Opportunities and Perspectives on the Design and Synthesis of PROTAC® Degraders

Tuesday, 05th July 2022

What is Targeted Protein Degradation?

Targeted Protein Degradation (TPD) broadly refers to strategies that re-direct a cell’s endogenous protein degradation machinery to degrade a target protein. An important strategy within TPD employs the use of heterobifunctional molecules, known generally as Degraders or PROTAC^® (PROteolysis TArgeting Chimera) Degraders, which are used to simultaneously bind an E3 ligase and a protein of interest (POI). Formation of a ternary complex between the E3 ligase, Degrader and POI facilitates polyubiquitination and subsequent degradation of the POI by the proteasome. There are a number of significant benefits to using small molecules to degrade, rather than simply inhibit a target protein. The event-driven, catalytic mechanism of action of Degraders/PROTAC Degraders (Figure 1) has the potential to become a transformative therapeutic platform.

PROTAC Degrader mode of action from ternary complex formation through polyubiquitination of the POI to proteasomal degradation of the POI

Figure 1: Schematic showing the mechanism of action of PROTAC Degraders.

Design and Synthesis of Degraders

Degraders can be designed in a modular process, utilizing three components, an E3 ligase ligand, a linker and a POI ligand. The majority of Degraders published to date harness the E3 ligases von Hippel-Lindau (VHL) and cereblon (CRBN) within the CRL2 and CRL4 E3 complexes, respectively. These large, ring-type E3 ligases display a level of conformational plasticity that facilitates ternary complex formation, and ultimately degradation, of a broad range of POI targets. It is crucial to identify a suitable site of attachment (“exit vector”) for the linker to both the POI and E3 ligase. The choice of attachment point is important, since binding affinity for both POI and E3 ligase must not be lost. Once a suitable POI ligand exit vector site has been identified, a linker is coupled to the POI or E3 ligase ligand, typically using established amide coupling, click chemistry, nucleophilic substitution, or reductive amination reactions. Typically for initial proof of concept, simple alkyl and PEG linkers are employed between the two ligands to determine the optimal linker length. Subsequent rounds of optimization often focus on linker rigidification strategies to improve the Degrader PK/PD properties.

A considerable bottleneck in TPD development is the synthesis and screening of a large panel of candidate Degraders. Synthesis can be carried out in parallel to generate Degraders with PEG and alkyl linkers of varying length and subsequently evaluated using cellular assays to determine the optimal length to promote degradation of the target protein. With so many variables to explore in the early stages of a Degrader development project, the synthetic chemistry resource requirements are substantial. Synthesis resources can be reduced by employing modular commercially available Degrader Building Blocks consisting of an E3 ligase ligand, pre-conjugated to a linker containing a reactive handle. Through enlisting a wide selection of Degrader Building Blocks, it is possible screen different: E3 ligases ligands (CRBN, VHL, IAP); exit vector type and position; and different linker lengths and types, in a streamlined manner. The use of Degrader Building Blocks can therefore reduce the time, cost and chemistry resources required to produce a library of candidate Degraders for a given POI. Our PROTAC^® Panel Builder online tool allows you to rapidly generate a bespoke collection of functionalized E3 ligase ligands plus linkers, by selecting any combination of E3 ligase ligand targeting cereblon, VHL or IAP, with PEG or alkyl linker, and functional group to couple to your POI ligand.

Cereblon-Recruiting Degraders

Immunomodulatory imide drugs (IMiD), such as Pomalidomide (Cat. No. 6302), Thalidomide (Cat. No. 0652) and Lenalidomide (Cat. No. 6305) bind to the CRBN substrate receptor of the E3 ubiquitin ligase, CUL4-RBX1-DDB1-CRBN. These CRBN ligands are extensively used in heterobifunctional Degrader development, including in Arvinas’ clinical candidates ARV-110 and ARV-471, as they are comprehensively characterized and display a favorable physicochemical profile. IMiDs have been shown to induce proximity between CRBN and proteins containing Zn-finger (ZF) motifs, which can cause polyubiquitination and degradation of these proteins. Proteins susceptible to degradation by IMiDs are known as ‘cereblon neosubstrates’. Important examples of these neosubstrates are the transcription factors IKZF1 and IKFZ3, which are known to serve important biological functions, so off-target degradation could have long-term implications.¹ It is possible to modulate and altogether avoid cereblon neosubstrate degradation by modifying the exit vector and linker used in IMiD-containing Degraders. 5'-modified phthalimides have been shown to generally display reduced ZF degradation relative to 4'-modified phthalimides, while incorporation of a fluoro group to the 6' position on the IMiD (Cat. No. 7470, Figure 2B), as featured in ARV-110, can reduce off-target ZF degradation for some exit vectors (Figure 2).

Ternary complex of a cereblon-recruiting PROTAC Degrader and chemical structures of selected cereblon ligands

Figure 2: A) Schematic showing the ternary complex of a cereblon-recruiting PROTAC Degrader. B) Cereblon ligand used in clinical candidate ARV-471. C,D) Potent and stable phenyl-glutarimide cereblon ligands.

IMiDs contain a phthalimide functional group which is reported to be hydrolytically unstable, potentially reducing the half-life of Degraders in a cellular environment. To address this matter, researchers from St Jude Children’s Research Hospital synthesized a novel cereblon ligand, without the phthalimide, called a phenyl-glutarimide (PG) (Figure 2C,D). PG derivatives retain CRBN affinity while exhibiting significantly improved chemical stability compared to IMiDs. An analog of the JQ-1 based BET bromodomain Degrader, dBET1 (Cat. No. 6327), in which the IMiD was replaced by a PG ligand, displayed a 6-fold higher degradation potency and increased the degradation efficiency after 24-hour incubation 15-fold.² Baricitinib-based JAK2 Degraders were shown to degrade the cereblon neosubstrate GSPT1 as an off-target, however analogous PG containing degraders maintained degradation potency whilst showing reduced off-target GSPT1 degradation.³ These promising in vitro results indicate PG ligands are worth further exploration as CRBN ligands in heterobifunctional degraders.

“Cereblon is likely to continue dominating targeted protein degradation translational research for many years to come. Consequently, developing novel ligands, such as PG, with different exit vectors and overall properties is critical for unlocking its full potential in drug discovery.”
– Zoran Rankovic, St Jude Children’s Research Hospital

5’-fluoro pomalidomide and phenyl-glutarimide building blocks are also available pre-conjugated to linkers through the Tocris PROTAC Panel Builder

VHL-Recruiting Degraders

The pioneering work of Alessio Ciulli, Craig Crews and co-workers, resulted in the development of small molecules that disrupt the von Hippel-Lindau (VHL)/HIF-1α interaction. This foundational work has ultimately resulted in the present availability of several potent and selective hydroxyproline containing VHL/ HIF-1α inhibitors, exemplified by VH 298 (Cat. No. 6156). Related inhibitors, e.g. VH 032 (Cat. No. 7774 - coming soon) and VH 101 are frequently used in the context of Degrader discovery to recruit the VHL substrate receptor, and as such have been made commercially available with convenient chemical ‘handles’ for conjugation to the linker-warhead ligand: VH 032, amine (Cat. No. 6462), VH 032, phenol (Cat. No. 6911), VH 101, phenol (Cat. No. 6952). These frequently used VHL ligands have a higher molecular weight than the IMiD based CRBN ligands, resulting in a substantial difference in physicochemical properties of the final Degraders. This is purported to be one of the key reasons for the lack of VHL-based Degraders that have progressed into the clinic. An interesting benefit of the core VHL ligand scaffold is the opportunity to explore several linker attachment sites (‘exit vectors’), which can impact the formation of the ternary complex, resulting in activity differences between Degrader analogs.

“Von Hippel-Lindau (VHL) is an important E3 ligase involved in fundamental biology that was recognized by the 2019 Nobel Prize in Physiology or Medicine. Our design and development of hydroxyproline-based small-molecule ligands for VHL has ushered a revolution in the PROTAC field, leading to very high-quality degraders with established mode of action. The recent findings that VHL-based PROTAC degraders can also be orally bioavailable, and show brain exposure, offers an exciting boost to unlocking their potential as clinical drugs.”
– Alessio Ciulli, Centre for Targeted Protein Degradation, University of Dundee

Ternary complex of a VHL-recruiting PROTAC Degrader including a representation of how different E3 ligand exit vectors can affect ternary complex formation

Figure 3: A) Schematic showing the ternary complex of a VHL-recruiting PROTAC Degrader. B) Schematic showing how the E3 ligand exit vector can affect ternary complex conformation. C) VHL-recruiting E3 ligase ligand used to generate PROTAC Degraders. D) Potent and selective VHL/HIF-1α interaction inhibitor.

Crews’ et al. demonstrated differential isoform-selectivity in p38 MAPK Degraders by using a single POI ligand and altering the orientation of the VHL ligand through alternate linker attachment sites between the amine and phenol site (Figure 3).⁴ Researchers at the University of Dundee and Boehringer Ingelheim selectively targeted BAF Chromatin Remodelling complex ATPase SMARCA2 with a VHL recuiting Degrader. By installing a linker attachment site at the benzylic position on VH 101 (Figure 3) they were able to demonstrate, through careful tuning of the exit vector, linker length and type, that orally bioavailable VHL-recruiting Degraders can indeed be developed. Another study at the University of Dundee employed a thiol exit vector on VH 101 to a Leucine Rich Repeat Kinase 2 (LRRK2) inhibitor, via a short cyclic linker. The resulting optimized LRRK2 degrader was orally-bioavailable and even blood brain barrier penetrant.

“By exploring the widest possible spectrum exit vectors and chemical functionalities used to connect to a linker, recent studies from CeTPD, together with our collaborators, have unearthed fascinating new opportunities for VHL based degraders. This has included oral bioavailability and activity as well as brain penetrant molecules, speaking to the value of continuing to develop synthetic approaches for connecting bifunctional molecules in diverse and efficient ways.”
– Dr Will Farnaby, Centre for Targeted Protein Degradation, University of Dundee

IAP-Recruiting Degraders

Inhibitor of apoptosis proteins (IAP) are a family of proteins that play crucial roles in inhibiting cellular apoptosis pathways, through regulating caspase activity. Five of the eight IAPs, such as cellular inhibitor of apoptosis-1 (cIAP1) and X-linked inhibitor of apoptosis protein (XIAP), also contain RING domains that function as E3 ligase complexes which have been harnessed for TPD by small molecule approaches. First generation heterobifunctional degraders employed aminopeptidase inhibitor Bestatin (Cat. No. 1956) to recruit IAP (Figure 4). These compounds, termed Specific and Nongenetic IAP-dependent Protein Erasers (SNIPERs), were able to degrade a number of targets, such as estrogen receptor-α (ERα), androgen receptor and BCR-ABL, however were only able to induce degradation when used at concentrations great than 10 µM. Subsequent optimization of bestatin resulted in the generation of potent and selective IAP ligands, such as LCL 161 and A 410099.1 (Cat. No. 6470). LCL 161 based SNIPERs were able to degrade ERα with a low nanomolar DC₅₀(<3 nM) and displayed in vivo activity in mouse xenograft models. LCL 161, phenol (Cat. No. 7178) and A 410099.1, amine (Cat. No. 6471) are IAP ligands functionalized with reactive handles, ready to conjugate to a linker or target protein ligand for Degrader development (Figure 4).

Ternary complex of IAP-recruiting SNIPERs and chemical structures of selected IAP ligands used in SNIPER development

Figure 4: Schematic showing the ternary complex of IAP-recruiting SNIPERs. A-D) IAP ligands used to develop SNIPERs.

KEAP1-Recruiting Degraders

KEAP1 is a member of the Kelch-like protein family and functions as a redox-regulated substrate adaptor for CUL3 E3 ligases. KEAP1 is more highly expressed in human cancers than VHL or CRBN and has distinctive tissue distributions, displaying high expression in lung, kidney, breast, and prostate carcinoma. Selectively targeting specific tissues can offer an increased therapeutic index by reducing off-target effects, therefore recruitment of E3 ligase activity by KEAP1-directed Degraders targeting oncogenic proteins is of significant interest. Two binding sites on the KEAP1/CRL3 complex are primarily targeted, the Kelch domain which directly blocks Nrf2 recruitment, and Cys-151 on the BTB domain, by non-covalent and covalent small molecule ligands, respectively. Researchers at Massachusetts General Hospital used their robust and versatile CoraFluor assay platform on wild-type, full-length KEAP1 to probe the relative affinities and domain selectivities of a range of the electrophilic reversible covalent KEAP1 inhibitors: CDDO-Me (Cat. No. 6646), DMF, and obtusaquinone, and the non-covalent inhibitor KI-696 (Figure 5).

Ternary complex with a KEAP1-recruiting PROTAC Degrader and chemical structures of selected KEAP1 ligands

Figure 5: Schematic showing the ternary complex of a KEAP1-recruiting PROTAC Degrader. A) Potent and selective KEAP1-NRF2 interaction inhibitor. B-D) Covalent KEAP1 ligands.

“Our highly versatile and modular CoraFluor TR-FRET assay platform enables the sensitive characterization of small molecule Keap1 ligands, including the domain-selective quantification of binding affinities and dissociation kinetics using untagged full-length protein or individual epitope-tagged domains.”
– Ralph Mazitschek, Massachusetts General Hospital

Future Outlook

PROTAC Degraders and the field of TPD represent an exciting platform for therapeutic development as well as an invaluable research tool to directly modulate protein levels by a small molecule approach. The chemical space within TPD is evolving rapidly as companies and academic institutes strive to recruit more of the 600 known E3 ligases in the human genome that have not been yet explored for TPD. There are significant and concerted efforts to expand this research area, discovery of each new E3 ligase ligand for TPD presents a wealth of new opportunities and challenges within this exciting field.

View Products