In order to use the PROTAC system, it is necessary to develop a PROTAC that can target the target protein from scratch or modify the latter with tags (as described above) to degrade the molecules by HaloPROTAC or dTAG. Indeed, before developing PROTAC against endogenous proteins, it may be advantageous to conduct preliminary studies with labeled target proteins to establish a proof of concept. In view of the latest advances in genome editing, it is relatively easy to label target proteins at endogenous sites to enable ligand-induced degradation without the need for target overexpression or the development of new ligands (Brand and Winter, 2019).
Target selection
Some ideal PROTAC targets are (1) proteins without enzymatic function that cannot be regulated by traditional small molecules, (2) proteins with other functions besides enzyme activity (such as FAK or BCR-ABL as a scaffold), or (3) After the protein is inhibited, it will be compensated up-regulated, so it is difficult to achieve the maximum loss of protein function. It may be useful to look at previous knockout experiments (for example, phenotypic genome-wide CRISPR screening or RNAi) or to understand cell type-specific dependence during target selection to gather information about the target protein (Tsherniak et al., 2017).
For some proteins, the inhibitory effect is sufficient to eliminate all functions and there is almost no increase in degradation from a biological point of view, but these proteins may be few and far apart. In these cases, PROTAC may still be beneficial in terms of reducing dose and duration of action. What's more exciting is that PROTAC can extend the drug-curable proteome to any ligandable protein. Of the approximately 17,470 observed proteins (Omenn et al., 2018), only 10% to 15% are considered pharmacologically (Hopkins and Groom, 2002), and many more proteins have been shown to have ligands features (Backus et al., 2016, Parker et al., 2017), but many of these small molecule binding events have no effect. The ability to impart biologically inert ligand activity through PROTAC transformation greatly expands the medicinal proteome.
PROTAC development
Subsequently, a comprehensive literature study should be conducted on the known target ligands. If a ligand is available, checking the co-crystal structure or the known structure-activity relationship may reveal a suitable location for the linker to connect. At this point, the PROTAC transformation can be started, using synthetic chemistry and medicinal chemistry methods to attach linkers and E3 recruitment elements to the targeting ligand. If no ligand has been reported before, or if the reported compound has a suspicious structure, then a ligand identification activity for the target protein can be initiated; or, it may be simpler to use HaloPROTAC or dTAG initially. We recommend using multiple connector lengths and compositions when developing PROTAC, especially if the synthesis method allows modularity. The length and composition of the joint can have a profound effect. For example, HaloPROTAC with the ethylene glycol unit removed cannot induce degradation (Buckley et al., 2015), and BCR-ABL PROTACs with ether instead of amide have higher cell permeability (Burslem et al., 2019), based on The PROTACs of lapatinib can be either dual EGFR/Her2 degraders or selective EGFR degraders. In terms of linker length (Burslem et al., 2018b).
When PROTAC candidate molecules or cell lines expressing marker proteins are available, it is important to first confirm that they actually induce target degradation. Although mass spectrometry or flow cytometry can be used, it is usually achieved by immunoblotting, depending on the characteristics of the target protein (Buckley et al., 2015). Cyclic screening may be necessary to synthesize a sufficiently effective PROTAC, or it may be necessary to explore several labeling methods and/or to incorporate the label into the target for maximum degradation. We usually recommend 24 hours for the initial treatment time of PROTAC, but kinetic characterization usually indicates that degradation occurs faster. Recently, customized techniques for studying PROTAC in cells have been developed, and these techniques may prove to help guide its development (Riching et al., 2018). The synthesis and analysis of candidates is usually the rate-limiting step using PROTAC and depends on the quality of the chemicals available for the target. Starting with well-defined small molecules provides clear advantages, and as more advantages can be obtained through the Target 2035 program (Carter et al., 2019), the development speed and simplicity of PROTAC will undoubtedly increase.
PROTAC verification
When PROTAC is identified, it is critical to carefully characterize the degradation event through controlled experiments (for example, confirming by qPCR that protein loss occurs at post-translational levels rather than a decrease in mRNA) (Bondeson et al., 2015). Some small molecules induce significant degradation of their target proteins, but actually work at the transcription level (Field et al., 2017). According to the toxicity of the compound, it is usually necessary to use the shortest incubation time required for significant
Importantly, degradation by application of small molecules enables the use of inactive analogs as control compounds. When using PROTAC derived from inhibitors, this is essential for discovering new biology. Generally, the E3 ligase ligand structure can be discretely modified to disrupt its activity to produce inactive PROTAC. The resulting molecule cannot induce degradation, but is as effective as PROTAC in inhibiting the target. We are in favor of using VHL recruitment ligands to carefully study the relationship between inhibition and degradation, because a simple inversion of the stereocenter on the VHL ligand will produce compounds with the same inhibitory activity and pharmacological properties (such as cell permeability), thereby enabling it to As a true control (Burslem et al., 2018b). The inversion of the stereocenter can also be used to generate inactive compounds as a control for PROTAC recruited by MDM2 (Hines et al., 2019). It is possible to impair the IMiD ligand recruited by CRBN by omitting the carbonyl group or by methylation of the imide functional group. However, this does fine-tune its physical and chemical properties (Burslem et al., 2018a, Huang et al., 2018). Care must be taken when using IMiD analogs, as they may still induce the degradation of new zinc finger substrates related to the pathophysiology of IMiD (Ishoey et al., 2018).
Inactivated PROTAC is an important experimental control. It can confirm or deny the degradation mechanism of PROTAC, because the ligand alone can make the target unstable without recruiting E3 ligase (Huang et al., 2017), such as hydrophobic tag or SERD (Gustafson Et al., 2015; Neklesa et al., 2011; Wardell et al., 2011).
Another optional experiment performed at this time is proteomics to characterize the effects induced by PROTAC treatment. For example, mass spectrometry or reversed-phase protein arrays allow users to obtain PROTAC selectivity data. Quantitative proteomics is a powerful tool that can identify proteins other than target proteins that are down-regulated after PROTAC treatment. Of course, this may be the biological result of the loss of the target protein, such as the loss of c-Myc when BRD4 is degraded (Lu et al., 2015; Winter et al., 2015), or the pharmacological result, such as the accidental degradation of foretinib-based PROTAC P38α (Bondeson et al., 2018; Smith et al., 2019).
To be continued in Part Two…