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Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USAThe Key Laboratory of Biomedical Information Engineering of Ministry of Education, Center for Mitochondrial Biology and Medicine, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an, China
Post-translational modifications (PTMs) regulate all aspects of protein function. Therefore, upstream regulators of PTMs, such as kinases, acetyltransferases, or methyltransferases, are potential therapeutic targets for human diseases, including cancer. To date, multiple inhibitors and/or agonists of these PTM upstream regulators are in clinical use, while others are still in development. However, these upstream regulators control not only the PTMs of disease-related target proteins but also other disease-irrelevant substrate proteins. Thus, nontargeted perturbing activities may introduce unwanted off-target toxicity issues that limit the use of these drugs in successful clinical applications. Therefore, alternative drugs that solely regulate a specific PTM of the disease-relevant protein target may provide a more precise effect in treating disease with relatively low side effects. To this end, chemically induced proximity has recently emerged as a powerful research tool, and several chemical inducers of proximity (CIPs) have been used to target and regulate protein ubiquitination, phosphorylation, acetylation, and glycosylation. These CIPs have a high potential to be translated into clinical drugs and several examples such as PROTACs and MGDs are now in clinical trials. Hence, more CIPs need to be developed to cover all types of PTMs, such as methylation and palmitoylation, thus providing a full spectrum of tools to regulate protein PTM in basic research and also in clinical application for effective cancer treatment.
Eukaryotic cells rely on posttranslational modifications (PTMs) to regulate protein activity, stability, subcellular localization, and protein–protein interactions (PPIs) (
). PTMs are present in all polar amino acids, such as phosphorylation, acetylation, methylation, ubiquitination, and glycosylation, whereas nonpolar amino acids like (Leu, L), isoleucine (Ile, I), and phenylalanine (Phe, F) lack such modifications (Fig. 1). However, when present at the N terminus of proteins, nonpolar amino acids, such as alanine (Ala, A), valine (Val, V), and methionine (Met, M) also undergo PTMs such as N-acetylation (
) (Fig. 1). Phosphorylation is the most widely studied PTM, occurring on the side-chain hydroxyl group of tyrosine (Tyr, Y), serine (Ser, S), and threonine (Thr, T), or on the side-chain amino group of histidine (His, H), or on the carboxyl group of aspartic acid (Asp, D) and glutamic acid (Glu, E) (
). PTMs primarily affect the side chain of amino acids but in some rare cases the N-terminal amino group of the peptide can also be modified, as seen in N-acetylation at Ala, cysteine (Cys, C), glycine (Gly, G), Met, Ser, Thr, and Val (
Figure 1Common PTMs on the side chain of amino acids. Protein posttranslational modifications (PTMs) often occur on the side chain of polar amino acids, while most nonpolar amino acids typically do not have PTMs on their side chains. The PTMs on the N-terminal amino group of the protein are not shown.
Protein PTMs are tightly regulated by a network of enzymatic regulators, including respective writers, erasers, and readers (Fig. 2). As PTMs play a critical role in almost all aspects of protein function, the upstream regulatory mechanism(s) and downstream effect(s) of these PTMs are being intensively studied for many key proteins. For instance, the p53 protein undergoes nearly all types of PTMs, such as phosphorylation, acetylation, methylation, ubiquitination, PARylation, and others (
). Consequently, these upstream regulators and downstream effectors serve as ideal therapeutic targets for human diseases, particularly cancer.
Figure 2Native and chemically proximity-induced engineered PTM of proteins.A, under physiological conditions, the posttranslational modification (PTM) is added to a target protein by enzymes (writers) and removed by other enzymes (erasers), where these protein PTMs could be specifically recognized by effector proteins (readers) to elicit downstream biological effects. B, to introduce a native or neo-PTM to a target protein, a chemical inducer of proximity (CIP) recruits the designed writer/eraser protein to be close enough to the protein of interest (POI) to subsequently facilitate the engineered PTM editing.
Several hundred small-molecule drugs that target these regulators of PTMs, such as kinase, deacetylase, and methyltransferase inhibitors, have been approved for cancer treatment, with many more in development. However, each upstream regulator typically has multiple substrates in addition to the therapeutic target. Therefore, indiscriminate inhibition of the PTM regulators may introduce unwanted side effects, limiting their application in the clinic. Therefore, drugs that can more precisely regulate the specific PTM of a single protein are in realistic need. In the past 2 decades, several types of chemical proximity-induced PTM methods, such as proteolysis-targeting chimera (PROTAC), have emerged as having high potential for clinical translation in the treatment of human diseases. Protein proximity is the foundation of PPIs, and chemically induced protein proximity is an emerging tool for manipulating PPI and regulating protein function in cells. Chemical inducers of proximity (CIPs) are drug-like small molecules that induce and/or remove native or new PPI (
) (Fig. 2). In this review, we summarize recent advances in CIP technologies that regulate PTMs, with a focus on CIPs that induce protein ubiquitination and degradation, as well as several novel CIPs for protein phosphorylation, acetylation, and O-linked-N-acetylglucosaminylation (O-GlcNAcylation) (Fig. 3).
Figure 3Native PTMs and chemically induced proximity for engineered PTM of proteins in cells. Various CIPs have been developed to modify the PTM of POIs, including ubiquitination by PROTAC, MGD, and LYTAC, deubiquitination by DUBTAC, phosphorylation by PHICS, dephosphorylation by PhoRC and PhosTAC, acetylation by AceTAG, O-GlcNAc by nanobody-OGT or Aptamer-OGT, and de-O-GlcNAc by nanobody-SplitOGA. Dashed arrows indicate CIP technology not yet been developed. Ac, acetylation; CIP, chemical inducers of proximity; DUBTAC, deubiquitinase-targeting chimera; LYTAC, lysosome-targeting chimera; MGD, molecular glue degrader; OGA, O-GlcNAcase; O-GlcNAc, O-linked N-acetylglucosamine; OGT, O-GlcNAc transferase; PHICS, phosphorylation-inducing chimeric small molecule; PhoRC, phosphatase recruiting chimeras; PhosTAC, phosphorylation targeting chimeras; POI, protein of interest; PROTAC, proteolysis-targeting chimera; PTM, posttranslational modifications.
CIPs promote protein ubiquitination and degradation
In eukaryotic cells, proteins undergo polyubiquitination and the ubiquitinated proteins are subsequently degraded by the proteasome or lysosome to transduce cell signals (
). This process involves the sequential catalysis of ubiquitin by the ubiquitin-activating enzyme E1, the ubiquitin-conjugating enzyme E2, and the ubiquitin ligase E3, and the transfer of ubiquitin onto the lysine residues of substrate proteins (
). There are different types of polyubiquitin chains with distinct biological functions due to the presence of seven lysine residues within the ubiquitin protein (
). For example, K11 and K48 polyubiquitin chains mainly promote protein degradation, whereas K63 polyubiquitin chain usually serves as a docking site for PPI to facilitate signal transduction events (
). E3 ubiquitin ligases dictate the specificity of the ubiquitination substrate, recognizing the degron within the substrate protein to confer substrate specificity (
). Ubiquitinated proteins are recruited to the 26S proteasome or lysosome for degradation, eliminating misfolded or unwanted proteins and governing intracellular protein homeostasis. There are several CIP approaches that facilitate targeted protein degradation (TPD), including PROTAC, molecular glue degraders (MGDs), lysosome-targeting chimera (LYTAC), autophagy-targeting chimera, autophagosome-tethering compound, cytokine receptor-targeting chimera (KineTAC), and proteolysis-targeting antibody (PROTAB) (Fig. 4). These methods introduce polyubiquitin modification of protein of interest (POI) or directly recruit POI to the 26S proteasome or lysosome for degradation.
Figure 4Targeted protein degradation approaches using small molecules or antibodies. Several methods have been devised to engineer the ubiquitination and subsequent degradation of proteins of interest (POIs). PROTAC, MGD, TF-PROTAC, RNA-PROTAC, and ATTEC are designed for the degradation of intracellular proteins. PROTAB, LYTAC, and KineTAC are used for transmembrane and extracellular proteins, while AUTAC for organelles. ATTEC, autophagosome-tethering compound; AUTAC, autophagy-targeting chimera; CIP, chemical inducers of proximity; KineTAC, cytokine receptor-targeting chimera; LYTAC, lysosome-targeting chimera; MGD, molecular glue degrader; PROTAB, proteolysis-targeting antibody; PROTAC, proteolysis-targeting chimera.
). PROTAC molecule consists of three functional modalities, including a POI ligand, an E3 ligase ligand, and a chemical linker that bridges the two ligands (
). The PROTAC molecule forms a ternary complex with the POI and the corresponding E3 ligase, thus facilitating the addition of the polyubiquitin chain to the POI (
). Compared to conventional small-molecule inhibitors, PROTAC acts catalytically, thereby making it more potent in suppressing the cellular function of the POI. Currently, two PROTAC compounds, ARV-110 and ARV-447, are undergoing Phase II clinical trials for the treatment of prostate cancer and breast cancer, through degrading androgen receptor (AR) and estrogen receptor (ER), respectively (
). Although PROTAC has the potential to target any intracellular protein for proteasomal degradation, the identification of a specific ligand for the target protein remains the primary challenge of PROTAC development. Interested readers can refer to other comprehensive reviews for further information on the progress of PROTAC (
MGDs are a class of monovalent compounds that bind both the E3 ubiquitin ligase and the target protein, resulting in the conversion of the target protein into a neo-substrate for ubiquitination and degradation. Unlike PROTAC, the discovery of MGD is usually serendipitous, with only a few examples reported to date, such as phthalidomides (
). The well-studied phthalidomide compounds, including thalidomide, pomalidomide, and lenalidomide, specifically bind to CRBN, an adaptor protein of the CUL4 E3 ubiquitin ligase complex (
). Sulfonamides, such as indisulam, tasisulam, and chloroquinoxaline sulfonamide, recruit the E3 ubiquitin ligase CUL4-DCAF15 to degrade RBM39, an mRNA splicing factor (
), which makes them promising splicing inhibitors that could induce antitumor immunity and synergize with immune checkpoint blockades in cancer immunotherapy (
) degrade Cyclin K in cells by recruiting the CDK12/Cyclin K complex onto the DDB1-CUL4-RBX1 E3 ligase, suggesting that TPD may also be initiated by a bridging protein that forms a stable complex with the target protein. Moreover, a bridge PROTAC MS28 has been developed to degrade Cyclin D1 by conjugating the VHL ligand to a ligand of CDK4/6, stable binding partner proteins of Cyclin D1 (
). As a result, MS28 effectively degrades both cyclin D1 and CDK4/6 in a VHL-dependent manner, making it more potent than CDK4/6 inhibitors and degraders (
). The studies suggest that it is possible to transform undruggable proteins into druggable targets through the utilization of POI binding proteins as bait and bridge during PROTAC development.
Other protein degraders
Besides PROTAC and MGD, other types of protein degraders have been developed to target-specific proteins for degradation, including LYTAC (
) that degrade lysosome-mediated protein degradation. LYTACs utilize the cation-independent mannose-6-phosphate receptor and the asialoglycoprotein receptor as docking sites to target protein for lysosomal degradation. KineTACs are bispecific antibodies that use a cytokine receptor as a docking site, containing a cytokine receptor-binding arm and a target-binding arm (
). In the first example of KineTAC, the cytokine CXCL12 arm specifically binds CXCR7, which triggers endocytosis and lysosomal degradation of target proteins (
). PROTAC is another type of bispecific antibody, which utilizes Zinc and Ring Finger 3, a plasma membrane E3 ubiquitin ligase, for the degradation of various transmembrane proteins and functions similarly to a PROTAC (
). Moreover, protein degraders that directly recruit protein targets into the proteasome have been developed, such as chemical inducers of degradation (
), which do not require a specific E3 ligase. In a proof-of-concept chemical inducers of degradation, the macrocyclic ligand of the 26S subunit PSMD2, MC1, is conjugated to the bromodomain (BRD)-4 ligand BETi, resulting in a ternary complex that facilitates the direct degradation of BRD4 by the 26S proteasome without prior ubiquitination (
Targeted protein stabilization is an increasing promising avenue for drug development, and inhibitors of E3 ligases or proteasome have been extensively tested for various human diseases (
). However, a major limitation of this approach is the broad range of substrates for a given E3 ubiquitin ligase. Therefore, a more specific strategy to stabilize only the target protein while sparing other downstream target proteins is ideal for therapeutic purposes. Abnormal degradation of certain proteins contributes to several pathologies, such as cystic fibrosis, which is caused by a single phenylalanine (F508) deletion within the cystic fibrosis transmembrane conductance regulator (CFTR), leading to its degradation (
). To address this issue, CFTR modulators, including ivacaftor, lumacaftor, tezacaftor, and elexacaftor have been developed to treat cystic fibrosis (CF) patients with the CFTR-dF508 mutant (
). Recently, Henning et al. have developed the first deubiquitinase-targeting chimera (DUBTAC) NJH-2-057 by conjugating a covalent ubiquitin thioesterase (OTUB1) ligand EN523 to lumacaftor. The CFTR-DUBTAC NJH-2-057 stabilizes the CFTR-dF508 mutant protein, providing a proof-of-concept for DUBTAC as a protein stabilizer to remove polyubiquitin chain from a specific POI (
). Similar to PROTAC, DUBTAC consists of three functional parts, a POI ligand, a DUB ligand, and a chemical linker region. To date, only OTUB1 and its ligand EN523 have been used for DUBTAC development (
). We have recently generated proof-of-concept lead compounds of TF-DUBTAC to stabilize transcription factors, such as FOXO3A and p53, by using EN523 to link to DNA oligomers that could be specifically recognized by tumor suppressor transcription factors (
). The development of more ligands for deubiquitinases other than OTUB1 may usher in a new era of DUBTAC, offering a promising approach to treat diseases, including cancer, where mutant proteins undergo uncontrolled degradation, such as GCase-related Gaucher disease (
Chemically induced proximity for other engineered PTMs
In addition to the extensively researched and commonly employed TPD technique, chemically induced proximity has also been utilized for various forms of engineered PTMs, such as phosphorylation/dephosphorylation (
Phosphorylation is the most abundant reversible protein PTM, with over two-thirds of the proteins encoded by the human genome being phosphorylated. Serine is the most frequently phosphorylated amino acid residue in the mammalian phosphoproteome, accounting for nearly 80% of all phosphorylated residues (
). PKs utilize ATP to catalyze the phosphorylation of the hydroxyl group of the amino acid side chain, while protein phosphatases (PPs) remove the phosphoryl group from phosphorylated proteins. There are approximately 500 PKs in the human genome, which can be classified into two types: serine/threonine kinases that phosphorylate serine and threonine residues and tyrosine kinases that phosphorylate the tyrosine residue (
). PKs can also be divided into nine distinct categories based on their structural and functional similarities, including AGC, CaMK, CK, PTK, with each PK group having a unique substrate spectrum and phosphorylation motifs (
). The human genome consists of 189 PPs that can be further divided into three major categories: phospho protein phosphatases, protein tyrosine phosphatases, and aspartate-based protein phosphatases (
). Phosphorylation introduces a negatively charged and hydrophilic group, which often serves as a docking site for protein–protein interactions or induces conformational changes that switch the enzymatic or receptor function of the protein on/off via the long-range allosteric effect. Reader proteins that contain phospho-Ser/Thr or Tyr binding domains, such as 14-3-3 (
To induce engineered phosphorylation on target proteins, Shoba et al. have developed a platform called phosphorylation-inducing chimeric small molecule (PHICS) that hijacks endogenous kinases like AMPK and PKC to phosphorylate target proteins, including those that are not their native substrates. The proof-of-concept compounds PHICS1 and PHICS2 are capable of recruiting AMPK and PKC, respectively, to phosphorylate BRD4 (
). BTK is a known PKC substrate that is phosphorylated at Ser180, which regulates its membrane localization and activation. The BTK-S180A mutant escapes PKC-mediated phosphorylation and inhibition, leading to the induction of B-cell malignancy (
). Notably, PHICS5-induced neo-phosphorylation of BTK inhibits its activation, which provides a promising avenue for the treatment of BTK-S180A–related cancers.
) have created a heterobifunctional molecule to recruit PP1 for the dephosphorylation of AKT. This was achieved by conjugating an Arg-Val-Ser-Phe (RVSF) tetrapeptide sequence as the PP1 ligand onto the allosteric and ATP competitive inhibitors of AKT, resulting in the formation of two phosphatase recruiting chimeras, namely 4a and 5a (
). Moreover, replacing RVSF with RVSA, a negative control peptide lacking PP1 binding affinity, completely abolished their effect on AKT dephosphorylation (
) have used an FKBP12(F36V)-PP2A fusion protein as a PP to generate a platform called phosphorylation targeting chimeras (PhosTACs) for dephosphorylation of protein targets. PhosTAC consists of an FKBP12(F36V) ligand and a HaloTag ligand, which recruit PP2A to remove the phosphoryl group from HaloTag-fused protein targets. PhosTAC7 can dephosphorylate HaloTag-PDCD4 at Ser67 in a dose- and time-dependent manner and also dephosphorylate HaloTag-FOXO3A at Ser318/Ser321. Recently, a PhosTAC has been developed that recruits PP2A to dephosphorylate tau protein and promotes its degradation (
). These advancements provide a promising approach to selectively manipulate protein phosphorylation and dephosphorylation events and have the potential to be used as research tools and therapeutics.
Acetylation is another common PTM that occurs at the ε-position of the lysine side chain within a protein (
). It is reversible and involves the transfer of an acetyl group from acetyl-Co-A onto lysine by protein acetyltransferases, including CBP/p300 and PCAF (
). Protein lysine acetylation is important for cell biology and tools that specifically regulate POI acetylation are valuable for biomedical research. Wang et al. have developed the first acetylation tagging system, called AceTAG, by hijacking the endogenous CBP/p300 acetyltransferase to induce acetylation of protein targets. AceTAG forms a ternary structure with the CBP/p300 and FKBP12(F36V) fused target protein, using a specific small-molecule ligand of the CBP/p300 BRD domain (
). Using Histone H3 as the target protein, AceTAG-1 rapidly induces its acetylation at multiple sites in as little as 5 min and the acetylation events are highly specific for the FKBP12(F36V) fused target protein but not for other endogenous untagged proteins or close family members (
), suggesting that AceTAG can generally induce selective acetylation on POIs and servers as a useful tool for studying the biological function of acetylation events.
O-GlcNAcylation occurs specifically at the serine and threonine residues (
Glycosylation of nuclear and cytoplasmic proteins. Purification and characterization of a uridine diphospho-N-acetylglucosamine: polypeptide beta-N-acetylglucosaminyltransferase.
Dynamic O-glycosylation of nuclear and cytosolic proteins: cloning and characterization of a neutral, cytosolic β-N-acetylglucosaminidase from human brain.
). Moreover, O-GlcNAc can modify the serine and threonine of kinases, which regulates their function and activity in cells. Schwein et al. recently have developed the first targeted O-GlcNAc editors, nanobody-OGT, and nanobody-splitOGA, to selectively write and erase O-GlcNAc, respectively (
). Using CK2α as the O-GlcNAc substrate, they successfully edited O-GlcNAc at Ser347 of CK2α and identified a dramatic change in the phosphoproteomics due to the alteration in CK2α kinase activity (
). Recently, a dual-specificity RNA-aptamer-based O-GlcNAc writing system has been developed by linking two RNA-aptamers for OGT and β-catenin, respectively (
). By incorporating O-GlcNAc onto β-catenin using this O-GlcNAc writing aptamer, they have found that O-GlcNAcylation stabilizes β-catenin protein and remodels the transcriptome by recruiting EZH2 to promoters (
). Therefore, these O-GlcNAc editors could be valuable tools for studying the downstream functions of protinen O-GlcNAcylation.
By taking advantage of proximity chemistry, the aforementioned heterobifunctional molecules are designed to introduce or remove native or neo-PTM on protein targets, thereby activating or deactivating the POIs (
). In addition, a recent preprint article has demonstrated the potential of a bifunctional molecule platform called regulated induced proximity targeting chimera (RIPTAC), which could elicit a designed PPI. The POI-RIPTAC-effector ternary complex abrogates the function of the effector protein, leading to the destruction of cancer cells (
Regulated induced proximity targeting chimeras (RIPTACs): a novel heterobifunctional small molecule therapeutic strategy for killing cancer cells selectively.
). These RIPTACs contain a POI ligand and a ligand for effector proteins crucial for cell survival such as BRD4, PLK1, and CDK. In the proof-of-concept design, RIPTAC suppresses the proliferation of cancer cells expressing HaloTag-FKBP12(F36V), but not of control cells without HaloTag-FKBP12(F36V) expression. By exploiting unique protein markers in certain cancers as POIs, RIPTACs can be used to target cancer cells while sparing normal cells without POI expression, resulting in a more precise treatment.
Conclusions and perspectives
Protein PTMs play a pivotal role in cellular function. However, gross inhibition of the PTM enzyme may lack specificity, affecting a broad range of substrates. To overcome this shortcoming, chemical proximity-induced PPI technology has been applied to various engineered PTMs, such as ubiquitination, phosphorylation, acetylation, and glycosylation. These lead compounds show promising effects in targeting disease-related proteins in preclinical and clinical studies. CIPs have several advantages over conventional small molecule inhibitors of upstream PTM enzymes. First, CIPs target a single PTM event on the target protein, avoiding unwanted effects on other proteins regulated by the same upstream PTM regulators. For example, AR-PROTAC only degrades AR protein, while inhibition of AR's upstream deubiquitinase USP14 reduces not only AR protein level, but also other substrates, such as FASN, therefore introducing off-target effect (
Proteasome-associated deubiquitinase ubiquitin-specific protease 14 regulates prostate cancer proliferation by deubiquitinating and stabilizing androgen receptor.
). Second, CIPs could also target undruggable targets by recruiting POI to the engineered PTM writer/eraser. Third, CIP can introduce or remove neo-PTM that may not be present under physiological conditions. However, current CIPs have some limitations, including the fact that they modify only a few types of PTMs, while many others are not amenable to engineering, such as methylation, palmitoylation, and proline hydroxylation (Fig. 3). Furthermore, more ligands of PTM upstream regulators need to be developed to introduce or remove the engineered PTM on POI, without affecting the activity of these PTM upstream regulators. Lastly, the molecular weight and structures of CIPs need to be optimized to achieve sufficient bioavailability and pharmacodynamic capability before being translated into clinical drugs. In summary, chemical proximity-induced PPI technology offers a promising approach for targeted modulation of specific PTM events on disease-related proteins, and further effects are needed to optimize these molecules for successful translation into clinical use.
Conflict of interest
W. W. is a cofounder and consultant for the ReKindle Therapeutics. Other authors declare no competing financial interests.
Author contributions
J. L. and W. W. conceptualization; Y. P. and J. L. resources; Y. P. and J. L. writing-original draft; Y. P., J. L., H. I., and W. W. writing-review and editing.
Funding and additional information
This work was supported by the NIH grants R35CA253027 and HL148667 to W. W. We apologize that due to space limitations, not all related studies are included in this review. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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