PLoS One

Targeting Acetylcholinesterase: Identification of Chemical Leads by High Throughput Screening, Structure Determination and Molecular Modeling

Lotta Berg1, C. David Andersson1, Elisabet Artursson2, Andreas Hörnberg2, Anna-Karin Tunemalm2,
Anna Linusson1*, Fredrik Ekström2*

1 Department of Chemistry, Umea° University, Umea°, Sweden, 2 Swedish Defence Research Agency, CBRN Defence and Security, Umea°, Sweden

Abstract
Acetylcholinesterase (AChE) is an essential enzyme that terminates cholinergic transmission by rapid hydrolysis of the neurotransmitter acetylcholine. Compounds inhibiting this enzyme can be used (inter alia) to treat cholinergic deficiencies (e.g. in Alzheimer’s disease), but may also act as dangerous toxins (e.g. nerve agents such as sarin). Treatment of nerve agent poisoning involves use of antidotes, small molecules capable of reactivating AChE. We have screened a collection of organic molecules to assess their ability to inhibit the enzymatic activity of AChE, aiming to find lead compounds for further optimization leading to drugs with increased efficacy and/or decreased side effects. 124 inhibitors were discovered, with considerable chemical diversity regarding size, polarity, flexibility and charge distribution. An extensive structure determination campaign resulted in a set of crystal structures of protein-ligand complexes. Overall, the ligands have substantial interactions with the peripheral anionic site of AChE, and the majority form additional interactions with the catalytic site (CAS). Reproduction of the bioactive conformation of six of the ligands using molecular docking simulations required modification of the default parameter settings of the docking software. The results show that docking-assisted structure-based design of AChE inhibitors is challenging and requires crystallographic support to obtain reliable results, at least with currently available software. The complex formed between C5685 and Mus musculus AChE (C5685NmAChE) is a representative structure for the general binding mode of the determined structures. The CAS binding part of C5685 could not be structurally determined due to a disordered electron density map and the developed docking protocol was used to predict the binding modes of this part of the molecule. We believe that chemical modifications of our discovered inhibitors, biochemical and biophysical characterization, crystallography and computational chemistry provide a route to novel AChE inhibitors and reactivators.

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY

Small-Molecule Screening Using a Whole-Cell Viral Replication Reporter Gene Assay Identifies 2-{[2-(Benzoylamino)Benzoyl]Amino}-Benzoic Acid as a Novel Antiadenoviral Compound

Emma K. Andersson,1 Mårten Strand,1 Karin Edlund,1 Kristina Lindman,1 Per-Anders Enquist,3 Sara Spjut,2 Annika Allard,1 Mikael Elofsson,2,3 Ya-Fang Mei,1 and Göran Wadell1*

Department of Virology, Umeå University, Umeå, Sweden1
Department of Chemistry, Umeå University, Umeå, Sweden2
Laboratories for Chemical Biology Umeå, Department of Chemistry, Umeå University, Umeå, Sweden3

Adenovirus infections are widespread in society and are occasionally associated with severe, but rarely with life-threatening, disease in otherwise healthy individuals. In contrast, adenovirus infections present a real threat to immunocompromised individuals and can result in disseminated and fatal disease. The number of patients undergoing immunosuppressive therapy for solid organ or hematopoietic stem cell transplantation is steadily increasing, as is the number of AIDS patients, and this makes the problem of adenovirus infections even more urgent to solve. There is no formally approved treatment of adenovirus infections today, and existing antiviral agents evaluated for their antiadenoviral effect give inconsistent results. We have developed a whole cell-based assay for high-throughput screening of potential antiadenoviral compounds. The assay is unique in that it is based on a replication-competent adenovirus type 11p green fluorescent protein (GFP)-expressing vector (RCAd11pGFP). This allows measurement of fluorescence changes as a direct result of RCAd11pGFP genome expression. Using this assay, we have screened 9,800 commercially available small organic compounds. Initially, we observed approximately 400 compounds that inhibited adenovirus expression by >80%, but  only 24 were later confirmed as dose-dependent inhibitors of adenovirus. One compound in particular, 2-{[2-(benzoylamino benzoyl]amino}-benzoic acid, turned out to be a potent inhibitor of adenovirus replication.