X-Ray Crystallography and Free Energy Calculations Reveal the Binding Mechanism of A2A Adenosine Receptor Antagonists.

We present a robust protocol based on iterations of free energy perturbation (FEP) calculations, chemical synthesis, biophysical mapping and X-ray crystallography to reveal the binding mode of an antagonist series to the A2A adenosine receptor (AR). Eight A2A AR binding site mutations from biophysical mapping experiments were initially analyzed with sidechain FEP simulations, performed on alternate binding modes. The results distinctively supported one binding mode, which was subsequently used to design new chromone derivatives. Their affinities for the A2A AR were experimentally determined and investigated through a cycle of ligand-FEP calculations, validating the binding orientation of the different chemical substituents proposed. Subsequent X-ray crystallography of the A2A AR with a low and a high affinity chromone derivative confirmed the predicted binding orientation. The new molecules and structures here reported were driven by free energy calculations, and provide new insights on antagonist binding to the A2A AR, an emerging target in immuno-oncology.

High-resolution structure of a BRICHOS domain and its implications for anti-amyloid chaperone activity on lung surfactant protein C.

BRICHOS domains are encoded in > 30 human genes, which are associated with cancer, neurodegeneration, and interstitial lung disease (ILD). The BRICHOS domain from lung surfactant protein C proprotein (proSP-C) is required for membrane insertion of SP-C and has anti-amyloid activity in vitro. Here, we report the 2.1 Å crystal structure of the human proSP-C BRICHOS domain, which, together with molecular dynamics simulations and hydrogen-deuterium exchange mass spectrometry, reveals how BRICHOS domains may mediate chaperone activity. Observation of amyloid deposits composed of mature SP-C in lung tissue samples from ILD patients with mutations in the BRICHOS domain or in its peptide-binding linker region supports the in vivo relevance of the proposed mechanism. The results indicate that ILD mutations interfering with proSP-C BRICHOS activity cause amyloid disease secondary to intramolecular chaperone malfunction.

Computer simulations of structure-activity relationships for HERG channel blockers.

The hERG potassium channel is of major pharmaceutical importance, and its blockade by various compounds, potentially causing serious cardiac side effects, is a major problem in drug development. Despite the large amounts of existing biochemical data on blockade of hERG by drugs and druglike compounds, relatively little is known regarding the structural basis of binding of blockers to the channel. Here, we have used a recently developed homology model of hERG to conduct molecular docking experiments with a series of channel blockers, followed by molecular dynamics simulations of the complexes and evaluation of binding free energies with the linear interaction energy method. The calculations yield a remarkably good agreement with experimental binding affinities and allow for a rationalization of three-dimensional structure-activity relationships in terms of a number of key interactions. Two main interaction regions of the channel are thus identified with implications for further mutagenesis experiments and design of new compounds.

Predicting Binding Free Energies of PDE2 Inhibitors. The Difficulties of Protein Conformation.

A congeneric series of 21 phosphodiesterase 2 (PDE2) inhibitors are reported. Crystal structures show how the molecules can occupy a 'top-pocket' of the active site. Molecules with small substituents do not enter the pocket, a critical leucine (Leu770) is closed and water molecules are present. Large substituents enter the pocket, opening the Leu770 conformation and displacing the waters. We also report an X-ray structure revealing a new conformation of the PDE2 active site domain. The relative binding affinities of these compounds were studied with free energy perturbation (FEP) methods and it represents an attractive real-world test case. In general, the calculations could predict the energy of small-to-small, or large-to-large molecule perturbations. However, accurately capturing the transition from small-to-large proved challenging. Only when using alternative protein conformations did results improve. The new X-ray structure, along with a modelled dimer, conferred stability to the catalytic domain during the FEP molecular dynamics (MD) simulations, increasing the convergence and thereby improving the prediction of ΔΔG of binding for some small-to-large transitions. In summary, we found the most significant improvement in results when using different protein structures, and this data set is useful for future free energy validation studies.

Acylguanidine Beta Secretase 1 Inhibitors: A Combined Experimental and Free Energy Perturbation Study.

A series of acylguanidine beta secretase 1 (BACE1) inhibitors with modified scaffold and P3 pocket substituent was synthesized and studied with free energy perturbation (FEP) calculations. The resulting molecules showed potencies in enzymatic BACE1 inhibition assays up to 1 nM. The correlation between the predicted activity from the FEP calculations and the experimental activity was good for the P3 pocket substituents. The average mean unsigned error (MUE) between prediction and experiment was 0.68 ± 0.17 kcal/mol for the default 5 ns lambda window simulation time improving to 0.35 ± 0.13 kcal/mol for 40 ns. FEP calculations for the P2' pocket substituents on the same acylguanidine scaffold also showed good agreement with experiment and the results remained stable with repeated simulations and increased simulation time. It proved more difficult to use FEP calculations to study the scaffold modification from increasing 5 to 6 and 7 membered-rings. Although prediction and experiment were in agreement for short 2 ns simulations, as the simulation time increased the results diverged. This was improved by the use of a newly developed "Core Hopping FEP+" approach, which also showed improved stability in repeat calculations. The origins of these differences along with the value of repeat and longer simulation times are discussed. This work provides a further example of the use of FEP as a computational tool for molecular design.

Free energy calculations of A(2A) adenosine receptor mutation effects on agonist binding.

A general computational scheme to evaluate the effects of single point mutations on ligand binding is reported. This scheme is applied to characterize agonist binding to the A2A adenosine receptor, and is found to accurately explain how point mutations of different nature affect the binding affinity of a potent agonist.

Computer-aided design of GPCR ligands.

The recent availability of several GPCR crystal structures now contributes decisively to the perspective of structure-based ligand design. In this context, computational approaches are extremely helpful, particularly if properly integrated in drug design projects with cooperation between computational and medicinal chemistry teams. Here, we present the pipelines used in one such project, devoted to the design of novel potent and selective antagonists for the different adenosine receptors. The details of the computational strategies are described, and particular attention is given to explain how these procedures can effectively guide the synthesis of novel chemical entities.

Structural and energetic effects of A2A adenosine receptor mutations on agonist and antagonist binding.

To predict structural and energetic effects of point mutations on ligand binding is of considerable interest in biochemistry and pharmacology. This is not only useful in connection with site-directed mutagenesis experiments, but could also allow interpretation and prediction of individual responses to drug treatment. For G-protein coupled receptors systematic mutagenesis has provided the major part of functional data as structural information until recently has been very limited. For the pharmacologically important A(2A) adenosine receptor, extensive site-directed mutagenesis data on agonist and antagonist binding is available and crystal structures of both types of complexes have been determined. Here, we employ a computational strategy, based on molecular dynamics free energy simulations, to rationalize and interpret available alanine-scanning experiments for both agonist and antagonist binding to this receptor. These computer simulations show excellent agreement with the experimental data and, most importantly, reveal the molecular details behind the observed effects which are often not immediately evident from the crystal structures. The work further provides a distinct validation of the computational strategy used to assess effects of point-mutations on ligand binding. It also highlights the importance of considering not only protein-ligand interactions but also those mediated by solvent water molecules, in ligand design projects.

Novel cruzain inhibitors for the treatment of Chagas' disease.

The protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas' disease, affects millions of individuals and continues to be an important global health concern. The poor efficacy and unfavorable side effects of current treatments necessitate novel therapeutics. Cruzain, the major cysteine protease of T. cruzi, is one potential novel target. Recent advances in a class of vinyl sulfone inhibitors are encouraging; however, as most potential therapeutics fail in clinical trials and both disease progression and resistance call for combination therapy with several drugs, the identification of additional classes of inhibitory molecules is essential. Using an exhaustive virtual-screening and experimental validation approach, we identify several additional small-molecule cruzain inhibitors. Further optimization of these chemical scaffolds could lead to the development of novel drugs useful in the treatment of Chagas' disease.

Computational identification of uncharacterized cruzain binding sites.

Chagas disease, caused by the unicellular parasite Trypanosoma cruzi, claims 50,000 lives annually and is the leading cause of infectious myocarditis in the world. As current antichagastic therapies like nifurtimox and benznidazole are highly toxic, ineffective at parasite eradication, and subject to increasing resistance, novel therapeutics are urgently needed. Cruzain, the major cysteine protease of Trypanosoma cruzi, is one attractive drug target. In the current work, molecular dynamics simulations and a sequence alignment of a non-redundant, unbiased set of peptidase C1 family members are used to identify uncharacterized cruzain binding sites. The two sites identified may serve as targets for future pharmacological intervention.