Selective Degradation Of Target Proteins Research Articles

Selective Elimination of Senescent Cancer Cells by Galacto-Modified PROTACs.

Although the selective and effective clearance of senescent cancer cells can improve cancer treatment, their development is confronted by many challenges. As part of efforts designed to overcome these problems, prodrugs, whose design is based on senescence-associated β-galactosidase (SA-β-gal), have been developed to selectively eliminate senescent cells. However, chemotherapies relying on targeted molecular inhibitors as senolytic drugs can induce drug resistance. In the current investigation, we devised a new strategy for selective degradation of target proteins in senescent cancer cells that utilizes a prodrug composed of the SA-β-gal substrate galactose (galacto) and the proteolysis-targeting chimeras (PROTACs) as senolytic agents. Prodrugs Gal-ARV-771 and Gal-MS99 were found to display senolytic indexes higher than those of ARV-771 and MS99. Significantly, results of in vivo studies utilizing a human lung A549 xenograft mouse model demonstrated that concomitant treatment with etoposide and Gal-ARV-771 leads to a significant inhibition of tumor growth without eliciting significant toxicity.

Abstract A047: Discovery of potent and paralog selective PROTAC degraders of CBP or p300 proteins for the treatment of various cancers

Abstract E1A binding protein (p300) and its paralog CREB binding protein (CBP or CREBBP) are ubiquitously expressed histone acetyl transferases (HAT). They act by scaffolding or as co-activator and enhancer of different transcription factors like HIF1a, BRCA-1, p53, c-Myc, PD-L1, Estrogen receptor (ER) and Androgen receptor (AR). CBP and p300 are multidomain proteins that harbour different functional units imperative for chromatin remodelling and transcription like Bromodomain (BD), Histone acetyl transferase (HAT) domain, KIX domain etc. These two closely related epigenetic modulators are known to play oncogenic role in a variety of cancers. Conventional CBP/p300 inhibitors targeting either BD, HAT or KIX domain do not discriminate between CBP and p300 proteins due to high homology. Because of the indiscriminate inhibition of both proteins, HAT domain inhibitors showed tolerability issues preventing their clinical advancement. In contrast, bromodomain inhibitors are better tolerated because they only block coactivation function at the transcription level without impacting the scaffolding or global histone acetylation function. They show less than desirable efficacy in various cancer models within very well tolerated doses. The implication of selective inhibition of either of these proteins in various cancer has been reported in the literature due to differential dependency on either of the paralogs. A synthetic lethal relationship between these paralogs in cancer set up has also been well established recently. However, none of the reported inhibitors of the various domains described above could selectively target CBP or p300 alone due to high sequence homology. Paralog selectivity in recent time has been proved to be possible with degrader approach due to differentiated ternary complex formation. Structure-guided modeling and iterative medicinal chemistry approaches were applied to identify paralog selective and potent first in class degraders with diverse linker length and linker composition. Identified compounds showed pronounced paralog selective degradation of target proteins and cellular effects in a panel of cell lines with loss of function mutation for either of the paralog proteins but not in wild type cells. The paralog selective lead degraders are currently being further optimized to identify development candidates. Selective degraders of CBP and p300 are expected to enhance efficacy of SOC drugs or immune checkpoint blockers by governing the acetylation and transcription of oncogenic factors and co-stimulatory molecules involved in tumor progression. Citation Format: Chandrasekhar Abbineni, Saravanan Thiyagarajan, Krishna Chaitanya T, Achala Apte, Naveen Kumar R, Avinash Kumar, Dabbeeru Madhu Babu, Shwetha S, Aishwarya B Kamath, Monalisha Mandal, Rituparna Panigrahi, Sivapriya Marappan, Subhendu Mukherjee, Kavitha Nellore, Shekar Chelur, Murali Ramachandra, Susanta Samajdar. Discovery of potent and paralog selective PROTAC degraders of CBP or p300 proteins for the treatment of various cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A047.

Recent Advances in PROTAC-Based Antiviral Strategies.

Numerous mysteries of cell and molecular biology have been resolved through extensive research into intracellular processes, which has also resulted in the development of innovative technologies for the treatment of infectious and non-infectious diseases. Some of the deadliest diseases, accounting for a staggering number of deaths, have been caused by viruses. Conventional antiviral therapies have been unable to achieve a feat in combating viral infections. As a result, the healthcare system has come under tremendous pressure globally. Therefore, there is an urgent need to discover and develop newer therapeutic approaches against viruses. One such innovative approach that has recently garnered attention in the research world and can be exploited for developing antiviral therapeutic strategies is the PROteolysis TArgeting Chimeras (PROTAC) technology, in which heterobifunctional compounds are employed for the selective degradation of target proteins by the intracellular protein degradation machinery. This review covers the most recent advancements in PROTAC technology, its diversity and mode of action, and how it can be applied to open up new possibilities for creating cutting-edge antiviral treatments and vaccines.

Proteolysis-Targeting Chimeras (PROTACs) in Cancer Therapy: Present and Future.

The PROteolysis TArgeting Chimeras (PROTACs) is an innovative technique for the selective degradation of target proteins via the ubiquitin-proteasome system. Compared with traditional protein inhibitor drugs, PROTACs exhibit advantages in the efficacy and selectivity of and in overcoming drug resistance in cancer therapy, providing new insights into the discovery of anti-cancer drugs. In the last two decades, many PROTAC molecules have been developed to induce the degradation of cancer-related targets, and they have been subjected to clinical trials. Here, we comprehensively review the historical milestones and latest updates in PROTAC technology. We focus on the structures and mechanisms of PROTACs and their application in targeting tumor-related targets. We have listed several representative PROTACs based on CRBN, VHL, MDM2, or cIAP1 E3 ligases, and PROTACs that are undergoing anti-cancer clinical trials. In addition, the limitations of the current research, as well as the future research directions are described to improve the PROTAC design and development for cancer therapy.

In vitro Auto- and Substrate-Ubiquitination Assays.

The precise regulation of the homeostasis of the cellular proteome is critical for the appropriate growth and development of plants. It also allows the plants to respond to various environmental stresses, by modulating their biochemical and physiological aspects in a timely manner. Ubiquitination of cellular proteins is one of the major protein degradation routes for maintaining cellular protein homeostasis, and ubiquitin E3 ligases, components of ubiquitin ligase complexes, play an important role in the selective degradation of target proteins via substrate-specific interactions. Thus, understanding the role of E3 ligases and their substrate regulation uncovers their specific cellular and physiological functions. Here, we provide protocols for auto- and substrate-ubiquitination analyses that utilize the combination of in vitro purified E3 ubiquitin ligase proteins and immunoprecipitation.

Cancer therapies based on targeted protein degradation - lessons learned with lenalidomide.

For decades, anticancer targeted therapies have been designed to inhibit kinases or other enzyme classes and have profoundly benefited many patients. However, novel approaches are required to target transcription factors, scaffolding proteins and other proteins central to cancer biology that typically lack catalytic activity and have remained mostly recalcitrant to drug development. The selective degradation of target proteins is an attractive approach to expand the druggable proteome, and the selective oestrogen receptor degrader fulvestrant served as an early example of this concept. Following a long and tragic history in the clinic, the immunomodulatory imide drug (IMiD) thalidomide was discovered to exert its therapeutic activity via a novel and unexpected mechanism of action: targeting proteins to an E3 ubiquitin ligase for subsequent proteasomal degradation. This discovery has paralleled and directly catalysed myriad breakthroughs in drug development, leading to the rapid maturation of generalizable chemical platforms for the targeted degradation of previously undruggable proteins. Decades of clinical experience have established front-line roles for thalidomide analogues, including lenalidomide and pomalidomide, in the treatment of haematological malignancies. With a new generation of 'degrader' drugs currently in development, this experience provides crucial insights into class-wide features of degraders, including a unique pharmacology, mechanisms of resistance and emerging therapeutic opportunities. Herein, we review these past experiences and discuss their application in the clinical development of novel degrader therapies.

PROTACs: Promising Approaches for Epigenetic Strategies to Overcome Drug Resistance.

Epigenetic modulation of gene expression is essential for tissue-specific development and maintenance in mammalian cells. Disruption of epigenetic processes, and the subsequent alteration of gene functions, can result in inappropriate activation or inhibition of various cellular signaling pathways, leading to cancer. Recent advancements in the understanding of the role of epigenetics in cancer initiation and progression have uncovered functions for DNA methylation, histone modifications, nucleosome positioning, and non-coding RNAs. Epigenetic therapies have shown some promise for hematological malignancies, and a wide range of epigenetic-based drugs are undergoing clinical trials. However, in a dynamic survival strategy, cancer cells exploit their heterogeneous population which frequently results in the rapid acquisition of therapy resistance. Here, we describe novel approaches in drug discovery targeting the epigenome, highlighting recent advances the selective degradation of target proteins using Proteolysis Targeting Chimera (PROTAC) to address drug resistance.

Arabidopsis F-box protein At1g08710 interacts with transcriptional protein ADA2b and imparts drought stress tolerance by negatively regulating seedling growth

Plants experience abiotic stresses throughout their life cycle and accordingly respond to tide over the unfavorable conditions. Plants adopt to drought stress through various molecular, biochemical, physiological and cellular processes. F-box protein subunit of the Skp1-Cullin-F-box (SCF) E3 ubiquitin ligases plays crucial role in imparting specificity for selective degradation of target proteins. Here we report the function of Arabidopsis F-box protein At1g08710 in drought stress adaptation. F-box protein is a constituent of SCF complex as it is shown interacting with ASK1 and Cullin 1. F-box protein localizes in both nucleus and membrane. F-box gene transcript is highly accumulated in root and altered in response to drought stress conditions. F-box protein interacts with a transcriptional co-activator protein ADA2b. F-box mutant plants growth is better under drought stress conditions compared to the wild type. Accumulation of H2O2 and malondialdehyde (MDA) content is reduced in mutant plants. Drought responsive genes RD29A, RD22, ABI3 expression is induced in F-box mutant plants. These results indicate F-box protein At1g08710 role in drought stress adaptation in Arabidopsis thaliana.

Ktx-120, a Novel Irakimid Degrader of IRAK4 and IMiD Substrates Shows Preferential Activity and Induces Regressions in MYD88-Mutant DLBCL CDX and PDX Models

An important goal of cancer therapy is to improve patient outcomes by driving to deep and durable tumor responses. The activity of single-agent targeted therapies, such as BTK inhibitors or IMiDs alone, has been modest in relapsed and refractory DLBCL, necessitating the use of combination therapy. Targeted protein degraders are heterobifunctional small molecules that co-opt the endogenous ubiquitin-proteasome system to drive the selective degradation of target proteins. Degraders of IRAK4, a key component of the myddosome complex, show potent and selective IRAK4 degradation and preferential activity in MYD88-mutant (MYD88MT) DLBCL models. Notably, the activity of IRAK4 degradation is superior to IRAK4 kinase inhibition, supporting the essential scaffolding role of IRAK4 in myddosome signaling. We have previously described IRAKIMiDs, novel IRAK4 degraders that utilize an IMiD as a pharmacologically active cereblon binder. These degraders simultaneously degrade both IRAK4 and IMiD substrates and show synergistic antitumor activity over either IRAK4 degraders or IMiDs alone, enabling a therapeutically relevant biological combination within a single small molecule. Here we describe KTX-120, a novel IRAKIMiD development candidate. KTX-120 is an equipotent degrader of both IRAK4 and the IMiD substrates Ikaros and Aiolos in lymphoma model systems with low single-digit nM DC50 for degradation of all substrates. The cell activity of KTX-120 has shown a high dependence on MYD88MT status: across a panel of MYD88MT cell lines, KTX-120 showed consistent and potent cell activity, with IC50 ranging from 7-29nM, whereas in MYD88WT lines, cell activity was poor ranging from 1800-3400nM. In the OCI-Ly10 ABC DLBCL cell line that harbors a MYD88L265P mutation, the cell activity of KTX-120 was associated with degradation of both IRAK4 and Ikaros, supporting the combined IRAK4 and IMiD targeting of this molecule as contributing to cell activity. Notably, the onset of cell death in OCI-Ly10 cells with KTX-120 treatment was rapid, with cells becoming committed to cell death within 72h of exposure, suggesting that continuous exposure to KTX-120 may not be necessary for antitumor activity. We explored the pharmacological activity of KTX-120 in several in vivo model systems. KTX-120 is orally bioavailable and shows dose-proportional exposure in several species. A single oral dose of KTX-120 (10 mg/Kg or 30 mg/Kg) showed significant degradation of both IRAK4 and Ikaros in a dose and time-dependent manner, with degradation of both substrates being sustained for >96h, further supporting the potential for intermittent dosing. To assess this, we have explored the antitumor efficacy of KTX-120 in intermittent dosing schedules. KTX-120 was well tolerated and showed potent antitumor activity in several CDX models of MYD88MT DLBCL, including OCI-Ly10, TMD8 and SUDHL2, achieving regressions in all models in as little as once every 2 weeks. As an example, in OCI-Ly10, a 30mpk dose Q2W drove >80% degradation of both IRAK4 and Ikaros and showed regressions (including CR) by D28. Similar activity and tolerability were seen with both PO and IV dosing at doses that achieve active exposure, enabling the potential for both oral and parenteral dosing. We have further explored the activity of KTX-120 in a collection of DLBCL patient derived xenograft models. KTX-120 shows robust activity (>85% TGI) in 4/5 models of MYD88MT DLBCL and shows no or modest activity in 2/2 models of MYD88WT DLBCL, supporting the preferential activity of this mechanism in patient samples harboring MYD88 activating mutations. Importantly, activity was observed in models with a variety of co-mutations that activate the NFkB pathway, including alterations in, CD79B and TNFAIP3, suggesting that KTX-120 has the potential for activity in MYD88MT lymphoma regardless of other mutations. Collectively, these data support the combined IRAK4 degradation and IMiD activity of KTX-120 has the potential to achieve robust and durable regressions in MYD88MT lymphomas with the increased convenience of an intermittently administered single agent and decreased potential for drug combination challenges. Disclosures Walker: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mayo:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Klaus:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Chen:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Bhaduri:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sharma:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Rusin:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. McDonald:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gollob:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mainolfi:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Weiss:Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company.

An IMiD-induced SALL4 degron system for selective degradation of target proteins

Regulating the amount of proteins in living cells is a powerful approach for understanding the functions of the proteins. Immunomodulatory drugs (IMiDs) induce the degradation of neosubstrates by interacting with celebron (CRBN) in the cullin E3 ubiquitin ligase complex (CRL4CRBN). Here, we developed the IMiD-dependent Sal-like protein 4 (SALL4) degron (S4D) system for chemical protein knockdown. In transient assays, an N- or C-terminal S4D tag induced the degradation of proteins localized to various subcellular compartments, including the plasma membrane. The activity of luciferase-S4D was reduced by 90% within 3 h of IMiD treatment. IMiD treatment reduced the expression of endogenous S4D-fused RelA and IκBα in knock-in (KI) experiments. Interestingly, the IκBα knockdown suggested that there may be another, unknown mechanism for RelA translocation to the nucleus. Furthermore, 5-hydroxythalidomide as a thalidomide metabolite specifically degradated S4D-tagged protein. These results indicate that the S4D system is a useful tool for cellular biology.

Abstract 5222: Degraders targeting both IRAK4 and IMiD substrates show combinatorial effects leading to broader activity with durable and complete regressions in MYD88 mutant lymphoma xenografts in vivo

Abstract Targeted protein degraders (TPD) are heterobifunctional small molecules that employ the ubiquitin degradation system to drive the selective degradation of target proteins. In approximately one third of B-cell lymphomas, gain of function mutations in MYD88 constitutively activate the myddosome and drive proliferation and survival through NFkB and AP1 signaling. IRAK4, is a key component of the myddosome and IRAK4 TPD show potent and selective IRAK4 degradation and preferential activity in MYD88 mutated B cell lymphoma models. Notably, the activity of IRAK4 degraders is superior to kinase inhibition, supporting the essential scaffolding role of IRAK4 in Myddosome signaling. An important goal of cancer therapy is to drive to deep and durable responses in patients, and this is frequently achieved by combination therapy. It has been suggested that blocking NFkB while simultaneously inducing a Type 1 IFN response with an IMiD (e.g. Lenalidomide Yang et al. (2012) Cancer Cell 21, 6, p723) may drive additive or synergistic antitumor activity. We have developed novel degraders that utilize an IMiD as a cereblon binder. These compounds simultaneously degrade both IRAK4 and IMiD substrates, enabling a therapeutically-relevant biological combination within a single small molecule. Here we show that dual IRAK4 and IMiD based degraders have potent antiproliferative activity across MYD88 mutant, but not MYD88WT lymphoma cell lines, with increased and broader antiproliferative and apoptosis activity relative to selective IRAK4 degraders or IMiDs alone. Several lines of evidence suggest that both IRAK4 and IMiD activity contribute to the overall superior activity of the dual degraders. Proteomic analysis demonstrates degradation of both both IRAK4 and key IMiD substrates, including Ikaros and Ailolos. This activity results in an enhanced Type 1 IFN response signature compared to selective IRAK4 degraders or IMiD alone, suggesting cooperative biological activity. Degradation of both IRAK4 and IMiD substrates corelates with cell activity, consistent with a requirement for both activities to drive cell death. In xenograft models of MYD88 mutant DLBCL, daily oral dosing of lead dual - degraders demonstrates strong antitumor activity in xenograft models of MYD88 mutant lymphoma, including durable complete regressions that can be maintained for several weeks following cessation of dosing. Regressions are associated with degradation of both IRAK4 and IMiD substrates, consistent with the dual-targeting activity of these molecules. Collectively, these data support a strategy to combine IRAK4 degradation and IMiD activity in a single molecule that will have the potential benefits of enhanced therapeutic activity, increased convenience and decreased potential for drug combination challenges, such as DDI. Lead degraders based on this synergistic profile are being advanced towards IND in 2020. Citation Format: Duncan H. Walker, Michele Mayo, Christine Klaus, Haojing Rong, Scott Rusin, Kirti Sharma, Alice McDonald, Veronica Campbell, Joseph Kelleher, Jared Gollob, Nello Mainolfi, Matt Weiss. Degraders targeting both IRAK4 and IMiD substrates show combinatorial effects leading to broader activity with durable and complete regressions in MYD88 mutant lymphoma xenografts in vivo [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5222.

Selective Degradation of Target Proteins by Chimeric Small-Molecular Drugs, PROTACs and SNIPERs.

New therapeutic modalities are needed to address the problem of pathological but undruggable proteins. One possible approach is the induction of protein degradation by chimeric drugs composed of a ubiquitin ligase (E3) ligand coupled to a ligand for the target protein. This article reviews chimeric drugs that decrease the level of specific proteins such as proteolysis targeting chimeric molecules (PROTACs) and specific and nongenetic inhibitor of apoptosis protein (IAP)-dependent protein erasers (SNIPERs), which target proteins for proteasome-mediated degradation. We cover strategies for increasing the degradation activity induced by small molecules, and their scope for application to undruggable proteins.

Significance of Selective Protein Degradation in the Development of Novel Targeted Drugs and Its Implications in Cancer Therapy

AbstractThe evolution of traditional chemotherapeutic drugs to targeted drugs has led to major changes in cancer treatment. However, it remains difficult to develop effective targeted drugs that can act against “undruggable” proteins, including some well‐known oncoproteins such as KRas. Moreover, mutations of target genes limit the use of targeted drugs. Although some strategies such as RNA interference and DNA editing have been adopted to solve these problems, their clinical use remains challenging. Therefore, new strategies are urgently needed to meet the dilemmas encountered in the use of targeted drugs. As protein degradation is a rapid and well‐regulated biological process in cells, targeting protein degradation by inducing cellular protein degradation machinery, regardless of the status of the target, is an attractive idea for developing novel targeted drugs. This review summarizes recent advances in targeted protein degradation, with a focus on the current reagents and strategies used for inducing selective degradation of target proteins to provide clues for the development of novel anticancer drugs and cancer therapies.

Development of Protein Knockdown Technology as Emerging Drug Discovery Strategy

Protein knockdown technologies based on small molecules are attracting considerable attention in the pharmaceutical industry as a strategy for novel drug discovery. We and others have developed such compounds, designated as Specific and Nongenetic Inhibitor of Apoptosis Protein (IAP)-dependent Protein Erasers (SNIPERs), proteolysis-targeting chimeras (PROTACs), and Degronimids, which induce selective degradation of target proteins. These compounds contain two different ligands, specific for an ubiquitin E3 ligase and for a target protein, respectively, connected by a linker. SNIPERs, PROTACs, and Degronimids are designed to cross-link E3 ligase and the target protein to induce polyubiquitylation and proteasomal degradation of the target protein within cells. To recruit the von Hippel-Lindau (VHL) E3 ligase complex and the cereblon (CRBN) E3 ligase complex, a VHL inhibitor and a thalidomide derivative have been integrated into PROTAC and Degronimid constructs, respectively. Similarly, an IAP antagonist has been incorporated into SNIPERs to recruit cellular inhibitor of apoptosis protein 1 (cIAP1) or X-linked inhibitor of apoptosis protein (XIAP) E3 ligase. To date, a range of such compounds have been developed, allowing selective degradation of a variety of proteins, including estrogen receptor α (ERα), oncogenic kinase BCR-ABL, and epigenetic regulator bromodomain-containing protein 4 (BRD4). Some compounds have also demonstrated ability to degrade target proteins in vivo, suggesting that this technology is feasible for use in novel drug development.

Sorting nexin 10 acts as a tumor suppressor in tumorigenesis and progression of colorectal cancer through regulating chaperone mediated autophagy degradation of p21Cip1/WAF1

Chaperone-mediated autophagy (CMA) characterized by the selective degradation of target proteins has been linked with tumorigenesis in recent years. Here, we explored the function of sorting nexin 10 (SNX10), a protein involved in maintaining endosome/lysosome homeostasis, in mediating CMA activity and its impact on the progression of mouse inflammation-driven colorectal cancer. Our results revealed that SNX10 deficiency increased the activation of CMA by preventing the degradation of lysosomal LAMP-2A. In SNX10 KO cells, we disclosed that p21Cip1/WAF1, a master effector in various tumor suppressor pathways, is a substrate of CMA, and decrease of p21Cip1/WAF1 caused by SNX10-mediated CMA activation contributes to HCT116 cell proliferation and survival. Moreover, we found that SNX10 KO promoted tumorigenesis in the mouse colorectum which could be restored by SNX10 over-expression. Furthermore, SNX10 was remarkably down-regulated in human CRC tissues which showed the increased activity of CMA and decreased expression of p21Cip1/WAF1. These findings suggest that SNX10 acts as a tumor suppressor in the mouse colorectum and drives inflammation-associated colorectal cancer by a chaperone-mediated autophagy mechanism.

Protein Knockdown Technology: Application of Ubiquitin Ligase to Cancer Therapy.

Selective degradation of pathogenic proteins by small molecules in cells is a novel approach for development of therapeutic agents against various diseases, including cancer. We and others have developed a protein knockdown technology with a series of hybrid small compounds, called SNIPERs (Specific and Nongenetic IAP-dependent Protein ERasers); and peptidic chimeric molecules, called PROTACs (proteolysis-targeting chimeric molecules), which induce selective degradation of target proteins via the ubiquitin-proteasome pathway. These compounds include two different ligands connected by a linker; one is a ligand for a ubiquitin ligase and the other is a ligand for the target protein, which are expected to crosslink these proteins in cells. Theoretically, any cytosolic protein can be targeted for degradation by this technology. To date, several SNIPERs and PROTACs against various oncogenic proteins have been developed, which specifically induce polyubiquitylation and proteasomal degradation of the oncogenic proteins, resulting in cell death, growth arrest, or impaired migration of cancer cells. Thus, this protein knockdown technology has a great potential for cancer therapy.

Cancer cell death induced by novel small molecules degrading the TACC3 protein via the ubiquitin-proteasome pathway.

The selective degradation of target proteins with small molecules is a novel approach to the treatment of various diseases, including cancer. We have developed a protein knockdown system with a series of hybrid small compounds that induce the selective degradation of target proteins via the ubiquitin–proteasome pathway. In this study, we designed and synthesized novel small molecules called SNIPER(TACC3)s, which target the spindle regulatory protein transforming acidic coiled-coil-3 (TACC3). SNIPER(TACC3)s induce poly-ubiquitylation and proteasomal degradation of TACC3 and reduce the TACC3 protein level in cells. Mechanistic analysis indicated that the ubiquitin ligase APC/CCDH1 mediates the SNIPER(TACC3)-induced degradation of TACC3. Intriguingly, SNIPER(TACC3) selectively induced cell death in cancer cells expressing a larger amount of TACC3 protein than normal cells. These results suggest that protein knockdown of TACC3 by SNIPER(TACC3) is a potential strategy for treating cancers overexpressing the TACC3 protein.

Regulation of Ubiquitination-Mediated Protein Degradation by Survival Kinases in Cancer

The ubiquitin–proteasome system is essential for multiple physiological processes via selective degradation of target proteins and has been shown to plays a critical role in human cancer. Activation of oncogenic factors and inhibition of tumor suppressors have been shown to be essential for cancer development, and protein ubiquitination has been linked to the regulation of oncogenic factors and tumor suppressors. Three kinases, AKT, extracellular signal-regulated kinase, and IκB kinase, we refer to as oncokinases, are activated in multiple human cancers. We and others have identified several key downstream targets that are commonly regulated by these oncokinases, some of which are regulated directly or indirectly via ubiquitin-mediated proteasome degradation, including FOXO3, β-catenin, myeloid cell leukemia-1, and Snail. In this review, we summarize these findings from our and other groups and discuss potential future studies and applications in the clinic.

Design, synthesis and biological evaluation of nuclear receptor-degradation inducers

Compounds that regulate the function(s) of nuclear receptors (NRs) are useful for biological studies and as candidate therapeutic agents. Most such compounds are agonists or antagonists. On the other hand, we have developed specific protein degradation inducers, which we designated as SNIPERs (Specific and Nongenetic IAPs-dependent Protein ERasers), for selective degradation of target proteins. SNIPERs are hybrid molecules consisting of an appropriate ligand for the protein of interest, coupled to a ligand for inhibitor of apoptosis proteins (IAPs), which target the bound protein for polyubiquitination and proteasomal degradation. We considered that protein knockdown with SNIPERs would be a promising alternative approach for modulating NR function. In this study, we designed and synthesized degradation inducers targeting retinoic acid receptor (RAR), estrogen receptor (ER), and androgen receptor (AR). These newly synthesized RAR, ER, and AR SNIPERs, 9, 11, and 13, respectively, were confirmed to significantly reduce the levels of the corresponding NRs in live cells.

Development of target protein-selective degradation inducer for protein knockdown

Our previous technique for inducing selective degradation of target proteins with ester-type SNIPER ( Specific and Nongenetic Inhibitor-of-apoptosis-proteins (IAPs)-dependent Protein ERaser) degrades both the target proteins and IAPs. Here, we designed a small-molecular amide-type SNIPER to overcome this issue. As proof of concept, we synthesized and biologically evaluated an amide-type SNIPER which induces selective degradation of cellular retinoic acid binding protein II (CRABP-II), but not IAPs. Such small-molecular, amide-type SNIPERs that induce target protein-selective degradation without affecting IAPs should be effective tools to study the biological roles of target proteins in living cells.