Trypanosoma brucei Glycogen Synthase Kinase-3, A Target for Anti-Trypanosomal Drug Development: A Public-Private Partnership to Identify Novel Leads

Over 60 million people in sub-Saharan Africa are at risk of infection with the parasite Trypanosoma brucei which causes Human African Trypanosomiasis (HAT), also known as sleeping sickness. The disease results in systemic and neurological disability to its victims. At present, only four drugs are available for treatment of HAT. However, these drugs are expensive, limited in efficacy and are severely toxic, hence the need to develop new therapies. Previously, the short TbruGSK-3 short has been validated as a potential target for developing new drugs against HAT. Because this enzyme has also been pursued as a drug target for other diseases, several inhibitors are available for screening against the parasite enzyme. Here we present the results of screening over 16,000 inhibitors of human GSK-3β (HsGSK-3) from the Pfizer compound collection against TbruGSK-3 short. The resulting active compounds were tested for selectivity versus HsGSK-3β and a panel of human kinases, as well as their ability to inhibit proliferation of the parasite in vitro. We have identified attractive compounds that now form potential starting points for drug discovery against HAT. This is an example of how a tripartite partnership involving pharmaceutical industries, academic institutions and non-government organisations such as WHO TDR, can stimulate research for neglected diseases

Broadband Multi-wavelength Properties of M87 during the 2017 Event Horizon Telescope Campaign

Abstract: In 2017, the Event Horizon Telescope (EHT) Collaboration succeeded in capturing the first direct image of the center of the M87 galaxy. The asymmetric ring morphology and size are consistent with theoretical expectations for a weakly accreting supermassive black hole of mass ∼6.5 × 109 M ⊙. The EHTC also partnered with several international facilities in space and on the ground, to arrange an extensive, quasi-simultaneous multi-wavelength campaign. This Letter presents the results and analysis of this campaign, as well as the multi-wavelength data as a legacy data repository. We captured M87 in a historically low state, and the core flux dominates over HST-1 at high energies, making it possible to combine core flux constraints with the more spatially precise very long baseline interferometry data. We present the most complete simultaneous multi-wavelength spectrum of the active nucleus to date, and discuss the complexity and caveats of combining data from different spatial scales into one broadband spectrum. We apply two heuristic, isotropic leptonic single-zone models to provide insight into the basic source properties, but conclude that a structured jet is necessary to explain M87’s spectrum. We can exclude that the simultaneous γ-ray emission is produced via inverse Compton emission in the same region producing the EHT mm-band emission, and further conclude that the γ-rays can only be produced in the inner jets (inward of HST-1) if there are strongly particle-dominated regions. Direct synchrotron emission from accelerated protons and secondaries cannot yet be excluded

Simultaneous Screening of Multiple Bacterial tRNA Synthetases Using an Escherichia coli S30-Based Transcription and Translation Assay

The search for novel antibiotics to combat the growing threat of resistance has led researchers to screen libraries with coupled transcription and translation systems. In these systems, a bacterial cell lysate supplies the proteins necessary for transcription and translation, a plasmid encoding a reporter protein is added as a template, and a complex mixture of amino acids and cofactors is added to supply building blocks and energy to the assay. Firefly luciferase is typically used as the reporter protein in high-throughput screens because the luminescent signal is strong and, since bacterial lysates contain no luciferase, the background is negligible. The typical coupled transcription and translation assay is sensitive to inhibitors of RNA polymerase and to compounds that bind tightly to the ribosome. We have found a way to increase the information content of the screen by making the assay more sensitive to inhibitors of tRNA synthetases. Restricting the concentration of amino acids added to the reaction mixture allows the simultaneous screening of multiple tRNA synthetase enzymes along with the classic transcription and translation targets. In addition, this assay can be used as a convenient way to determine if an antibacterial compound of unknown mechanism inhibits translation through inhibition of a tRNA synthetase, and to identify which synthetase is the target.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63335/1/adt.2007.061.pd

Synthesis and Structure-Activity Relationship Study of Potent Trypanocidal

this paper, we report a new series of mechanism-based smallmolecule inhibitors discovered during a collaborative screening effect, the thio semicarbazones. Peptidyl semicarbazones inhibit cysteine proteases through the formation of a reversible tetrahedral adduct by attack of the thiolate on the C-5 carbo

GSK-3 Enzyme, Antiparasitic and cytotoxicity testing (values in µM).

<p>Values are a mean of at least 2 replicates. NT  =  not tested due to limited compound availability.</p><p>*Due to limited compound availability, these compounds were only tested at a single concentration of 1 µM and showed >50% inhibition at this concentration.</p

Kinase selectivity screening.

<p>Compounds were screened in one of two kinase panels at a concentration of 10 µM. The degree of inhibition of each kinase is indicated by shading as follows: white <29% inhibition, light grey 30-49% inhibition, dark grey 50-69% inhibition, black >70% inhibition. Key to compounds: (1) 0181276, (2) CE-160042, (3) 0180532, (4) PF-4903528, (5) PF-4936572, (6) PF-4995633, (7) AG-24290, (8) PF-4279731, (9) CE-317112, (10) PF-1242377, (11) PF-744923, (12) PF-2368935, (13) PF-956933. NT  =  not tested.</p

Modelling of the binding-site residues with putative inhibitors.

<p>Compounds (orange) docked into the catalytic domain of the crystal structure of <i>Hs</i>GSK3 beta in their binding modes. A: CE-317112 shows preference for <i>Hs</i>GSK-3 beta. B: PF-4903528 shows preference for <i>Tbru</i>GSK-3 short. The residues that differ between human and <i>Tbru</i>GSK-3 short are shown in magenta, with only L132M (top centre of the image) directly lining the pocket. Images were created using the Pfizer molecule-modelling package MoViT.</p