Ligand Binding Kinetics Research Articles

Ligand Gaussian Accelerated Molecular Dynamics (LiGaMD) for Characterization of Ligand Binding Thermodynamics and Kinetics

Calculations of ligand binding free energies and kinetic rates are important for drug design. However, such tasks have proven challenging in computational chemistry and biophysics. To address this challenge, we have developed a new computational method “LiGaMD”, which selectively boosts the ligand non-bonded interaction potential energy based on the Gaussian accelerated molecular dynamics (GaMD) enhanced sampling technique. Another boost potential could be applied to the remaining potential energy of the entire system in a dual-boost algorithm (LiGaMD_Dual) to facilitate ligand binding.

Understanding the impacts of cellular environments on ligand binding of membrane receptors by computational simulations.

Binding of cell surface receptors with their extracellular ligands initiates various intracellular signaling pathways. However, our understanding of the cellular functions of these receptors is very limited due to the fact that in vivo binding between ligands and receptors has only been successfully measured in a very small number of cases. In living cells, receptors are anchored on surfaces of the plasma membrane, which undergoes thermal undulations. Moreover, it has been observed in various systems that receptors can be organized into oligomers prior to ligand binding. It is not well understood how these cellular factors play roles in regulating the dynamics of ligand-receptor interactions. Here, we tackled these problems by using a coarse-grained kinetic Monte Carlo simulation method. Using this method, we demonstrated that the membrane undulations cause a negative effect on ligand-receptor interactions. We further found that the preassembly of membrane receptors on the cell surface can not only accelerate the kinetics of ligand binding but also reduce the noises during the process. In general, our study highlights the importance of membrane environments in regulating the function of membrane receptors in cells. The simulation method can be potentially applied to specific receptor systems involved in cell signaling.

Direct coupling of oligomerization and oligomerization-driven endocytosis of the dopamine transporter to its conformational mechanics and activity

Dopamine transporter (DAT) mediates the reuptake of synaptically released dopamine, and thus controls the duration and intensity of dopamine neurotransmission. Mammalian DAT has been observed to form oligomers, although the mechanisms of oligomerization and its role in DAT activity and trafficking remain largely unknown. We discovered a series of small molecule compounds that stabilize trimers and induce high-order oligomers of DAT and concomitantly promote its clathrin-independent endocytosis. Using a combination of chemical cross-linking, fluorescence resonance energy transfer microscopy, antibody-uptake endocytosis assay, live-cell lattice light sheet microscopy, ligand binding and substrate transport kinetics analyses, and molecular modeling and simulations, we investigated molecular basis of DAT oligomerization and endocytosis induced by these compounds. Our study showed that small molecule–induced DAT oligomerization and endocytosis are favored by the inward-facing DAT conformation and involve interactions of four hydrophobic residues at the interface between transmembrane (TM) helices TM4 and TM9. Surprisingly, a corresponding quadruple DAT mutant displays altered dopamine transport kinetics and increased cocaine-analog binding. The latter is shown to originate from an increased preference for outward-facing conformation and inward-to-outward transition. Taken together, our results demonstrate a direct coupling between conformational dynamics of DAT, functional activity of the transporter, and its oligomerization leading to endocytosis. The high specificity of such coupling for DAT makes the TM4-9 hub a new target for pharmacological modulation of DAT activity and subcellular localization.

Conformational Dynamics and Binding Kinetics Drive Mutant Selective Degradation of EGFR

Targeted degradation of proteins by chimeric chemical inducers of degradation (CIDEs) has emerged as a major drug discovery paradigm. Despite the increased interest in this approach, the criteria dictating target protein degradation by a CIDE remain poorly understood, and potent target engagement by a CIDE does not strongly correlate with target degradation. In this study, we present the biochemical characterization of an EGFR mutant-selective degrader that potently binds to wildtype and mutant EGFR but only degrades EGFR mutant variants. Mechanistic studies reveal that ternary complex half-life is the primary determinant driving mutant-selective degradation. We present a cryo-EM structure of a CRL2VHL:CIDE:EGFR complex and show that potent target degradation can be achieved in the absence of CIDE-induced protein-protein interactions. These results highlight the importance of considering target conformation during CIDE development as well as leveraging ligand binding kinetics to achieve robust target degradation.

Fluorescence Anisotropy-Based Assay for Characterization of Ligand Binding Dynamics to GPCRs: The Case of Cy3B-Labeled Ligands Binding to MC4 Receptors in Budded Baculoviruses.

During the past decade, fluorescence methods have become valuable tools for characterizing ligand binding to G protein-coupled receptors (GPCRs). However, only a few of the assays enable studying wild-type receptors and monitor the ligand binding in real time. One of the approaches that is inherently suitable for this purpose is the fluorescence anisotropy (FA) assay. In the FA assay, the change of ligand's rotational freedom connected with its binding to the receptor can be monitored with a conventional fluorescence plate reader equipped with suitable optical filters. To achieve the high receptor concentration required for the assay and the low autofluorescence levels essential for reliable results, budded baculoviruses that display GPCRs on their surfaces can be used. The monitoring process generates a substantial amount of kinetic data, which is usually stored as a proprietary file format limiting the flexibility of data analysis. To solve this problem, we propose the use of the data curation software Aparecium ( http://gpcr.ut.ee/aparecium.html ), which integrates experimental data with metadata in a Minimum Information for Data Analysis in Systems Biology (MIDAS) format. Aparecium enables data export to different software packages for fitting to suitable kinetic or equilibrium models. A combination of the FA assay with the novel data analysis strategy is suitable for screening new active compounds, but also for modeling complex systems of ligand binding to GPCRs. We present the proposed approach using different fluorescent probes and assay types to characterize ligand binding to melanocortin 4 (MC4) receptor.

An Appraisal of Computer Simulation Approaches in Elucidating Biomolecular Recognition Pathways.

Computer simulation approaches in biomolecular recognition processes have come a long way. In this Perspective, we highlight a series of recent success stories in which computer simulations have played a remarkable role in elucidating the atomic resolution mechanism of kinetic processes of protein-ligand binding in a quantitative fashion. In particular, we show that a robust combination of unbiased simulation, harnessed by a high-fidelity computing environment, and Markov state modeling approaches has been instrumental in revealing novel protein-ligand recognition pathways in multiple systems. We also elucidate the role of recent developments in enhanced sampling approaches in providing the much-needed impetus in accelerating simulation of the ligand recognition process. We identify multiple key issues, including force fields and the sampling bottleneck, which are currently preventing the field from achieving quantitative reconstruction of experimental measurements. Finally, we suggest a possible way forward via adoption of multiscale approaches and coarse-grained simulations as next steps toward efficient elucidation of ligand binding kinetics.

Cellular Dynamics of Transition Metal Exchange on Proteins: A Challenge but a Bonanza for Coordination Chemistry.

Transition metals interact with a large proportion of the proteome in all forms of life, and they play mandatory and irreplaceable roles. The dynamics of ligand binding to ions of transition metals falls within the realm of Coordination Chemistry, and it provides the basic principles controlling traffic, regulation, and use of metals in cells. Yet, the cellular environment stands out against the conditions prevailing in the test tube when studying metal ions and their interactions with various ligands. Indeed, the complex and often changing cellular environment stimulates fast metal–ligand exchange that mostly escapes presently available probing methods. Reducing the complexity of the problem with purified proteins or in model organisms, although useful, is not free from pitfalls and misleading results. These problems arise mainly from the absence of the biosynthetic machinery and accessory proteins or chaperones dealing with metal / metal groups in cells. Even cells struggle with metal selectivity, as they do not have a metal-directed quality control system for metalloproteins, and serendipitous metal binding is probably not exceptional. The issue of metal exchange in biology is reviewed with particular reference to iron and illustrating examples in patho-physiology, regulation, nutrition, and toxicity.

Drug binding dynamics of the dimeric SARS-CoV-2 main protease, determined by molecular dynamics simulation

We performed molecular dynamics simulation of the dimeric SARS-CoV-2 (severe acute respiratory syndrome corona virus 2) main protease (Mpro) to examine the binding dynamics of small molecular ligands. Seven HIV inhibitors, darunavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, and tipranavir, were used as the potential lead drugs to investigate access to the drug binding sites in Mpro. The frequently accessed sites on Mpro were classified based on contacts between the ligands and the protein, and the differences in site distributions of the encounter complex were observed among the ligands. All seven ligands showed binding to the active site at least twice in 28 simulations of 200 ns each. We further investigated the variations in the complex structure of the active site with the ligands, using microsecond order simulations. Results revealed a wide variation in the shapes of the binding sites and binding poses of the ligands. Additionally, the C-terminal region of the other chain often interacted with the ligands and the active site. Collectively, these findings indicate the importance of dynamic sampling of protein–ligand complexes and suggest the possibilities of further drug optimisations.

Dynamics of Ligand Binding to a Rigid Glycosidase**

AbstractThe single‐domain GH11 glycosidase from Bacillus circulans (BCX) is involved in the degradation of hemicellulose, which is one of the most abundant renewable biomaterials in nature. We demonstrate that BCX in solution undergoes minimal structural changes during turnover. NMR spectroscopy results show that the rigid protein matrix provides a frame for fast substrate binding in multiple conformations, accompanied by slow conversion, which is attributed to an enzyme‐induced substrate distortion. A model is proposed in which the rigid enzyme takes advantage of substrate flexibility to induce a conformation that facilitates the acyl formation step of the hydrolysis reaction.

Dynamics of Ligand Binding to a Rigid Glycosidase*.

The single‐domain GH11 glycosidase from Bacillus circulans (BCX) is involved in the degradation of hemicellulose, which is one of the most abundant renewable biomaterials in nature. We demonstrate that BCX in solution undergoes minimal structural changes during turnover. NMR spectroscopy results show that the rigid protein matrix provides a frame for fast substrate binding in multiple conformations, accompanied by slow conversion, which is attributed to an enzyme‐induced substrate distortion. A model is proposed in which the rigid enzyme takes advantage of substrate flexibility to induce a conformation that facilitates the acyl formation step of the hydrolysis reaction.

Quantitative modeling of the function of kinetically driven transcriptional riboswitches

Riboswitches are cis-acting regulatory mRNA elements in bacteria, that modulate the expression of their associated genes in response to a cognate metabolite, operating either on the level of translation or transcription. Transcriptional riboswitches have to fold into functional structures as they are being synthesized and, only if transcription rates and ligand binding kinetics match, structured transcription intermediates are enabled to undergo ligand-dependent conformational refolding as a prerequisite for ligand-mediated gene expression. Therefore, transcription rates are of essential importance for functional riboswitch-mediated gene regulation. Here, we propose a generalized modeling framework for the kinetic mechanisms of transcriptional riboswitches. The formalism accommodates time-dependent transcription rates and changes of metabolite concentration and permits incorporation of variations in transcription rate depending on transcript length. We derive explicit analytical expressions for the fraction of transcripts that determine repression or activation of gene expression as a function of pause site location and its slowing down of transcription for the case of the (2’dG)-sensing riboswitch from Mesoplasma florum. Our modeling challenges the current view on the exclusive importance of metabolite binding to transcripts containing only the aptamer domain. Numerical simulations of transcription proceeding in a continuous manner under time-dependent changes of metabolite concentration further suggest that rapid modulations in concentration result in a reduced dynamic range for riboswitch function regardless of transcription rate, while a combination of slow modulations and small transcription rates ensures a wide range of finely tuneable regulatory outcomes.

Ligand Gaussian Accelerated Molecular Dynamics (LiGaMD): Characterization of Ligand Binding Thermodynamics and Kinetics.

Calculations of ligand binding free energies and kinetic rates are important for drug design. However, such tasks have proven challenging in computational chemistry and biophysics. To address this challenge, we have developed a new computational method, ligand Gaussian accelerated molecular dynamics (LiGaMD), which selectively boosts the ligand nonbonded interaction potential energy based on the Gaussian accelerated molecular dynamics (GaMD) enhanced sampling technique. Another boost potential could be applied to the remaining potential energy of the entire system in a dual-boost algorithm (LiGaMD_Dual) to facilitate ligand binding. LiGaMD has been demonstrated on host-guest and protein-ligand binding model systems. Repetitive guest binding and unbinding in the β-cyclodextrin host were observed in hundreds-of-nanosecond LiGaMD_Dual simulations. The calculated guest binding free energies agreed excellently with experimental data with <1.0 kcal/mol errors. Compared with converged microsecond-time scale conventional molecular dynamics simulations, the sampling errors of LiGaMD_Dual simulations were also <1.0 kcal/mol. Accelerations of ligand kinetic rate constants in LiGaMD simulations were properly estimated using Kramers' rate theory. Furthermore, LiGaMD allowed us to capture repetitive dissociation and binding of the benzamidine inhibitor in trypsin within 1 μs simulations. The calculated ligand binding free energy and kinetic rate constants compared well with the experimental data. In summary, LiGaMD provides a powerful enhanced sampling approach for characterizing ligand binding thermodynamics and kinetics simultaneously, which is expected to facilitate computer-aided drug design.

Noncooperative thermodynamics and kinetic models of ligand binding to polymers: Connecting McGhee-von Hippel model with the Tonks gas model.

Ligand binding to polymers modifies the physical and chemical properties of the polymers, leading to physical, chemical, and biological implications. McGhee and von Hippel obtained the equilibrium coverage as a function of the ligand affinity, through the computation of the possible binding sites for the ligand. Here, we complete this theory deriving the kinetic model for the ligand-binding dynamics and the associated equilibrium chemical potential, which turns out to be of the Tonks gas model type. At low coverage, the Tonks chemical potential becomes the Fermi chemical potential and even the ideal gas chemical potential. We also discuss kinetic models associated with these chemical potentials. These results clarify the kinetic models of ligand binding, their relations with the chemical potentials, and their range of validity. Our results highlight the inaccuracy of ideal and simplified kinetic approaches for medium and high coverages.

Vibrational Spectroscopic Map, Vibrational Spectroscopy, and Intermolecular Interaction.

Vibrational spectroscopy is an essential tool in chemical analyses, biological assays, and studies of functional materials. Over the past decade, various coherent nonlinear vibrational spectroscopic techniques have been developed and enabled researchers to study time-correlations of the fluctuating frequencies that are directly related to solute-solvent dynamics, dynamical changes in molecular conformations and local electrostatic environments, chemical and biochemical reactions, protein structural dynamics and functions, characteristic processes of functional materials, and so on. In order to gain incisive and quantitative information on the local electrostatic environment, molecular conformation, protein structure and interprotein contacts, ligand binding kinetics, and electric and optical properties of functional materials, a variety of vibrational probes have been developed and site-specifically incorporated into molecular, biological, and material systems for time-resolved vibrational spectroscopic investigation. However, still, an all-encompassing theory that describes the vibrational solvatochromism, electrochromism, and dynamic fluctuation of vibrational frequencies has not been completely established mainly due to the intrinsic complexity of intermolecular interactions in condensed phases. In particular, the amount of data obtained from the linear and nonlinear vibrational spectroscopic experiments has been rapidly increasing, but the lack of a quantitative method to interpret these measurements has been one major obstacle in broadening the applications of these methods. Among various theoretical models, one of the most successful approaches is a semiempirical model generally referred to as the vibrational spectroscopic map that is based on a rigorous theory of intermolecular interactions. Recently, genetic algorithm, neural network, and machine learning approaches have been applied to the development of vibrational solvatochromism theory. In this review, we provide comprehensive descriptions of the theoretical foundation and various examples showing its extraordinary successes in the interpretations of experimental observations. In addition, a brief introduction to a newly created repository Web site (http://frequencymap.org) for vibrational spectroscopic maps is presented. We anticipate that a combination of the vibrational frequency map approach and state-of-the-art multidimensional vibrational spectroscopy will be one of the most fruitful ways to study the structure and dynamics of chemical, biological, and functional molecular systems in the future.

Predicting Ligand Binding Kinetics Using a Markovian Milestoning with Voronoi Tessellations Multiscale Approach

Accurate and efficient computational predictions of ligand binding kinetics can be useful to inform drug discovery campaigns, particularly in the screening and lead optimization phases. Simulation enabled estimation of kinetic rates (SEEKR) is a multiscale molecular dynamics, Brownian dynamics, and milestoning simulation approach for calculating receptor-ligand association and dissociation rates. Here, we present the implementation of a Markovian milestoning with Voronoi tessellations approach that significantly reduces the simulation cost of calculations as well as further improving their parallelizability. The new approach is applied to a host-guest system to assess its effectiveness for rank-ordering compounds by kinetic rates and to the model protein system, trypsin, with the noncovalent inhibitor benzamidine. For both applications, we demonstrate that the new approach requires up to a factor of 10 less simulation time to achieve results with comparable or increased accuracy.

Comprehensive characterization of tense and relaxed quaternary state glutaraldehyde polymerized bovine hemoglobin as a function of cross‐link density

Previously, our lab developed high molecular weight (MW) tense (T) quaternary state glutaraldehyde polymerized bovine hemoglobins (PolybHbs) that exhibited reduced vasoactivity in several small animal models. In this study, we prepared PolybHb in the T and relaxed (R) quaternary state with ultrahigh MW (>500 kDa) with varying cross-link densities, and investigated the effect of MW on key biophysical properties (i.e., O2 affinity, cooperativity (Hill) coefficient, hydrodynamic diameter, polydispersity, polymer composition, viscosity, gaseous ligand-binding kinetics, auto-oxidation, and haptoglobin [Hp]-binding kinetics). To further optimize current PolybHb synthesis and purification protocols, we performed a comprehensive meta-data analysis to evaluate correlations between procedural parameters (i.e., cross-linker:bovine hemoglobin (bHb) molar ratio, gas-liquid exchange time, temperature during sodium dithionite addition, and number of diafiltration cycles) and the biophysical properties of both T- and R-state PolybHbs. Our results showed that, the duration of the fast-step auto-oxidation phase of R-state PolybHb increased with decreasing glutaraldehyde:bHb molar ratio. Additionally, T-state PolybHbs exhibited significantly higher bimolecular rate constants for binding to Hp and unimolecular O2 offloading rate constants compared to R-state PolybHbs. The methemoglobin (metHb) level in the final product was insensitive to the molar ratio of glutaraldehyde to bHb for all PolybHbs. During tangential flow filtration processing of the final product, 14 diafiltration cycles was found to yield the lowest metHb level.

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.

Ranking of Ligand Binding Kinetics Using a Weighted Ensemble Approach and Comparison with a Multiscale Milestoning Approach.

To improve lead optimization efforts in finding the right ligand, pharmaceutical industries need to know the ligand's binding kinetics, such as binding and unbinding rate constants, which often correlate with the ligand's efficacy in vivo. To predict binding kinetics efficiently, enhanced sampling methods, such as milestoning and the weighted ensemble (WE) method, have been used in molecular dynamics (MD) simulations of these systems. However, a comparison of these enhanced sampling methods in ranking ligands has not been done. Hence, a WE approach called the concurrent adaptive sampling (CAS) algorithm that uses MD simulations was used to rank seven ligands for β-cyclodextrin, a system in which a multiscale milestoning approach called simulation enabled estimation of kinetic rates (SEEKR) was also used, which uses both MD and Brownian dynamics simulations. Overall, the CAS algorithm can successfully rank ligands using the unbinding rate constant koff values and binding free energy ΔG values, as SEEKR did, with reduced computational cost that is about the same as SEEKR. We compare the CAS algorithm simulations with different parameters and discuss the impact of parameters in ranking ligands and obtaining rate constant and binding free energy estimates. We also discuss similarities and differences and advantages and disadvantages of SEEKR and the CAS algorithm for future use.

Novel Bioluminescent Tools to Study GPCR Pharmacology in Living Cells

G‐protein coupled receptors (GPCRs) continue to be prominent targets for new therapeutics. To support these efforts, we have developed a suite of bioluminescent assays to study GPCR pharmacology in real time in living cells. Quantification of ligand binding and receptor internalization relies on N‐terminal fusion to High BiT (HiBiT), an 11 a.a. peptide. HiBiT binds with high affinity to Large BiT (LgBiT) to form NanoBiT Luciferase. For both applications, a non‐lytic detection reagent is added containing cell‐impermeable LgBiT and furimazine substrate. Ligand binding dynamics at the cell surface can be measured in real time through bioluminescent resonance energy transfer (BRET) between NanoBiT Luciferase and a fluorescent tracer. Receptor internalization and recycling are monitored in real time by measuring changes in the cell surface density of HiBiT‐tagged GPCRs. Notably, the bright signal of NanoBiT Luciferase allows both assays to be run at endogenous levels of expression using CRISPR/Cas9 to introduce the HiBiT tag. Similarly, fusion proteins to LgBiT and Small BiT (SmBiT), an 11 a.a. peptide with low affinity binding to LgBiT, can be used to measure β‐arrestin‐1/2 recruitment to activated GPCRs in real time. Furthermore, changes in intracellular cAMP concentration can be sensitively measured using endpoint (cAMP‐Glo) or real time formats (GloSensor cAMP) using thermal stable or circularly permuted forms of luciferase, respectively. These homogenous, cell‐based assays, which are readily adaptable to laboratory automation, should be immensely useful in the study of GPCR pharmacology.Support or Funding InformationPromega Corporation

Fluorescent ligands: Bringing light to emerging GPCR paradigms.

In recent years, several novel aspects of GPCR pharmacology have been described, which are thought to play a role in determining the in vivo efficacy of a compound. Fluorescent ligands have been used to study many of these, which have also required the development of new experimental approaches. Fluorescent ligands offer the potential to use the same fluorescent probe to perform a broad range of experiments, from single-molecule microscopy to in vivo BRET. This review provides an overview of the in vitro use of fluorescent ligands in further understanding emerging pharmacological paradigms within the GPCR field, including ligand-binding kinetics, allosterism and intracellular signalling, along with the use of fluorescent ligands to study physiologically relevant therapeutic agents.