DISCUSSION
If the conditions for UV-pH measurements coincide, then
this method is the fastest way for the determination of pK
■
HSA-Binding Measurements by UV-pH Titration. We
investigated the possibility of measuring HSA binding of APIs
based on changes of proton dissociation constants of their free
and complexed molecular forms using UV-pH titration. First,
we collected UV−vis absorbance data of HSA during the UV-
pH titration assay to determine if the presence of HSA would
interfere with the signal of APIs. As can be seen in Figure 1
(left), HSA has two pK values (pK = 3.87 and pK = 11.56),
a
values (approximately 20−25 min/3 parallel measurements).
Furthermore, the sample requirement of built-in UV-pH
methods of the SiriusT3 instrument is also minuscule, and
only 5 μL of a 10 mM stock solution (DMSO, MeCN, etc.) of
the API is needed in a titration volume of 1 mL. Also,
measurements in the presence of co-solvents, macromolecules,
or additives may be possible, making it an ideal choice for
complexation studies as well.
a
a1
a2
which are measurable by UV-pH titration. If we add HSA to
the blank UV-pH titration assay (Figure 1, right), it annuls the
Upon complexation with macromolecules (e.g., cyclo-
dextrins, crown ethers, proteins, etc.), the dissociation
spectral changes belonging to the first pK value; however, the
a
1
765
https://dx.doi.org/10.1021/acs.jmedchem.0c00046
J. Med. Chem. 2020, 63, 1763−1774

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 2. Absorbance spectra and detectable pK values of diclofenac (DIC), phenylbutazone (PHB), piroxicam (PIR), and procaine (PRC) in the
a
presence and absence of HSA.
second pKa value remains detectable even after blank
correction. Therefore, even though the intensity of the signals
varying extent; we proposed that this might indicate which part
of the molecule contributes more to the binding to HSA. This
was later confirmed by molecular docking studies (see the next
section for further details). In some cases, absorption bands of
HSA suppressed the absorbance of the API; therefore, pKa,HSA
could not be determined. This usually occurred in the case of
APIs with only minuscule pH-dependent absorbance changes
in their spectrum in aqueous medium (e.g., diltiazem,
imipramine, indomethacin, and propranolol) or in the case
of pK values higher than ∼10 (e.g., furosemide pK and
of the second pK value is decreased, we must consider the
a
presence of overlapping absorption bands during titrations of
APIs.
Next, we investigated HSA binding of APIs by measuring
their pK values in aqueous medium in the absence of HSA and
a
in the presence of HSA (pKa,HSA). Figure 2 shows some
examples of the acquired spectra and experimental pK values
a
in the presence and absence of HSA. We observed significant
a
a2
pK shifts (ΔpK ) for several APIs, while others showed no
amodiaquine pK ) where the signals of pK of HSA
a
a
a3
a2
change of pK values upon addition of HSA (Table 2 and
interfered. We could avoid this latter problem if the API had
significant absorption at wavelengths higher than 320 nm (e.g.,
chloroquine pK , nitrazepam pK , and oxazepam pK ),
a
Table S1, Supporting Information). In the case of molecules
with multiple protonation centers, their pK values changed to
a
a2
a2
a2
1
766
https://dx.doi.org/10.1021/acs.jmedchem.0c00046
J. Med. Chem. 2020, 63, 1763−1774

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Table 2. Literature Data and Experimental Values of HSA Binding by Chromatographic, Rapid Equilibrium Dialysis (RED),
and UV-pH Titration Measurements
literature data
experimental data
UV-pH titration
4
,45−47
48,49
API name
amodiaquine
cefuroxime
chloroquine
PPB%
HSA% (HPLC)
HSA% (HPLC)
HSA% (RED)
ΔpKa1
ΔpKa2
78.8
40.1
50.5
0.12 ± 0.04
0.64 ± 0.03
a
a
33 ± 3; 31.5
(S): 66.6 ± 3.3
R): 42.7 ± 2.1
98.7 ± 0.2; 99
>99.5; 99.5
n.d.
n.d.
b
0.21 ± 0.03
0.75 ± 0.05
(
c
diazepam
diclofenac
diclofenac ethyl ester
diflunisal
diflunisal ethyl ester
diltiazem
famotidine
furosemide
imipramine
indomethacin
isoniazid
isoxicam
lornoxicam
meloxicam
O-methyl meloxicam
metronidazole
nitrazepam
93.2
99.0
89.2
100.0
98.1
100.0
97.5
56.6
25.1
99.4
84.9
100.0
10.9
97.3
100.0
99.9
84.2
11.9
76.7
79.9, 89.5
99.9
85.0
n.d.
0.09 ± 0.03
0.47 ± 0.01
95.8 ± 0.2
c
n.d.
a
99
98.7
∼100
0.38 ± 0.04
n.d.
c
n.d.
a
78
20
98.4
53.9
14.5
63.8
83.1
99.5
6.8
n.d.
4.4 ± 2.6
0.02 ± 0.03
0.10 ± 0.01
a
n.d.
a
90.1 ± 1.4; 92.6
90; 92−99
∼0
n.d.
a
95.8 ± 0.2
n.d.
a
0.05 ± 0.03
0.22 ± 0.05
0.87 ± 0.23
0.16 ± 0.23
n.d.
80.4 ± 0.8
98.0 ± 0.2
96.7 ± 0.1
65.9 ± 1.8
d
d
1.00 ± 0.07
0.55 ± 0.05
99
10; 11 ± 1
5.4
82.3
94.2
98.4
0.00 ± 0.01
0.05 ± 0.03
0.06 ± 0.03
0.53 ± 0.05
c
n.d.
n.d.
0.98 ± 0.06
0.27 ± 0.03
c
oxazepam
98.4
phenylbutazone
C-methyl phenylbutazone
O-methyl phenylbutazone
physostigmine
piroxicam
procaine
propranolol
sulindac
tenoxicam
97.8; 98−99
96.3 ± 0.5
64.1 ± 1.8
92.9 ± 1.9
9.7 ± 6.6
93.3 ± 0.3
6.4 ± 0.4
92.9
20.0
100.0
21.0
62.5
98.2
100.0
37.3
0.05 ± 0.03
0.00 ± 0.04
0.02 ± 0.06
99; 99
6
96.8
36.0
62.0
92.0
0.55 ± 0.09
0.00 ± 0.06
a
87 ± 6; 87
94; 93.5
n.d.
64.9 ± 6.9
96.8 ± 0.0
3.1 ± 4.6
91.5 ± 0.7
0.16 ± 0.06
0.13 ± 0.11
0.01 ± 0.02
0.41 ± 0.05
d
0.72 ± 0.09
trimethoprim
warfarin
37.5; 41.5
99 ± 1; 99
37.6
97.9
99.9, 100.0
a
b
Experimental data has been measured using a racemic compound. HSA binding could not be determined due to chemical decomposition during
incubation. HSA suppressed the absorbance of the API, and pK
c
d
could not be determined. High standard deviations originate from
a,HSA
extrapolated pK values, out of the measurement range of UV-pH titration.
a
where no interference with HSA absorption bands was
observed and thus evaluation of data was possible.
ΔpK 0.53), piroxicam (PIR; ΔpK , 0.55), and tenoxicam
a,
a2
(TEN; ΔpK , 0.72). In each case, the pK shifted toward a
a2
a
Comparison of the Results with Reference Values. To
compare our results with data from orthogonal methods, we
also measured HSA binding of the APIs using chromatographic
measurements on an immobilized HSA column using rapid
equilibrium dialysis (RED) and carried out molecular docking
of some APIs (Figure 3) into the crystal structure of HSA. We
found that in the case of compounds with high HSA binding,
more acidic value, favoring the deprotonated form of the APIs
when bound to HSA. To understand the structural basis of
these changes, the available X-ray structures of human serum
albumin (HSA) were scrutinized. From the mentioned APIs,
co-crystallized structures with HSA exist for DIC (PDB:
50
51
51
4Z69 ), DIF (PDB: 2BXE ), and PHB (PDB: 2BXC ).
Notably, DIC and PHB bind to drug site I (“warfarin site”),
while DIF binds to drug site II (indole-benzodiazepine site).
Although the exact hydrogen positions are not available at the
resolution of these structures, the immediate vicinity of the
ligand protonatable groups to positively charged amino acids
significant shifts (ΔpK > 0.1) could be observed for at least
a
one pK value, while no change of pK values can be expected
a
a
for APIs with lower HSA binding (HSA% less than ∼40%).
However, in some cases (e.g., diazepam (DZP)), the results of
UV-pH titration (ΔpK = 0.09) contradicted the data from
a
(
such as the K195-K199-R218-R222 cluster in drug site I or
orthogonal methods (HSA binding, >90%).
R410-K414 in drug site II) strongly implies the preference
toward the deprotonated forms of the APIs. The experimental
binding modes were evaluated with the extra precision (XP)
mode of Glide for both the protonated and deprotonated
forms (refinement only): per-residue Coulomb interaction
Among the studied drugs, pK shifts (from free to HSA-
bound form) around or greater than 0.4 units were observed
a
for chloroquine (CHQ; ΔpK , 0.75), diclofenac (DIC; ΔpK
a2
a,
0
0
.47), diflunisal (DIF; ΔpK , 0.38), meloxicam (MEL; ΔpK ,
a2
a2
.55), nitrazepam (NZP; ΔpK , 0.98), phenylbutazone (PHB;
a2
1
767
https://dx.doi.org/10.1021/acs.jmedchem.0c00046
J. Med. Chem. 2020, 63, 1763−1774

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 3. Structure of APIs investigated by molecular modeling studies. CHQ, chloroquine; DIC, diclofenac; DIF, diflunisal; DZP, diazepam; MEL,
meloxicam; NZP, nitrazepam; PHB, phenylbutazone; PIR, piroxicam; and TEN, tenoxicam.
Figure 4. Refined experimental binding modes for the deprotonated (green) and protonated (magenta) forms of (A) diclofenac, (B)
phenylbutazone, and (C) diflunisal (C). Coulomb interaction scores with the most important interacting residues are shown in matching colors,
the smaller the better. (While in phenylbutazone, formally, the ring CH gets deprotonated, and practically, the negative charge is located on one of
the oxo groups due to tautomerization).
scores between the ligands and the mentioned charged
residues confirm this implication (Figure 4).
For the remaining five APIs, Glide XP was used to predict
their binding modes in the two drug sites of HSA by docking
to the X-ray structures 2BXC and 2BXE. The poses were
inspected with regard to the vicinity of the protonatable groups
sites (K195-K199-R218-R222 in drug site I and R410-K414 in
drug site II, see also Figure 4).
Due to the contradictory UV-pH titration results of DZP, to
find possible explanations, we compared X-Ray structures
51
(
where available) and predicted binding modes of DZP and its
structural analogue NZP (Figure 6). In the X-ray structure
BXD, DZP occupies drug site II (A), with its only
2
(
specifically, the ones with the significant pK shifts) to
a
protonatable nitrogen being too far from the R485 residue to
make direct contact. Although its predicted binding mode in
potential ionic interaction partners. For four APIs, such a
binding pose was identified in drug site I (as the most favorable
binding pose in each case), and the differences of the Coulomb
interaction scores confirm the preference toward the
deprotonated forms (Figure 5). For CHQ (the only API
with a formal positive charge in its protonated form), no such
binding mode was identified in either drug site. We propose
that in the case of CHQ, the deprotonated form is favored
because ligand entry to both drug sites is hindered for the
protonated form due to the repulsive interactions with the
positively charged residue clusters at the entry points of the
site I (B) would justify a pK shift (the same nitrogen being
a
only 3.0 Å away from R257), binding to this site is not
observed in the crystal structure, suggesting that site II is
clearly preferred. Thus, the protonatable nitrogen will not be
able to directly interact with HSA, which can explain the
smaller than expected pK shift. On the contrary, in the
a
predicted binding mode of NZP to drug site II (C), the
deprotonatable amide nitrogen is too far from R410 to make
direct contact, but the predicted binding mode in drug site I
(D) nicely supports the observed pK shift of 0.56 units (by
a
1
768
https://dx.doi.org/10.1021/acs.jmedchem.0c00046
J. Med. Chem. 2020, 63, 1763−1774

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 5. Refined predicted binding modes for the deprotonated (green) and protonated (magenta) forms of (A) meloxicam, (B) nitrazepam, (C)
piroxicam, and (D) tenoxicam. Coulomb interaction scores with the most important interacting residues are shown in matching colors, the smaller
the better.
Figure 6. Comparison of crystallographic and predicted binding modes of (A, B) diazepam and (C, D) nitrazepam, with distances of the relevant
protonatable groups to the closest charged residues.
placing the amide nitrogen 3.4 Å away from R257), suggesting
the preference of NZP toward drug site I.
increase their bioavailability. For validation purposes, some of
the APIs with considerable pK shifts (DIC, DIF, MEL, and
a
Use of UV-pH Titration To Assist Medicinal Chem-
istry. As the previous examples show, UV-pH titration
combined with molecular modeling can be used to identify
the role of ionization centers in the formation of strong API−
HSA interactions. The information gathered this way might be
used to help chemists to design molecules with decreased HSA
binding, which might improve the API pharmacokinetics and
PHB) were modified by ester formation or alkylation to
neutralize their protonatable groups; these analogues were
evaluated with the same protocols as described above.
The chromatographic measurements showed a significant
decrease in retention time for each modified API, indicating a
lower HSA binding affinity (Table 2 and Figure 7). We
observed moderate decreases in binding for DIC ethyl ester
1
769
https://dx.doi.org/10.1021/acs.jmedchem.0c00046
J. Med. Chem. 2020, 63, 1763−1774

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 7. Chromatograms of diclofenac (DIC), diflunisal (DIF), meloxicam (MEL), phenylbutazone (PHB), and their modified forms with
neutralized ionization centers. (Due to low absorbance of O-methyl MEL, its extracted ion chromatogram (EIC) (m/z = 366.0) was also used for
peak identification).
(
100% → 98.1%) and DIF ethyl ester (100% → 97.5%) and
extensive decreases in the case of O-methyl MEL (99.9% →
4.2%), O-methyl PHB (99.9% → 92.9%), and C-methyl PHB
99.9% → 85.0%). RED measurements also showed significant
to help the design of novel molecules with favorable
pharmacokinetic behavior.
8
(
CONCLUSIONS
■
decreases in the binding of modified MEL (99.9% → 65.9%)
and PHB (99.9% → 92.9% (O-methyl PHB) and 99.9% →
In this study, we demonstrated the applicability of UV-pH
titration as an orthogonal method for the identification of APIs
with high-affinity binding. This fast and cost-effective method
can be used as a screening assay in the case of molecules
containing ionization centers, providing binding data faster
than conventional methods. We showed that the observed pKa
8
5.0% (C-methyl PHB)), while HSA binding could not be
determined this way for the esters of DIC and DIF due to their
instability under the measurement conditions of RED (Table
2
).
To further support the experimental results, molecular
shifts (ΔpK ) are proportional to HSA binding of APIs. In the
a
docking studies have also been carried out to compare binding
modes of the analogues. For the ethyl ester of DIF, the O- and
C-methylated PHB, and the ethyl ester of DIC, the top binding
modes predicted by Glide XP docking were closely similar to
their respective unmodified counterparts. Nonetheless, the
Coulomb interaction scores against the charged residues were
small, as expected (Figure 8). For O-methyl MEL, unrestrained
Glide XP docking could not identify a binding mode similar to
that of the unmodified API. When restricted to the reference
binding mode of meloxicam, the modified analogue exhibits
repulsive Coulomb interaction scores, while in its unrestricted
binding mode, the interaction scores confer slightly and in one
case (K199), moderately attractive contributions, similar to the
other analogues.
As the results of experimental measurements and molecular
modeling show, we succeeded in using UV-pH titration as a
screening tool for identifying structural moieties that make
major contribution in formation of complexes with strong
binding. By the modification of the APIs, the ionization centers
responsible for strong interactions with HSA could be
neutralized, resulting in a lower HSA binding. Since even a
few percent decreases have a considerable effect on the
case of multiprotic molecules, by means of molecular docking,
we demonstrated that the pK shifts of different sizes provide
a
structural information on the binding mode of the API. To
elucidate the significance of ionization centers of molecules, we
also investigated modified analogues with neutralized proto-
nation centers. The results clearly showed that decreased
protein binding can be achieved by this approach, resulting in
molecules with improved pharmacokinetics. Therefore, the
UV-pH titration method combined with an in silico support
might be used as a novel medicinal chemistry tool to assist
researchers in the rational drug design to decrease the high
attrition rate in later stages of drug discovery. In the future,
besides screening for APIs with high-affinity binding, this
method might also be used for comparison of binding of a
specific API with different kinds of modified HSA derivatives
or in the case of different formulations of HSA.
EXPERIMENTAL SECTION
■
Materials. Analytical grade solvents such as acetonitrile (MeCN),
dimethyl sulfoxide (DMSO), ethanol (absolute), formic acid,
trifluoroacetic acid (TFA), and potassium hydroxide (KOH) were
purchased from Merck KGaA (Darmstadt, Germany). Analytical
grade 2-propanol (IPA), 0.5 M hydrochloric acid, and 0.5 M
potassium hydroxide were purchased from Honeywell International
5
distribution of APIs, the observed decreases in HSA binding
(
from 2−3 to 10−35%) support the technique’s applicability
1
770
https://dx.doi.org/10.1021/acs.jmedchem.0c00046
J. Med. Chem. 2020, 63, 1763−1774

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 8. Predicted binding modes for (A) diflunisal ethyl ester, (B) C-methyl phenylbutazone, (C) O-methyl phenylbutazone, (D) O-methyl
meloxicam, and (E) diclofenac ethyl ester. For methoxy-meloxicam, the binding mode with the core position restricted to the binding mode of
meloxicam is shown in orange. Coulomb interaction scores with the most important charged residues are shown in matching colors (the smaller the
better): these are mostly slightly attractive (and in some cases, slightly repulsive), similar to the protonated forms of the original APIs. By
comparison, the deprotonated forms of the APIs exhibit large negative (strongly attractive) Coulomb contributions (see Figures 4 and 5).
Inc. (New Jersey, USA). Albumin from human serum (lyophilized
Diflunisal ethyl ester and C-methyl phenylbutazone were synthesized
at Gedeon Richter Plc. Compounds possessed a purity of >95% by
means of HPLC. (Diflunisal ethyl ester: SHIMADZU prominence
modular HPLC system equipped with a photodiode array detector
powder, ≥97% (agarose gel electrophoresis) cat. no.: A9511),
ammonium acetate (NH OAc), dichloromethane (DCM), ethyl
4
acetate (EtOAc), methyl iodide (MeI), phosphate-buffered saline
(
(
PBS) powder, potassium chloride, and the reference materials
acetaminophen, amodiaquine dihydrochloride dihydrate, amoxicillin,
(
PDA) and a mass spectrometer equipped with an electrospray
ionization source (ESI); column: CORTECS C18+, 90 Å, 2.7 μm
3.0 × 50 mm); retention time = 3.89 min; peak area, 98.76%; eluent
ampicillin, antipyrine, aspirin, atenolol, captopril, carbamazepine,
cefotaxime sodium, cefuroxime sodium, cephalexin, chloroquine
diphosphate, chlorothiazide, chlorpromazine hydrochloride, cimeti-
dine hydrochloride, clonidine hydrochloride, diazepam, diclofenac
sodium, diflunisal, diltiazem hydrochloride, diphenhydramine hydro-
chloride, famotidine, furosemide, imipramine hydrochloride, indome-
thacin, isoniazid, isoxicam, ketoconazole, meloxicam, metronidazole,
naproxen, nifedipine, nitrazepam, oxazepam, phenylbutazone, pheny-
toin, physostigmine, piroxicam, procaine hydrochloride, propranolol
hydrochloride, ranitidine hydrochloride, rifampin, sulfamerazine,
sulfamethoxazole, sulindac, terbutaline hemisulfate, trimethoprim,
and warfarin) were purchased from Sigma-Aldrich Co., Ltd.
(
A, 0.1% (v/v) formic acid in water; eluent B, MeCN/water 95:5 (v/v)
with 0.1% (v/v) formic acid; gradient: 0−4.5 min/2−100% B and
4
.5−5.6 min/100% B; flow rate of 1.25 mL/min; detection at 220 ± 4
nm; column temperature: 40 °C. C-methyl phenylbutazone: Agilent
200 liquid chromatography system equipped with a diode array
1
detector and coupled with an Agilent 6410 triple quadrupole mass
spectrometer (QQQ-MS) equipped with an ESI source; column:
Kinetex EVO C18, 100 Å, 2.6 μm (3.0 × 100 mm); retention time =
8
.60 min; peak area, 99.77%; eluent A, 0.1% (v/v) TFA in water;
eluent B, MeCN/water 95:5 (v/v) with 0.1% (v/v) TFA; gradient:
−15 min/0−100% B and 15−18 min/100% B; flow rate of 1 mL/
0
(Budapest, Hungary). Diclofenac ethyl ester, ketorolac tromethamine,
min; detection at 220 ± 4 nm; column temperature: 45 °C.) For
detailed information on their syntheses and confirmation of structures
(NMR and HRMS), see the Supporting Information (Sections 4−6).
Neutral linear buffer (NLB) was purchased from Pion Inc. (U.K.) Ltd.
(Forrest Row, United Kingdom). In all experiments, distilled water
and O-methyl meloxicam were purchased from Toronto Research
Chemicals Inc. (North York, Toronto, Canada). Lornoxicam and
tenoxicam were purchased from Carbosynth Ltd. (Compton,
Berkshire, United Kingdom). O-methyl phenylbutazone was pur-
chased from Ambinter c/o Greenpharma S.A.S. (Orleans, France).
́
1
771
https://dx.doi.org/10.1021/acs.jmedchem.0c00046
J. Med. Chem. 2020, 63, 1763−1774

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
was purified by the Millipore Milli-Q 140 gradient water purification
system.
UV-pH Titration. The UV-pH titrations were performed using a
Authors
Gergo Dargo − Department of Chemical and Environmental
Process Engineering, Budapest University of Technology and
Economics, 1111 Budapest, Hungary; Chemistry Department,
Gedeon Richter Plc., 1107 Budapest, Hungary
́
David Bajusz − Medicinal Chemistry Research Group, Research
Centre for Natural Sciences, 1117 Budapest, Hungary;
̋
́
fully automated SiriusT3 instrument (Pion Inc., Forest Row, U.K.).
Spectrophotometric determination of pK values was performed using
a
the built-in fast UV pK method, the absorbance changes during the
a
titrations were monitored by the dip-probe absorption spectroscopy
52,53
(D-PAS) method,
and evaluation and calculation of pK values
a
orcid.org/0000-0003-4277-9481
were performed by the SiriusT3Refine software (version 1.1.3.0., Pion
Kristof Simon − Department of Organic Chemistry and
́
Inc., Forest Row, U.K.). For the determination of pK values in
a
aqueous media, 5 μL of 10 mM DMSO solution of the samples was
titrated from pH 2.0 to pH 12.0 in 1.5 mL ionic strength adjusted
water (ISA-water: 0.15 M KCl; the initial sample concentration is
Technology, Budapest University of Technology and Economics,
1111 Budapest, Hungary
Judit Mu
̈
ller − Medicinal Chemistry Laboratory II, Gedeon
approximately 33.3 μM). In the case of determination of pK values in
a
Richter Plc., 1107 Budapest, Hungary
the presence of HSA (pKa,HSA), a modified fast UV assay was used:
ISA-water solutions containing HSA (33.3 μM, with a 1:1 nominal
molar ratio of HSA and APIs) were added manually in advance to the
titration vials. All measurements were performed under a nitrogen
atmosphere at T = 25.0 ± 0.1 °C. The pH region 2.0−10.0 and the
spectral region of 250−450 nm were used in the analysis, and the
results were calculated from a minimum of three replicates in each
case.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c00046
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Molecular Docking. Ligand and protein structures were prepared
5
4−56
Notes
with the standard tools of the Schrodinger software package,
̈
57
The authors declare no competing financial interest.
based on the OPLS3 force field. For ligand docking and the
generation of per-residue interaction scores, the extra precision mode
58,59
ACKNOWLEDGMENTS
G.D. thanks the Gedeon Richter Talentum Foundation for the
financial support. We are grateful to Gyula Beke for his
(
2
XP) of Glide was used.
BXC, 2BXE, and 4Z69 were used for evaluating the experimental
The publicly available PDB structures
■
51
50
binding modes of PHB, DIF, and DIC, respectively. For the additional
five APIs, the structures 2BXC and 2BXE were used to generate
predicted binding modes with Glide XP, after validating them by
redocking their cognate ligands into the respective binding pockets.
́
assistance in synthetizing modified APIs, to Aron Szigetvar
and Prof. Csaba Szantay Jr. for NMR measurements, and to
Janos Koti for HRMS measurements.
́
i
́
́
́
(
RMSD values between experimental and predicted poses were 1.40
and 1.01 Å for 2BXC and 2BXE, respectively, and 5.60 Å for 4Z69;
therefore, this structure was omitted from further use.) The best
identified binding pose was refined for both the protonated and
deprotonated forms of the APIs.
For detailed description of HSA-binding measurement by RED and
biomimetic chromatography, see the Supporting Information.
ABBREVIATIONS
■
CE, capillary electrophoresis; CHQ, chloroquine; DIC,
diclofenac; DIF, diflunisal; DZP, diazepam; DAD, diode
array detector; ED, equilibrium dialysis; EIC, extract ion
chromatogram; EtOAc, ethyl acetate; HT, high throughput;
IPA, 2-propanol; ISA, ionic strength adjusted; KOH,
potassium hydroxide; LT, low throughput; MeCN, acetoni-
trile; MeI, methyl iodide; MEL, meloxicam; MT, medium
throughput; NLB, neutral linear buffer; NSB, nonspecific
binding; NZP, nitrazepam; PDA, photodiode array (detector);
PHB, phenylbutazone; PIR, piroxicam; RED, rapid equilibrium
dialysis; SPME, solid-phase microextraction; TEN, tenoxicam;
TFA, trifluoroacetic acid; UF, ultrafiltration
ASSOCIATED CONTENT
■
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c00046.
Results of UV-pH titrations in the presence and absence
of HSA, results of HSA-binding measurements by RED
and biomimetic chromatography (CHIRALPAK HSA
column), comparison of the HPLC systems used for
HPLC-HSA measurements, and synthesis and structural
confirmation (NMR and HRMS) of diflunisal ethyl ester
and C-methyl phenylbutazone (PDF)
REFERENCES
■
(1) Di, L.; Kerns, E. H. Drug-Like Properties: Concepts, Structure
Design and Methods from ADME to Toxicity Optimization; Elsevier
Inc., 2016.
(
2) Fessey, R. E.; Austin, R. P.; Barton, P.; Davis, A. M.; Wenlock,
M. C. The Role of Plasma Protein Binding in Drug Discovery. In
Pharmacokinetic Profiling in Drug Research; Wiley-VCH Verlag GmbH
Molecular formula strings of different APIs (CSV)
&
(
Co. KGaA: Weinheim, Germany, 2007; pp 119−141.
3) Trainor, G. L. The Importance of Plasma Protein Binding in
AUTHOR INFORMATION
■
Drug Discovery. Expert Opin. Drug Discov. 2007, 2, 51−64.
Corresponding Author
̈
Gyorgy T. Balogh − Department of Chemical and
(4) Goodman & Gilman’s The Pharmacological Basis of Therapeutics,
1
3th ed.; Brunton, L. L., Hilal-Dandan, R., Knollmann, B. C., Eds.;
McGraw-Hill Education, 2018.
5) Sun, H.; Zhao, H. Physiologic Drug Distribution and Protein
Environmental Process Engineering, Budapest University of
Technology and Economics, 1111 Budapest, Hungary;
Chemistry Department, Gedeon Richter Plc., 1107 Budapest,
Hungary; Department of Pharmacodynamics and Biopharmacy,
University of Szeged, 6720 Szeged, Hungary; orcid.org/
(
Binding. In Applied Biopharmaceutics & Pharmacokinetics; Shargel, L.,
Yu, A. B. C., Eds.; McGraw-Hill Education, 2016.
(6) Fox, S., Blood, I. Heart, and Circulation. In Human Physiology;
McGraw-Hill Education, 2016.
(7) Betts, J. G.; Desaix, P.; Johnson, E. W.; Johnson, J. E.; Korol, O.;
Kruse, D.; Poe, B.; Wise, J.; Womble, M. D.; Young, K. A. The
0
000-0001-8273-1760; Phone: +36 1 463 2174;
Email: gytbalogh@mail.bme.hu
1
772
https://dx.doi.org/10.1021/acs.jmedchem.0c00046
J. Med. Chem. 2020, 63, 1763−1774

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Cardiovascular System: Blood. In Anatomy & Physiology; OpenStax,
(27) Ronzetti, M.; Baljinnyam, B.; Yasgar, A.; Simeonov, A. Testing
for Drug-Human Serum Albumin Binding Using Fluorescent Probes
and Other Methods. Expert Opin. Drug Discov. 2018, 13, 1005−1014.
(28) Sebille, B. Methods of Drug Protein Binding Determinations.
Fundam. Clin. Pharmacol. 1990, 4, 151s−161s.
2
(
013.
8) Fanali, G.; di Masi, A.; Trezza, V.; Marino, M.; Fasano, M.;
Ascenzi, P. Human Serum Albumin: From Bench to Bedside. Mol.
Aspects Med. 2012, 33, 209−290.
(9) Di, L.; Kerns, E. H.; Di, L.; Kerns, E. H. Chapter 14 − Plasma
(29) Wan, H.; Rehngren, M. High-Throughput Screening of Protein
Binding by Equilibrium Dialysis Combined with Liquid Chromatog-
raphy and Mass Spectrometry. J. Chromatogr. A 2006, 1102, 125−
and Tissue Binding. In Drug-Like Properties; Academic Press, 2016; pp
19−228.
10) Quinlan, G. J.; Martin, G. S.; Evans, T. W. Albumin:
Biochemical Properties and Therapeutic Potential. Hepatology 2005,
1, 1211−1219.
11) Kragh-Hansen, U.; Chuang, V. T. G.; Otagiri, M. Practical
2
(
1
(
34.
30) Blanchard, J.; Harvey, S. Comparison of Ultrafiltration Devices
for Assessing Theophylline Protein Binding. Ther. Drug Monit. 1990,
2, 398−403.
31) Cole, J. L.; Lary, J. W.; Moody, T. P.; Laue, T. M. Analytical
4
(
1
(
Aspects of the Ligand-Binding and Enzymatic Properties of Human
Ultracentrifugation: Sedimentation Velocity and Sedimentation
Equilibrium. In Methods in Cell Biology; Elsevier, 2008; Vol. 84, pp
Serum Albumin. Biol. Pharm. Bull. 2002, 25, 695−704.
(12) Jahanban-Esfahlan, A.; Davaran, S.; Moosavi-Movahedi, A. A.;
1
(
43−179.
32) Wan, H.; Bergstro
Protein Binding in Drug Discovery. J. Liq. Chromatogr. Relat. Technol.
007, 30, 681−700.
33) Li, Y.-F.; Zhang, X.-Q.; Hu, W.-Y.; Li, Z.; Liu, P.-X.; Zhang, Z.-
Dastmalchi, S. Investigating the Interaction of Juglone (5-Hydroxy-1,
-Naphthoquinone) with Serum Albumins Using Spectroscopic and
in Silico Methods. J. Iran. Chem. Soc. 2017, 14, 1527−1540.
13) Roufegarinejad, L.; Amarowicz, R.; Jahanban-Esfahlan, A.
̈
m, F. High Throughput Screening of Drug-
4
2
(
(
Characterizing the Interaction between Pyrogallol and Human Serum
Albumin by Spectroscopic and Molecular Docking Methods. J.
Biomol. Struct. Dyn. 2018, 37, 2766−2775.
Q. Rapid Screening of Drug-Protein Binding Using High-Performance
Affinity Chromatography with Columns Containing Immobilized
Human Serum Albumin. J. Anal. Methods Chem. 2013, 2013, 1−7.
(14) Christensen, H.; Baker, M.; Tucker, G. T.; Rostami-Hodjegan,
(34) Noctor, T. A. G.; Diaz-Perez, M. J.; Wainer, I. W. Use of a
A. Prediction of Plasma Protein Binding Displacement and Its
Implications for Quantitative Assessment of Metabolic Drug-Drug
Interactions from in Vitro Data. J. Pharm. Sci. 2006, 95, 2778−2787.
Human Serum Albumin-Based Stationary Phase for High-Perform-
ance Liquid Chromatography as a Tool for the Rapid Determination
of Drug−Plasma Protein Binding. J. Pharm. Sci. 1993, 82, 675−676.
(15) Birkett, D. J.; Myers, S. P.; Sudlow, G. Effects of Fatty Acids on
(35) Danon, A.; Chen, Z. Binding of Imipramine to Plasma Proteins:
Two Specific Drug Binding Sites on Human Serum Albumin. Mol.
Pharmacol. 1977, 13, 987−992.
Effect of Hyperlipoproteinemia. Clin. Pharmacol. Ther. 1979, 25,
3
16−321.
(16) Sudlow, G.; Birkett, D. J.; Wade, D. N. Further Character-
(36) Lucas, L. H.; Larive, C. K. Measuring Ligand-Protein Binding
Using NMR Diffusion Experiments. Concepts Magn. Reson. Part A.
2004, 20A, 24−41.
ization of Specific Drug Binding Sites on Human Serum Albumin.
Mol. Pharmacol. 1976, 12, 1052−1061.
(17) Yamasaki, K.; Maruyama, T.; Kragh-Hansen, U.; Otagiri, M.
(37) Gallo, M.; Matteucci, S.; Alaimo, N.; Pitti, E.; Orsale, M. V.;
Summa, V.; Cicero, D. O.; Monteagudo, E. A Novel Method Using
Nuclear Magnetic Resonance for Plasma Protein Binding Assessment
in Drug Discovery Programs. J. Pharm. Biomed. Anal. 2019, 167, 21−
29.
(38) Zhu, L.; Yang, F.; Chen, L.; Meehan, E. J.; Huang, M. A New
Drug Binding Subsite on Human Serum Albumin and Drug−Drug
Interaction Studied by X-Ray Crystallography. J. Struct. Biol. 2008,
Characterization of Site I on Human Serum Albumin: Concept about
the Structure of a Drug Binding Site. Biochim. Biophys. Acta - Protein
Struct. Mol. Enzymol. 1996, 1295, 147−157.
(18) Zhivkova, Z. Studies on Drug − Human Serum Albumin
Binding: The Current State of the Matter. Curr. Pharm. Des. 2015, 21,
817−1830.
19) Bohnert, T.; Gan, L.-S. Plasma Protein Binding: From
Discovery to Development. J. Pharm. Sci. 2013, 102, 2953−2994.
20) Rolan, P. Plasma Protein Binding Displacement Interactions-
1
(
1
(
62, 40−49.
39) Wan, H.; Thompson, R. A. Capillary Electrophoresis
(
Technologies for Screening in Drug Discovery. Drug Discov. Today
Technol. 2005, 2, 171−178.
Why Are They Still Regarded as Clinically Important? Br. J. Clin.
Pharmacol. 1994, 37, 125−128.
(40) Lambrinidis, G.; Vallianatou, T.; Tsantili-Kakoulidou, A. In
(21) Bowman, C. M.; Benet, L. Z. An Examination of Protein
Vitro, in Silico and Integrated Strategies for the Estimation of Plasma
Binding and Protein-Facilitated Uptake Relating to in Vitro-in Vivo
Protein Binding. A Review. Adv. Drug Delivery Rev 2015, 86, 27−45.
Extrapolation. Eur. J. Pharm. Sci. 2018, 123, 502−514.
(41) Herbst, Z. M.; Shibata, S.; Fan, E.; Gelb, M. H. An Inexpensive,
(22) Baker, M.; Parton, T. Kinetic Determinants of Hepatic
In-House-Made, Microdialysis Device for Measuring Drug−Protein
Clearance: Plasma Protein Binding and Hepatic Uptake. Xenobiotica
007, 37, 1110−1134.
23) Talbert, A. M.; Tranter, G. E.; Holmes, E.; Francis, P. L.
Binding. ACS Med. Chem. Lett. 2018, 9, 279−282.
2
(
(42) Kieltyka, K.; McAuliffe, B.; Cianci, C.; Drexler, D. M.; Shou,
W.; Zhang, J. Application of Cassette Ultracentrifugation Using Non-
Labeled Compounds and Liquid Chromatography-Tandem Mass
Spectrometry Analysis for High-Throughput Protein Binding
Determination. J. Pharm. Sci. 2016, 1036.
Determination of Drug−Plasma Protein Binding Kinetics and
Equilibria by Chromatographic Profiling: Exemplification of the
Method Using l−Tryptophan and Albumin. Anal. Chem. 2002, 74,
4
(
46−452.
(43) Salgado, L. E. V.; Vargas-Hernandez, C. Spectrophotometric
́
24) Svennebring, A. The Connection Between Plasma Protein
Determination of the PKa, Isosbestic Point and Equation of
Absorbance vs. PH for a Universal PH Indicator. Am. J. Anal.
Chem. 2014, 05, 1290−1301.
Binding and Acute Toxicity as Determined by the LD 50 Value. Drug
Dev. Res. 2016, 77, 3−11.
(25) Ramanathan, V.; Vachharajani, N. Protein Binding in Drug
(44) Connors, K. A. Optical Absorption Spectroscopy. In Binding
Discovery and Development. In Evaluation of Drug Candidates for
Preclinical Development; John Wiley & Sons, Inc.: Hoboken, NJ, USA,
Constants: The Measurement of Molecular Complex Stability; Wiley-
Interscience, 1987; pp 141−187.
2
(
010; Vol. 13, pp 135−167.
(
45) Goodman and Gilman’s The Pharmacological Basis of
26) Buscher, B.; Laakso, S.; Mascher, H.; Pusecker, K.; Doig, M.;
Therapeutics, 10th ed.; Hardman, J. G., Limbird, L. E., Gilman, A.
G., Eds.; McGraw-Hill, 2001.
(46) Zhang, F.; Xue, J.; Shao, J.; Jia, L. Compilation of 222 Drugs’
Plasma Protein Binding Data and Guidance for Study Designs. Drug
Discovery Today 2012, 475−485.
Dillen, L.; Wagner-Redeker, W.; Pfeifer, T.; Delrat, P.; Timmerman,
P. Bioanalysis for Plasma Protein Binding Studies in Drug Discovery
and Drug Development: Views and Recommendations of the
European Bioanalysis Forum. Bioanalysis 2014, 6, 673−682.
1
773
https://dx.doi.org/10.1021/acs.jmedchem.0c00046
J. Med. Chem. 2020, 63, 1763−1774

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(
47) Singh, S. S.; Mehta, J. Measurement of Drug−Protein Binding
by Immobilized Human Serum Albumin-HPLC and Comparison with
Ultrafiltration. J. Chromatogr., B 2006, 834, 108−116.
(48) Valko, K.; Nunhuck, S.; Bevan, C.; Abraham, M. H.; Reynolds,
D. P. Fast Gradient HPLC Method to Determine Compounds
Binding to Human Serum Albumin. Relationships with Octanol/
Water and Immobilized Artificial Membrane Lipophilicity. J. Pharm.
Sci. 2003, 92, 2236−2248.
(49) Hollosy, F.; Valko, K.; Hersey, A.; Nunhuck, S.; Keri, G.;
́ ́ ́
Bevan, C. Estimation of Volume of Distribution in Humans from
High Throughput HPLC-Based Measurements of Human Serum
Albumin Binding and Immobilized Artificial Membrane Partitioning.
J. Med. Chem. 2006, 49, 6958−6971.
(50) Zhang, Y.; Lee, P.; Liang, S.; Zhou, Z.; Wu, X.; Yang, F.; Liang,
H. Structural Basis of Non-Steroidal Anti-Inflammatory Drug
Diclofenac Binding to Human Serum Albumin. Chem. Biol. Drug
Des. 2015, 86, 1178−1184.
(51) Ghuman, J.; Zunszain, P. A.; Petitpas, I.; Bhattacharya, A. A.;
Otagiri, M.; Curry, S. Structural Basis of the Drug-Binding Specificity
of Human Serum Albumin. J. Mol. Biol. 2005, 353, 38−52.
(52) Allen, R. I.; Box, K. J.; Comer, J. E. A.; Peake, C.; Tam, K. Y.
Multiwavelength Spectrophotometric Determination of Acid Dissoci-
ation Constants of Ionizable Drugs. J. Pharm. Biomed. Anal. 1998, 17,
6
(
99−712.
53) Tam, K. Y.; Takacs-Novak, K. Multi-Wavelength Spectropho-
́
́
tometric Determination of Acid Dissociation Constants: A Validation
Study. Anal. Chim. Acta 2001, 434, 157−167.
(54) Madhavi Sastry, G.; Adzhigirey, M.; Day, T.; Annabhimoju, R.;
Sherman, W. Protein and Ligand Preparation: Parameters, Protocols,
and Influence on Virtual Screening Enrichments. J. Comput.-Aided
Mol. Des. 2013, 27, 221−234.
(
55) Schro
Schrodinger, LLC: New York, NY, 2018.
56) Schrodinger Release 2018−4: LigPrep; Schro
York, NY, 2018.
57) Harder, E.; Damm, W.; Maple, J.; Wu, C.; Reboul, M.; Xiang, J.
̈
dinger Release 2018−4: Protein Preparation Wizard;
̈
(
̈
̈
dinger, LLC: New
(
Y.; Wang, L.; Lupyan, D.; Dahlgren, M. K.; Knight, J. L.; Kaus, J. W.;
Cerutti, D. S.; Krilov, G.; Jorgensen, W. L.; Abel, R.; Friesner, R. A.
OPLS3: A Force Field Providing Broad Coverage of Drug-like Small
Molecules and Proteins. J. Chem. Theory Comput. 2016, 12, 281−296.
(58) Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.;
Greenwood, J. R.; Halgren, T. A.; Sanschagrin, P. C.; Mainz, D. T.
Extra Precision Glide: Docking and Scoring Incorporating a Model of
Hydrophobic Enclosure for Protein-Ligand Complexes. J. Med. Chem.
2
(
006, 49, 6177−6196.
59) Schrodinger Release 2018−4: Glide; Schro
York, NY, 2018.
̈
̈
dinger, LLC: New
1
774
https://dx.doi.org/10.1021/acs.jmedchem.0c00046
J. Med. Chem. 2020, 63, 1763−1774