Discovering Protein Ligands to Probe and Alter Function
Examples of our research and methods include
Site-directed chemistry via disulfide trapping
Fragment-based drug discovery screens pieces of small molecules for binding against protein targets, and then combines the best of these to generate drug leads. However, the advantage of this combinatorial testing can be offset by the challenge of detecting weaker bonding.
To counter this, department researchers developed a novel technique to enhance the efficiency of fragment-binding detection. This technique has also proven to be a prime tool for investigating allosteric sites and regulation.
Disulfide trapping, or tethering, takes advantage of the reversible covalent bond formed between the sulfur atoms of two thiol groups. A cysteine residue with a thiol side chain is found on or engineered into a target protein adjacent to the site of interest. Then fragments modified to include thiol groups are tested for binding via high-throughput screening. The disulfide bond holds the fragment in place, giving it the opportunity to bind to the selected site. A reagent with a free thiol group then competes for the disulfide bond, thus selecting for fragments with stronger affinity, which maintain their bond and are identified via mass spectrometry. Effective fragment binders at allosteric sites can be combined and optimized to create allosteric modulators and potential drug leads.
Allosteric regulation of caspases and protein kinases
Department researchers have used site-directed chemistry to identify allosteric regulators and investigate such regulation in two important types of enzymes: caspases and kinases.
The dozen types, or isoforms, of human caspases are all cysteine protease enzymes that cleave other proteins to induce vital biological processes such as apoptosis and inflammation. The former is the programmed self-destruction of cells that fails to occur in cancers and over-occurs in neurodegenerative disease, The latter is vital to innate immune function but destructive in inflammatory and autoimmune diseases.
Caspases are prime examples of a role for exploratory allosteric regulation. Their active sites are structurally very similar and their substrate specificities are virtually identical, so it is difficult to find small molecules that will selectively inhibit separate isoforms. Also, the molecules necessary to bind their highly charged active sites are typically not drug-like, since their electrostatics would prevent them from readily penetrating cell membranes. Taking a different tack, department scientists used site-directed chemistry to screen libraries of thousands of thiol-containing fragments against accessible cysteine residues to discover an allosteric site common to several caspase isoforms.
This research has discovered distinctive allosteric inhibitors, for apoptotic caspases-3 and -7 and for inflammatory caspases-1 and -5, which trap the enzyme into an inactive conformation, disabling its active site. Further analysis of this inhibition in caspase-1 revealed the intramolecular circuitry connecting the allosteric and active sites.
There are more than 500 protein kinases, which post-translationally modify up to 30 percent of human proteins by adding phosphate groups (phosphorylation), which regulates many biological pathways. But targeting allosteric sites of kinases has proven challenging.
Department scientists used disulfide trapping to discover a variety of fragments that can either activate or inhibit a particular kinase isoform at an allosteric site. They followed up with x-ray crystallography and biochemical analysis to analyze the allosteric mechanisms. This offered proof of concept for a new way to modulate kinases and study their roles, particularly in cell proliferative and apoptotic pathways.