Heterobifunctional Molecules Research Articles

Bifunctional Molecules beyond PROTACs.

Heterobifunctional molecules, which recruit E3 ligases to ubiquitinate a target protein of interest, have found wide application as both biological tools and molecules with the potential to have clinical effects. In their recent paper, Yamazoe et al. report a heterobifunctional molecule that recruits the phosphatase PP1 to promote the dephosphorylation of pAKT to give AKT. This Viewpoint seeks to place this work in the wider context of heterobifunctional molecules and looks ahead to new possibilities presented by these results.

Current strategies for the design of PROTAC linkers: a critical review.

PROteolysis TArgeting Chimeras (PROTACs) are heterobifunctional molecules consisting of two ligands; an “anchor” to bind to an E3 ubiquitin ligase and a “warhead” to bind to a protein of interest, connected by a chemical linker. Targeted protein degradation by PROTACs has emerged as a new modality for the knock down of a range of proteins, with the first agents now reaching clinical evaluation. It has become increasingly clear that the length and composition of the linker play critical roles on the physicochemical properties and bioactivity of PROTACs. While linker design has historically received limited attention, the PROTAC field is evolving rapidly and currently undergoing an important shift from synthetically tractable alkyl and polyethylene glycol to more sophisticated functional linkers. This promises to unlock a wealth of novel PROTAC agents with enhanced bioactivity for therapeutic intervention. Here, the authors provide a timely overview of the diverse linker classes in the published literature, along with their underlying design principles and overall influence on the properties and bioactivity of the associated PROTACs. Finally, the authors provide a critical analysis of current strategies for PROTAC assembly. The authors highlight important limitations associated with the traditional “trial and error” approach around linker design and selection, and suggest potential future avenues to further inform rational linker design and accelerate the identification of optimised PROTACs. In particular, the authors believe that advances in computational and structural methods will play an essential role to gain a better understanding of the structure and dynamics of PROTAC ternary complexes, and will be essential to address the current gaps in knowledge associated with PROTAC design.

Heterobifunctional Molecules Induce Dephosphorylation of Kinases-A Proof of Concept Study.

Heterobifunctional molecules have proven powerful tools to induce ligase-dependent ubiquitination of target proteins. We describe here a chemical strategy for controlling a different post-translational modification (PTM): phosphorylation. Heterobifunctional molecules were designed to promote the proximity of a protein phosphatase (PP1) to protein targets. The synthesized molecules induced the PP1-dependent dephosphorylation of AKT and EGFR. To our knowledge, this work represents the first examples of small molecules recruiting non-native partners to induce removal of a PTM.

Assessing the Cell Permeability of Bivalent Chemical Degraders Using the Chloroalkane Penetration Assay.

Bivalent chemical degraders provide a catalytic route to selectively degrade disease-associated proteins. By linking target-specific ligands with E3 ubiquitin ligase recruiting ligands, these compounds facilitate targeted protein ubiquitination and degradation by the proteasome. Due to the complexity of this multistep mechanism, the development of effective degrader molecules remains a difficult, lengthy, and unpredictable process. Since degraders are large heterobifunctional molecules, the efficacy of these compounds may be limited by poor cell permeability, and an efficient and reliable method to quantify the cell permeability of these compounds is lacking. Herein, we demonstrate that by the addition of a chloroalkane tag on the BRD4 specific degrader, MZ1, cell permeability can be quantified via the chloroalkane penetration assay. By extending this analysis to individual components of the degrader molecule, we have obtained structure-permeability relationships that will be informative for future degrader development, particularly as degraders move into the clinic as potential therapeutics.

Abstract LB-A11: Quantitative and qualitative characterization of spontaneous resistance to targeted protein degradation

Abstract Targeted protein degradation (TPD) by heterobifunctional small molecules commonly called PROTACs has recently emerged as a promising novel strategy in drug development. PROTACs hijack the endogenous cellular degradation machinery by recruiting E3 ubiquitin ligases to a protein of interest, resulting in its ubiquitination and degradation by the proteasome. The first two PROTACs have entered clinical investigation this year, but resistance mechanisms to this novel pharmacology are not yet thoroughly understood. Here, we set out to systematically quantify and characterize spontaneous resistance to TPD. For this, we made use of the well-characterized PROTACs dBET6 and ARV-771, that induce degradation of BET bromodomain proteins by recruitment of the E3 ligase CRBN or VHL, respectively. We estimated spontaneous resistance rates by exposing cells to lethal doses of drug and monitoring for the emergence of resistant clones. We detected important quantitative differences between the two PROTACs, with resistance rates to the VHL-based degrader ARV-771 being significantly lower than to dBET6. To characterize the mechanism of resistance, we implemented a customized hybrid-capture sequencing pipeline for known determinants of PROTAC sensitivity and applied it to drug-resistant cells, revealing several resistance-conferring mutations. Mapping of the mutations on crystal structures of the E3 ligase complexes highlighted their clustering at the drug binding pocket, as well as on important protein-protein interaction surfaces in the complex. The majority of identified mutations affected the hijiacked E3 ligase complex directly and not the general ubiquitination machinery, thereby not conferring cross-resistance to different PROTACS. This suggests combination treatment with degraders that exploit different E3 ligases as a viable strategy to limit emergence of PROTAC resistance. Citation Format: Matthias Brand, Georg E. Winter. Quantitative and qualitative characterization of spontaneous resistance to targeted protein degradation [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A11. doi:10.1158/1535-7163.TARG-19-LB-A11

Bioavailable Dual-Protein Degraders of CK1α and Transcriptional Kinase CDK9 As Potential Therapeutics for Hematological Malignancies

BioTheryx's small-molecule kinase inhibitor, BTX-A51 (the ditosylated salt of A51), has recently received FDA approval of its IND application to initiate a Phase I clinical trial in relapsed or refractory acute myeloid leukemia (AML). A51 (API of BTX-A51) is a multi-kinase inhibitor that blocks the leukemic stem cell target, Casein Kinase 1α (CK1α), as well as the super-enhancer regulator, Cyclin-Dependent Kinase 9 (CDK9), thus preventing the transcription of key oncogenic genes. This molecule has demonstrated remarkable preclinical animal efficacy inferring the eradication of AML stem cells and its potential for use in treating multiple malignancies. To exploit the unique properties of this multi-kinase inhibitor in the context of kinase protein degradation, Proteolysis-Targeting Chimeras (PROTACs) of A51 were investigated. PROTACs utilize the cell's natural ubiquitin-proteasome system to induce the selective and sustained degradation of unwanted disease-causing proteins. PROTACs are heterobifunctional molecules which are comprised of an E3-ubiquitin-ligase ligand which is covalently linked to a target-protein ligand. Through concomitant binding to an E3 ligase and to a target protein, a PROTAC promotes ubiquitination and ultimately degradation of the target protein via the proteasome. As a reversible kinase inhibitor, A51 binds stoichiometrically to CK1α and CDK9 and requires continuous occupancy of these proteins to sustain its unique inhibitory activity. A PROTAC of A51, however, would act catalytically and ablate the target proteins. In this study, PROTACs were assembled through chemically linking A51 to BioTheryx's proprietary Protein Homeostatic Modulators (PHMs™). PHMs™ are a new class of small molecules which bind to the E3 ubiquitin ligase, Cereblon (CRBN), and promote proteasomal degradation of known as well as unreported clinically-relevant CRBN neo-substrates. For the discovery of PHM®-A51 PROTACs, CDK9 crystal structure (6GZD.pdb) and CRBN crystal structure (5FQD.pdb) were used independently for computational drug design. Upon testing in AML and lymphoma cell lines, the PHM®-A51 PROTACs rapidly induced the degradation of both CK1α and CDK9. Additionally, these PROTACs had diminished effect on the protein levels of other A51 targets while inducing the degradation of other desirable targets unaffected by A51. This distinct selectivity arises from the PROTACs' ability to facilitate formation of a ternary complex that enables subsequent ubiquitination of the bound target protein - a selectivity not inherent to any kinase inhibitor. Further, the PHM®-A51 PROTACs have demonstrated low-nanomolar inhibition of cell proliferation in both AML and lymphoma cell lines yet have significantly less toxicity in fibroblast cells and PBMCs. In addition, this class of PROTACs has good pharmaceutical properties, as demonstrated by pharmacokinetic studies in mice, and will advance into AML and lymphoma in vivo studies. The targeted degradation of CK1α and CDK9 using safe, bioavailable drugs designed using strategically-selected PHMs™ with unique biological properties of their own is a very promising approach to treating hematological malignancies and other devasting cancers. Disclosures No relevant conflicts of interest to declare.

Protein targeting chimeric molecules specific for dual bromodomain 4 (BRD4) and Polo-like kinase 1 (PLK1) proteins in acute myeloid leukemia cells

Proteolysis targeting chimeras (PROTACs) are hetero-bifunctional molecules that could simultaneously bind to the target protein and the E3 ubiquitin ligase, thereby leading to selective degradation of the target protein. Polo-like kinase 1 (PLK1) and bromodomain 4 (BRD4) are both attractive therapeutic targets in acute myeloid leukemia (AML). Here, we developed a small-molecule BRD4 and PLK1 degrader HBL-4 based on PROTAC technology, which leads to fast, efficient, and prolonged degradation of BRD4 and PLK1 in MV4-11 cells tested in vitro and vivo, and potent anti-proliferation and BRD4 and PLK1 degradation ability in human acute leukemia MOLM-13 and KG1 cells. Meanwhile, HBL-4 more effectively suppresses c-Myc levels than inhibitor BI2536, resulting in more effective inducing apoptosis activity in MV4-11 cells. At the same time, HBL-4 induced dramatically improved efficacy in the MV4-11 tumor xenograft model as compared with BI2536. This study is, to our knowledge, the first reports about dual PLK1 and BRD4 degraders, which potentially represents an important therapeutic advance in the treatment of cancer.

Design, Synthesis, and Evaluation of Highly Potent FAK-Targeting PROTACs.

Focal adhesion kinase (FAK), a cytoplasmic protein tyrosine kinase, exerts kinase-dependent enzymatic functions and kinase-independent scaffolding functions, both of which are crucial in cancer development, early embryonic development, and reproduction. However, previous efforts for FAK blocking mainly focus on kinase inhibitors. Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that allow direct post-translational knockdown of proteins via ubiquitination of a target protein by E3 ubiquitin ligase and subsequent proteasomal degradation. Here, we designed and synthesized a FAK PROTAC library with FAK inhibitor (PF562271 or VS6063) and CRBN E3 ligand. A novel FAK-targeting PROTAC, FC-11, showed a rapid and reversible FAK degradation with a picomolar of DC50 in various cell lines in vitro, which imply that FAK-PROTACs could be useful as expand tools for studying functions of FAK in biological system and as potential therapeutic agents.

Inducing the Degradation of Disease-Related Proteins Using Heterobifunctional Molecules

Current drug development strategies that target either enzymatic or receptor proteins for which specific small molecule ligands can be designed for modulation, result in a large portion of the proteome being overlooked as undruggable. The recruitment of natural degradation cascades for targeted protein removal using heterobifunctional molecules (or degraders) provides a likely avenue to expand the druggable proteome. In this review, we discuss the use of this drug development strategy in relation to degradation cascade-recruiting mechanisms and successfully targeted disease-related proteins. Essential characteristics to be considered in degrader design are deliberated upon and future development challenges mentioned.

Targeted degradation of aberrant tau in frontotemporal dementia patient-derived neuronal cell models.

Tauopathies are neurodegenerative diseases characterized by aberrant forms of tau protein accumulation leading to neuronal death in focal brain areas. Positron emission tomography (PET) tracers that bind to pathological tau are used in diagnosis, but there are no current therapies to eliminate these tau species. We employed targeted protein degradation technology to convert a tau PET-probe into a functional degrader of pathogenic tau. The hetero-bifunctional molecule QC-01-175 was designed to engage both tau and Cereblon (CRBN), a substrate-receptor for the E3-ubiquitin ligase CRL4CRBN, to trigger tau ubiquitination and proteasomal degradation. QC-01-175 effected clearance of tau in frontotemporal dementia (FTD) patient-derived neuronal cell models, with minimal effect on tau from neurons of healthy controls, indicating specificity for disease-relevant forms. QC-01-175 also rescued stress vulnerability in FTD neurons, phenocopying CRISPR-mediated MAPT-knockout. This work demonstrates that aberrant tau in FTD patient-derived neurons is amenable to targeted degradation, representing an important advance for therapeutics.

Abstract P5-04-18: ARV-471, an oral estrogen receptor PROTAC degrader for breast cancer

Abstract ARV-471, an estrogen receptor (ER) alpha PROTAC, is a hetero-bifunctional molecule that facilitates the interactions between estrogen receptor alpha and an intracellular E3 ligase complex, leading to the ubiquitylation and subsequent degradation of estrogen receptors via the proteasome. ARV-471 robustly degrades ER in ER-positive breast cancer cell lines with a half-maximal degradation concentration (DC50) of ˜ 2 nM. PROTAC-mediated ER degradation decreases the expression of classically-regulated ER-target genes (PR, GREB1, TFF) and inhibits cell proliferation of ER-dependent cell lines (MCF7, T47D). Additionally, ARV-471 degrades clinically-relevant ESR1 variants (Y537S and D538G) and inhibits growth of cell lines expressing those variants. In an immature rat uterotrophic model, ARV-471 degrades rat uterine ER and demonstrates no agonist activity. Daily, oral-administration of single agent ARV-471 (3, 10, and 30 mpk) leads to significant tumor volume regressions of estradiol-dependent MCF7 xenografts and concomitant tumor ER protein reductions of >90% at study termination. Moreover, when a CDK4/6 inhibitor is combined with ARV-471 in the MCF7 model, even more pronounced tumor growth inhibition is observed (˜130% TGI), accompanied by significant reductions in ER protein levels. In an ESR1 Y537S, hormone-independent patient-derived xenograft model, ARV-471 at 10 mpk completely inhibited growth and also reduced mutant ER protein levels. Taken together, the preclinical data of ARV-471 supports its continued development as a best-in-class oral ER PROTAC-degrader. Citation Format: Flanagan JJ, Qian Y, Gough SM, Andreoli M, Bookbinder M, Cadelina G, Bradley J, Rousseau E, Willard R, Pizzano J, Crews CM, Crew AP, Taylor I, Houston J. ARV-471, an oral estrogen receptor PROTAC degrader for breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-18.

Differential PROTAC substrate specificity dictated by orientation of recruited E3 ligase

PROteolysis-TArgeting Chimeras (PROTACs) are hetero-bifunctional molecules that recruit an E3 ubiquitin ligase to a given substrate protein resulting in its targeted degradation. Many potent PROTACs with specificity for dissimilar targets have been developed; however, the factors governing degradation selectivity within closely-related protein families remain elusive. Here, we generate isoform-selective PROTACs for the p38 MAPK family using a single warhead (foretinib) and recruited E3 ligase (von Hippel-Lindau). Based on their distinct linker attachments and lengths, these two PROTACs differentially recruit VHL, resulting in degradation of p38α or p38δ. We characterize the role of ternary complex formation in driving selectivity, showing that it is necessary, but insufficient, for PROTAC-induced substrate ubiquitination. Lastly, we explore the p38δ:PROTAC:VHL complex to explain the different selectivity profiles of these PROTACs. Our work attributes the selective degradation of two closely-related proteins using the same warhead and E3 ligase to heretofore underappreciated aspects of the ternary complex model.

A MedChem toolbox for cereblon-directed PROTACs.

A modular chemistry toolbox was developed for cereblon-directed PROTACs. A variety of linkers was attached to a CRBN ligand via the 4-amino position of pomalidomide. We used linkers of different constitution to modulate physicochemical properties. We equipped one terminus of the linker with a set of functional groups, e.g. protected amines, protected carboxylic acids, alkynes, chloroalkanes, and protected alcohols, all of which are considered to be attractive for PROTAC design. We also highlight different opportunities for the expansion of the medicinal chemists' PROTAC toolbox towards heterobifunctional molecules, e.g. with biotin, fluorescent, hydrophobic and peptide tags.

Cereblon as a primary target of IMiDs

Various derivatives of thalidomide, a drug that is well-known for its teratogenicity, have recently been developed; among them, lenalidomide and pomalidomide, known as immunomodulatory drugs (IMiDs), have potent anticancer activity. These drugs have been approved by Food and Drug Administration for the treatment of several diseases, including multiple myeloma, under strict control. The primary direct target protein of thalidomide and IMiDs is cereblon (CRBN), a substrate receptor of Cullin-RING ligase 4 (CRL4). CRL4CRBN is a unique E3 ubiquitin ligase because its substrate selectivity is altered by ligands such as IMiDs. Each IMiD induces degradation of neosubstrates, such as Ikaros or CK1a. Because the activity of new CRBN-binding compounds is not limited to immunomodulation, the designation of "cereblon modulators" has been proposed for these small CRBN-binding compounds. Currently, researchers are investigating CC-122 (avadomide) and CC-220 (iberdomide) for the treatment of diffuse large B-cell lymphoma and systemic lupus erythematosus, respectively. Other recent studies have been investigating heterobifunctional molecules called proteolysis-targeting chimeras (PROTACs) for protein of interest degradation. Moreover, several proteins, such as BRD4, CDK9, or Tau, have already been successfully degraded by CRBN-based PROTACs. In this review, recent advances in CRBN and its binding compounds have been discussed.

The one-pot nonhydrolysis Staudinger reaction and Staudinger or SPAAC ligation.

The combined usage of two bioorthogonal reactions can provide hetero-bifunctional molecules under physiological conditions for various applications. Based on the Nonhydrolysis Staudinger Reaction (NSR), we design and develop a bisazido linker 1 for chemoselective dual-functionalization without the need of protection using catalyst-free and one-pot procedures. The NSR is much faster with tetrafluorinated aromatic azide than that the Staudinger-Bertozzi or SPAAC ligation with alkyl azide, as revealed by HPLC analysis and fluorescence kinetics. Based on the tandem NSR and Staudinger-Bertozzi ligation, we prepare a molecular beacon 7 from 1 in one-pot synthesis with a recovery yield of 32%. When a faster SPAAC ligation is used instead of the Staudinger-Bertozzi ligation, compound 8 is prepared in the tandem NSR and SPAAC reactions with a recovery yield of 59%. As a proof-of-concept study, the tandem NSR and SPAAC ligation is further used to produce a FRET-based dyad in living cells, as revealed by dual-color bioimaging. This work shows that NSR can be combined with other bioorthogonal reactions without the need of protection in one-pot.

MDM2-Recruiting PROTAC Offers Superior, Synergistic Antiproliferative Activity via Simultaneous Degradation of BRD4 and Stabilization of p53.

Although the number of proteins effectively targeted for posttranslational degradation by PROTAC has grown steadily, the number of E3 ligases successfully exploited to accomplish this has been limited to the few for which small-molecule ligands have been discovered. Although the E3 ligase MDM2 is bound by the nutlin class of small-molecule ligands, there are few nutlin-based PROTAC. Because a nutlin-based PROTAC should both knockdown its target protein and upregulate the tumor suppressor p53, we examined the ability of such a PROTAC to decrease cancer cell viability. A nutlin-based, BRD4-degrading PROTAC, A1874, was able to degrade its target protein by 98% with nanomolar potency. Given the complementary ability of A1874 to stabilize p53, we discovered that the nutlin-based PROTAC was more effective in inhibiting proliferation of many cancer cell lines with wild-type p53 than was a corresponding VHL-utilizing PROTAC with similar potency and efficacy to degrade BRD4. This is the first report of a PROTAC in which the E3 ligase ligand and targeting warhead combine to exert a synergistic antiproliferative effect. Our study highlights the untapped potential that may be unlocked by expanding the repertoire of E3 ligases that can be recruited by PROTAC. SIGNIFICANCE: These findings present the first BRD4-targeting MDM2-based PROTAC that possesses potent, distinct, and synergistic biological activities associated with both ends of this heterobifunctional molecule.

Targeted Degradation of IRAK4 Protein Via Heterobifunctional Small Molecules for Treatment of MYD88 Mutant Lymphoma

Recurrent mutations in the scaffolding protein MYD88 are present in 30-40% of activated B cell diffuse lymphocytic B cell lymphoma (ABC-DLBCL)(Ngo et al. Nature 2011). MYD88 links activated interleukin 1 receptor (IL1R) and Toll-like receptors (TLRs) to downstream effectors by nucleating assembly of the Myddosome, a multi-protein complex containing MYD88, the protein kinases IRAK4 and IRAK1 and the pseudokinase IRAK2, via oligomerization of the N-terminal Death Domains in each of these proteins (Motshwene et al. JBC 2009; Lin, Lo and Wu. Nature 2010). The most prevalent MYD88 mutation, L265P, constitutively activates assembly of the Myddosome, causing IRAK4-dependent NFκB and MAP kinase signaling and leading to lymphoma survival and proliferation (Ngo et al. Nature 2011).Constitutive activation of the Myddosome has proven to be difficult to drug. MYD88 and IRAK2 lack enzymatic activity and cannot be targeted by conventional small molecule catalytic inhibitors. There are no IRAK1 inhibitors in clinical trials. Specific inhibitors of the kinase activity of IRAK4 have shown limited activity as single agents in preclinical models of MYD88 mutant lymphoma (Lim et al. Blood 2012; Booher et al. Blood 2014). This is consistent with the observations that there is kinase-independent activity of IRAK4 in mediating downstream signals, such as those leading to induction of NFκB (Qin et al. JBC 2004; Fraczek et al. JBC 2008; Sun et al. Sci Signal. 2016). IRAK4 kinase inhibitors thus might not provide optimal blockade of NFκB-dependent tumor survival signals driven by MYD88 mutations in ABC-DLBCL.Kymera Therapeutics is advancing a new class of drugs based on targeted protein degradation: heterobifunctional molecules that recruit disease-causing proteins to specific E3 ubiquitin ligases, resulting in their ubiquitination and subsequent degradation. We have developed a portfolio of potent and specific degraders of IRAK4 with drug-like propertiesOur lead molecules cause potent and specific degradation of IRAK4 in the ABC-DLBCL cell lines OCI-LY10 (MYD88 L265P) and U2932 (MYD88 WT) while showing selective cytotoxic effects on OCI-LY10 vs U2932. The apoptotic response is triggered within 24-48 h in OCI-LY10, demonstrated by appearance of cleaved caspase 3 and PARP. Quantitative proteomic studies show selective degradation of IRAK4 vs five to six thousand other proteins detected in OCI-LY10.IRAK4 degraders were tested in an OCI-LY10 xenograft model with daily dosing for 28 days, to determine the level of IRAK4 degradation and impact of that degradation on tumor growth in vivo. We observed that degraders mediated decrease in IRAK4 in vivo and produced a statistically significant decrease in tumor growth rate.Here we will present in vitro and in vivo activity and mechanism of action of our most advanced IRAK4 degraders. These IRAK4 degrader molecules represent a new therapeutic modality and provide an exciting opportunity to treat MYD88 mutation-driven ABC-DLBCL. DisclosuresKelleher:Kymera Therapeutics: Employment, Equity Ownership. Audoly:Kymera Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Campbell:Kymera Therapeutics: Employment, Equity Ownership. Chen:Kymera Therapeutics: Employment, Equity Ownership. Ji:Kymera Therapeutics: Employment, Equity Ownership. Kamadurai:Kymera Therapeutics: Employment, Equity Ownership. Li:Kymera Therapeutics: Employment, Equity Ownership. Loh:Kymera Therapeutics: Employment, Equity Ownership. Rong:Kymera Therapeutics: Employment, Equity Ownership. Vigil:Kymera Therapeutics: Employment, Equity Ownership. Weiss:Kymera Therapeutics: Employment, Equity Ownership. Yuan:Kymera Therapeutics: Employment, Equity Ownership. Zhang:Kymera Therapeutics: Employment, Equity Ownership. Mainolfi:Kymera Therapeutics: Employment, Equity Ownership.

Induced protein degradation of anaplastic lymphoma kinase (ALK) by proteolysis targeting chimera (PROTAC)

Recently, proteolysis targeting chimera (PROTAC) technology is highlighted in drug discovery area as a new therapeutic approach. PROTAC as a heterobifunctional molecule is comprised of two ligands, which recruit target protein and E3 ligase, respectively. To degrade the anaplastic lymphoma kinase (ALK) fusion protein, such as NPM-ALK or EML4-ALK, we generated several ALK-PROTAC molecules consisted of ceritinib, one of the ALK inhibitors, and ligand of von Hippel-Lindau (VHL) E3 ligase. Among these molecules, TD-004 effectively induced ALK degradation and inhibited the growth of ALK fusion positive cell lines, SU-DHL-1 and H3122. We also confirmed that TD-004 significantly reduced the tumor growth in H3122 xenograft model.

Carboxylic acid-modified metal oxide catalyst for selectivity-tunable aerobic ammoxidation

Controlling the reaction selectivity of a heterobifunctional molecule is a fundamental challenge in many catalytic processes. Recent efforts to design chemoselective catalysts have focused on modifying the surface of metal nanoparticle materials having tunable properties. However, precise control over the surface properties of base-metal oxide catalysts remains a challenge. Here, we show that green modification of the surface with carboxylates can be used to tune the ammoxidation selectivity toward the desired products during the reaction of hydroxyaldehyde on manganese oxide catalysts. These modifications improve the selectivity for hydroxynitrile from 0 to 92% under identical reaction conditions. The product distribution of dinitrile and hydroxynitrile can be continuously tuned by adjusting the amount of carboxylate modifier. This property was attributed to the selective decrease in the hydroxyl adsorption affinity of the manganese oxides by the adsorbed carboxylate groups. The selectivity enhancement is not affected by the tail structure of the carboxylic acid.

Abstract P4-04-04: Identification and development of oral estrogen receptor PROTAC degraders for breast cancer

Abstract ER-positive breast cancers comprise approximately 70-80% of all newly diagnosed cases. Downregulation or degradation of ER is a treatment approach currently used in the clinic to target estrogen receptor signaling. Faslodex, the only clinically-approved ER-downregulator, is administered as a monthly intramuscular injection with limiting pharmaceutical properties. Reasoning that an orally-available estrogen receptor degrader would be beneficial to patients, we have leveraged our experience in targeted protein degradation to generate and characterize novel proteolysis targeting chimeras (PROTACs) against estrogen receptor alpha. PROTACs are heterobifunctional molecules that facilitate the formation of a “trimer complex” comprised of the PROTAC, a pathogenic target protein of interest and an E3 ligase, which catalyzes the ubiquitylation and subsequent degradation of the target protein via the proteasome. To identify novel ER degraders (ER PROTACs), we have used several in vitro assays to characterize the extent of target engagement and receptor degradation. Potent ER PROTACs with good oral exposure and other pharmaceutical properties in multiple pre-clinical species were further evaluated in breast cancer xenograft models. Orally-administered ER PROTACs achieved >80% degradation of estrogen receptor alpha and demonstrated single agent tumor growth inhibition in these disease models. Further, combination with a CDK4/6 inhibitor resulted in the expected improvement in anti-proliferative activity. Citation Format: Flanagan JJ, Qian Y, Gough SM, Andreoli M, Bookbinder M, Bradley J, Rousseau E, Willard R, Crews CM, Crew AP, Taylor I, Houston J. Identification and development of oral estrogen receptor PROTAC degraders for breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-04-04.