Principal Research Interests
 Many
biological responses are triggered when small chemical
moieties bind to cavities or surfaces of proteins. Attention
typically focuses on small molecules binding to the main
functional site, which is termed the protein active site.
In other cases, binding of small molecules to external
sites, called allosteric sites, dramatically influences
the activity of the protein. Some well known allosteric
sites like those in hemoglobin, citrate synthase and glycogen
phosphorylase were discovered many years ago; however,
new allosteric sites like the one in caspase-7 (right,
red) are being uncovered serendipitously all the time.
We aim to understand and exploit allosteric regulation
by designing allosteric binding sites for alternative small
molecules. We redesign both known and novel allosteric
sites to be controlled by the small molecule of our choice. In
the figure at the right a novel allosteric site cavity
in caspase-7 (red) is occupied by a small molecule
we chose based on its size, solubility and exceptional
drug-like properties (yellow sticks). Redesigning this
novel allosteric site to bind to an alternative small molecule
will allow us to regulate caspase-7 activity independently
of all other caspases.
 Designed
allosteric proteins are of great utility because of their
exquisite innate specificity. Very often new small molecules
that interact with protein active or allosteric sites have
a broad specificity because identical sites exist on other
members of that family of proteins. The diagram at the
left illustrates the situation where all of the family
members have identically shaped active sites. Thus active-site
inhibitors typically interact with all the members of a
family of proteins. Designing allosteric sites to bind
to the small molecule we choose allows that small molecule
to serve as an exquisitely specific switch to turn on or
turn off activity of the designed allosteric protein independent
of any related family members. Because none of the wild-type  family
members contain the designed site, they have no affinity
for the designed inhibitor, and thus are unaffected by
the presence of the novel inhibitor.
We use computational protein design methods to engineer
new allosteric sites, molecular biology to introduce these
sites into proteins of interest, biochemical and cell-based
assays to determine the inhibitory ability of our designed
small molecule-protein pair, and x-ray crystallography
to determine that our designed mode of binding occurs.
Once we are convinced that we have designed an optimal
pair, we use the small molecule as a chemical genetics
agent in any relevant biological situation.
In principle, designed allosteric sites
can be engineered into proteins in any family in any biological
system. Apoptosis (programmed cell death) is a fascinating
biological system of great therapeutic importance. Blocking
apoptosis is a useful way of treating cells that are routed
to death following ischemic events such as stroke or heart
attack or in long-term diseases such as Alzheimer’s
Disease. Activating apoptosis is the best known way to induce
proliferating cancer cells to die, and many of the best known
cancer therapies work via this mechanism. While a great deal
of research effort has focused on the regulation of apoptosis,
many important details remain to be discovered. Designed
allosteric sites will allow us to understand the precise
roles of caspases, phosphatases and other large families
of proteins involved in apoptosis. This understanding will
lead us to discover new drug targets and give us new methods
of controlling apoptosis and treating disease.
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| Representative Publications
“A Link Means a Lot: Disulfide Tethering in Structure-Based Drug Design,” J.A. Hardy, Computational Approaches to Structure Based Drug Design, publishers Royal Society of Chemistry, 318-347 (2007).
“Discovery and Exploitation of Allosteric Sites for Control of Protein Function,” J.A. Hardy, Nanomedicine 2(4), 291 (2006).
Hardy, J. A. and Wells, J. “Searching for Allosteric
Sites in Enzymes.” Curr. Opin. Str. Biol. 14(6), 706-715
(2004).
Hardy, J. A., Lam, J., Nguyen, J. T.,
O’Brien, T.
and Wells, J. “Discovery of an allosteric site in caspases.” Proc.
Natl. Acad. Sci. U S A. 101(34), 12461-6 (2004).
Erlanson, D.A., Hansen, S.K., Hardy,
J. A., Lam, J., O’Brien,
T. “Methods for Identifying Allosteric Sites.” World
Patent WO 03/087051 A2, April 11, 2003.
Cicero, M. P., Hubl, S. T., Harrison, C.
J., Littlefield, O., Hardy, J. A., Nelson, H. C. “The wing in yeast heat shock
transcription factor (HSF) DNA-binding domain is required for
full activity.” Nucleic Acids Res., 29(8), 1715-23 (2001). Hardy, J. A. and Nelson, H. C. M. “Proline in an α-helical kink
is required for folding kinetics but not for kinked structure, function or
stability of heat shock transcription factor” Protein Science, 9(11),
2128-2141, (2000).
Hardy, J. A., Walsh, S. T. R., and Nelson,
H. C. M. “Role of an α-Helical
Bulge in the Yeast Heat Shock Transcription Factor” J. Mol. Biol., 295(3),
393-409 (2000).
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