21 research outputs found
Computational Workflow for Refining AlphaFold Models in Drug Design Using Kinetic and Thermodynamic Binding Calculations: A Case Study for the Unresolved Inactive Human Adenosine A<sub>3</sub> Receptor
A structure-based
drug design pipeline that considers
both thermodynamic
and kinetic binding data of ligands against a receptor will enable
the computational design of improved drug molecules. For unresolved
GPCR-ligand complexes, a workflow that can apply both thermodynamic
and kinetic binding data in combination with alpha-fold (AF)-derived
or other homology models and experimentally resolved binding modes
of relevant ligands in GPCR-homologs needs to be tested. Here, as
test case, we studied a congeneric set of ligands that bind to a structurally
unresolved G protein-coupled receptor (GPCR), the inactive human adenosine
A3 receptor (hA3R). We tested three available
homology models from which two have been generated from experimental
structures of hA1R or hA2AR and one model was
a multistate alphafold 2 (AF2)-derived model. We applied alchemical
calculations with thermodynamic integration coupled with molecular
dynamics (TI/MD) simulations to calculate the experimental relative
binding free energies and residence time (Ï)-random accelerated
MD (Ï-RAMD) simulations to calculate the relative residence
times (RTs) for antagonists. While the TI/MD calculations produced,
for the three homology models, good Pearson correlation coefficients,
correspondingly, r = 0.74, 0.62, and 0.67 and mean
unsigned error (mue) values of 0.94, 1.31, and 0.81 kcal molâ1, the Ï-RAMD method showed r = 0.92 and 0.52
for the first two models but failed to produce accurate results for
the multistate AF2-derived model. With subsequent optimization of
the AF2-derived model by reorientation of the side chain of R1735.34 located in the extracellular loop 2 (EL2) that blocked
ligandâs unbinding, the computational model showed r = 0.84 for kinetic data and improved performance for thermodynamic
data (r = 0.81, mue = 0.56 kcal molâ1). Overall, after refining the multistate AF2 model with physics-based
tools, we were able to show a strong correlation between predicted
and experimental ligand relative residence times and affinities, achieving
a level of accuracy comparable to an experimental structure. The computational
workflow used can be applied to other receptors, helping to rank candidate
drugs in a congeneric series and enabling the prioritization of leads
with stronger binding affinities and longer residence times
Free Energy Calculations Reveal the Origin of Binding Preference for Aminoadamantane Blockers of Influenza A/M2TM Pore
Aminoadamantane derivatives, such as amantadine and rimantadine,
have been reported to block the M2 membrane protein of influenza A
virus (A/M2TM), but their use has been discontinued due to reported
resistance in humans. Understanding the mechanism of action of amantadine
derivatives could assist the development of novel potent inhibitors
that overcome A/M2TM resistance. Here, we use Free Energy Perturbation
calculations coupled with Molecular Dynamics simulations (FEP/MD)
to rationalize the thermodynamic origin of binding preference of several
aminoadamantane derivatives inside the A/M2TM pore. Our results demonstrate
that apart from crucial proteinâligand intermolecular interactions,
the flexibility of the protein, the water network around the ligand,
and the desolvation free energy penalty to transfer the ligand from
the aqueous environment to the transmembrane region are key elements
for the binding preference of these compounds and thus for lead optimization.
The high correlation of the FEP/MD results with available experimental
data (R<sup>2</sup> = 0.85) demonstrates that this methodology holds
predictive value and can be used to guide the optimization of drug
candidates binding to membrane proteins
Interpreting Thermodynamic Profiles of Aminoadamantane Compounds Inhibiting the M2 Proton Channel of Influenza A by Free Energy Calculations
The development of novel anti-influenza
drugs is of great importance
because of the capability of influenza viruses to occasionally cross
interspecies barriers and to rapidly mutate. One class of anti-influenza
agents, aminoadamantanes, including the drugs amantadine and rimantadine
now widely abandoned due to virus resistance, bind to and block the
pore of the transmembrane domain of the M2 proton channel (M2TM) of
influenza A. Here, we present one of the still rare studies that interprets
thermodynamic profiles from isothermal titration calorimetry (ITC)
experiments in terms of individual energy contributions to binding,
calculated by the computationally inexpensive implicit solvent/implicit
membrane molecular mechanics PoissonâBoltzmann surface area
(MM-PBSA) approach, for aminoadamantane compounds binding to M2TM
of the avian âWeybridgeâ strain. For all eight pairs
of aminoadamantane compounds considered, the trend of the predicted
relative binding free energies and their individual components, effective
binding energies and changes in the configurational entropy, agrees
with experimental measures (ÎÎ<i>G</i>, ÎÎ<i>H</i>, <i>T</i>ÎÎ<i>S</i>) in
88, 88, and 50% of the cases. In addition, information yielded by
the MM-PBSA approach about determinants of binding goes beyond that
available in component quantities (Î<i>H</i>, Î<i>S</i>) from ITC measurements. We demonstrate how one can make
use of such information to link thermodynamic profiles from ITC with
structural causes on the ligand side and, ultimately, to guide decision
making in lead optimization in a prospective manner, which results
in an aminoadamantane derivative with improved binding affinity against
M2TM<sub>Weybridge</sub>
Dual A1/A3 Adenosine Receptor Antagonists: Binding Kinetics and StructureâActivity Relationship Studies Using Mutagenesis and Alchemical Binding Free Energy Calculations
Drugs targeting adenosine receptors
(AR) can provide treatment
for diseases. We report the identification of 7-(phenylamino)-pyrazolo[3,4-c]âpyridines L2âL10, A15, and A17 as low-micromolar to low-nanomolar A1R/A3R dual antagonists, with 3-phenyl-5-cyano-7-(trimethoxyphenylamino)-pyrazolo[3,4-c]âpyridine (A17) displaying the highest
affinity at both receptors with a long residence time of binding,
as determined using a NanoBRET-based assay. Two binding orientations
of A17 produce stable complexes inside the orthosteric
binding area of A1R in molecular dynamics (MD) simulations,
and we selected the most plausible orientation based on the agreement
with alanine mutagenesis supported by affinity experiments. Interestingly,
for drug design purposes, the mutation of L2506.51 to alanine
increased the binding affinity of A17 at A1R. We explored the structureâactivity relationships against
A1R using alchemical binding free energy calculations with
the thermodynamic integration coupled with the MD simulation (TI/MD)
method, applied on the whole G-protein-coupled receptorâmembrane
system, which showed a good agreement (r = 0.73)
between calculated and experimental relative binding free energies
Dual A1/A3 Adenosine Receptor Antagonists: Binding Kinetics and StructureâActivity Relationship Studies Using Mutagenesis and Alchemical Binding Free Energy Calculations
Drugs targeting adenosine receptors
(AR) can provide treatment
for diseases. We report the identification of 7-(phenylamino)-pyrazolo[3,4-c]âpyridines L2âL10, A15, and A17 as low-micromolar to low-nanomolar A1R/A3R dual antagonists, with 3-phenyl-5-cyano-7-(trimethoxyphenylamino)-pyrazolo[3,4-c]âpyridine (A17) displaying the highest
affinity at both receptors with a long residence time of binding,
as determined using a NanoBRET-based assay. Two binding orientations
of A17 produce stable complexes inside the orthosteric
binding area of A1R in molecular dynamics (MD) simulations,
and we selected the most plausible orientation based on the agreement
with alanine mutagenesis supported by affinity experiments. Interestingly,
for drug design purposes, the mutation of L2506.51 to alanine
increased the binding affinity of A17 at A1R. We explored the structureâactivity relationships against
A1R using alchemical binding free energy calculations with
the thermodynamic integration coupled with the MD simulation (TI/MD)
method, applied on the whole G-protein-coupled receptorâmembrane
system, which showed a good agreement (r = 0.73)
between calculated and experimental relative binding free energies
Discovery of Novel Adenosine Receptor Antagonists through a Combined Structure- and Ligand-Based Approach Followed by Molecular Dynamics Investigation of Ligand Binding Mode
An intense effort is made by pharmaceutical
and academic research
laboratories to identify and develop selective antagonists for each
adenosine receptor (AR) subtype as potential clinical candidates for
âsoftâ treatment of various diseases. Crystal structures
of subtypes A<sub>2A</sub> and A<sub>1</sub>ARs offer exciting opportunities
for structure-based drug design. In the first part of the present
work, Maybridge HitFinder library of 14400 compounds was utilized
to apply a combination of structure-based against the crystal structure
of A<sub>2A</sub>AR and ligand-based methodologies. The docking poses
were rescored by CHARMM energy minimization and calculation of the
desolvation energy using PoissonâBoltzmann equation electrostatics.
Out of the eight selected and tested compounds, five were found positive
hits (63% success). Although the project was initially focused on
targeting A<sub>2A</sub>AR, the identified antagonists exhibited low
micromolar or micromolar affinity against A<sub>2A</sub>/A<sub>3</sub>, ARs, or A<sub>3</sub>AR, respectively. Based on these results,
19 compounds characterized by novel chemotypes were purchased and
tested. Sixteen of them were identified as AR antagonists with affinity
toward combinations of the AR family isoforms (A<sub>2A</sub>/A<sub>3</sub>, A<sub>1</sub>/A<sub>3</sub>, A<sub>1</sub>/A<sub>2A</sub>/A<sub>3</sub>, and A<sub>3</sub>). The second part of this work
involves the performance of hundreds of molecular dynamics (MD) simulations
of complexes between the ARs and a total of 27 ligands to resolve
the binding interactions of the active compounds, which were not achieved
by docking calculations alone. This computational work allowed the
prediction of stable and unstable complexes which agree with the experimental
results of potent and inactive compounds, respectively. Of particular
interest is that the 2-amino-thiophene-3-carboxamides, 3-acylamino-5-aryl-thiophene-2-carboxamides,
and carbonyloxycarboximidamide derivatives were found to be selective
and possess a micromolar to low micromolar affinity for the A<sub>3</sub> receptor
Dual A1/A3 Adenosine Receptor Antagonists: Binding Kinetics and StructureâActivity Relationship Studies Using Mutagenesis and Alchemical Binding Free Energy Calculations
Drugs targeting adenosine receptors
(AR) can provide treatment
for diseases. We report the identification of 7-(phenylamino)-pyrazolo[3,4-c]âpyridines L2âL10, A15, and A17 as low-micromolar to low-nanomolar A1R/A3R dual antagonists, with 3-phenyl-5-cyano-7-(trimethoxyphenylamino)-pyrazolo[3,4-c]âpyridine (A17) displaying the highest
affinity at both receptors with a long residence time of binding,
as determined using a NanoBRET-based assay. Two binding orientations
of A17 produce stable complexes inside the orthosteric
binding area of A1R in molecular dynamics (MD) simulations,
and we selected the most plausible orientation based on the agreement
with alanine mutagenesis supported by affinity experiments. Interestingly,
for drug design purposes, the mutation of L2506.51 to alanine
increased the binding affinity of A17 at A1R. We explored the structureâactivity relationships against
A1R using alchemical binding free energy calculations with
the thermodynamic integration coupled with the MD simulation (TI/MD)
method, applied on the whole G-protein-coupled receptorâmembrane
system, which showed a good agreement (r = 0.73)
between calculated and experimental relative binding free energies
Dual A1/A3 Adenosine Receptor Antagonists: Binding Kinetics and StructureâActivity Relationship Studies Using Mutagenesis and Alchemical Binding Free Energy Calculations
Drugs targeting adenosine receptors
(AR) can provide treatment
for diseases. We report the identification of 7-(phenylamino)-pyrazolo[3,4-c]âpyridines L2âL10, A15, and A17 as low-micromolar to low-nanomolar A1R/A3R dual antagonists, with 3-phenyl-5-cyano-7-(trimethoxyphenylamino)-pyrazolo[3,4-c]âpyridine (A17) displaying the highest
affinity at both receptors with a long residence time of binding,
as determined using a NanoBRET-based assay. Two binding orientations
of A17 produce stable complexes inside the orthosteric
binding area of A1R in molecular dynamics (MD) simulations,
and we selected the most plausible orientation based on the agreement
with alanine mutagenesis supported by affinity experiments. Interestingly,
for drug design purposes, the mutation of L2506.51 to alanine
increased the binding affinity of A17 at A1R. We explored the structureâactivity relationships against
A1R using alchemical binding free energy calculations with
the thermodynamic integration coupled with the MD simulation (TI/MD)
method, applied on the whole G-protein-coupled receptorâmembrane
system, which showed a good agreement (r = 0.73)
between calculated and experimental relative binding free energies
Dual A1/A3 Adenosine Receptor Antagonists: Binding Kinetics and StructureâActivity Relationship Studies Using Mutagenesis and Alchemical Binding Free Energy Calculations
Drugs targeting adenosine receptors
(AR) can provide treatment
for diseases. We report the identification of 7-(phenylamino)-pyrazolo[3,4-c]âpyridines L2âL10, A15, and A17 as low-micromolar to low-nanomolar A1R/A3R dual antagonists, with 3-phenyl-5-cyano-7-(trimethoxyphenylamino)-pyrazolo[3,4-c]âpyridine (A17) displaying the highest
affinity at both receptors with a long residence time of binding,
as determined using a NanoBRET-based assay. Two binding orientations
of A17 produce stable complexes inside the orthosteric
binding area of A1R in molecular dynamics (MD) simulations,
and we selected the most plausible orientation based on the agreement
with alanine mutagenesis supported by affinity experiments. Interestingly,
for drug design purposes, the mutation of L2506.51 to alanine
increased the binding affinity of A17 at A1R. We explored the structureâactivity relationships against
A1R using alchemical binding free energy calculations with
the thermodynamic integration coupled with the MD simulation (TI/MD)
method, applied on the whole G-protein-coupled receptorâmembrane
system, which showed a good agreement (r = 0.73)
between calculated and experimental relative binding free energies
Dual A1/A3 Adenosine Receptor Antagonists: Binding Kinetics and StructureâActivity Relationship Studies Using Mutagenesis and Alchemical Binding Free Energy Calculations
Drugs targeting adenosine receptors
(AR) can provide treatment
for diseases. We report the identification of 7-(phenylamino)-pyrazolo[3,4-c]âpyridines L2âL10, A15, and A17 as low-micromolar to low-nanomolar A1R/A3R dual antagonists, with 3-phenyl-5-cyano-7-(trimethoxyphenylamino)-pyrazolo[3,4-c]âpyridine (A17) displaying the highest
affinity at both receptors with a long residence time of binding,
as determined using a NanoBRET-based assay. Two binding orientations
of A17 produce stable complexes inside the orthosteric
binding area of A1R in molecular dynamics (MD) simulations,
and we selected the most plausible orientation based on the agreement
with alanine mutagenesis supported by affinity experiments. Interestingly,
for drug design purposes, the mutation of L2506.51 to alanine
increased the binding affinity of A17 at A1R. We explored the structureâactivity relationships against
A1R using alchemical binding free energy calculations with
the thermodynamic integration coupled with the MD simulation (TI/MD)
method, applied on the whole G-protein-coupled receptorâmembrane
system, which showed a good agreement (r = 0.73)
between calculated and experimental relative binding free energies