Induced protein degradation: an emerging drug discovery paradigm.

Therapeutic targeting of RNA-binding protein by RNA-PROTAC

An E3 ligase guide to the galaxy of small-molecule-induced protein degradation

Traditional drug discovery strategies are usually focused on occupancy of binding sites that directly affect functions of proteins. Hence, proteins that lack such binding sites are generally considered pharmacologically intractable. Modulators of protein activity, especially inhibitors, must be applied in appropriate dosage regimens that often lead to high systemic drug exposures in order to maintain sufficient protein inhibition in vivo. Consequently, there is a risk of undesirable off-target drug binding and side effects. Recently, PROteolysis TArgeting Chimera (PROTAC) technology has emerged as a new pharmacological modality that exploits PROTAC molecules for induced protein degradation. PROTAC molecule is a heterobifunctional structure consisting of a ligand that binds a protein of interest (POI), a ligand for recruiting an E3 ubiquitin ligase (an enzyme involved in the POI ubiquitination) and a linker that connects these two. After POI-PROTAC-E3 ubiquitin ligase ternary complex formation, the POI undergoes ubiquitination (an enzymatic post-translational modification in which ubiquitin is attached to the POI) and degradation. By merging the principles of photopharmacology and PROTAC technology, photocontrollable PROTACs for spatiotemporal control of induced protein degradation have recently emerged. The main advantage of photocontrollable over conventional PROTACs is the possible prevention of off-target toxicity thanks to local photoactivation.

Advances and opportunities in targeted protein degradation

Targeted protein degradation compounds, including molecular glues or proteolysis targeting chimeras, are an exciting new therapeutic modality in small molecule drug discovery. This class of compounds induces protein degradation by bringing into proximity the target protein and the E3 ligase machinery proteins required to ubiquitinate and ultimately degrade the target protein through the ubiquitin-proteasomal pathway (UPP). Profiling of target protein degradation in a high-throughput fashion, however, remains highly challenging given the complexity of cellular pathways required to achieve degradation. Here we present a protocol and screening strategy based on the use of CRISPR/Cas9 endogenous tagging of target proteins with the 11 amino acid HiBiT tag which complements with high affinity to the LgBiT protein, to produce a luminescent protein. These CRISPR targeted cell lines with endogenous tags can be used to measure compound induced degradation in either real-time, kinetic live cell or endpoint lytic modes by monitoring luminescent signal using a luminescent plate-based reader. Here we outline the recommended screening protocols for the different formats, andalso describe the calculation of key degradation parameters of rate, Dmax, DC50, Dmax50, as well as multiplexing with cell viability assays. These approaches enable rapid discovery and triaging of early stage compounds while maintaining endogenous expression and regulation of target proteins in relevant cellular backgrounds, allowing for efficient optimization of lead therapeutic compounds.

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.

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

Targeted protein degradation (TPD) marks an emerging novel technology within the chemical biology and drug discovery communities. The classical drug discovery process has focused on target driven inhibition, where a small molecule is designed to bind to the protein of interest (POI) and then interfere with its function. For enzymes, inhibitors are often designed to target the active site (orthosteric), inhibiting the protein´s enzymatic activity by cofactor or substrate competitive binding. However, approximately 75% of human proteins lack typical active sites and are considered “undruggable” by conventional inhibitors, including a large number of proteins linked to diseases, such as e.g. the transcription factors p53 and MYC. TPD is an emerging approach capable to target a much larger fraction of the human proteome. Here, target proteins are not inhibited at a functional binding site but they are degraded, leading to a complete elimination of all protein functions, including enzymatic activities and scaffolding functions, which is impossible to achieve by conventional inhibitors. To target a protein for degradation, so-called PROteolysis TArgeting Chimeras (PROTACs) can be used, which comprise bi-functional small-molecules containing two binding moieties, connected via a chemical linker. This arrangement induces proximity between both targeted proteins. In PROTACs, one of the targeted proteins marks an E3 ligase, while the other ligand recruits a POI as a non-natural substrate (neosubstrate) to the E3 ligase. Since E3 ligases act as substrate recruitment sites of the ubiquitin system, the chemically induced recruitment to the E3 can trigger ubiquitination and subsequent degradation of the POI. To undergo degradation, a complex and highly controlled mechanism known as the ubiquitin proteasomal system (UPS), consisting of a cascade of enzymatic reactions for ubiquitin transfer, is required. Due to the complexity of the mechanism, many developed proximity-inducing compounds do not lead to final protein degradation because they fail to mediate all required steps in the UPS cascade. Therefore, this thesis focused on the investigation why and where PROTAC mediated degradation can fail and how to rationally optimize small molecule degraders. To address this question, bespoke cellular assays for each of the different key steps within the PROTAC-mediated degradation cycle have been developed. Starting with the establishment of assay systems for binary complexes, a model system focusing on the baculoviral IAP repeat-containing (BIRC) proteins was established using a variety of biochemical measurements and the development of a cell-based selectivity screening platform. This system was crucial for the validation of the live-cell assays using NanoBRET-based technologies which were subsequently developed and yielded good correlation between biophysical in vitro assays and live-cell assays. Therefore, live-cell assays were chosen as the most promising assay system for the evaluation of novel degrader and E3 ligase ligands (e.g. for GID4), since this assay system additionally enabled information about the fraction of compounds entering living cells. Moreover, live cells allowed the use of full-length protein together with all binding partners which was previously shown to be crucial for robust small molecule-protein interaction (e.g. the CRBN-DDA1-DDB1 complex). Using NanoBRET experiments evaluating a variety of PROTACs, cell penetration was identified as an important parameter which was successfully assessed by measuring live-cell target engagement in comparison with permeabilized cells. Using this assay system, a relative ratio of cell penetration was determined which correlated well with data measured on well-established but work-intensive systems (e.g. Caco2 or PAMPA assay). The use of BRET-based assay technology also enabled measurements in relevroperties, demonstrated by significantly weaker live cell target engagement compared to permeabilized cells. During this work, it became obvious that a database for tracer molecules – ligands of POIs labelled with fluorescent dyes – which are necessary key reagents for cellular target engagement assays (e.g. for TR-FRET or NanoBRET), would have significantly sped up the assay development process. Therefore, an online database, called “tracerDB” was developed. Within this crowdsourced project, we established a repository of experimentally validated tracers that can be identified in a web-based searchable format. In addition, tracers developed by the community can be submitted which resulted in worldwide submissions from numerous institutions. Generally, this database project received an overall great acceptance and support from the scientific community. Due to the weak binding of some PROTACs, for one of the POIs (WDR5) a tracer-free alternative technique called HiBiT-based cellular thermal shift assay (HiBiT-CETSA) was established for binary complex assessment. HiBiT-CETSA (also called BiTSA), a Split-NLuc system, measures the thermal stability of HiBiT-tagged target proteins which in theory increases when bound by a compound. The increase in stability of the protein-compound complex compared to the unbound state is a measure for the interaction affinity of the compound with its target protein. In the case of WDR5, HiBiT-CETSA was found to be an excellent tool to rank PROTACs according to their affinity. However, due to the high melting temperature, it was not possible to unambiguously distinguish between measurements in intact and already thermally lysed cells.

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.

Targeted protein degradation has emerged as a promising strategy for developing novel therapeutics, particularly for "undruggable" disease-related proteins. One approach is the use of PROteolysis TArgeting Chimeras (PROTACs) degraders, which induce the formation of ternary complexes between the target protein and E3 ligase, leading to ubiquitination and degradation of the target protein. Understanding the conformational behavior of PROTACs in solutions and how it relates to their pharmacokinetic properties and membrane permeability is crucial for optimizing PROTAC design and efficacy. Due to the large size and flexibility of PROTACs and their chameleonic character, it is essential to understand their conformational ensembles, and how they depend on the environment. Here, we introduce a novel methodology for exploring PROTAC conformational behavior using atomistic simulations. We employ the enhanced sampling method parallel bias metadynamics, where we bias generic local collective variables, specifically all rotatable dihedral angles, thereby avoiding the considerable challenge of identifying suitable global collective variables for biasing. The methodology allows for obtaining free energy surfaces of global CVs via reweighting. We apply the method to the prototypical case of the MZ1 PROTAC degrader, which targets bromodomain-containing protein-4 (BRD4) for degradation via the von Hippel-Lindau (VHL) E3 ligase, and elucidate its conformational behavior in different solvents, allowing us to gain insights into the chameleonic property of MZ1. Our results confirm that MZ1 adopts distinct conformations depending on the solvent, exhibiting collapsed conformations in water and chloroform, and extended conformations in DMSO. Collapsed conformations in chloroform have been correlated to increased cell permeability. Thus, our results show that MZ1 takes on conformations suitable for membrane permeation in apolar environments. Our methodological framework is generally applicable to large flexible molecules like PROTACs, and the results demonstrate its efficiency, laying the groundwork for similar investigations for other PROTACs and other "beyond-rule-of-5" drug candidates. This work provides valuable insights into the design and optimization of PROTACs, ultimately contributing to developing novel therapeutics for "undruggable" proteins.

Proteolysis targeting chimeras (PROTACs) in targeted protein degradation (TPD) is an exciting emerging therapeutic modality in drug discovery. PROTACs include three components: a target-specific warhead for the protein of interest; a chemical linker; and an E3 ubiquitin ligase ligand connected to the target warhead-linker combination. The resulting induced proximity results in an E3 ubiquitin ligase like Cereblon or pVHL, ubiquitinating the protein of interest for selective proteasomal degradation resulting in the depletion of cellular levels of the target protein. There are two key steps in developing a PROTAC against a target protein, which are identifying an appropriate small molecule warhead for the target and introducing an appropriate linker for the E3 Ligase-ligand portion. We have developed methods that can vastly simplify and speed up the discovery and development of PROTACs for TPD, providing a range of options and opportunities for new drug discovery research.Our approach starts with our hit generation by our proprietary high-throughput protein X-ray crystallography-based library screening of hundreds of compounds in a few days as a primary screen. Hits are then evolved rapidly into lead warheads in a few weeks. The prioritized warheads are coupled to our proprietary E3 ligase library in a parallel fashion to afford hundreds of putative degraders, which are tested in bioassays without intermediate purification. This high-throughput Direct-to-Biology (D2B) approach provides means to generate and characterize a large array of putative PROTACs in a matter of days, hence accelerating the PROTAC discovery process by several orders magnitude compared to traditional methodologies.We will discuss this approach in the context of drug discovery on three DNA Damage Response proteins APE1, LINE-1 EN, POLH and FEN1.

The rapid and ever-growing advancements from within the field of proteolysis-targeting chimeras (PROTAC)-induced protein degradation have driven considerable development to gain a deeper understanding of their mode of action. The ternary complex formed by PROTACs with their target protein and E3 ubiquitin ligase is the key species in their substoichiometric catalytic mechanism. Here, we describe the theoretical framework that underpins ternary complexes, including a current understanding of the three-component binding model, cooperativity, hook effect and structural considerations. We discuss in detail the biophysical methods used to interrogate ternary complex formation in vitro, including X-ray crystallography, AlphaLISA, FRET, FP, ITC and SPR. Finally, we provide detailed ITC methods and discuss approaches to assess binary and ternary target engagement, target ubiquitination and degradation that can be used to obtain a more holistic understanding of the mode of action within a cellular environment.

Development of decoy oligonucleotide-warheaded chimeric molecules targeting STAT3

Targeted protein degradation attracts attention as a novel modality for drug discovery, as well as for basic research. Various types of degrader molecules have been developed so far, which include proteolysis-targeting chimaeras (PROTACs) and specific and nongenetic IAP-dependent protein erasers (SNIPERs), E3 modulators, hydrophobic tagging molecules, IAP antagonists and deubiquitylase inhibitors. PROTACs and SNIPERs are chimeric degrader molecules consisting of a target ligand linked to another ligand that binds to an E3 ubiquitin ligase. In the cells, they recruit an E3 ligase to the target protein, thereby inducing ubiquitylation and proteasomal degradation of the target protein. Because of their modular structure, novel PROTACs and SNIPERs targeting proteins of your interest can be rationally developed by substituting target ligands. In this article, various compounds capable of inducing protein degradation were overviewed, including SNIPER compounds developed in our laboratory.

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