Cisbio eBook - 14

Trends in Protein-Protein Interactions Research | Targeting Protein-Protein Interactions Yields a Cornucopia of New Therapies

The OncoPPi uncovered hundreds of previously
unknown interactions stemming from important
"protein interaction hubs," including a connection
between MYC (a famously intractable oncogene)
and nuclear receptor binding SET domain protein
3 (NSD3; an epigenetic modulator).
To search for compounds that disrupt this interaction, Yuhong Du, PhD, associate professor
of pharmacology and associate director of
the Emory Chemical Biology Discovery Center
(ECBDC) for assay development and HTS, Emory
University, and her colleagues developed an
"ultrahigh-throughput" screening assay using
time-resolved fluorescence resonance energy
transfer (TR-FRET). The TR-FRET uses distancedependent energy transfer measurements to
detect whether two proteins are interacting. One
protein is labeled with a donor fluorophore, the
other with an acceptor, and when the two proteins
get close enough, the donor emits a photon that
is absorbed by the acceptor. The acceptor then
emits its own photon at a specific wavelength. If
that glow is interrupted, that means the protein-
protein interaction has been inhibited.
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After many rounds of optimization, Du's team
devised a labeling configuration that generated
robust signals, allowing for miniaturization to
5 microliters per sample in 1536-well plates. "If
we miniaturize to a 5-microliter assay, we can
decrease the cost by more than half, so that
makes screening a large number of compounds
feasible," Du says. Further simplifying the process,
the assay uses cell lysate rather than purified
protein, a move that both saves labor and
better preserves the native cellular environment
of the PPi.
The pilot screen of 2000 compounds identified 1
compound, validated with GST pull-down assay,
that reliably interfered with NDS3-MYC interaction. The molecule, TF-3, has been previously
reported to inhibit tumorigenesis by reducing
MYC expression. "No MYC inhibitor has advanced
to the clinic yet," Du points out. "NDS3 is a very
interesting target, it's an epigenetic modulator.
This epigenetic regulator can interact with MYC
and impact MYC functions, so it might provide
us with a potential new mechanism for how MYC
drives tumorigenesis."

Untangling ALS
Similar strategies are afoot in the realm of neurodegenerative disease. A team of researchers led
by Hidenori Ichijo, PhD, in the Laboratory of Cell
Signaling at the University of Tokyo, used TR-FRET
to find small molecules that inhibit the interaction between two proteins, SOD1 and Derlin-1.
Inhibiting this interaction, they found, staves off
symptoms of ALS (amyotrophic lateral sclerosis)
in mice.
Mutations in SOD1 account for a significant
fraction of ALS cases, apparently due to toxic
gain-of-function activity. Ten years ago, Ichijo
and colleagues first identified a link between
ALS and the interaction between mutant SOD1
and Derlin-1. Endoplasmic reticulum (ER) stress
contributes to ALS by activating the ASK1-dependent apoptosis pathway, and activation of that
pathway is associated with mutant SOD1. In that
study, Ichijo's group showed that by disrupting
the interaction between Derlin-1 and mutant
SOD1, they could halt ER stress, activation of the
ASK1 pathway, and motor neuron degeneration.

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