TY  - JOUR
A1  - Wu, Yu
A1  - Pons, Valérie
A1  - Goudet, Amélie
A1  - Panigai, Laetitia
A1  - Fischer, Annette
A1  - Herweg, Jo-Ana
A1  - Kali, Sabrina
A1  - Davey, Robert A.
A1  - Laporte, Jérôme
A1  - Bouclier, Céline
A1  - Yousfi, Rahima
A1  - Aubenque, Céline
A1  - Merer, Goulven
A1  - Gobbo, Emilie
A1  - Lopez, Roman
A1  - Gillet, Cynthia
A1  - Cojean, Sandrine
A1  - Popoff, Michel R.
A1  - Clayette, Pascal
A1  - Le Grand, Roger
A1  - Boulogne, Claire
A1  - Tordo, Noël
A1  - Lemichez, Emmanuel
A1  - Loiseau, Philippe M.
A1  - Rudel, Thomas
A1  - Sauvaire, Didier
A1  - Cintrat, Jean-Christophe
A1  - Gillet, Daniel
A1  - Barbier, Julien
T1  - ABMA, a small molecule that inhibits intracellular toxins and pathogens by interfering with late endosomal compartments
JF  - Scientific Reports
N2  - Intracellular pathogenic microorganisms and toxins exploit host cell mechanisms to enter, exert their deleterious effects as well as hijack host nutrition for their development. A potential approach to treat multiple pathogen infections and that should not induce drug resistance is the use of small molecules that target host components. We identifed the compound 1-adamantyl (5-bromo-2-methoxybenzyl) amine (ABMA) from a cell-based high throughput screening for its capacity to protect human cells and mice against ricin toxin without toxicity. This compound efciently protects cells against various toxins and pathogens including viruses, intracellular bacteria and parasite. ABMA provokes Rab7-positive late endosomal compartment accumulation in mammalian cells without affecting other organelles (early endosomes, lysosomes, the Golgi apparatus, the endoplasmic reticulum or the nucleus). As the mechanism of action of ABMA is restricted to host-endosomal compartments, it reduces cell infection by pathogens that depend on this pathway to invade cells. ABMA may represent a novel class of broad-spectrum compounds with therapeutic potential against diverse severe infectious diseases.
KW  - biology
KW  - antimicrobials
KW  - high-throughput screening
KW  - infectious diseases
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-173170
VL  - 7
ER  - 
TY  - JOUR
A1  - Wandrey, Georg
A1  - Wurzel, Joel
A1  - Hoffmann, Kyra
A1  - Ladner, Tobias
A1  - Büchs, Jochen
A1  - Meinel, Lorenz
A1  - Lühmann, Tessa
T1  - Probing unnatural amino acid integration into enhanced green fluorescent protein by genetic code expansion with a high-throughput screening platform
JF  - Journal of Biological Engineering
N2  - Background

Genetic code expansion has developed into an elegant tool to incorporate unnatural amino acids (uAA) at predefined sites in the protein backbone in response to an amber codon. However, recombinant production and yield of uAA comprising proteins are challenged due to the additional translation machinery required for uAA incorporation.

Results

We developed a microtiter plate-based high-throughput monitoring system (HTMS) to study and optimize uAA integration in the model protein enhanced green fluorescence protein (eGFP). Two uAA, propargyl-L-lysine (Plk) and (S)-2-amino-6-((2-azidoethoxy) carbonylamino) hexanoic acid (Alk), were incorporated at the same site into eGFP co-expressing the native PylRS/tRNAPyl CUA pair originating from Methanosarcina barkeri in E. coli. The site-specific uAA functionalization was confirmed by LC-MS/MS analysis. uAA-eGFP production and biomass growth in parallelized E. coli cultivations was correlated to (i) uAA concentration and the (ii) time of uAA addition to the expression medium as well as to induction parameters including the (iii) time and (iv) amount of IPTG supplementation. The online measurements of the HTMS were consolidated by end point-detection using standard enzyme-linked immunosorbent procedures.

Conclusion

The developed HTMS is powerful tool for parallelized and rapid screening. In light of uAA integration, future applications may include parallelized screening of different PylRS/tRNAPyl CUA pairs as well as further optimization of culture conditions.
KW  - protein engineering
KW  - amber codon suppression
KW  - online monitoring system
KW  - high-throughput screening
KW  - unnatural amino acid
KW  - bio-orthogonal chemistry
Y1  - 2016
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166304
VL  - 10
IS  - 11
ER  -