Bradley L. Pentelute

A reagent binding to the cysteine site of an amino acid sequence (pi-clamp) to modify the protein's structure

Selecting a cellular site and modifying its characteristics to perform specific functions is one of the most complex studies done in chemistry. Typical modification techniques involved using a catalyst or reaction pairs to change a site of interest. Cysteine residues were used in modifying proteins via bioconjugation because they acted as natural catalysts, however they lacked the ability to target specific sites. Pentelute was inspired to create a new site-selecting approach by altering an amino acid's environment in a peptide sequence. Hence, Pentelute and his lab created an amino acid sequence consisting of phenylalanine, cysteine, proline, and phenylalanine, known as the pi-clamp, to selectively modify a cysteine site in proteins. Having being made from natural compounds, the pi-clamp reacts with a perfluoroaromatic reagent and the cysteine thiol site, thus causing an overall decrease in the reaction's activation energy. Additional advantages of this pi-clamping technique compared to non-natural methods include the clamp being of small size and being able to have direct interaction with the site. This new approach for modification of cells helped researchers target site-specific cells and label proteins without the use of enzymes, which makes the modification process more efficient. A significant use of this method has been applied through the successful killing of breast cancer cells.[6][7]

Moving peptide and protein therapeutics through the plasma membrane of cells has been made more efficient through the use of a platform made from the anthrax lethal toxin (PA/LFN), which arises from the bacterium Bacillus anthracis.[8] Pentelute's lab took more than two decades to develop this delivery vehicle. His studies of intracellular delivery help us understand the movements of proteins and to explore different biological functions within cells. Previous techniques to transport molecules through the plasma membrane of mammalian cells proved to be less effective and required higher concentrations of substance to be useful. When compared, the anthrax lethal toxin based delivery method was proved to transport proteins faster and more efficiently. Through the use of chemical ligation (NCL) and enzyme-mediated ligation using Sortase A (SrtA), non-native cargos that contain functionalities that don't naturally occur can be created that provide benefits such as increased stability to internal degradation of the cell, added use of affinity handles, and adjusted connective affinities to target molecules. These fusions also attach the resulting peptides to the N-terminus of the native lethal factor (LFN).[9][7]