PROTACS- targeted protein degradation as a path to precision cancer therapeutics.

Advances and opportunities in targeted protein degradation

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

Therapeutic targeting of RNA-binding protein by RNA-PROTAC

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.

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

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 target 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 anticancer 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 include breast cancer, where PROTAC targets CDK4/6 (cyclin-dependent kinase) and Estrogen Receptors (ER), prostate cancer, addressing Androgen Receptor (AR) degradation, hematologic malignancies, 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 comprehensive review explores recent advancements in PROTAC and related technologies, emphasizing the mechanisms and structures of PROTAC and their applications in proteins targeting cancer.

Proteolysis targeting chimeras (PROTACs) technology has emerged as a novel therapeutic paradigm in recent years. PROTACs are heterobifunctional molecules that degrade target proteins by hijacking the ubiquitin–proteasome system. Currently, about 20–25% of all protein targets are being studied, and most works focus on their enzymatic functions. Unlike small molecules, PROTACs inhibit the whole biological function of the target protein by binding to the target protein and inducing subsequent proteasomal degradation. PROTACs compensate for limitations that transcription factors, nuclear proteins, and other scaffolding proteins are difficult to handle with traditional small-molecule inhibitors. Currently, PROTACs have successfully degraded diverse proteins, such as BTK, BRD4, AR, ER, STAT3, IRAK4, tau, etc. And ARV-110 and ARV-471 exhibited excellent efficacy in clinical II trials. However, what targets are appropriate for PROTAC technology to achieve better benefits than small-molecule inhibitors are not fully understood. And how to rationally design an efficient PROTACs and optimize it to be orally effective poses big challenges for researchers. In this review, we summarize the features of PROTAC technology, analyze the detail of general principles for designing efficient PROTACs, and discuss the typical application of PROTACs targeting different protein categories. In addition, we also introduce the progress of relevant clinical trial results of representative PROTACs and assess the challenges and limitations that PROTACs may face. Collectively, our studies provide references for further application of PROTACs.

Potential application of proteolysis targeting chimera (PROTAC) modification technology in natural products for their targeted protein degradation

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.

Target protein degradation (TPD) is an emerging approach to mitigate disease-causing proteins. TPD contains several strategies, and one of the strategies that gained immersive importance in recent times is Proteolysis Targeting Chimeras (PROTACs); the PROTACs recruit small molecules to induce the poly-ubiquitination of disease-causing protein by hijacking the ubiquitin–proteasome system (UPS) by bringing the E3 ligase and protein of interest (POI) into appropriate proximity. The steps involved in designing and evaluating the PROTACs remain critical in optimising the PROTACs to degrade the POI. It is observed that using in-silico and biochemical methods to study the ternary complexes (TCs) of the POI-PROTAC-E3 ligase is essential to understanding the structural activity, cooperativity, and stability of formed TCs. A better understanding of the above-mentioned leads to an appropriate rationale for designing the PROTACs targeting the disease-causing proteins. In this review, we tried to summarise the approaches used to design the ternary complexes, i.e., in-silico and in-vitro methods, to understand the behaviour of the PROTAC-induced ternary complexes.

Expansion of targeted degradation by Gilteritinib-Warheaded PROTACs to ALK fusion proteins

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 (BRD4 BD1 ), 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 BRD4 BD1 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.

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 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 (BRD4 BD1 ), 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 BRD4 BD1 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.

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.