PROTAC Degraders with Ligands Recruiting MDM2 E3 Ubiquitin Ligase: An Updated Perspective

Abstract

Mouse double minute 2 (MDM2) is an E3 ubiquitin ligase which effectively degrades tumor suppressor p53. In the past two decades, many MDM2 inhibitors that disrupt the MDM2-p53 binding have been discovered and developed. Given that the MDM2-p53 forms auto-regulatory loop in which p53 is a substrate of MDM2 for targeted degradation, while MDM2 is a p53 target for transcriptional upregulation, these MDM2 inhibitors have limited efficacy due to p53 degradation by accumulated MDM2 upon rapid in vivo clearance of the MDM2 inhibitors. Fortunately, proteolysis targeting chimeras (PROTACs), a novel therapeutic strategy, overcome the limitations of MDM2 inhibitors. Some of MDM2 inhibitors developed in the past two decades have been used in PROTAC technology for two applications: 1) as component 1 to bind with endogenous MDM2 as a target for PROTAC-based degradation of MDM2; and 2) as component 2 to bind with endogenous MDM2 as a PROTAC E3 ligand for PROTAC-based degradation of other oncogenic proteins. In this review, we summarize current progress in the discovery and development of MDM2-based PROTAC drugs with future perspectives and challenges for their applications in effective treatment of human cancer.

1. Introduction

The p53 tumor suppressor is a potent transcription factor that plays key roles in cancer prevention^{1, 2}. Upon activation by a variety of stresses internally or externally, p53 induces growth arrest, if damage is repairable or apoptosis to eliminate unrepairable cells^{3, 4}. The loss of p53 tumor-suppressor activity by point mutations or gene deletion, which is most frequently seen in many human cancers, escapes the p53 role as a genome guardian and allows abnormal proliferation of cells carrying damaged genome⁵. Such uncontrolled proliferation leads to cancer development. Thus, restoring p53 activity or function in cancer cells has been a long-term goal in the field of cancer drug discovery⁶.

MDM2 has been well characterized as a negative regulator of p53 by targeting p53 via direct binding⁷. A variety of stress signals disrupt the MDM2-p53 interaction, leading to p53 activation and subsequent cellular responses, such as growth arrest and apoptosis induction^{8, 9}. MDM2 effective inactivation of p53 through three general mechanisms^10–12: 1) MDM2 directly binds to the transcriptional activation domain of p53, thus inhibiting p53-mediated transcriptional activation; 2) MDM2 contains a nuclear export signal sequence, that induces p53 nuclear export upon binding to prevent p53 from binding to the target DNAs; 3) most effectively, MDM2 is an E3 ubiquitin ligase that promotes ubiquitylation and degradation of p53. Thus, the disruption of the MDM2-p53 interaction had become an effective strategy in the past two decades for the discovery and development of potent MDM2 inhibitors that disrupt the MDM2-p53 binding, leading to stabilization and restoration of p53 function for the treatment of human cancers, harboring a wild-type p53^13–16.

In the past two decades, PROTAC strategies have gained momentum and shown promise in the discovery and development of new types of small-molecule therapeutics by inducing targeted protein degradation^17–22. A PROTAC molecule consists of three components^23–27: 1) a small molecule that specifically binds to targeted proteins; 2) another small molecule or peptide that binds to an E3 ligase as an E3 ligand; and 3) a chemical linker the connects the first two components. To date, PROTAC technology has been used to target various proteins, including transcription factors, skeleton proteins, nuclear receptors, enzymes, and regulatory proteins^28–37. In the cancer therapy, many studies have shown that degrading a protein is more effective than inhibiting it^38–41.

In this review, we will introduce a battery of MDM2 inhibitors, and then describe how some of these inhibitors are used to build-up several MDM2-recruiting PROTAC degraders (Table 1) for 1) the disruptors of the MDM2-p53 binding to stabilize p53, and 2) acting as E3 ligand component of PROTAC for degradation of other targeted oncogenic proteins (Figure 1).

Table 1.

The summary of MDM2 inhibitors and degraders

No.	Name	Structure	Category	E3 ligase ligand	Target protein
1	Nutlin-3		Inhibitor	--a	--
2	RG7388		Inhibitor	--	--
3	RG7112		Inhibitor	--	--
4	MI-77301 / SAR405838		Inhibitor	--	--
5	HDM201		Inhibitor	--	--
6	DS-3032b		Inhibitor	--	--
7	APG-115		Inhibitor	--	--
8	MK-8242		Inhibitor	--	--
9	NVP-CGM097		Inhibitor	--	--
10	AMG-232		Inhibitor	--	--
11	WB214		Degrader	CRBN	MDM2
12	TW-32		Degrader	CRBN	MDM2
13	MD-224		Degrader	CRBN	MDM2
14	MG-277		Degrader	CRBN	MDM2
15	--		Degrader	Nutlin-3	AR
16	A1874		Degrader	RG-7388	BRD4
17	--		Degrader	Nutlin-3	PARP1
18	--		Degrader	Nutlin-3	MDM2
19	--		Degrader	RG-7388	EGFR
20	--		Degrader	Nutlin-3	TRKC

^aNot Applicable

Figure 1.

Working model of MDM2-recruiting PROTAC degraders. A) MDM2 promotes the degradation of p53 with overexpression of MDM2. B) MDM2 inhibitors block the protein-protein interaction of MDM2-p53. C) MDM2 degraders block the protein-protein interaction of MDM2-p53 and degrade the POI (protein of interest).

2. MDM2 inhibitors

The MDM2 E3 ubiquitin ligase was found overexpression in a number of human cancers, particularly in soft tissue sarcomas^42–45. The main function of MDM2 is to ubiquitylate tumor suppressor p53 for proteasomal degradation, thus acting as an oncogenic protein to promote tumorigenesis⁴⁶. Thus, targeting MDM2 via disrupting the MDM2-p53 binding was an effective approach for p53 activation and targeted anti-cancer therapy⁴⁷. Toward this end, many small-molecule inhibitors targeting the p53-MDM2 protein-protein interactions have been discovered in past two decades with Nutlin-3 as the first one², and few are currently in clinical development may be effective in the treatment of cancer and other related diseases, including RG7388 (NCT02407080)⁴⁸, RG7112 (NCT01605526, NCT01143740, NCT01677780)^{49, 50}, SAR405838 (NCT01636479, NCT01985191)^{5, 51, 52}, HDM201 (NCT02143635, NCT02343172)^53–55, DS-3032b (NCT01877382, NCT02579824, NCT02319369, NCT03634228)^{56, 57}, APG-115 (NCT03781986, NCT02935907)^{58, 59}, MK-8242 (NCT01451437, NCT01463696)^60–62, NVP-CGM097 (NCT01760525)^63–65, and AMG-232 (NCT03217266, NCT03107780, NCT03041688, NCT03031730)^66–68 (Figure 2 and Table 1).

Figure 2.

Representative MDM2-p53 inhibitors in clinical trials.

While p53 is a substrate of MDM2 for targeted degradation, MDM2 itself is a p53 target, subjected to p53 upregulation⁶⁹. This auto-regulatory feedback loop produces a potential side-effect for MDM2 inhibitors, which disrupt the MDM2-p53 binding to free up p53, accumulated p53 then transactivates MDM2 to cause MDM2 accumulation, leading to p53 degradation when MDM2 inhibitors are rapidly cleared in vivo, thus compromising the therapeutic effect of MDM2 inhibitors. In fact, studies have shown that p53 protein accumulates in xenograft tumor tissue for only several hours after a single dose of MDM2 inhibitor is administered. Furthermore, the accumulation of MDM2 protein in normal tissues may have deleterious effects, because MDM2 is itself oncogenic⁵. To overcome these potential drawbacks of MDM2 inhibitors, new strategies are needed to target MDM2 more effectively.

3. PROTACs and MDM2-associated degraders

Targeted protein degradation (TPD) via the ubiquitin proteasome system has received substantial interest among medicinal chemists and biologists, and is an emerging direction in the field of drug development^70–73. Toward this end, the molecules, designated as proteolysis-targeting chimeras (PROTACs), have been developed, which are bi-functional molecules that hijack cellular ubiquitin proteasome system to achieve the degradation of a disease-related target protein^74–77. Structurally, PROTACs are new chimeric molecules, consisting of three components: one end binds to the targeted protein, also known as protein of interest (POI), and the other end is a ligand for recruitment of an E3 ubiquitin ligase, and a linker in between to connect both end (Figure 3)^{78, 79}.

Figure 3.

Mode of action of PROTAC degraders.

After multiple rounds of recruiting ubiquitin to the target protein to form polyubiquitinated target, the PROTAC-target molecule is recognized by the 26S proteasome for target degradation⁸⁰. PROTAC molecule is then recycled for next round of POI targeting. Thus, PROTACs target POIs through an “event-driven” mode rather than an “occupation-driven” mode for small-molecule drugs^{81, 82}. The advantage of PROTAC molecules include the ability to overcome drug resistance and target undruggable targets with low toxicity, high efficiency and selectivity^{83, 84}. It is worth-noting that drug resistance develops to varying degrees after a certain period of clinical use of almost all traditional small-molecule drugs. PROTACs effectively overcome this problem via degrading the target proteins. Furthermore, whereas most traditional small-molecule drugs work by binding to the active site of an enzyme or receptor, PROTACs work efficiently as long as there is effective binding with POI at essentially any site (Figure 3).

Human genome encodes more than 600 E3 ligases, but only a small fraction has been used in designing PROTACs to date, including Cereblon (CRBN), von Hippel-Lindau (VHL), MDM2, and cellular IAP1 (cIAP1)^85–87. Two types of MDM2-related PROTAC degraders have been developed. One type is to recruit MDM2 inhibitor as MDM2 binding partner for MDM2 degradation. The second type is to recruit MDM2 as E3 ligand to target other POIs for degradation, although it is less effective than those recruiting CRBN and VHL.

4. PROTAC degraders targeting MDM2

The first type PROTACs, based on nutlin or idasanutlin (RG7388), have been developed with representative CRBN-based MDM2 degraders shown in Figure 4. However, the number of these MDM2 degraders is still limited, owing to their challenging physicochemical profiles and limited degradation activities (e.g. WB214 and TW-32)⁸⁸. Recently, Wang group disclosed a series potent PROTAC MDM2 degraders, including MD-224 as a lead that targets MDM2 protein to CRBN for degradation (Figure 4)⁸⁹. MD-224 is effectively in rapidly degrading MDM2 in leukemia cells. Intravenous administration of MD-224 was found to achieve complete and durable tumor regression in a RS4–11 xenograft model. Moreover, MD-224 inhibits the growth of only leukemia cells carrying wild-type p53 but not p53 mutants. On the basis of the previously reported PROTAC MDM2 degrader MD-224, Wang group have disclosed additional analogues. Interestingly and unexpectedly, MG-277, an analogues of MD-224, showed conversion of the PROTAC into a “molecular glue” (Figure 4)⁹⁰. MG-277 induced only moderate MDM2 degradation and failed to activate wild-type p53. However, it shows highly potent inhibition of tumor cell growth in a p53-independent manner. This compound provides the first example demonstrating that a simple structural modification can turn a bona fide PROTAC degrader into a molecular glue compound⁹⁰.

Figure 4.

Representative CRBN-based MDM2 degraders and molecular glue.

5. PROTAC degraders recruiting MDM2 to target other POIs

5.1. MDM2-based PROTAC AR degrader

Nutlin-3 is a potent MDM2 inhibitor that bind to the p53-binding pocket of MDM2⁹¹. Interestingly, Nutlin-3 were also used for the design of the second type PROTACs to recruit endogenous MDM2 for targeting androgen receptor (AR) by Crews group in 2008⁹². This was the first report of an all-small-molecule PROTAC degrader, consisting of an AR antagonist and the nutlin motif, that disrupted the interaction of MDM2 with p53 without affecting the E3 ligase activity of MDM2. Specifically, this first synthetic all-small-molecule PROTAC AR degrader was a heterobifunctional compound consisting of a bicalutamide analogue (non-steroidal androgen receptor ligand) and MDM2 inhibitor joined by a PEG-based linker (Figure 5, compound 15). This cell permeable PROTAC successfully recruits the androgen receptor to MDM2 for ubiquitination and proteasomal degradation, but with weak potency⁹².

Figure 5.

Schematic design of an MDM2 (nutlin-3)-based PROTAC AR degrader. A) Structure of the AR antagonist bicalutamide and its working model with androgen receptor (AR). B) Structure of the MDM2 inhibitor nutlin-3 and its crystal structure with MDM2.

RG7388, another typical MDM2-p53 inhibitor, was also found to bind to MDM2 and used in PROTACs design⁹³. However, the poor physiochemical properties of nutlin-3 were exacerbated after incorporation into PROTACs. Recent efforts have identified MDM2-p53 PPI inhibitors with better solubility and activity may increase the applications of MDM2 in PROTACs (Figure 6)^93–95.

Figure 6.

Representative MDM2 E3 ligase ligands and the corresponding PROTACs.

5.2. MDM2-based PROTAC BRD4 degrader

Recently, numerous BET PROTAC degraders have been generated and tested in vitro and in vivo^{96, 97}. Various E3 ligases, including VHL, CRBN, IAP, MDM2, aryl hydrocarbon receptor (AHR), DDB1-cullin 4 associated factor 16 (DCAF16), RING finger protein 114 (RNF114), and RNF4, have been used as E3 ligands to degrade BET proteins^98–105. In 2019, Crews group reported an MDM2/nutlin-based BRD4 PROTAC (compound 15, A1874)⁹³, which not only degrades BRD4 protein but also stabilizes p53 gene, and thus exhibiting strong anti-proliferative effects in several tumor cell lines, such as myeloid leukemia cells (Figure 7). Moreover, it increases p53 levels in HCT116 colon cancer cells harboring a wild-type p53 due to the activity of RG7338 against MDM2. A1874 potently inhibits the proliferation of p53-wild-type cancer cells, presumably through dual inhibition of BRD4 and MDM2.

Figure 7.

Schematic design of an MDM2 (nutlin-3)-based PROTAC BRD4 degrader. A) Structure of the BRD4/BET inhibitor JQ1 and its crystal structure with BRD4. B) Structure of the MDM2 inhibitor Idasanutlin (RG7388) and its crystal structure with MDM2.

5.3. MDM2-based PROTAC PARP1 degrader

The PARP1 poly(ADP-ribose) polymerase is a ubiquitously expressed DNA-dependent nuclear poly(ADP-ribosyl)transferase that regulates multiple nuclear events, such as transcription, rRNA biogenesis, and DNA repair^{106, 107}. Owing to its essential role in the DNA-damage response, PARP1 is considered a potent cancer therapeutic target. Several PARP1 inhibitors, such as niraparib, iniparib and olaparib, are in various stages of clinical development; however, cytotoxicity and drug resistance are the primary problems that restrict their use in patients^{108, 109}. Thus, additional therapeutic methods to overcome these obstacles are demanded.

In 2018, Rao group reported the first PARP1-targeting PROTAC by linking the PARP1 inhibitor niraparib and the MDM2 inhibitor nutlin-3. Compound 17 was obtained after detailed degradation screening in several triple-negative breast cancer cell lines (Figure 8)⁹⁵. Impressively, the compound 17 selectively induced significant PARP1 degradation and cell apoptosis in MDA-MB-231 cells with fivefold more potent in antiproliferative activity than the PARP1 inhibitors niraparib, olaparib, and veliparib, while showing no cytotoxicity in normal cells.

Figure 8.

Schematic design of an MDM2 (nutlin-3a)-based PROTAC PARP1 degrader. A) Structure of the PARP1 inhibitors and the crystal structure of niraparib bound to the PARP1 catalytic domain (PDB code: 4R6E). A click chemistry strategy was used to construct the PROTAC candidates targeting PARP1. B) Structure of the MDM2 inhibitor nutlin-3a and its crystal structure with MDM2.

5.4. MDM2-based PROTAC homo-MDM2 degrader

Inspired by previous efforts to design small molecules targeting the MDM2-p53 interaction, the first homo-PROTAC, targeting MDM2 by inducing its self-degradation was recently reported by Sheng group (Figure 9)⁹⁴. The compound 18 efficiently induced MDM2 dimerization with highly competitive binding activity, and induced proteasome-dependent self-degradation of MDM2 in A549 non-small cell lung cancer (NSCLC) cells. Impressively, compound 18 effectively inhibited the growth of tumor in a xenograft mouse model derived from A549 cells, the first demonstration of the in vivo efficacy of homo-PROTAC, which could be an alternative therapeutic tool for effective targeting human cancers with overexpressed MDM2.

Figure 9.

Homo-PROTAC design strategy: A) Chemical structures of nutlin-3. B) Binding model of compound nutlin-3 with MDM2 (PDB: 4IPF).

5.5. MDM2-based PROTAC EGFR degrader and TrkC degrader

In 2020, Ding group reported a series of PROTAC EGFR degraders based on different E3 ligase ligands, one of which was MDM2 inhibitor RG7388¹¹⁰. The compound 19 has moderate degradation on EGFR^L858R/T790M mutant (Table 1). In 2019, Burgess group reported a class of TrkC-targeted kinase PROTACs by linking the TrkC-targeted kinase inhibitor IY−IY and the MDM2 ligand nutlin-3¹¹¹. However, compound 20 has very weak potency in degrading TrkC protein (Table 1).

All 20 compounds described here are summarized in Table 1.

6. Summary and outlook

As we enter the third decade of TPD strategy, which was first reported in 2001¹¹², PROTACs will remain at the forefront of research for targeted degradation of POIs, and have begun to move from vertical to horizontal development. A variety of developed TPD technologies have been discovered and developed, including photo‐controlled PROTACs, homo‐PROTACs, covalent PROTACs, dual-PROTACs, antibody‐PROTACs, lysosome‐targeting chimeras (LYTACs), autophagy‐targeting chimeras (AUTACs) and peptide-based PROTACs^113–118. MDM2-based PROTACs selectively binds to the p53 site on the surface of MDM2 stabilizing p53 and degrading target protein with better anti-cancer activity. These PROTACs enable the degradation of a variety of undruggable proteins. However, the major bottleneck in the development of effective PROTAC drugs lies in achieving good oral bioavailability, given that PROTAC molecules are “beyond the rule of 5” for small molecule inhibitors due to their higher molecular weight (M.W.), poorer solubility (LogP), higher topological polar surface area (tPSA) and other poor physicochemical properties. Compounds with favorable physiochemical properties, such as lower M.W. (<800 Da), good water solubility, ideal tPSA, and fewer aromatic groups, must be discovered to increase oral bioavailability. The application of molecule glue could be an effective approach^{119, 120}. The second challenge is to develop more effective E3 ligase, acting as the ligands for PROTACs, given human genome encodes more than 600 E3 ligase. Currently several oral-available PROTAC drugs are in few clinical trials for cancer treatment^{19, 21, 22, 121, 122}. The knowledge gained from basic research and clinical trials in combination of technology development will certainly advance the field of cancer therapy using PROTAC strategy.