Ligand-induced protein degradation has emerged as a compelling approach to promote the targeted elimination of proteins from cells by directing these proteins to the ubiquitin-proteasome machinery. So far, only a limited number of E3 ligases have been found to support ligand-induced protein degradation, reflecting a dearth of E3-binding compounds for proteolysis-targeting chimera (PROTAC) design. Here, we describe a functional screening strategy performed with a focused library of candidate electrophilic PROTACs to discover bifunctional compounds that degrade proteins in human cells by covalently engaging E3 ligases. Mechanistic studies revealed that the electrophilic PROTACs act through modifying specific cysteines in DCAF11, a poorly characterized E3 ligase substrate adaptor. We further show that DCAF11-directed electrophilic PROTACs can degrade multiple endogenous proteins, including FBKP12 and the androgen receptor, in human prostate cancer cells. Our findings designate DCAF11 as an E3 ligase capable of supporting ligand-induced protein degradation via electrophilic PROTACs.
To mL eedom, Mark Foster,a nd Curt W. Bradshaw [a] Oligonucleotides are important therapeutic approaches, as evidenced by recent clinicals uccesses with antisense oligonucleotides (ASOs)a nd double-stranded short interfering RNAs (siRNAs). Phosphorothioate (PS) modifications are as tandard feature in the current generation of oligonucleotide therapeutics, but generatei someric mixtures, leadingt o2 n isomers. All currently marketed therapeutic oligonucleotides (ASOs and siRNAs)a re complex isomeric mixtures. Recent chemical methodologiesf or stereopure PS insertions have resulted in preliminary rules for ASOs, with multiple stereopure ASOs moving into clinicald evelopment. Although siRNAs have comparatively fewer PSs, the field has yet to embrace the idea of stereopure siRNAs.H erein, it has been investigated whether the individual isomersc ontributee qually to the in vivo activity of ar epresentative siRNA. The results of as ystematic evaluation of stereopure PS incorporation into antithrombin-3 (AT3) siRNA are reported and demonstrate that individualP Sisomersd ramatically affect in vivo activity.Astandard siRNA design with six PS insertions was investigated and it was found that only about 10 %o ft he 64 possible isomersw ere as efficacious as the stereorandomc ontrol.B ased on this data, it can be concluded that G1R stereochemistry is critical, G2R is important,G 21S is preferable, and G22 and P1/P2 tolerate both isomers. Surprisingly,t he disproportionate loss of efficacy for most isomers does not translate into significant gain for the productive isomers, and thus, warrants further mechanistic studies.Since the discovery by Fire and Mello in 1998 that doublestranded short interfering RNA (siRNA) can cleave mRNA and inhibit protein translation, [1] this new class of therapeutics has moved towards clinical utility.T he advent of double-stranded siRNAs as am odality to harnessanatural catalytic pathway, called RNA interference (RNAi), excited scientific and business communities alike because of its implications in therapeutics, particularly for targets difficult to drug with small-molecules and proteins. [2] Interesti ns iRNAs has had periodic boom and bust cycles following the recognition of and solutions for technical challenges. One of the significant challenges is delivery of these large, charged siRNA molecules across the cell membrane.B oth lipid nanoparticle and ligand-based approaches are clinically validated, with ar ecent approvalo ft he first RNAi therapeutic, Patisiran, al ipid complex from Alnylam, and nu-merousG alNAc-targeteds iRNAs in clinical trials. [3] Naturally occurring siRNA molecules have two complementary strands (19b ase pairs), with an additional two unpaired bases at the 3'-ends, so-called 21/21. Aw ide variety of variations weree xploredi ne arly optimization, includings ignificant modifications in size (19-to 27-mers) and shape (Dicer-substrate siRNA, asymmetric and blunt-ended designs). [4] In addition, siRNA components, such as sugars,n ucleobases, and the phosphate backbon...
Introduction: Targeted protein degradation is a novel therapeutic modality that holds great promise. Current bispecific degraders have been mostly built using reversible ligands against two well characterized E3 ligases, CRBN and VHL. Features unique to each particular E3 ligase, such as its tissue expression pattern or its intrinsic catalytic efficiency could ultimately impact the in vivo bioactivity of any bifunctional drug based on it. Therefore, discovering ligands to novel E3s has the potential to enable a new class of drugs with substantially differentiated pharmacology compared to CRBN- or VHL-based degraders. Our unique covalent small molecule library and proteomics platform is an ideal tool for discovering ligands against novel E3s. Experimental plan: Utilize Vividion's chemical proteomics platform to screen for ligands capable of covalently engaging novel E3s and driving targeted degradation when configured in bifunctional formats. Summary of Results: Initial screens identified covalent ligands capable of potently engaging numerous E3 ligases, including members of the Cullin E3s, other RING-containing ligases and HECT E3 ligases. We describe here a representative molecule from this screening effort that was advanced to engagement potencies of < 10 nM while maintaining a selectivity window of over a thousand-fold relative to the next closest target. Proof-of-concept studies were designed by incorporating this E3 ligand into bifunctional degraders using 3 different target recruiting ligands capable of binding to FKBP12, BRD4 or to multiple kinases. These bifunctional molecules demonstrated profound and sustained degradation of all tested targets in vitro, with near complete degradation observable within 4 hours and potency ranges of < 10 nM. These covalent degraders were consistently more potent compared to several published CRBN- and VHL-based degraders designed to target the same proteins. When tested in vivo, a representative molecule from this series was capable of achieving near complete degradation of the intended target across multiple tissues. These molecules consistently outperformed similarly configured CRBN- and VHL-based degraders when assessed head to head in mouse degradation studies, and several molecules demonstrated > 50% degradation the target proteins at doses < 1 mg/kg. The current data suggest covalent ligands to novel E3 engagers could prove useful for future degrader-based drug design against multiple cancer targets. Further, Vividion's chemical proteomic platform is well suited for discovering novel covalent E3 ligands capable of supporting targeted degradation applications in cancer. Citation Format: Kristen Baltgalvis, Shota Kikuchi, Kent Symons, Joseph Klebba, Lena Luukkonen, Yuta Naro, Colin Walsh, Joon Chang, Charles Chapman, Ali Tabatabaei, Brian Nordin, Christie Eissler, Joel Chick, Landon Whitby, Jaclyn Brannon, Gabe Simon, Matt Patricelli, Dean Stamos, Larry Burgess, Todd Kinsella. Discovery of covalent ligands to novel E3 ligases enables bispecific degraders with highly differentiated protein degradation across a broad range of targets [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6411.
170 Background: CRC is the 4th leading cause of global cancer-related deaths, and novel therapeutic strategies for advanced CRC are urgently needed. Adoptive cell therapy (ACT) is effective in treating hematological malignancies; however, ACT in solid tumors is hindered by target antigen identification, restricted migration into tumors, and survival in the tumor microenvironment (TME) due to immunosuppressive signals and scarcity of nutrients. NK cells are central to anti-tumor immunity and can directly eliminate tumor cells without prior sensitization. Through cytokine reprogramming, NK cells can also gain memory-like features that augment their anti-tumor potential. WU-NK-101 is a cytokine-reprogrammed, expanded, cryopreserved, off-the-shelf NK cell product derived from peripheral blood mononuclear cells, with no additional engineering. Methods: WU-NK-101 ± Ctx was evaluated in vitro in 2D cytotoxicity assays in complete (N) and TME-aligned medias. WU-NK-101 cytotoxicity was further assessed against primary CRC surgical samples in native-TME-aligned 3D assays. Proteomic analysis was performed using tandem-mass spectrometry. In vivo efficacy of WU-NK-101 ± Ctx was evaluated in NSG mice bearing LoVo xenograft CRC tumors. Cell trafficking/penetration to TME was measured by tracking labeled WU-NK-101 ± trastuzumab in NSG mice bearing subcutaneous SKOV-3 xenografts. Results: Compared to conventional NK cells (cNK), WU-NK-101 had a unique phenotype consistent with rapid activation and proliferation (higher expression of activating receptors, Ki67, and GZMB), and showed enhanced in vitro cytotoxicity. WU-NK-101 also exhibited potent cytotoxicity against LoVo CRC tumors in vivo, which was further enhanced in combination with Ctx. Antibody combination improved WU-NK-101 penetration, and persistence in TME. WU-NK-101’s metabolic profile was consistent with aerobic (Warburg) glycolysis, potentially facilitating effector functions in the TME. WU-NK-101 also showed enhanced metabolic fitness and flexibility, as proteins in several metabolic pathways were upregulated in TME vs N media. Consistent with this, WU-NK-101 had increased cell-surface expression of nutrient transporters. While cNK and T cells cytotoxicity was significantly suppressed in TME-aligned media, WU-NK-101’s function was not impacted. Conclusions: We show that WU-NK-101 exerted potent activity against CRC, and in combination with Ctx showed improved intra-tumor infiltration/persistence and anti-tumor activity. Also, WU-NK-101 cells had enhanced metabolic fitness/flexibility and decreased susceptibility to immunosuppression, overcoming limitations encountered by ACT for solid tumors. A Phase 1b clinical trial is in development, which may reshape ACT in CRC and other EGFR-expressing tumors. [Table: see text]
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