Innovative Strategies for the Targeted Degradation of Viral Proteins: Paving the Way for Next-Generation Therapeutics

Advances and opportunities in targeted protein degradation

Therapeutic targeting of RNA-binding protein by RNA-PROTAC

Targeted Protein Degradation (TPD) offers a solution, eliminating disease-related proteins and overcoming challenges associated with unintended toxicity and lack of precision. PROTACs (Proteolysis Targeting Chimeras) represent an innovative strategy for the specific degradation of tar-get proteins through the UPS (Ubiquitin-Proteasome System). In comparison to conventional protein inhibitor medications, PROTAC offers advantages in terms of efficacy, selectivity, and the ability to overcome drug resistance in cancer treatment, contributing novel perspectives to the field of anti-cancer drug discovery. Proteins play vital roles in an organism's health, and misfolded contributes to diseases like neurodegenerative disorders and cancer. Cells maintain protein balance through quality control systems, primarily the UPS and autophagy. Protac, a Targeted Protein Degradation (TPD) strategy, utilizes UPS, employing small molecules to induce targeted protein degradation. PROTAC exhibits promise in preclinical studies and clinical trials for diverse cancers. Notable examples in-clude breast cancer, where PROTAC targets CDK4/6 (cyclin-dependent kinase) and Estrogen Recep-tors (ER), prostate cancer, addressing Androgen Receptor (AR) degradation, hematologic malignan-cies, focusing on AURORA-A and CDKs, and NSCLC (Non-Small-Cell Lung Cancer), targeting Estimated Glomerular Filtration Rate (EGFR), and KRAS. Despite their potential, PROTAC faces challenges, including compensatory protein expression in response to targeted therapies. This com-prehensive review explores recent advancements in PROTAC and related technologies, emphasizing the mechanisms and structures of PROTAC and their applications in proteins targeting cancer.

Colorectal cancer (CRC) is a leading cause of cancer-related mortality worldwide, with treatment challenges arising from late-stage diagnosis, therapy resistance, and the presence of undruggable targets. The advent of proteolysis-targeting chimeras (PROTACs) offers a novel therapeutic avenue by leveraging the ubiquitin-proteasome system (UPS) for targeted protein degradation. Unlike conventional inhibitors that act like temporary “off switches” for cancer-related proteins, PROTACs function as “disposal tags,” marking harmful proteins for complete removal by the cell, enabling sustained oncogenic protein degradation. PROTACs address key drivers of CRC progression, including Kirsten rat sarcoma viral oncogene homolog (KRAS) , the Wnt/β-catenin pathway, bromodomain-containing protein 4 (BRD4), cyclin-dependent kinase 4/6 (CDK4/6), signal transducer and activator of transcription 3 (STAT3), and DNA repair regulators. This review explores the mechanistic foundation of PROTACs, their application in CRC therapy, and the ongoing advancements in optimizing their selectivity, bioavailability, and clinical translation. While challenges such as E3 ligase selection, intracellular delivery, and resistance mechanisms remain, recent innovations in linker chemistry, nanocarrier systems, and artificial intelligence (AI)-driven drug design are enhancing the clinical feasibility of PROTAC therapeutics. As research progresses, PROTAC-based therapies hold significant promise for overcoming current treatment limitations, paving the way for a new era of precision medicine in CRC management.

Modeling the CRL4A ligase complex to predict target protein ubiquitination induced by cereblon-recruiting PROTACs

Small molecular drugs have been an important part of disease treatment, but there are also some problems along with time and intrinsic disadvantages of small molecular drugs. In recent years, the emergence of proteolysis targeting chimeras (PROTACs) is expected to overcome some defeats of traditional small molecules. PROTACs, also known as hetero-bifunctional compounds, are a new kind of therapeutic, that consists of a specific ligand to bind the target protein, a suitable linker, and an E3 ubiquitin ligase substrate. After binding to target proteins, PROTACs recruit E3 ligase and induce degradation of target proteins through the ubiquitin-proteasome system. Therefore, PROTACs are a good choice for some protein targets that have small molecular binding ligands but poor small molecular efficacy, which expands druggable targets. Meanwhile, the protein degradation induced by PROTACs destroys not only the enzymatic but also the non-enzymatic functions of targets, rather than only inhibiting the protein activity, which reduces drug resistance. In this study, in order to detect the degradation of intracellular target proteins by PROTACs, Kyinno has designed and constructed several HiBiT-tagged cell line models for different target proteins and their mutations, including KRA, CDK2, LDHA, and HPK. HiBiT is a small tag with 11 amino acids that don’t influence the expression, folding, and localization of target proteins, and luminesces with a detection buffer. All these cell lines with endogenous protein mutations and HiBiT knock-ins are constructed by CRISPR-Cas9 technology and validated by siRNA and PROTACs. Compared with overexpressed exogenous HiBiT-tagged target proteins, endogenous proteins can more accurately and stably reflect the efficacy of PROTACs. Our cell models can be used for high-throughput screening not only for PROTAC drugs but also for molecular glues, lysosome-targeting chimeras (LYTACs), and antibody-based PROTACs (AbTACs). Citation Format: Yunpeng Zhai, Jiayi Ma, Yu Wang, Meng Liang, Yao Peng, Jinying Ning, Feng Hao. Cell line panel with HIBIT-tagged endogenous proteins to accelerate PROTAC drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6919.

Introduction Target protein degradation (TPD) provides a novel therapeutic modality, other than inhibition, through the direct depletion of target proteins. Two primary human protein homeostasis mechanisms are exploited: the ubiquitin-proteasome system (UPS) and the lysosomal system. TPD technologies based on these two systems are progressing at an impressive pace. Areas Covered This review focuses on the TPD strategies based on UPS and lysosomal system, mainly classified into three types: Molecular Glue (MG), PROteolysis Targeting Chimera (PROTAC), and lysosome-mediated TPD. Starting with a brief background introduction of each strategy, exciting examples and perspectives on these novel approaches are provided. Expert Opinion MGs and PROTACs are two major UPS-based TPD strategies that have been extensively investigated in the past decade. Despite some clinical trials, several critical issues remain, among which is emphasized by the limitation of targets. Recently developed lysosomal system-based approaches provide alternative solutions for TPD beyond UPS’ capability. The newly emerging novel approaches may partially address issues that have long plagued researchers, such as low potency, poor cell permeability, on-/off-target toxicity, and delivery efficiency. Comprehensive considerations for the rational design of protein degraders and continuous efforts to seek effective solutions are imperative to advance these strategies into clinical medications.

PROteolysis TArgeting Chimeras (PROTACs) technology is a new protein-degradation strategy that has emerged in recent years. It uses bifunctional small molecules to induce the ubiquitination and degradation of target proteins through the ubiquitin–proteasome system. PROTACs can not only be used as potential clinical treatments for diseases such as cancer, immune disorders, viral infections, and neurodegenerative diseases, but also provide unique chemical knockdown tools for biological research in a catalytic, reversible, and rapid manner. In 2019, our group published a review article “PROTACs: great opportunities for academia and industry” in the journal, summarizing the representative compounds of PROTACs reported before the end of 2019. In the past 2 years, the entire field of protein degradation has experienced rapid development, including not only a large increase in the number of research papers on protein-degradation technology but also a rapid increase in the number of small-molecule degraders that have entered the clinical and will enter the clinical stage. In addition to PROTAC and molecular glue technology, other new degradation technologies are also developing rapidly. In this article, we mainly summarize and review the representative PROTACs of related targets published in 2020–2021 to present to researchers the exciting developments in the field of protein degradation. The problems that need to be solved in this field will also be briefly introduced.

Introduction Targeted protein degradation (TPD) has emerged as an innovative therapeutic strategy through selective degradation of specific proteins by harnessing the cellular ubiquitin-proteasome system (UPS), which involves over 600 E3 ubiquitin ligases. Recent proteome profiling reported tumor-specific E3 ligases in human. Development of those tumor-specific E3 ligase ligands would provide a solution for tumor-specific TPD for effective cancer treatment. Areas covered This review provides a comprehensive list of E3 ligases found only in specific types of tumor from public databases and highlights examples of their ligands discovered through fragment-based approaches. It details their discovery process and potential applications for precise TPD and effective cancer treatments. Expert opinion Current TPD strategies using proteolysis-targeting chimeras (PROTACs) primarily utilize general E3 ligases, such as CRBN and VHL. Since these E3 ligases demonstrate effective protein degradation activity in most human cell types, CRBN and VHL-based PROTACs can exhibit undesired TPD in off-target tissues, which often leads to the side effects. Therefore, developing tumor-specific E3 ligase ligands can be crucial for effective cancer treatments. Fragment-based ligand discovery (FBLD) approaches would accelerate the identification of these tumor-specific E3 ligase ligands and associated PROTACs, thereby advancing the field of targeted cancer therapies.

Special Focus Issue - Targeted protein degradation: a new paradigm in medicinal chemistry.

The SLAS Discovery Editor's Top 10 for 2021.

Recent progress in degradation of membrane proteins by PROTACs and alternative targeted protein degradation techniques

Proteolysis-targeting chimeras (PROTACs) have emerged as a promising technology for inducing targeted protein degradation by leveraging the intrinsic ubiquitin-proteasome system (UPS). While the potential druggability of PROTACs toward undruggable proteins has accelerated their rapid development and the wide-range of applications across diverse disease contexts, off-tissue effects and side-effects of PROTACs have recently received attentions to improve their efficacy. To address these issues, spatial or temporal target protein degradation by PROTACs has been spotlighted. In this review, we explore chemical strategies for modulating protein degradation in a cell type-specific (spatio-) and time-specific (temporal-) manner, thereby offering insights for expanding PROTAC applications to overcome the current limitations of target protein degradation strategy.

Targeted protein degradation: elements of PROTAC design

PROteolysis TArgeting Chimeras (PROTACs) are small molecules that induce target protein degradation via the ubiquitin-proteasome system. PROTACs recruit the target protein and E3 ligase; a critical first step is forming a ternary complex. However, while the formation of a ternary complex is crucial, it may not always guarantee successful protein degradation. The dynamics of the PROTAC-induced degradation complex play a key role in ubiquitination and subsequent degradation. In this study, we computationally modelled protein complex structures and dynamics associated with a series of PROTACs featuring different linkers to investigate why these PROTACs, all of which formed ternary complexes with Cereblon (CRBN) E3 ligase and the target protein bromodomain-containing protein 4 (BRD4BD1), exhibited varying degrees of degradation potency. We constructed the degradation machinery complexes with Culling-Ring Ligase 4A (CRL4A) E3 ligase scaffolds. Through atomistic molecular dynamics simulations, we illustrated how PROTAC-dependent protein dynamics facilitating the arrangement of surface lysine residues of BRD4BD1 into the catalytic pocket of E2/ubiquitin cascade for ubiquitination. Despite featuring identical warheads in this PROTAC series, the linkers were found to affect the residue-interaction networks, and thus governing the essential motions of the entire degradation machine for ubiquitination. These findings offer a structural dynamic perspective on ligand-induced protein degradation, providing insights to guide future PROTAC design endeavors.