Structural and dynamic insights into the energetics of activation loop rearrangement in FGFR1 kinase

Protein tyrosine kinases differ widely in their propensity to undergo rearrangements of the N-terminal Asp- Phe-Gly motif of the activation loop, with some, including FGFR1 kinase, appearing refractory to this so called ‘DFG flip’. Recent inhibitor-bound structures have unexpectedly revealed FGFR1 for the first time in a ‘DFG-out’ state. Here, we use conformationally-selective inhibitors as chemical probes for interrogation of the structural and dynamic features that appear to govern the DFG flip in FGFR1. Our detailed structural and biophysical insights identify contributions from altered dynamics in distal elements, including the ?H helix towards the outstanding stability of the DFG-out complex with the inhibitor ponatinib. We conclude that the ?C-?4 loop and ‘molecular brake’ regions together impose a high energy barrier for this conformational rearrangement, and that this may have significance for maintaining autoinhibition in the non-phosphorylated basal state of FGFR1

Exploiting Ligand-Protein Conjugates to Monitor Ligand-Receptor Interactions

We introduce three assays for analyzing ligand-receptor interactions based on the specific conjugation of ligands to SNAP-tag fusion proteins. Conjugation of ligands to different SNAP-tag fusions permits the validation of suspected interactions in cell extracts and fixed cells as well as the establishment of high-throughput assays. The different assays allow the analysis of strong and weak interactions. Conversion of ligands into SNAP-tag substrates thus provides access to a powerful toolbox for the analysis of their interactions with proteins

Exploring the Trypanosoma brucei Hsp83 Potential as a Target for Structure Guided Drug Design

Human African trypanosomiasis is a neglected parasitic disease that is fatal if untreated. The current drugs available to eliminate the causative agent Trypanosoma brucei have multiple liabilities, including toxicity, increasing problems due to treatment failure and limited efficacy. There are two approaches to discover novel antimicrobial drugs--whole-cell screening and target-based discovery. In the latter case, there is a need to identify and validate novel drug targets in Trypanosoma parasites. The heat shock proteins (Hsp), while best known as cancer targets with a number of drug candidates in clinical development, are a family of emerging targets for infectious diseases. In this paper, we report the exploration of T. brucei Hsp83--a homolog of human Hsp90--as a drug target using multiple biophysical and biochemical techniques. Our approach included the characterization of the chemical sensitivity of the parasitic chaperone against a library of known Hsp90 inhibitors by means of differential scanning fluorimetry (DSF). Several compounds identified by this screening procedure were further studied using isothermal titration calorimetry (ITC) and X-ray crystallography, as well as tested in parasite growth inhibitions assays. These experiments led us to the identification of a benzamide derivative compound capable of interacting with TbHsp83 more strongly than with its human homologs and structural rationalization of this selectivity. The results highlight the opportunities created by subtle structural differences to develop new series of compounds to selectively target the Trypanosoma brucei chaperone and effectively kill the sleeping sickness parasite

Characterisation of radioiodinated flavonoid derivatives for SPECT imaging of cerebral prion deposits

Prion diseases are fatal neurodegenerative diseases characterised by deposition of amyloid plaques containing abnormal prion protein aggregates (PrPSc). This study aimed to evaluate the potential of radioiodinated flavonoid derivatives for single photon emission computed tomography (SPECT) imaging of PrPSc. In vitro binding assays using recombinant mouse PrP (rMoPrP) aggregates revealed that the 4-dimethylamino-substituted styrylchromone derivative (SC-NMe2) had higher in vitro binding affinity (Kd = 24.5 nM) and capacity (Bmax = 36.3 pmol/nmol protein) than three other flavonoid derivatives (flavone, chalcone, and aurone). Fluorescent imaging using brain sections from mouse-adapted bovine spongiform encephalopathy (mBSE)-infected mice demonstrated that SC-NMe2 clearly labelled PrPSc-positive prion deposits in the mice brain. Two methoxy SC derivatives, SC-OMe and SC-(OMe)2, also showed high binding affinity for rMoPrP aggregates with Ki values of 20.8 and 26.6 nM, respectively. In vitro fluorescence and autoradiography experiments demonstrated high accumulation of [125I]SC-OMe and [125I]SC-(OMe)2 in prion deposit-rich regions of the mBSE-infected mouse brain. SPECT/computed tomography (CT) imaging and ex vivo autoradiography demonstrated that [123I]SC-OMe showed consistent brain distribution with the presence of PrPSc deposits in the mBSE-infected mice brain. In conclusion, [123I]SC-OMe appears a promising SPECT radioligand for monitoring prion deposit levels in the living brain

Exploiting bacterial DNA gyrase as a drug target: current state and perspectives

DNA gyrase is a type II topoisomerase that can introduce negative supercoils into DNA at the expense of ATP hydrolysis. It is essential in all bacteria but absent from higher eukaryotes, making it an attractive target for antibacterials. The fluoroquinolones are examples of very successful gyrase-targeted drugs, but the rise in bacterial resistance to these agents means that we not only need to seek new compounds, but also new modes of inhibition of this enzyme. We review known gyrase-specific drugs and toxins and assess the prospects for developing new antibacterials targeted to this enzyme