PROTACs: An Emerging Therapeutic Modality in Precision Medicine

Abstract

Targeted protein degradation (TPD) has emerged as an exciting new era in chemical biology and drug discovery. PROteolysis TArgeting Chimera (PROTAC) technology targets cellular proteins for degradation by co-opting the ubiquitin-proteasome system. Over the last 5 years, numerous studies have expanded our understanding of the unique mode of action and advantages of PROTACs, which has in turn spurred interest in both academia and industry to explore PROTACs as a novel therapeutic strategy. In this review, we first highlight the key advantages of PROTACs and then discuss the spatiotemporal regulation of protein degradation. Next, we explore current chemically tractable E3 ligases focusing on expanding the existing repertoire with novel E3 ligases to uncover the full potential of TPD. Collectively, these studies are guiding the development of the PROTAC technology as it emerges as a new modality in precision medicine.

Keywords: E3 ligase; PROTACs; PhotoPROTACs; covalent ligands; proteasome; targeted protein degradation.

Figure 1-. Targeted Protein Degradation by PROTACs.

PROTACs are heterobifunctional molecules that simultaneously bind to a Protein of Interest (POI) and an E3 ubiquitin ligase complex, leading to ubiquitination and degradation of the POI via the ubiquitin proteasome system. Ub: Ubiquitin. E2: Ub conjugating enzyme, E3: E3 ligase/ substrate adaptor protein.

Figure 2-. Advantages of the PROTAC Technology.

A) PROTACs induce isoform-selective protein degradation. Pan inhibitors bind and inhibit multiple isoforms due to sequence and structural similarities among isoforms. In the depicted cartoon, the stable ternary complex between the E3 ligase and isoform 2 leads to ubiquitination and degradation, while sparing other isoforms. B) PROTACs induce degradation of multi domain proteins to eliminate both enzymatic and non-enzymatic/scaffolding roles. In contrast, small molecule inhibitors block only enzymatic functions. C) PROTACs convert promiscuous ligands into selective degraders. Promiscuous inhibitors bind to multiple proteins, however PROTACs derived from the promiscuous inhibitor do not induce degradation of bound proteins. As depicted in the carton, only protein C forms a stable ternary complex with E3 ligase, leading to effective ubiquitination and degradation. D) PROTACs can target multicomponent complexes for proteasomal degradation. Inhibitors that bind to a single protein in a complex are ineffective in blocking scaffolding functions of the protein complex. PROTACs bind to the protein complex via a single protein subunit and induce degradation of multiple proteins in the complex. E2- Ub conjugating enzyme. Orange circle on E2 represents ubiquitin.

Figure 3-. Light-induced Protein Degradation by Photoswitchable PROTACs.

A) Schematic of photoswitchable PROTACs. Upon light irradiation, the PROTAC interconverts between its cis and trans conformers, leading to changes in PROTAC functionality. As depicted, the cis photoPROTAC is unable to form the stable ternary complex between E3 ligase and POI. Upon light irradiation, the cis isomer converts to the active trans isomer, leading to stable ternary complex formation, effective POI ubiquitination and degradation. B) PhotoPROTAC1 comprised of BRD-targeting JQ1 and a VHL ligand linked via a photoswitchable tetra fluoro azobenzene moiety. Light irradiation converts the inactive cis photoPROTAC1 into its active trans isomer, and vice versa. C) PHOTAC-I-3 consists of BRD targeting JQ1 and CRBN recruiting ligands. Light irradiation converts inactive trans PHOTAC-I-3 to active cis isomer and vice versa. D) Azo-PROTAC-4C consists of BCR-abl targeting ligand and CRBN recruiting ligands. Light irradiation converts active trans azo-PROTAC-4C to inactive cis isomer and vice versa. cis azobenzene and trans azobenzene are highlighted in green and orange ovals, respectively.

Figure 4-. Light-induced Protein Degradation by Photocaged PROTACs.

A) Schematic of photocaged PROTAC. Upon light irradiation, the photocaged group is removed to form the active PROTAC. As depicted in the cartoon, the inactive photocaged PROTACs (E3 ligand side) are unable to bind to the E3 ligase thereby blocking the formation of stable ternary complex between E3 ligase and POI. Upon light irradiation, the photocaging group will be removed allowing the formation of ternary complex between POI-PROTAC-E3 ligase, leading to ubiquitination and degradation of the target protein. B) Photocaged PROTAC-1 (pcPROTAC-1) comprised of BRD-targeting JQ1 and CRBN-recruiting ligand thalidomide. The inactive pcPROTAC-1 has a DMNB as the photocaging group on the POI ligand (JQ1) side. Upon light irradiation, the DMNB group is released, producing the active PROTAC-1. C) Photocaged PROTAC-3 (pcPROTAC-3) comprised of a BTK-targeting ligand and thalidomide. Inactive pcPROTAC-3 has a NVOC as the photocaging group on thalidomide. Upon light irradiation NVOC group is released, generating active PROTAC-3. Photocaging groups (DMNB: 4,5-Dimethoxy-2-nitrobenzyl, NVOC: 6-nitroveratryloxycarbonyl) are shown as brown ovals.

Figure 5-. Molecular Glue Degraders.

A) Pomalidomide binds to CRBN, stabilizes interactions with IKZF1, causing ubiquitination and degradation of latter. B) Indisulam binds to DCAF15 and induce protein-protein interactions with RBM39, causing ubiquitination and degradation of RBM39.

Figure 6-. Structures of Covalent E3 ligase Ligands and Their Application in TPD.

A) KB02 is a covalent binder of the nuclear localized E3 ligase- DCAF16. KB02-SLF targets nuclear FKBP12 for degradation. B) Nimbolide covalently binds to the E3 ligase RNF114. BT1 is a PROTAC derived from a BCR-Abl ligand and nimbolide. C) CCW-16 covalently binds to RNF4 E3 ligase. CCW-28–3 is a PROTAC linking JQ1 to CCW-16 and targets BRD4 for degradation by recruiting RNF4. D) Bardoxolone-me (CDDO-me) is a reversible covalent ligand for KEAP1 E3 ligase. CDDO-JQ1 PROTAC links JQ1 to CDDO-me and targets BRD4 for proteasomal degradation by recruiting KEAP1. POI ligands and E3 ligase recruiting ligands are highlighted with orange and blue ovals, respectively.

Figure 7-. Conventional Non-covalent PROTACs and Covalent PROTACs.

A) Conventional PROTACs derived from non-covalent ligands (POI-yellow oval and E3-gray oval) that reversibly engage both POI and E3 ligase, leading to a stable ternary complex formation to achieve successful degradation. These PROTACs maintain the catalytic nature, a unique feature of PROTAC technology compared to small molecule inhibitors. B) Irreversible covalent PROTACs can be based on covalent targeting of the POI (left) or the E3 ligase (right). These PROTACs form a stable binary complex given that one side of the PROTAC is irreversibly bound to the POI or E3 ligase. While covalent interaction of the PROTAC with the POI (left, green oval) can compromise the catalytic feature of the PROTAC, covalent targeting of the E3 ligase (right, orange oval) retains its catalytic nature. C) Reversible covalent PROTACs are derived from a reversible covalent ligand that binds to the POI (left, magenta oval) or E3 ligase (right, purple oval), therefore they retain the catalytic nature of non-covalent PROTACs. The representative examples of POI and E3 ligases exploited in each of these PROTAC categories are listed in pink and gray colors, respectively.