biomolecules
Review
Central Nervous System Targeted Protein Degraders
Bedwyr ab Ion Thomas 1 , H. Lois Lewis 1 , D. Heulyn Jones 1,2 and Simon E. Ward 1, *
1
2
*
Medicines Discovery Institute, Cardiff University, Cardiff CF10 3AT, UK; lewisl33@cardiff.ac.uk (H.L.L.)
Chemistry Department, Cardiff University, Cardiff CF10 3AT, UK
Correspondence: wards10@cardiff.ac.uk
Abstract: Diseases of the central nervous system, which once occupied a large component of the
pharmaceutical industry research and development portfolio, have for many years played a smaller
part in major pharma pipelines—primarily due to the well cited challenges in target validation,
valid translational models, and clinical trial design. Unfortunately, this decline in research and
development interest has occurred in tandem with an increase in the medical need—in part driven
by the success in treating other chronic diseases, which then results in a greater overall longevity
along with a higher prevalence of diseases associated with ageing. The lead modality for drug
agents targeting the brain remains the traditionally small molecule, despite potential in gene-based
therapies and antibodies, particularly in the hugely anticipated anti-amyloid field, clearly driven
by the additional challenge of effective distribution to the relevant brain compartments. However,
in recognition of the growing disease burden, advanced therapies are being developed in tandem
with improved delivery options. Hence, methodologies which were initially restricted to systemic
indications are now being actively explored for a range of CNS diseases—an important class of which
include the protein degradation technologies.
Keywords: PROTACs; targeted protein degraders; CNS
1. Proteolysis-Targeting Chimeras (PROTACs)
Citation: Thomas, B.a.I.; Lewis, H.L.;
Jones, D.H.; Ward, S.E. Central
Nervous System Targeted Protein
Degraders. Biomolecules 2023, 13,
1164. https://doi.org/10.3390/
biom13081164
Academic Editor: Christopher
L. Cioffi
Received: 1 June 2023
Revised: 5 July 2023
Accepted: 11 July 2023
Published: 25 July 2023
Research on central nervous system diseases has decreased due to challenges in target
validation and clinical trials, despite a growing medical need [1]. Small molecule drugs still
dominate brain-targeted therapies, but interest in gene-based and antibody treatments is
rising to address the disease burden [2,3]. Furthermore, advanced therapies and improved
delivery options are being explored, including protein degradation technologies [3].
Hijacking the ubiquitin proteasome system (UPS) has been seen as a potential tool
for therapeutic use for nearly twenty years [4]. The primary technology used for this
targeted protein degradation approach is the utilisation of proteolysis-targeting chimeras, or
PROTACs, which are heterobifunctional molecules that are capable of bringing the protein
of interest (POI) into proximity with the UPS mechanism. PROTACs are composed of
three domains, namely a POI-binding moiety, an E3 ligase-binding moiety, and a molecular
linker connecting the two (Figure 1).
1.1. Necessary Steps for Targeted Protein Degradation
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
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4.0/).
A specific set of events must take place correctly in order to mimic or harness this
process, the failure of any one of which will prevent the targeted degradation of the targeted
protein (Figure 2) [5]:
1.
2.
Cellular uptake of the PROTAC to the appropriate intracellular compartment containing the ubiquitination machinery and the POI;
Ternary complex formation to enable ubiquitin transfer once the PROTAC is inside
the cell. This relies on simultaneously binding to the POI and E3 ligase, which
in turn is reliant on the binding affinity of the PROTAC to both proteins. Albeit
Biomolecules 2023, 13, 1164. https://doi.org/10.3390/biom13081164
https://www.mdpi.com/journal/biomolecules
Biomolecules 2023, 13, 1164
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well described, the thermodynamics of ternary complex formation are less intuitively simple compared to a two-body system. Specifically, a “hook effect”, characterised by a bell-shaped dose–response curve, which occurs when the PROTAC forms
1:1 ligand-bound complexes with either the POI or the E3 ligase, leads to ineffectual
complex formation at higher concentrations of the PROTAC, and may give rise to
possible issues around in vivo dose selection. Secondary protein–protein interactions
(PPIs) may also favour or hinder ternary complex formation through cooperativity or
steric clashes, respectively. Figure 3 graphically depicts the concentration-dependent
“hook effect”;
Figure 1. The constituent parts of a PROTAC.
Figure 2. Schematic of a PROTAC bringing the POI into proximity with the UPS.
Biomolecules 2023, 13, 1164
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Figure 3. (A) Hook effect depicting the proportion of degraded POI when the systemic concentration
of PROTAC is both too high and too low. (B) PPIs between the POI and E3 ligase. Importantly, even
when PROTAC–POI affinity is weak, favourable PPIs can stabilise the ternary complex, leading to
effective ubiquitination and consequent degradation.
3.
4.
5.
6.
Once formed, the ternary complex must accommodate the two bound proteins to
occupy a favourable conformation, such that ubiquitin transfer may take place to
a suitable acceptor site, commonly a surface lysine. Ubiquitin transfer must occur
quickly, at a rate faster than dissociation of the ternary complex;
Targeted induced polyubiquitination should also kinetically outcompete deubiquitinases,
which belong to a large family encompassing wide-ranging substrate specificities;
Furthermore, the motif of the transferred ubiquitin residues should facilitate facile
recognition through the proteasome to bring about actual degradation;
Even if all the previous steps are successful, and the POI is degraded by the proteasome, this does not guarantee a decreased steady state level of protein. The de novo
resynthesis rate of the POI, which may vary significantly between cell types, must be
markedly slower than the rate of induced degradation. Likewise, the initial reduction
of equilibrium protein levels may not persist over time if the loss of mature protein
triggers the induction of feedback mechanisms that upregulate either the translation
or transcription of the new protein.
The Hook Effect
PROTACs sometimes display atypical bell-curve type dose–response curves, which
means that they must be evaluated over a range of concentrations. At low concentrations
of PROTAC, only a small proportion of POI- and E3 ligase-binding sites are occupied,
indicating that only a small number of ternary complexes can form, leading to only some
degradation of the POI. At high concentrations of PROTAC, most binding sites of both the
POI and E3 ligases are occupied by the PROTAC, again leading to only a small amount
of ternary complex formation, and consequently to only some degradation of the POI.
An optimum concentration of PROTAC represents a 50% occupancy of all binding sites
Biomolecules 2023, 13, 1164
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at 1:1 for POI:E3 ligase, leading to the greatest amount of ternary complex formation, and
thus the largest degree of degradation of the POI.
Small-molecule inhibitors act through an occupancy-driven pharmacological model
where direct inhibition stops the protein’s function. In contrast, PROTACs work via an
event-driven pharmacology model where removal controls protein abundance, indicating
that protein function is governed by decreasing the cellular protein level [6].
A key feature of PROTACs that is considered advantageous with respect to smallmolecule inhibitors is an added layer of specificity due to the ternary complex formation
and their ubiquitin transfer-dependent mechanism of action (MoA). Another advantage
is their increased potency compared to a PROTAC’s component inhibitor, along with a
sub-stoichiometric drug requirement owing to the catalytic MoA and PPI during ternary
complex formation. Furthermore, the degradation of the POI results in prolonged pharmacodynamic suppression in vivo due to protein re-synthesis being required to restore target
activity [7]. A range of PROTACs have demonstrated their efficacy at nM concentrations,
achieving a high degradation potency due to their MoA [8].
Many proteins, including those which possess non-catalytic functions, namely pseudokinases, transcription factors, non-enzymatic proteins, and scaffolding proteins, lack
an active site suitable for an inhibitor and are thus deemed “undruggable”. Moreover, in
cancer, inhibition may be less effective or ineffective for small molecules due to the presence
of mutations close to the inhibitor binding pocket. PROTACs, however, can allosterically
instigate ubiquitination of the POI, circumventing this phenomenon due to their ability to
form transient interactions with their targets if ubiquitination occurs quickly within the
timescale of ternary complex formation [9].
1.2. Blood–Brain Barrier Permeability
Access to the CNS necessitates permeation across the blood–brain barrier. This is a
challenge for traditional small-molecule drug discovery, requiring careful consideration
of the physiochemical properties e.g., through the use of the MultiParameter Optimisation (MPO) guidelines [10]. This challenge is further amplified for PROTACs due to their
increased molecular weight and may be a major limiting factor for their use in the CNS.
Radical solutions, such as developing antibody conjugates, have been proposed [11]. However, there are isolated reports of brain penetrable PROTACs. Limited blood–brain barrier
permeability was observed for a Tau-targeting PROTAC [12]. However, XL01126, a PROTAC which has been discussed in greater length in Section 2.3, has been shown to be present
in the CNS at meaningful concentrations [13]. This is especially surprising, considering the
total polar surface area of XL01126 (194.3 Å2 ) relative to the maximum value suggested for
reliably entering the CNS (90 Å2 ) [14]. This example gives hope to the field in that access
to the CNS is achievable, but the level of the task must not be understated. Additional
examples are required in order to spot linker motifs that may allow a common strategy to
promote permeability to the CNS.
1.3. Solubility of PROTACs
Solubility is a crucial parameter for drug delivery, especially in the context of the CNS,
where poor solubility could hamper drug distribution. The proteins of interest that require
degradation are intracellularly localised, and thus PROTAC solubility and cell permeability
are paramount in their therapeutic success. PROTACs are, by design, large molecules, and
an increased MW is often correlated with a poor solubility and a decreased permeability
due to larger molecules having a higher propensity for aggregation or precipitation [1].
The molecular structure of the PROTAC itself can determine the extent of its solubility with
the presence of polar functional groups, including the hydroxyl (-OH), amino (-NH2 ), or
carboxyl (-COOH) groups, increasing its solubility by facilitating hydrogen bond formation
with water molecules. Lipophilicity is another factor that needs considering, where the
balance between lipophilicity and hydrophilicity needs to be considered in order to achieve
an optimal solubility profile. Presently, the current choice for E3 ligase-binding ligands
Biomolecules 2023, 13, 1164
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is limited, as explored in Section 1.4, whereas the POI-binding ligand is preliminarily
determined by its binding affinity to the POI. Therefore, to modulate the solubility of the
PROTAC, varying the linker length and type may be the best course of action [15]. A study
by Jiménez et al. of 21 commercial degraders proposed BRlogD and TPSA as key indicators
of PROTAC solubility, where 2.58 and 289 Å2 were the irrespective calculated thresholds
for an experimental solubility classification [16]. However, it should be noted that the
PROTACs which display a poor or moderate solubility may benefit from pharmaceutical
excipients and formulations.
1.4. Choice of E3 Ligase
Currently, only a handful of the broad family of E3 ligases have been exploited, with
the most favoured being von Hippel–Lindau (VHL) and cereblon (CRBN), which are
part of the Cullin-RING E3 ubiquitin ligase complexes (CRLs) CRL2VHL and CRL4ACRBN ,
respectively [17]. CRBN is the primary target of immunomodulatory imide drugs, whereas
VHL mediates the degradation of its substrate HIF-1α [18,19]. Several specialised E3
ligases for potential PROTAC applications have been reported to have an increased tissue
specificity, which could be harnessed by the targeted protein degradation modalities to
enable the tissue- and cell-type-specific targeting of the disease-causing proteins within
the CNS [4,20–22]. Among many others, these include the TRIM9 and RNF182 E3 ligases,
which are part of the TRIM and RNF families of the E3 ligases, respectively.
Whilst the notion of developing a universal ubiquitin ligase binder would at first seem
beneficial, presenting the possibility of a “one-size-fits-all” PROTAC with a customisable
POI ligand, this approach is considered as impractical.
Localisation of the PROTAC to specific brain regions poses a potential limitation, with
the most widely utilised E3 ligases, CRBN and VHL, being indiscriminately expressed
throughout the brain. [23] Many specialised E3 ligases have shown an increased tissue
specificity with the potential to be used in a tissue- and cell-specific manner by TPD to
degrade the proteins that are involved with diseases in the CNS, but only preliminary steps
have been made to develop ligands with the potential to exploit them [4,20–22].
As of today, only ~1% of roughly 600 E3 ligases have been successfully targeted by
small-molecule degraders (CRBN, VHL, IAPs, MDM2, DCAF15, DCAF16, and RNF114,
respectively) [24]. Of the 623 E3 ligases in the human proteasome, around 270 of those
are involved with the UPS [25]. PROTACs, with their ability to recruit E3 ligases with
tissue-specific expression profiles, have a unique opportunity within a therapeutic context,
as in theory they should not degrade the target protein in tissues where the E3 ligase is
not expressed. This is particularly important in the context of neurodegenerative diseases
because of the clear advantage to only degrading proteins in the brain without causing serious off-target effects in other tissues. Roughly twenty E3 ligases have a narrow expression
across human tissues, and of these ligases it is known that four promote the degradation
of their substrates (ASB9, KLH10, KLH41, and TRIM69, respectively) [24]. For example,
TRIM69 is expressed in the pancreas and testicles, while FBXL16 is solely expressed in the
cerebral cortex. Small ligands that bind with a sufficient affinity to one of these E3 ligases
can be linked to several ligands that target substrates for degradation within a specific
tissue [24].
1.5. Role of the Linker
It is known that the PROTAC’s linker plays an important role in protein degradation,
the formation of the ternary complex, and the drug’s ADME properties [15,26–28]. The
first generation of PROTACs used alkyl chains as their linkers [29]. It is vital that drugs
possess a sufficient lipophilicity to traverse cell membranes; however, the high lipophilicity
of the alkyl chains within the PROTACs reduce their solubility in water. To increase
the solubility of the PROTACs, the use of polyethylene glycol (PEG) chains has become
commonplace [15,30,31]. This shift from alkyl to PEG chains increases the number of
hydrogen-bond donors, thus increasing the solubility. It is incredibly challenging to predict
Biomolecules 2023, 13, 1164
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an optimum length of linker—there is no clear correlation between the length, nor the
the increasedofrigidity
obtained
byitsopting
for a effect
1-(piperidin-4-ylmethyl)piperazine
linker
composition,
the PROTAC
and
degrading
[32,33]. Two recent examples of
compared
to
the
first
few
generations
of
PROTACs.
It
could
be
implied
that
keeping
linkers that deviated from the common alkyl and PEG-type chains include ARV-110 and the
number ofboth
rotational
bonds
low withinrespect
toclinical
the alkyl
and[34,35].
PEG linkers
may present
ARV-471,
of which
are currently
phase II
trials
It is interesting
to an
advantage.
However,
it is obtained
wise to be
when
drawing such a conclusion, as there
note
the increased
rigidity
bycautious
opting for
a 1-(piperidin-4-ylmethyl)piperazine
linker
compared
to theexamples
first few generations
couldundergoing
be implied that
keeping
are only
two known
of this kindofofPROTACs.
PROTAC Itlinker
clinical
trials. It
the
number
of rotational
bonds
low with respect
to the
alkyl
and
linkers may
present
is also
possible
that other
physiochemical
factors
play
a role
inPEG
the relative
success
of these
an
advantage.
is wise to be
cautiousdue
when
drawing such
a conclusion,
as introducthere
PROTACs;
forHowever,
example,itincreased
solubility
to lowering
the logD
7.4 and the
are
two known examples
of this kind of PROTAC linker undergoing clinical trials.
tiononly
of protonatable
basic centres.
It is also possible that other physiochemical factors play a role in the relative success of
these
PROTACs;
for example, increased
solubility
due to lowering the logD7.4 and the
2. Treating
Neurodegenerative
Diseases
using PROTACs
introduction of protonatable basic centres.
Recently, several studies have conducted research in exploring the potential use of
PROTACs
as therapeutic toolsDiseases
for neurodegenerative
2.
Treating Neurodegenerative
Using PROTACsdiseases, namely for Alzheimer’s
Disease
(AD),
Parkinson’s
Disease
(PD),
frontotemporal
amyotrophic
Recently, several studies have conducted
research in dementia
exploring (FTD),
the potential
use of lateral
sclerosis
(ALS),
and
Huntington’s
Disease
(HD).
A
selection
of
the
most
promising
PROTACs as therapeutic tools for neurodegenerative diseases, namely for Alzheimer’s
studies has
been
discussedDisease
in the following
sections. dementia (FTD), amyotrophic
Disease
(AD),
Parkinson’s
(PD), frontotemporal
lateral sclerosis (ALS), and Huntington’s Disease (HD). A selection of the most promising
studies
discussed
in the(AD)
following sections.
2.1. Tauhas
andbeen
Alzheimer’s
Disease
The Tau protein plays a central role in neuronal cells to maintain their cell shape,
stabilise microtubules, and provide routes for the transport of cargo proteins. An imbalThe Tau protein plays a central role in neuronal cells to maintain their cell shape,
ance in the level of the Tau protein is a principal common factor for a range of neurodegenstabilise microtubules, and provide routes for the transport of cargo proteins. An imbalance
erative diseases, for example FTD and AD, and mediates the toxicity of amyloid-β (Aβ).
in the level of the Tau protein is a principal common factor for a range of neurodegenerative
The pathology
of these
diseases
hasand
been
heavilythe
associated
the toxic(Aβ).
accumulation
diseases,
for example
FTD
and AD,
mediates
toxicity with
of amyloid-β
The
of
aberrant
Tau
species
aggregating
into
paired
helical
filaments
and
neurofibrillary
pathology of these diseases has been heavily associated with the toxic accumulation of tangles, leading
to neuronal
cell death.
Currently,
are no
moleculestangles,
which are
aberrant
Tau species
aggregating
into paired
helicalthere
filaments
andsmall
neurofibrillary
able
to
modulate
its
dysregulation
as
Tau
is
a
non-enzymatic
(lacking
active
leading to neuronal cell death. Currently, there are no small molecules which arean
able
site/pocket)
protein
[36].
Hence,
Tau
downregulation
is
a
desired
therapeutic
strategy
to modulate its dysregulation as Tau is a non-enzymatic (lacking an active site/pocket) [4].
Peptide
PROTACs
have recently been
developed
to targetstrategy
the Tau[4].
protein for degraprotein
[36]. Hence,
Tau downregulation
is a desired
therapeutic
Peptide
PROTACs
been developed
to target
the Tau
protein forofdegradadation,
however,
thesehave
haverecently
been found
to suffer from
the usual
limitations
linear peption,
however,
these
have
been
found
to
suffer
from
the
usual
limitations
of
linear
peptides
tides targeting the CNS indications. TH006—a PROTAC peptide (Figure 4)—was revealed
targeting
CNS
indications.
PROTAC peptideN2a
(Figure
revealedblotting
to
to inducethe
Tau
degradation
in TH006—a
Tau-EGFP-overexpressed
cells4)—was
with Western
induce
Tau
degradation
in
Tau-EGFP-overexpressed
N2a
cells
with
Western
blotting
analanalysis, albeit at high concentrations (200 µM). Following this, TH006 was used in a
ysis,
albeit
at high
concentrations
(200 µM). Following
this, TH006
wasreduced
used in levels
a mouse
mouse
model
of AD
(15 mg/kg intranasal
and IV), which
revealed
of Tau
model of AD (15 mg/kg intranasal and IV), which revealed reduced levels of Tau in the
in the cerebral cortex and hippocampus [37]. However, the combined use of intranasal
cerebral cortex and hippocampus [37]. However, the combined use of intranasal and intraand intravenous administration for 10 days to take advantage of olfactory transfer prevenous administration for 10 days to take advantage of olfactory transfer presumable is
sumable is required due to poor blood-brain barrier (BBB) permeability, which is a comrequired due to poor blood-brain barrier (BBB) permeability, which is a common limitation
mon
limitation
for[37,38].
such peptides.
Notably,
this work
demonstrated
a method
for
such
peptides.
Notably, [37,38].
this work
demonstrated
a method
to regulate,
and to
regulate,
and
ultimately
degrade,
the
Tau
protein
on
a
post-translational
level,
which
ultimately degrade, the Tau protein on a post-translational level, which in turn offers a in
turn offers
a cellular quality-control
system
to rescue the
of Aβ.
cellular
quality-control
system to rescue
the neurotoxicity
of neurotoxicity
Aβ.
2.1. Tau and Alzheimer’s Disease (AD)
Figure
PROTAC
TH006.
Figure4.4.Representation
Representationofofthe
thepeptide
peptide
PROTAC
TH006.
An
from
thethe
abovementioned
study
demonstrated
Anapproach
approachwhich
whichtook
tookinspiration
inspiration
from
abovementioned
study
demonstrated
that a Keap1-dependent peptide PROTAC enables Tau knockdown via the
that a Keap1-dependent peptide PROTAC enables Tau knockdown via the UPS (Figure 5)
[39]. A tetramethylrhodamine (TAMRA) analogue was shown to be cell permeable
through measurements of fluorescence intensity, taking 12 h to reach its maximal cellular
concentration.
Biomolecules 2023, 13, 1164
7 of 23
UPS (Figure 5) [39]. A tetramethylrhodamine (TAMRA) analogue was shown to be cell
permeable through measurements of fluorescence intensity, taking 12 h to reach its maximal
cellular concentration.
Figure 5. Representation of a Keap1 peptide PROTAC.
Figure
peptide
PROTAC.
Figure 5.
5. Representation
Representationofofa aKeap1
Keap1
peptide
PROTAC.
A series of new small-molecule PROTACs have also been developed for targeted Tau
degradation.
For example,
QC-01-175,
a Tau-degrading
PROTAC
based
on theTau
PET tracer
A
PROTACs
have
also
been
developed
for targeted
A series
seriesof
ofnew
newsmall-molecule
small-molecule
PROTACs
have
also
been
developed
for targeted
Tau
T807, has been used
extensively in vivo
to visualisePROTAC
various based
tauopathies
(Figure
6) [40,41]
degradation.
on the
tracer
degradation.For
Forexample,
example,QC-01-175,
QC-01-175,a Tau-degrading
a Tau-degrading
PROTAC based
on PET
the PET
tracer
QC-01-175
was
shown
to degrade
Tau
at concentrations
ranging from
100
to 1 µM in
T807,
has been
used
extensively
in vivo
to visualise
various tauopathies
(Figure
6) nM
[40,41].
T807, has been used extensively in vivo to visualise various tauopathies (Figure 6) [40,41].
QC-01-175
was shown
to degrade
Tau at concentrations
fromvariant
100 nMfrom
to 1 µM in patien
patient-derived
neuronal
cell models,
the ranging
Tau-A152T
QC-01-175
was shown
to degrade
Tau atincluding
concentrations
ranging from
100 nM toa 1PSP
µM in
patient-derived
neuronal
cell models,
including
the
Tau-A152T variant from
a PSP
patient
and
the
Tau-P301L
variant
obtained
from
a
behavioural-variant
FTD
[42]. Cru
patient-derived neuronal cell models, including the Tau-A152T variant frompatient
a PSP patient
and
the
Tau-P301L
variant
obtained
from
a
behavioural-variant
FTD
patient
[42].
Crucially,
cially,
such
levels ofvariant
degradation
using
QC-01-175
were not observed
for the
normal
Tau
and
the
Tau-P301L
obtained
from
a behavioural-variant
FTD patient
[42].
such
levels
of degradation
using
QC-01-175
were
not observed for the normal
Tau and
lowCruand
low
levels
of
P-Tau
observed
in
wild-type
cells,
as
demonstrated
in
three
iPSC-derived
cially,ofsuch
levels
of degradation
werein
not
observed
for theneuronal
normal Tau
levels
P-Tau
observed
in wild-typeusing
cells, QC-01-175
as demonstrated
three
iPSC-derived
neuronal
cell
models.
andmodels.
low levels of P-Tau observed in wild-type cells, as demonstrated in three iPSC-derived
cell
neuronal cell models.
Figure
tracer
and
PROTAC
analogue.
Figure6.6.Tau
TauPET
PET
tracer
and
PROTAC
analogue.
Figure 6. Tau PET tracer and PROTAC analogue.
A
study
proposed
improvements
to thetolead
culminating
in two in two
Afollow-up
follow-up
study
proposed
improvements
thePROTAC
lead PROTAC
culminating
A follow-up
studyPROTACs
proposedwith
improvements
to the lead
PROTAC
culminating
in two
promising
Tau-targeting
improved activities,
namely
FMF-06-038
and FMFpromising Tau-targeting PROTACs with improved activities, namely FMF-06-038 and
06-049,
respectively
(FigurePROTACs
7) [43]. Thewith
complexities
evaluatingnamely
these Tau-degrading
promising
Tau-targeting
improvedof activities,
FMF-06-038 and
FMF-06-049,
respectively
(Figure
7) [43].
The complexities
of evaluating
these Tau-degrad
PROTACs
was
highlighted,(Figure
with these
PROTACs
performing
across
Tau-A152T,
FMF-06-049,
respectively
7) [43].
The complexities
ofvariably
evaluating
these
Tau-degrading
PROTACs
was
highlighted,
with
these
PROTACs
performing
variably
across Tau
Tau-P301L,
and
WT
Tau.
Second-generation
PROTACs
displayed
a
consistently
poor
ing PROTACs was highlighted, with these PROTACs performing variably across
TauA152T, Tau-P301L,
andWT
WTTau.
Tau.Second-generation
Second-generation
PROTACs
displayed
a consistently
permeability
in a CRBN
engagement assay;PROTACs
with
permeability
presenting
a
A152T, Tau-P301L,
and competitive
displayed
a consistently
poor permeability
permeability
aCRBN
CRBN
competitive
engagement
assay;
with
permeability
present
major
challenge thatinin
isamore
broadly
for PROTAC
optimisation.
Encouragingly,
reduced
poor
competitive
engagement
assay;
with
permeability
presentlevels
of
phospho-Tau
were
observed
for
up
to
8
days
following
the
cessation
of
treatment,
ing aa major
majorchallenge
challengethat
thatisismore
morebroadly
broadly
PROTAC
optimisation.
Encouragingly,
ing
forfor
PROTAC
optimisation.
Encouragingly,
re- re
showing
that the
regeneration
of were
phosphor-Tau
is for
a slow
process.
Potentially
poor
brain
duced
levels
of
phospho-Tau
observed
up
to
8
days
following
the
cessation
duced levels of phospho-Tau were observed for up to 8 days following the cessation of o
penetration,
lack of clinical
biomarkers,
and of
evaluation
of off-target
binding
were Potentially
all
treatment, showing
showing
thatthe
the
regeneration
phosphor-Tau
a slow
process.
treatment,
that
regeneration
of phosphor-Tau
is aisslow
process.
Potentially
explicitly mentioned as challenges to onward development.
poor brain
brain penetration,
penetration,lack
lackofofclinical
clinical
biomarkers,
and
evaluation
of off-target
binding
poor
biomarkers,
and
evaluation
of off-target
binding
were all
all explicitly
explicitlymentioned
mentionedasaschallenges
challenges
onward
development.
were
to to
onward
development.
Figure 7. Second-generation Tau PROTAC degraders.
Figure 7. Second-generation Tau PROTAC degraders.
Biomolecules 2023, 13, 1164
A152T, Tau-P301L, and WT Tau. Second-generation PROTACs displayed a consistently
poor permeability in a CRBN competitive engagement assay; with permeability presenting a major challenge that is more broadly for PROTAC optimisation. Encouragingly, reduced levels of phospho-Tau were observed for up to 8 days following the cessation of
treatment, showing that the regeneration of phosphor-Tau is a slow process. Potentially
8 of 23
poor brain penetration, lack of clinical biomarkers, and evaluation of off-target binding
were all explicitly mentioned as challenges to onward development.
Figure
7. Second-generation
Second-generation
Tau PROTAC
PROTAC
degraders.
the proteasomal
pathway
(Figure
8) [44].
Figure
7.
Tau
degraders.
C004019 exhibited an exceptionall
icity in both the HEK293 and SH-SY5Y cell lines, with an DC50 of 7.9 nM
a PROTAC
that
waswas
demonstrated
to degrade
the Tau
In 2021, Wang
Wang et
etal.
al.designed
designed
a PROTAC
that
demonstrated
to degrade
the
hTau
overexpressed
cell
line.
Intracerebroventricular
administration
Tau
protein
both
in
vitro
and
in
vivo,
preferentially
removing
pathological
Tau
proteins
protein both in vitro and in vivo, preferentially removing pathological Tau proteins via promo
8) [44].
exhibited anadministrations
exceptionally low at 3 m
via
theof
proteasomal
pathway
ance
pathological
Tau(Figure
in vivo,
andC004019
subcutaneous
cell toxicity in both the HEK293 and SH-SY5Y cell lines, with an DC50 of 7.9 nM in a
strated a robust and sustained Tau clearance. Most impressively, improved
HEK293-hTau overexpressed cell line. Intracerebroventricular administration promoted
cognitive
were
AD-like hTau
3× Tg transgenic
the
clearancefunctions
of pathological
Tauobserved
in vivo, andin
subcutaneous
administrations
at 3 mg/kg mice m
demonstrated
and sustained
Tau clearance.
Most impressively,
improved
particularlya robust
notable,
as the brain
concentration
of C004019
wassynaptic
low followin
and cognitive functions were observed in AD-like hTau 3× Tg transgenic mice models.
neous administration (10.8 ng/mL, which translates to a Kpuu value of on
This is particularly notable, as the brain concentration of C004019 was low following its
illustrates that
despite (10.8
the ng/mL,
inherent
challenges
achieving
good
BBB perm
subcutaneous
administration
which
translates toin
a Kpuu
value ofaonly
0.009).
This
illustrates meaningful
that despite thetherapeutic
inherent challenges
in achieving acan
good
permeability
PROTACs,
interventions
beBBB
observed.
Howeve
with
PROTACs,
meaningful
therapeutic
interventions
can
be
observed.
However,
it
is
portant to note, that administration by intragastrical gavage (20 mg/kg)
did
also important to note, that administration by intragastrical gavage (20 mg/kg) did not
reduction
in Tau,
indicating
thatsignificant
significant
optimisation
needed
lead
to a reduction
in Tau,
indicating that
optimisation
is needed is
to achieve
anto achi
appropriate
exposurefollowing
following oral
administration.
priate exposure
oral
administration.
Figure
8. Effective
in vitro
in vivo
PROTAC.
Figure
8. Effective
inand
vitro
andTau-degrading
in vivo Tau-degrading
PROTAC.
Several patents have also been published concerning the treatment of AD using
of which have
shown
PROTAC
technology
to target
the also
Tau protein
Several
patents
have
been [12,42,45,46],
published all
concerning
the
treatment
promise
so
far.
However,
replication
of
the
results
from
other
laboratories
is
needed,
PROTAC technology to target the Tau protein [12,42,45,46], alland
of which
further exploration of their viability as a therapeutic treatment for AD is required.
promise
far. However,
the
other
i
A pressso
release
by Arvinas inreplication
2019 claimedof
that
oneresults
of their from
PROTACs
in alaboratories
preclinical
modelexploration
was able to remove
95%viability
of pathological
and successfully
cross the
further
of their
as a Tau
therapeutic
treatment
forBBB,
AD is req
crucially
without
altering
the
wild-type
Tau
in
the
mouse
brain
24
h
after
parenteral
adA press release by Arvinas in 2019 claimed that one of their PROTACs i
ministration [47]. However, neither the structure of the small molecule PROTACs nor their
model was experimental
able to remove
95%
ofdisclosed.
pathological Tau and successfully cross
corresponding
data have
been
cially without altering the wild-type Tau in the mouse brain 24 h after pare
istration [47]. However, neither the structure of the small molecule PROT
corresponding experimental data have been disclosed.
2.2. Huntingtin and HD
Biomolecules 2023, 13, 1164
9 of 23
2.2. Huntingtin and HD
Huntington’s disease is an autosomal dominant neurodegenerative disorder that is
caused by an excessive expansion of a CAG trinucleotide repeat, leading to the formation of
polyglutamine-expanded mutant huntingtin (mHTT) protein aggregates which accumulate
and eventually lead to cell death. Although multiple different strategies, of varying degrees
of success, to develop effective therapies for HD are currently under investigation, a recent
small-molecule PROTAC strategy developed by Tomoshige et al. is currently of interest
(Figure 9, Tomoshige 1) [48]. Two PROTACs recruiting E3 ligases from the IAP family were
shown to induce the ubiquitination and subsequent degradation of mHTT in fibroblast cells
derived from two individuals with HD. Further validation with in vivo studies is necessary
to obtain a deeper understanding of the full potential for this approach in treating HD,
primarily due to wild-type Htt also being degraded. This could suggest that wild-type
Htt also forms small oligomers that can be recognised by aggregate binders, leading to
PROTAC-mediated degradation [49]. A follow-up study looked at switching to a more
potent E3 ligand; however, this did not lead to more potent PROTACs (Figure 9, Tomoshige
7)
[50].
further
of PROTAC
design—it
is not the
binding
but
is This
rather
the highlights
stability the
of complexity
the ternary
complex,
as depicted
previously
affinity for the E3 ligase nor the protein of interest that is important, but is rather the
[51,52].
stability of the ternary complex, as depicted previously in Figure 3B [51,52].
in Fi
Figure
generations
of mHTT-degrading
PROTACsPROTACs
by Tomoshige
al., which showed
Figure9.9.Two
Two
generations
of mHTT-degrading
by etTomoshige
et al.,an
which
initial
promise.
initial promise.
sh
2.3. LRRK2
2.3. Mutations
LRRK2 within leucine-rich repeat kinase 2 (LRRK2) encoded by PARK8—an impli-
cated gene
for PD—have
been
associated with
idiopathic
late-onset
PD. Toencoded
date, numerous
Mutations
within
leucine-rich
repeat
kinase
2 (LRRK2)
by PARK8—
missense mutations have been linked to PD pathogenesis, including R1441C, R1441G,
plicatedY1699C,
gene for
PD—have
been
with
late-onset
PD. To d
R1441H,
G2019S,
and I2020T,
andassociated
as such LRRK2
has idiopathic
been a popular
choice of tarmerous
missense
mutations
have
beena linked
PD pathogenesis,
including R
get
for therapeutic
modulation
[53,54].
Recently,
study by to
Konstantinidou
et al. disclosed
that
work had
commenced
on designing
andand
synthesising
PROTAC
capable
of degrading
R1441G,
R1441H,
Y1699C,
G2019S,
I2020T,a and
as such
LRRK2
has been a
LRRK2
Although
permeabilitymodulation
and target binding
wereRecently,
observed, these
PROTACs
choice[55].
of target
forcell
therapeutic
[53,54].
a study
by Konstan
were unable to induce ubiquitination and subsequent degradation, with difficulties in
et al. disclosed that work had commenced on designing and synthesising a PROT
assembling the ternary complex having been postulated as a possible reason. A follow-up
pable was
of degrading
LRRK2selective
[55]. Although
cell
permeability
and targetsugbinding w
patent
later filed claiming
modulators
of mutant
LRRK2 proteolysis,
served,that
these
PROTACs
unable totheinduce
ubiquitination
and subsequent d
gesting
progress
has beenwere
made regarding
CRBN-based
G2019S-LRRK2-PROTAC
degraders
(wit
G2019S-LRRK2
being
the
most
common
LRRK2
pathogenic
mutation)
[56].
tion, with difficulties in assembling the ternary complex having been postulated
a
Recently, Ciulli et al. published a substantial piece of work on LRRK2 PROTAC
sible reason. A follow-up patent was later filed claiming selective modulators of
degraders, culminating in XL01126 (Figure 10) [13]. Not only is the lead compound a
LRRK2
proteolysis,
suggesting
that progress
hasproperties
been made
regarding
the CRBN
potent
degrader
of LRRK2,
but it also exhibited
favourable
in both
in vitro and
G2019S-LRRK2-PROTAC
(wit
G2019S-LRRK2
being
the most
common
in
vivo experiments (C57BL/6 degraders
mice). Despite
having
a poor solubility,
XL01126
has an
pathogenic mutation) [56].
Recently, Ciulli et al. published a substantial piece of work on LRRK2 PROT
graders, culminating in XL01126 (Figure 10) [13]. Not only is the lead compound a
degrader of LRRK2, but it also exhibited favourable properties in both in vitro and
Biomolecules 2023, 13, 1164
G2019S-LRRK2-PROTAC degraders (wit G2019S-LRRK2 being the most common
pathogenic mutation) [56].
Recently, Ciulli et al. published a substantial piece of work on LRRK2 PROT
graders, culminating in XL01126 (Figure 10) [13]. Not only is the lead compound
of 23
degrader of LRRK2, but it also exhibited favourable properties in both10in
vitro and
experiments (C57BL/6 mice). Despite having a poor solubility, XL01126 has an or
vailability of 15% in rats with a half-life of 21.9 h—most likely protected from met
oral bioavailability of 15% in rats with a half-life of 21.9 h—most likely protected from
by its high degree of plasma protein binding. Moreover, XL01126 was detected a
metabolism by its high degree of plasma protein binding. Moreover, XL01126 was detected
centration
of 14ofnM
in both
the
theCSF.
CSF.
This
ability
to cross
theis BBB is
at
a concentration
14 nM
in both
thebrain
brainand
and the
This
ability
to cross
the BBB
2
2
unexpected
considering
its its
high
surface
area
perhaps
unexpected
considering
hightotal
totalpolar
polar surface
area
(194(194
Å ). Å ).
Figure
PROTAC
targeting
LRRK2LRRK2
for degradation.
Figure10.10.
PROTAC
targeting
for degradation.
2.4. Alpha-Synuclein
2.4.
Alpha-Synuclein
Another opportunity
opportunityto
totarget
targetaarange
rangeofofneurological
neurological
diseases
where
PROTAC
techAnother
diseases
where
PROTAC
technolnology
offer
improvements
current
therapeutic
methods
in targeting
α-synuogy
maymay
offer
improvements
overover
current
therapeutic
methods
is inistargeting
α-synuclein.
clein. α-Synuclein
is a protein
that accumulates
within
the neurons
of individuals
with
α-Synuclein
is a protein
that accumulates
within the
neurons
of individuals
with PD, leadPD,to
leading
to the formation
of Lewy
bodies
[57].
It is an intrinsically
ing
the formation
of Lewy bodies
[57].
It is an
intrinsically
disordereddisordered
protein thatprotein
drives
thatpathology
drives theof
pathology
of PD,
andtargeted
for which
targeted
protein degradation
via the may
prothe
PD, and for
which
protein
degradation
via the proteasome
teasome
may
offer
a
therapeutic
route.
offer a therapeutic route.
syntheticpeptide
peptidePROTAC
PROTAChas
hasrecently
recently
shown
a selective
degradation
of α-synuA synthetic
shown
a selective
degradation
of α-synuclein
cleinthe
viaproteasome
the proteasome
in a timeand dose-dependent
manner
within
neuroblastoma
via
in a timeand dose-dependent
manner
within
neuroblastoma
cells
ThisThis
study
demonstrated
that,
functionally,
thethe
reduction
in
and
neurons
[58].[58].
cells primary
and primary
neurons
study
demonstrated
that,
functionally,
reduction
α-synuclein
rescued
mitochondrial
dysfunction
and
cellular
defects,
which
were
consein α-synuclein
rescued
mitochondrial
dysfunction
and
cellular
defects,
which
were
conquential
of of
α-synuclein
overexpression,
indicating
at the
possibility
of utilising
thethe
peptide
sequential
α-synuclein
overexpression,
indicating
at the
possibility
of utilising
pepPROTAC
as
a
potential
strategy
to
treat
PD.
Further
validation
is
required
to
determine
tide PROTAC as a potential strategy to treat PD. Further validation is required to deterwhether
this method
has potential
clinical
applications.
mine whether
this method
has potential
clinical
applications.
In 2020, Kargbo summarised a series of small-molecule PROTACs
PROTACs (Figure
(Figure 11)
11) develdeveloped by Arvinas that are capable of degrading up to 65% of α-synuclein
α-synuclein at a concentration
of
These small-molecule
small-molecule PROTACs
PROTACs consisted
consisted of
of an
an ααof 11 µM
µM in
in various
various cell
cell lines
lines [59,60].
[59,60]. These
synuclein-binding
domain,
a
linker,
and
different
E3
ligase-binding
domains,
namely
for
synuclein-binding domain, a linker, and different E3 ligase-binding domains, namely for
the
of the
the VHL,
VHL, CRBN,
CRBN, IAP,
IAP, and
and MDM2
MDM2 E3
E3 ligases.
ligases. Independent
Independent reproduction
reproduction of
the results
results is
is
required
in
order
to
fully
realise
the
claims
made
therein;
however,
it
is
an
encouraging
required in order to fully realise the claims made therein; however, it is an encouraging
starting
starting point
point in
in exploring
exploring aa targeted
targeted protein
protein degradation
degradation therapeutic
therapeutic approach
approach using
using
small-molecule
PROTACs
in
tackling
PD.
small-molecule PROTACs in tackling PD.
Figure 11.
11. α-synuclein-degrading
Figure
α-synuclein-degrading PROTACs.
PROTACs.
2.5. C-TDP-43
The TAR DNA-binding protein 43 (TDP-43) is another example of a misfolded protein implicated in a number of neurodegenerative diseases, such as amyotrophic lateral
sclerosis (ALS), frontotemporal dementia (FTD), and limbic-predominant age-associated
TDP-43 encephalopathy (LATE) [61]. The successful removal of the toxic C-terminal form
Figure 11. α-synuclein-degrading PROTACs.
Biomolecules 2023, 13, 1164
2.5. C-TDP-43
11 of 23
The TAR DNA-binding protein 43 (TDP-43) is another examp
2.5. C-TDP-43
tein
implicated in a number of neurodegenerative diseases, such a
The TAR(ALS),
DNA-binding
protein 43 (TDP-43)
is another example
of a and
misfolded
protein
sclerosis
frontotemporal
dementia
(FTD),
limbic-predom
implicated in a number of neurodegenerative diseases, such as amyotrophic lateral sclerosis
TDP-43
encephalopathy
(LATE)
[61]. The successful
removal
(ALS),
frontotemporal
dementia (FTD),
and limbic-predominant
age-associated
TDP-43of the
encephalopathy
(LATE)
[61]. The successful
removal of the
toxic C-terminal
form
by theJMF4560
use
by
the use of
a PROTAC
has recently
been
reported
[62].
of a PROTAC has recently been reported [62]. JMF4560 (Figure 12) displayed selectivity over
selectivity
the
endogenous
TDP-43,
Westerndepletion
blot analysis
the endogenousover
TDP-43,
with
Western blot analysis
showingwith
a near-complete
at
5
µM
in
a
cellular
model.
plete depletion at 5 µM in a cellular model.
Figure 12. C-TDP-43-degrading PROTAC, JMF4560.
Figure 12. C-TDP-43-degrading PROTAC, JMF4560.
As with non-brain diseases, PROTACs are the leading technology that is currently
being exploited, with other modalities still at significantly earlier stages. This pattern is
replicated in the field of CNS diseases, with wider non-PROTAC technologies only recently
starting to be reported.
3. Antibody PROTACs
PROTACs have shown great promise in the treatment of a range of CNS diseases.
However, they are not generally tissue specific due to the inclusion of E3 ligases with
expansive expression. Despite the discovery of numerous ligases with an attested tissue
specificity, including brain specific ligases, none of these ligases have yet been utilised
to develop an effective tissue-specific PROTAC [63–66]. The development of a tissue- or
cell-specific PROTAC could prevent protein degradation in normal cells and significantly
reduce the side effects of these compounds.
The inclusion of antibodies in PROTAC technology is a new and emerging field of
research, which has proven to be effective in providing these compounds with an enhanced
tissue and cell specificity. There are two major categories of antibody PROTACs—antibody–
PROTAC conjugates and antibody-based PROTACs (AbTACs) [67].
Antibody–PROTAC conjugates were derived from the existing antibody–drug conjugates but have the added benefits of the small-molecule PROTACs [68]. This new class of
drugs consists of PROTACs connected to cell-specific antibodies through a cleavable linker
and directs the PROTACs to specific cells for the internalisation and degradation of the
POI. Maneiro and colleagues demonstrated the specificity of these drugs by creating an
antibody–PROTAC conjugate which degraded bromodomain-containing protein 4 (BRD4)
specifically in HER2-positive breast cancer cell lines without affecting the BRD4 levels in
HER2-negative cells. Their antibody-dependent specificity was further confirmed by assessing the same PROTAC molecule with the antibody removed. This PROTAC demonstrated
an effective degradation in both cell lines, thereby confirming the antibody’s ability to provide cell specificity [69]. Dragovich and others also developed several antibody–PROTAC
conjugates targeting ERα in HER2-positive cells and gave evidence for efficient intracellular
degrader release in the HER2 cells alone [70]. In addition to tissue selectivity, there a num-
Biomolecules 2023, 13, 1164
12 of 23
ber of other potential advantages to these compounds, such as improved pharmacokinetic
properties, and simpler routes for administration [71,72].
AbTACs are recombinant bispecific antibodies and differ from antibody–PROTAC
conjugates since they possess the ability to bind to both the POI and an E3 ubiquitin
ligase without the need for additional components. These antibody-based PROTACs
use the lysosomal pathway for the degradation of their targets and could allow a wider
range of applications due to their ability in being able to degrade challenging membrane
proteins [73]. Cotton and colleagues recently reported the first AbTAC, which allowed the
degradation of the cell-surface protein PD-L1 (programmed death-ligand 1). This study
presented recombinant AbTACs with the ability to degrade 63% of the protein in vitro
through its colocalisation with the RNF43-membrane-bound E3 ligase [73,74]. Despite the
capability of both types of antibody compounds, there are currently no reports of AbTACs
targeting the central nervous system. This may be due to the difficulty of developing
cell-specific antibodies which can penetrate the blood–brain barrier along with the high
manufacturing costs of these constructs [71]. However, there have been a number of
brain penetrant antibodies showing great promise in clinical trials, which could aid the
development of specific and brain penetrant antibody PROTACs [75].
4. The Potential of New Degrader Technology
Despite the potential ability of PROTAC technology in being able to target many of
the proteins mentioned above, there are many limitations to their use as pharmaceutical
compounds, particularly with regard to targeting CNS diseases. PROTACs are classically
large compounds with high molecular weights (>800 kDa) and high polar surface areas,
which gives rise to particular limitations, including their low solubility, poor cell permeability, low oral bioavailability, and critically, poor penetration of the BBB [76]. Another
clear limitation with PROTACs is their inherent dependence on the choice of E3 ligase subunit, which evidently can limit their application in particular cell types. BBB permeability
and efficiency may be impacted by varying molecular weights, modulated by a range of
linker lengths and types, different POI-binding ligands, and differing E3 ligase-binding
ligands. Localisation of the PROTAC to specific brain regions could also be a potential
limitation, with the most widely utilised E3 ligases, CRBN and VHL, being indiscriminately
expressed throughout the brain [23]. The dependence on E3 ligase subunits can also lead to
cancer cell resistance following chronic PROTAC treatment, as demonstrated in a study by
Zhang et al., where cancer cells acquired resistance to both VHL-based and CRBN-based
BET PROTACs [77,78]. Lastly, as the PROTAC technology depends on degradation via the
intracellular UPS system, many proteins, including extracellular protein targets, cannot be
pursued by PROTACs [79]. Extracellular proteins, which account for approximately 40% of
the proteome, and other biomolecules cannot be targeted through accessing the UPS system.
These biomolecules come in a variety of significant classes with differing roles, such as
growth factors, proteins with expanded repeat sequences, protein aggregates, and other
non-protein molecules, which are of high importance in disease progression [77,80,81].
5. Hydrophobic Tags
The use of hydrophobic tags (HyTs) presents an alternative approach to PROTACs for
the degradation of proteins, but also utilises the proteasomal pathway to conduct its activity.
Hydrophobic tag degraders are bifunctional molecules with a ligand for the protein of
interest (POI) attached to a hydrophobic tag, such as adamantane or tert-butyl carbamateprotected arginine (Boc3 Arg). Partially denatured proteins, where the core hydrophobic
residues are exposed, are quickly degraded by the UPS system [82,83]. The hydrophobic
tag, which mimics the exposed hydrophobic core residues of a denatured protein, interacts
with the chaperone proteins, heat shock proteins 70 and 90 (HSP70/90), and these direct
them to the CHIP E3 ligase for protein ubiquitination and proteasomal degradation [84].
HyTs offer advantages over PROTACs as they usually have smaller molecular weights and
fewer hydrogen bond donors and hydrogen bond acceptors, leading to more favourable
Biomolecules 2023, 13, 1164
13 of 23
properties for CNS drugs (in particular with regard to the penetration of the blood–brain
barrier) [85].
Several hydrophobic tag degraders have been developed for the treatment of CNS
diseases. Gao and colleagues synthesised a hydrophobic degrader which targeted the
Tau protein associated with Alzheimer’s disease and other tauopathies. This degrader
contained adamantane as the hydrophobic tag, YQQYQDATADEQG peptide as the Tau
binding ligand, a GSGS peptide linker, and a poly-D-arginine cell-penetrating peptide. A
reduction in the level of Tau in vitro and in the brain of AD mouse models was observed
by this degrader, as well as an effective cell penetration in wild-type N2a cells and their
ability to cross the BBB [86].
In addition to this, Gao and colleagues also developed several single and double
hydrophobic tag degraders to target the amyotrophic lateral sclerosis (ALS)-associated
protein, TDP-43. Among these degraders was the peptide-based degrader D4, which
showed the strongest degradation potential in cells and in a transgenic drosophila model,
as well as a low cytotoxicity. D4 is composed of two adamantane groups as hydrophobic
tags, an EDLIIKGISV peptide TDP-43 recognition motif, a KGSGS peptide linker, and
a GRKKKRRWRRR cell-penetrating peptide. Despite the degradation activity achieved
in vivo in this study, high doses of 20–150 µM were required for the different hydrophobic
tag degraders activity in cells, and several cytotoxicity issues were also observed for
some of these degraders [49,87]. Hirai and colleagues have also developed a hydrophobic
tag degrader targeting the disease-causing mutant huntingtin protein, from what was
originally a PROTAC [85]. The conversion of this PROTAC to a hydrophobic tag degrader
decreased the molecular weight and reduced the number of hydrogen bond donors and
acceptors, which in turn improved the permeability into the CNS whilst retaining the drug’s
potency. This study allowed for the discovery of a potentially brain-penetrant degrader
of mHTT through IAM chromatography analysis and an in vivo brain penetration assay.
This hydrophobic tag consisted of an adamantane hydrophobic tag and a known mHTT
aggregate-binding moiety. Their results proved to be successful with the development of a
brain permeable HyT capable of inducing the selective degradation of mHTT of up to 60%
at concentrations of 5 µM. Selective degradation, however, could not be achieved at higher
concentrations. This study shows promise and highlights the advantages of hydrophobic
tag degraders as an alternative approach for targeting CNS diseases. However, as was the
case for the initial PROTAC work, further studies are required to demonstrate the potential
of this approach, and in particular to replicate the current single-case examples that have
been published.
6. The Lysosomal Pathway
In order to target the biomolecules that cannot be degraded by the UPS pathway, efforts
have been made to create alternative degraders that can access the lysosomal degradation
pathway, which include the lysosomal-targeting chimeras (LYTACs), autophagy-targeting
chimeras (AUTACs), and autophagosome-tethering compounds (ATTECs). The lysosomal
pathway is important in regulating extracellular and intracellular homeostasis in cells, as
well as for many other processes. Degradation by the lysosome can occur using two major
pathways: autophagy and the endosome–lysosomal pathways [88]. For the endosome–
lysosomal pathway, substrates, such as extracellular and membrane-bound proteins, enter
the cell by interacting with a recycling receptor triggering endocytosis, a process in which
the plasma membrane invaginates to enclose the substrate (bound to the receptor) in a
vesicle [89]. Then, early endosomes are fully formed in the cytoplasm, and their slightly
acidic pH means that the receptors dissociate from the cargo and are returned by the
recycling endosomes to the surface, where they can be reused and allow for the endocytosis
of further cargo. Meanwhile, the internalised cargo is transported to the late endosomes
and then to the lysosome [90]. The lysosome contains numerous acidic hydrolases that
allow for the breakdown of the cargo substrate by hydrolysis. This process is summarised
in Figure 13.
Biomolecules 2023, 13, 1164
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Figure 13. The degradation of a substrate via the endosome–lysosomal pathway.
The degradation of cargo through the autophagy pathway uses a slightly different
mechanism, but also ends with the hydrolysis of cargo by the lysosome. Autophagy is a
complex process involving numerous molecules and signalling events. Initially, various
triggers can lead to the nucleation of a phagophore, where certain cytoplasmic compounds
are enveloped by a double membrane. LC3 receptor-type proteins insert themselves into
the phagophore membrane, and the cargo is degraded both non-selectively and selectively.
In selective autophagy, specific cargo (K63-ubiquitinated proteins) will interact indirectly
with the LC3-II membrane-bound proteins through adaptor proteins, such as p62/SQSTM1,
and become enclosed as the phagophore develops [91,92]. In bulk (non-specific) autophagy,
random cytoplasm containing non-specific cargo is isolated and fully enclosed as the
phagophore elongates and the edges fuse to give an autophagosome vesicle [93]. Following
the elongation of the phagophore to give the autophagosome, this structure fuses with the
lysosomes to form an autolysosome. The lysosomal acidic hydrolases then degrade the
enclosed cargo and the inner membrane; the resulting products are recycled back to the
cytosol [94–96]. This process is summarised in Figure 14.
Figure 14. The non-specific bulk and specific degradation of cargo via the autophagy pathway.
Biomolecules 2023, 13, 1164
15 of 23
7. Lysosomal-Based Degraders
Degraders that use the lysosomal pathway are based on a similar concept to PROTACs
in the way that they are composed of a ligand that binds to the molecule of interest and
a ligand that triggers its degradation, usually tethered together by a linker. There are
different types of compounds that have different properties which can be used to degrade
the different types of biomolecules, which will be summarised in this review.
7.1. LYTACs
Different types of LYTACs (lysosomal-targeting chimeras) have been developed which
can target extracellular and membrane-bound proteins [77]. These LYTACs consist of
a moiety that binds to the substrate molecule, which is attached to a ligand that can
bind to a lysosome-shuttling receptor [76]. The substrate protein bound to these LYTAC
compounds can undergo internalisation via clathrin-mediated endocytosis; this is triggered
by the LYTAC’s interaction with a membrane-bound receptor and involves the membrane
forming around the protein–LYTAC–receptor complex [88]. The substrate then progresses
through to the early endosomes and late endosomes, and this then fuses with the lysosome
where the substrate is degraded, as previously described. The Bertozzi lab developed the
first-generation LYTAC compounds that contained a 20- or 90-mer of mannose-6-phosphate
(M6P), a glycan ligand, as the receptor binding ligand. This LYTAC has been stated to bind
non-covalently to the substrate and allow its transport into the cell via interactions with the
M6P ligands with the cation-independent M6P receptors (CI-M6PR) [97].
This concept was further explored with the development of LYTACs with targetspecific antibodies; these different antibodies were conjugated to a poly(M6Pn)-bearing
glycoprotein to form target-specific LYTACs. Bertozzi and colleagues further demonstrated
the potential of LYTACs as therapeutics, as they showed their ability to degrade the
membrane protein epidermal growth factor receptor (EGFR), CD71, and programmed
death-ligand 1 (PDL1) [97]. In addition to this, a liver-specific LYTAC was developed by
Tang and colleagues that permitted endocytosis and lysosomal degradation by binding
to the asialoglycoprotein receptor (ASGPR) [98]. Whilst LYTACs are uniquely able to
degrade extracellular proteins, they suffer from major practical considerations related to
their significant size, as shown in Figure 15, resulting in a poor tissue permeability and a
low CNS penetration. In addition to this, the LYTACs’ receptor binding ligand (M6P) is
challenging to synthesise, and their peptide-like structure mean that immune responses
were hypothesised to be an issue in vivo [43,68,69]. The antibody-based LYTACs also have
some additional challenges, including the high cost, potential immunogenicity, and lack of
understanding about the endocytosis mechanism [99–101].
Figure 15. The degradation of substrates using LYTACs.
Biomolecules 2023, 13, 1164
16 of 23
7.2. AUTACs
Autophagy-targeting chimeras (AUTACs) are bifunctional molecules that degrade
their targets by accessing the previously mentioned autophagy pathway. Similar to the
PROTAC’s structure, they contain a specific ligand for the target molecule that is attached
by a linker to a ligand that triggers ubiquitination. However, instead of triggering the K48
polyubiquitination of the substrate as for the PROTACs, AUTACs trigger K63 polyubiquitination; this type of polyubiquitination leads to the degradation of the substrate through the
autophagy pathway. The degradation tag commonly used in these molecules is a guanine
derivative; this mimics the S-guanylation of the invading group A streptococci (GAS) by
the endogenous nucleotide 8-nitroguanosine 3′ ,5′ -cyclic monophosphate (8-nitro-cGMP).
S guanylation is a post-translational modification that marks the invading GAS for K63
polyubiquitination and subsequent degradation [76,102]. Once the substrate has been ubiquitinated it is recognised by p62/SQSTM1, a ubiquitin-binding receptor, and is shuttled
to the phagophore. In the autophagosome, the p62/SQSTM1 receptor binds to LC3-II, a
ubiquitin-like membrane-bound protein which docks the ubiquitinated substrate into the
phagophore [103].
In a study by Takahashi and colleagues, AUTACs were developed which downregulated cytosolic proteins and improved mitochondrial turnover by degrading the small,
fragmented mitochondria. The mitochondria-degrading AUTAC was able to significantly
improve mitochondrial activities in down syndrome (DS)-derived fibroblast cells [102,104].
Chang and colleagues developed different a AUTAC technology, which allows for the
binding to the ZZ domain of the p62/SQSTM1 receptor [105]. This technology allowed
for the degradation of various oncoproteins and degradation-resistant aggregates under
conditions of neurodegeneration at nanomolar DC50 values in vitro and in vivo. One application of this technology was to degrade the aggregation prone P301L Tau mutant to
treat Alzheimer’s disease. The AUTACs PBA-1105 and PBA-1106 (Figure 16) were able
to induce the degradation of mutant Tau in SH-SY5Y cells at a DC50 of approximately
1–10 nM and effectively removed aggregated Tau from mouse brain. In addition to this,
these AUTACs (shown in Figure 16) were also able to induce the lysosomal degradation
of both the nucleus- and cytosol-resistant mutant huntingtin, suggesting that AUTACs
could be applied to target a range of protein aggregates in CNS diseases [105]. Similar
types of AUTAC degraders have also been developed to degrade many other proteins, such
as MetAP2, FKBP12, BET, and TSPO, demonstrating the potential of this technology in
cancer, neurodegenerative diseases, and several other diseases [106]. In spite of the success
and many advantages of AUTACS, including its broad and specific spectrum of targets,
this technology is in its infancy and independent replications of these key findings are
required. Currently, the mechanism of selective degradation is still not fully understood,
and it is still to be determined whether AUTACs affect global autophagy or any other
cellular functions [101].
7.3. Molecular Glues and ATTECs
Molecular glues are an intriguing alternative to PROTACs that have several distinct
advantages due to their mechanism of action [107]. Molecular glues do not require a
lipophilic pocket in either the POI or the ligase, but rather promote direct protein–protein
interactions between both. This can result in simplified chemical structures that adhere
more closely to the typical drug discovery physiochemical properties. This has obvious
benefits considering their pharmacodynamic properties, but perhaps more importantly
in the context of this review, they stand a greater chance of achieving blood–brain barrier permeability due to their lower MW and TPSA. One critical drawback of molecular
glues is that their rational design is currently near-impossible as they are often found
serendipitously. Indeed, there are instances where they have been discovered by accident
in PROTAC research [108,109]. Care is needed so as not to assume biological changes
following treatment with PROTACs are always due to their intended mechanism of action,
in addition to maximising the potential of molecular glues when they are found.
Biomolecules 2023, 13, 1164
such as MetAP2, FKBP12, BET, and TSPO, demonstrating the potential of this technology
in cancer, neurodegenerative diseases, and several other diseases [106]. In spite of the success and many advantages of AUTACS, including its broad and specific spectrum of targets, this technology is in its infancy and independent replications of these key findings
are required. Currently, the mechanism of selective degradation is still not fully under17 of 23
stood, and it is still to be determined whether AUTACs affect global autophagy or any
other cellular functions [101].
Figure
Figure 16.
16. AUTACs targeting mutant Tau for degradation.
Autophagy-tethering
compounds (ATTECs) are a specific class of molecular glues
7.3. Molecular
Glues and ATTECs
that can degrade proteins and other molecules through the autophagy pathway but access
Molecular glues are an intriguing alternative to PROTACs that have several distinct
the autophagy pathway using a different mechanism to the AUTACs. Unlike AUTACs,
advantages due to their mechanism of action [107]. Molecular glues do not require a lipothe ATTECs’ activities are not dependent on ubiquitination, and they offer a more direct
philic pocket in either the POI or the ligase, but rather promote direct protein–protein
approach for degrading the molecules of interest, including biological molecules other than
interactions between both. This can result in simplified chemical structures that adhere
proteins by autophagy [76]. These molecules exert their role by tethering the molecule of
interest directly to the autophagosome via the LC3 membrane protein present on nascent
autophagosomes (phagophores) [106]. This technology has been applied to target Huntington’s disease and the mHTT (mutant huntingtin) protein. A few different ‘molecular glues’
like ATTECs were developed by Zhaoyang and colleagues that were able to degrade the
mHTT protein and not the wild-type protein due to their interactions with the extended
polyglutamine (PolyQ) sequence on the mHTT protein. Two of these compounds were
found to be effective for the degradation of mHTT in vivo HD mouse models at nanomolar
concentrations and were also able to cross the BBB (10O5 and AN2, Figure 17) [110]. These
compounds were investigated further to see whether they could target other pathogenic
species with extended polyQ sequences, and 10O5 and AN2 were both found to be effective
in degrading the mutant ataxin-3 (ATXN3) protein in fibroblasts derived from patients with
the neurodegenerative disease spinocerebellar ataxia type 3 [111]. In addition to this, these
compounds have been applied to degrade lipid droplets, which function as lipid-storing
locations in the cell. Yuhua and colleagues developed bifunctional ATTEC molecules consisting of the LC3-binding motifs (used to degrade mHTT) and a lipid droplet-binding
motif, derived from oil red O, which were connected by linkers [112]. These compounds
were able to degrade lipid droplets in cells, which was not possible with the use of the
PROTACs or AUTACs. This demonstrates the potential of this technology to degrade
non-protein molecules [113,114]. These molecules offer a large advantage over other technologies when targeting CNS diseases, due to their low molecular weights and drug-like
properties, meaning that they are more likely to penetrate the BBB. The ability to target a
wider range of molecules, including those other than proteins, is additionally attractive.
However, there have only been a few reports published on this topic, and further examples
are needed to gain confidence in the ultimate utility of this technology. Furthermore, additional LC3-bound chemical moieties need to be discovered, and further studies need to be
conducted on designed chimeras for the advancement of this technology [76].
Biomolecules 2023, 13, 1164
ular weights and drug-like properties, meaning that they are more likely to penetrate the
BBB. The ability to target a wider range of molecules, including those other than proteins,
is additionally attractive. However, there have only been a few reports published on this
topic, and further examples are needed to gain confidence in the ultimate utility of this
technology. Furthermore, additional LC3-bound chemical moieties need to be discovered,
18 of 23
and further studies need to be conducted on designed chimeras for the advancement
of
this technology [76].
Figure
Figure 17.
17. Effective ATTECs targeting mHTT shown to cross the BBB.
7.4. Chaperone-Mediated Autophagy Degraders
Chaperone-mediated autophagy degraders have also been developed to target CNS
diseases [84]. These degraders contain a specific protein-binding ligand and a chaperonebinding ligand, such as the pentapeptide KFERQ motif and the VKKDQ peptide found
on α-synuclein. The chaperone-binding motif present on these degraders bind to the
heat shock cognate 71 KDa (HSC70) chaperone protein and other co-chaperones, which
allows for the delivery of the degrader and the associated protein to the lysosome for
degradation [115]. A few examples of chaperone-mediated autophagy degraders have
been developed, with one example by Fan and colleagues targeting α-synuclein [116,117].
This degrader contained a cell-penetrating peptide (CPP) sequence TAT, a short amino
acid sequence binding α-synuclein (βsyn36), and the chaperone-mediated autophagy
targeting motif (CTM) KFERQ sequence. This chaperone-mediated autophagy degrader
demonstrated an effective degradation of the protein in rat neuronal cultures and in rats’
brains in vivo. Chaperone-mediated autophagy degraders have also been employed by
Bauer and colleagues to degrade mHTT in the mouse brain. Expression of a 46 amino
acid peptide adaptor molecule compromising two copies of the polyglutamine-binding
peptide 1 (QBP1) sequence and the two KFERQ and VKKDQ HSC70-binding motifs were
used to achieve this degradation and improve motor impairment [49]. This technology
has potential to be adapted to therapeutics for various CNS diseases but is still at its
earliest stages.
As with all new technologies, the ultimate successful application requires navigation
of the well-described hype-hope cycles, and this is particularly true in areas of disease
where there are no chances of disease-modifying therapies in the near- to mid-term. The
ability to remove proteins by the use of PROTAC technologies has clearly opened enormous opportunities within various cancers—with a number of programmes having been
evaluated in patient-based studies. The extension of protein degradation to aggregate
removal is of particular interest in the neurodegenerative field, and the small-molecule
approaches here may well offer distinct advantages over the current antibody approaches.
However, the additional challenges involved in successfully progressing these PROTAC
oncology projects to the clinic should not be overlooked—and a clear appreciation is required of the uphill struggle that is needed to be able to bring a CNS-targeted PROTAC to
the same development stage. However, the growing efforts in this area are encouraging,
and hopefully through this collective effort, breakthroughs will be made that will allow for
the therapeutic potential of these approaches to be realised.
Author Contributions: Writing—original draft preparation, B.a.I.T., H.L.L., D.H.J. and S.E.W.;
writing—review and editing, B.a.I.T., H.L.L., D.H.J. and S.E.W. All authors have read and agreed to
the published version of the manuscript.
Funding: B.a.I.T. is funded by a PhD studentship 50% from by the Coleg Cymraeg Cenedlaethol;
H.L.L. is funded through a PhD studentship from Janssen.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Biomolecules 2023, 13, 1164
19 of 23
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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