International Journal for Parasitology Drugs and Drug Resistance,
Год журнала:
2024,
Номер
25, С. 100538 - 100538
Опубликована: Апрель 16, 2024
Leishmaniasis,
a
vector-borne
disease,
is
caused
by
the
infection
of
Leishmania
spp.,
obligate
intracellular
protozoan
parasites.
Presently,
human
vaccines
are
unavailable,
and
primary
treatment
relies
heavily
on
systemic
drugs,
often
presenting
with
suboptimal
formulations
substantial
toxicity,
making
new
drugs
high
priority
for
LMIC
countries
burdened
but
low
in
agenda
most
pharmaceutical
companies
due
to
unattractive
profit
margins.
New
ways
accelerate
discovery
new,
or
repositioning
existing
needed.
To
address
this
challenge,
our
study
aimed
identify
potential
protein
targets
shared
among
clinically-relevant
species.
We
employed
subtractive
proteomics
comparative
genomics
approach,
integrating
high-throughput
multi-omics
data
classify
these
based
different
druggability
metrics.
This
effort
resulted
ranking
6502
ortholog
groups
across
14
pathogenic
Among
top
20
highly
ranked
groups,
metabolic
processes
known
be
attractive
drug
targets,
including
ubiquitination
pathway,
aminoacyl-tRNA
synthetases,
purine
synthesis,
were
rediscovered.
Additionally,
we
unveiled
novel
promising
such
as
nicotinate
phosphoribosyltransferase
enzyme
dihydrolipoamide
succinyltransferases.
These
exhibited
appealing
features,
less
than
40%
sequence
identity
host
proteome,
predicted
essentiality,
structural
classification
druggable
druggable,
expression
levels
above
50th
percentile
amastigote
form.
The
resources
presented
work
also
represent
comprehensive
collection
integrated
regarding
trypanosomatid
biology.
Nature Communications,
Год журнала:
2021,
Номер
12(1)
Опубликована: Фев. 23, 2021
Abstract
Differentiation
between
distinct
stages
is
fundamental
for
the
life
cycle
of
intracellular
protozoan
parasites
and
transmission
hosts,
requiring
stringent
spatial
temporal
regulation.
Here,
we
apply
kinome-wide
gene
deletion
tagging
in
Leishmania
mexicana
promastigotes
to
define
protein
kinases
with
transition
roles.
Whilst
162
are
dispensable,
44
kinase
genes
refractory
likely
core
required
parasite
replication.
Phenotyping
pooled
mutants
using
bar-seq
projection
pursuit
clustering
reveal
functional
phenotypic
groups
involved
differentiation
from
metacyclic
promastigote
amastigote,
growth
survival
macrophages
mice,
colonisation
sand
fly
motility.
This
unbiased
interrogation
function
allows
targeted
investigation
organelle-associated
signalling
pathways
successful
parasitism.
ACS Bio & Med Chem Au,
Год журнала:
2022,
Номер
3(1), С. 32 - 45
Опубликована: Дек. 15, 2022
Targeted
protein
degradation
(TPD)
is
emerging
as
one
of
the
most
innovative
strategies
to
tackle
infectious
diseases.
Particularly,
proteolysis-targeting
chimera
(PROTAC)-mediated
may
offer
several
benefits
over
classical
anti-infective
small-molecule
drugs.
Because
their
peculiar
and
catalytic
mechanism
action,
PROTACs
might
be
advantageous
in
terms
efficacy,
toxicity,
selectivity.
Importantly,
also
overcome
emergence
antimicrobial
resistance.
Furthermore,
have
potential
(i)
modulate
"undruggable"
targets,
(ii)
"recycle"
inhibitors
from
drug
discovery
approaches,
(iii)
open
new
scenarios
for
combination
therapies.
Here,
we
try
address
these
points
by
discussing
selected
case
studies
antiviral
first-in-class
antibacterial
PROTACs.
Finally,
discuss
how
field
PROTAC-mediated
TPD
exploited
parasitic
Since
no
antiparasitic
PROTAC
has
been
reported
yet,
describe
parasite
proteasome
system.
While
its
infancy
with
many
challenges
ahead,
hope
that
diseases
lead
development
next-generation
Medicinal Research Reviews,
Год журнала:
2023,
Номер
44(3), С. 1055 - 1120
Опубликована: Дек. 24, 2023
Leishmaniasis
is
a
group
of
neglected
tropical
diseases
caused
by
at
least
20
species
Leishmania
protozoa,
which
are
spread
the
bite
infected
sandflies.
There
three
main
forms
disease:
cutaneous
leishmaniasis
(CL,
most
common),
visceral
(VL,
also
known
as
kala-azar,
serious),
and
mucocutaneous
leishmaniasis.
One
billion
people
live
in
areas
endemic
to
leishmaniasis,
with
an
annual
estimation
30,000
new
cases
VL
more
than
1
million
CL.
New
treatments
for
urgent
need,
existing
ones
inefficient,
toxic,
and/or
expensive.
We
have
revised
experimental
structure-based
drug
design
(SBDD)
efforts
applied
discovery
drugs
against
grouped
explored
targets
according
metabolic
pathways
they
belong
to,
key
achieved
advances
highlighted
evaluated.
In
cases,
SBDD
studies
follow
high-throughput
screening
campaigns
secondary
pharmacokinetic
optimization,
due
majoritarian
belief
that
there
few
validated
However,
some
strategies
significantly
contributed
candidates
bigger
number
holds
promise
future
development.
The
ability
to
analyse
the
function
of
all
genes
in
a
genome
is
highly
desirable,
yet
challenging
Leishmania
due
repetitive
genome,
limited
DNA
repair
mechanisms
and
lack
RNA
interference
most
species.
While
our
introduction
cytosine
base
editor
(CBE)
demonstrated
potential
overcome
these
limitations
(Engstler
Beneke
(2023)),
challenges
remained,
including
low
transfection
efficiency,
variable
editing
rates
across
species,
parasite
growth
effects,
competition
between
deleterious
non-deleterious
mutations.
Here,
we
present
an
optimized
approach
addressing
issues.We
identified
T7
RNAP
promoter
variant
ensuring
high
species
without
compromising
growth.
A
revised
CBE
single-guide
RNAs
(sgRNAs)
scoring
system
was
developed
prioritize
STOP
codon
generation.
Additionally,
triple-expression
construct
created
for
stable
integration
sgRNA
expression
cassettes
into
safe
harbor
locus
using
AsCas12a
ultra-mediated
double-strand
breaks,
increasing
efficiency
by
∼400-fold
one
transfectant
per
70
transfected
cells.
Using
this
improved
small-scale
proof-of-principle
pooled
screen,
successfully
confirmed
essential
fitness-associated
functions
CK1.2,
CRK2,
CRK3,
AUK1/AIRK,
TOR1,
IFT88,
IFT139,
IFT140
RAB5A
L.
mexicana
,
demonstrating
significant
improvement
over
previous
method.
Lastly,
show
utility
co-expressing
ultra,
hybrid
CRISPR
gene
replacement
within
same
cell
line.Overall,
improvements
will
broaden
range
possible
applications
enable
variety
loss-of-function
screens
near
future.
The
ability
to
analyze
the
function
of
all
genes
in
a
genome
is
highly
desirable,
yet
challenging
Leishmania
due
repetitive
genome,
limited
DNA
repair
mechanisms,
and
lack
RNA
interference
most
species.
While
our
introduction
cytosine
base
editor
(CBE)
demonstrated
potential
overcome
these
limitations
(Engstler
Beneke,
2023),
challenges
remained,
including
low
transfection
efficiency,
variable
editing
rates
across
species,
parasite
growth
effects,
competition
between
deleterious
non-deleterious
mutations.
Here,
we
present
an
optimized
approach
addressing
issues.
We
identified
T7
RNAP
promoter
variant
ensuring
high
species
without
compromising
growth.
A
revised
CBE
single-guide
RNAs
(sgRNAs)
scoring
system
was
developed
prioritize
STOP
codon
generation.
Additionally,
triple-expression
construct
created
for
stable
integration
sgRNA
expression
cassettes
into
safe
harbor
locus
using
AsCas12a
ultra-mediated
double-strand
breaks,
increasing
efficiency
by
~400-fold
1
transfectant
per
70
transfected
cells.
Using
this
improved
small-scale
proof-of-principle
pooled
screen,
successfully
confirmed
essential
fitness-associated
functions
CK1.2,
CRK2,
CRK3,
AUK1/AIRK,
TOR1,
IFT88,
IFT139,
IFT140,
RAB5A
mexicana
,
demonstrating
significant
improvement
over
previous
method.
Lastly,
show
utility
co-expressing
ultra,
RNAP,
hybrid
CRISPR
gene
replacement
within
same
cell
line.
Overall,
improvements
will
broaden
range
possible
applications
enable
variety
loss-of-function
screens
near
future.
bioRxiv (Cold Spring Harbor Laboratory),
Год журнала:
2025,
Номер
unknown
Опубликована: Март 1, 2025
Abstract
In
Leishmania
parasites,
as
for
their
hosts,
the
ubiquitin
proteasome
system
is
important
cell
viability.
As
part
of
a
systematic
gene
deletion
study,
it
was
discovered
that
four
cysteine
protease
type
deubiquitinases
(DUBs)
are
essential
parasite
survival
in
promastigote
stage,
including
DUB16.
Here
we
have
purified
and
characterised
recombinant
DUB16
from
donovani
,
which
belongs
to
C-terminal
hydrolase
(UCH)
family.
efficiently
hydrolyses
aminocoumarin
rhodamine
conjugates
consistent
with
proposed
cellular
roles
UCH-type
DUBs
regenerating
free
monomeric
small
molecule
adducts
arising
adventitious
metabolic
processes.
The
crystal
structure
reveals
typical
deubiquitinase
fold,
relatively
short
disordered
crossover
loop
appears
restrict
access
catalytic
cysteine.
At
close
stoichiometric
enzyme
substrate
ratios,
exhibits
activity
towards
diubiquitins
linked
through
isopeptide
bonds
between
Lys11,
Lys48
or
Lys63
residues
proximal
C-terminus
distal
ubiquitin.
With
100-1000-fold
higher
turnover
rates,
cleaves
ubiquitin-ribosomal
L40
fusion
protein
give
mature
products.
A
DUB-targeting
cysteine-reactive
cyanopyrrolidine
compound,
IMP-1710,
inhibits
activity.
IMP-1710
shown
viability
assays
killing
EC
50
values
1−2
μM,
comparable
anti-leishmanial
drug,
miltefosine.
L.
mexicana
parasites
engineered
overproduce
showed
modest
increase
resistance
providing
evidence
vivo
.
Together
these
results
suggest
on-target
may
be
druggable
target
develop
new
anti-leishmania
compounds.
Frontiers in Cellular and Infection Microbiology,
Год журнала:
2021,
Номер
11
Опубликована: Ноя. 10, 2021
Until
2015,
loss-of-function
studies
to
elucidate
protein
function
in
Leishmania
relied
on
gene
disruption
through
homologous
recombination.
Then,
the
CRISPR/Cas9
revolution
reached
these
protozoan
parasites
allowing
efficient
genome
editing
with
one
round
of
transfection.
In
addition,
development
LeishGEdit,
a
PCR-based
toolkit
for
generating
knockouts
and
tagged
lines
using
CRISPR/Cas9,
allowed
more
straightforward
effective
editing.
this
system,
plasmid
pTB007
is
delivered
episomal
expression
or
integration
β-tubulin
locus
stable
T7
RNA
polymerase
Cas9.
South
America,
especially
Brazil,
(
Viannia
)
braziliensis
most
frequent
etiological
agent
tegumentary
leishmaniasis.
The
L.
presents
significant
sequence
divergence
comparison
major
,
which
precludes
To
overcome
limitation,
sequences,
present
pTB007,
were
replaced
by
conserved
pTB007_Viannia
plasmid.
This
modification
successful
cassette
M2903
genome,
silico
predictions
suggest
that
can
also
be
achieved
other
species.
activity
Cas9
was
evaluated
knocking
out
flagellar
PF16,
caused
phenotype
immobility
transfectants.
Endogenous
PF16
successfully
mNeonGreen,
an
in-locus
complementation
strategy
employed
return
C-terminally
copy
original
locus,
resulted
recovery
swimming
capacity.
modified
both
provided
important
tool
study
biology
parasite.
Molecular and Biochemical Parasitology,
Год журнала:
2024,
Номер
258, С. 111619 - 111619
Опубликована: Март 29, 2024
In
eukaryotic
cells,
molecular
fate
and
cellular
responses
are
shaped
by
multicomponent
enzyme
systems
which
reversibly
attach
ubiquitin
ubiquitin-like
modifiers
to
target
proteins.
The
extent
of
the
proteasome
system
in
Leishmania
mexicana
its
importance
for
parasite
survival
has
recently
been
established
through
deletion
mutagenesis
life-cycle
phenotyping
studies.
conjugating
E2
UBC2,
variant
UEV1,
with
it
forms
a
stable
complex
vitro,
were
shown
be
essential
differentiation
promastigote
parasites
infectious
amastigote
form.
To
investigate
further,
we
used
immunoprecipitation
Myc-UBC2
or
Myc-UEV1
identify
interacting
proteins
L.
promastigotes.
interactome
UBC2
comprises
multiple
ubiquitin-proteasome
components
including
UEV1
four
RING
E3
ligases,
as
well
potential
substrates
predicted
have
roles
carbohydrate
metabolism
intracellular
trafficking.
smaller
six
proteins,
shared
consistent
presence
UBC2-UEV1
complexes.
Recombinant
RING1,
RING2
RING4
ligases
support
transfer
reactions
involving
E1,
UBA1a,
available
substrate
unanchored
chains.
These
studies
define
additional
UBC2-dependent
ubiquitination
pathway
previously
differentiation.
Current Opinion in Microbiology,
Год журнала:
2022,
Номер
70, С. 102202 - 102202
Опубликована: Сен. 11, 2022
In
eukaryotic
cells,
reversible
attachment
of
ubiquitin
and
ubiquitin-like
modifiers
(Ubls)
to
specific
target
proteins
is
conducted
by
multicomponent
systems
whose
collective
actions
control
protein
fate
cell
behaviour
in
precise
but
complex
ways.
trypanosomatids,
Ubls
regulates
the
cycle,
endocytosis,
sorting
degradation,
autophagy
various
aspects
infection
stress
responses.
The
extent
these
trypanosomatids
has
been
surveyed
recent
reports,
while
Leishmania
mexicana,
essential
roles
have
defined
for
many
ubiquitin-system
genes
deletion
mutagenesis
life-cycle
phenotyping
campaigns.
first
steps
elucidate
pathways
transfer
among
ubiquitination
components
define
acceptor
substrates
downstream
deubiquitinases
are
now
being
taken.
Scientific Reports,
Год журнала:
2021,
Номер
11(1)
Опубликована: Ноя. 8, 2021
Abstract
The
inducible
Di-Cre
system
was
used
to
delete
the
putative
ubiquitin-conjugating
enzyme
13
gene
(
ubc13)
of
Plasmodium
falciparum
study
its
role
in
ubiquitylation
and
functional
consequence
during
parasite
asexual
blood
stage
.
Deletion
resulted
a
significant
reduction
growth
vitro
,
reduced
Lys63
residue
ubiquitin
attached
protein
substrates,
an
increased
sensitivity
both
mutagen,
methyl
methanesulfonate
antimalarial
drug
dihydroartemisinin
(DHA),
but
not
chloroquine.
also
sensitive
UBC13
inhibitor
NSC697923.
data
suggest
that
this
does
code
for
conjugating
responsible
K63
ubiquitylation,
which
is
important
DNA
repair
pathways
as
previously
demonstrated
other
organisms.
DHA
absence
ubc13
function
indicates
may
act
primarily
through
pathway
inhibitors
enhance
efficacy
directly
inhibit
growth.