Targeted protein degradation using thalidomide and its derivatives

Abstract

Thalidomide, a small-molecule drug, caused a global drug disaster more than half a century ago. Thalidomide derivatives are widely used to treat several hematological cancers. Recent studies have revealed that thalidomide and its derivatives act as protein degraders that bind to cereblon (CRBN), the substrate-recognition receptor of a complex-type E3 ubiquitin ligase, and degrade neo-substrates that are not their original substrates. Furthermore, they are currently being used in proteolysis-targeting chimeras (PROTACs), which are chimeric protein degraders. Protein degraders open up a new mechanism of drug action and proteolysis and have the potential to target various therapeutic proteins, including undruggable proteins. Analysis of drug-dependent protein-protein interactions (PPIs) is crucial for the development and use of this new class of protein degraders. We developed an in vitro and intracellular protein degrader-dependent PPI analysis method using a cell-free protein synthesis system and proximity-dependent biotinylation. Using these analytical methods, we successfully identified several neo-substrates involved in the mechanism of action of thalidomide. Furthermore, we developed thalidomide derivatives and PROTACs with enhanced selectivity for neo-substrates or target proteins involved in the anti-hematological cancer effects. In this paper, we review the history of thalidomide and the research achievements related to protein degraders, as represented by thalidomide derivatives.

Highlights

Thalidomide is a small molecule drug that caused a global drug disaster more than half a century ago. Currently, thalidomide derivatives are used to treat hematological cancers. Thalidomide and its derivatives are drugs that induce targeted protein degradation and are applied to heterobifunctional protein degraders called proteolysis-targeting chimeras (PROTACs). Thalidomide derivatives and PROTACs are promising drug discovery approaches for undruggable proteins. However, the development of selective protein degraders that do not degrade off-target proteins remains a challenge. This review introduces the history of thalidomide, recent studies on protein degraders, and its future prospects.

Introduction

History of the thalidomide drug disaster

Thalidomide was developed in the late 1950s by the German pharmaceutical company Chemie Grünenthal as a non-toxic, non-barbiturate sedative [1, 2]. Thalidomide has proven to be highly effective and has been used in at least 40 countries, particularly as an effective treatment for morning sickness in pregnant women [1,2,3]. Thalidomide was considered safe as it showed no significant side effects in in vivo studies [3,4,5,6]. However, shortly after its release, reports of peripheral neuropathy due to thalidomide use and severe birth defects in babies born to mothers who had taken thalidomide began to emerge, leading to its withdrawal from the market in the early 1960s [1, 7]. Congenital defects caused by thalidomide are most commonly reported in the limbs, but also affect multiple tissues and organs, such as the eyes, ears, and heart [1,2,3,4,5, 8, 9]. Therefore, thalidomide is one of the largest drug disasters worldwide, highlighting, for the first time, species differences in drug responses and prompting a review of pharmaceutical testing methods.

Thalidomide and its derivatives today

Research on the mechanism of action of thalidomide has continued even after its withdrawal from the market, and it has been reported that thalidomide exhibits various effects in the body. In 1965, it was reported that thalidomide was effective against erythema nodosum leprosum, a complication of leprosy [10, 11]. Subsequently, from the 1980s to the 1990s, thalidomide was reported to be effective against autoimmune diseases, such as rheumatoid arthritis [12], to have anti-inflammatory effects [1, 13] to inhibit angiogenesis [1, 2, 14, 15]. In 1999, it was shown to be effective in the treatment of multiple myeloma [16]. Based on these findings, thalidomide was approved by the U.S. Food and Drug Administration (FDA) in 1998 and 2006 for the treatment of erythema nodosum leprosum and multiple myeloma, respectively. Subsequently, thalidomide derivatives, such as lenalidomide and pomalidomide, were developed as immunomodulatory drugs (IMiDs) [17, 18]. IMiDs are highly effective against several hematological cancers, including multiple myeloma [19,20,21,22], and have become representative small-molecule drugs with annual sales exceeding USD 10 billion.

Mechanism of action of thalidomide and its derivatives

The mechanism of action of thalidomide has remained unclear for more than half a century. In 2010, Handa et al. identified cereblon (CRBN) as a target protein involved in thalidomide-induced teratogenicity [23]. CRBN was known to function as a substrate receptor for the E3 ubiquitin ligase complex CRL4 [23, 24]. However, there were no reports of CRBN-associated genetic disorders with phenotypes similar to thalidomide teratogenicity [25], suggesting the presence of target proteins other than CRBN. In 2014, Kaelin Jr., et al. elucidated that the transcription factors Ikaros family zinc finger protein 1 (IKZF1) and Ikaros family zinc finger protein 3 (IKZF3), which are not original substrates of CRL4^CRBN, induced protein degradation by lenalidomide [26, 27]. Therefore, thalidomide and its derivatives (immunomodulatory drugs/IMiDs) act as molecular glues and induce protein degradation of “neo-substrates” as molecular glue-type protein degraders [28, 29]. Subsequently, various neo-substrates involved in the pharmacological actions and side effects of thalidomide and its derivatives were reported, and their mechanisms of action were gradually elucidated [30,31,32,33,34,35,36,37].

Targeted protein degradation using thalidomide and its derivatives

Proteins without enzymatic activity, including transcription factors, are considered “undruggable” and difficult to target [38]. IMiDs induce the degradation of several transcription factors [30,31,32,33,34,35,36,37], suggesting that protein degraders may be a promising approach for targeting undruggable proteins that account for more than 80% of the human proteome [39]. Furthermore, IMiDs are currently used to develop chimeric protein degraders known as proteolysis-targeting chimeras (PROTACs), which induce the degradation of therapeutic target proteins [40, 41]. PROTACs consist of two functional compounds: an E3 binder, which binds to an E3 ubiquitin ligase, such as IMiDs, and a target binder, which binds to the therapeutic target protein [40, 41]. PROTACs theoretically have the potential to target any protein for therapy by altering the target binder [42, 43]. Since the mechanism of action of thalidomide and its derivatives was elucidated, targeted protein degradation (TPD) using drugs has progressed rapidly, and several protein degraders are currently in clinical trials [44].

Development of an Analysis Method for Identifying Neo-substrates of Protein Degraders

When neo-substrates were first discovered, the mainstream method of identification was quantitative mass spectrometry using cultured cells [26, 30, 31]. However, no neo-substrates involved in the teratogenicity of thalidomide have been discovered, indicating the need for new analytical methods. Therefore, we developed a method for analyzing biochemical interactions using recombinant proteins based on a wheat cell-free system. This system uses wheat germ extract for in vitro protein synthesis [45, 46], which allows the high-throughput synthesis of multiple proteins [45, 46]. Furthermore, the synthesized recombinant proteins can be used directly in protein-protein interaction analysis based on AlphaScreen technology without purification [47, 48]. Through interaction analysis using CRBN synthesized using the wheat cell-free system and the neo-substrate IKZF1, we successfully detected thalidomide-dependent CRBN–IKZF1 interactions (Fig. 1A). Additionally, through comprehensive screening using a transcription factor protein array constructed with a wheat cell-free system, we identified the promyelocytic leukemia zinc finger protein (PLZF/ZBTB16) as a neo-substrate involved in thalidomide teratogenicity [36], demonstrating that this method is useful for identifying neo-substrates.

Fig. 1.

(A) Thalidomide-dependent Interaction Analysis Method Based on the Wheat Cell-free System. N-terminally biotinylated CRBN and N-terminally FLAG-GST-tagged IKZF1 were synthesized using the wheat cell-free system. The thalidomide-dependent interaction between biotinylated CRBN and FLAG-GST-IKZF1 was detected using AlphaScreen technology. A high luminescence signal is detected when thalidomide is present in the reaction mixture. The Relative AS signal is represented by the AlphaScreen signal with DMSO as one. (B) Thalidomide-dependent Interaction Analysis Method Using the BioID Method. In cultured-cells expressing AirID-fused CRBN, thalidomide treatment induces the biotinylation of IKZF1 by AirID-fused CRBN. The biotinylated peptides are analyzed by LC-MS/MS.

Many proteins function by forming complexes within cells. However, it is challenging to analyze the interactions between drugs and complex proteins using recombinant proteins. Therefore, we developed a protein-protein interaction (PPI) analysis method that is dependent on protein degraders within cells, using BioID, a proximity-dependent biotinylation method. The BioID method allows biotin labeling under physiological conditions, and by fusing the BioID enzyme to a target protein, it can comprehensively biotinylate proteins in proximity to the target protein [49]. Biotinylated proteins can be identified for various applications using avidin-like proteins. In particular, it is possible to comprehensively identify biotinylated proteins using mass spectrometry [49, 50]. However, conventional BioID enzymes exhibit low biotinylation activity and require long labeling times. In 2018, TurboID, a high-activity BioID enzyme, was developed [51]. In 2020, we developed a new BioID enzyme, AirID, which is useful for PPI analysis [52]. Using AirID-fused CRBN, we performed a thalidomide-derivative-dependent PPI analysis and successfully detected the biotinylation of neo-substrates (Fig. 1B) [53]. Comprehensive mass spectrometry analysis identified zinc finger mym protein2 (ZMYM2) as a POM neo-substrate, suggesting that this method is useful for neo-substrate identification [53].

Thus, we successfully developed two interaction analysis methods for protein degrader-dependent interactions between E3 ubiquitin ligases and neo-substrates using recombinant proteins and cultured cells.

Proposed Molecular Mechanism of Thalidomide-Induced Teratogenicity

In 2014 and 2015, IKZF1, IKZF3, and casein kinase I isoform alpha (CK1α) were reported as neo-substrates involved in the anti-multiple myeloma effect and the anti-5q myelodysplastic syndrome (5q MDS) effect of IMiDs [26, 27, 30]. Degradation of IKZF1 and IKZF3 by IMiDs leads to the downregulation of interferon regulatory factor 3 (IRF3) and Myc proto-oncogene protein (MYC), which are critical for multiple myeloma cell proliferation [26, 27]. In 5q MDS, lenalidomide induces megakaryocyte differentiation by causing the degradation of IKZF1 and induces cell death by causing the degradation of CK1α [54].

Regarding the neo-substrates involved in thalidomide teratogenicity, in 2018, the transcription factor Sal-like protein 4 (SALL4), the causative gene of Duane-radial ray syndrome, was reported to be degraded in a thalidomide-dependent manner [33, 34]. Duane-radial ray syndrome is a genetic disorder that exhibits teratogenic phenotypes in various tissues and organs, including the limbs, similar to thalidomide teratogenicity [55]. However, we showed that SALL4 was not degraded by thalidomide in chicken embryos exhibiting thalidomide teratogenicity [36], suggesting the existence of additional neo-substrates. Through screening using a wheat cell-free system, we identified the promyelocytic leukemia zinc finger protein (PLZF) as a neo-substrate involved in thalidomide teratogenicity [36]. It has been reported that the deletion of PLZF results in teratogenic phenotypes in the limbs [56, 57]. Importantly, it has been reported that SALL4 and PLZF double knockout mice exhibit severe thalidomide-like teratogenic phenotypes in the hind limbs [58].

Thalidomide is a typical drug that shows species specificity with no phenotypic effects in rodents, such as mice and rats [3,4,5,6, 34]. In contrast, chickens and rabbits are species known to exhibit teratogenic phenotypes upon exposure to thalidomides [23]. Previous studies have shown that rodent Crbn cannot induce protein degradation of neo-substrates because of differences in the amino acid sequence of CRBN [30, 33, 34, 36]. Therefore, IMiDs do not exhibit anti-hematological or teratogenic effects in rodents. In addition, a 2016 study reported that mouse embryos expressing human cytochrome P450 3A (CYP3A) exhibited thalidomide teratogenic phenotypes [59]. This suggests that thalidomide metabolites are crucial for the species specificity of thalidomide teratogenicity. Thalidomide is metabolized by CYP450s to 5-hydroxythalidomide (5-HT) and 5′-hydoroxythaldiomide (5′-HT) [60]. 5′-HT cannot interact with CRBN due to modification on the glutarimide ring, which is important for the interaction between IMiDs and CRBN. Notably, we found that 5-HT, a metabolite of thalidomide, does not degrade IKZF1, but strongly induces SALL4 [36]. Furthermore, we elucidated the structural basis of selective and potent degradation of SALL4 by 5-HT [61]. Indeed, a previous study showed that 5′-HT is the main metabolite in the plasma of rats [62]. In summary, the dual degradation of SALL4 and PLZF by thalidomide and 5-HT has been proposed to cause severe teratogenicity in highly thalidomide-sensitive species, such as humans and rabbits [36].

Thus, the molecular mechanisms underlying the anti-hematological effects and teratogenicity of thalidomide and its derivatives are currently being elucidated (Fig. 2). In contrast, the neo-substrates involved in the sedative and anti-angiogenic effects remain undiscovered, and the mechanisms underlying this mechanism of actions are still unknown.

Fig. 2.

Schematic Diagram of the Mechanism of Action of Thalidomide, Thalidomide Derivatives, and Thalidomide Metabolites. (A) Mechanism of Action of Anti-hematological Cancer Effects by Thalidomide and Its Derivatives. IMiDs (Thalidomide/Tha, Lenalidomide/Len, and Pomalidomide/Pom) bind to CRBN, leading to the ubiquitination and degradation of neo-substrates involved in anti-hematological cancer effects. The degradation of IKZF1 and IKZF3 is involved in the anti-multiple myeloma effect, the degradation of IKZF1 and CK1α is involved in the anti-5q-deletion myelodysplastic syndrome effect. (A) Mechanism of Action of Teratogenic Effects by Thalidomide and Thalidomide Metabolites. Thalidomide/Tha and the thalidomide metabolite (5-hydroxythalidomide; 5-HT) bind to CRBN, leading to the ubiquitination and degradation of neo-substrates involved in teratogenic effects. 5-HT strongly induces protein degradation of SALL4. The dual degradation of SALL4 and PLZF caused severe teratogenicity in thalidomide-sensitive species.

Development of Thalidomide Derivatives and PROTACs for the Selective Degradation of Neo-substrates

It has been reported that the selectivity of thalidomide and its derivatives towards neo-substrates depends on their chemical structures [30, 31, 33, 36]. IMiDs consist of two ring structures, a glutarimide ring and a phthalimide ring (Fig. 3A). Structural studies have shown that the former is critical for interactions with CRBN, while the latter is important for neo-substrate selectivity [63,64,65]. Indeed, lenalidomide induces the degradation of CK1α, whereas thalidomide and pomalidomide do not [30, 65]. In addition, thalidomide derivatives, such as CC-122 and CC-220, which are currently under development, modify the phthalimide ring of thalidomide [66]. Based on this background, we hypothesized that chemical modification of the phthalimide ring of thalidomide could alter neo-substrate selectivity, leading to the development of thalidomide derivatives that selectively degrade the neo-substrates involved in anti-hematological cancer effects. We screened various thalidomide derivatives with different chemical modifications to the phthalimide ring. We found that modifying the 6-position of lenalidomide with small substituents enhanced its selectivity for neo-substrates involved in the anti-hematological cancer effects [67]. Specifically, 6-fluoro-lenalidomide exhibits higher antiproliferative activity than lenalidomide against cultured cells derived from multiple myeloma and 5q MDS cells [67]. Importantly, 6-position-modified lenalidomide showed reduced degradation activity towards neo-substrates involved in teratogenicity [67]. Detailed intracellular analysis further demonstrated that 6-position-modified lenalidomide has high selectivity for neo-substrates involved in anti-hematological cancers [67] (Fig. 3B).

Fig. 3.

(A) Chemical Structures of Thalidomide and Thalidomide Derivatives. Thalidomide consists of two ring structures: a glutarimide ring and a phthalimide ring. The chemical structure of the phthalimide ring is modified in lenalidomide and pomalidomide. (B) Schematic Diagram of Thalidomide Derivatives Effective Against Hematological Cancers with Reduced Teratogenicity. The 6-position-modified lenalidomides strongly induce the degradation of neo-substrates (IKZF1, IKZF3, and CK1α) involved in anti-hematologic cancer effects but has a low ability to induce the degradation of neo-substrates (SALL4 and PLZF) involved in teratogenicity. (C) Chemical Structure of PROTACs Using 6-position-modified Lenalidomide. PROTACs were synthesized using JQ1, an inhibitor of BET proteins, as the target binder. (D) Selective Degradation of Target Proteins by PROTACs Using 6-position-modified Lenalidomide. PROTACs using 6-position-modified lenalidomide have a low ability to induce the degradation of neo-substrates (IKZF1, IKZF3, CK1α, SALL4, and PLZF) but can strongly induce the degradation of target proteins.

Currently, the development of various PROTACs using thalidomide derivatives is actively pursued, and several PROTACs are undergoing clinical trials [44]. However, PROTACs using thalidomide derivatives induce the degradation of both neo-substrates in addition to the target proteins [34, 53]. Therefore, the use of thalidomide derivatives in PROTACs raises concerns regarding thalidomide-induced teratogenicity as a side effect. In addition, because IKZF1 and IKZF3 are crucial for the development and differentiation of blood cells, there is a risk of hematotoxicity. Therefore, we applied 6-position-modified lenalidomides to PROTACs for the selective degradation of target proteins. It was expected that the modification of the 6-position of lenalidomide with bulky substituents would reduce its degradation activity towards neo-substrates. Indeed, lenalidomide modified at the 6-position with a trifluoromethyl group did not induce the degradation of the neo-substrates tested, including IKZF1 and IKZF3 [67]. Furthermore, a comprehensive proteomic analysis using quantitative mass spectrometry showed that this modification did not induce degradation of the reported neo-substrates [67]. Using 6-position-modified lenalidomide and the BET protein inhibitor JQ1, we synthesized PROTACs (Fig. 3C) and analyzed the degradation of BET proteins and neo-substrates. PROTACs using 6-position-modified lenalidomide strongly induced the degradation of BET proteins, demonstrating that 6-position-modified lenalidomide is a viable CRBN binder for PROTACs [67]. More importantly, PROTACs using 6-position-modified lenalidomide exhibit neo-substrate selectivity similar to that of 6-position-modified lenalidomide, showing improved selectivity for target protein degradation [66]. These results suggest that the use of 6-position-modified lenalidomide allows for the control of neo-substrate degradation, enabling the selective degradation of target proteins (Fig. 3D).

Discussion

In this paper, we review the recent research on thalidomide and its derivatives, including their mechanisms of action, based on the history of thalidomide. Previous studies have elucidated the mechanisms of action related to thalidomide-induced teratogenicity and its anti-hematological effects on cancer cells [26, 27, 30, 33, 34, 36]. Consequently, the neo-substrates that should be avoided to prevent thalidomide-induced teratogenicity have become clearer. We previously developed novel thalidomide derivatives with enhanced selectivity for neo-substrates with anti-hematological cancer effects [67]. However, our analysis was only performed on cultured cells. Therefore, bioactivity and bioavailability studies are required in the future.

Thalidomide is a typical drug that exhibits species specificity, but an evaluation system for thalidomide teratogenicity in mice, which is commonly used in drug evaluation, has not yet been developed. Previous studies have reported that mouse Crbn cannot induce the degradation of neo-substrates because of differences in amino acid sequences between mouse Crbn and human CRBN [30, 33, 34, 36]. Genome-edited mice sensitive to thalidomide are capable of inducing neo-substrate degradation. However, these genome-edited mice did not exhibit the teratogenic phenotypes of thalidomide [68, 69]. Although mice are insensitive to thalidomide, embryos expressing human cytochrome P450 3A (CYP3A) exhibit thalidomide-induced teratogenic phenotypes [59]. Previous studies reported that human microsomal CYP3A is involved in the production of 5-hydroxythalidomide (5-HT) [70]. In contrast, the primary thalidomide metabolite in rats was 5′-hydroxythalidomide (5′-HT) [62]. Furthermore, the concentration of 5-HT was higher in rabbits and humanized-liver mice than in rats [62]. Thus, in addition to the differences in amino acid sequences, thalidomide metabolites play a crucial role in thalidomide-induced teratogenicity. Indeed, we have demonstrated that 5-HT strongly and selectively induces the degradation of SALL4 [36, 61]. These findings suggest that detailed studies using mice with humanized-CRBN and -metabolism are required for elucidating thalidomide-induced teratogenicity and species-specificity. Moreover, we believe that applying the interaction analysis method using AirID-fused CRBN in mice will contribute to understanding the mechanisms of thalidomide-induced teratogenicity.

The mechanisms underlying the teratogenicity and anti-hematological effects of thalidomide derivatives have been elucidated [26, 27, 30, 33, 34, 36]. However, the mechanisms underlying the other effects of thalidomide, such as angiogenesis inhibition and sedative effects, remain unclear. To elucidate these mechanisms, it is important to identify the neo-substrates involved. Several analytical methods for protein degradation have been established in recent years [31, 35, 36, 53, 71]. Using these analytical methods to elucidate unresolved mechanisms will be important for the future development and use of thalidomide derivatives and PROTACs.

Previous studies reported that the anticancer drug indisulam is a molecular glue-type protein degrader similar to thalidomide [72, 73]. Indisulam binds to DDB1- and CUL4-associated factor 15 (DCAF15), the substrate recognition receptor of CRL4, and induces the degradation of neo-substrate RNA-binding protein 39 (RBM39) [72, 73], exhibiting antitumor effects. The cyclin-dependent kinase (CDK) inhibitor CR8 functions as a molecular glue-type protein degrader and induces the degradation of cyclin K by the CRL4 complex [74]. In addition, the B-cell lymphoma 6 protein (BCL6) inhibitor BI-3802 induces the polymerization of BCL6, leading to its degradation by the E3 ubiquitin protein ligase SIAH1 (SIAH1) [75]. Various small molecule drugs have been reported to exert pharmacological effects as protein degraders. Therefore, we expect that additional protein degraders will be discovered using the analytical methods introduced in this review.

Conclusion

Thalidomide is an infamous small-molecule drug that caused a global drug disaster more than half a century ago and is now widely used in the treatment of several hematological cancers under strict control. Recent studies revealed that thalidomide and its derivatives represent a new class of drugs that induce targeted protein degradation. Protein degraders have attracted global attention as a next-generation drug discovery approach, opening doors for various diseases that pose challenges to the development of therapeutic drugs. This review focuses on protein degraders, and introduces targeted protein degradation using thalidomide derivatives. To date, various protein degrader-dependent PPI–protein interaction (PPI) analysis methods using cultured cells and recombinant proteins have been developed to elucidate the mechanisms of action. Using these analytical methods, we developed novel thalidomide derivatives and PROTACs for selective, targeted protein degradation. These findings have the potential to contribute to the development of novel protein degraders.

Conflict of Interest

The authors declare that this study was conducted without any commercial or financial relationships that could be construed as conflicts of interest.

Acknowledgments

The authors thank all the staff and collaborators involved in this research. This work was mainly supported by the Project for Cancer Research and Therapeutic Evolution (P-CREATE) from the Japan Agency for Medical Research and Development (AMED) under grant number JP21cm0106181h0006, the Project for Promotion of Cancer Research and Therapeutic Evolution (P-PROMOTE) from AMED under grant number JP22cm0106181h0002, the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Numbers JP19am0101077 and 22ama121010j0001, and a Grant-in-Aid for Scientific Research on Innovative Areas (21H00285 and JP16H06579) from the Japan Society for the Promotion of Science (JSPS). This work was supported by JSPS KAKENHI (JP17J08477, JP16H04729, and JP19H03218), a Grant-in-Aid for JSPS Research Fellows (JP17J08477) from the JSPS, and the Takeda Science Foundation.

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