YSM Issue 93.4 Full Magazine

FOCUS
Biochemistry
postdoctoral fellow and the first author of
the study published in Science Signaling.
Alternatively, virtual screenings use
structural biological information to
create docking sites on a desired protein.
Then, simulations can dock millions of
compounds to test their interactions.
Both screening methods have their
benefits and drawbacks. Physical
screening requires a significant amount
of protein, an optimized experiment,
and is also more expensive and time
intensive than computational methods.
However, when you get a hit, you
know the compound is an inhibitor.
On the other hand, computational
screenings require researchers to have
an idea of the protein structure and
the structure of the sites where small
molecules can interact. After conducting
computational models, researchers are
required to eventually physically test
the compounds with the most promise.
A positive is not necessarily guaranteed
to be an inhibitor, and the highest hit
rates are still fairly low. With insufficient
information about docking sites on
MKP5 for computational screenings, the
research team opted to conduct physical
screenings. The screening tests revealed
that a molecule, denoted by Compound
1, showed promise in inhibiting MKP5.
The How’s and the Why’s
With initial screenings showing the
inhibitory properties of Compound 1,
Bennett’s team wanted to find out how
the molecule interacts with MKP5.
Understanding the molecular interactions
between MKP5 and Compound 1 required
acquiring a crystal structure—a repeated
lattice of stable protein interactions—
of the two interacting molecules, which
would help researchers determine the
structure of the proteins and their
interaction. “This was the first crystal
structure of an MKP in a complex with a
small molecule,” Gannam said. The lack of
previous procedures for crystallography
meant that the team had to test out the
MKP5-Compound 1 complex in solutions
of various combinations of buffers, salts
and precipitants. “It’s very idiosyncratic
and there are not many set rules to follow,”
Gannam said. Rounds and rounds of
screening for crystallization were required
to finally develop the crystal structure.
The structure revealed that Compound
1 fundamentally shifts the shape of
MKP5. Notably, a distinct allosteric site
on the protein shifts to interact with
Compound 1. These shifts cause the
volume of the active site to decrease by
eighteen percent. Analyzing the specific
residues which Compound 1 interacts
with also showed its selectivity for
MKP5 as opposed to other MKPs within
the molecule family. Specifically, the
research team showed that methionine
and threonine residues on MKP5’s
allosteric site were unique to it. Further
tests revealed that Compound 1 was less
effective at inhibiting a mutated MKP5
with altered methionine and threonine
residues. Thus, Compound 1 seems to
selectively bind to MKP5 due to these
two residues.
Moving to Cells
So far, research on Compound 1
had been conducted outside of the
cell. To make sure that Compound 1
behaves predictably within a biological
context, the team investigated the
effect of Compound 1 in mice cells.
Since MKP5 inhibits MAPK and
JNK, introducing Compound 1 would
inhibit MKP5, therefore increasing the
phosphorylation of MAPK and JNK. Not
only did Compound 1 increase MAPK
ABOUT THE AUTHOR
and JNK activities, it had no effect on
other kinases such as ERK1/2, which is
not regulated by MKP5. These results
showed that even in a cellular context,
Compound 1 seems to only inhibit
MKP5, displaying the specificity that is
crucial for viability as a drug.
What’s Next?
Discovering Compound 1 represents
the crucial first step to developing a
treatment for DMD. However, there
is still a long way to go to produce a
viable drug. “Essentially, we have a
drug development project to make a
compound that is ideally highly potent,
orally viable and fits the once-a-day pill
treatment,” Bennett said.
Compound 1 may also have applications
beyond DMD. Compound 1 targets the
pathway leading to tissue fibrosis or the
thickening of scarring on tissue. For
example, postoperative fibrosis is a type of
complication that occurs after surgeries,
involving excess tissue scarring as a result
of the surgery. “Fibrosis accounts for
forty-five percent of deaths worldwide in
various clinical presentations,” Bennett
said. These include cardiac, lung, liver,
and kidney fibrosis. Compound 1 can
potentially address these fibrosis diseases
and complications.
The path to a workable drug requires
meticulous and thorough work. It allows
for innovations like Compound 1 to have
the potential to help millions. ■
JENNY TAN
JENNY TAN is a sophomore in Saybrook majoring in Chemistry. She is from northern Virginia just
outside of Washington D.C. Outside of school, she likes baking and listening to music.
THE AUTHOR WOULD LIKE TO THANK Zachary Gannam and Anton Bennett for their time and
illuminating discussions about their research.
FURTHER READING
AM;, M. (n.d.). Loss of MKP-5 promotes myofiber survival by activating STAT3/Bcl-2 signaling during
regenerative myogenesis. Retrieved November 26, 2020, from https://pubmed.ncbi.nlm.nih.
gov/29047406/
Bennett, A. M. (2019, January 01). MKP5 in Dystrophic Muscle Disease. Retrieved November 26, 2020,
from https://grantome.com/grant/NIH/R01-AR066003-05
Gannam, Z. T., Min, K., Shillingford, S. R., Zhang, L., Herrington, J., Abriola, L., . . . Bennett, A. M. (2020).
An allosteric site on MKP5 reveals a strategy for small-molecule inhibition. Science Signaling, 13(646).
doi:10.1126/scisignal.aba3043
Zhang, W., & Liu, H. (n.d.). MAPK signal pathways in the regulation of cell proliferation in mammalian
cells. Retrieved November 26, 2020, from https://www.nature.com/articles/7290105
18 Yale Scientific Magazine December 2020 www.yalescientific.org