Journal of Biomolecular Screening

Pawel Linke, Kwame Amaning, Melanie Maschberger, Francois Vallee, Valerie Steier, Philipp Baaske, Stefan Duhr, Dennis Breitsprecher, Alexey Rak

Abstract
Fragment-based lead discovery has proved to be an effective alternative to high-throughput screenings in identifying chemical matter that can be developed into robust lead compounds. The search for optimal combinations of biophysical techniques that can correctly and efficiently identify and quantify binding can be challenging due to the physicochemical properties of fragments. In order to minimize the time and costs of screening, optimal combinations of biophysical techniques with maximal information content, sensitivity, and robustness are needed. Here we describe an approach utilizing automated microscale thermophoresis (MST) affinity screening to identify fragments active against MEK1 kinase. MST identified multiple hits that were confirmed by X-ray crystallography but not detected by orthogonal methods. Furthermore, MST also provided information about ligand-induced aggregation and protein denaturation. The technique delivered a large number of binders while reducing experimentation time and sample consumption, demonstrating the potential of MST to execute and maximize the efficacy of fragment screening campaigns.

Keywords
Monolith NT.Automated, Protein-Fragment Interaction, Screening, Mulitple Component Interactions, Biomolecular Interaction

ACS Chemical Biology

Zhizhou Fang, Jeffrey R. Simard, Dennis Plenker, Hoang D. Nguyen, Trang Phan, Patrik Wolle, Stefan Baumeister, Daniel Rauh

Abstract

In addition to the catalytically active kinase domain, most kinases feature regulatory domains that govern their activity. Modulating and interfering with these interdomain interactions presents a major opportunity for understanding biological systems and developing novel therapeutics. Therefore, small molecule inhibitors that target these interactions through an allosteric mode of action have high intrinsic selectivity, as these interactions are often unique to a single kinase or kinase family. Here we report the development of iFLiK (interface-Fluorescent Labels in Kinases), a fluorescence-based assay that can monitor such interdomain interactions. Using iFLiK, we have demonstrated selective detection of allosteric Akt inhibitors that induce an inactive closed conformation unique to Akt. This methodology easily distinguished small molecule allosteric inhibitors from classic ATP-competitive inhibitors. Screening an in-house compound library with iFLiK, we were able to identify novel compounds with a scaffold that has not been previously described for allosteric Akt inhibitors.

Keywords

Monolith NT.115; Protein-Small Molecule; Biomolecular Interaction

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Nature

Xiaoli Xiong, Peter J. Coombs, Stephen R. Martin, Junfeng Liu, Haixia Xiao, John W. McCauley, Kathrin Locher, Philip A. Walker, Patrick J. Collins, Yoshihiro Kawaoka, John J. Skehel & Steven J. Gamblin

Abstract

Cell-surface-receptor binding by influenza viruses is a key determinant of their transmissibility, both from avian and animal species to humans as well as from human to human. Highly pathogenic avian H5N1 viruses that are a threat to public health have been observed to acquire affinity for human receptors, and transmissible-mutant-selection experiments have identified a virus that is transmissible in ferrets1, 2, 3, the generally accepted experimental model for influenza in humans. Here, our quantitative biophysical measurements of the receptor-binding properties of haemagglutinin (HA) from the transmissible mutant indicate a small increase in affinity for human receptor and a marked decrease in affinity for avian receptor. From analysis of virus and HA binding data we have derived an algorithm that predicts virus avidity from the affinity of individual HA-receptor interactions. It reveals that the transmissible-mutant virus has a 200-fold preference for binding human over avian receptors. The crystal structure of the transmissible-mutant HA in complex with receptor analogues shows that it has acquired the ability to bind human receptor in the same folded-back conformation as seen for HA from the 1918, 1957 (ref. 4), 1968 (ref. 5) and 2009 (ref. 6) pandemic viruses. This binding mode is substantially different from that by which non-transmissible wild-type H5 virus HA binds human receptor. The structure of the complex also explains how the change in preference from avian to human receptors arises from the Gln226Leu substitution, which facilitates binding to human receptor but restricts binding to avian receptor. Both features probably contribute to the acquisition of transmissibility by this mutant virus.

Keywords

Animals; Birds/metabolism/virology; Chick Embryo; Crystallography, X-Ray; Ferrets/*virology; Hemagglutinin Glycoproteins; Influenza Virus/*chemistry/genetics/*metabolism; *Host Specificity; 
Humans; Influenza A Virus; H5N1 Subtype/chemistry/*genetics/*metabolism/pathogenicity; Models, Biological; Models, Molecular; Mutation; Orthomyxoviridae Infections/*transmission/*virology; Protein Conformation; Receptors, Virus/*metabolism; Species Specificity; Monolith NT.115; Protein-Protein