Introduction to PROTACs
PROTACs (PROteolysis-TArgeting Chimeras) are heterobifunctional small molecules that induce targeted protein degradation. Each PROTAC molecule contains two distinct ligands: one is a target protein ligand that binds specifically to the intracellular protein of interest (POI), and the other is a ligand that recruits an E3 ubiquitin ligase. These two ligands are covalently connected by a chemical linker.
After entering the cell, a PROTAC first engages the POI via its target protein ligand, while its E3 ligand simultaneously recruits an E3 ubiquitin ligase to form a ternary POI–PROTAC–E3 complex. The E3 ligase then mediates the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to the POI, resulting in its polyubiquitination. The polyubiquitinated POI is subsequently recognized and degraded by the 26S proteasome. Because the PROTAC molecule itself is not consumed in this process and only needs to transiently assemble the ternary complex to complete a single ubiquitination cycle, it can act in a “catalytic-like” manner in cells, driving multiple rounds of target protein degradation.
Schematic of the mechanism of PROTAC-mediated target protein degradation:
This figure illustrates the typical intracellular mechanism by which a designed PROTAC exerts its function.
Selection of E3 Ligase Ligands
The E3 ligases most widely used in current PROTAC design include CRBN (Cereblon), VHL (von Hippel–Lindau), IAP (Inhibitor of Apoptosis Proteins) and MDM2. Among these, CRBN ligands represented by thalidomide, lenalidomide and pomalidomide, together with various small-molecule VHL ligands, constitute the two most extensively applied classes.
In practical design, E3 ligands based on CRBN or VHL with established drug-like properties are usually preferred. At the same time, several factors need to be considered in an integrated manner, including:
(1) the expression level of the corresponding E3 ligase in the target cell or tissue,
(2) the impact on overall physicochemical properties of the PROTAC (e.g., polar surface area, molecular weight, LogP), and
(3) potential risks of resistance.
Selection of Linkers
The linker physically connects the E3 ligand and the target protein ligand. Its length, flexibility, polarity and topology can all significantly influence ternary complex formation, cell membrane permeability and overall pharmacokinetic behavior.
Common linker backbones include polyethylene glycol (PEG) chains, alkyl chains, and rigid fragments containing aromatic or heteroaromatic rings. An ideal linker should adopt a spatial conformation that facilitates simultaneous engagement of the E3 ligase and the POI by the PROTAC, thereby promoting formation of a stable ternary complex, while avoiding excessive polarity or molecular weight.
The attachment point of the linker (exit vector) is likewise critical. Systematic optimization is typically required, guided by crystal structures, computational modeling and structure–activity relationship (SAR) studies.
Selection of Target Protein Ligands
The target protein ligand serves as the “guiding module” of a PROTAC, responsible for specific recognition and capture of the POI. It therefore generally needs to exhibit high binding affinity and good selectivity.
In practice, reported small-molecule inhibitors, agonists or ligands of the target are usually the first candidates considered. For novel targets without known ligands, binders can be identified by high-throughput screening (HTS), fragment-based screening or virtual screening.
Unlike traditional inhibitors, target ligands used in PROTACs do not necessarily need to exert inhibitory activity or competitively occupy the active site. It is sufficient that they bind specifically to the target protein and permit installation of a suitable linker.
Key Structural Modules for PROTACs and Reagent Selection
E3 Ligase Ligands and Ligand–Linker Conjugates: Summary of Common E3 Ligase Ligands and Building Blocks Used in PROTAC Design
E3 type | Product name | CAS No. | Aladdin Cat. No. | Grade & purity | Type | Typical use / description |
CRBN (Cereblon) | Thalidomide | 50-35-1 | Moligand™ ≥98% | Classic CRBN ligand (IMiD) | One of the earliest CRBN ligands used in PROTACs; often employed as a reference scaffold or for early structure–activity relationship (SAR) exploration. | |
CRBN | Lenalidomide | 191732-72-6 | Moligand™ ≥99% | CRBN ligand (IMiD) | Second-generation clinical IMiD with improved affinity and drug-like properties versus thalidomide; a mainstream E3-recruiting ligand in many CRBN-based PROTACs. | |
CRBN | Pomalidomide | 19171-19-8 | Moligand™ ≥99% | CRBN ligand (IMiD) | Third-generation clinical IMiD with higher affinity; currently one of the most widely used CRBN ligands in PROTAC design. | |
CRBN | Thalidomide-O-COOH (Cereblon ligand 3) | 1061605-21-7 | ≥98% (HPLC) | CRBN ligand–COOH | Carboxyl group introduced at the 4-position of thalidomide via –O-CH₂–COOH; can be coupled to NH₂-containing linkers or target ligands. A very classic CRBN-PROTAC building block. | |
CRBN | Thalidomide-O-amido-PEG4-azide | 2411681-89-3 | ≥98% | CRBN ligand–PEG4–N₃ | Pre-installed PEG4 linker; one end is a CRBN ligand and the other an azide. Suitable for CuAAC/SPAAC coupling with alkyne/DBCO fragments, enabling rapid construction of CRBN-based PROTACs. | |
CRBN | Pomalidomide-PEG3-azide | 2267306-15-8 | – | CRBN ligand–PEG3–N₃ | Pomalidomide-based CRBN ligand–linker conjugate incorporating a three-unit PEG chain and an N₃ terminus; suitable for click coupling with target protein ligands. | |
VHL (von Hippel–Lindau) | VH032 | 1448188-62-2 | – | – | Classic VHL ligand | Hydroxyproline-based VHL/HIF-1α interaction inhibitor; one of the most commonly used VHL ligands in PROTAC design. |
VHL | (S,R,S)-AHPC (VH032-NH₂; VHL ligand 1) | 2055344-67-5 | – | VHL ligand–NH₂ | VHL ligand derived from VH032 bearing a free amine; can be coupled to a variety of carboxyl-containing linkers or warheads and is a mainstream E3 end in current VHL-PROTACs. | |
VHL | (S,R,S)-AHPC-PEG3-N₃ (also known as VH032-PEG3-N₃) | 1797406-80-4 | ≥95% | VHL ligand–PEG–N₃ | AHPC extended with a PEG3/PEG4 chain and an N₃ terminus; suitable for click coupling with alkyne/DBCO-containing target ligands. Widely used in kinase-targeting VHL-PROTAC design. | |
VHL | (S,R,S)-AHPC-PEG4-N₃ (VH032-PEG4-N₃) | 1797406-81-5 | ≥95% | VHL ligand–PEG–N₃ | Same as above, with a longer PEG4 linker to further adjust spacer length. | |
VHL | (S,R,S)-AHPC-PEG3-NH₂·HCl (VH032-PEG3-NH₂ hydrochloride) | 2097971-11-2 | ≥95% | VHL ligand–PEG–NH₂ | Termini such as NH₂/COOH allow flexible coupling to different warheads or further linker extension; part of a versatile family of VHL building blocks. | |
VHL | (S,R,S)-AHPC-PEG4-NH₂·HCl (VH032-PEG4-NH₂ hydrochloride) | 2010159-57-4 | ≥98% | VHL ligand–PEG–NH₂ | As above, with PEG4 to provide a longer, more flexible spacer for ternary complex optimization. | |
VHL | VH032-PEG3-acetylene (also known as VH032-PEG3-alkyne) | 2098799-80-3 | ≥95% (HPLC) | VHL ligand–PEG–alkyne | VH032 coupled to a short PEG linker terminating in an alkyne; can be connected to N₃-containing warheads via CuAAC, enabling modular synthesis of VHL-based PROTACs. | |
IAP (cIAP1/2, XIAP, etc.) | Bestatin-amido-Me (PROTAC IAP binding moiety 1) | 339186-54-8 | – | IAP ligand | Bestatin-based IAP ligand; the classic IAP-recruiting moiety in SNIPER-type degraders and can be linked via suitable linkers to kinase inhibitors and other warheads. | |
IAP | LCL161 | 1005342-46-0 | ≥99% | IAP inhibitor/ligand | Smac mimetic that binds and inhibits multiple IAP proteins; commonly used as a starting scaffold for IAP-related PROTACs or other bifunctional molecules. | |
IAP | BV6 | 1001600-56-1 | Moligand™ | IAP ligand (Smac mimetic) | Classic cIAP1/XIAP antagonist and one of the most widely used Smac mimetics; often employed as the E3-recruiting end in IAP-PROTACs. | |
IAP | Xevinapant (AT-406) | 1071992-99-8 | Moligand™ ≥98% | IAP ligand (Smac mimetic) | Smac mimetic that antagonizes XIAP and cIAP1/2; tested in combination with radio-/chemotherapy in multiple tumor models and can also serve as a warhead/E3 end in IAP-PROTACs. | |
MDM2 | Nutlin-3 / Nutlin-3a | 548472-68-0 | ≥98% | MDM2 ligand | Classic small-molecule p53–MDM2 interaction inhibitor; one of the first ligands developed as an E3-recruiting end for MDM2-PROTACs. Typically modified on the Nutlin scaffold to introduce a linker attachment site. | |
MDM2 | Idasanutlin (RG7388) | 1229705-06-9 | ≥98% | MDM2 ligand | Potent and selective p53–MDM2 antagonist; multiple MDM2-PROTACs based on this scaffold have been reported, making it a suitable starting point for further functionalization as an MDM2 ligand. | |
MDM2 | Nutlin-3a | 675576-98-4 | ≥97% | MDM2 ligand | Classic MDM2 inhibitor that blocks MDM2–p53 binding; the parent scaffold for many Nutlin-type MDM2-PROTACs. | |
MDM2 | Nutlin carboxylic acid (MDM2 ligand 1) | 2249750-27-2 | ≥98% | MDM2 ligand–linker building block | Nutlin-3-based MDM2 ligand bearing a carboxylic acid handle; can be coupled to linkers or target ligands and is a commonly used “linker-ready” MDM2 building block in PROTAC design. |
Common PROTAC Linkers: Linker Types and Representative Building Blocks Used in PROTAC Design
Linker category | Product name | CAS No. | Aladdin Cat. No. | Grade & purity | Functional groups / structural features | Typical use / application |
PEG backbone linkers | PEG3 (triethylene glycol, PEG3) | 67439-82-1 | ≥97% (GC) | HO-(CH₂CH₂O)₃-H; hydrophilic, flexible, moderate length | Basic short PEG backbone used to customize terminal functional groups and to fine-tune overall linker length and polarity; commonly serves as a “mother chain” unit in PROTACs, ADCs and related conjugates. | |
Amino / carboxyl PEG linkers | Amino-PEG4-acid (NH₂-PEG4-COOH) | 663921-15-1 | ≥98% | Primary amine at one end and carboxylic acid at the other (PEG4); good aqueous solubility | Classic “NH₂/COOH dual-headed” PEG4 linker. The two termini can be coupled to –COOH/–NH₂ groups on the E3 ligand and target ligand, respectively; a very versatile building block in PROTAC and ADC applications. | |
| Fmoc-NH-PEG2-COOH (Fmoc-8-amino-3,6-dioxaoctanoic acid) | 166108-71-0 | ≥97% | Fmoc-protected amino group + PEG2 + carboxylic acid; Fmoc can be removed to reveal a free –NH₂ | Widely used as an ADC linker and recommended as a PEG-based PROTAC linker. Suitable for solid-phase or stepwise solution synthesis to precisely tune linker length and attachment position. | |
| Fmoc-NH-PEG3-COOH | 867062-95-1 | ≥97% | Fmoc-protected amino group + PEG3 + carboxylic acid; slightly longer than PEG2 | Also used as a degradable linker in ADCs and PROTACs. Well suited for gradually extending PEG chains in solid-phase or solution synthesis to optimize ternary complex geometry. | |
| Fmoc-PEG4-NHS ester | 1314378-14-7 | ≥95% | Fmoc-protected amino group at one end and NHS active ester at the other (PEG4) | The NHS terminus can first be coupled to an NH₂-containing fragment; subsequent Fmoc deprotection reveals a second amine, enabling construction of advanced linkers bearing a “masked” amino group. | |
Click-type linkers: azide / alkyne / DBCO | Azido-PEG3-amine (Amino-PEG3-azide) | 134179-38-7 | ≥98% | Primary amine at one end and azide at the other; PEG3 backbone | Classic bifunctional linker combining click reactivity and amide coupling: the N₃ terminus can undergo CuAAC/SPAAC with alkyne or DBCO, while the NH₂ terminus couples to –COOH/NHS. Widely used in PROTAC and general bioconjugation. | |
| Azido-PEG4-NHS ester | 944251-24-5 | ≥95% | Azide at one end and NHS ester at the other; PEG4 | Widely used heterobifunctional PEG linker: the NHS terminus is first coupled to an NH₂-containing ligand, followed by click coupling of the N₃ terminus with an alkyne/DBCO counterpart. Ideal for modular assembly of PROTACs. | |
| Alkyne-PEG4-NHS ester | 1428629-70-2 | ≥97% | Terminal alkyne (–C≡CH) at one end and NHS ester at the other; PEG4 | Complementary to Azido-PEG4-NHS: the NHS terminus couples to amine-containing fragments, and the terminal alkyne is then clicked with azido-functionalized E3 or target ligands. Commonly used to construct alkyne–azide-type PROTAC linkers. | |
| DBCO-PEG4-NHS ester (dibenzocyclooctyne-PEG4-NHS ester) | 1427004-19-0 | ≥95% | DBCO + PEG4 + NHS; typical copper-free click reagent explicitly labeled as a PROTAC linker | Very common DBCO-type PROTAC linker. The NHS terminus couples to amine-containing ligands, and the DBCO terminus undergoes SPAAC with N₃-containing E3 or target ligands. Copper-free, making it well suited for cell-compatible construction or post-modification. | |
| Biotin-PEG3-azide | 875770-34-6 | ≥95% | Biotin + PEG3 + azide; click-compatible with alkyne | Frequently used as a “tool linker” in PROTAC design (e.g., pull-down assays, probe construction). Enables biotinylation of PROTACs or ligands for streptavidin-based enrichment and detection. | |
Thiol / maleimide linkers | Maleimide-PEG4-NHS ester | 756525-99-2 | ≥98% | Maleimide (thiol-reactive) at one end and NHS (amine-reactive) at the other; PEG4 | Classic –SH/–NH₂ cross-linker. Enables rapid bidirectional connection of Cys-containing peptides/proteins to small molecules (or E3/POI ligands) and can be used to build thiol-containing PROTACs or bispecific molecules. | |
Simple alkyl / rigid aromatic linkers | Adipic acid (1,6-hexanedioic acid) | 124-04-9 | Ultra pure grade ≥99.5% (HPLC) | Linear C₆ dicarboxylic acid providing a short, moderately flexible hydrophobic alkyl linker | Simple, low-cost C₆ alkyl spacer that can form aliphatic amide linkers via condensation with diamines. Helps reduce polarity and increase hydrophobicity; can be combined with PEG-based linkers. | |
| 1,6-Hexanediamine | 124-09-4 | ≥99.5% | Linear C₆ diamine; reacts with dicarboxylic acids or activated esters | Used as a flexible alkyl diamine linker, often paired with dicarboxylic acids or activated esters to build linear alkyl spacers and adjust PROTAC hydrophobicity and flexibility. | |
| p-Phenylenediamine | 106-50-3 | ≥99% (GC) | 1,4-disubstituted benzene diamine; rigid aromatic structure | Representative rigid aromatic linker unit. Can be further extended with dicarboxylic acids or isocyanates to form “para-phenylene”-type rigid linkers, useful when conformational restriction and increased spatial separation between the E3 ligase and target protein are required. |
Summary of Common and Important Target Protein Ligands in PROTAC Design
Target family | Representative target | Product name | CAS No. | Aladdin Cat. No. | Grade & purity | Role / description (as PROTAC warhead) |
BET bromodomains | BRD2/3/4 | (+)-JQ1 | 1268524-70-4 | ≥98% (HPLC) | Classic small-molecule BET bromodomain inhibitor used as a warhead in multiple BRD4/BET PROTACs; one of the most commonly used BET target ligands. | |
BET bromodomains | BRD4 | JQ-1 (carboxylic acid) | 202592-23-2 | ≥96% | Carboxylic acid derivative of JQ1 with a pre-installed –COOH handle as a linker exit vector; specifically designed for BRD4 PROTAC synthesis and a classic BRD4 warhead building block. | |
Tyrosine kinases | BTK | Ibrutinib | 936563-96-1 | Moligand™ ≥98% | Standard BTK inhibitor and the most widely used warhead in BTK-PROTACs. Forms a covalent bond with BTK Cys481 and has been adopted in numerous BTK degrader studies. | |
Multi-target kinases | BCR-ABL / Src / c-Kit, etc. | Dasatinib | 302962-49-8 | Moligand™ ≥99% | Oral multi-target kinase inhibitor frequently used as a warhead in kinase PROTACs for degradation of BCR-ABL, Src and other kinases. | |
CDKs | CDK4 / CDK6 | Palbociclib | 571190-30-2 | ≥99% | Selective CDK4/6 inhibitor widely employed as a warhead in CDK4/6-PROTACs for selective CDK6 degradation or dual CDK4/6 degradation. | |
CDKs | CDK2 / CDK7 / CDK9 | SNS-032 (BMS-387032) | 345627-80-7 | Moligand™ ≥98% | Potent CDK2/7/9 inhibitor, with particularly strong inhibition of CDK9; commonly used as a warhead in CDK9 PROTACs such as THAL-SNS-032. | |
Receptor tyrosine kinases | EGFR | Gefitinib | 184475-35-2 | Moligand™ ≥99% | Classic EGFR TKI; several EGFR PROTACs use Gefitinib as the EGFR-binding warhead, coupled to VHL/CRBN ligands to drive EGFR degradation. | |
Receptor tyrosine kinases | HER2 / EGFR | Lapatinib | 231277-92-2 | Moligand™ ≥99% | Dual HER2/EGFR TKI that can serve as a HER2/EGFR warhead scaffold in PROTAC design for degradation of HER family receptors. | |
Androgen receptor | AR | Enzalutamide | 915087-33-1 | Moligand™ ≥98% | AR antagonist. Enzalutamide derivatives are widely used as warheads in AR-PROTACs to construct AR degraders and help overcome resistance to traditional AR inhibitors. | |
Estrogen receptor | ERα | 4-Hydroxytamoxifen (4-OHT) | 68047-06-3 | Moligand™ ≥98% | Active metabolite of tamoxifen and a high-affinity ERα ligand. Commonly used as a warhead in ER-PROTACs for selective ERα degradation. | |
BCL-2/BCL-XL | BCL-2 / BCL-XL | ABT-263 (Navitoclax) | 923564-51-6 | ≥98% | Dual BCL-2/BCL-XL inhibitor and a typical warhead starting point for BCL-XL PROTACs (e.g., DT2216). Coupling to VHL ligands enables selective BCL-XL degradation. | |
BCL-2 | BCL-2 | ABT-199 (Venetoclax) | 1257044-40-8 | Moligand™ ≥99% | Highly selective BCL-2 inhibitor used to design BCL-2-targeting PROTACs, enhancing cytotoxicity against BCL-2–dependent hematologic malignancies. | |
HDACs | HDAC1/2/3/6, etc. | Vorinostat (SAHA) | 149647-78-9 | Moligand™ ≥99% | First approved broad-spectrum HDAC inhibitor with a simple, easily modifiable structure; frequently used as a warhead in HDAC-PROTACs, especially HDAC6 degraders. | |
HDACs | Multiple HDAC isoforms | Panobinostat (LBH589) | 404950-80-7 | Moligand™ ≥98% | Highly potent pan-HDAC inhibitor commonly used as a warhead targeting HDAC3/HDAC6 and other isoforms in PROTAC designs to explore downstream HDAC biology. |