Expert Opinion on Drug Discovery

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Binding affinity in drug design: experimental and

computational techniques

Visvaldas Kairys, Lina Baranauskiene, Migle Kazlauskiene, Daumantas

Matulis & Egidijus Kazlauskas

To cite this article: Visvaldas Kairys, Lina Baranauskiene, Migle Kazlauskiene, Daumantas
Matulis & Egidijus Kazlauskas (2019) Binding affinity in drug design: experimental and
computational techniques, Expert Opinion on Drug Discovery, 14:8, 755-768, DOI:
10.1080/17460441.2019.1623202

To link to this article: https://doi.org/10.1080/17460441.2019.1623202

Published online: 31 May 2019.

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EXPERT OPINION ON DRUG DISCOVERY
2019, VOL. 14, NO. 8, 755–768
https://doi.org/10.1080/17460441.2019.1623202

REVIEW

Binding affinity in drug design: experimental and computational techniques

Visvaldas Kairys a, Lina Baranauskiene b
, Migle Kazlauskiene c
, Daumantas Matulis b

and Egidijus Kazlauskas b

a
Department of Bioinformatics, Institute of Biotechnology, Life Sciences Center, Vilnius University, Vilnius, Lithuania; bDepartment of
Biothermodynamics and Drug Design, Institute of Biotechnology, Life Sciences Center, Vilnius University, Vilnius, Lithuania; cDepartment of
Biochemistry, University of Zurich, Zurich, Switzerland

ABSTRACT ARTICLE HISTORY

Introduction: In pharmaceutical design where future drugs are developed by targeting a specific Received 2 April 2019
chosen protein, the evaluation of ligand affinity is crucial. For this very purpose are a multitude of Accepted 21 May 2019
diverse methods which are continuously being improved, which, in turn, makes it difficult to choose KEYWORDS
which techniques to use in practice. Drug design; ligand;
Areas covered: In this review, the authors discuss both experimental and computational approaches inhibitor; affinity; free
for affinity evaluation. Basic principles, general limitations and advantages, as well as main areas of energy; Gibbs energy;
application in drug discovery, are overviewed for some of the most popular ligand binding assays. The binding; ITC; DSC; DSF; FTSA;
authors further provide a guide to affinity predictions, collectively covering several techniques that are TSA; AUC; electrophoresis;
used in the first stages of rational drug design. MST; thermophoresis;
Expert opinion: All affinity estimation methods have limitations and advantages that partially overlap fluorescence; ICD; BLI; SPR;
NMR; QSAR; molecular
and complement one another. Some of the suggested best practices include cross-verification of data
dynamics; QM/MM; MM-
using at least two different techniques and careful data interpretation. PBSA

1. Introduction Therefore, here, we will briefly overview experimental and

computational techniques devised for the evaluation of bind-
Target-oriented drug design aims to find a high affinity ligand
ing affinity. Hopefully, it will allow the reader to get a general
that would bind the target protein in order to block its dis-
idea of where a particular method would shine or fail as well
ease-associated function – catalytic activity or interaction with
as to help familiarize themselves with the most recent devel-
other molecules. This process usually starts with the screening
opments in the field.
of an initial library of ligands or their fragments to identify
All of the applicable methods reviewed here aim to evaluate
binders of the target protein, either experimentally or compu-
binding affinity. While pharmacologists often refer to biological
tationally (Figure 1). Often computational screens are used
activity or efficacy, which is the ability of a drug to produce
first to narrow the library down before proceeding with the
a functional response in living matter, rational drug design
experimental screening. Successful hits are then characterized
usually simplifies the studied system into only the ligand and
and further developed. At each such stage, the ligand binding
its target protein. The strength of the interaction between these
to the target protein is reevaluated.
two molecules is defined by either binding and dissociation
Correct and precise estimation of the binding affinity is
constants (Kb and Kd) or Gibbs energy of binding (ΔGb) and is
crucial throughout these essential drug design stages, as
commonly referred to as affinity. In a simple, reversible one
they commonly serve as the main factor for choosing promis-
protein – one ligand interaction case, equilibrium exists between
ing leads and their optimization strategies [1,2]. This high
the free molecules (P, protein, and L, ligand) and their complex
demand has facilitated the development of a number of dif-
(PL) that associate and dissociate at certain rates (described by
ferent techniques to assess or predict ligand binding. They all
rate constants kon and koff, respectively):
have their unique set of applications and limitations, and
several different methods are usually employed in parallel to kon
ensure data reliability. Multiple attempts have been made to P þ L ! PL (1)
systemize experimental [3,4] or computational [5,6] koff
approaches, but some of the methods have seen significant
Experimental researchers tend to define the binding affinity
improvements since. Since collaborations between bioinfor-
using equilibrium dissociation (Kd) or binding (Kb) constants:
maticians and biophysicists are becoming more and more
the norm, as illustrated by a variety of successful examples ½PL 1 kon
Kb ¼ ¼ ¼ (2)
from both industry and academia [1,7], both sides would ½P½L Kd koff
benefit from a better understanding of each other’s tools.

CONTACT Egidijus Kazlauskas ekazl@ibt.lt Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Life Sciences Center, Vilnius
University, Saulėtekio al. 7, Vilnius LT-10257, Lithuania
© 2019 Informa UK Limited, trading as Taylor & Francis Group
756 V. KAIRYS ET AL.
Article highlights
● Affinity determination is one of the cornerstones of modern drug
design.
● There are a lot of experimental and computational techniques avail-
able to evaluate a drug’s affinity towards its target, each with their
own pros and cons.
● Isothermal titration calorimetry (ITC) measures the binding energy of
unmodified interactors directly at physiological temperature but is
costly both materials- and time-wise.
● High-throughput stability, mobility, or spectroscopic shift assays
allow for rapid affinity evaluation, which is readily exploited for
screening, but often are limited target-wise.
● In silico binding affinity prediction methods which encompass a vast
range of algorithms are advanced enough to be able to reduce the
Root Mean Square Error of prediction for a series of compounds to ~5
kJ/mol.
● Specialized competitions for calculating the binding affinity show the
latest trends in the computational methods and their performance,
compared to older, venerable approaches.
This box summarizes the key points contained in the article.
At equilibrium under standard conditions, the Gibbs energy
(previously also known as free energy) of binding describes
the energy difference between the two states:
½PL
ΔGb ¼ RTlnð½P½LÞ  RTln½PL ¼ RTln ¼ RTlnKb
½P½L
¼ RTlnKd ;
where R is the gas constant and T is the absolute temperature
at which the binding occurs. ΔGb can also be dissected into its
components enthalpy (ΔHb) and entropy (ΔSb):
ΔGb ¼ ΔHb  TΔSb
In computational approaches, ΔGb is often approximated to the
protein–ligand interaction energies (i.e. essentially ΔHb), adding
additional terms appraising entropy. While researchers from dif-
ferent fields tend to use either ΔGb or Kd/Kb to define the strength
of binding, equation (3) clearly shows the direct relation between
the binding (or dissociation) constant and Gibbs energy of bind-
ing, with only temperature as an additional variable. Therefore,
while we will be discussing mainly binding constant determina-
tion in the section on experimental approaches and Gibbs energy
calculations in the computational section, both of them refer to
the same affinity and can be compared with minimal calculations.
100...000 compounds
Hit Lead
Target selection
generation optimisation
screening: analysis:
Docking, ... QSAR, ...
FTSA, MST, ITC, SPR,
CE, NMR... NMR...
Figure 1. Affinity estimation methods in drug discovery process. Protein-ligand affinity determination methods are heavily relied upon during the early stages of
drug design and throughout the entire lead optimization process in order to produce drug candidates for preclinical and clinical trials from initial library of millions
of compounds.
2. Experimental approaches to evaluate affinity
Experimentalists can choose from a wide range of techniques
designed to determine the affinity of two molecules. They vary
significantly in the underlying principles, suitability for differ-
ent cases, and experimental procedures. Majority of such
approaches that are well established in drug design are sum-
marized in Table 1. However, as the review space is quite
limited in comparison to the plethora of viable methods,
some of them are inevitably omitted.
Except for ITC, which records the binding energetics directly,
and structural approaches, most of the methods discussed here
detect what portion of the ligand is being bound at certain
concentrations, and calculate the Kb from there. As seen from
Table 1, the methods significantly vary not only in their principles
but also their detection limits. Typical experiment is performed
by preparing at least 8–16 samples in serial dilutions: the con-
centration of one of the interacting molecules (e.g. protein) is
kept constant while the other molecule (e.g. ligand) is diluted
over a wide range of concentrations. The techniques for
a specific case are chosen based on properties of the target
protein and ligands – the detectability and stability. In this
chapter, we will discuss the most prominent experimental
approaches for evaluating ligand binding to a protein target.
For convenience, we grouped these methods based on
what binding event or product properties they are exploiting:
released energy, changes in stability, mobility, or spectro-
(3) scopic characteristics upon binding, and structure-orientated
approaches. Notably, this sorting is arbitrary as a lot of these
techniques can fit several categories.
2.1. Isothermal titration calorimetry
(4)
Isothermal titration calorimetry (ITC) can measure thermodynamic
energetics of binding directly, without any need of labeling,
immobilization, or any other modification of the interactors.
Moreover, ITC is highly versatile as any binding results in either
consumed or released heat, and there are no limitations for the
size, optical or other properties of the binders. Due to its generally
reliable data, wide target applicability, lack of modifications or
reporters, and multiple output binding parameters, ITC is being
considered the ‘golden standard’ of protein–ligand interaction
measurements. ITC experiments provide a thermodynamic picture
of binding, encompassing ΔGb and its enthalpic (ΔHb) and thus
calculated entropic (ΔSb) components.
Preclinical
testing
Clinical trials 1 drug
Table 1. Overview of selected methods for experimental determination of the binding Gibbs energy.
Required
Protein amount of Typical Application
Short name Full name Information interactor Priciple samplea Kb range Typical Kd range example
ITC Isothermal titration calorimetry Kd, ΔHb, ΔGb, ΔSb, Any Heat release or uptake Medium-high 103–109 mM–nM [124]
stoichiometry
Stability shift assays:
ICD Isothermal chemical denaturation Kd Small ligand Change in chemical denaturation profile, as Medium 103–1012 mM–pM [8]
detected by fluorescence
FPSA Fluorescence pressure shift assay Kd, ΔV, Δβ Small ligand Change in unfolding point, as detected by High 103–1010 mM–sub-nM [9]
(PressureFluor) fluorescent probe
3 12
DSC Differential scanning calorimetry Kd, ΔCp, ΔHunfolding Small ligand Change in melting point, as detected by heat High 10 –10 mM–sub-pM [10]
release or uptake
FTSA, TSA, Fluorescence thermal shift assay (differential Kd, Tm Small ligand Change in melting point, as detected by Low 103–1013 mM– sub-pM [11]
DSF scanning fluorimetry, ThermoFluor) fluorescent probe
Mobility shift (separative) assays:
ED Equilibrium dialysis Kd, stoichiometry Small ligand Separation of bound and free ligands High 101–1012 mM–pM [12]
SEC Size exclusion chromatography Kd, size, stoichiometry Any Change in hydrodynamic size High 104–1013 µM–sub-pM [13]
WAC Weak affinity chromatography Kd Small ligands, Change in retention time due to binding Medium 102–107 mM–μM [14]
fragments
AUC Analytical ultracentrifugation Any
AUC sedimentation velocity Kd, shape, Change in sedimentation properties Medium 103–109 mM–nM [39]
conformation
changes
AUC sedimentation equilibrium Kd, size, stoichiometry Change in solution mass Medium 103–109 mM–nM [15]
EMSA Electrophoretic mobility shift assay Kd Nucleic acid Change in size and/or charge, as detected by Low 104–109 µM–nM [42]
mobility in electric field
CE Capillary electrophoresis Kd, (kon, koff), Any Change in size, shape, or charge, as detected by Low 102–107 mM–µM [16]
stoichiometry mobility in electric field
MST Microscale thermophoresis Kd, stoichiometry Any Change in size, charge, or hydration shell, as Low 103–1012 mM–pM [17]
detected by mobility in temperature gradient
Spectroscopic assays:
UV-vis UV-vis spectroscopy Kd Any Change in absorbance Medium <109 >nM [18]
Fluorescence Different applications of fluorescence Kd Any Change in fluorescence properties Low <1012 >pM [50]
spectroscopy
ICD Induced circular dichroism spectroscopy Kd Small ligand Change in elipticity Low 103–107 mM–µM [47]
SEC-MALS Size-exclusion chromatography with multi- Kd, molecule size Any with >3% Change in molecular size (complex formation) Medium reported Reported µM [19]
angle light scattering mass change 105–107
SPR Surface plasmon resonance Kd, kon, koff Any, >100 Da Changes in the refractive index Low 103–1011 mM–pM [20]
BLI Bio-layer interferometry Kd, kon, koff Any, >150 Da Shift in interferometric light reflection Low 103–1011 mM–pM [20]
NMR Nuclear magnetic resonance Any,
one <40 kDa
b b
Ligand-observed NMR Kd, (kon, koff) Change in chemical shift and relaxation or Medium 102–1010 mM–nM [21]
diffusion rates of the ligand(s)
b b
Protein-observed NMR Kd, structural change, Change in chemical shift and relaxation or High 103–109 mM – nM [22]
interacting residues diffusion rates of the protein
Kd – dissociation constant; ΔGb – Gibbs energy of binding; ΔHb – binding enthalpy change; ΔSb – binding entropy change; ΔV – volume change; Δβ – compressibility; ΔCp – heat capacity change; ΔHunfording – enthalpy change
of unfolding; Tm – melting temperature; kon – association rate constant; koff – dissociation rate constant.
a
EXPERT OPINION ON DRUG DISCOVERY

≤10 ng of protein required for Kd determination is considered low sample consumption, ≥1 mg – high.
b
In NMR, the reliably detectable affinity range significantly depends on kinetics.
757
758 V. KAIRYS ET AL.
During the measurement, one of the binding partners is
titrated with aliquots of the other under constant temperature,
and the released or absorbed heat is measured. Construction
of a binding isotherm of interaction heat as a function of
titrated ligand (Figure 2(a)) yields ΔHb, Kb (and therefore
ΔGb) with ΔSb calculated from equation (4), and usually stoi-
chiometry (n). The range for accurate ITC measurement win-
dow for a given reaction is defined by c value [23] but too
tight affinities can be tackled using competition ITC assays.
One approach is to premix a moderate affinity ligand with
excess of the target protein and titrate this mixture with the
high-affinity ligand [24]. While this approach also shows
whether both ligands compete for the same site, the data
interpretation is not always straightforward. For example, the
difference between the affinities has to be sufficient to result
in two distinct isotherm transition points.
Notably, due to its versatility, ITC detects the total heat
exchange, which can be caused not only by the binding
event itself but also other simultaneous events, including
protonation, desolvation, and dilution. The true Kb values,
obtained after subtracting energies of other events occurring
in the measurement cell, are called ‘intrinsic’ and are better
suited for correlations with structural data [25,26]. These addi-
tional events can be unraveled using correct control experi-
ments, further increasing the required experimental time.
Thus, the raw determined Kb values, often referred to as
‘observed’, are still widely used to rank compounds.
Since it has been observed that more enthalpy-driven ligand
binding is common for natural inhibitors and correlates with
higher selectivity [27], dissection of ΔGb into enthalpic and entro-
pic components is useful for planning lead optimization. While
van’t Hoff relationship allows to indirectly calculate enthalpy
change from thermal stability assays (see next section), it relies
on sometimes erroneous assumption that enthalpy change is
temperature-independent and thus does not always correlate
with values directly measured using ITC [28]. Overall, ITC is not
a standard technique in the initial high-throughput drug screen-
ing stages due to its relatively high requirement of material and
time, but it has proven its usability in the later drug development
stages [29].
2.2. Stability shift assays
Before ITC was well established, another calorimetric technique
had gained the trust of pharmacologists – differential scanning
calorimetry (DSC). In general, stability shift assays rely on ligand’s
effect on the stability of the protein. Usually, drug leads stabilize
the target protein and a favorable stability shift is observed. In
general, the stability is evaluated by denaturing the protein using
temperature, pressure [30], or chemical agents [31]. The stability
shift per ligand added can be correlated to the Kb and thus ΔGb.
However, complex multi-domain proteins and their assemblies are
a challenge for any stability shift assay, as unfolding of different
domains and other substructures often happens independently
and thus yields multiple denaturation signals that can be hard to
interpret. Therefore for Kb determination, single-domain and sin-
gle-binding site systems are favored as targets for this group of
methods.
2.1.1. Differential scanning calorimetry
In case of DSC, large amount of protein is subjected to increasing
temperature and its unfolding energetics profile (enthalpy, heat
capacity, and melting temperature Tm) is obtained. Determining
Kb via protein unfolding profiles enables precision over a much
wider range of binding constants as compared to the ITC
approach. Just as ITC, DSC is a label and reporter free assay. On
the other hand, it also involves a number of assumptions and
thus should be considered carefully [32].
2.1.2. Fluorescence thermal shift assay
A considerably more efficient technique exploiting the profiles
of protein unfolding is the fluorescence thermal shift assay
(FTSA; also referred to as TSA, DSF – differential scanning
fluorimetry, and ThermoFluor [33]). During the experiment,
the protein samples with varying ligand concentrations are
being subjected to a constantly increasing temperature.
Protein denaturation is tracked indirectly, by measuring the
fluorescence of a solvatochromic dye molecule, which reports
on unfolded protein regions (Figure 2(b)). Thus instead of the
full thermodynamic denaturation profile, it yields only Kb and
Tm but is significantly less time and reagent consuming.
Further, fluorescence-monitoring PCR machines can be easily
repurposed for FTSA experiments. While the assay has been
around for some time, its ease and high-throughput makes it
a highly popular technique, with improvements still being
made [34]. It is especially widely used in the screening stages
(Figure 1). However, for precise affinity calculations, the
method imposes some target limitations and works best for
simple single domain proteins.
2.1.3. Cellular thermal shift assay
Interestingly, protein Tm shift upon ligand binding can be also
exploited to evaluate target engagement in cells. In the
recently discovered cellular thermal shift assay (CETSA [35],),
various complex protein samples, including tissues, are ali-
quoted with and without ligand and subjected to different
temperatures. Soluble fractions are isolated from the lysates
and quantified using antibody-based assays or mass spectro-
metry (MS). This approach allows to evaluate ligand efficacy in
the desired environment and is being rapidly developed [36].
However, one has to keep in mind that the data is heavily
influenced by off-target binding and, in some setups, mem-
brane permeability and thus should not be considered as
actual affinity.
2.3. Mobility shift assays
To determine the binding affinity, mobility shift assays physi-
cally separate the ligand or protein from the protein-ligand
complex and quantify these fractions. To this end, they exploit
the differences in size, charge, hydrodynamic or other proper-
ties between these molecules and their assemblies. The ligand
(or protein) is usually detected using UV, fluorescence signal,
or MS. Determined bound fractions are plotted against target
concentrations to calculate the Kd.
As these techniques rely on separability, they are inapplic-
able when the difference between the complex and free
 EXPERT OPINION ON DRUG DISCOVERY 759

a ITC b FTSA c MST

8 1.05
Time, s Data Data
0 1000 2000 3000 4000 Fit
19.8
0.95
6

Fluorescence, a.u.
19.7

Fluorescence, a.u.
19.6
Power, µJxs-1
0.85
19.5
4
19.4
0.75
19.3
19.2 2
0.65
19.1
19.0
Data 0 0.55
18.9 40 45 50 55 60 65 70 0 10 20 30 40
Baseline
18.8 Temperature, C Time, s
0 64 650

Relative fluorescence, a.u.

Data Data
-10 62 Fit Fit
640

temperature, C
Protein melting
H, kJxmol-1

-20
60
630
-30
58
-40 620
56
b

-50
Data
-60 Fit 54 // 610

-70 52 // 600
102 104 106
0 0.5 1 1.5 2 0
1.E-07 10-6
1.E-06 10-5
1.E-05 10-4
1.E-04 10-3
1.E-03 1
1.E+00 1.E+02 1.E+04 1.E+06
Molar ratio Total concentration of the added ligand, M Titrant, nM

Figure 2. Exemplary data of protein-ligand affinity measurements. The same protein-ligand pair was studied using three different approaches: (a) isothermal
titration calorimetry, (b) fluorescence thermal shift assay, and (c) microscale thermophoresis. The upper panels contain raw data, the lower panels – processed data
as used for calculating binding or dissociation constants. The ITC data comes from a single experiment while in case of FTSA and MST several measurements with
different ligand concentrations were conducted in parallel (12, with only 4 of them displayed in the raw data panel, and 16, respectively).

molecules is too insignificant based on the chosen separation throughout experiment measuring absorbance, refraction, or
criteria. Another general drawback for these assays is protein more sensitive fluorescence [40]. This distribution depends
or ligand interactions with the instrumentation (such as dia- solely on the molecular mass and not shape. Kd can thus be
lysis membrane or capillary walls) that distort the results. On calculated based on how the curvature changes at different
the other hand, most of these techniques are easy to use. protein: ligand ratios [41]. Naturally, it can only be applied for
Most notable examples of such techniques include equilibrium interactions with sufficient difference in molecular weight (the
dialysis (ED), analytical size exclusion chromatography (SEC), precision is usually 1–2%).
affinity selection chromatography, analytical ultracentrifuga-
tion (AUC), electrophoresis (CE, MCE, EMSA), and microscale 2.3.2. Electrophoresis
thermophoresis (MST). Another commonly used force to separate molecules is the
Chromatography techniques, relying on hydrodynamic size, electric field. While electrophoretic mobility shift assay (EMSA)
are usually reserved for studying inhibitors of protein–protein is still a popular technique to study protein affinity to nucleic
interactions or in combination with various spectroscopic acids and has been successfully applied to study inhibitors of
techniques for improved precision. Equilibrium dialysis, such interactions [42], the capillary and microchip electrophor-
where the semipermeable membrane between two reservoirs esis (CE and MCE; reviewed in [43]) offer more versatility and
ensures that only the small ligand can diffuse between two higher throughput. In typical affinity studies, the capillary is
connected chambers, nowadays is mostly used for assaying filled with the target protein and varying amounts of ligand is
drug interaction with plasma proteins [37]. injected (or vice versa), and electricity is applied. The techni-
que is widely used in lead screening. If the target protein is
2.3.1. Analytical ultracentrifugation exposed on the cell surface, living cells can be used in the
In the more widely used AUC, samples are subjected to cen- screening instead of purified proteins [44]. Furthermore, capil-
trifugal force. There are two types of AUC experiments – lary electrophoresis can also be used to study binding kinetics.
sedimentation velocity and sedimentation equilibrium. The
high-speed sedimentation velocity experiments separate 2.3.3. Microscale thermophoresis
molecular assemblies in the solution based on their mass While MST has been around for some time, it has gained much
and shape. Therefore, they are mostly used to characterize wider acceptance over the last several years as a routine
protein–protein interaction inhibitors and ligand effect on method for Kd determination (Figure 2(c)). The measurements
conformations of the targets (see Table 1 for examples). For are carried out in small glass capillaries, ensuring low sample
this and other hydrodynamics-based methods, solution prop- consumption. During the experiment, molecules move
erty prediction software suites such as HYDRO [38] are often through infrared laser-induced temperature gradients depend-
used to correlate observed data with high- and low-resolution ing on their size, charge, and hydration shell. Since at least one
structural information. of the parameters is altered upon binding of a ligand, this
While sedimentation velocity experiments can be used for method is highly versatile. Intrinsic Trp fluorescence or higher-
Kb measurements [39], sedimentation equilibrium is better sensitivity extrinsic fluorescent labels can be used to track the
suited for it. In these experiments, the sample is ultracentri- protein. Further, it can be used in cell lysates, with added
fuged until equilibrium between the centrifugal force and purified labeled protein [45] or by labeling the selected pro-
diffusion is reached. The distribution of molecules is tracked tein in the lysate [46]. However, stability of some targets inside
760 V. KAIRYS ET AL.
the capillary remains a challenge, and its results are still
usually required to be verified using other, more established,
methods.
2.4. Spectroscopic assays
While spectroscopy is often used as detection component in
various other previously discussed assays, some spectroscopic
methods, such as UV-vis, fluorescence, CD, IR, or NMR, are
capable of ligand-binding analysis on their own. Their use is
based on changes in spectroscopic properties of the ligand or
protein upon binding, as observed at equilibrium. Contrary to
the mobility shift assays, no physical separation of bound and
free fractions is required.
Circular dichroism (CD) spectroscopy is based on the ability
of the chiral molecule (e.g. protein) to absorb left and right
circularly polarized light at different extents. This technique is
generally used for secondary (far UV) and tertiary (near UV)
protein structure analysis. Upon binding to protein, small
molecule ligand acquires induced CD spectrum (ICD). Varying
protein: ligand ratio in samples the ICD spectra can be used to
quantify binding affinity [47].
Absorption spectroscopy for ligand binding is limited to
instances where protein or its ligand has a chromophore in UV
or visible range that is altered during binding. Most common
examples are proteins with cofactors, such as heme or flavin.
2.4.1. Fluorescence-based assays
Fluorescence spectroscopy for protein–ligand interaction ana-
lysis is much more widely applicable [48]. Fluorescence-based
detection is significantly more sensitive compared to absorp-
tion due to higher signal-to-noise ratio. It enables lowering the
working concentration range down to nanomolar scale and
thus analysis of tight interactions. Molecule binding can induce
changes in fluorescence intensity and/or emission maximum
(center of mass). In practice, relative fluorescence intensity is
used for analysis more often than the absolute one. Analysis
using center of mass is often applied when the properties of
fluorophore depend on the environment (e.g. hydrophobicity).
Either intrinsic fluorescence of the interacting molecules or the
extrinsic fluorescence probe can be used to monitor interactions.
The most common intrinsic fluorophore is Trp amino acid in
proteins. Trp spectral properties depend on its surroundings and
can have emission maximum from approx. 308 nm for deeply
buried amino acids to approx. 350 nm for water accessible Trp
[49]. Extrinsic fluorophores can be either covalently attached to
one of the interacting molecules or produce the observed signal in
its free form. Different techniques, such as the use of concurrent
fluorescent ligands, solvatochromic probes, etc., can be employed.
Other variations of signal detection, such as differences in fluores-
cence anisotropy or fluorescence polarization are also used [50].
Förster resonance energy transfer (FRET) is yet another techni-
que for detection of intermolecular binding. It is based on the non-
radiative energy transfer between two fluorophores: one fluoro-
phore (donor) is excited by the external light source, and the
energy is transferred to another fluorophore (acceptor). The key
requirements to observe FRET are 1) overlap of donor emission
and acceptor excitation spectrum, and 2) close proximity (approx.
10–100 nm) between the two fluorophores during the protein–
ligand interaction. The fluorophores are generally artificially
attached at suitably designed positions, for example, to monitor
conformational changes or interaction with nucleic acids.
2.4.2. Light-scattering-based assays
Light-scattering-based analysis of binding interaction uses
dynamic light scattering (DLS) or static light scattering (SLS)
to detect the size of molecular species in solution [51] and can
be used to determine the absolute stoichiometry of multi-
component interaction. As it can detect both hetero- and
homo-association events, it is an excellent tool for analysis of
protein oligomerization and aggregation.
The most popular version is multi-angle light scattering
(MALS), especially when coupled with size exclusion chroma-
tography (SEC-MALS). Instead of relying on retention times,
here SEC is used to separate free and ligand-bound species
while the exact mass/size is detected by MALS. The measured
difference of Rayleigh factor at a given angle is related to the
molecular weight of the solute. The affinity of the interacting
molecules can be determined according to the ratio of free
and ligand-bound protein.
2.4.3. Optical spectroscopy assays
Optical spectroscopy assays, namely surface plasmon reso-
nance (SPR) and bio-layer interferometry (BLI), are highly
regarded for their reliable binding affinity and kinetics data
gained with relatively low sample consumption. These two
major real-time reporter-free sensor-based techniques allow
for both kinetic and thermodynamic characterization of pro-
tein – ligand binding. They are used to determine the kinetics
of molecule binding by measuring the binding and dissocia-
tion rates (kon and koff constants). The affinity can be obtained
either from the ratio of kinetic constants (equation (2)) or by
steady-state calculations according to signal intensity. The
binding assay is performed with one binding partner being
immobilized on the sensor surface, and the other is free in
solution. The molecule in the solution can be either in pure
form or in a complex mixture, such as cell lysate. The main
difference between these two methods is the principle used
for signal detection.
BLI [52] uses white light and measures the interference
between two waves: one is reflected from internal reference
layer, and the other from bio-compatible layer with immobi-
lized molecules. The spectral pattern depends on the thickness
of the molecular layer. Therefore, the signal is increasing with
increasing size of the binding molecule. While other assay
formats are available, it is commonly performed in 96 well
plate format simultaneously measuring interactions in 8 wells.
The sample is reusable.
The SPR [53] measures changes in the refractive index at
the surface of the sensor. Molecule binding at the surface
changes the refractive index proportionally to the mass
bound. The resonance angle shift is monitored as a function
of time during the binding event. In typical SPR experiment,
one binding partner is coupled to a sensor while solution of
the other flows through the sensor using microfluidics system.

However, a microfluidics-free, 96-well plate version of SPR (FO-
SPR) is also available [54].
The main issues concerning BLI and SPR come from possi-
ble immobilization effects. While SPR is much more sensitive
than BLI, for precise small molecule binding determination it
still usually requires quite sophisticated experimental setups.
2.5. Structural biology techniques
First of all, determination of structures of the targets and
especially their complexes with lead compounds serve as the
basis for most of the bioinformatic analysis tools outlined in
the next chapter. They are also a vital component for the
rational drug design, allowing to see what part of the ligand
interacts with which residues of the target protein as well as to
which direction the ligand could be expanded. Furthermore,
nuclear magnetic resonance (NMR) spectroscopy is also cap-
able of a more direct Kb determination.
X-ray crystallography has always been the go-to method
for gaining structural information on protein–ligand interac-
tions (reviewed in [55]), with established and emerging dedi-
cated consortiums [56,57]. However, not all targets can be
readily crystallized and even obtained diffracting protein crys-
tals cannot reveal conformational and chemical heterogeneity.
Rapid recent advancements in cryo-electron microscopy (cryo-
EM) has brought its resolution to sufficient for analysis of
ligand interactions, as exemplified by various recent studies
[58], and can sometimes be used interchangeably with X-ray
crystallography. Small angle X-ray or neutron scattering (SAXS
or SANS) allow to glimpse at general solution shape in a high-
throughput manner, which can be exploited for studying
inhibitors of protein–protein interaction and conformational
dynamics [59,60]; however, so far they have remained under
the radar of most drug design studies and are commonly
applied mainly for drug carrier characterization.
2.5.1. Nuclear magnetic resonance
NMR is the only technique capable of obtaining atomic-
resolution structures in solution, albeit its targets are of limited
size (<40 kDa). However, it has much more uses for assaying
ligand binding. In principle, during an NMR experiment,
a magnetic field is applied to the sample, which affects the
spins of the nuclei. The energy released by these nuclear spins
coming back to their original states is detected and analyzed.
The energy differs for the same atom nuclei placed in different
chemical environments – between different compounds or
between the bound and unbound states.
Based on which interactor’s spectra is being recorded, rele-
vant NMR techniques are classified into two major groups:
ligand-observed and protein-observed (reviewed in [61,62]).
Protein-observed techniques, requiring large amounts of isoto-
pically labeled protein, can be used to determine solution
structures of small proteins and their complexes with ligands.
Titration experiments can be used to precisely quantify Kd.
Ligand-observed techniques, while unable to yield high resolu-
tion, are cheaper as they require less protein and no isotopic
labeling. Moreover, in these assays larger size of proteins is only
an advantage and not a limitation. They are well suited for
ligand and especially fragment screening, while Kd
EXPERT OPINION ON DRUG DISCOVERY 761
determination usually requires a competitor and is limited by
binding kinetics.
Overall, the usage of structural biology techniques in
drug design has been on the rise over the last decade
due to significant technological advances and gaining
popularity of computational approaches that rely on initial
structural data.
3. In silico calculation of ligand-receptor binding
free energies
Binding affinity prediction is one of the ‘holy grails’ of compu-
tational chemistry [6]. To calculate binding free energies of
ligands to receptors a wide range of methods have been
developed, from chemometrics-based approaches to quantum
mechanics. As this is a vast topic, only a cursory introduction is
presented, to serve as a starting guide for a more in-depth
exploration of the field.
3.1. QSAR and other chemoinformatics-based
approaches
Quantitative Structure–Activity Relationship (QSAR) and the clo-
sely related Quantitative Structure–Property Relationship (QSPR)
are based on a hypothesis that a change in the structure of
a compound has a proportional effect on the biological activity
[63]. In practice, QSAR builds a (usually linear) equation with
compound properties (so-called descriptors) being variables, and
the compound activity (or binding affinity) as the target [64]. An
attractive advantage of QSAR is that in its simplest form it does not
require knowledge of the receptor structure or the ligand’s bind-
ing mode. Probably the main disadvantage is that for the
unknown compound, the knowledge of binding affinities of
a series of related compounds needs to be known. Because of
this, and because the method relies heavily on a variety of differ-
ent statistical measures, a vast array of QSAR protocols (often
referred to as 2D QSAR as they generally use two-dimensional
structures of compounds), often with underlying weaknesses, exist
[63,65–67].
A variation of QSAR in which spatial structure of the ligand
(and in some methods, the structure of the receptor with the
bound ligand) is used is known as 3D QSAR [68,69], and even
of higher ‘dimensionalities’ [70]. Often these approaches are
derived from the 3D pharmacophore search [64]. Without
going too deep into the wide QSAR field, it will be sufficient
to say QSAR-based methods are very well researched and are
widely used in drug discovery process due to many successes,
both in academia and in industry.
Due to a historic connection with cheminformatics, it is a good
opportunity to mention the recent rise of popularity of Machine
Learning methods, which include Neural Networks, Support Vector
Machines, Random Forest and others [71]. It should be noted that
some Machine Learning approaches make use of receptor infor-
mation. The applications of Machine Learning in a variety of drug
discovery areas have been reviewed elsewhere [72–74]. It should
also be mentioned that the recent Deep Learning and related
methods can be partially attributed to the emerging use of
Graphical Processing Units (GPU), which allowed to significantly
speed up the calculations [72,74]. Among the recent success
762 V. KAIRYS ET AL.
stories, Deep Belief Networks were shown to dramatically outper-
form QSAR models generated by Deep Neural Networks [75], and
remarkable performance of Random Forest-based scoring function
ΔvinaRF20 [76] is further described in Section 3.3.
3.2. Quantum mechanics-based methods
On a theoretical level, probably the most accurate representa-
tion of atomic systems is based on quantum mechanics
because they take into account possible changes of electronic
states. Unfortunately, quantum calculations, also known as ab
initio calculations, are very CPU-intensive, since they take into
consideration all electrons. The methods used to compute QM
wavefunction vary from semiempirical models to Density
Functional Theory (DFT) functionals or more rigorous and
time-consuming methods such as Coupled Cluster calculations
[77]. The calculations become somewhat more manageable by
converting less important atoms to a non-quantum represen-
tation, and these methods are referred to as QM/MM
(Quantum Mechanics/Molecular Mechanics). The solvation
effects are taken into account via cheap but not very accurate
implicit (continuum) solvation models or by using computa-
tionally much more expensive explicit water molecules [6].
The electronic structure-based methods in principle are
able to achieve absolute accuracy of 1–3 kcal/mol [6]. It should
be noted that the calculation of relative binding affinities of
a series of compounds is generally easier and more accurate
than calculation of the absolute binding affinities. A recent
very thorough review of QM binding affinity calculations by
Ryde and Söderhjelm [77] concludes with an interesting fact
that MM methods performed better than QM in the SAMPL4
host-guest blind binding affinity prediction challenge [78] due
to a variety of reasons, one of them being a simple cancella-
tion of errors. Since MM parameters are often QM-derived, it is
obvious that QM calculations still have a large leeway for
development. Indeed, the speed and accuracy of QM calcula-
tions are slowly but constantly being improved. Development
of efficient and accurate hybrid Density Functional Theory
functionals by Grimme’s group [79] is but one example of
such progress. The main challenges and perspectives of the
quantum methods in computational chemistry have been
recently comprehensively outlined by Grimme and
Schreiner [80].
3.3. MM-PBSA and other related Force Field-based
methods. Scoring functions
One of the oldest and often used approaches to calculate free
energies of binding is known as MM-PBSA (Molecular
Mechanics Poisson-Boltzmann Surface Area) or its variant MM-
GBSA (Molecular Mechanics Generalized Born Surface Area)
[81]. In fact, MM-GBSA served as the ‘null model’ against
which other results were compared in the latest SAMPL bind-
ing affinity prediction challenge [82]. In its simplified form, the
binding energy between a ligand and a protein in MM-PBSA is
computed using the following expression, written in form
which emphasizes the role of the surface area:
ΔGbind ¼ ΔEvdw þ ΔEelec þ γΔSA þ δ (5)
where ΔEvdw, ΔEelec, and ΔSA are changes in van der Waals
energy, electrostatic energy, and solvent accessible surface
area upon ligand binding, γ, δ – empirical fitting coefficients
which can vary to some extent [83]. ΔSA serves as an approx-
imation for the nonpolar solvation energy. ΔEelec above often
includes polar solvation energy obtained by solving electro-
static Poisson-Boltzmann (PB) differential equation [84] or by
using generalized Born (GB) approximation [85]. There exist
many variations and extensions of the method which differ by
the way individual terms are handled, or by inclusion of addi-
tional terms, often empirically tuned. The conformations for
the calculations are normally generated using molecular
dynamics, but the equation can also be used to score docked
conformations [86,87]. Mining Minima is one of the more
interesting relatively new methods which goes beyond MM-
PB(GB)SA while trying to maintain sound physical grounds. In
Mining Minima, the local potential energy surface is explored
computationally, leading to reasonable estimates to the con-
formational entropy and the free energy of binding [88]. MM-
PB(GB)SA applications in calculation of binding free energies
have been recently reviewed by Genheden and Ryde [83].
Because of the convenience and computational speed,
a great number of ‘scoring functions’ was developed. They
are computational estimates for the binding affinity for
a given conformation of ligand and protein [89,90]. Docking
programs use scoring functions by necessity to evaluate the
quality of the docked pose. Molecular docking is an immen-
sely popular tool allowing to explore ligand-protein binding,
and this lead to creation of a great number of docking pro-
grams and web servers, as reviewed elsewhere [91–93].
However, the performance of the docking programs largely
hinges on the quality of the underlying scoring functions.
Scoring functions are generally classified by the principles
they were built upon: empirical, physics-based, knowledge-
based, and machine-learning-based [90,94,95]. It also should
be borne in mind that the success of the scoring functions is
often system dependent [96]. A recent CASF-2016 test of 25
scoring functions applied to many receptor-ligand complexes
[97] showed that Machine Learning-based scoring function
ΔvinaRF20 [76] performed strikingly better at scoring compared
to others (its Pearson correlation coefficient in the scoring
power test was 0.816, while the correlation coefficient of the
runner-up, X-score [98], was only 0.631). However, some cau-
tion needs to be exercised as it is not clear to what extent
CASF-2016 test overlaps with the very large ΔvinaRF20 training
set, thereby reducing the value of the test.
3.4. Enhanced sampling MD methods
It is possible to obtain accurate distance vs. free energy pro-
files from a molecular dynamics calculation analyzing the
thermally driven (i.e. Poisson-Boltzmann) distribution of the
distances during the simulation [99]. Unfortunately, the system
itself can easily become too big to traverse the conformational
space during the limited time available for the calculation. In
addition, the potential energy barriers could be too high to be
surmounted during MD: for example, the ligand-protein com-
plex is often likely to stay bound during the simulation. This
can be overcome by enhanced sampling techniques [100,101].

One of such techniques is called umbrella sampling [100]. In
this method, the whole reaction path (for example, dissocia-
tion/association coordinate of a complex) is split into a series
of windows, and a harmonic bias is imposed for each window.
The bias helps to overcome potential barriers on the reaction
path. At the end of many relatively short simulations for each
window, the resulting distributions are combined and ana-
lyzed, for example, using the Weighted Histogram Analysis
Method (WHAM) [102]. Umbrella sampling has often been
used to explore the energetic profile of transport of various
species through transmembrane channels (e.g. ref [103].), but
this method can also be successfully applied to investigate
ligand-enzyme binding. For example, umbrella sampling
showed good agreement with experimental results for benzo-
nitrile association with wild type and mutant lysozyme T4, and
also with MM-PBSA calculations [104].
Another MD based enhanced sampling approach is called
Steered Molecular Dynamics. As its name suggests, the ligand
is dragged (‘steered’) by additional force out of the binding
pocket until both molecules can be considered separated, and
the work needed to such separation can be used to estimate
the free energy. Moreover, energy vs. reaction coordinate
profile can be built, also known as PMF (Potential of Mean
Force), where the reaction coordinate can be, for example, the
distance between the protein and the ligand [105]. In a recent
study using three protein-ligand systems, Okimoto et al. [106]
found that Steered MD overestimated the binding affinities,
but ranked ligands quite accurately.
Among the relatively new enhanced sampling methods
metadynamics and its many variants should be certainly men-
tioned [107,108]. In metadynamics, every n-th MD step a small
Gaussian type ‘hill’ (bias potential) is deposited along the
chosen internal coordinate. Since the simulation tends to
move to low energy regions, the bias potential gradually fills
deep energy wells first. At the end of simulation, the depos-
ited bias potential is explored, and the energy vs. coordinate
profile is built. Metadynamics based approaches can be used
not only to explore ligand binding to the protein but also to
build more complex free energy landscapes along various
chosen internal coordinates [109,110]. The binding affinity
prediction accuracy using metadynamics can reach less than
1 kcal/mol RMSE [111].
As a side note, the conformational sampling statistics can
be obtained not only from simulations but also from experi-
mental data (so-called ‘knowledge-based’ approaches [112]).
The knowledge-based scoring functions are able to compete
rather well against many other scoring functions, e.g. a recent
Convex-PL scoring function example [113].
3.5. Free energy methods
A group of approaches is known, somewhat tautologically, as
‘free energy methods’ and is based on rigorous statistical
thermodynamics as applied to Molecular Dynamics or Monte
Carlo simulations. In the free energy perturbation (FEP) theory,
the change of the free energy between states A and B can be
written as
ΔGAB ¼ kTlnheðVB VA Þ=kT iA (6)
EXPERT OPINION ON DRUG DISCOVERY 763
where k is the Boltzmann’s constant, and the angular brackets
denote ensemble average that was sampled using the VA poten-
tial [114–116]. For this formula to be of practical use, the poten-
tials VA and VB should have a significant thermally accessible
overlap. To be able to achieve this, a series of purely artificial
intermediate states (windows) is created between A and B, and
ΔGA–B is calculated as the sum of relatively small changes of G for
each window. This method is often used to calculate changes of
the ligand binding energy to the receptor where one functional
group is gradually transformed into another one [117]. Because
of this, the method is also known as alchemical perturbation.
When the width of the window approaches zero, the sum
changes to integral, and the approach is being referred to as
Thermodynamic Integration (TI) [115]. The free energy methods
are considered as the ‘cutting edge’ approaches and have been
successfully applied for biochemical systems [117], even though
the results can be somewhat system dependent [118].
A significantly simplified version of FEP and therefore rela-
tively easy to implement is LIE (Linear Interaction Energy)
[119]. The method requires performing two MD simulations,
one of the ligand in pure solvent, and another one of ligand/
receptor complex, also in solvent. The binding affinity is com-
puted as an average change of the interactions felt by the
ligand when it changes environment from the pure solvent to
the receptor/solvent (with some scaling factors for van der
Waals and Coulombic energy components). In spite of relative
simplicity, the method shows a relatively good ~1 kcal/mol
RMSE accuracy with respect to the experimental binding affi-
nities [120,121].
4. Conclusions
Binding affinity is one of the major factors in target-oriented
drug design. The main goal is to produce a ligand that would
bind with high affinity to the target protein at functionally
important site and would exhibit low off-target binding. As
this brief overview shows, while there is a wide variety of
experimental (see Table 1) and computational methods to
estimate ligand affinity to the target protein, neither of them
is perfect. Therefore, the recommended techniques vary with
the chosen protein-ligand system. While computational meth-
ods are much cheaper, they are tightly intertwined with the
experimental approaches. The development and assessment of
most computational techniques relies heavily on experimental
data. Therefore, its quality strongly influences the development
of theoretical approaches. Moreover, the predictions still have
to be verified using an experimental approach. The balancing
between high-throughput and accuracy is often achieved by
using faster but less reliable techniques to screen the initial
long list of putative candidates and then relying on more pre-
cise methods to characterize promising leads. As exemplified by
inhibitor design of human carbonic anhydrases, renin, and
Plasmodium falciparum proteases [122–124], often docking is
combined with structural studies and affinity determination
using ITC or FTSA. In general, drug design field is a great exam-
ple of how combination of different approaches drives the
discoveries forward.
764 V. KAIRYS ET AL.
5. Expert opinion
While lead optimization cannot rely on binding affinity
(defined as either Gibbs energy or the binding/dissociation
constant) alone, it serves as a major factor. Therefore, it is
critical to evaluate it correctly before making decisions con-
cerning further ligand design. This involves choosing suitable
methods, correctly planning the experiment, and properly
interpreting the data.
While this review might serve as a general guideline for which
method to choose, it is often hard to predict which approach will
be suitable for each selected system. Therefore, to obtain reliable
affinity data, we promote the application of several complemen-
tary techniques. It is most convenient to use a high-throughput
method (e.g. FTSA) to sort through potential ligands in combina-
tion with a more in-depth analysis of the promising hits using ITC
or SPR. ITC provides detailed thermodynamic information, which
helps to design enthalpically beneficial improvements, while SPR
describes binding kinetics.
When choosing which methods to use, one must also keep in
mind that most of the methods are not as straightforward as
advertised. Some might require more sophisticated equipment
or experimental planning for drug discovery-related applications
or a well-trained specialist. A common mistake in experimental
planning is working with unsuitable concentrations of ligand and/
or protein. The concentrations should be chosen depending on
the strength of interaction and signal-to-noise ratio. When plan-
ning the dilution series for stability, mobility, or spectroscopic
assays, it is recommended to cover a range of 6 orders of
magnitude.
As this brief overview shows, most modern computational
methods are able to make useful predictions, reaching ~5 kJ/mol
(~1 kcal/mol) RMSE. However, the success of an in silico method is
not always guaranteed to work on your system due to several
reasons: data overfitting when calibrating methods; success is
often system-dependent; lastly, the negative results are rarely
published. For this reason, it can be important to use methods
with a realistic underlying physical model. Some additional limita-
tions may arise due to computing power restrictions, e.g. most
molecular dynamics and quantum mechanics-based approaches
can be CPU-demanding, but in some cases the accuracy is worth it.
Also, much improved accuracy is expected when comparing rela-
tive binding affinities, instead of absolute ones. Of course, in the
absence of the receptor structure, the chemoinformatics-based
methods are the most helpful. Among those, AI-based approaches
show clear promise, but one should be aware that they work best
on systems which are included in their training set.
A useful resource for comparing performances of various com-
putational methods is through the D3R Grand Challenge [125],
a regularly held competition in which various scientific groups
contest the computational prediction of various aspects of host/
guest binding. Additionally, SAMPL is a closely related computa-
tional prediction challenge involving host-guest systems [82],
which can be considered as ‘miniature models’ for protein–ligand
interactions. Because of the smaller sizes of these systems,
amongst SAMPL entrants are many groups who use CPU-
intensive methods, such as quantum mechanics and MD-based
approaches, while D3R is far more focused on docking and
scoring. These competitions are useful resources for comparing
the performance of various methods, and also for discovering
emerging new trends in computing.
Regardless of the experimental or computational approach,
data analysis and critical evaluation of the results remain an
important issue. In the end, all data are only as good as its analysis
and interpretation. For data processing using stability and mobility
shift assays where a large range of concentrations are plotted, we
recommend using a logarithmic scale as it makes it easier to
evaluate the fit and Kd estimation. Global fit analysis is encouraged
for calculating the Kd from replicates. Nowadays, fully automated
data processing software is often available, especially for the more
popular techniques. For example, SEDPHAT [126] is often used for
processing data, including that of AUC and ITC, and is highly
regarded for the global fitting functionality. NITPIC is another
highly useful tool for processing raw ITC data [127]. However,
this simplification should not be regarded as lack of necessity for
the ability to critically evaluate the results. In fact, a lot of issues
arise from the careless and uneducated use of automatically
processed data, as explained in the case of X-ray crystallography
in a recent review [55]. To ensure data reproducibility, we also
encourage the inclusion of raw data in the publications in addition
to the provision of sufficient experimental details in the methods
sections.
Finally, when integrating data from diverse approaches, their
differences have to be kept in mind. For example, lysate and
purified protein data might differ significantly due to additional
cellular interactors. While this might lead to incomparable affinity
values, it also provides more insight into the interaction in ques-
tion. For the simple ranking of ligands by best performance,
experimental conditions that mimic drug use (such as CETSA
using tissues) are best suited. However, for rational design studies
that try to draw upon structural, computational, and biophysical
data, it is especially important to ensure that the data being
compared shed light on intermolecular interactions. For good
correlation with structural data, ‘intrinsic’ binding parameters
should be used [25,26], with the binding-linked events such as
ligand protonation subtracted from the ‘observed’ data.
To sum up, there is a wide variety of experimental and
computational methods that are constantly expanding both
because of high demand for reliable and precise affinity quan-
tification tools and because, so far, none of the described
methods are considered perfect for all possible situations.
For now, the main goals of future technique improvements
remain enhancing throughput, accuracy, and accessibility.
Finally, the current amount of experimental affinity mea-
surements is required because, despite enormous efforts, the
general trends of Structure–Affinity Relationship (SAR) are still
not well understood. Therefore, systematization of existing
experimental data, especially databases that compare affinities
of chemically and/or structurally similar compounds [128], is
important to identify correlations and to pinpoint the impact
of small chemical changes. Furthermore, additional thermody-
namic parameters (enthalpy and entropy) should also be
regarded, and the influence of binding-linked events should
be omitted in order to facilitate our understanding of the
molecular recognition phenomenon.

Acknowledgements
We apologise to all authors whose work we were unable to mention due
to space constraints. M. Kazlauskiene is a European Molecular Biology
Organization Fellow (ALTF 1087–2018).
Declaration of interest
The authors have no other relevant affiliations or financial involvement with
any organization or entity with a financial interest in or financial conflict with
the subject matter or materials discussed in the manuscript.
Reviewer Disclosures
Peer reviewers on this manuscript have no relevant financial or other
relationships to disclose.
ORCID
Visvaldas Kairys http://orcid.org/0000-0002-5427-0175
Lina Baranauskiene http://orcid.org/0000-0002-9924-9177
Migle Kazlauskiene http://orcid.org/0000-0003-2329-0410
Daumantas Matulis http://orcid.org/0000-0002-6178-6276
Egidijus Kazlauskas http://orcid.org/0000-0001-8430-7349
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