1. Structural Characteristics of PROTAC
PROTAC consists mainly of three key structures: on one side is the ligand structure targeting the Protein of Interest (POI); on the other side is the ligand structure of the E3 ligase; and in the middle is the linker connecting the two ligands.
1.1 POI Ligand
Currently, more than 360 POI ligands have been reported in PROTAC molecules. The targeted proteins include androgen receptor (AR), estrogen receptor (ER), bromodomain-containing protein (BRD), Bruton’s tyrosine kinase (BTK), and others. The POI ligand of PROTAC is generally selected from active inhibitors reported in the literature, such as the BSJ-03-204 developed by the Gray group in 2019 based on CDK4/6 inhibitor palbociclib, as well as the B03 developed by Bian’s team in 2021 based on CDK9 inhibitor BAY-1143572.
The target of PROTAC drugs not only includes the targets of conventional small molecule drugs, but also extends to non-druggable proteins, scaffold proteins, protein aggregates, drug-resistant mutant proteins, and specific subtypes of proteins. For example, the Signal Transducer and Activator of Transcription 3 (STAT3) is a transcription factor that is closely related to cancer and is considered a non-druggable protein.
1.2 E3 Ligase Ligands
Currently, there are over 600 human genome-encoded E3 ligases. However, in the field of drug design related to PROTAC, the E3 ubiquitin ligases mainly relevant are cereblon (CRBN, 71 subtypes), von Hippel-Lindau (VHL, 47 subtypes), cellular inhibitor of apoptosis protein (cIAP, 7 subtypes), and mouse double minute 2 (MDM2, 4 subtypes). Among the PROTACs that have entered the clinical stage and have disclosed structures, molecules with CRBN ligands account for over 39%, and the only PROTAC containing a VHL ligand is DT2216.
Exploring novel E3 ligase targets is one of the hot topics in the field of PROTAC research. In recent years, researchers have also discovered some structurally novel E3 ligases, including DCAF11, DCAF15, DCAF16, RNF114, RNF4, AhR, FEM1B, and KEAP1, among others. Currently, over 80 new E3 ligase ligands have been reported, and it is believed that future researchers will find more diverse E3 ligases and ligands with smaller molecular weights and higher activities.
1.3 PROTAC Linkers
The linker is an important structure that connects the two active groups of PROTAC drugs and plays a crucial role in the activity, metabolic stability, and pharmacokinetic properties of PROTAC molecules.
Currently, over 1500 different linker structures have been reported. From the perspective of molecular variability, linkers can be divided into two types: flexible and rigid. Relevant literature has shown that linkers that are too short or too long are not conducive to the efficacy of PROTAC. When the linker is too short, the steric hindrance will hinder the binding of the two ligands on both sides, making it difficult to generate a ternary complex. When the linker is too long, the E3 ligase cannot approach the target protein closely, and the ubiquitination process cannot occur. Additionally, the large molecular weight will reduce the membrane permeability of PROTAC. Studies have shown that linkers with rigidity (such as the pyridine group) or polar groups (such as polyethylene glycol) can improve the pharmacokinetic properties of PROTAC.
In addition to the structure of the linker, the connection site also affects the selectivity and activity of PROTAC. The general principles for selecting connection sites are: 1) not to reduce the binding ability between E3 ligase ligands or POI ligands and their receptors; 2) to choose the solvent-exposed area of the ligand binding pocket; 3) to ensure the integrity of the POI ligand as much as possible when connecting the linker to avoid its binding ability being affected. Figure 5 lists common binding sites for E3 ligase ligands (red arrow part) and several connection sites for POI ligands.
2. Metabolic characteristics of PROTAC
Due to its unique structural features, the molecular weight and physicochemical properties of PROTAC often exceed the limitations of Lipinski’s Rule of Five, and its metabolic processes differ significantly from traditional small molecule drugs. A deeper understanding of its metabolic characteristics will help scientists better address the challenges encountered in studying its metabolites.
2.1 Impact of E3 ligand on PROTAC metabolism
The Cruciani team found, after comparing three groups of data, that the metabolic stability of PROTAC targeting BET, CK2, and PARP proteins was significantly lower when the E3 ligand was Saldemidine compared to VHL ligand under the scenario where POI ligand remained unchanged and linker was similar. The Cruciani team speculated that the possible reason was that Saldemidine and its analogs contained multiple internal amides, which may undergo non-enzymatic hydrolysis. Another reason could be that the VHL ligand had a larger molecular weight, which led to the corresponding PROTAC’s poor cell permeability, and thus a more accurate estimation of its half-life could not be achieved. Therefore, for the study of the metabolic stability of PROTACs containing VHL ligands, liver microsomes may be a more suitable in vitro metabolic research system.
2.2 Impact of POI ligand on PROTAC metabolism
The research team of Cruciani also found that when the linker is unchanged and connected to the E3 ligase ligand, the metabolic stability of the PROTAC molecule will change with the variation of the POI ligand. For the PROTAC with the AR receptor antagonist as the POI ligand, regardless of how the linker changes with the E3 ligase ligand, its metabolic stability is lower compared to other POI ligands. Metabolite identification studies found that the metabolic soft spots of these compounds are mainly in the POI ligand, rather than the E3 ligase ligand. Considering that the POI ligand itself has a faster metabolic rate (with a half-life of 18.3 minutes), this suggests that we need to consider the metabolic stability of the POI ligand when designing PROTAC drugs, and cannot have any obvious shortcomings. For example, the PROTAC drug ARV-110, which has already been conducted in Phase II clinical trials, uses an AR receptor antagonist (It has better metabolic stability.) as the POI ligand, with a half-life of up to 110 hours.
2.3 Impact of linker on PROTAC metabolism
In PROTAC molecules, the linker is the most easily metabolized part, with the main metabolic site being the position where the linker connects with the ligand. The length, connection site, and flexibility of the linker can affect the overall metabolic stability of the molecule.
(1) The influence of the length and connection site of the linker on metabolic stability
For most PROTACs, as the length of the linker increases, the metabolic stability of the molecule decreases. As shown in Figure 7, R1 is a PROTAC designed based on JQ1 (a BET inhibitor) and thalidomide. When its straight chain alkyl linker is extended from 4 methylene units to 8 to form R2, the half-life is reduced from 135 min to 18.2 min. This may be because shorter linkers have more steric hindrance, which prevents PROTAC from entering the catalytic site of the metabolic enzyme.
Studies have also found that changes in the connection site between the linker and the E3 ligase ligand can also affect the metabolic stability of PROTAC, as shown in Figure 8.
Wang’s team reported in 2021 on PROTAC molecule 6e, which has high degradation activity towards the BTK protein but has low metabolic stability (with a half-life of 1.3 min in mouse liver microsomes). In 2023, they conducted structure-activity relationship studies on a series of rigid linkers and CRBN ligands by using a strategy of increasing linker rigidity, replacing the chain-like polyethylene glycol linker with a rigid linker containing two pyridine rings, and obtaining molecule 3e. Its metabolic stability was significantly improved (with a half-life greater than 145 min in mouse liver microsomes), and its activity was better than that of 6e.
2.4 Relationship between metabolic characteristics of PROTAC components and the whole molecule
Regarding PROTAC, which is composed of three key structures, can we predict its metabolic characteristics by the metabolic characteristics of each ligand? To answer this question, the Cruciani team conducted a systematic study. As shown in Fig. 13, the POI ligand and CRBN ligand undergo corresponding metabolism reactions of alkyl hydroxylation and amide hydrolysis, respectively. However, when they are connected by a linker to form the PROTAC molecule, the two ligand molecules do not undergo corresponding metabolic reactions, and the main metabolism occurs in the linker. In addition, the metabolic rate of the PROTAC molecule is also different from that of a single ligand. Therefore, as a new chemical entity, the metabolic characteristics of the PROTAC molecule cannot be predicted by the metabolic characteristics of its components, and metabolic product research of the overall molecule is needed.
2.5. Summary of metabolic characteristics of PROTAC
From the above analysis, it can be seen that the type of E3 ligase, stability of the POI ligand, length of linker, connection site, and changes in rigidity/flexibility can all affect the metabolism of PROTAC. The metabolic characteristics of the PROTAC molecule are different from the sum of the metabolic characteristics of its various parts, and the metabolic rate may also be completely different.
The metabolic reaction types of PROTAC include hydroxylation, acylamide hydrolysis, O-dealkylation, etc. for the ligand part, and hydroxylation, N-dealkylation, and acylamide hydrolysis, etc. for the linker part. If the linker is PEG-like, there are also a large number of O-dealkylation reactions. The enzymes involved in PROTAC metabolism mainly include Phase I metabolism enzymes (CYP enzymes) and common Phase II metabolism enzymes (uridine diphosphate glucose aldehyde transferase (UGTs), sulfotransferases (SULTs), etc.). In addition, the Cruciani team found that for PROTAC containing VHL ligands, aldehyde oxidase (hAOX) will participate in metabolism, catalyzing the hydroxylation of its thiazole ring. Since aldehyde oxidase is expressed more in the human body, research on metabolism targeting this enzyme deserves attention.
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