Attempts to identify potential drugs that interfere
with the action of one particular enzyme linked to heart
disease and similar health problems led scientists at Johns
Hopkins to create a new tool and new experimental approach
that allow them to see multiple, real-time chemical
reactions in living cells. Their report on the work is
published July 21 in the journal ACS Chemical
Most current drug development operations test
chemicals on enzymes isolated from their normal environs
and then take further steps to see if the chemical can get
into the cell to do its work, and figure out how poisonous
the chemical is to a cell.
"Living cells are critical to our work because they
show us how and what is actually happening in a normal
context and time span when a chemical is added," said Jin
Zhang, an assistant professor of
pharmacology and molecular sciences in Johns Hopkins'
Institute for Basic Biomedical Sciences.
Testing chemicals on enzymes in living cells provides
the opportunity to find potential drugs that work in new
ways. For example, using living cells allows researchers to
"see" where chemicals do their work in the cell. Scientists
could then design new drugs to go to specific places within
cells to work more efficiently. Also, streamlining the
one-at-a-time approach offers the chance to study —
and rule out or in — many potentially useful
chemicals at once.
What Zhang's team developed is a biosensor and simple
testing procedure that tells if a particular enzyme called
PKA that acts like a "switch" is "on" or "off" in a living
cell. The group has been focused on trying to understand
and interfere with this "switch," because if the enzyme is
turned on at the wrong time or at the wrong place within
cells, it can lead to cells' misbehaving, which ultimately
can lead to heart disease.
In the course of their work, the team built a protein
biosensor that indicates if an enzyme located nearby is
turned on or off. The sensor is made from a protein that
glows, originally isolated from jellyfish. When PKA is
turned off, the biosensor glows blue. When PKA is turned on
and is physically close to a biosensor, PKA itself changes
the shape of the biosensor, causing it to glow green
Manipulating the sensor allows the researchers to
direct it to specific locales within cells. That allows the
researchers to see where in the cell the active enzyme is
located. So this PKA sensor not only indicates whether the
enzyme is on or off but also locates where PKA is being
turned on or off within the cell. "Proteins aren't spread
out evenly in cells," Zhang said, "but tend to cluster
together in order to do specific jobs, and we now can see
how different clusters are regulated differently."
When the researchers put their new sensor into living
mammalian cells growing in the lab, they were able to test
the effects of 160 different chemicals at once and, by
looking for green or blue glowing cells, see if any of
these chemicals could turn on or off the PKA enzyme.
Of the 160 chemicals tested, three caused cells to
turn on the switch, and two others caused cells to turn off
The 160 chemicals tested are from the Johns Hopkins
Clinical Compound Library, a collection of about 3,300
chemicals. Most of them are drugs already approved by the
U.S. Food and Drug Administration, while others are
approved by regulatory agencies in other countries or are
other clinically relevant chemicals.
"If we can find a new activity for a known drug, this
may lead to a new use or a new way of thinking about that
drug," said Zhang, who hopes to test the rest of the
chemicals in the collection soon for their ability to
interfere with the enzyme tested in this study. Finding a
drug that can tame this enzyme could lead to new treatments
for heart disease, diabetes, memory disorders and certain
cancers, for example.
Zhang says the "high throughput" potential of the
sensor may have wide-reaching applications that could be
adapted to testing various chemicals for their ability to
interfere with other enzymes related to PKA — which
as a family are known as kinases — that are widely
implicated in diseases and an emerging class of drug
The researchers were funded by the American Heart
Association and the National Institutes of Health.
Authors on this paper are Michael Allen, Lisa
DiPilato, Meghdad Rahdar, Yunzhao Ren, Curtis Chong, Jun
Liu and Zhang, all of Johns Hopkins.