Broad based label-free systems identify protein stabilizers at physiological or near physiological conditions that specifically inhibit Bacterial Toxin or Viral entry into cells.
A wide array of bacterial protein toxins (e.g. tetanus, anthrax, C. difficile, C. botulinum toxins) and viral coat proteins (e.g. from Zika, Influenza, Ebola) have to undergo large-scale acid-dependent protein transformations on and through endosome membranes prior to cell entry. Specifically targeting these protein transformations at their crucial entry points on the endosomal membrane enables one to combat acute infections using unique first-response and preventative prophylactic measures. Up until now, specific acidic entry reactions have not been targeted because these traversing membrane proteins rapidly aggregate in current screening systems. KU researchers have constructed numerous novel biosensor platform technologies capable of obtaining binding affinities and stability measurements simultaneously while avoiding acidic aggregation. Compounds are rationally and rapidly identified using newly constructed GPU based in silico computational identification algorithms [(through-put times are around 20 million compounds within 15 min computed at the KU Computational Chemistry Core. The root compounds identified by these combined methodologies are provided to and or are optimized by Medicinal Chemistry collaborators at KU and/or through a CDA agreement with Frontier Scientific Inc. (~20 million compound library on hand)]. The biosensor target platform construction uses either purified toxin endosome assembly components or biosensor coupled toxins that are evaluated using patent pending 1) denaturant-pulse chaperone-based stability and 2) toxin assembly methodologies. In summary, both of these systems avoid the protein aggregation bottleneck that has plagued searches for stabilizers of these pH induced toxin or viral protein transitions.
Targeted drug screening platforms to rapidly identify stabilizing compounds (i.e. small molecules) to inhibit protein conformational changes for bacterial and viral soluble to membrane inserted systems.
How it works:
The basis for this highly automated method takes advantage of a truly unique ability control and target aggregation prone protein conformational changes. Specific toxin or viral orientation chemistries are used to recapitulate in vivo cellular endosomal assembly states on label free biosensor surfaces. This allows one to observe previously untargeted conformational changes and prevent these transitions from occurring, ultimately blunting bacterial toxin lethality and viral invasion.
One can directly determine candidate compounds that halt toxin and viral mechanisms of cell entry. This reduces off-target compounds (false positives), improves compound specificity and most importantly, can be rapidly and easily designed to accommodate any protein based cell entry system.
Why it is better:
These combined computational/stability assessment systems significantly accelerates lead compound discovery. This method is also more cost effective since high-throughput screening is avoided and rationally designed targeted compounds binding to unique dynamic sites may be less likely to fail.
Pharmacological chaperone identification for common diseases (e.g. p53 misfolding in cancer), common neurological misfolding diseases (e.g. Parkinson’s) or most importantly, rare protein misfolding diseases (e.g. cystic fibrosis variants (common/rare missense mutants), ALS, anti trypsin inhibitor non-smoker emphysema, von Willebrand diseases, Maple syrup Urine Disease)