Autophagy mediated targeting degradation, a promising strategy in drug development.

Targeted protein degradation (TPD) technologies have become promising therapeutic approaches through degrading disease-causing proteins via the protein degradation system. Autophagy is a fundamental biological process with a high relationship to protein degradation, which belongs to one of two main protein degradation pathways, the autophagy-lysosomal system. Recently, various autophagy-based TPD techniques ATTECs, AUTACs, and AUTOTACs, etc, have also been gradually developed, and they have achieved efficient degradation potency for the targeted protein, expanding the potential of degradation for large-size proteins or protein aggregates. Herein, we introduce the machinery of autophagy and its relation to protein degradation, and multiple methods for using autophagy to specifically degrade target proteins.

Toward effective Atg8-based ATTECs: Approaches and perspectives.

Induction of Atg8-family protein (LC3/GABARAP proteins in human) interactions with target proteins of interest by proximity-inducing small molecules offers the possibility for novel targeted protein degradation approaches. However, despite intensive screening campaigns during the last 5 years, no potent ligands for LC3/GABARAPs have been developed, rendering this approach largely unexplored and unsuitable for therapeutic exploitation. In this Viewpoint, we analyze the reported attempts identifying LC3/GABARAP inhibitors and provide our own point of view why no potent inhibitors have been found. Additionally, we designate reasonable directions for the identification of potent and probably selective LC3/GABARAP inhibitors for alternative therapeutic applications.

Advancing targeted protein degradation for metabolic diseases therapy.

The development and application of traditional drugs represented by small molecule chemical drugs and biological agents, especially inhibitors, have become the mainstream drug development. In recent years, targeted protein degradation (TPD) technology has become one of the most promising methods to remove specific disease-related proteins using cell self-destruction mechanisms. Many different TPD strategies are emerging based on the ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP), including but not limited to proteolysis-targeting chimeras (PROTAC), molecular glues (MG), lysosome targeting chimeras (LYTAC), chaperone-mediated autophagy (CMA)-targeting chimeras, autophagy-targeting chimera (AUTAC), autophagosome-tethering compound (ATTEC), and autophagy-targeting chimera (AUTOTAC). The advent of targeted degradation technology can change most protein targets in human cells from undruggable to druggable, greatly expanding the therapeutic prospect of refractory diseases such as metabolic syndrome. Here, we summarize the latest progress of major TPD technologies, especially in metabolic syndrome and look forward to providing new insights for drug discovery.

Direct degradation and stabilization of proteins: New horizons in treatment of nonalcoholic steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) is featured with excessive hepatic lipid accumulation and its global prevalence is soaring. Nonalcoholic steatohepatitis (NASH), the severe systemic inflammatory subtype of NAFLD, is tightly associated with metabolic comorbidities, and the hepatocytes manifest severe inflammation and ballooning. Currently the therapeutic options for treating NASH are limited. Potent small molecules specifically intervene with the signaling pathways that promote pathogenesis of NASH. Nevertheless they have obvious adverse effects and show long-term ineffectiveness in clinical trials. It poses the fundamental question to efficiently and safely inhibit the pathogenic processes. Targeted protein degradation (TPD) belongs to the direct degradation strategies and is a burgeoning strategy. It utilizes the small molecules to bind to the target proteins and recruit the endogenous proteasome, lysosome and autophagosome-mediated degradation machineries. They effectively and specifically degrade the target proteins. It has exhibited promising therapeutic effects in treatment of cancer, neurodegenerative diseases and other diseases in a catalytic manner at low doses. We critically discuss the principles of multiple direct degradation strategies, especially PROTAC and ATTEC. We extensively analyze their emerging application in degradation of excessive pathogenic proteins and lipid droplets, which promote the progression of NASH. Moreover, we discuss the opposite strategy that utilizes the small molecules to recruit deubiquinases to stabilize the NASH/MASH-suppressing proteins. Their advantages, limitations, as well as the solutions to address the limitations have been analyzed. In summary, the innovative direct degradation strategies provide new insights into design of next-generation therapeutics to combat NASH with optimal safety paradigm and efficiency.

DDHD2 promotes lipid droplet catabolism by acting as a TAG lipase and a cargo receptor for lipophagy.

Mutations in the DDHD2 (DDHD domain containing 2) gene cause autosomal recessive spastic paraplegia type 54 (SPG54), a rare neurodegenerative disorder characterized by the early childhood onset of progressive spastic paraplegia. DDHD2 is reported as the principal brain triacylglycerol (TAG) lipase whose dysfunction causes massive lipid droplet (LD) accumulation in the brains of SPG54 patients. However, the precise functions of DDHD2 in regulating LD catabolism are not yet fully understood. In a recent study, we demonstrate that DDHD2 interacts with multiple members of the Atg8-family proteins (MAP1LC3/LC3s, GABARAPs), which play crucial roles in lipophagy. DDHD2 possesses two LC3-interacting region (LIR) motifs that contribute to its LD-eliminating activity. Moreover, DDHD2 enhances the colocalization between LC3B and LDs to promote lipophagy. LD·ATTEC, a compound that tethers LC3 to LDs to enhance their macroautophagic/autophagic clearance, effectively counteracts DDHD2 deficiency-induced LD accumulation. These findings provide insights into the dual functions of DDHD2 as a TAG lipase and cargo receptor for lipophagy in neuronal LD catabolism, and also suggest a potential therapeutic approach for treating SPG54 patients.

Targeted protein modification as a paradigm shift in drug discovery.

Targeted Protein Modification (TPM) is an umbrella term encompassing numerous tools and approaches that use bifunctional agents to induce a desired modification over the POI. The most well-known TPM mechanism is PROTAC-directed protein ubiquitination. PROTAC-based targeted degradation offers several advantages over conventional small-molecule inhibitors, has shifted the drug discovery paradigm, and is acquiring increasing interest as over ten PROTACs have entered clinical trials in the past few years. Targeting the protein of interest for proteasomal degradation by PROTACS was the pioneer of various toolboxes for selective protein degradation. Nowadays, the ever-increasing number of tools and strategies for modulating and modifying the POI has expanded far beyond protein degradation, which phosphorylation and de-phosphorylation of the protein of interest, targeted acetylation, and selective modification of protein O-GlcNAcylation are among them. These novel strategies have opened new avenues for achieving more precise outcomes while remaining feasible and minimizing side effects. This field, however, is still in its infancy and has a long way to precede widespread use and translation into clinical practice. Herein, we investigate the pros and cons of these novel strategies by exploring the latest advancements in this field. Ultimately, we briefly discuss the emerging potential applications of these innovations in cancer therapy, neurodegeneration, viral infections, and autoimmune and inflammatory diseases.

TACkling Cancer by Targeting Selective Protein Degradation.

Targeted protein degradation has emerged as an alternative therapy against cancer, offering several advantages over traditional inhibitors. The new degrader drugs provide different therapeutic strategies: they could cross the phospholipid bilayer membrane by the addition of specific moieties to extracellular proteins. On the other hand, they could efficiently improve the degradation process by the generation of a ternary complex structure of an E3 ligase. Herein, we review the current trends in the use of TAC-based technologies (TACnologies), such as PROteolysis TArgeting Chimeras (PROTAC), PHOtochemically TArgeting Chimeras (PHOTAC), CLIck-formed Proteolysis TArgeting Chimeras (CLIPTAC), AUtophagy TArgeting Chimeras (AUTAC), AuTophagosome TEthering Compounds (ATTEC), LYsosome-TArgeting Chimeras (LYTAC), and DeUBiquitinase TArgeting Chimeras (DUBTAC), in experimental development and their progress towards clinical applications.

Novel approaches to targeted protein degradation technologies in drug discovery.

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.

Targeting lipid droplets for autophagic degradation by ATTEC.

Targeted protein degradation (TPD) provides unprecedented drug discovery strategies, but it is incapable of degrading non-protein pathogenic biomolecules. We have previously developed the concept of autophagosome-targeting compounds (ATTEC), which can target pathogenic proteins to autophagic degradation. Since macroautophagy (autophagy hereafter) is capable of degrading a wide spectrum of substrates including non-protein biomolecules, ATTEC should also be capable of targeting those non-protein biomolecules for autophagic degradation. Here in our most recent study, we have demonstrated this possibility using lipid droplets (LDs) as an exemplar target. LDs are intracellular structures storing neutral lipids, which can be degraded by autophagy. Based on the concept of ATTEC, compounds binding with both the LDs and the key phagophore and autophagosome protein LC3 may target LDs to autophagic degradation. We designed and synthesized such compounds by connecting the identified LC3-binding molecules to known LD-binding probes via a chemical linker. At micromolar concentrations, these compounds drastically reduced LDs via autophagy through the predicted mechanism, and rescued LD-related phenotypes in cells and in two independent mouse models with hepatic lipidosis. Our proof-of-concept study demonstrates the possibility of harnessing autophagy to degrade non-protein biomolecules by ATTEC.

Selective autophagy as the basis of autophagy-based degraders.

Degrader technologies, which enable the chemical knockdown of disease-causing proteins, are promising for drug discovery. After two decades of research, degraders using the ubiquitin-proteasome system (UPS) are currently in clinical trials. However, the UPS substrates are mainly limited to soluble proteins. Autophagy-targeting chimeras and autophagosome-tethering compounds are degraders that use autophagy, which has functions complementary to the UPS. They can degrade organelles and aggregate-prone proteins, making them promising treatments against age-related conditions such as mitochondrial dysfunction and neurodegenerative diseases. The molecular mechanism of selective autophagy is an ongoing research topic, which explains why autophagy-based degraders were not available until recently. In this review, we introduce four classifications of selective autophagy mechanisms to facilitate the understanding of the degrader design.

Targeting autophagy using small-molecule compounds to improve potential therapy of Parkinson's disease.

Parkinson's disease (PD), known as one of the most universal neurodegenerative diseases, is a serious threat to the health of the elderly. The current treatment has been demonstrated to relieve symptoms, and the discovery of new small-molecule compounds has been regarded as a promising strategy. Of note, the homeostasis of the autolysosome pathway (ALP) is closely associated with PD, and impaired autophagy may cause the death of neurons and thereby accelerating the progress of PD. Thus, pharmacological targeting autophagy with small-molecule compounds has been drawn a rising attention so far. In this review, we focus on summarizing several autophagy-associated targets, such as AMPK, mTORC1, ULK1, IMPase, LRRK2, beclin-1, TFEB, GCase, ERRα, C-Abelson, and as well as their relevant small-molecule compounds in PD models, which will shed light on a clue on exploiting more potential targeted small-molecule drugs tracking PD treatment in the near future.

Emerging New Concepts of Degrader Technologies.

Traditional drug discovery focuses on identifying direct inhibitors of target proteins. This typically relies on a measurable biochemical readout and accessible binding sites whose occupancy influences the function of the target protein. These requirements preclude many disease-causing proteins from being 'druggable' targets, and these proteins are categorized as 'undruggable'. The proteolysis-targeting chimera (PROTAC) technology provides powerful tools to degrade these undruggable targets and has become a promising approach for drug discovery. However, the PROTAC technology has some limitations, and emerging new degrader technologies may greatly broaden the spectrum of targets that could be selectively degraded by harnessing a second major degradation pathway in cells. We review key emerging technologies that exploit the lysosomal degradation pathway and discuss their potential applications and limitations.