Formation Of Ternary Complex Research Articles

Alkylamine-tethered molecules recruit FBXO22 for targeted protein degradation

Targeted protein degradation (TPD) relies on small molecules to recruit proteins to E3 ligases to induce their ubiquitylation and degradation by the proteasome. Only a few of the approximately 600 human E3 ligases are currently amenable to this strategy. This limits the actionable target space and clinical opportunities and thus establishes the necessity to expand to additional ligases. Here we identify and characterize SP3N, a specific degrader of the prolyl isomerase FKBP12. SP3N features a minimal design, where a known FKBP12 ligand is appended with a flexible alkylamine tail that conveys degradation properties. We found that SP3N is a precursor and that the alkylamine is metabolized to an active aldehyde species that recruits the SCFFBXO22 ligase for FKBP12 degradation. Target engagement occurs via covalent adduction of Cys326 in the FBXO22 C-terminal domain, which is critical for ternary complex formation, ubiquitylation and degradation. This mechanism is conserved for two recently reported alkylamine-based degraders of NSD2 and XIAP, thus establishing alkylamine tethering and covalent hijacking of FBXO22 as a generalizable TPD strategy.

Potential of Native Cyclodextrins and L-Lysine for Enhancing Ellagic Acid Aqueous Solubility

This study aimed to examine the influence of β cyclodextrin (βCD) and γ cyclodextrin (γCD) alone and in combination with L-lysine on ellagic acid (EA) solubility. Indeed, complexation with cyclodextrins can be used to improving the solubility of drugs poorly soluble in water such as EA. EA is a Biopharamceutical Classification System (BCS) IV bioactive polyphenol with numerous therapeutic activities, including antimalarial activities. However, its unfavorable physicochemical properties limit its therapeutic use. Therefore, after a phase solubility study, we successfully prepared EA-βCD and γ CD binary solid complexes using the freeze-drying technique. Methods including proton nuclear magnetic resonance (1H-NMR) analysis and fourier-transform infrared (FTIR) spectroscopy were used to characterize the inclusion behaviors of the complexes of EA with cyclodextrins both in solution and solid forms. The results of the phase solubility study indicated Ap-type diagrams of EA with the two cyclodextrins (CDs). The solubility of EA was multiplied by 9.92 and 2.98 in the presence of γCD and βCD respectively. Moreover, the complexes characterization by FTIR spectroscopy revealed the involvement of the C=O and EA OH groups in the interaction with the CDs. The results of NMR spectroscopic characterization revealed that the formation of EA inclusion complexes with βCD was partial. However, these results did not indicate the appearance of EA inclusion complexes with γCD. These relatively modest results in terms of increased EA solubility, obtained with cyclodextrins, prompted us to use a third compound, l-lysine, to enhance CDs complexation. Thus, the formation of ternary complexes led to a very significant increase EA solubility. Indeed, the incorporation of EA into EA-L-lysine-βCD and γ CD complexes increased its water solubility at pH 7.4 by a factor of 555 and 663, respectively.

Abstract A005: A live cell PRMT5 NanoBRET™ target engagement assay querying competitive and uncompetitive modes of inhibition

Abstract PRMT5 is an essential arginine methyltransferase that regulates a wide spectrum of cellular processes through the methylation of histone and non-histone substrates. In PRMT5 catalysis, SAM serves as the cofactor and methyl group donor, generating a methylated guanidinium moiety on the target substrate. In normal cells, the SAM pools are maintained through the methionine salvage pathway. In 15% of cancers, a key enzyme in this pathway (MTAP) is deleted, leading to an accumulation of the intermediate MTA, which inhibits PRMT5 activity. This genetic loss of function has therefore been pursued as a collateral vulnerability in MTAP deleted cancers. To exploit this synthetic lethality, a number of novel inhibitors (such as MRTX1719) have been developed that bind cooperatively at the PRMT5/MTA complex, offering a compelling pathway to precision medicine. Here we describe a novel, live cell NanoBRET™ Target Engagement assay that enables mechanistic studies on a variety of PRMT5 inhibitors. Using a cell-permeable NanoBRET probe directed to the substrate pocket of PRMT5, both substrate- and cofactor-competitive engagement can be quantified in cells. Moreover, this method can be used to quantify MTA-uncompetitive target engagement in cells, providing a platform to measure the potency of PRMT5-MTA-drug ternary complex formation. This method serves as a first-in-class method to quantify uncompetitive target engagement in live cells, which can be applied to other model systems. Citation Format: Kelly A. Teske, Ani Michaud, Elisabeth Rothweiler, Cesear Corona, Kaitlin Dunn Hoffman, Jennifer Wilkinson, Michael Beck, James Vasta, Kilian Huber, Matthew Robers. A live cell PRMT5 NanoBRET™ target engagement assay querying competitive and uncompetitive modes of inhibition [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr A005.

Mechanistic computational modeling of monospecific and bispecific antibodies targeting interleukin-6/8 receptors.

The spread of cancer from organ to organ (metastasis) is responsible for the vast majority of cancer deaths; however, most current anti-cancer drugs are designed to arrest or reverse tumor growth without directly addressing disease spread. It was recently discovered that tumor cell-secreted interleukin-6 (IL-6) and interleukin-8 (IL-8) synergize to enhance cancer metastasis in a cell-density dependent manner, and blockade of the IL-6 and IL-8 receptors (IL-6R and IL-8R) with a novel bispecific antibody, BS1, significantly reduced metastatic burden in multiple preclinical mouse models of cancer. Bispecific antibodies (BsAbs), which combine two different antigen-binding sites into one molecule, are a promising modality for drug development due to their enhanced avidity and dual targeting effects. However, while BsAbs have tremendous therapeutic potential, elucidating the mechanisms underlying their binding and inhibition will be critical for maximizing the efficacy of new BsAb treatments. Here, we describe a quantitative, computational model of the BS1 BsAb, exhibiting how modeling multivalent binding provides key insights into antibody affinity and avidity effects and can guide therapeutic design. We present detailed simulations of the monovalent and bivalent binding interactions between different antibody constructs and the IL-6 and IL-8 receptors to establish how antibody properties and system conditions impact the formation of binary (antibody-receptor) and ternary (receptor-antibody-receptor) complexes. Model results demonstrate how the balance of these complex types drives receptor inhibition, providing important and generalizable predictions for effective therapeutic design.

β-Amino Acids Reduce Ternary Complex Stability and Alter the Translation Elongation Mechanism.

Templated synthesis of proteins containing non-natural amino acids (nnAAs) promises to expand the chemical space available to biological therapeutics and materials, but existing technologies are still limiting. Addressing these limitations requires a deeper understanding of the mechanism of protein synthesis and how it is perturbed by nnAAs. Here we examine the impact of nnAAs on the formation and ribosome utilization of the central elongation substrate: the ternary complex of native, aminoacylated tRNA, thermally unstable elongation factor, and GTP. By performing ensemble and single-molecule fluorescence resonance energy transfer measurements, we reveal that both the (R)- and (S)-β2 isomers of phenylalanine (Phe) disrupt ternary complex formation to levels below in vitro detection limits, while (R)- and (S)-β3-Phe reduce ternary complex stability by 1 order of magnitude. Consistent with these findings, (R)- and (S)-β2-Phe-charged tRNAs were not utilized by the ribosome, while (R)- and (S)-β3-Phe stereoisomers were utilized inefficiently. (R)-β3-Phe but not (S)-β3-Phe also exhibited order of magnitude defects in the rate of translocation after mRNA decoding. We conclude from these findings that non-natural amino acids can negatively impact the translation mechanism on multiple fronts and that the bottlenecks for improvement must include the consideration of the efficiency and stability of ternary complex formation.

Experimental and theoretical study of novel polyamine bis(nitrophenylureylbenzamide) receptors with anion and cation interaction capacity

In this work the interaction of three new heterotopic polyamine bis(nitrophenylureylbenzamide) receptors (R = 3A-3C) with anions (A) and cations (M) was analyzed by UV–Vis spectroscopy and theoretical calculations. These receptors are highly pre-organized and form supramolecular complexes R-A at different ratios depending on the anion characteristics (1:1, 2:1, 3:1 and 4:3). The calculated log K values based on UV–Vis absorbance profiles and the structural information obtained from geometry optimizations using Density Functional Theory indicate strong stability in these complexes due to multiple intermolecular interactions between both species. Changes in the absorption band of receptors evidence the interaction with metallic ions forming complexes R-M (1:1) and the theoretical calculations predict the coordination with polyamine and amide groups adopting an aza-crown type conformation surrounding the Cu or Zn atoms. The subsequent formation of ternary complexes R-M-A with acetate showed different spectral changes for R-Cu and R-Zn ligands. The spectral changes with R-Cu ligands allowed to identified and calculated log K values for R-Cu-A and R-Cu-A2 complexes. Moreover, the optimized structures for R-Cu-A and R-Cu-A2 complexes show the coordination of one acetate ion with the metal center and a subsequent interaction of a second acetate with the urea groups in agreement with the experimental results.

A molecular glue RBM39-degrader induces synthetic lethality in cancer cells with homologous recombination repair deficiency

E7820 and Indisulam (E7070) are sulfonamide molecular glues that modulate RNA splicing by degrading the splicing factor RBM39 via ternary complex formation with the E3 ligase adaptor DCAF15. To identify biomarkers of the antitumor efficacy of E7820, we treated patient-derived xenograft (PDX) mouse models established from 42 patients with solid tumors. The overall response rate was 38.1% (16 PDXs), and tumor regression was observed across various tumor types. Exome sequencing of the PDX genome revealed that loss-of-function mutations in genes of the homologous recombination repair (HRR) system, such as ATM, were significantly enriched in tumors that responded to E7820 (p = 4.5 × 103). Interestingly, E7820-mediated double-strand breaks in DNA were increased in tumors with BRCA2 dysfunction, and knockdown of BRCA1/2 transcripts or knockout of ATM, ATR, or BAP1 sensitized cancer cells to E7820. Transcriptomic analyses revealed that E7820 treatment resulted in the intron retention of mRNAs and decreased transcription, especially for HRR genes. This induced HRR malfunction probably leads to the synthetic lethality of tumor cells with homologous recombination deficiency (HRD). Furthermore, E7820, in combination with olaparib, exerted a synergistic effect, and E7820 was even effective in an olaparib-resistant cell line. In conclusion, HRD is a promising predictive biomarker of E7820 efficacy and has a high potential to improve the prognosis of patients with HRD-positive cancers.

Formation and non-covalent interactions of binary and ternary complexes based on β-casein, Lentinus edodes mycelia polysaccharide, and taxifolin

Polyphenols, polysaccharides, and proteins are essential nutrients and functional substances present in food, and when present together these components often interact with each other to influence their structure and function. Proteins and polysaccharides are also excellent carrier materials for polyphenols. In this context, this study investigated the non-covalent interactions between taxifolin (TAX), Lentinus edodes mycelia polysaccharide (LMP), and β-casein (β-CN). β-CN and LMP spontaneously formed nanocomplexes by hydrogen bonds and van der Waals forces. The quenching constant and binding constant were (1.94 ± 0.02) × 1013 L mol−1 s−1 and (3.22 ± 0.17) × 105 L mol−1 at 298 K, respectively. The altered conformation of β-CN, resulting from the binding to LMP, affected the interaction with TAX. LMP significantly enhanced the binding affinity of TAX and β-CN, but did not change the static quenching binding mode. The binding constant for β-CN-TAX was (3.96 ± 0.09) × 1013 L mol−1, and that for the interaction between TAX and β-CN-LMP was (32.06 ± 0.05) × 1013 L mol−1. In summary, β-CN-LMP nanocomplexes have great potential as a nanocarrier for polyphenols, and this study provides a theoretical foundation for the rational design of non-covalent complexes involving LMP and β-CN, both in binary and ternary configurations.

Luciferase- and HaloTag-based reporter assays to measure small-molecule-induced degradation pathway in living cells.

The rational development of small-molecule degraders (e.g., proteolysis targeting chimeras) remains a challenge as the rate-limiting steps that determine degrader efficiency are largely unknown. Standard methods in the field of targeted protein degradation mostly rely on classical, low-throughput endpoint assays such as western blots or quantitative proteomics. Here we applied NanoLuciferase- and HaloTag-based screening technologies to determine the kinetics and stability of small-molecule-induced ternary complex formation between a protein of interest and a selected E3 ligase. A collection of live-cell assays were designed to probe the most critical steps of the degradation process while minimizing the number of required expression constructs, making the proposed assay pipeline flexible and adaptable to the requirements of the users. This approach evaluates the underlying mechanism of selective target degraders and reveals the exact characteristics of the developed degrader molecules in living cells. The protocol allows scientists trained in basic cell culture and molecular biology to carry out small-molecule proximity-inducer screening via tracking of the ternary complex formation within 2 weeks of establishment, while degrader screening using the HiBiT system requires a CRISPR-Cas9 engineered cell line whose generation can take up to 3 months. After cell-line generation, degrader screening and validation can be carried out in high-throughput manner within days.

Abstract P05: Novel PROTAC-based EZH2 Degraders Effectively Target Lymphoid Malignancies by Depleting Enzyme-Independent Functions of EZH2

Abstract Overexpression or mutation of the epigenetic silencer EZH2 correlates with invasive cancer growth, resistance, and poor survival outcomes. Previously, we have established a methylation-independent role for EZH2 in the transcriptional activation of oncogenes in NKTL. As EZH2 enzymatic inhibitors have proven ineffective against NKTL, we embarked on the development of novel EZH2 Proteolysis Targeting Chimera (PROTAC) compounds. EZH2 PROTACs consist of an EZH2- and E3-ligase binding domain which promotes targeted ubiquitination of the EZH2 protein thereby marking it for proteasomal degradation. Our initial efforts focused on the iterative synthesis of EZH2 PROTAC candidates optimizing factors such as permeability, solubility and stable ternary complex formation. Through extensive screening, we identified C2911 and C1311, which successfully trigger the dose-dependent degradation of EZH2 and PRC2 components concomitant with a significant loss of survival and induction of apoptosis in both NKTL and DLBCL. These effects were rescued by pre-treatment with proteasome inhibitor Bortezomib or when E3 ligase activity was disrupted through siRNA knockdown. In NKTL, we show that EZH2 PROTAC treatment decreased JAK3 and phospho-STAT5 protein expression and identified the suppression of the JAK-STAT-MYC axis as a potential mechanism of action. EZH2 PROTAC treatment effectively reactivates transcription of canonical EZH2 target genes such as MYT1 and ANPEP and downregulates expression of oncogenes c-Myc and E2F1. Pharmacokinetic studies in vivo demonstrate that these PROTACs exhibit good plasma stability, half-life (>4h) and low clearance thereby establishing potential for future clinical development. The efficacy of EZH2 PROTACs in xenograft mice are currently being evaluated. Collectively, our data supports the discovery of novel EZH2 PROTACs and the critical role it plays in successfully targeting the oncogenic role of EZH2 in NKTL or other non-canonical EZH2-driven cancers through protein depletion where enzymatic-only inhibition was ineffective. We are further investigating the mechanisms stabilizing EZH2 protein levels to enhance PROTAC-mediated cytotoxicity. Citation Format: Nurulhuda Mustafa, Zubaida Talal Alnaseri, Darrel Tan Egk Jian, David Quach Thanh Truong, Wan Hsin Lim, Wee Joo Chng. Novel PROTAC-based EZH2 Degraders Effectively Target Lymphoid Malignancies by Depleting Enzyme-Independent Functions of EZH2 [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P05.

Magnetically recyclable mesoporous carbon/zirconia composites for highly efficient removal of Cr(III) organic complexes from high salinity water: adsorption behavior and mechanism

ABSTRACT In this study, zirconia-functionalized magnetic core-shell mesoporous carbon composites (Fe3O4@C-ZrO2) were synthesized for the effective removal of Cr(III)-EDTA in high-salinity wastewater. Fe3O4@C-ZrO2 with Fe3O4 as a magnetic core and ZrO2 embedded in a mesoporous carbon skeleton were prepared by the sol-gel method in the nano-casting stage, which can be magnetically separated. The presence of ZrO2 on the adsorbents enhanced the adsorption of Cr(III)-EDTA and the maximum adsorption capacity of Fe3O4@C-ZrO2 was 16.21 mg·g−1 at pH = 3.0, which is remarkably higher than that of Fe3O4@C (3.65 mg·g−1). Fe3O4@C-ZrO2 has a strong anti-interference capability for cations in water, which can exhibit effective adsorption properties in presence of high concentration cations and anions (80 mmol·L−1) and high salt/complexant agent environments. Cr(III)-EDTA removal by adsorption of Fe3O4@C-ZrO2 was mainly through the formation of ternary complexes between ZrO2 in Fe3O4@C-ZrO2 and carboxylic acid groups in Cr(III)-EDTA. The Cr(III)-EDTA saturated Fe3O4@C-ZrO2 can be easily regenerated in 0.1 mol·L−1 NaOH than in 0.1 mol·L−1 HCl and still demonstrated an effective adsorption capacity for Cr(III)-EDTA after three repeated cycles. The results reflect the effective removal of Cr(III)-EDTA by Fe3O4@C-ZrO2 in high-salinity water, providing a new method for removing heavy metals in complex water.

Drugs Form Ternary Complexes with Human Liver Fatty Acid Binding Protein 1 (FABP1) and FABP1 Binding Alters Drug Metabolism.

Liver fatty acid binding protein 1 (FABP1) binds diverse endogenous lipids and is highly expressed in the human liver. Binding to FABP1 alters the metabolism and homeostasis of endogenous lipids in the liver. Drugs have also been shown to bind to rat FABP1, but limited data are available for human FABP1 (hFABP1). FABP1 has a large binding pocket, and up to two fatty acids can bind to FABP1 simultaneously. We hypothesized that drug binding to hFABP1 results in formation of ternary complexes and that FABP1 binding alters drug metabolism. To test these hypotheses, native protein mass spectrometry (MS) and fluorescent 11-(dansylamino)undecanoic acid (DAUDA) displacement assays were used to characterize drug binding to hFABP1, and diclofenac oxidation by cytochrome P450 2C9 (CYP2C9) was studied in the presence and absence of hFABP1. DAUDA binding to hFABP1 involved high (Kd,1 = 0.2 μM) and low (Kd,2 > 10 μM) affinity binding sites. Nine drugs bound to hFABP1 with equilibrium dissociation constant (Kd) values ranging from 1 to 20 μM. None of the tested drugs completely displaced DAUDA from hFABP1, and fluorescence spectra showed evidence of ternary complex formation. Formation of DAUDA-hFABP1-diclofenac ternary complex was verified with native MS. Docking predicted diclofenac binding in the portal region of FABP1 with DAUDA in the binding cavity. The catalytic rate constant of diclofenac hydroxylation by CYP2C9 was decreased by ∼50% (P < 0.01) in the presence of FABP1. Together, these results suggest that drugs form ternary complexes with hFABP1 and that hFABP1 binding in the liver will alter drug metabolism and clearance. SIGNIFICANCE STATEMENT: Many commonly prescribed drugs bind fatty acid binding protein 1 (FABP1), forming ternary complexes with FABP1 and the fluorescent fatty acid 11-(dansylamino)undecanoic acid. These findings suggest that drugs will bind to apo-FABP1 and fatty acid-bound FABP1 in the human liver. The high expression of FABP1 in the liver, together with drug binding to FABP1, may alter drug disposition processes in vivo.

P53 upregulation by USP7-engaging molecular glues

Molecular glues are typically small chemical molecules that act at the interface between a target protein and degradation machinery to trigger ternary complex formation. Identifying molecular glues is challenging. There is a scarcity of target-specific upregulating molecular glues, which are highly anticipated for numerous targets, including P53. P53 is degraded in proteasomes through polyubiquitination by specific E3 ligases, whereas deubiquitinases (DUBs) remove polyubiquitination conjugates to counteract these E3 ligases. Thus, small-molecular glues that enhance P53 anchoring to DUBs may stabilize P53 through deubiquitination. Here, using small-molecule microarray-based technology and unbiased screening, we identified three potential molecular glues that may tether P53 to the DUB, USP7, and elevate the P53 level. Among the molecular glues, bromocriptine (BC) is an FDA-approved drug with the most robust effects. BC was further verified to increase P53 stability via the predicted molecular glue mechanism engaging USP7.Consistent with P53 upregulation in cancer cells, BC was shown to inhibit the proliferation of cancer cells in vitro and suppress tumor growth in a xenograft model. In summary, we established a potential screening platform and identified potential molecular glues upregulating P53. Similar strategies could be applied to the identification of other types of molecular glues that may benefit drug discovery and chemical biology studies.

Abstract 5777: Rewiring EWS/FLI1 with transcriptional chemical inducers of proximity (TCIPs)

Abstract The purpose of this study was to determine if EWS/FLI1 (E/F), the oncogenic fusion protein that drives Ewing Sarcoma (ES), can be rewired to enhance expression of BCL6-controlled genes using Transcriptional Chemical Inducers of Proximity (TCIPs), a novel bivalent small molecule platform. TCIP molecules recruit a transcriptional regulator, such as BRD4, to the transcriptional repressor BCL6. Upon binding, a ternary complex is formed on chromatin at BCL6-bound loci, causing transcriptional upregulation of BCL6 target genes. Since E/F does not have a bona fide ligand, we have used an exogenously expressed N terminal FKBPF36V-EWS/FLI1 (N-FK-E/F) fusion system to study how modulation of E/F by TCIPs impacts the transcriptome of ES cells. To eliminate background signal from endogenous E/F, we used CRISPR/Cas9 to knockout (KO) endogenous E/F. Our exogenously expressed N-FK-E/F has mutated PAM motifs and is resistant to KO. In our proof-of-concept study we used TCIP1 consisting of ortho-AP1867 (oAP), a synthetic ligand for FKBPF36V, linked to the BCL6 inhibitor BI3812 (BI) to rewire N-FK-E/F. We have also tested a negative control TCIP (TCIP NEG) that links oAP to a BI derivative that abolishes BCL6 binding. TCIP NEG has similar physiochemical properties to our active TCIP1; however, it cannot bind BCL6, serving as a negative control for ternary complex formation. TCIP1 rapidly and dose-dependently increases the RNA and protein expression of several BCL6 target genes, like p21, in multiple ES models that stably express N-FK-E/F. TCIP NEG does not increase the expression of BCL6 target genes in ES cells. TCIP1 induces ternary complex formation in ES cell lysates, and ternary complex formation is necessary for activity in ES cells. TCIP1 is specific and does not increase BCL6 target gene levels to the same extent in ES cells expressing a FKBP-GFP construct or in parental ES cells lacking N-FK-E/F. RNA sequencing of EWS502 N-FK-E/F expressing cells shows that TCIP1 treatment induces a larger increase in expression of BCL6 target gene RNA than BCL6 inhibition. Moreover, CUT&amp;RUN experiments in EWS502 N-FK-E/F expressing cells suggest TCIP1 leads to an accumulation of N-FK-E/F at the promoters of BCL6 target genes. Motif analysis showed that TCIP1 increases the likelihood of N-FK-E/F binding to BCL6 consensus sequences as wells as increases the likelihood of BCL6 binding to E/F recognition sequences. In future studies we will conduct RNA sequencing, CUT&amp;RUN, and other ‘omics’ approaches (e.g. DisP-seq) to compare TCIP1 to TCIP NEG in multiple cell lines at multiple concentrations and time points. Our data suggests that N-FK-E/F can be rewired to induce the expression of BCL6 target genes in ES. Since ES is driven solely by E/F, TCIPs may be promising next generation therapeutics. Our study provides a proof-of-concept that will be the foundation for the development of future TCIP molecules that recruit endogenous E/F once suitable ligands are developed. Citation Format: Michael J. Bond, Ryan P. Golden, Roman C. Sarott, Giulia DiGiovanni, Basel A. Karim, Briana Howard, Kenneth Ross, Nathanael S. Gray, Kimberly Stegmaier. Rewiring EWS/FLI1 with transcriptional chemical inducers of proximity (TCIPs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5777.

Abstract 6044: EPriL: A platform for the rapid identification of degraders of inhibitor-resistant kinases

Abstract Heterobifunctional kinase degraders (HKDs) are an important new class of therapeutic modalities, of which the first have recently entered clinical trials. HKDs are small molecules that comprise a kinase-of-interest binding moiety and an E3 ligase binding moiety connected via a spacer. HKDs are excellently suited to target kinases that have a scaffolding function, or that are resistant against conventional inhibitors. A HKD brings a kinase and an E3 ligase in proximity forming a ternary complex leading to ubiquitin-dependent catalytic degradation of the kinase of interest. This ternary complex formation is unique for each kinase which can make HKDs highly specific. To discover novel HKDs, we have developed a platform called Energetically Privileged Ligands (EPriLs), which is based on a macrocycle scaffold that binds non-covalently in the kinase ATP pocket with near-covalent affinity, and allows binding to kinases that have acquired known inhibitor resistance mutations. The macrocyclic structure allows attachment of E3 ligase ligands with multiple exit vectors. With the EPriL platform, we have developed novel HKDs for various clinically relevant kinase targets, including Bruton’s Tyrosine Kinase (BTK). BTK inhibitor drugs are used as first line treatment for chronic lymphocytic leukemia (CLL), Waldenström macroglobulinemia (WM) and as second line treatment for mantle cell lymphoma (MCL). Despite their great success in the clinic, many patients on BTK inhibitors relapse eventually, mostly because of the emergence of resistance mutations in the BTK protein. These include BTKC481S, which abrogates binding of covalent drugs, and BTKT474I, BTKL528W and BTKV416L which preclude binding of non-covalent inhibitors like pirtobrutinib. HKDs can combat resistance by degrading such BTK variants, and by removing the scaffold function of BTK. BTK degraders such as NX-5948 and NX-2127 are currently in clinical Phase 1. To show the applicability of the EPriL platform for the discovery of novel BTK degraders, we screened a library of more than 100 EPriL HKDs for biochemical binding to BTK, and tested their propensity to induce degradation of HiBiT-tagged BTKWT, BTKC481S and BTKT474I in HEK293 cell lines. The degrading activity of hits was verified through western blot on the BTK-dependent DLBCL cell line TMD8. The best degraders - based on potency and efficacy - were tested in cell proliferation assays on a panel of TMD8 BTK mutant cell lines. In these lines, endogenous BTK was mutated by CRISPR to BTKC481S, BTKT474I, BTKL528W or BTKV416L. Several BTK degraders had potent (&amp;lt; 20 nM IC50) antiproliferative activity on most TMD8 lines, including difficult-to-target variants such as BTKV416L and BTKL528W. The unique binding mode of the EPriL scaffold thus translates to broad activity on resistance variants, while its selectivity ensures minimal degradation of off-target tyrosine kinases as observed for other degraders. Citation Format: Michelle Muller, Jos de Man, Joost C. Uitdehaag, Freek van Cauter, Sander van Gemert, Milan Hoffmann, Yvonne G. van Mil, Winfried R. Mulder, Martine B. Prinsen, Jan Gerard Sterrenburg, Diep Vu, Joeri de Wit, Erik Ensing, Rogier C. Buijsman. EPriL: A platform for the rapid identification of degraders of inhibitor-resistant kinases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6044.

Abstract 4501: First reported PIM kinase degraders: Design, profiling &amp; optimization

Abstract The PIM serine/threonine kinases (PIM1/PIM2/PIM3) are downstream effectors of ABL, JAK2 and Flt-3 oncogenes and are required for tumorigenesis. Overexpression has been reported in hematological and solid tumors, myeloma, lymphoma, leukemia and adenocarcinoma. The first generation of PIM inhibitors to make it to the clinic, including SGI-1776, AZD1208 and PIM447, are known to be pan-PIM inhibitors, while the aim of this project was to identify novel &amp; selective PIM3 inhibitors. To this end, several Hit-finding approaches were employed to rapidly generate novel chemical matter, which were then assessed for their activity against PIM3 using a robust ADP-Glo™ assay. In parallel to this Hit-findings activity, we used AZD1208 scaffold as a starting point to generate PIM3 bifunctional protein degraders and designed a specific screening cascade in order to profile such modalities. The PIM degrader library, synthetized using Automated Robotics Lab, was characterized using ADP-Glo assays. It was then screened on our binding and activity kinase platforms, KINOMEscan® and KinaseProfiler™, and then fully profiled including ternary complex formation with biophysics and in vitro Safety Pharmacology Profiling Panels. One example is the AZD1208 derivate degrader, i.e. EC108154-1, which showed nanomolar range affinity for PIM3 and the E3-ligase CRBN-DDB1, and its affinity range was also confirmed with the ternary complex in SPR and MST-TRIC. This comprehensive screening cascade ensures an in-depth characterization and optimization of protein degraders, with the possibility of monitoring the ternary complex formation with biophysics methods. It is a good illustration of how this combination of tests can be instrumental in selecting the best degraders to progress to the next step of drug discovery. Citation Format: Celine Legros, Paul Ratcliffe, Vanessa Porkolab, Michele Modugno, Olivier Mirguet. First reported PIM kinase degraders: Design, profiling &amp; optimization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4501.

Abstract 2044: A live cell PRMT5 NanoBRET Target Engagement Assay querying competitive and uncompetitive modes of inhibition

Abstract PRMT5 is an essential arginine methyltransferase that regulates a wide spectrum of cellular processes through the methylation of histone and non-histone substrates. In PRMT5 catalysis, SAM serves as the cofactor and methyl group donor, generating a methylated guanidinium moiety on the target substrate. In normal cells, the SAM pools are maintained through the methionine salvage pathway. In 15% of cancers, a key enzyme in this pathway (MTAP) is deleted, leading to an accumulation of the intermediate MTA, which inhibits PRMT5 activity. This genetic loss of function has therefore been pursued as a collateral vulnerability in MTAP deleted cancers. To exploit this synthetic lethality, a number of novel inhibitors have been developed that bind cooperatively at the PRMT5/MTA complex, offering a compelling pathway to precision medicine. Here we describe a novel, live cell NanoBRET Target Engagement assay that enables mechanistic studies on a variety of PRMT5 inhibitors. Using a cell-permeable NanoBRET probe directed to the substrate pocket of PRMT5, both substrate- and cofactor-competitive engagement can be quantified in cells. Moreover, this method can be used to quantify MTA-uncompetitive target engagement in cells, providing a platform to measure the potency of PRMT5-MTA-drug ternary complex formation. This method serves as a first-in-class method to quantify uncompetitive target engagement in live cells, which can be applied to other model systems. Citation Format: Ani Michaud, Elisabeth Rothweiler, Cesear Corona, Michael Beck, Jennifer Wilkinson, James Vasta, Kilian Huber, Matthew Robers. A live cell PRMT5 NanoBRET Target Engagement Assay querying competitive and uncompetitive modes of inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2044.

Abstract 3309: Discovery of potent and selective bivalent CDK2 degraders that demonstrate activity in CCNE1amp driven tumors

Abstract Introduction: The Cyclin-Dependent Kinases (CDKs) with their cyclin binding partners, are associated with cell cycle progression and transcriptional regulation. Targeting CDKs is a key oncology therapeutic strategy with CDK4/6 inhibitors demonstrating significant clinical benefit in HR+/HER2− metastatic breast cancer, the most prevalent subtype. However, 30% of patients develop acquired resistance in the clinic with cyclin E1 (CCNE1) amplification/overexpression and CDK2 activation implicated as a major resistance mechanism to CDK4/6i breast cancer therapy. Additionally, CCNE1/CDK2 activation has also been associated with poor prognosis in ovarian and endometrial cancers. Selective targeting of CDK2 using small molecule inhibitor-based approaches have recently advanced into the clinic. Targeted protein degradation of CDK2 provides an alternative strategy that has the potential to eliminate the activity of the CCNE1/CDK2 complex, as well as CDK2 complexed with other cyclins, including cyclin A, and provide improved clinical benefit. Results: Discovery efforts at Plexium have identified CDK2 bivalent degraders that consist of a CDK2-binding moiety, a linker and a high affinity cereblon-binding ligand. Cereblon binding potency was found to correlate with potent and deep degradation of CDK2. Degradation was blocked in the presence of a proteasome inhibitor as well as in a cereblon knock-out cell line, confirming that CDK2 degradation is mediated by the ubiquitin proteasome system and through engaging cereblon. Proteome-wide analysis demonstrated that CDK2 was selectively depleted without significantly modulating other CDKs or known cereblon neo-substrates. Selective degradation was achieved despite lack of selectivity for binding/inhibition of other CDKs and was confirmed to be dependent on the formation of a CRBN-CDK2 ternary complex. Dose dependent CDK2 degradation resulted in dose dependent inhibition of Rb phosphorylation, cell cycle arrest, senescence-associated phenotypes and antiproliferative activity in CCNE1 amplified cancer cell lines. The in vitro data was used to evaluate the PK/PD drug exposure-response relationship in vivo and deep CDK2 degradation was demonstrated both in vitro and in vivo. Conclusions: These data provide validation for CDK2 degradation as a therapeutic approach. Potent and selective CDK2 bivalent degraders were exploited as tools for studying the sensitivity of CCNE1 amplified tumor models to CDK2 degradation and dependence on E3 ligase. This proof-of concept study supports Plexium’s current approach of discovering novel CDK2 molecular glue degraders for the treatment of CDK4/6 inhibitor-naïve and -resistant HR+/HER2− breast cancer, and CCNE1 amplified ovarian and endometrial cancers. Citation Format: Adina Gerson-Gurwitz, Pengyu Yang, Sarah Fish, Gabrielle Blanco, Hongfeng Gao, Kyohei Hayashi, Mike Hocker, Aleks Jamborcic, Andre Richters, Mary E. Spalding, Julia Toth, Duc Tran, Linette Yang, Shu You, Andrew Burritt, Alex Campos, Gregory Parker, Kevin Freeman-Cook, Peggy A. Thompson, Simon Bailey. Discovery of potent and selective bivalent CDK2 degraders that demonstrate activity in CCNE1amp driven tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3309.

Abstract 6567: Affinity and cooperativity modulate ternary complex formation to drive degradation of SMARCA2

Abstract Small molecules that hijack the ubiquitin-proteasome system to degrade target proteins have gained traction as tools for chemical biology and as promising clinical candidates. Targeted degradation strategies encompass relatively large hetero-bifunctional small molecules (hSMs) that are built from target and effector ligands joined by a linker to much smaller molecular glues that have limited affinity for the target or effector proteins outside of a ternary complex. There is good reason for medicinal chemists to move from linkered hSMs toward glue-like scaffolds. Molecular glues can be considerably smaller, e.g., the IMiDs, and thus are less likely to possess atoms and functional motifs that add up to poor permeability and higher protein binding. Guided by the principle that hSMs with reduced molecular weight will have superior druglike properties, we have used SMARCA2, a potential therapeutic target in patients that have both copies of SMARCA4 inactivated, as a test of our own ability to drive rapid target degradation in relevant models. The hetero-bifunctional degraders that resulted from our work trade conventional linkers for chemical spacers that orient SMARCA2 and VHL to promote fortuitous protein-protein contacts in the ternary complex. Following in the footsteps of previously published works, we employed surface plasmon resonance (SPR), x-ray crystallography, and molecular modeling to understand the relationships between our cell-based metrics of degrader activity and the biophysical parameters that describe the ternary complex. Consequently, we found that cooperativity (α) and ternary complex affinity (KLPT) correlated well with both DC50 and initial rate of degradation. Within our series of novel SMARCA2 degraders, compound 1 exhibited exceptional cooperativity and ternary complex affinity, α = 12.8 and KLPT = 4.7 ± 1. The result of high cooperativity and enhanced affinity was rapid and near complete SMARCA2 degradation. Treatment of a cancer cell line with compound 1 resulted in 50% of SMARCA2 being degraded in under 30 minutes, and greater than 90% degradation in 2 hours. Most importantly, the near complete degradation of SMARCA2 resulted in potent effects on SWI/SNF dependent transcription in vitro and in inhibition of tumor cell growth both in vitro and in vivo. Citation Format: Ivonne Archibeque, Ken Dellamaggiore, Karen Rex, Sudipa Ghimire-Rijal, Kaylee Choi, Smither Kate, Albert Amegadzie, Ning Chen, Xiaofen Li, Ryan Wurz, Amit Vaish, Paul Hughes, Dane Mohl. Affinity and cooperativity modulate ternary complex formation to drive degradation of SMARCA2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6567.

Abstract 6065: Identification and characterization of paralogue selective CBP degraders for potential use in cancer therapy

Abstract CBP and p300 are closely related epigenetic modulators that participate in chromatin remodeling and transcription and play an oncogenic role in a variety of cancers. Studies have indicated that the selective targeting of CBP is a highly attractive therapeutic strategy for cancers with p300 mutations because of synthetic lethality, those that are dependent on Wnt/β-catenin signaling and ERα and HER2-positive breast cancers. Prevalence of high mutation frequency of p300 in several cancers including skin cancers (27%), small cell lung cancers (8%), lymphoma (8-13%) and bladder cancer (9%) further highlight an opportunity for selective inactivation of CBP in cancer therapy. However, it has not been possible to identify selective inhibitors of p300 or CBP because of the conserved sequence and structure of functional domains such as HAT domain or bromodomain. We sought to achieve selective CBP targeting by utilizing the degrader approach, which in the recent time has proven to be useful in achieving paralog selectivity due to differentiated ternary complex formation. Additionally, the degrader approach offers several inherent advantages including stronger phenotype due to greater potency, sustained action due to catalytic mode of action, overcoming resistance and greater selectivity. Utilizing a combination of structure-guided modelling and iterative medicinal chemistry strategies, we identified first-in-class selective degraders targeting CBP with varying linker lengths and compositions. Our lead compounds exhibit remarkable bias in degrading CBP in cell lines harboring loss-of-function mutations in p300, while sparing wild-type cells. Selective CBP degraders disrupted β-catenin transactivation function in a dose-dependent manner and demonstrated good oral bioavailability and PK profile in preclinical species. Further optimization of potency, selectivity, ADME properties while evaluating in vivo efficacy in appropriate models and tolerability in higher species is ongoing towards identifying a development candidate. Citation Format: Saravanan Thiyagarajan, Chandrasekhar Abbineni, Krishna Chaitanya T, Achala Apte, Iram Khan Iqbal, Naveen Kumar R, Aravind A B, Avinash Kumar, Girish A R, Aishwarya B Kamath, Deepak Dattatray Mane, Gouri Raj A, Reshma Reghu, Subhasmita Mohapatra, Sivapriya Marappan, Suraj T Gore, Subhendu Mukherjee, Girish Daginakatte, Kavitha Nellore, Shekar Chelur, Murali Ramachandra, Susanta Samajdar. Identification and characterization of paralogue selective CBP degraders for potential use in cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6065.