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Can you imagine a world in which we could selectively erase disease-causing proteins from our bodies, leaving healthy cells unaffected? This revolutionary concept is not science fiction; it’s the promise of targeted protein degradation (Protac Services), a cutting-edge approach that holds the potential to transform medicine as we know it.

Targeted Protein Degradation (TPD) with it’s PROTAC Services, promisingly offers a novel way to eliminate toxic or disease-associated targets from cells. New medicines are being developed that leverage the cell’s intrinsic destruction machinery (ubiquitin-proteasome system), enabling them to surmount many of the conventional obstacles encountered in drug discovery. This empowering approach holds the potential to unlock fresh treatment avenues for a wide range of diseases that currently lack effective therapeutic solutions.

TPD is like precision molecular assassination. It focuses on specific proteins, tagging them for destruction while sparing the rest of the cell. This opens the opportunity to treat a wide range of diseases, from cancer and neurological disorders to infectious diseases, by eliminating the root causes at a molecular level.

 

(Graphical representation of the degradation mechanism of proteolysis-targeting chimeras (PROTACs) Image references - https://journals.sagepub.com/doi/full/10.1177/2472555220965528

          (Graphical representation of the degradation mechanism of proteolysis-targeting chimeras (PROTACs)
                        (Image references – https://journals.sagepub.com/doi/full/10.1177/2472555220965528)

 

Targeted Protein Degradation (TPD) involves employing heterobifunctional small molecule “degraders,” such as PROTAC molecules, to effectively reduce the levels of specific proteins within cells. These degraders, also referred to as Active Degraders, are composed of binding components for an E3 ubiquitin ligase and a target protein, connected by a linker. When both binding components bind their targets,  they form a ternary complex involving the target protein and E3 ligase. This complex initiates a process of polyubiquitination by proximity, where the target protein becomes tagged with multiple ubiquitin molecules. Consequently, the proteasome identifies and degrades the ubiquitinated target protein, leading to its effective removal from the cellular environment.

TPD, driven by the precision of E3 ligases, is revolutionising the fields of biotechnology and medicine. At its core lies the intricate machinery of E3 Ligases, enzymes with the remarkable ability to tag specific proteins for destruction. 

The PROTAC services offered by o2h provide cutting-edge solutions for harnessing the power of targeted protein degradation. In this blog, we will deep dive into the world of TPD and try to understand the transformative power of E3 ligases in detail.

  1. Table of Contents

    The Ubiquitin-Proteasome System (UPS):

The ubiquitin-proteasome system is a highly regulated and intricate cellular machinery responsible for the targeted degradation of specific proteins within a cell. It plays a fundamental role in maintaining cellular health by controlling protein levels, regulating various processes, and disposing of damaged or unnecessary proteins. Dysregulation of this system has been implicated in various diseases, making it an attractive target for therapeutic intervention.

Ubiquitination: The Tagging Process

Ubiquitination is the key event in the UPS. It involves the covalent attachment of a small protein called ubiquitin to target proteins. This process marks the proteins for degradation.

Ubiquitination occurs in a three-step enzymatic cascade: activation (E1 enzymes), conjugation (E2 enzymes), and ligation (E3 ligases). E3 ligases are the determinants of specificity in this process.

E1, E2, and E3: The Three Essential Enzymes

In the intricate machinery of the Ubiquitin-Proteasome System (UPS), three essential enzymes play pivotal roles: E1, E2, and E3. These enzymes work in a coordinated manner to tag specific proteins for degradation. Let’s break down their functions and significance:

E1 (Ubiquitin-Activating Enzyme)

E1 is the initiator of the ubiquitination process. It’s responsible for activating ubiquitin molecules, which are small proteins that serve as tags for marking other proteins for degradation. E1 binds to ubiquitin and transfers it to an E2 enzyme.

E2 (Ubiquitin-Conjugating Enzyme)

E2 acts as an intermediary between E1 and E3 in the ubiquitination cascade. Once E1 has activated ubiquitin, E2 accepts the ubiquitin molecule and carries it to the target protein. E2 enzymes are crucial for determining the specificity of ubiquitination since different E2 enzymes can interact with various E3 ligases and substrates.

E3 (Ubiquitin-Protein Ligase)

E3 is the conductor of this molecular orchestra. It plays a central role in recognizing specific protein substrates and facilitating the transfer of ubiquitin from the E2 enzyme to the target protein. Each E3 ligase has a unique substrate specificity, allowing it to select its protein targets carefully. This specificity is what makes the UPS highly precise and essential for regulating various cellular processes.

 

The Ubiquitin-Proteasome System
(Image reference - https://lifesensors.com/ubiquitinproteasomesystem/)

The Ubiquitin-Proteasome System (Image reference – https://lifesensors.com/ubiquitinproteasomesystem/)

Selective Protein Degradation: E3 Ligases’ Significance

The presence of numerous E3 ligases in the cell allows for selective degradation of specific proteins. Different E3 ligases recognize different substrates, ensuring that only the intended proteins are tagged for degradation.

This selectivity is critical for maintaining cellular health. It makes it possible for broken, improperly folded, or extra proteins to be quickly removed, preventing their accumulation, which could be harmful to the cell.

The Diverse Landscape of E3 Ligases:

The landscape of E3 ligases is incredibly diverse, with numerous members and subclasses that allow for precise regulation of ubiquitination and degradation of specific target proteins. It enables cells to respond dynamically to changes in their environment, maintain protein homeostasis, and participate in a wide range of cellular processes. Dysregulation of E3 ligases can lead to various diseases, making them important targets for research and therapeutic development. The complex landscape of E3 ligases continues to be a rich area of exploration in the field of molecular biology and cellular regulation. Here’s a glimpse into this diverse landscape:

RING E3 Ligases:

The Cullin-RING E3 Ligases (CRLs)

Constitute the largest family of E3 ligases, exceeding 200 members, and in certain cell types, they govern up to 20% of proteasome-dependent proteome degradation. The evolutionarily conserved Cullin family comprises seven key members—Cul1, Cul2, Cul3, Cul4A, Cul4B, Cul5, and Cul7—sharing similar structural architecture. Classification of CRLs is based on the specific Cullin protein within the complex, leading to designations such as SCF (Skp1–Cdc53–F-box Cdc4), ECS (EloBC–Cul2/5–SOCS-box, with EloBC representing the ElonginB–ElonginC complex and SOCS denoting suppressor of cytokine signaling), and BCR (BTB–Cul3–Rbx1, where BTB refers to bric-a-brac/tramtrack/broad complex). CRLs are involved in the degradation of a wide range of proteins, including those involved in cell cycle regulation, signal transduction, and DNA repair.

Single-Subunit RING E3 Ligases:

These E3 ligases function as single entities and include well-known examples like MDM2, which regulates the tumor suppressor p53, and TRIM (Tripartite Motif) proteins, with roles in immunity, antiviral defense, and cell cycle control.

HECT-like RING E3 Ligases:

HECT-type E3 ubiquitin ligases, comprising a modest subfamily of E3 enzymes with 28 identified members in humans, exhibit inherent enzymatic activity. Notably, these E3 ligases are commonly found to be dysregulated in various human cancers, underscoring their significance as potential targets for understanding and addressing oncogenic pathways.

CRBN and VHL: Key Players in the PROTAC Field

In the dynamic world of PROTACs (Proteolysis Targeting Chimeras), CRBN and VHL ligands are key contributors, celebrated for their robust, target-specific binding, favorable physicochemical properties, and precisely defined structural interactions. These properties have not only accelerated the development of numerous PROTACs, but have also elevated them to the status of powerful modulators in biological systems. The success of CRBN and VHL ligands in enabling exceptional in vitro and in vivo degradation capabilities has not gone unnoticed, leading to their widespread use in the search for novel therapeutics.

Aside from their efficacy, the distinct mechanisms of action associated with CRBN and VHL ligands have broadened the scope of PROTAC technology application. The CRL4CRBN E3 ligase complex, in particular, interacts with CRBN, orchestrating the ubiquitination and subsequent degradation of target proteins. VHL ligands, on the other hand, interact with the VHL E3 ligase complex, providing a distinct pathway for targeted protein degradation. Because of the versatility of these ligands, there has been an increase in research and development, resulting in a diverse portfolio of PROTACs with diverse target specificities.

Furthermore, the impact of PROTACs containing CRBN or VHL ligands extends beyond the laboratory, having a significant impact on intellectual property landscapes. The success of these ligands has resulted in a significant increase in patent filings, indicating an appreciation for their transformative potential in therapeutic interventions. As a result, clinical development efforts for PROTACs utilizing CRBN or VHL ligands continue to expand, highlighting their role as pioneers in the pursuit of next-generation, targeted therapeutics.

                                                                       E3 Ligase (CRBN generic scaffold)E3 Ligase (VHL generic scaffold)

HECT E3 Ligases:

NEDD4 (Neural Precursor Cell Expressed Developmentally Down-Regulated 4)-like E3 Ligases

This subclass includes NEDD4, NEDD4-2, and ITCH, among others. NEDD4-like ligases regulate membrane protein trafficking, endocytosis, and the degradation of various cellular proteins.

HERC (HECT and RLD Domain-Containing E3 Ubiquitin Protein Ligases)

HERC family members participate in diverse cellular processes, including DNA damage repair, cellular trafficking, and protein homeostasis.

Other HECT E3 Ligases

There are several additional HECT E3 ligases, such as E6-AP (E6-associated protein), SMURF1 (SMAD Ubiquitination Regulatory Factor 1), and UBE3C (Ubiquitin Protein Ligase E3C), each with unique substrate specificities and roles.

RBR E3 Ligases (Really Interesting New Gene-like RING E3 Ligases)

These E3 ligases have a distinctive combination of RING and HECT-like domains. They participate in various processes, including protein degradation and quality control. RBR ligases are structurally distinct from both HECT and other RING-type E3 ligases. They contain two RING domains separated by an in-between-RING (IBR) domain, which gives them their name.

The Complex Network of E3 Ligases: From Specificity to Function

The E3 ligases form a complex network within cells, each with its own substrate specificity and biological function. This complexity arises from the diversity of E3 ligases and their ability to recognize and tag specific target proteins for degradation. Here, we explore the intricacies of this complex network:

Substrate Specificity

E3 ligases are highly selective in choosing their substrate proteins. This selectivity is often governed by specific protein-protein interactions between the E3 ligase and its target protein. Some E3 ligases are highly specialized, targeting only a single protein, while others are more promiscuous, targeting multiple substrates with similar structural features.

Biological Function

E3 ligases play crucial biological roles by regulating various cellular processes through the targeted ubiquitination and degradation of specific proteins. For example, some E3 ligases are involved in cell cycle control, ensuring that certain proteins are degraded at specific times during the cell cycle. Others are implicated in DNA repair, immune response regulation, and protein quality control etc.

Ubiquitin Chain Types

E3 ligases can also determine the type of ubiquitin chains that are added to target proteins. Ubiquitin chains can vary in length and linkage type, and these variations can have different functional consequences. For instance, K48-linked ubiquitin chains typically target proteins for proteasomal degradation, while K63-linked chains can have signaling roles.

Interactions with E2 Enzymes

E3 ligases collaborate with E2 enzymes to transfer ubiquitin to target proteins. Different E3 ligases interact with specific E2 enzymes, adding another layer of specificity to the ubiquitination process.

The Cullin-RING E3 Ligase (CRL) complex has drawn a lot of attention in recent years because of its important role in protein degradation and cellular homeostasis regulation. The CRL complex, which includes several subtypes such as SCF complexes and BTB complexes, orchestrates the targeted ubiquitination and subsequent degradation of a diverse set of proteins, influencing critical cellular processes such as cell cycle progression, signal transduction, and DNA repair.

Advancing Therapeutic Applications:

The ongoing advancement of targeted protein degradation, including the use of PROTACs and other innovative approaches, is reshaping therapeutic strategies across a spectrum of diseases. As research continues to unveil the intricacies of the ubiquitin-proteasome system and our understanding of E3 ligases deepens, we can anticipate increasingly precise, effective, and personalized treatments that have the potential to improve the lives of countless individuals affected by a wide variety of medical conditions. This field represents a promising frontier in the pursuit of better healthcare and disease management. The below graphic describes the pipeline of PROTACs from 2001 to the future aspect.

    (Timetable describing the evolution of PROTACs (2001–2016) and publication numbers during several years)
                                                      (Source - https://www.mdpi.com/1420-3049/28/10/4014)

(Timetable describing the evolution of PROTACs (2001–2016) and publication numbers during several years)                                                                             (Source – https://www.mdpi.com/1420-3049/28/10/4014)

PROTACs in Cancer Therapy: Overcoming Drug Resistance and Reducing Side Effects

In the realm of cancer therapy, Proteolysis Targeting Chimeras (PROTACs) offers an effective avenue. These molecules present a novel approach to tackling drug resistance, a formidable challenge in cancer treatment. By leveraging the power of E3 ligases, PROTACs can selectively degrade proteins associated with drug resistance, allowing for the resensitization of cancer cells to conventional therapies. Moreover, the precision of PROTACs minimizes off-target effects, potentially reducing the often debilitating side effects experienced by cancer patients undergoing treatment.

Unleashing the Potential in Neurodegenerative Disorders and Autoimmune Diseases

Beyond cancer, the therapeutic potential of targeted protein degradation extends to neurodegenerative disorders and autoimmune diseases. Here, the precision of PROTACs shines brightly. These conditions often involve misfolded or overactive proteins that drive pathology. PROTACs can be designed to specifically target these aberrant proteins, offering a new approach to mitigating disease progression. By addressing the root causes with such precision, PROTACs hold the promise of halting or even reversing the debilitating effects of these conditions.

PROTACs Beyond the Ordinary: Addressing “Undruggable” Targets

The term “undruggable” has long haunted drug discovery efforts, referring to proteins that have been notoriously difficult to target with traditional small-molecule drugs. However, PROTACs are rewriting the rules of the game. Their ability to harness E3 ligases to degrade even the most challenging proteins opens doors previously considered firmly shut. Whether it’s tackling elusive transcription factors or mutant proteins, PROTACs are at the forefront of making formerly undruggable targets accessible for therapeutic intervention.

Read more – https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9499226/#funding-group-1title

Future Challenges and Perspectives

The field of targeted protein degradation, including PROTACs and related approaches, is on the cusp of transformative breakthroughs in medicine and biology, but it will require ongoing innovation, collaboration, and a commitment to rigorous scientific exploration. Most of the target proteins created for PROTACs are within the druggable proteome. It’s possible to consider making compounds using PROTAC technology that can influence difficult-to-address non-traditional therapeutic targets. PROTACs only need temporary binders to facilitate compound formation, allowing for less potent ligands for the protein of interest. The below graphic depicts the role and future pipeline of PROTAC molecule in drug discovery – 

(The Future of Drug Discovery with PROTAC Technology)
(Source - https://www.mdpi.com/1420-3049/28/10/4014)

(The Future of Drug Discovery with PROTAC Technology) (Source – https://www.mdpi.com/1420-3049/28/10/4014)

 

The following challenges will be crucial for realising the full potential of targeted protein degradation in medicine and biotechnology. However, as with any emerging field, several challenges and future perspectives need to be addressed-

Unraveling the E3 Ligase-Substrate Code: Identifying Optimal E3 Ligases for Specific Targets

One of the ongoing challenges in the field of targeted protein degradation is deciphering the E3 ligase-substrate code. While we understand the broad specificity of many E3 ligases, identifying the optimal E3 ligase for specific protein targets remains a complex puzzle. Researchers are working diligently to uncover the rules that govern these interactions. Once we crack this code, we can design more precise and efficient PROTACs tailored for specific diseases, improving their efficacy and safety.

Enhancing PROTAC Pharmacokinetics: Designing for Clinical Success

As PROTACs progress towards clinical applications, optimising their pharmacokinetic properties becomes paramount. This involves fine-tuning aspects such as stability, half-life, and bioavailability to ensure that PROTACs can navigate the complexities of the human body effectively. Multiple studies are under way to explore various strategies, from modifying the linker chemistry to encapsulating PROTACs in nanoparticles, to enhance their pharmacokinetics and make them viable candidates for clinical trials. Advancements in structural biology and computational modelling may aid in rational PROTAC design.

Combining Forces: Integrating PROTACs with CRISPR-Based Targeting

In the pursuit of precision medicine, the integration of PROTACs with CRISPR-based targeting represents a thrilling frontier. While PROTACs can degrade specific proteins, CRISPR allows for precise gene editing. Combining these two cutting-edge technologies holds the potential to not only eliminate problematic proteins but also correct genetic mutations at their source. Genome-wide CRISPR-Cas9 knockout screens can be used to identify E3 ligases that, when knocked out, prevent the degradation of a target protein.

This synergy offers a holistic approach to treating diseases at both the protein and genetic levels, opening new doors for therapeutic intervention.

o2h discovery’s expertise in Targeted Protein Degradation and PROTAC synthesis services

o2h discovery (o2h) is a fully integrated Anglo-Indian medicinal chemistry CRO, supporting biotech companies in the design, synthesis, and testing of novel PROTAC molecules for about 10 years with collaborators in the UK, US,  and now, across the world. o2h discovery offers a range of E3 ligase ligands from an “off the shelf toolbox” for the quick synthesis of PROTAC equipped with a variety of linkers  with different length and composition for a quick assembly of degrader analogues to accelerate your drug discovery and development process.

Here is a recent testimonial from one of our clients to whom we provided PROTAC services and helped their team deliver a range of personalised, cutting-edge Targeted Protein Degradation/PROTAC services:

    (Julian & Martin’s feedback about o2h discovery’s PROTAC/targeted protein degradation services)

Expertise in PROTAC synthesis

At o2h, we have extensive experience and expertise in the design, synthesis and purification of PROTACs acquired over a number of years by working on various Protein Degradation collaborations. The o2h PROTAC Toolbox allows us to build and test molecules quickly and efficiently using off-the-shelf molecules building blocks. Novel and complex PROTAC molecules can be accessed by engaging our well-established and experienced discovery and custom synthesis team. 

Our PROTAC chemistry expert team leverages the innovative Proteolysis Targeting Chimeras (PROTAC) technology to design customised strategies to degrade specific proteins of interest (POI) for your research or drug development project. 

Our investment in analytical and biological capabilities and our expertise to support the synthesis and delivery of high-quality PROTAC molecules is ingrained in our PROTAC delivery model. Below are some of our key offerings: 

  • PROTAC toolbox of 100+ different off-the-shelf building blocks with different phys-chem properties for a quick and efficient PROTAC matrix assembly and discovery
  • A range of personalised, cutting-edge PROTAC design and synthetic  services to optimise the profile of your degrader molecule
  • Analytical expertise and customizable solutions to isolate and purify each PROTAC compounds 
  • Dedicated lab in Cambridge, UK, where degraders can be further characterised for biological activity and cell permeability

Our proprietary o2h PROTAC ToolboxTM

o2h discovery has a customisable “off-the-shelf” PROTAC Toolbox™ consisting of a diverse set of linkers, E3 ligase ligands and E3 ligand-linker compounds, all variously functionalized, to JUMPSTART design, synthesis and testing/screening novel Protein Degraders.

Our customisable o2h PROTAC toolboxTM kit of 100+ building blocks includes:

  • Different VHL and Lenalidomide derivatives E3 Ligase ligands (with different vectors and functional groups);
  • Different bi-functionalised linkers of different sizes and shape (Alkyl, PEG, cyclic) with different phys-chem properties;
  • A selected combination of the above already connected E3 Ligase ligands and linkers.

The quick assembly of PROTAC molecules aims to provide you with a quick tool to evaluate a range of molecules that will help prove and de-risk your protein degradation approach.

o2h protac toolbox

(o2h protac toolboxTM)

PROTAC – biology expertise

o2h biologists have many years of experience in basic and applied research, with a proven track record of working with “me too” and “undruggable” class of targets. This coupled with a good understanding of potency to efficacy translation, we can provide an integrated suite of high-quality biophysical, biochemical & cellular-assay platforms and in delivering an optimal solution in the evaluation of targeted protein degraders. 

  1. Is my target degraded? 
  2. Do they form a ternary complex? 
  3. Are they soluble and cell-permeable? 
  4. What’s the phenotypic consequence of target degradation?
Targeted Protein Degradation Process

                                               Targeted Protein Degradation Process

PROTAC Purification Synthesis – Analytical 

o2h discovery’s analytical team has effectively purified PROTAC compounds through preparative high-performance liquid chromatography (Prep. HPLC) at various scales (mg/g). Some of our PROTAC expertise includes: 

  • Purification of multiple PROTAC compounds containing various Linkers/Ligands like VHL, CRBN or Thalidomide
  • >5 years of hands-on experience on PROTAC compounds purification and different column chemistry available for the purification of PROTAC molecules.
  • Successfully enriched the purity level from ~20% crude purity to >95%
  • Well experienced Analytical team to handle purification of PROTAC compounds on gm scale.
  • Achieved up to 50% practical recovery from prep purification, even from crude reaction mixtures containing solvents like DMSO or DMF.

And many more…

o2h discovery has an experienced, cross-functional team of experts in medicinal chemistry, synthesis, computational chemistry, biology, pharmacology, process development, along with specialized knowledge in PROTAC (Proteolysis Targeting Chimeras), PROTAC chemistry and formulation with high-throughput screening techniques. Our in-house capability to execute hit-lead-optimization programs aims to provide you with a comprehensive, integrated approach to enable you to accelerate your Targeted Protein Degradation and drug discovery programs.

To discover the full extent of the o2h PROTAC Toolbox™ offer, please contact us at discovery@o2h.com

To know more about o2h discovery’s PROTAC services and PROTAC chemistry capabilities or to get a quote, please visit: https://o2h.com/protac-synthesis-services/

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