Identifying
target
proteins
for
bioactive
molecules
is
essential
understanding
their
mechanisms,
developing
improved
derivatives,
and
minimizing
off-target
effects.
Despite
advances
in
identification
(target-ID)
technologies,
significant
challenges
remain,
impeding
drug
development.
Most
target-ID
methods
use
cell
lysates,
but
maintaining
an
intact
cellular
context
vital
capturing
specific
drug–protein
interactions,
such
as
those
with
transient
protein
complexes
membrane-associated
proteins.
To
address
these
limitations,
we
developed
POST-IT
(Pup-On-target
Small
molecule
Target
Identification
Technology),
a
non-diffusive
proximity
tagging
system
live
cells,
orthogonal
to
the
eukaryotic
system.
utilizes
engineered
fusion
of
proteasomal
accessory
factor
A
HaloTag
transfer
Pup
proximal
upon
directly
binding
small
molecule.
After
optimization
eliminate
self-pupylation
polypupylation,
minimize
depupylation,
optimize
chemical
linkers,
successfully
identified
known
targets
discovered
new
binder,
SEPHS2,
dasatinib,
VPS37C
hydroxychloroquine,
enhancing
our
drugs’
mechanisms
action.
Furthermore,
demonstrated
application
zebrafish
embryos,
highlighting
its
potential
broad
biological
research
Identifying
target
proteins
for
bioactive
molecules
is
essential
understanding
their
mechanisms,
developing
improved
derivatives,
and
minimizing
off-target
effects.
Despite
advances
in
identification
(target-ID)
technologies,
significant
challenges
remain,
impeding
drug
development.
Most
target-ID
methods
use
cell
lysates,
but
maintaining
an
intact
cellular
context
vital
capturing
specific
drug-protein
interactions,
such
as
those
with
transient
protein
complexes
membrane-associated
proteins.
To
address
these
limitations,
we
developed
POST-IT
(Pup-On-target
Small
molecule
Target
Identification
Technology),
a
non-diffusive
proximity
tagging
system
live
cells,
orthogonal
to
the
eukaryotic
system.
utilizes
engineered
fusion
of
proteasomal
accessory
factor
A
(PafA)
HaloTag
transfer
Pup
proximal
upon
directly
binding
small
molecule.
After
optimization
eliminate
self-pupylation
polypupylation,
minimize
depupylation,
optimize
chemical
linkers,
successfully
identified
known
targets
discovered
new
binder,
SEPHS2,
dasatinib,
VPS37C
hydroxychloroquine,
enhancing
our
drugs’
mechanisms
action.
Furthermore,
demonstrated
application
zebrafish
embryos,
highlighting
its
potential
broad
biological
research
Identifying
target
proteins
for
bioactive
molecules
is
essential
understanding
their
mechanisms,
developing
improved
derivatives,
and
minimizing
off-target
effects.
Despite
advances
in
identification
(target-ID)
technologies,
significant
challenges
remain,
impeding
drug
development.
Most
target-ID
methods
use
cell
lysates,
but
maintaining
an
intact
cellular
context
vital
capturing
specific
drug–protein
interactions,
such
as
those
with
transient
protein
complexes
membrane-associated
proteins.
To
address
these
limitations,
we
developed
POST-IT
(Pup-On-target
Small
molecule
Target
Identification
Technology),
a
non-diffusive
proximity
tagging
system
live
cells,
orthogonal
to
the
eukaryotic
system.
utilizes
engineered
fusion
of
proteasomal
accessory
factor
A
HaloTag
transfer
Pup
proximal
upon
directly
binding
small
molecule.
After
optimization
eliminate
self-pupylation
polypupylation,
minimize
depupylation,
optimize
chemical
linkers,
successfully
identified
known
targets
discovered
new
binder,
SEPHS2,
dasatinib,
VPS37C
hydroxychloroquine,
enhancing
our
drugs’
mechanisms
action.
Furthermore,
demonstrated
application
zebrafish
embryos,
highlighting
its
potential
broad
biological
research
Chemical Communications,
Год журнала:
2024,
Номер
60(96), С. 14208 - 14211
Опубликована: Янв. 1, 2024
Our
study
shows
efficient
tyrosine
labeling
using
1-methyl-4-arylurazole
(MAUra)
with
laccase
under
mild
conditions.
This
method
achieves
a
high
efficiency
(
k
cat
/
K
m
=
7.88
×
10
4
M
−1
s
),
selectively
targeting
exposed
sites
on
proteins.
Identifying
target
proteins
for
bioactive
molecules
is
essential
understanding
their
mechanisms,
developing
improved
derivatives,
and
minimizing
off-target
effects.
Despite
advances
in
identification
(target-ID)
technologies,
significant
challenges
remain,
impeding
drug
development.
Most
target-ID
methods
use
cell
lysates,
but
maintaining
an
intact
cellular
context
vital
capturing
specific
drug-protein
interactions,
such
as
those
with
transient
protein
complexes
membrane-associated
proteins.
To
address
these
limitations,
we
developed
POST-IT
(Pup-On-target
Small
molecule
Target
Identification
Technology),
a
non-diffusive
proximity
tagging
system
live
cells,
orthogonal
to
the
eukaryotic
system.
utilizes
engineered
fusion
of
proteasomal
accessory
factor
A
(PafA)
HaloTag
transfer
Pup
proximal
upon
directly
binding
small
molecule.
After
optimization
eliminate
self-pupylation
polypupylation,
minimize
depupylation,
optimize
chemical
linkers,
successfully
identified
known
targets
discovered
new
binder,
SEPHS2,
dasatinib,
VPS37C
hydroxychloroquine,
enhancing
our
drugs’
mechanisms
action.
Furthermore,
demonstrated
application
zebrafish
embryos,
highlighting
its
potential
broad
biological
research
ACS Chemical Biology,
Год журнала:
2024,
Номер
unknown
Опубликована: Дек. 18, 2024
Cell-cell
interactions
are
fundamental
in
biology
for
maintaining
physiological
conditions
with
direct
contact
being
the
most
straightforward
mode
of
interaction.
Recent
advancements
have
led
to
development
various
chemical
tools
detecting
or
identifying
these
interactions.
However,
use
exogenous
cues,
such
as
toxic
reagents,
bulky
probes,
and
light
irradiation,
can
disrupt
normal
cell
physiology.
For
example,
toxicity
hydrogen
peroxide
(H
Identifying
target
proteins
for
bioactive
molecules
is
essential
understanding
their
mechanisms,
developing
improved
derivatives,
and
minimizing
off-target
effects.
Despite
advances
in
identification
(target-ID)
technologies,
significant
challenges
remain,
impeding
drug
development.
Most
target-ID
methods
use
cell
lysates,
but
maintaining
an
intact
cellular
context
vital
capturing
specific
drug–protein
interactions,
such
as
those
with
transient
protein
complexes
membrane-associated
proteins.
To
address
these
limitations,
we
developed
POST-IT
(Pup-On-target
Small
molecule
Target
Identification
Technology),
a
non-diffusive
proximity
tagging
system
live
cells,
orthogonal
to
the
eukaryotic
system.
utilizes
engineered
fusion
of
proteasomal
accessory
factor
A
HaloTag
transfer
Pup
proximal
upon
directly
binding
small
molecule.
After
optimization
eliminate
self-pupylation
polypupylation,
minimize
depupylation,
optimize
chemical
linkers,
successfully
identified
known
targets
discovered
new
binder,
SEPHS2,
dasatinib,
VPS37C
hydroxychloroquine,
enhancing
our
drugs’
mechanisms
action.
Furthermore,
demonstrated
application
zebrafish
embryos,
highlighting
its
potential
broad
biological
research
