Bioluminescence-Based Determination of Cytosolic Accumulation of Antibiotics in Escherichia coli
ACS Infectious Diseases,
Journal Year:
2024,
Volume and Issue:
10(5), P. 1602 - 1611
Published: April 9, 2024
Antibiotic
resistance
is
an
alarming
public
health
concern
that
affects
millions
of
individuals
across
the
globe
each
year.
A
major
challenge
in
development
effective
antibiotics
lies
their
limited
ability
to
permeate
cells,
noting
numerous
susceptible
antibiotic
targets
reside
within
bacterial
cytosol.
Consequently,
improving
cellular
permeability
often
a
key
consideration
during
development,
underscoring
need
for
reliable
methods
assess
molecules
membranes.
Currently,
used
measure
fail
discriminate
between
arrival
cytoplasm
and
overall
association
with
cell.
Additionally,
these
techniques
typically
possess
throughput
limitations.
In
this
work,
we
describe
luciferase-based
assay
designed
assessing
cytosolic
compartment
Gram-negative
bacteria.
Our
findings
demonstrate
robust
system
can
elucidate
kinetics
intracellular
accumulation
live
cells
real
time.
Language: Английский
Fluorescence-Based Enzyme Activity Assay: Ascertaining the Activity and Inhibition of Endocannabinoid Hydrolytic Enzymes
International Journal of Molecular Sciences,
Journal Year:
2024,
Volume and Issue:
25(14), P. 7693 - 7693
Published: July 13, 2024
The
endocannabinoid
system,
known
for
its
regulatory
role
in
various
physiological
processes,
relies
on
the
activities
of
several
hydrolytic
enzymes,
such
as
fatty
acid
amide
hydrolase
(FAAH),
N-acylethanolamine-hydrolyzing
amidase
(NAAA),
monoacylglycerol
lipase
(MAGL),
and
α/β-hydrolase
domains
6
(ABHD6)
12
(ABHD12),
to
maintain
homeostasis.
Accurate
measurement
these
enzymes’
is
crucial
understanding
their
function
development
potential
therapeutic
agents.
Fluorometric
assays,
which
offer
high
sensitivity,
specificity,
real-time
monitoring
capabilities,
have
become
essential
tools
enzymatic
studies.
This
review
provides
a
comprehensive
overview
principles
behind
substrates
fluorophores
used,
advances
assay
techniques
used
not
only
determination
kinetic
mechanisms
enzyme
reactions
but
also
setting
up
assays
high-throughput
screening
each
critical
involved
degradation.
Through
this
review,
we
aim
highlight
strengths
limitations
current
fluorometric
suggest
future
directions
improving
activity
system.
Language: Английский
Methotrexate-based PROTACs as DHFR-specific chemical probes
Cell chemical biology,
Journal Year:
2023,
Volume and Issue:
31(2), P. 221 - 233.e14
Published: Oct. 23, 2023
Methotrexate
(MTX)
is
a
tight-binding
dihydrofolate
reductase
(DHFR)
inhibitor,
used
as
both
an
antineoplastic
and
immunosuppressant
therapeutic.
MTX,
like
folate
undergoes
folylpolyglutamate
synthetase-mediated
γ-glutamylation,
which
affects
cellular
retention
target
specificity.
Mechanisms
of
MTX
resistance
in
cancers
include
decrease
poly-γ-glutamylation
upregulation
DHFR.
Here,
we
report
series
potent
MTX-based
proteolysis
targeting
chimeras
(PROTACs)
to
investigate
DHFR
degradation
pharmacology
one-carbon
biochemistry.
These
on-target,
cell-active
PROTACs
show
proteasome-
E3
ligase-dependent
activity,
selective
multiple
cancer
cell
lines.
By
comparison,
treatment
with
increases
protein
expression.
Importantly,
these
produced
distinct,
less-lethal
phenotypes
compared
MTX.
The
chemical
probe
set
described
here
should
complement
conventional
inhibitors
serve
useful
tools
for
studying
biochemistry
dissecting
complex
polypharmacology
related
drugs.
Such
compounds
may
also
leads
potential
autoimmune
therapeutics.
Language: Английский
Bioluminescence-Based Determination of Cytosolic Accumulation of Antibiotics inEscherichia coli
bioRxiv (Cold Spring Harbor Laboratory),
Journal Year:
2023,
Volume and Issue:
unknown
Published: Dec. 7, 2023
Abstract
Antibiotic
resistance
is
an
alarming
public
health
concern
that
affects
millions
of
individuals
across
the
globe
each
year.
A
major
challenge
in
development
effective
antibiotics
lies
their
limited
ability
to
permeate
into
cells,
noting
numerous
susceptible
antibiotic
targets
reside
within
bacterial
cytosol.
Consequently,
improving
cellular
permeability
often
a
key
consideration
during
development,
underscoring
need
for
reliable
methods
assess
molecules
membranes.
Currently,
used
measure
fail
discriminate
between
arrival
cytoplasm
and
overall
association
with
cell.
Additionally,
these
techniques
typically
possess
throughput
limitations.
In
this
work,
we
describe
luciferase-based
assay
designed
assessing
cytosolic
compartment
Gram-negative
bacteria.
Our
findings
demonstrate
robust
system
can
elucidate
kinetics
intracellular
accumulation
live
cells
real
time.
Language: Английский