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.

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.

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.

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.

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 A2A 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.

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.

T he recent progress in the determination of three-dimensional (3D) structures of biological ion channels holds great promise for obtaining a structure-based quantitative view of interactions between channels and ligands of biological and pharmacological importance. Notwithstanding the increasing number of ion channel structures that have been determined, 1À6 there are, however, still relatively few complexes with ligands, such as channel blockers, that have been described. 7,8 From a pharmaceutical viewpoint, several of the most relevant channels have also not been structurally characterized at the atomic level. In the K + channel field, a number of key structures have been obtained 1À6 that in some cases allow for relatively reliable homology modeling of related channels and their interactions with ligands. 1,3,5 Of particular interest among the human K + channels is the human ether-a-go-go-related gene (hERG) channel that is associated with both inherited and drug-induced long QT syndrome. 9,10 The latter problem, which is a side effect caused by blockade of hERG by various compounds, is a major obstacle for drug development and is presently receiving intense attention. 11 In the cardiac action potential, the hERG channel carries the rapid delayed rectifier (I Kr ) current, and its blockade leads to a prolongation of the QT interval, with severe risks for arrhythmias and sudden death. 12,13 Most compounds causing such blockade apparently become trapped in the relatively unspecific internal pore cavity of the channel, and it is therefore of major interest to investigate the binding properties of this cavity. Unfortunately, the pore-forming helices of hERG show only relatively weak homology to K + channels with known 3D structure and particularly with members of the Shaker-related family. 5,14 The short pore helix, the selectivity filter, and the innermost S6 helices lining the pore can, however, be confidently aligned on the basis of conservation of the filter region and a glycine hinge in S6. 15,16 The situation is more ambiguous for the S5 helices that pack against the S6 helices in the tetrameric structure, and several alignments of S5 against K + channels of known structure have been published. 15À18 We have recently reported an analysis of seven different alignments and 3D pore models utilizing conventional 3D structure quality validation methods as well as molecular dynamics (MD) simulations. From that work, one model (model 6 of ref 19) emerged as the most consistent, and it also provided a rationalization of the results from mutation scanning experiments. 20 Here, we investigate the performance of our best hERG pore model with respect to prediction of binding affinities for a series of sertindole analogues listed in Table 1. 21 Sertindole is an indolylpiperidine antipsychotic agent that has nanomolar affinities for dopamine D 2 , serotonin 5-HT 2 , and R 1 adrenergic ABSTRACT: The hERG potassium channel is of major pharmaceutical importance, and its blockade by various compounds, potentially causin...

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.