Discovering Protein Ligands to Probe and Alter Function

There are four research areas in the Department of Pharmaceutical Chemistry. Discovering protein ligands to probe and alter function is a research challenge within chemical biology and medicinal chemistry.

The challenge

In addition to enzymes’ active sites, proteins have alternative (allosteric) sites where small molecules can bind and alter their activity by causing changes in their shapes (conformations). Researchers seek to tap this natural regulatory process to better understand the mechanisms underlying allostery, to modulate enzyme activity instead of competitively binding to active sites, and to more precisely parse the roles of different enzymes. Understanding allostery could ultimately allow for therapeutics that alter protein activity with greater subtlety and specificity.

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.

Schematic of disulfide trapping (tethering)

Schematic of disulfide trapping (tethering), as thiol(SH)-containing fragments bind with thiol group in target’s natural or engineered cysteine side chain to reveal allosteric site (round) near active site (square).

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.

A human caspase-7 enzyme. (Stick elements represent some of the enzyme’s active site side chains with elements color-coded including blue nitrogens and yellow sulfurs.) Molecular image made with UCSF Chimera developed by UCSF Resource for Biocomputing, Visualization, and Informatics.

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.


An inhibitor (spheres at center) discovered via disulfide trapping bound to allosteric site of a human caspase-1 enzyme. Orange spheres denote active sites 15 angstroms away.

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.


Fragments (activators and inhibitors) linked to allosteric site on a kinase protein via disulfide trapping. This figure shows the effect of small molecule modular binding on the positioning (arrows) of secondary structures (B and C helices), thus altering the conformation of active site residues.

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.