Use of “Tethering” for the Identification of a Small Molecule that Binds to a Dynamic Hot Spot on the Interleukin‐2 Surface

The modulation of protein±protein interactions by small organic molecules represents one of the most rewarding yet challenging topics of current research at the interface of organic chemistry and biochemistry. Since the biological function of most proteins depends on their interactions with other macromolecules, disruption or enhancement of these interactions by cell-permeable molecules provides a means of influencing protein function. Cell-permeable molecules that allow a given protein to be turned on or off with high temporal and spatial control are therefore desirable tools for the analysis of complex biological systems in basic research. However, the following difficulties need to be overcome: 1) protein±protein interfaces are significantly larger than the surface areas of small molecules, 2) many protein±protein interfaces lack obvious binding pockets for small molecules, and 3) mechanismbased or natural product-based lead structures rarely exist. A solution for the problem of size difference between small molecules and protein±protein interfaces was offered in 1995 by the group of J. Wells, who proposed the presence of TMhot spots∫ in protein±protein interfaces. Hot spots are subregions of protein±protein interfaces that contribute significantly to the overall free energy of binding between the proteins, and whose size is comparable to the surface area of drug-like molecules. Further research by Wells and other scientists recently provided additional evidence that the problem originating from the frequent absence of obvious binding pockets for small molecules in flat protein surfaces can be overcome. The articles by Wells, Braisted, and Oslob highlighted here point to an aspect of hot spots that encourages the initial screening of diverse chemical libraries: flexible protein surfaces. These articles describe the discovery of small-molecule inhibitors of the interactions between interleukin-2 (IL-2) and its receptor IL-2Ra, and elucidate the inhibitors' mechanisms of action. Compound 1, a micromolar inhibitor of the IL-2/IL-2Ra interaction, acts by binding to the IL2Ra-binding region of IL-2. This region of IL-2 had previously been defined by mutational studies, which analyzed the importance of individual amino acids for binding to IL-2Ra, and consists of a rigid and a flexible region. Efforts to optimize 1 by structure-based design and parallel synthesis led to novel lead structures 2 and 3 (Scheme 1), whose potency did not exceed the potency of the original inhibitor 1. X-ray analysis revealed that inhibitor 3 binds to the hot spot of the IL-2/IL-2Ra interaction in a similar manner to the parent compound 1. In order to identify more active inhibitors, a fragment-assembly method referred to as TMtethering∫ was applied. Tethering can identify low-affinity fragments that bind to a specific site of a protein. It involves generating protein mutants in which cysteine mutations are introduced at the perimeter of the protein region of interest. Subsequently, the mutant proteins are probed with disulfide-containing chemical libraries under conditions that facilitate thiol±disulfide exchange, and molecules that bind to the site near the cysteine mutation (even if the affinity is low) are captured by disulfide bonds. The identity of the small molecules covalently attached to the protein is then analyzed by mass spectrometry (Scheme 2).