Keywords PROTACs - targeted protein degradation - chemistry-driven - integrated innovations
Introduction
Since its inception in the early 2000s, proteolysis-targeting chimera (PROTAC) technology
has revolutionized therapeutic strategies by introducing a novel event-driven mechanism.
Unlike conventional small molecule therapeutics that rely on high-affinity binding
through an occupancy-driven mechanism, PROTACs catalyze the degradation of target
proteins via the ubiquitin-proteasome system. This allows for potent biological activity
even at sub-stoichiometric concentrations of the targeting ligand.[1 ]
[2 ]
[3 ]
[4 ] Compared with traditional inhibitors, PROTACs can eliminate proteins that lack deep,
well-defined ligandable pockets, such as transcription factors (TFs, e.g., MYC, E2F1),
scaffolding proteins, and mutant or overexpressed oncoproteins that have evolved resistance
to orthosteric blockade.[5 ]
[6 ]
[7 ]
[8 ]
Despite their therapeutic potential, PROTACs face substantial challenges in their
development. It is well known that the optimization process is demanding and often
benefits from high-throughput chemical synthesis coupled to systematic cellular degradation,
selectivity, and ADME/Tox assays, although such workflows are not strictly indispensable.[9 ]
[10 ] A PROTAC is a heterobifunctional molecule composed of three main components: a ligand
that binds to the target protein, a linker, and a recruitment ligand that binds to
an E3 ubiquitin ligase. The molecular architecture of PROTACs is a critical aspect
of their design. Specifically, the optimization of linker length, flexibility, and
chemical composition is essential for achieving optimal ternary complex formation.
The ternary complex refers to the complex formed between the target protein, the PROTAC,
and the E3 ubiquitin ligase. This complex is necessary for efficient ubiquitination
and subsequent degradation of the target protein by the proteasome.[11 ] Moreover, the substantial molecular weight characteristic of PROTACs presents significant
pharmacological challenges, particularly in terms of cellular membrane permeability,
aqueous solubility, and overall drug-likeness. These physicochemical limitations impose
additional constraints on the development of effective degraders, necessitating innovative
strategies to optimize their pharmacokinetic profiles while maintaining degradation
efficacy.[12 ]
To address these challenges, a multidisciplinary innovation strategy is essential.
Critical to this endeavor is the development of advanced synthetic methodologies capable
of efficiently constructing these complex heterobifunctional molecules. The rapid
assembly of diverse PROTAC libraries can be achieved by implementing modular synthetic
approaches, coupled with combinatorial chemistry strategies. This may involve the
establishment of novel synthetic routes or strategic adaptation of existing organic
transformations to enhance synthetic efficiency, scalability, and structural diversity.[13 ]
[14 ]
[15 ]
On the other hand, high-throughput screening methods have been developed to accelerate
the evaluation of the biological activity of newly synthesized PROTACs.[16 ] The design of PROTACs needs to be refined to improve their drug-like properties,
ensuring optimal molecular weight for cellular permeability and solubility.[17 ] And identifying suitable ligands for undruggable targets is another critical area
of interest, requiring a deep understanding of protein–ligand interactions to minimize
any potential toxic effects.[18 ] Computational modeling can be utilized to forecast the impact of structures on activities,
thereby reducing reliance on empirical methods and saving substantial time and resources.[19 ]
This perspective does not intend to provide a comprehensive overview of PROTAC applications
in therapeutic interventions, as this aspect has been extensively reviewed in the
literature.[4 ]
[7 ] Rather, it focuses on elucidating the pivotal role of innovative chemical strategies
in advancing PROTAC technology, with particular emphasis on the integration of cutting-edge
synthetic methodologies and rational molecular design principles to overcome existing
limitations in the field ([Fig. 1 ]).
Fig. 1 Illustration of chemical technologies and proteolysis-targeting chimera strategy.
Modular Reactions-Enabled PROTACs
Modular Reactions-Enabled PROTACs
Click Chemistry
The construction of a PROTAC molecule must incorporate two indispensable binding moieties,
one that binds to the E3 ubiquitin ligase and the other that binds to the protein
of interest (POI). Unlike many designs that rely on preidentified ligands, the design
of PROTAC molecules is largely dependent on the linkers, specifically the linkage
site, length, flexibility, and chemical composition of the linker.[17 ] Consequently, linker optimization constitutes a critical step in the chemical synthesis
of PROTACs. However, due to the structural heterogeneity and diverse binding topologies
exhibited by potential target proteins, linker design presents significant challenges.
This necessitates not only extensive synthetic efforts in conventional laboratory
settings but also exploration through in situ approaches, thereby underscoring the critical need to establish robust and versatile
linking strategies. In this context, “click chemistry” has emerged as a particularly
effective platform for the parallel synthesis of diverse PROTAC libraries ([Fig. 2 ]).[20 ]
[21 ] For example, the formation of a new triazole ring through the coupling of azides
and alkynes through the copper-catalyzed Huisgen 1,3-dipolar cycloaddition reaction.
It proved to be ideal due to its biological orthogonality and rapid reaction characteristics.
Fig. 2 General strategy using “click chemistry” for the parallel synthesis of proteolysis-targeting
chimeras.
In 2018, Wurz et al proposed a “click chemistry” methodology to assemble PROTACs.[22 ] As shown in [Fig. 3 ], this technique can be applied to synthesize bromodomain and extra-terminal domain-4
(BRD4) degraders, utilizing the ligand JQ-1 (1 ), along with binders for the ligases Von Hippel-Lindau (VHL) and Cereblon (CRBN).
In this process, an AlphaScreen proximity assay was employed to assess the formation
of the required ternary complex consisting of ligase, PROTAC, and target protein.
Additionally, a Meso Scale Discovery platform and a commercially produced detection
antibody were implemented to quantify the level of target protein degradation mediated
by the PROTACs in cells.
Fig. 3 The employment of “Click Reaction” for the synthesis of PROTACs degrading the BET
bromodomain BRD4 protein. BRD4, member of the BET bromodomain family; PROTACs, proteolysis-targeting
chimeras.
In 2022, Cross et al utilized “click chemistry” to develop a series of PROTACs based
on entinostat, a class I histone deacetylase (HDAC) inhibitor under clinical investigation
for solid tumors and hematological malignancies ([Fig. 4 ]).[23 ] Among them, a novel compound, JMC-137 (2 ) demonstrated effective degradation activity against HDAC1/2 and HDAC3 in HCT116
cells. However, comparative analysis revealed its degradation efficacy to be substantially
lower than that of previously reported class I HDAC-targeting PROTACs. This finding
highlights the critical importance of three key design parameters: optimal selection
of HDAC-targeting warheads, strategic positioning of linker attachment points, and
precise spatial orientation in the ternary complex architecture, collectively underscoring
the intricate nature of PROTAC optimization. Notably, the application of click chemistry
has recently been extended to the development of degraders targeting oncologically
relevant proteins, including glutathione peroxidase 4 (GPX4) and peptidyl-prolyl cis–trans
isomerase NIMA-interacting 1 (PIN1), demonstrating its versatility in PROTAC discovery
(3 and 4 , [Fig. 4 ]).[24 ]
[25 ]
Fig. 4 The employment of “Click Reaction” for the synthesis of representative proteolysis-targeting
chimeras.
Each triazole formed by CuAAC constitutes a geometrically defined and metabolically
stable linker whose length and flexibility were systematically varied in the reported
libraries. The degradation structure–activity relationship (SAR) across these series
directly demonstrates that incremental one-carbon or polyethylene glycol extensions,
and the rigidity imparted by the triazole itself, can modulate the stability and cellular
potency of the ternary complex, thereby illustrating how click chemistry rapidly probes
and optimizes linker parameters without resynthesizing entire molecules.
Multicomponent Reactions
“Click chemistry” allows for the efficient production of VHL- and CRBN-based degraders.
However, the incorporation of the triazole ring may yield poorly soluble products
and posing challenges in the scaling-up process.[26 ] The synthetic assembly of PROTACs requires precise asymmetric functionalization
of linker architectures and their regioselective conjugation with protein-binding
motifs, presenting a significant chemical challenge. This synthetic complexity typically
mandates the employment of orthogonal chemical strategies or multistep protection/deprotection
protocols to ensure controlled molecular assembly and structural integrity.
In 2022, Bhela et al reported an innovative multicomponent reaction-based platform,
based on the modular characteristics of multicomponent reaction systems coupled with
targeted protein degraders to facilitate the synthesis of structurally elaborated
PROTACs.[27 ] Aldehyde, isocyanide, and carboxylic acid building blocks are combined using a one-pot
Passerini three-component reaction to form the entire linker–warhead segment, generating
BRD4-degrading PROTAC (9 ) and congeners in a single operation. As a proof of concept, the platform has been
utilized for the synthesis of PROTAC-based BRD4 degraders, leading to the identification
of a series of degraders exhibiting remarkable degradation efficiency ([Fig. 5 ]).
Fig. 5 The versatile and sustainable multicomponent platform for the synthesis of protein
degraders.
In 2023, Zhu et al developed a novel activity-based PROTAC probe PD-Q2 via the Ugi
reaction, assembling the GPX4 inhibitor ML-162 with features of the cereblon ligand
pomalidomide.[28 ] As illustrated in [Fig. 6 ], the desired compound was synthesized as a racemic mixture through a four-component
Ugi reaction. Validation studies confirmed the covalent engagement of GPX4 by PD-Q2,
while revealing limitations in degradation efficiency that prompted further optimization.
Mechanistically, these compounds induced a significant accumulation of lipid reactive
oxygen species (ROS), resulting in profound suppression of clonogenic potential and
proliferation kinetics in cancer cells.
Fig. 6 The design of PROTAC-like active probes using a Ugi reaction. PROTAC, proteolysis-targeting
chimera.
In addition to the aforementioned one-pot synthesis method, Arndt et al reported a
one-pot, visible-light-driven decarboxylative C(sp2
)–C(sp3
) coupling that directly joins a brominated CRBN ligand to a JQ-1-derived carboxylic
acid, furnishing a single PROTAC in 55% yield.[29 ] As shown in [Fig. 7 ], this consolidated methodology enables streamlined generation of PROTAC libraries
in one pot by covalently linking a CRBN E3 ubiquitin ligase recruiter to JQ-1 (serving
as the target protein binder) through various linker types-flexible aliphatic chains,
aromatic systems, and rigidified bridging units. Notably, this streamlined protocol
significantly accelerates the construction of targeted protein degradation tools.
Fig. 7 One-pot synthesis of PROTAC from three components via photoinduced C(sp2 )-C(sp3 ) coupling and amidation. PROTACs, proteolysis-targeting chimeras.
Late-Stage Functionalization
In the last several years, late-stage functionalization has made significant progress
and emerged as a powerful approach for the diversification and assembly of increasingly
complex molecules.[30 ] Recent progress in transition metal-catalyzed C–H functionalization has unlocked
novel bond-forming strategies with enhanced atom- and step-efficiency, significantly
broadening medicinal chemistry's synthetic repertoire. Antermite et al presented a
robust ruthenium-catalyzed late-stage C–H amidation strategy that allows for the synthesis
of fully elaborated heterobifunctional compounds ([Fig. 8 ]).[31 ] The strategic utilization of commercially accessible dioxazolone reagents enables
site-selective C–H amidation on structurally complex bioactive molecules, guided by
inherent directing groups. This transformation exhibits remarkable functional group
compatibility and regioselectivity, facilitating the late-stage incorporation of linker
moieties equipped with orthogonal reactive handles for downstream derivatization.
Notably, the authors successfully demonstrated the versatility of this approach through
the efficient one-pot synthesis of both CRBN-recruiting and biotinylated conjugates,
underscoring the methodology's potential for streamlined production of sophisticated
therapeutic agents and chemical probes.
Fig. 8 Synthesis of PROTAC-like compounds via late-stage C–H amidation. PROTAC, proteolysis-targeting
chimera.
Microscale Synthesis-Enabled PROTACs
Microscale Synthesis-Enabled PROTACs
The bottleneck in developing PROTAC workflows lies not only in efficient synthesis
but also in the subsequent purification steps, which are often time-consuming and
resource-intensive while simultaneously limiting the speed of iterative optimization.
To address this challenge, Stevens et al developed an ultra-high-throughput experimentaion
(ultraHTE) system. This represents a core innovation of microscale synthesis for PROTACs,
integrating two critical functions: facilitating rapid PROTAC synthesis and enabling
immediate biological evaluation of unpurified reaction products in cellular degradation
assays ([Fig. 9 ]).[32 ] This innovative “direct-to-biology” (D2B) strategy was successfully validated through
a medicinal chemistry initiative focused on discovering new BRD4-targeting PROTACs.
Via the strategy, researchers synthesized 650 PROTACs in a 1,536-well plate and performed
biological evaluations within less than 1 month. The process involved dosing stock
solutions onto a 384-well plate, followed by robotic dispensing into the 1,536-well
reaction plate; aliquots were analyzed by liquid chromatography–mass spectrometry
using PyParse, and successful reactions were directly advanced to cellular assays
without additional purification steps. Compounds of interest were resynthesized for
further profiling, with SAR guiding the next iteration of the compound design. Given
its remarkable capability to significantly expedite new degrader optimization processes,
it is anticipated that this platform will revolutionize both synthesis and testing
aspects associated with PROTAC development.
Fig. 9 A fully automated discovery platform of PROTACs from crude reaction mixtures. PROTACs,
proteolysis-targeting chimeras. Reproduced with permission from Stevens et al.[32 ]
The precise determination of length, shape, and linker attachment points is crucial
for designing effective PROTACs. To expedite the identification of potential hits,
Plesniak et al exploited a library of established E3–ligand–linker intermediates,
introducing a parallel synthesis and purification procedure for PROTAC screening ([Fig. 10 ]).[33 ] A high-throughput nanomole-scale synthesis platform has been developed utilizing
amide coupling chemistry enabled by a low-loading palladium catalyst and green solvent
system that suppresses side reactions and boosts conversion efficiency. This platform
enables direct biological evaluation of crude reaction mixtures and simultaneous determination
of critical physicochemical parameters, including lipophilicity (logD) and exposed
polar surface area (PSA). This innovative approach dramatically accelerated PROTAC
SAR exploration, reducing the optimization cycle from several weeks to merely 5 days
while eliminating the need for extensive purification procedures. Relying on the above
amide coupling innovations, the implementation of this cost-effective and sustainable
nanoscale synthesis paradigm has significantly expedited the discovery of PROTAC leads,
as the platform's efficiency stems from the coupling system's selectivity, exemplifying
how such synthetic chemistry advancements facilitate the realization of PROTAC technology's
therapeutic potential.
Fig. 10 Rapid PROTACs discovery platform based on the nanomole-scale array synthesis and
direct screening of reaction mixtures. PROTACs, proteolysis-targeting chimeras.
In 2024, Tian et al introduced a miniaturized, high-throughput platform integrating
solid-phase synthesis and biological screening for PROTACs.[34 ] The developed on-chip methodology uses ultraviolet (UV)-induced release in direct
experimental procedures and has facilitated the successful synthesis and characterization
of 132 novel PROTAC-like molecules with minimal consumption of reagents. In addition,
it is compatible with cancer cell cultivation and mitogen-activated protein kinase
kinase inhibition determinations, and therefore, provides an efficient approach to
accelerate PROTAC optimization and drug development.
Guo et al described a novel Rapid-TAC platform that streamlines PROTAC synthesis through
direct coupling between the O -dibenzaldehyde group on a protein ligand and amine groups in a commercial PROTAC
library.[35 ] This approach enables quick assembly of functional PROTACs for immediate screening,
with lead degraders (e.g., AR-RapidTAC-1, BRD4-RapidTAC-2) featuring diimine linkers
that support ternary complex formation. The platform's efficacy was demonstrated through
the rapid identification of potent androgen receptor (AR) and BRD4 degraders. The
inherent versatility of the Rapid-TAC platform holds promise for significantly expanding
its utility in drug discovery paradigms, streamlining PROTAC accessibility, and optimizing
SAR investigations. This technological advancement exemplifies how innovative chemical
methodologies can enhance the therapeutic landscape of targeted protein degradation,
potentially accelerating the translation of PROTAC-based therapeutics from bench to
bedside.
Yan et al proposed a direct-to-biology platform for the efficient synthesis of plate-based
PROTAC libraries. This platform eliminates post-synthesis purification by ensuring
reaction mixtures have low toxic byproducts and good biocompatibility, thereby enabling
direct application in cell-based assays.[36 ] Specifically, it integrates amide coupling (to link ligand fragments with a molecular
linker) and light-induced primary amines and o-nitrobenzyl alcohols cyclization photoclick
chemistry, forming a modular PROTAC assembly workflow. This workflow facilitated the
rapid identification of potent PROTACs that selectively degrade the kinase CDK9 and
suppress triple-negative breast cancer cell growth. By streamlining the process, the
approach enhances chemical space exploration, accelerates novel PROTAC discovery,
and thus improves drug development efficiency.
Dynamic Combinatorial Chemistry-Enabled PROTACs
Dynamic Combinatorial Chemistry-Enabled PROTACs
The dynamic combinatorial chemistry (DCC) platform enables efficient generation of
molecular libraries with sophisticated architectures, demonstrating broad applicability
in both medicinal chemistry and supramolecular science.[37 ]
[38 ] DCC builds reversible libraries ([Fig. 11 ]) from interchangeable building blocks under thermodynamic control. The introduction
of a target macromolecule as a template shifts the equilibrium, selectively amplifying
the strongest binders. Enrichment factors that quantitatively rank affinity and selectivity
can be obtained by comparing templated and nontemplated mixtures, allowing rapid identification
of PROTACs or other ligands for enzymes, membrane receptors, transmembrane proteins,
and nucleic acids alike.
Fig. 11 The schematic representation of the DCC concept for PROTAC screening. DCC, dynamic
combinatorial chemistry; PROTAC, proteolysis-targeting chimera.
In recent years, the DCC platform has been progressively adapted for PROTAC discovery,
utilizing its capacity to selectively stabilize the most effective ternary complexes
from dynamic libraries. Diehl et al created a novel DCC strategy for PROTAC selection,
utilizing thermodynamic stabilization of the target–E3 ligase–PROTAC ternary assembly
as the key criterion.[39 ] Their focus lies in utilizing VHL-targeting Homo-PROTACs as a model system. They
demonstrated that the reversible assembly of a VHL Homo-PROTAC ternary complex through
thiol-disulfide exchange chemistry leads to potent amplification of VHL Homo-PROTACs
with degradation activities strongly correlated with their biophysical capacity to
dimerize VHL.
Self-Assembly-Enabled PROTACs
Self-Assembly-Enabled PROTACs
The development of intracellular self-assembled PROTACs technology addresses challenges
in clinical translation, such as limited penetration, inadequate degradation, and
potential DNA damage caused by UV radiation. Lebraud et al proposed the concept of
click-formed proteolysis targeting chimeras (CLIPTACs, [Fig. 12 ]), wherein two small precursor molecules capable of undergoing click reactions within
cells exhibit enhanced cellular membrane permeability compared with a single large
compound.[40 ] They created a thalidomide derivative labeled with tetrazine, which quickly reacts
with a ligand of the target protein tagged with trans-cyclo-octene within cells to
produce a cereblon E3 ligase recruiting a PROTAC molecule. In this work, the developed
CLIPTAC precursors exhibit substantially lower molecular weight and reduced PSA relative
to literature-reported BRD4-targeting PROTACs, as demonstrated by a comparative analysis.
The successful degradation of BRD4 and ERK1/2 in three different cell lines, along
with corresponding control experiments, confirms the hypothesis that CLIPTACs can
be generated within cells through click reactions involving trans-cyclooctene (TCO)-ligands
(17 ) and Tz-thalidomide (19 ).
Fig. 12 The scheme explaining the mode of action of click-formed proteolysis-targeting chimeras
CLIPTACs.
In 2023, Gui et al addressed PROTAC challenges such as cellular permeability, solubility,
and “hook effect” with the aim of optimizing PROTACs' therapeutic properties.[41 ] Self-assembled PROTACs were designed to efficiently degrade the VHL E3 ubiquitin
ligase via a rapid, reversible covalent bonding mechanism between hydroxylamine and
aldehyde groups, thus enhancing their in vivo therapeutic potential without the “hook effect.” Recently, Giardina et al modified
their Coferon platform to create small molecule degraders called Combinatorial Ubiquitination
REal-time PROteolysis (CURE-PROs).[42 ] These CURE-PROs self-assemble into covalent heterodimers through reversible bio-orthogonal
linkers, incorporating phenylboronic acid and diol/catechol moieties into ligands
for VHL, CRBN, mouse double minute 2 (MDM2), etc. This approach enabled the degradation
of BRD4 both in vitro and in vivo . The combinatorial design facilitated compound synthesis, optimized linker length,
and identified suitable E3 ligase partners for each target, making it ideal for screening
new targets.
Concerns have arisen regarding the possible adverse effects of compounds generated
using this approach due to uncontrolled protein degradation and undesired off-target
effects mediated by ligases. The precise manipulation of the PROTAC degradation activity
could mitigate their potential toxicity and side effects. Chang et al have developed
a bio-orthogonal prodrug strategy called click-release “crPROTACs” that enables targeted
activation of PROTAC prodrugs and selective release of PROTACs in cancer cells ([Fig. 13 ]).[43 ] Inactive TCO-DT2216 and TCO-ARV-771 PROTAC prodrugs were rationally designed by
incorporating a bio-orthogonal TCO group into the VHL E3 ubiquitin ligase ligand.
The tetrazine (Tz)-modified RGD peptide, c(RGDyK)-Tz, which targets integrin αv β3 biomarkers in cancer cells, serves as the activating component for click-release
of PROTAC prodrugs to achieve targeted degradation of POIs, specifically in cancer
cells rather than noncancerous normal cells.
Fig. 13 The bio-orthogonal PROTAC prodrug strategy enabled by on-target activation. PROTAC,
proteolysis-targeting chimera.
Epigenetic regulation has been successfully used in therapeutic targeting of BET proteins
to correct pathological protein imbalances in cancer treatment. Nevertheless, creating
tumor microenvironment-responsive BET degraders that combine precise targeting with
optimal drug-like properties remained a significant challenge. In 2022, Do et al reported
enzyme-derived clicking PROTACs (ENCTACs), capable of orthogonally cross-linking two
distinct small molecule warhead ligands. These specifically recognize BRD4 protein
and E3 ligase within tumors only upon hypoxia-induced activation of nitroreductase
enzyme ([Fig. 14 ]).[44 ] The spatially restricted generation of these bifunctional degraders enables targeted
BRD4 suppression, leading to coordinated epigenetic reprogramming and controlled manipulation
of hypoxia response pathways across cellular, zebrafish, and murine tumor models.
Fig. 14 Enzyme-derived click formation of heterobifunctional degraders of BRD4. BRD4, a member
of the BET bromodomain family.
TFs are proteins that regulate gene expression by binding to specific DNA sequences.[45 ] TFs dysregulation is associated with cancer, neurodegenerative disorders, cardiovascular
diseases, and other human diseases. TFs targeting offer potential therapeutic benefits
by modulating gene expression in a disease-specific manner; however, for several decades,
despite remarkable progress in understanding their biological functions and even elucidating
their crystal structures, targeting the majority of TFs has remained an insurmountable
challenge. Liu et al devised a TF-PROTACs platform in which a DNA oligonucleotide
is linked to a VHL ligand via a copper-free SPAAC click reaction between an azide-functionalized
DNA (N3-ODN) and a DBCO-bearing VHL ligand (VHLL) ([Fig. 15 ]).[45 ] The resulting triazole conjugate selectively recruits VHL to the TF recognized by
the DNA sequence, triggering degradation. Notably, two series of VHL-based TF-PROTACs
named E2F-PROTAC (dE2F) and NF-κB-PROTAC (dNF-κB) were designed. These compounds were
effective in degrading endogenous E2F1 and p65 proteins in cellular models, exhibiting
remarkable antiproliferative effects. These findings highlight the potential utility
of TF-PROTACs as a versatile platform for achieving targeted TF degradation and provide
a universal strategy for addressing previously “undruggable” TF targets.
Fig. 15 The schematic diagram of the TF-PROTAC strategy.
DNA-Encoded Library-Enabled PROTAC
DNA-Encoded Library-Enabled PROTAC
The DNA-encoded library (DEL) represents a highly efficient technology for the parallel
synthesis and screening of millions or billions of compounds through affinity selection.
This efficiency stems from its ability to rapidly identify high-affinity binders from
large chemical spaces, making it a powerful tool for drug discovery. The DEL affinity
selection process fully meets the requirements for affinity-based PROTAC discovery.
Chen et al employed DEL technology to synthesize a broad range of potential PROTAC
molecules while presenting a parallel screening approach that employs DNA barcodes
as indicators for ternary complex formation and cooperative binding ([Fig. 16 ]).[46 ] The process involves synthesizing a diverse library of PROTACs, each tagged with
a unique DNA barcode, and then incubating the library with the target protein and
E3 ligase complex to form ternary complexes. Through affinity selection and next-generation
sequencing of the DNA barcodes, the most effective PROTACs that stabilize the ternary
complex are identified, thereby optimizing the design for target protein degradation.
Additionally, they employed a designed PROTAC DEL targeting CRBN and BRD4 to demonstrate
a dual protein affinity selection method, leading to the direct discovery of novel
and potent BRD4 PROTACs. This approach facilitated the simultaneous evaluation of
all potential PROTACs while overcoming issues related to solubility and permeability
interference.
Fig. 16 The optimization of PROTAC ternary complex using DNA encoded library approach. PROTACs,
proteolysis-targeting chimeras. Reproduced with permission from Chen et al.[46 ]
Therapeutically relevant small molecules that stimulate protein complex formation
offer precise means to modulate essential cellular functions. Mason et al have developed
a platform for the direct discovery of compounds capable of inducing association between
any two preselected proteins, utilizing the E3 ligase VHL and bromodomains as test
systems.[47 ] By harnessing the screening capabilities of DELs, they synthesized approximately
one million compounds with a VHL-targeting ligand, various connectors, and a diversity
element generated through split-and-pool combinatorial chemistry. Through screening
the DEL against bromodomains in the presence and absence of VHL, they successfully
identified molecules that bind to VHL while simultaneously bound to bromodomains.
The research provided a methodological framework for utilizing DEL screening to discover
heterobifunctional small molecules.
Solid-Phase Synthesis-enabled PROTACs
Solid-Phase Synthesis-enabled PROTACs
The development of HDAC degraders has attracted significant interest, with a few HDAC
degraders being disclosed in recent years. In 2022, Sinatra et al used a combination
of solution- and solid-phase parallel synthesis protocols to obtain a series of HDAC6
degraders based on nonselective and HDAC6-selective ligands, which involved preparing
key intermediates through solution-phase synthesis, constructing peptide chains and
coupling CRBN ligands on resin through solid-phase synthesis, and finally releasing
the target PROTAC through cleavage reaction. The most potent compounds were efficiently
screened ([Fig. 17 ]), combined with the parallel synthesis strategy.[48 ] According to the previously published approach, PROTAC synthesis was accomplished
by coupling the E3 ligase component using hydroxamic acids immobilized on resins,
followed by cleavage from the resin under gentle conditions that generated hydroxamic
acids with excellent crude purities up to 91%. Before biological evaluation, compounds
were further purified above 95% purity, using preparative reversed-phase high-performance
liquid chromatography, rendering PROTAC derivatives with total yields ranging from
27 to 71%. Overall, this synthetic strategy facilitated the quick and efficient generation
of PROTAC libraries.
Fig. 17 Solid-phase synthesis of HDAC6 PROTACs. The hydroxamic acid warhead is first attached
to Rink-amide resin via an oxygen linker, followed by Fmoc-based peptide elongation,
on-resin CRBN ligand coupling, and TFA cleavage to release the final PROTAC. CRBN,
cereblon; HDAC6, histone deacetylase 6; PROTACs, proteolysis-targeting chimeras.
Sulfonyl Fluoride-Based Covalent PROTACs
Sulfonyl Fluoride-Based Covalent PROTACs
Covalent ligand-directed PROTACs targeting E3 ubiquitin ligases have emerged as a
promising strategy in targeted protein degradation. Structural studies have elucidated
that the hydroxyproline moiety in VHL ligands forms a critical hydrogen bonding interaction
with the Ser110 residue, which is essential for molecular recognition. To expand the
substrate range of covalent E3 ligase PROTACs, Shah et al developed VHL-SF2 (27 , [Fig. 18A ]), a heterobifunctional degrader that replaces the native hydroxyl with a sulfonyl
fluoride warhead, covalently targeting Ser110 of VHL, linked via a flexible alkyl
chain to an APT1-directed hydroxamate.[49 ] This study first demonstrated covalent VHL ligands that can be seamlessly integrated
into bifunctional degrader architectures, thereby significantly expanding the targetable
proteome for covalent E3 ligase-engaging PROTACs. The development of these novel covalent
warheads strategically expands the chemical toolbox for targeted protein degradation,
enabling access to previously undruggable protein targets through covalent engagement
strategies.
Fig. 18 (A ) The structure of covalent targeted-VHL (26 ) probe VHL-SF2 (27 ) with a sulfonyl fluoride moiety. (B ) General model of sulfonyl fluoride PROTACs. PROTACs, proteolysis-targeting chimeras;
VHL, Von Hippel-Lindau.
PRosettaC software has been recently used to optimize the design of a sulfonyl-fluoride-based
PROTAC linker targeting acyl protein thioesterase 1 (APT1).[50 ] The computational approach proposed alkyl linkers for APT1 ([Fig. 18B ]), which were experimentally validated for effective APT1 degradation and selectivity
among serine hydrolases. The integration of PRosettaC with chemoproteomics provides
a strategy for efficient PROTAC candidate prioritization and assessment, enhancing
the design process and accelerating targeted protein degrader development.
Photocontrolled PROTACs
PROTAC molecules may exhibit an event-driven behavior at low doses, thereby partially
mitigating high-dose-related toxicity. Nevertheless, controlling their activity through
dosage remains challenging due to their potent catalytic mechanism. Furthermore, PROTACs
retain the risk of off-target effects like small molecule inhibitors. To address this
issue, researchers are endeavoring to use photoregulation techniques to precisely
regulate protein degradation with high spatiotemporal resolution, thereby minimizing
potential systemic side effects.[51 ]
Li et al synthesized a series of pomalidomide analogs by substituting the photocage
group 6-nitroveratryloxycarbonyl with the glutarimide group at the C-terminal of pomalidomide
ligand CRBN of E3 ubiquitin ligase and subsequently conjugating them to BET inhibitors
through appropriate linkers to obtain a series of PC-PROTACs ([Fig. 19 ]).[52 ] The presence of the photocage group hindered protein degradation, but upon irradiation
with 365 nm UV light, it could be reactivated and induce degradation again. At a concentration
of 1 μmol/L, PC-PROTAC completely degraded BRD3 protein in HEK293FT cells after 15 minutes
of UV irradiation, demonstrating superior in vitro antitumor activity compared with the nonirradiated control group.
Fig. 19 Representative photo-controlled proteolysis-targeting chimeras.
Nicotinamide adenine dinucleotide (NAD+ ) is an indispensable coenzyme in DNA synthesis and possesses diverse biological functions.
Nicotinamide phosphoribosyltransferase (NAMPT) is the key rate-limiting enzyme involved
in NAD+ biosynthesis in mammals.[53 ] Cheng et al developed a pioneering chemical tool for the optical manipulation of
NAMPT and NAD+ in biological systems using photoswitchable proteolysis-targeting chimeras (PS-PROTACs,
[Fig. 19 ]).[54 ] By employing an NAMPT activator and a dimethylpyrazolazobenzene photoswitch, highly
efficient PS-PROTACs were designed, enabling reversible up- and downregulation of
NAMPT and NAD+ in a light-dependent manner while mitigating the toxicity associated with inhibitor-based
PS-PROTACs. Under irradiation at 620 nm, PS-PROTAC was activated, thereby achieving
optical control of antitumor activity, NAMPT, and NAD+ through in vivo manipulation.
Jin et al developed azobenzene-proteolysis targeting chimeras (Azo-PROTACs, [Fig. 19 ]) as a novel small molecule tool to manipulate the protein degradation process using
UV light.[55 ] By employing the lenalidomide-Azo-dasatinib trifunctional system, they demonstrated
distinct differences in protein degradation activity between the trans- and cis-isomers
of Azo-PROTACs. This system enables precise degradation of ABL and BCR-ABL proteins
through configuration changes induced by UV-C light exposure. Azo-PROTACs combine
potent protein knockdown capabilities and efficient cell uptake properties with reversible
photo-switchability, proposing a promising chemical knockdown strategy based on light-induced
reversible on/off modulation.
Naro et al pioneered light-responsive PROTAC systems through targeted modification
of VHL- and CRBN-recruiting ligands with photolabile caging groups, enabling optical
regulation of protein degradation activity ([Fig. 19 ]).[56 ] By installing photolabile caging groups onto the E3 ligase ligands of PROTACs, the
activity of protein degradation can be regulated by light. This method employs UV/VIS
light of a specific wavelength (365 nm) to remove the caging groups, thereby activating
the protein degradation function of PROTACs. This approach provides a powerful tool
for studying protein function and developing new therapeutic strategies.
Artificial Intelligence-Driven Innovations in PROTAC Development
Artificial Intelligence-Driven Innovations in PROTAC Development
PROTACs have revolutionized targeted protein degradation, providing a novel therapeutic
strategy for previously “undruggable” proteins; however, their development is hindered
by challenges like complex linker design, pharmacokinetic property optimization, and
the demand for high-throughput screening. In recent years, artificial intelligence
(AI) and machine learning (ML) have emerged as powerful tools to address these issues,
accelerating PROTAC discovery and optimization. Specifically, AI-driven approaches
optimize PROTAC design by using computational models to predict how structural modifications
affect activity-reducing reliance on empirical methods and saving time and resources.
For instance, the PROTACable platform integrates three-dimensional structural modeling
with advanced ML algorithms for automated PROTAC design, enabling efficient identification
of lead compounds with improved ADME characteristics and therapeutic potential.[19 ] This approach leverages AI to analyze large datasets, yielding insights into linker
design, target specificity, and degradation efficiency.
AI-driven predictive modeling has also been applied to enhance the drug-like properties
of PROTACs. AI-driven predictive modeling can use the data from techniques like nuclear
magnetic resonance spectroscopy, molecular dynamics simulations, and principal component
analysis to predict the cell permeability of PROTACs based on their structural properties
([Fig. 20 ]). These models help in the rational design of PROTACs with improved pharmacokinetic
profiles, addressing challenges such as high molecular weight and poor solubility.[12 ] By integrating AI with chemoproteomics, researchers can further refine PROTAC candidates,
ensuring they meet the necessary criteria for clinical translation.
Fig. 20 Artificial intelligence-driven predictive modeling. Reproduced with permission from
Poongavanam et al.[12 ]
Summary
PROTACs represent a transformative therapeutic modality, yet their clinical translation
is hindered by challenges in molecular design, synthesis, and biological evaluation.
This section focuses on chemistry-driven innovations that address these hurdles, paving
the way for next-generation protein degraders.
Click chemistry and multicomponent reactions address critical challenges in linker
design, where length, flexibility, and composition determine therapeutic efficacy.
Those techniques enable precise modulation of linker properties. Microscale platforms
and direct-to-biology approaches streamlined labor-intensive purification in traditional
synthesis, accelerating high-throughput production. Selectivity and off-target effects
are mitigated via chemistry-based strategies, e.g., DCC, self-assembled PROTACs, and
covalent PROTACs. DELs and solid-phase synthesis overcome limitations in cellular
permeability and solubility, while photocontrolled PROTACs achieve spatiotemporal
degradation control, reducing systemic toxicity.[57 ]
The integration of chemical-driven innovations with advanced computational and biological
tools plays a key role. The application of AI and deep learning in PROTAC design holds
the potential to revolutionize the field by enabling the rational design of degraders
with optimized pharmacokinetic and pharmacodynamic properties. AI-driven models can
predict the impact of structural modifications on PROTAC activity, reducing the reliance
on empirical methods and accelerating the discovery process. Moreover, the combination
of PROTACs with other drug discovery strategies, such as multivalency,[58 ] multitargeting,[59 ]
[60 ] prodrug strategy,[61 ] aptamer,[62 ] antibody conjugation,[63 ]
[64 ] etc., has expanded the scope of protein degradation beyond traditional small molecule
inhibitors. The integration of PROTACs with other drug discovery strategies showcases
the versatility of this technology in addressing complex biological challenges, thereby
presenting a novel paradigm characterized by “integrated medicinal chemistry.” Integrated
innovation will be a prominent trend in contemporary drug discovery, where pioneering
research findings often arise from the combination of two or more strategies or technologies.[65 ] This tendency is particularly evident within the field of PROTAC technology.
In summary, chemistry-driven strategies are the cornerstone of PROTAC advancement.
To date, innovations in linker chemistry, synthetic methodologies, and controlled
degradation have overcome some key barriers, bringing clinical translation closer.
Nevertheless, ongoing progress in chemical design remains essential and will be pivotal
to addressing remaining obstacles and unlocking PROTACs' full therapeutic potential.
