Pharmacological characterisation of novel adenosine A3 receptor antagonists

SummaryBackground and PurposeThe adenosine A3 receptor (A3R) belongs to a family of four adenosine receptor (AR) subtypes which all play distinct roles throughout the body. A3R antagonists have been described as potential treatments for numerous diseases including asthma. Given the similarity between ARs orthosteric binding sites, obtaining highly selective antagonists is a challenging but critical task.Experimental approach39 potential A3R, antagonists were screened using agonist-induced inhibition of cAMP. Positive hits were assessed for AR subtype selectivity through cAMP accumulation assays. The antagonist affinity was determined using Schild analysis (pA2 values) and fluorescent ligand binding. Further, a likely binding pose of the most potent antagonist (K18) was determined through molecular dynamic (MD) simulations and consistent calculated binding free energy differences between K18 and congeners, using a homology model of A3R, combined with mutagenesis studies.Key ResultsWe demonstrate that K18, which contains a 3-(dichlorophenyl)-isoxazole group connected through carbonyloxycarboximidamide fragment with a 1,3-thiazole ring, is a specific A3R (<1 µM) competitive antagonist. Structure-activity relationship investigations revealed that loss of the 3-(dichlorophenyl)-isoxazole group significantly attenuated K18 antagonistic potency. Mutagenic studies supported by MD simulations identified the residues important for binding in the A3R orthosteric site. Finally, we introduce a model that enables estimates of the equilibrium binding affinity for rapidly disassociating compounds from real-time fluorescent ligand-binding studies.Conclusions and ImplicationsThese results demonstrate the pharmacological characterisation of a selective competitive A3R antagonist and the description of its orthosteric binding mode. Our findings may provide new insight for drug discovery.What is already knownThe search for AR subtype specific compounds often leads to ones with multiple subtype bindingWhat this study addsThis study demonstrates the pharmacological characterisation of a selective competitive A3R antagonistMD simulations identified the residues important for binding in the A3R orthosteric siteClinical significanceThis study offers insight into A3R antagonists that may provide new opportunities for drug discovery

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Improving Small-Molecule Force Field Parameters in Ligand Binding Studies

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.760283 ◽

2021 ◽

Vol 8 ◽

Author(s):

Stefano Raniolo ◽

Vittorio Limongelli

Keyword(s):

Ligand Binding ◽

Force Field ◽

Small Molecules ◽

Binding Mode ◽

Free Energy Calculations ◽

Binding Studies ◽

Torsion Angles ◽

Molecular Binding ◽

Future Drug ◽

Force Field Parameters

Small molecules are major players of many chemical processes in diverse fields, from material science to biology. They are made by a combination of carbon and heteroatoms typically organized in system-specific structures of different complexity. This peculiarity hampers the application of standard force field parameters and their in silico study by means of atomistic simulations. Here, we combine quantum-mechanics and atomistic free-energy calculations to achieve an improved parametrization of the ligand torsion angles with respect to the state-of-the-art force fields in the paradigmatic molecular binding system benzamidine/trypsin. Funnel-Metadynamics calculations with the new parameters greatly reproduced the high-resolution crystallographic ligand binding mode and allowed a more accurate description of the binding mechanism, when the ligand might assume specific conformations to cross energy barriers. Our study impacts on future drug design investigations considering that the vast majority of marketed drugs are small-molecules.

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Binding Modes of Ligands Using Enhanced Sampling (BLUES): Rapid Decorrelation of Ligand Binding Modes Using Nonequilibrium Candidate Monte Carlo

10.26434/chemrxiv.5406907.v2 ◽

2017 ◽

Author(s):

Samuel Gill ◽

Nathan M. Lim ◽

Patrick Grinaway ◽

Ariën S. Rustenburg ◽

Josh Fass ◽

...

Keyword(s):

Monte Carlo ◽

Ligand Binding ◽

Binding Mode ◽

Free Energy Calculations ◽

Dynamics Simulation ◽

Binding Modes ◽

Enhanced Sampling ◽

Recent Success ◽

Multiple Binding ◽

Proper Sampling

<div>Accurately predicting protein-ligand binding is a major goal in computational chemistry, but even the prediction of ligand binding modes in proteins poses major challenges. Here, we focus on solving the binding mode prediction problem for rigid fragments. That is, we focus on computing the dominant placement, conformation, and orientations of a relatively rigid, fragment-like ligand in a receptor, and the populations of the multiple binding modes which may be relevant. This problem is important in its own right, but is even more timely given the recent success of alchemical free energy calculations. Alchemical calculations are increasingly used to predict binding free energies of ligands to receptors. However, the accuracy of these calculations is dependent on proper sampling of the relevant ligand binding modes. Unfortunately, ligand binding modes may often be uncertain, hard to predict, and/or slow to interconvert on simulation timescales, so proper sampling with current techniques can require prohibitively long simulations. We need new methods which dramatically improve sampling of ligand binding modes. Here, we develop and apply a nonequilibrium candidate Monte Carlo (NCMC) method to improve sampling of ligand binding modes.</div><div><br></div><div>In this technique the ligand is rotated and subsequently allowed to relax in its new position through alchemical perturbation before accepting or rejecting the rotation and relaxation as a nonequilibrium Monte Carlo move. When applied to a T4 lysozyme model binding system, this NCMC method shows over two orders of magnitude improvement in binding mode sampling efficiency compared to a brute force molecular dynamics simulation. This is a first step towards applying this methodology to pharmaceutically relevant binding of fragments and, eventually, drug-like molecules. We are making this approach available via our new Binding Modes of Ligands using Enhanced Sampling (BLUES) package which is freely available on GitHub.</div>

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Binding Modes of Ligands Using Enhanced Sampling (BLUES): Rapid Decorrelation of Ligand Binding Modes Using Nonequilibrium Candidate Monte Carlo

10.26434/chemrxiv.5406907 ◽

2018 ◽

Author(s):

Samuel Gill ◽

Nathan M. Lim ◽

Patrick Grinaway ◽

Ariën S. Rustenburg ◽

Josh Fass ◽

...

Keyword(s):

Monte Carlo ◽

Ligand Binding ◽

Binding Mode ◽

Free Energy Calculations ◽

Dynamics Simulation ◽

Binding Modes ◽

Enhanced Sampling ◽

Recent Success ◽

Multiple Binding ◽

Proper Sampling

<div>Accurately predicting protein-ligand binding is a major goal in computational chemistry, but even the prediction of ligand binding modes in proteins poses major challenges. Here, we focus on solving the binding mode prediction problem for rigid fragments. That is, we focus on computing the dominant placement, conformation, and orientations of a relatively rigid, fragment-like ligand in a receptor, and the populations of the multiple binding modes which may be relevant. This problem is important in its own right, but is even more timely given the recent success of alchemical free energy calculations. Alchemical calculations are increasingly used to predict binding free energies of ligands to receptors. However, the accuracy of these calculations is dependent on proper sampling of the relevant ligand binding modes. Unfortunately, ligand binding modes may often be uncertain, hard to predict, and/or slow to interconvert on simulation timescales, so proper sampling with current techniques can require prohibitively long simulations. We need new methods which dramatically improve sampling of ligand binding modes. Here, we develop and apply a nonequilibrium candidate Monte Carlo (NCMC) method to improve sampling of ligand binding modes.</div><div><br></div><div>In this technique the ligand is rotated and subsequently allowed to relax in its new position through alchemical perturbation before accepting or rejecting the rotation and relaxation as a nonequilibrium Monte Carlo move. When applied to a T4 lysozyme model binding system, this NCMC method shows over two orders of magnitude improvement in binding mode sampling efficiency compared to a brute force molecular dynamics simulation. This is a first step towards applying this methodology to pharmaceutically relevant binding of fragments and, eventually, drug-like molecules. We are making this approach available via our new Binding Modes of Ligands using Enhanced Sampling (BLUES) package which is freely available on GitHub.</div>

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Engineered interdomain disulfide in the periplasmic receptor for sulfate transport reduces flexibility. Site-directed mutagenesis and ligand-binding studies.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)67775-6 ◽

1991 ◽

Vol 266 (8) ◽

pp. 5220-5225

Author(s):

B L Jacobson ◽

J J He ◽

P S Vermersch ◽

D D Lemon ◽

F A Quiocho

Keyword(s):

Ligand Binding ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Sulfate Transport ◽

Binding Studies

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Interbase-FRET binding assay for pre-microRNAs

Scientific Reports ◽

10.1038/s41598-021-88922-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Mattias Bood ◽

Anna Wypijewska del Nogal ◽

Jesper R. Nilsson ◽

Fredrik Edfeldt ◽

Anders Dahlén ◽

...

Keyword(s):

Ligand Binding ◽

Drug Targets ◽

Structural Changes ◽

Small Sample Size ◽

Resonance Energy ◽

Small Sample ◽

Titration Calorimetry ◽

Drug Candidate ◽

Binding Studies ◽

Aberrant Expression

AbstractThe aberrant expression of microRNAs (miRs) has been linked to several human diseases. A promising approach for targeting these anomalies is the use of small-molecule inhibitors of miR biogenesis. These inhibitors have the potential to (i) dissect miR mechanisms of action, (ii) discover new drug targets, and (iii) function as new therapeutic agents. Here, we designed Förster resonance energy transfer (FRET)-labeled oligoribonucleotides of the precursor of the oncogenic miR-21 (pre-miR-21) and used them together with a set of aminoglycosides to develop an interbase-FRET assay to detect ligand binding to pre-miRs. Our interbase-FRET assay accurately reports structural changes of the RNA oligonucleotide induced by ligand binding. We demonstrate its application in a rapid, qualitative drug candidate screen by assessing the relative binding affinity between 12 aminoglycoside antibiotics and pre-miR-21. Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) were used to validate our new FRET method, and the accuracy of our FRET assay was shown to be similar to the established techniques. With its advantages over SPR and ITC owing to its high sensitivity, small sample size, straightforward technique and the possibility for high-throughput expansion, we envision that our solution-based method can be applied in pre-miRNA–target binding studies.

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Exploring aromatic cage flexibility of the histone methyllysine reader protein Spindlin1 and its impact on binding mode prediction: an in silico study

Journal of Computer-Aided Molecular Design ◽

10.1007/s10822-021-00391-9 ◽

2021 ◽

Author(s):

Chiara Luise ◽

Dina Robaa ◽

Wolfgang Sippl

Keyword(s):

Molecular Dynamic ◽

In Silico ◽

Binding Pocket ◽

Binding Mode ◽

Molecular Dynamic Simulations ◽

Flexible Docking ◽

Dynamic Simulations ◽

Binding Mode Prediction ◽

Aromatic Cage ◽

The Right

AbstractSome of the main challenges faced in drug discovery are pocket flexibility and binding mode prediction. In this work, we explored the aromatic cage flexibility of the histone methyllysine reader protein Spindlin1 and its impact on binding mode prediction by means of in silico approaches. We first investigated the Spindlin1 aromatic cage plasticity by analyzing the available crystal structures and through molecular dynamic simulations. Then we assessed the ability of rigid docking and flexible docking to rightly reproduce the binding mode of a known ligand into Spindlin1, as an example of a reader protein displaying flexibility in the binding pocket. The ability of induced fit docking was further probed to test if the right ligand binding mode could be obtained through flexible docking regardless of the initial protein conformation. Finally, the stability of generated docking poses was verified by molecular dynamic simulations. Accurate binding mode prediction was obtained showing that the herein reported approach is a highly promising combination of in silico methods able to rightly predict the binding mode of small molecule ligands in flexible binding pockets, such as those observed in some reader proteins.

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Monitoring the Ligand Binding Mode by Proton NMR Chemical Shift Differences

ChemMedChem ◽

10.1002/cmdc.200600175 ◽

2006 ◽

Vol 1 (11) ◽

pp. 1197-1199 ◽

Cited By ~ 1

Author(s):

Srisunder Subramaniam ◽

Stephen L. Briggs ◽

Allen D. Kline

Keyword(s):

Chemical Shift ◽

Ligand Binding ◽

Proton Nmr ◽

Binding Mode ◽

Nmr Chemical Shift

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The identification of cytoplasmic membrane calcium-entry channels by ligand-binding studies

Biochemical Society Transactions ◽

10.1042/bst0120941 ◽

1984 ◽

Vol 12 (6) ◽

pp. 941-943 ◽

Cited By ~ 3

Author(s):

H. GLOSSMANN ◽

A. GOLL ◽

D. R. FERRY ◽

M. ROMBUSCH

Keyword(s):

Ligand Binding ◽

Cytoplasmic Membrane ◽

Calcium Entry ◽

Binding Studies

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Comparative studies on peptides representing the so-called tachykinin-like region of the Alzheimer Aβ peptide [Aβ(25–35)]

Biochemical Journal ◽

10.1042/bj3360419 ◽

1998 ◽

Vol 336 (2) ◽

pp. 419-427 ◽

Cited By ~ 18

Author(s):

Omar M. A. EL-AGNAF ◽

G. Brent IRVINE ◽

Geraldine FITZPATRICK ◽

W. Kenneth GLASS ◽

David J. S. GUTHRIE

Keyword(s):

Electron Microscopy ◽

Ligand Binding ◽

Comparative Studies ◽

Aβ Peptide ◽

Amyloid Protein ◽

Binding Studies ◽

Nk1 Receptors ◽

Ligand Displacement ◽

Β Amyloid

In an attempt to answer the question of whether or not the so-called tachykinin-like region of the Alzheimer β-amyloid protein [Aβ(25–35)] can act as a tachykinin, the sequences Aβ(25–35), Aβ(25–35)amide and their norleucine-35 and phenylalanine-31 analogues were synthesized. These peptides were examined with ligand binding studies, electron microscopy, CD and NMR. In all cases some differences were found between the Aβ(25–35) analogue and the corresponding Phe31 peptide. In addition, in ligand displacement studies on tachykinin NK1 receptors, only the Phe31 analogue showed activity comparable to that of genuine tachykinins. We conclude that peptides based on Aβ(25–35) but with a Phe residue at position 31 do display properties typical of a tachykinin, but that peptides with Ile at this position do not.

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