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PROTAC In Vitro Evaluation

As a novel frontier for drug discovery, PROTACs utilize bifunctional small molecules to remove disease-causing proteins, and it has huge potential to offer therapeutic interventions not achievable with existing approaches. Creative Biolabs now possesses one-stop PROTAC service platform to provide discovery and development services. After PROTAC molecule design is determined, we offer a comprehensive set of in vitro evaluation modules to verify the efficacy of PROTACs.

Background

PROTAC is composed of two functional molecules: one is to bind target protein, and the other is to recruit specific E3 ligase to play the role of ubiquitination-mediated degradation for target protein (see Fig.1). The first PROTAC was described by Crews and Deshaies in 2001. In light of various advantages of this technique, it has been applied by a variety of drug discovery to various E3 ligases including pVHL, MDM2, beta-TrCP1, cereblon, and c-IAP1. PROTACs had stepped into clinical trial stages for ovarian cancer, prostate cancer in ARVINAS. It indicates that PROTAC is becoming a promising and attractive therapeutic strategy in pharmaceutical industry.

Schematic of the PROteolysis TArgeting Chimera (PROTAC) technology. Fig.1 Schematic of the PROteolysis TArgeting Chimera (PROTAC) technology. (Neklesa, 2017)

Introduction of PROTAC In Vitro Evaluation Service

Modern drug discovery begins with the identification/design and chemistry optimization of PROTAC. Once a compound is designed and synthesized, it follows a series of in vitro evaluation assays from administration to intracellular target degradation to verify the efficacy of PROTACs in the drug development stage prior to clinical trials. Creative Biolabs is capable of offering a comprehensive set of in vitro evaluation assays to monitor the specific parameters in each stage (see Fig.2). We divide our services into seven modules, a package service or any individual modules are all available in Creative Biolabs.

Schematic diagram of <em>in vitro</em> evaluation assays to assess PROTACs. Fig.2 Schematic diagram of in vitro evaluation assays to assess PROTACs.

Solubility and Chemical Stability

Among the chemical properties of small molecules, solubility and stability are crucial to determine if the PROTAC candidate can be a potential drug. Both computational and experimental approaches can be applied to estimate or measure the solubility and stability. Creative Biolabs is dedicated to helping our clients to evaluate the solubility and stability of PROTAC candidate in a wide range of conditions with different pH, buffer type, chemical additives, and excipients.

Cell Permeability Efficacy

PROTAC application is critically limited by permeability of PROTAC candidate, hence, monitoring cell permeability efficacy is essential prior to subsequent structural and functional assays. Creative Biolabs offers our clients with specific assays to determine the permeability by PAMPA, Caco-2, and MDCK.

Binary Target Engagement

Once chemical properties and permeability are confirmed, PROTAC will undergo binary engagement assays to detect the affinities with either target protein or specific E3 ligase. Creative Biolabs provides a variety of biochemical techniques such as isothermal titration calorimetry (ITC), fluorescence polarization (FP), surface plasmon resonance (SPR), etc. to monitor IC50 and Kd values. In addition, Creative Biolabs is also capable of offering structure-based and whole cell ligand binding assays to detect the interaction between PROTAC and target protein or E3 ligase.

Ternary Complex Formation

Binary target engagement can result in ternary complex formation which is structurally critical to PROTAC efficacy. Creative Biolabs is committed to different assays to detect the ternary complex formation, such as ITC, FRET, NMR, Cell-free proximity assays, BRET, and Co-IP, etc. We aim to tailor the most optimal assay for our clients to fit the specific requirements and project budgets.

Cell Cytotoxicity Test

Following ternary complex formation, E3 ligase will degrade target proteins via proteasome machinery. After target protein degradation, PROTAC is released and continues the degradation process. This contributes to the low cytotoxicity of PROTAC benefited by low dosage. Creative Biolabs owns a variety of assays to evaluate cell cytotoxicity through enzyme leakage assays, membrane impermeable dyes, amine-reactive dyes, dye combination assays, cell viability assays, apoptosis assays, cell proliferation and cell cycle assays.

Target Ubiquitination

After cell cytotoxicity test, we come to the final step of in vitro evaluation - degradation efficacy. The initiation signal of degradation is ubiquitination of target protein. Herein, Creative Biolabs offers target ubiquitination assays through a variety of biochemical approaches, such as MS, gel shift assay, and western blot assay treated with or without proteasome or ubiquitination inhibitors.

Target Degradation

Target degradation is the final goal of in vitro evaluations. The dominant feature of PROTAC technique is target degradation rather than blocking. Creative Biolabs provides our clients with different assays to evaluate the degradation efficacy including western blots, reporter gene assay, and downstream effects.

Highlight Features of Our Services

  • A wide range of assays available
  • Assay design and conduct
  • Ensure timely response
  • Experienced scientists and advanced one-stop platform

Creative Biolabs is dedicated to covering all assays to help our clients to evaluate the efficacy of PROTAC. We aim to utilize our knowledge and passion to promote the development of drug discovery field and contribute to a healthier world. If you are interested in any individual assay or a package service, please don't hesitate to contact us.

Reference

  1. Neklesa, T.K., et al. Targeted protein degradation by PROTACs. Pharmacol Ther. 2017, 174, 138-144.
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