Measurement Of Ligand Binding Research Articles

A general method for quantifying ligand binding to unmodified receptors using Gaussia luciferase

Reliable measurement of ligand binding to cell surface receptors is of outstanding biological and pharmacological importance. Resonance energy transfer–based assays are powerful approaches to achieve this goal, but the currently available methods are hindered by the necessity of receptor tagging, which can potentially alter ligand binding properties. Therefore, we developed a tag-free system to measure ligand‒receptor interactions in live cells using the Gaussia luciferase (GLuc) as a bioluminescence resonance energy transfer donor. GLuc is as small as the commonly applied Nanoluciferase but has enhanced brightness, and its proper substrate is the frequently used coelenterazine. In our assay, bystander bioluminescence resonance energy transfer is detected between a GLuc-based extracellular surface biosensor and fluorescent ligands bound to their unmodified receptors. The broad spectrum of applications includes equilibrium and kinetic ligand binding measurements for both labeled and competitive unlabeled ligands, and the assay can be utilized for different classes of plasma membrane receptors. Furthermore, the assay is suitable for high-throughput screening, as evidenced by the identification of novel α1 adrenergic receptor ligands. Our data demonstrate that GLuc-based biosensors provide a simple, sensitive, and cost-efficient platform for drug characterization and development.

Quantitative analysis of fluorescent ligand binding to dopamine D3 receptors using live-cell microscopy.

Dopamine receptors are G protein-coupled receptors that have several essential functions in the central nervous system. A better understanding of the regulatory mechanisms of ligand binding to the receptor may open new possibilities to affect the downstream signal transduction pathways. The majority of the available ligand binding assays use either membrane preparations, cell suspensions, or genetically modified receptors, which may give at least partially incorrect understanding of ligand binding. In this study, we implemented an assay combining fluorescence and bright-field microscopy to measure ligand binding to dopamine D3 receptors in live mammalian cells. For membrane fluorescence intensity quantification from microscopy images, we developed a machine learning-based user-friendly software membrane tools and incorporated it into a data management software aparecium that has been previously developed in our workgroup. For the experiments, a fluorescent ligand NAPS-Cy3B was synthesized by conjugating a dopaminergic antagonist N-(p-aminophenethyl)spiperone with a fluorophore Cy3B. The subnanomolar affinity of NAPS-Cy3B makes it a suitable ligand for the characterization of D3 receptors in live HEK293 cells. Using a microplate compatible automated widefield fluorescence microscope, together with the membrane tools software, enables the detection and quantification of ligand binding with a high-throughput. The live cell assay is suitable for the characterization of fluorescent ligand binding and also in the competition experiments for the screening of novel unlabeled dopaminergic ligands. We propose that this simple yet more native-like approach is feasible in GPCR research, as it enables the detection of ligand binding in an environment containing more components involved in the signal transduction cascade.

Abstract 6408: E3scan™ ligand binding assay platform for targeted protein degradation and PROTAC discovery

Abstract E3 ligases have emerged as pivotal targets for a next generation protein degradation-based drug discovery paradigm. This new paradigm includes both ligand binding-directed “reprogramming” of E3 substrate specificity approaches and a more directed approach, using small molecule proteolysis-targeting chimeras (PROTACs), to selectively degrade disease-driving proteins. As there are hundreds of diverse putative E3 ligases with differentiated tissue expression, this new paradigm may well define a next dimension of precision medicine defined by an axis of tissue-specific activity. While there have been some early successes, the E3 drug discovery field has a significant unmet need for a standardized biochemical ligand binding assay platform. A platform is required that: 1) Can measure ligand binding across the E3 family using a standardized method enabling “apples to apples” comparisons; 2), Is highly scalable and rapid; 3) Has an exquisite dynamic range for the measurement of accurate KD values as low as digit picomolar (pM). Eurofins DiscoverX herein presents its novel E3scan™ technology that addresses each of these unmet needs. E3scan, based upon well-established KINOMEscan® technology, has been successfully applied to diverse E3 ligases, including CRBN, VHL, MDM2, MDMX, cIAP1, cIAP2, and XIAP, with many other E3 assays in progress. We shall present assay validation data for these targets, including data for ligands with KD values in the low to mid pM range. In conclusion, we present Eurofins DiscoverX's novel E3scan platform that shall enable accelerated screening and SAR analysis in the E3 drug discovery field, with rapid turnaround times for discovery library screens (20 business day TAT) and weekly SAR (5 business day TAT) and the largest assay panel available on a single technology platform. Citation Format: Ksenya Cohen Katsenelson, Luis Gonzalez, Gabriel Pallares, Alastair J. King, Daniel K. Treiber. E3scan™ ligand binding assay platform for targeted protein degradation and PROTAC discovery [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6408.

Detailed In Vitro Pharmacological Characterization of Clinically Tested Negative Allosteric Modulators of the Metabotropic Glutamate Receptor 5

Negative allosteric modulation of the metabotropic glutamate 5 (mGlu5) receptor has emerged as a potential strategy for the treatment of neurologic disorders. Despite the success in preclinical studies, many mGlu5 negative allosteric modulators (NAMs) that have reached clinical trials failed due to lack of efficacy. In this study, we provide a detailed in vitro pharmacological characterization of nine clinically and preclinically tested NAMs. We evaluated inhibition of l-glutamate-induced signaling with Ca2+ mobilization, inositol monophosphate (IP1) accumulation, extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, and real-time receptor internalization assays on rat mGlu5 expressed in HEK293A cells. Moreover, we determined association rates (kon) and dissociation rates (koff), as well as NAM affinities with [3H]methoxy-PEPy binding experiments. kon and koff values varied greatly between the nine NAMs (34- and 139-fold, respectively) resulting in long receptor residence times (>400 min) for basimglurant and mavoglurant, medium residence times (10-30 min) for AZD2066, remeglurant, and (RS)-remeglurant, and low residence times (<10 mins) for dipraglurant, F169521, F1699611, and STX107. We found that all NAMs inhibited l-glutamate-induced mGlu5 receptor internalization, generally with a similar potency to IP1 accumulation and ERK1/2 phosphorylation, whereas Ca2+ mobilization was less potently inhibited. Operational model of allosterism analyses revealed that dipraglurant and (RS)-remeglurant were biased toward (affinity) receptor internalization and away (cooperativity) from the ERK1/2 phosphorylation pathway, respectively. Our study is the first to measure mGlu5 NAM binding kinetics and negative allosteric modulation of mGlu5 receptor internalization and adds significant new knowledge about the molecular pharmacology of a diverse range of clinically relevant NAMs. SIGNIFICANCE STATEMENT: The metabotropic glutamate 5 (mGlu5) receptor is important in many brain functions and implicated in several neurological pathologies. Negative allosteric modulators (NAMs) have shown promising results in preclinical models but have so far failed in human clinical trials. Here we provide the most comprehensive and comparative molecular pharmacological study to date of nine preclinically/clinically tested NAMs at the mGlu5 receptor, which is also the first study to measure ligand binding kinetics and negative allosteric modulation of mGlu5 receptor internalization.

Equilibrium and Kinetic Measurements of Ligand Binding to HiBiT-tagged GPCRs on the Surface of Living Cells.

G-protein coupled receptors (GPCRs) remain at the forefront of drug discovery efforts. Detailed assessment of features contributing to GPCR ligand engagement in a physiologically relevant environment is imperative to the development of new therapeutics with improved efficacy. Traditionally, binding properties such as affinity and kinetics were obtained using biochemical radioligand binding assays. More recently, the high specificity of resonance energy transfer has been leveraged toward the development of homogeneous cell-based proximity assays with capacity for real-time kinetic measurements. This suite of ligand binding protocols couples the specificity of bioluminescent resonance energy transfer (BRET) with the sensitivity afforded by the luminescent HiBiT peptide. The BRET format is used to quantify dynamic interactions between ligands and their cognate HiBiT-tagged GPCRs through competitive binding with fluorescent Tracers. At the same time, high affinity complementation of HiBiT with the cell impermeable LgBiT limits the bright bioluminescence donor signal to the cell surface and eliminates luminescence background from unoccupied receptors present in intracellular compartments.

Detection of Ligand-binding to Membrane Proteins by Capacitance Measurements.

In multi-cellular organisms, cells communicate with each other utilizing chemical messengers. For many of these messenger molecules, the membrane is an insurmountable barrier. Yet, they act by binding to surface proteins often triggering a cascade of reactions inside the cell. Accordingly, studying ligand-receptor interactions at the cellular surface is key to understanding important aspects of membrane biology. However, despite a multitude of approaches to study membrane features, there is a need for developing techniques that can measure ligand binding with high temporal resolution and on a single cellular level. We recently developed a label-free approach to study ligand binding in real time. This methodology capitalizes on changes of the membrane's surface potential induced by the adsorption of a charged ligand. The resulting apparent alteration of membrane capacitance is measurable by capacitance recordings. Herein, we describe the implementation of the same using recordings obtained from HEK293 cells stably expressing the human serotonin transporter (SERT), which were challenged with the inhibitor cocaine.

Erfassung der Internalisierung von GPCRs mit CRISPR/Cas9

Receptors are prevalent drug targets as they translate signals from outside the cell to cellular reactions. In drug finding and characterization it is not only required to measure ligand binding, but just as well subsequent regulatory steps including desensitizing protein interactions. Here, we show how CRISPR/Cas9 allowed endogenous expression of a G-protein coupled receptor (GPCR) and how that was used to study protein inter - actions and internalization of a cancer progressing receptor.

Membrane (and Soluble) Protein Stability and Binding Measurements in the Lipid Cubic Phase Using Label-Free Differential Scanning Fluorimetry.

Label-free differential scanning fluorimetry (DSF) is a relatively new method for evaluating the stability of proteins. It can be used as a screening tool for downstream applications such as crystallization. The method is attractive in that it requires miniscule quantities of proteins, it can be performed using intrinsic tryptophan and tyrosine fluorescence, and, with the right equipment, it is easy to perform. To date, the method has been used with proteins in liquid solutions and dispersions. It was of interest to determine if DSF could be used with membrane proteins in the lipid cubic phase (LCP), which increasingly is being used for crystallization in support of structure-function studies. The cubic phase is viscous. Furthermore, in coexistence with excess aqueous solution, as happens during crystallization trials, it can become turbid and scatter light. The concern was that these features may render the mesophase unsuitable for DSF analysis. However, using lysozyme and four integral membrane proteins we demonstrate that the method works with all tested proteins in solution and in the LCP. Of note is the observation that some of the test membrane proteins are more stable while others are less so in the mesophase. The method also works in ligand binding measurements. Thus, DSF should prove useful as an analytical tool for identifying host and additive lipids, detergents, precipitants and chemical probes that support the generation of quality crystals by the cubic phase method. Microscale thermophoresis was used to supplement the DSF study and was also shown to work with proteins in the mesophase. Measurements with lysozyme highlight the utility of the cubic mesophase as a model system in which to perform confinement studies.

Measuring Ligand Binding Kinetics to Membrane Proteins Using Virion Nano-oscillators.

Membrane proteins play vital roles in cellular signaling processes and serve as the most popular drug targets. A key task in studying cellular functions and developing drugs is to measure the binding kinetics of ligands with the membrane proteins. However, this has been a long-standing challenge because one must perform the measurement in a membrane environment to maintain the conformations and functions of the membrane proteins. Here, we report a new method to measure ligand binding kinetics to membrane proteins using self-assembled virion oscillators. Virions of human herpesvirus were used to display human G-protein-coupled receptors (GPCRs) on their viral envelopes. Each virion was then attached to a gold-coated glass surface via a flexible polymer to form an oscillator and driven into oscillation with an alternating electric field. By tracking changes in the oscillation amplitude in real-time with subnanometer precision, the binding kinetics between ligands and GPCRs was measured. We anticipate that this new label-free detection technology can be readily applied to measure small or large ligand binding to any type of membrane proteins and thus contribute to the understanding of cellular functions and screening of drugs.

Thermodynamic and NMR Assessment of Ligand Cooperativity and Intersubunit Communication in Symmetric Dimers: Application to Thymidylate Synthase.

Thymidylate synthase (TS) is a homodimeric enzyme with evidence for negative regulation of one protomer while the other protomer acts on substrate, so called half-the-sites reactivity. The mechanisms by which multisubunit allosteric proteins communicate between protomers is not well understood, and the simplicity of dimeric systems has advantages for observing conformational and dynamic processes that functionally connect distance-separated active sites. This review considers progress in overcoming the inherent challenges of accurate thermodynamic and atomic-resolution characterization of interprotomer communication mechanisms in symmetric protein dimers, with TS used as an example. Isothermal titration calorimetry (ITC) is used to measure ligand binding cooperativity, even in cases where the two binding enthalpies are similar, and NMR spectroscopy is used to detect site-specific changes occurring in the two protomers. The NMR approach makes use of mixed-labeled dimers, enabling protomer-specific detection of signals in the singly ligated state. The rich informational content of the NMR signals from the singly ligated state, relative to the apo and saturated states, requires new considerations that do not arise in simple cases of 1:1 protein-ligand interactions.

Ion Binding to Transport Proteins using Isothermal Titration Calorimetry.

Isothermal titration calorimetry (ITC) is an emerging, label-free technology used to measure ligand binding to membrane proteins. This technology utilizes a titration calorimeter to measure the heat exchange upon ligands binding to proteins, the magnitude of which is based on the overall enthalpy of the reaction. In this protocol, the steps we and others use to measure ion binding to ion transport proteins are described.

Specific Capture of Peptide-Receptive Major Histocompatibility Complex Class I Molecules by Antibody Micropatterns Allows for a Novel Peptide-Binding Assay in Live Cells.

Binding assays with fluorescently labeled ligands and recombinant receptor proteins are commonly performed in 2D arrays. But many cell surface receptors only function in their native membrane environment and/or in a specific conformation, such as they appear on the surface of live cells. Thus, receptors on live cells should be used for ligand binding assays. Here, it is shown that antibodies preprinted on a glass surface can be used to specifically array a peptide receptor of the immune system, i.e., the major histocompatibility complex class I molecule H-2Kb , into a defined pattern on the surface of live cells. Monoclonal antibodies make it feasible to capture a distinct subpopulation of H-2Kb and hold it at the cell surface. This patterned receptor enables a novel peptide-binding assay, in which the specific binding of a fluorescently labeled index peptide is visualized by microscopy. Measurements of ligand binding to captured cell surface receptors in defined confirmations apply to many problems in cell biology and thus represent a promising tool in the field of biosensors.

Mapping transmembrane residues of proteinase activated receptor 2 (PAR2) that influence ligand-modulated calcium signaling

Proteinase-activated receptor 2 (PAR2) is a G protein-coupled receptor involved in metabolism, inflammation, and cancers. It is activated by proteolysis, which exposes a nascent N-terminal sequence that becomes a tethered agonist. Short synthetic peptides corresponding to this sequence also activate PAR2, while small organic molecules show promising PAR2 antagonism. Developing PAR2 ligands into pharmaceuticals is hindered by a lack of knowledge of how synthetic ligands interact with and differentially modulate PAR2. Guided by PAR2 homology modeling and ligand docking based on bovine rhodopsin, followed by cross-checking with newer PAR2 models based on ORL-1 and PAR1, site-directed mutagenesis of PAR2 was used to investigate the pharmacology of three agonists (two synthetic agonists and trypsin-exposed tethered ligand) and one antagonist for modulation of PAR2 signaling. Effects of 28 PAR2 mutations were examined for PAR2-mediated calcium mobilization and key mutants were selected for measuring ligand binding. Nineteen of twenty-eight PAR2 mutations reduced the potency of at least one ligand by >10-fold. Key residues mapped predominantly to a cluster in the transmembrane (TM) domains of PAR2, differentially influence intracellular Ca2+ induced by synthetic agonists versus a native agonist, and highlight subtly different TM residues involved in receptor activation. This is the first evidence highlighting the importance of the PAR2 TM regions for receptor activation by synthetic PAR2 agonists and antagonists. The trypsin-cleaved N-terminus that activates PAR2 was unaffected by residues that affected synthetic peptides, challenging the widespread practice of substituting peptides for proteases to characterize PAR2 physiology.

The Cytochrome P450-Catalyzed Oxidative Rearrangement in the Final Step of Pentalenolactone Biosynthesis: Substrate Structure Determines Mechanism.

The final step in the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone (1) is the highly unusual cytochrome P450-catalyzed, oxidative rearrangement of pentalenolactone F (2), involving the transient generation and rearrangement of a neopentyl cation. In Streptomyces arenae this reaction is catalyzed by CYP161C2 (PntM), with highly conserved orthologs being present in at least 10 other Actinomycetes. Crystal structures of substrate-free PntM, as well as PntM with bound substrate 2, product 1, and substrate analogue 6,7-dihydropentalenolactone F (7) revealed interactions of bound ligand with three residues, F232, M77, and M81 that are unique to PntM and its orthologs and absent from essentially all other P450s. Site-directed mutagenesis, ligand-binding measurements, steady-state kinetics, and reaction product profiles established there is no special stabilization of reactive cationic intermediates by these side chains. Reduced substrate analogue 7 did not undergo either oxidative rearrangement or simple hydroxylation, suggesting that the C1 carbocation is not anchimerically stabilized by the 6,7-double bond of 2. The crystal structures also revealed plausible proton relay networks likely involved in the generation of the key characteristic P450 oxidizing species, Compound I, and in mediating stereospecific deprotonation of H-3re of the substrate. We conclude that the unusual carbocation intermediate results from outer shell electron transfer from the transiently generated C1 radical to the tightly paired heme-•Fe(3+)-OH radical species. The oxidative electron transfer is kinetically dominant as a result of the unusually strong steric barrier to oxygen rebound to the neopentyl center C-1si, which is flanked on each neighboring carbon by syn-axial substituents.

Interaction between 2-(p-toluidino)-6-naphthalenesulfonic acid sodium salt (TNS) and β-lactoglobulin

The major bovine milk protein β-lactoglobulin (β-LG), a member of the lipocalin superfamily, can bind a wide range of ligands and act as a transporter. In the present study, the combination of the hydrophobic molecule 2-(p-toluidino)-6-naphthalenesulfonic acid sodium salt (TNS) with β-LG was analyzed using fluorescence spectroscopy and AutoDock modeling to discern the major binding sites of the protein and to determine the capacity of other small ligands to bind with β-LG by utilizing TNS as a reference. The experimental data indicate that in a neutral pH environment, TNS is located in the hydrophobic domain of the protein, 2.5 nm away from the Trp19 residues of β-LG. The binding constant of the small molecule to β-LG is (3.30 ± 0.32) × 106 (mol L–1)−1. An interaction model between the ligand and β-LG was developed, and AutoDock modeling also demonstrates that the ligand is located in the central hydrophobic calyx of β-LG within the regions covered by the Förster radius of the Trp19–ligand pair. Although the interaction between the ligand and β-LG is affected by increasing ion strength, pH change, and heat treatment, the complex is maintained until the secondary structure of β-LG is destroyed. Additionally, the ligand binding stabilizes the folding of β-LG. The binding constants of sodium dodecyl sulfate (SDS) and sodium dodecylbenzene sulfonate (SDBS) to β-LG were obtained using competitive ligand binding measurements. With a sensitive fluorescence signal and stable complex, the ligand could be utilized as a reference to detect the binding of other small ligands to β-LG.

MgF3- and AlF4- transition state analogue complexes of yeast phosphoglycerate kinase.

The phospho-transfer mechanism of yeast phosphoglycerate kinase (PGK) has been probed through formation of trifluoromagnesate (MgF3-) and tetrafluoroaluminate (AlF4-) transition state analogue complexes and analyzed using 19F, 1H waterLOGSY and 1H chemical shift perturbation NMR spectroscopy. We observed the first 19F NMR spectroscopic evidence for the formation of metal fluoride transition state analogues of yeast PGK and also observed significant changes to proton chemical shifts of PGK in the presence, but not in the absence, of fluoride upon titration of ligands, providing indirect evidence of the formation of a closed ternary transition state. WaterLOGSY NMR spectroscopy experiments using an uncompetitive model were used in an attempt to measure ligand binding affinities within the transition state analogue complexes.

Deciphering the function of the CNGB1b subunit in olfactory CNG channels.

Olfactory cyclic nucleotide-gated (CNG) ion channels are key players in the signal transduction cascade of olfactory sensory neurons. The second messengers cAMP and cGMP directly activate these channels, generating a depolarizing receptor potential. Olfactory CNG channels are composed of two CNGA2 subunits and two modulatory subunits, CNGA4, and CNGB1b. So far the exact role of the modulatory subunits for channel activation is not fully understood. By measuring ligand binding and channel activation simultaneously, we show that in functional heterotetrameric channels not only the CNGA2 subunits and the CNGA4 subunit but also the CNGB1b subunit binds cyclic nucleotides and, moreover, also alone translates this signal to open the pore. In addition, we show that the CNGB1b subunit is the most sensitive subunit in a heterotetrameric channel to cyclic nucleotides and that it accelerates deactivation to a similar extent as does the CNGA4 subunit. In conclusion, the CNGB1b subunit participates in ligand-gated activation of olfactory CNG channels and, particularly, contributes to rapid termination of odorant signal in an olfactory sensory neuron.

Preparation To Minimize Buffer Mismatch in Isothermal Titration Calorimetry Experiments.

There is a growing need to study ligand binding to proteins in native or complex solution using isothermal titration calorimetry (ITC). For example, it is desirable to measure ligand binding to membrane proteins in more native lipid-like environments such as bicelles, where ligands can access both sides of the membrane in a homogeneous environment. A critical step to obtain high signal-to-noise is matching the reaction chamber solution to the ligand solution, typically through a final dialysis or gel filtration step. However, to obtain reproducible bicelles, the lipid concentrations must be carefully controlled which eliminates the use of dialysis that can disrupt these parameters. Here, we report and validate a rapid preparation ITC (RP-ITC) approach to measure ligand binding without the need for a dialysis step. This general approach is used to quantify ion binding to a K(+) channel embedded in bicelles and can be applied to complex, less defined systems.

The Role of Annular Lipids in the Mechanism of Membrane Protein Solubilization and Formation of Functionally Competent Proteomicelles

The “solubilization” procedure in which membrane proteins are extracted from their native lipid membranes and transferred into detergent micelles (proteomicelles) is a critical step in the purification of the protein for subsequent structure/function studies. In some notable examples it has become clear, however, that the properties of the proteins in proteomicelles depend on the detergent type and its concentration relative to the residual membrane lipids originating from the preparation. To reveal the molecular mechanism of the solubilization process and the effect of various lipid-to-detergent (L-D) ratios on the properties of the system, we performed extensive atomistic molecular dynamics simulations. We previously explored the properties of micelles formed by dodecyl-β-maltoside (DDM) detergent around the leucine transporter (LeuT), a bacterial homolog of the neurotransmitter:sodium symporter family, and showed how the DDM concentration determines the extent of detergent penetration into the functionally relevant secondary substrate (S2) binding site in LeuT. Here we present computational studies of LeuT embedded in proteomicelles at different L-D ratios exploring configurations of LeuT proteomicelles at different stages of the protein solubilization process. Our results reveal that at a sparse lipid annulus around the transporter, consisting of no more than ∼10 lipids, DDM partitions into the LeuT, engaging with the residues in the S2 site. Higher lipid concentrations protect the S2 site from DDM penetration. In contrast to DDM, MNG-3 (lauryl maltose-neopentyl glycol) detergent, an amphiphile known to better stabilize membrane proteins, does not penetrate the transporter even in the absence of lipids. Parallel experimental measurements of ligand binding to LeuT in the presence of increasing MNG-3 concentrations revealed that the functionality of the S2 site was fully preserved even at the highest (0.5%) MNG-3 concentration probed.

Thermodynamic characterization of new positive allosteric modulators binding to the glutamate receptor A2 ligand-binding domain: combining experimental and computational methods unravels differences in driving forces.

Positive allosteric modulation of the ionotropic glutamate receptor GluA2 presents a potential treatment of cognitive disorders, for example, Alzheimer's disease. In the present study, we describe the synthesis, pharmacology, and thermodynamic studies of a series of monofluoro-substituted 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides. Measurements of ligand binding by isothermal titration calorimetry (ITC) showed similar binding affinities for the modulator series at the GluA2 LBD but differences in the thermodynamic driving forces. Binding of 5c (7-F) and 6 (no-F) is enthalpy driven, and 5a (5-F) and 5b (6-F) are entropy driven. For 5d (8-F), both quantities were equal in size. Thermodynamic integration (TI) and one-step perturbation (OSP) were used to calculate the relative binding affinity of the modulators. The OSP calculations had a higher predictive power than those from TI, and combined with the shorter total simulation time, we found the OSP method to be more effective for this setup. Furthermore, from the molecular dynamics simulations, we extracted the enthalpies and entropies, and along with the ITC data, this suggested that the differences in binding free energies are largely explained by the direct ligand-surrounding enthalpies. Furthermore, we used the OSP setup to predict binding affinities for a series of polysubstituted fluorine compounds and monosubstituted methyl compounds and used these predictions to characterize the modulator binding pocket for this scaffold of positive allosteric modulators.