An efficient auxin-inducible degron system with low basal degradation in human cells

Auxin-inducible degron (AID) technology allows rapid and controlled protein depletion, revealing acute effects of protein loss in cells. A major caveat of the commonly used AID system is the severe degradation of target proteins in the absence of an inducer (known as ‘basal degradation’). Here we solved the issue by identifying an efficient auxin receptor-degron pair with low basal degradation.

Authors: Shiqian Li, Veijo T. Salo, Elina Ikonen

 

Small-molecule inhibitors of the E3 ubiquitin-ligase Hakai against cancer

At early stages of carcinoma progression, a highly conserved cellular process, named epithelial-mesenchymal transition (EMT), takes place. It is characterized by a decreased expression of E-cadherin, a tumour suppressor that is responsible for cell-cell adhesion in the epithelium. During EMT, E-cadherin is downregulated at a posttranscriptional and a posttranslational level. The requirement of the E3 ubiquitin-ligase Hakai for the ubiquitination and subsequent degradation of E-cadherin has been associated not only with an enhanced epithelial-to-mesenchymal transition (EMT) but also with an increased cell proliferation and invasiveness, thus playing a key role during tumour progression and metastasis. In addition, recent studies have highlighted the importance of Hakai during tumour progression in colorectal cancer, as it has been shown to be increased in advanced TNM stages compared to adenoma or adjacent healthy colon tissues.
So far, most of the reported EMT-related inhibitors indirectly affect the EMT, as they were not designed for anti-EMT purposes. On the other hand, as the specific inhibition of E3 ubiquitin-ligase enzymes prevents broader side effects, they have recently emerged as promising therapeutic targets. Taking into account this background, a virtual screening was carried out to further identify novel specific inhibitors of Hakai, by targeting its phosphotyrosine-binding pocket, where phosphorylated-E-cadherin specifically binds. As a result of virtual screening, some inhibitor compounds were selected. These inhibitor compounds showed an important effect on Hakai-induced ubiquitination and were also able to reduce carcinoma growth and tumour progression, both in vitro in and in vivo. Therefore, these results show for the first time small-molecule compounds that target the E3 ubiquitin-ligase Hakai, which reduce Hakai-dependent ubiquitination of E-cadherin affecting the EMT process. This represents an important step forward in further development of an effective therapeutic drug that would prevent or inhibit carcinoma tumour progression.

 

Authors: Alba Casas-Pais, Andrea Rodríguez-Alonso, Olaia Martinez-Iglesias, Daniel Roca-Lema, Ángel Concha, Begoña Graña, Gabriela Romay, Álvaro Cortés, Federico Gago and Angélica Figueroa

 

 

 

Physicochemical property-based design of new oral PROTACs: which descriptors?

Pharmaceutical scientists have huge expectations from proteolysis-targeting chimeras (PROTACs) to treat diseases with serious unmet medical need and improve human health. However, much of the fundamental chemistry and biology in the PROTAC field is still unknown and therefore the identification of robust drug candidates to move into the clinic is a very uncertain step. For instance, PROTACs are large and flexible molecules and could pose significant challenges in terms of cellular uptake and bioavailability (1). An efficient physicochemical property-based design is needed to optimize the ADME profile of drug candidates and to discover new oral PROTAC medicines. Molecular properties, lipophilicity among others (2), can significantly help PROTAC early developability screen. Here for the first time we provide information about PROTACs molecular properties. Firstly, we show the non‑transferability of in silico descriptors routinely implemented in small molecule drug discovery programs (e.g. clogP, TPSA) to the PROTAC chemical space and provide insights on how to develop new ad hoc tools. Then we report experimental values of ionization, lipophilicity in different environments, and polarity of a series of seven freely available (through the opnMe platform, https://opnme.com/) PROTACs and verify their relationship with cell permeability. 

Authors: Giuseppe Ermondi, Maura Vallaro, and Giulia Caron

 

 

Exhaustive Yeast n-Hybrid Screening Technology to support Targeted Protein Degradation Drug Discovery Projects

Targeted protein degradation with small molecules or bifunctional ones such as PROteolysis-TArgeting Chimeras (PROTACs) has evolved as an exciting and promising approach to modulate and inhibit disease-causing proteins that are difficult to target with conventional methods. Examples of successful PROTACs include molecules that interact with the E3 ubiquitin ligases CRBN or VHL on one side of the chimeric molecule, and selected target proteins like the Androgen Receptor or BRD4 on the other side of the molecule, leading eventually to the degradation of the target proteins by the ubiquitin-proteasome pathway.

 

Authors: Marie-Edith Gourdel, Petra Tafelmeyer, Gisèle Guimèse, Fanny Moisant, Kais Aloui, Jean-Christophe Rain

 

 

Efficient Targeted Degradation via Reversible and Irreversible Covalent PROTACs

Proteolysis targeting chimeras (PROTACs) represent an exciting inhibitory modality with many advantages, including substoichiometric degradation of targets. Their scope, though, is still limited to date by the requirement for a sufficiently potent target binder. 
A solution that proved useful in tackling challenging targets is the use of electrophiles to allow irreversible binding to the target. However, such binding will negate the catalytic nature of PROTACs. 
Reversible covalent PROTACs potentially offer the best of both worlds. They possess the potency and selectivity associated with the formation of the covalent bond, while being able to dissociate and regenerate once the protein target is degraded. 
Using Bruton’s tyrosine kinase (BTK) as a clinically relevant model system, we show efficient degradation by noncovalent, irreversible covalent, and reversible covalent PROTACs, with <10 nM DC50’s and >85% degradation. Our data suggest that part of the degradation by our irreversible covalent PROTACs is driven by reversible binding prior to covalent bond formation, while the reversible covalent PROTACs drive degradation primarily by covalent engagement. The PROTACs showed enhanced inhibition of B cell activation compared to ibrutinib and exhibit potent degradation of BTK in patient-derived primary chronic lymphocytic leukemia cells. The most potent reversible covalent PROTAC, RC-3, exhibited enhanced selectivity toward BTK compared to noncovalent and irreversible covalent PROTACs. 
These compounds may pave the way for the design of covalent PROTACs for a wide variety of challenging targets.

Authors: Ronen Gabizon, Amit Shraga, Paul Gehrtz, Ella Livnah, Yamit Shorer, Neta Gurwicz, Liat Avram, Tamar Unger, Hila Aharoni, Shira Albeck, Alexander Brandis, Ziv Shulman, Ben-Zion Katz, Yair Herishanu, and Nir London

 

 

 

 

Targeted degradation of endogenously tagged proteins for phenotypic studies using HaloPROTAC3 and HaloTag® technologies

To understand a protein's function inside the cell, studies are often done to remove it using CRISPR-mediated knockout or RNAi knockdown methods. However, these approaches have challenges that include obtaining efficient loss of the targeted protein, or cell death if the protein is found to be essential for cell growth. To overcome these hurdles, we have employed a highly precise and temporally controlled target protein degradation strategy utilizing HaloPROTAC3, a HaloTag® proteolysis-targeting chimera small molecule which specifically degrades HaloTag fusion proteins in live cells. HaloPROTAC3, developed by Prof. Craig Crews at Yale University, recruits HaloTag® fusion proteins to VHL E3 ligase complexes, resulting in ubiquitination and degradation of the HaloTag® fusion via the ubiquitin-proteasomal pathway. Endogenous HaloTag® fusion proteins are developed via CRISPR/Cas9 gene editing into either the N- or C-terminal loci of any target protein. Additionally, we've appended the 11 amino acid HiBiT to the HaloTag® fusion protein, allowing for highly quantitative kinetic monitoring of degradation in live cells with the use of luminescence instead of antibodies. Shown here is the rapid and sustained degradation (80-90% loss) of key therapeutic HiBiT-HaloTag® fusion proteins, BRD4 and β-catenin, after treatment with HaloPROTAC3 in HEK293 LgBiT stable cells. For BRD4, 95% of the protein fusion was lost after 48 hours of treatment with HaloPROTAC3. For β-catenin, phenotypic studies were done to show a muted response to Wnt3a stimulation and TCF/LEF promoter gene activation after degradation of the HiBiT-HaloTag® fusion protein. Together, we demonstrate how these technologies can be used to elicit robust degradation of target proteins, with control over the protein level as well as the time frame for protein degradation. Also, we provide new opportunities for phenotypic studies in order to investigate the function of essential proteins without the need of protein specific PROTACs.

 

Authors: Elizabeth A. Caine, Sarah Mahan, Kristin Riching, Cesear Corona, Nancy Murphy, Chris Eggers, Chris Heid, Danette L. Daniels, and Marjeta Urh

Real-time Monitoring of PROTAC and Molecular Glue Targeted Degradation in Living Cells

The emergence of targeted protein degradation as a broad, new therapeutic modality has greatly expanded the opportunities for treatments of many diseases. Currently, small molecule degrader compounds fall under two major categories; molecular glues and heterobifunctional PROTACs.  The two types of compounds both facilitate and induce a ternary complex consisting of an E3 ligase-degrader-target protein, bringing into proximity the machinery proteins required to ubiquitinate and ultimately degrade the target protein. Significant challenges exist in characterization and rank-ordering of degradation compounds in live cells given the differences in dynamics of protein loss and recovery among compounds. Currently, the availability of technologies to interrogate real-time protein degradation is severely lacking. Here, we present a live-cell, luminescence-based technology platform with these capabilities. We use CRISPR/Cas9 endogenous tagging of target proteins with the small peptide, HiBiT, which has high affinity for and can complement with the LgBiT protein to produce NanoBiT luminescence. This allows for sensitive detection of endogenous protein levels in living cells and can also serve as a BRET energy donor to study protein:protein or protein:small molecule interactions. We demonstrate the power of this technology in continuous 24-hour monitoring of endogenous target protein levels, and the ability to quantify key degradation parameters for compound ranking including rate, Dmax, and Dmax50. We further show the ability to measure the mechanistic steps important for the degradation including kinetics of PROTAC- or molecular glue-induced ternary complex formation and target ubiquitination. These studies facilitate discernment of individual parameters required for successful degradation, ultimately enabling chemical design strategies for optimization and rank ordering of therapeutic degradation compounds.

Kristin M. Riching, Sarah Mahan, Elizabeth A. Caine, James Vasta, Cesear Corona, Matt Robers, Danette L. Daniels, and Marjeta Urh