Cellular Target Engagement Research Articles

Coupling cellular drug-target engagement to downstream pharmacology with CeTEAM

Cellular target engagement technologies enable quantification of intracellular drug binding; however, simultaneous assessment of drug-associated phenotypes has proven challenging. Here, we present cellular target engagement by accumulation of mutant as a platform that can concomitantly evaluate drug-target interactions and phenotypic responses using conditionally stabilized drug biosensors. We observe that drug-responsive proteotypes are prevalent among reported mutants of known drug targets. Compatible mutants appear to follow structural and biophysical logic that permits intra-protein and paralogous expansion of the biosensor pool. We then apply our method to uncouple target engagement from divergent cellular activities of MutT homolog 1 (MTH1) inhibitors, dissect Nudix hydrolase 15 (NUDT15)-associated thiopurine metabolism with the R139C pharmacogenetic variant, and profile the dynamics of poly(ADP-ribose) polymerase 1/2 (PARP1/2) binding and DNA trapping by PARP inhibitors (PARPi). Further, PARP1-derived biosensors facilitated high-throughput screening for PARP1 binders, as well as multimodal ex vivo analysis and non-invasive tracking of PARPi binding in live animals. This approach can facilitate holistic assessment of drug-target engagement by bridging drug binding events and their biological consequences.

Abstract 4135531: The Ubiquitin-Specific Protease 30 Inhibitor, MTX652, Attenuates Cardiac Dysfunction And Remodelling In a Murine Model Of Transverse Aortic Constriction

Introduction: Mitochondrial abnormalities are a characteristic of heart failure leading to energy deprivation and disease worsening. The mitochondrial deubiquitinating enzyme USP30, removes ubiquitin and represses PINK1/PARKIN-mediated mitophagy leading to accumulation of dysfunctional mitochondria. In this study we investigated the role of MTX652, a clinical stage USP30 inhibitor, in a mouse model of cardiac hypertrophy using transverse aortic constriction (TAC). Hypothesis: Inhibition of USP30 will promote mitophagy resulting in clearance of dysfunctional mitochondria and improved cellular health leading to cardioprotective effects. Aims: To determine if MTX652 can protect from TAC induced cardiac hypertrophy and remodelling. Methods: Male C57BL/6 mice, 8 weeks old, were subjected to sham surgeries or TAC using a 26G needle to induce cardiac pressure overload. MTX652 (1.5 or 5 mg/kg, n =12-13) or vehicle ( n =11) was administered orally, twice daily, starting 2 days post-surgery. Captopril (30 mg/kg/d, n =13) was also included as a positive control. After 4 weeks, echocardiography assessed left ventricle (LV) function and morphology. Cell based assays assessed MTX652 target engagement and TOM20 ubiquitination in vitro. Results: LV hypertrophy and wall thickening was observed in TAC animals. Treatment with MTX652 decreased systolic LV volume (1.5 mg/kg p<0.05 and 5 mg/kg p<0.01) and diameter (1.5 mg/kg p<0.01 and 5 mg/kg p<0.05,) demonstrating comparable effects to Captopril. A reduction in anterior wall thickness in diastole was also observed with MTX652 (5 mg/kg, p<0.01). Cardiac dysfunction was also evident in TAC animals with MTX652 showing improvements in both ejection fraction (1.5 mg/kg and 5 mg/kg, p<0.0001) and fractional shortening (1.5 mg/kg and 5 mg/kg, p<0.0001). Cellular target engagement for MTX652 was confirmed in HeLa-YFP-Parkin cells with an EC 50 of 2.7 nM ( n =2). Furthermore, the USP30 substrate TOM20 was ubiquitinated following MTX652 treatment in vitro (EC 50 =6.1 nM, n =4) providing evidence of mitophagy activation, a finding also confirmed in mouse heart tissue (MTX652, 5mg/kg). Conclusions: The USP30 inhibitor, MTX652 significantly protected mice from TAC induced cardiac hypertrophy and LV dysfunction. Furthermore, MTX652 potentiates ubiquitylation of TOM20 as a mechanism to promote mitophagy. These data suggest our clinical stage molecule could provide a novel therapeutic approach for the treatment of cardiac hypertrophy and heart failure.

From Virtual Screens to Cellular Target Engagement: New Small Molecule Ligands for the Immune Checkpoint LAG-3.

Herein, we performed a virtual screening study to discover new scaffolds for small molecule-based ligands of the immune checkpoint lymphocyte-activation gene 3 (LAG-3). Molecular dynamics (MD) simulations using the LAG-3 structure revealed two putative binding sites for small molecules: the antibody interface and the lipophilic canyon. A 3D pharmacophore screening resulted in the identification of potential ligands for these binding sites and afforded a library of 25 compounds. We then evaluated the screening hits for LAG-3 binding via microscale thermophoresis (MST) and surface plasmon resonance (SPR). Our biophysical screening identified two binders with K D values in the low micromolar range, compounds 3 (antibody interface) and 25 (lipophilic canyon). Furthermore, we investigated the ability of LAG-3 hits to engage LAG-3 on a cellular level using a cellular thermal shift assay (CETSA). In summary, compound 3 shows potential as a lead but is not yet a development candidate.

Structure-Based Optimization of a Series of Covalent, Cell Active Bfl-1 Inhibitors.

Bfl-1, a member of the Bcl-2 family of proteins, plays a crucial role in apoptosis regulation and has been implicated in cancer cell survival and resistance to venetoclax therapy. Due to the unique cysteine residue in the BH3 binding site, the development of covalent inhibitors targeting Bfl-1 represents a promising strategy for cancer treatment. Herein, the optimization of a covalent cellular tool from a lead-like hit using structure based design is described. Informed by a reversible X-ray fragment screen, the strategy to establish interactions with a key glutamic acid residue (Glu78) and optimize binding in a cryptic pocket led to a 1000-fold improvement in biochemical potency without increasing reactivity of the warhead. Compound (R,R,S)-26 has a kinact/KI of 4600 M-1 s-1, shows <1 μM caspase activation in a cellular assay and cellular target engagement, and has good physicochemical properties and a promising in vivo profile.

Development of new pyrazoles as class I HDAC inhibitors: Synthesis, molecular modeling, and biological characterization in leukemia cells.

Class I histone deacetylases (HDACs) are considered promising targets in current cancer research. To obtain subtype-selective and potent HDAC inhibitors, we used the aminobenzamide scaffold as the zinc-binding group and prepared new derivatives with a pyrazole ring as the linking group. The synthesized compounds were analyzed in vitro using an enzymatic assay against HDAC1, -2, and -3. Compounds 12b, 15b, and 15i were found to be potent HDAC1 inhibitors, also in comparison to the reference compounds entinostat and tacedinaline, with IC50 values of 0.93, 0.22, and 0.68 μM, respectively. The best compounds were measured for their cellular effect and target engagement in acute myeloid leukemia (AML) cells. In addition, we studied the interaction of the compounds with HDAC subtypes using docking and molecular dynamic simulations. In summary, we have developed a new chemotype of HDAC1 inhibitors that can be used for further structure-based optimization.

Development of SYK NanoBRET cellular target engagement assays for gain–of–function variants

Development of SYK NanoBRET cellular target engagement assays for gain–of–function variants

Development of Nurr1 agonists from amodiaquine by scaffold hopping and fragment growing

The neuroprotective transcription factor nuclear receptor-related 1 (Nurr1) has shown great promise as a therapeutic target in Parkinson’s and Alzheimer’s disease as well as multiple sclerosis but high-quality chemical tools for pharmacological target validation of Nurr1 are rare. We have employed the weak Nurr1 modulator amodiaquine (AQ) and AQ-derived fragments as templates to design a new Nurr1 agonist chemotype by scaffold hopping and fragment growing strategies. Systematic structural optimization of this scaffold yielded Nurr1 agonists with nanomolar potency and binding affinity. Comprehensive in vitro profiling revealed efficient cellular target engagement and compliance with the highest probe criteria. In human midbrain organoids bearing a Parkinson-driving LRRK2 mutation, a novel Nurr1 agonist rescued tyrosine hydroxylase expression highlighting the potential of the new Nurr1 modulator chemotype as lead and as a chemical tool for biological studies.

Development of SYK NanoBRET Cellular Target Engagement Assays for Gain-of-Function Variants.

Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase that is activated by phosphorylation events downstream of FcR, B-cell and T-cell receptors, integrins, and C-type lectin receptors. When the tandem Src homology 2 (SH2) domains of SYK bind to phosphorylated immunoreceptor tyrosine-based activation motifs (pITAMs) contained within these immunoreceptors, or when SYK is phosphorylated in interdomain regions A and B, SYK is activated. SYK gain-of-function (GoF) variants were previously identified in six patients that had higher levels of phosphorylated SYK and phosphorylated downstream proteins JNK and ERK. Furthermore, the increased SYK activation resulted in the clinical manifestation of immune dysregulation, organ inflammation, and a predisposition for lymphoma. The knowledge that the SYK GoF variants have enhanced activity was leveraged to develop a SYK NanoBRET cellular target engagement assay in intact live cells with constructs for the SYK GoF variants. Herein, we developed a potent SYK-targeted NanoBRET tracer using a SYK donated chemical probe, MRL-SYKi, that enabled a NanoBRET cellular target engagement assay for SYK GoF variants, SYK(S550Y), SYK(S550F), and SYK(P342T). We determined that ATP-competitive SYK inhibitors bind potently to these SYK variants in intact live cells. Additionally, we demonstrated that MRL-SYKi can effectively reduce the catalytic activity of SYK variants, and the phosphorylation levels of SYK(S550Y) in an epithelial cell line (SW480) stably expressing SYK(S550Y).

Potency-Enhanced Peptidomimetic VHL Ligands with Improved Oral Bioavailability.

The von Hippel-Lindau (VHL) protein plays a pivotal role in regulating the hypoxic stress response and has been extensively studied and utilized in the targeted protein degradation field, particularly in the context of bivalent degraders. In this study, we present a comprehensive peptidomimetic structure-activity relationship (SAR) approach, combined with cellular NanoBRET target engagement assays to enhance the existing VHL ligands. Through systematic modifications of the molecule, we identified the 1,2,3-triazole group as an optimal substitute of the left-hand side amide bond that yields 10-fold higher binding activity. Moreover, incorporating conformationally constrained alterations on the methylthiazole benzylamine moiety led to the development of highly potent VHL ligands with picomolar binding affinity and significantly improved oral bioavailability. We anticipate that our optimized VHL ligand, GNE7599, will serve as a valuable tool compound for investigating the VHL pathway and advancing the field of targeted protein degradation.

Unexpected Noncovalent Off-Target Activity of Clinical BTK Inhibitors Leads to Discovery of a Dual NUDT5/14 Antagonist.

Cofactor mimicry represents an attractive strategy for the development of enzyme inhibitors but can lead to off-target effects due to the evolutionary conservation of binding sites across the proteome. Here, we uncover the ADP-ribose (ADPr) hydrolase NUDT5 as an unexpected, noncovalent, off-target of clinical BTK inhibitors. Using a combination of biochemical, biophysical, and intact cell NanoBRET assays as well as X-ray crystallography, we confirm catalytic inhibition and cellular target engagement of NUDT5 and reveal an unusual binding mode that is independent of the reactive acrylamide warhead. Further investigation of the prototypical BTK inhibitor ibrutinib also revealed potent inhibition of the largely unstudied NUDIX hydrolase family member NUDT14. By exploring structure-activity relationships (SARs) around the core scaffold, we identify a potent, noncovalent, and cell-active dual NUDT5/14 inhibitor. Cocrystallization experiments yielded new insights into the NUDT14 hydrolase active site architecture and inhibitor binding, thus providing a basis for future chemical probe design.

Abstract ND06: First disclosure of AZD8421, a highly selective CDK2 inhibitor to address resistance to CDK4/6 inhibitors in breast and CCNE1-high cancers

Abstract Cyclin-dependent kinases (CDKs) are an attractive class of cancer targets due to their role in cell-cycle regulation. CDK2 is a Ser/Thr kinase activated via interaction with its cyclin partners CCNE and CCNA, driving G1/S progression of the cell cycle. CDK2 inhibition has the potential to address multiple resistance mechanisms to CDK4/6 inhibitors in breast cancer. In addition, CCNE1 is frequently amplified and over-expressed in cancers including ovarian, with pre-clinical data linking CDK2 inhibition sensitivity to high CCNE1. First-generation inhibitors targeting CDK2 suffered from poor tolerability in the clinic likely due to off-target activities across both the CDK family and across the protein kinome. As well as achieving CDK family selectivity in cells versus key off-targets (CDK1, CDK4/6, CDK9), it’s noteworthy that AZD8421 had no significant kinase inhibition outside the CDK family. X-ray co-crystal structures of AZD8421 with CDK2 provided the basis for the observed selectivity among the CDK-family. For example, hydrogen bonding interaction of AZD8421 with Lys89 (a CDK2-specific residue) near the solvent region of the ATP-binding pocket was the key requisite for achieving &amp;gt;300-fold selectivity over CDK9. In nanoBRET assays measuring cellular target engagement, AZD8421 had an IC50 against CDK2 of 9nM with selectivity over CDK1, CDK4 and CDK6. Further kinetic characterization of AZD8421 binding revealed a long residence time at CDK2 (2 hours) but not CDK1 (7 minutes). These kinetic studies showed that AZD8421 has a low Kd,app for CDK2 and thus even higher selectivity vs CDK1 than was measured by single timepoint assays. In a CCNE1 amplified cell line, AZD8421 potently inhibited cell proliferation (69nM, OVCAR3), correlated with inhibition of pRB, arrest in G1/S phase of the cell cycle and induction of senescence. In vitro combination assays in CDK4/6 inhibitor resistant breast cancer cell lines showed combination benefit with AZD8421 plus approved CDK4/6 inhibitors. In vivo, AZD8421 potently suppressed phosphorylation of Rb, and demonstrated robust monotherapy and CDK4/6i combination activity in breast and ovarian in vivo models. In vivo PD marker suppression and efficacy was demonstrated in CDK4/6 inhibitor resistant breast PDXs in combination with palbociclib (CDK4/6i). AZD8421 showed robust monotherapy activity in a CCNE1 amplified ovarian model OVCAR3 with regressions seen with monotherapy and in combination with palbociclib. These data, along with suitable physical and pharmacokinetic properties, supported progression of AZD8421 into clinical studies [NCT06188520]. Citation Format: Christopher R. Denz, Michael Grondine, Jun Fan, Jessie Hao-Ru Hsu, Anne Jackson, James Robinson, Grace Guo, Wen Li, Yanjun Wang, Maryann San Martin, Laura Prickett, Jeffrey Johannes, Avipsa Ghosh, Kun Song, Dhivya Sudhan, Christina Vasalou, Ryan Richards, Kevin Beaumont, Matthew Peters, Lisa Drew, Stephen Fawell, Frederick W. Goldberg. First disclosure of AZD8421, a highly selective CDK2 inhibitor to address resistance to CDK4/6 inhibitors in breast and CCNE1-high cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr ND06.

Abstract 4507: Discovery of a smart molecular glue: A small-molecular compound selectively degrades Nrf2 in KEAP1-mutated tumor cells, by restoring the broken KEAP1 mutant/Nrf2 interaction

Abstract Overcoming therapeutic resistance without incurring prohibitive normal tissue toxicity is a great challenge in anticancer therapies. Transcription factor NRF2 is a master regulator of cellular protective response, which provides protective effects in normal tissues. KEAP1, the endogenous NRF2 inhibitor, binds NRF2 and redirect it towards proteasome-dependent degradation. KEAP1/NRF2 interaction is therefore critical for maintaining NRF2 at a basal level. Accumulated studies revealed cancer cells could hijack NRF2 pathway to confer drug resistance. A number of clinically-relevant KEAP1 mutations were shown to disrupt the KEAP1/NRF2 interaction, leading to elevated NRF2 level and confer drug resistance. Here, by structure-based drug design approach, we discovered a small-molecule NRF2 inhibitor, R16, which selectively binds KEAP1 mutants and restores their NRF2-inhibitory function in tumor cells. We performed in silico screening against the NCI-open database. Top-ranking candidates were initially evaluated by ARE-luc reporter assays, leading to discovery of R16. Effect of R16 in restoring interaction between KEAP1 mutants and NRF2 were evaluated by BRET2 assays. Among 14 KEAP1 mutations that result in disruption of KEAP1/NRF2 interaction, R16 is active against 11 of them. A variety of assays, including tryptophan quenching, cellular target engagement, BRET2 and GST-pull down assays, indicated that R16 selectively engage KEAP1 mutants and restore their interaction with NRF2, leading to NRF2 degradation. R16 at 0.5 uM substantially sensitizes KEAP1-mutated tumor cells to cisplatin and gefitinib, with no obvious effect in wild-type KEAP1 cells. Importantly, R16 showed significant in-vivo efficacy in sensitizing A549 xenograft bearing KEAP1 G333C mutation to cisplatin, while it has no effect on xenograft with wild-type KEAP1. KEAP1-mutated tumors are emerging as a sub-group with severe resistance to currently available therapies. Here, we have identified a KEAP1 mutant-selective NRF2 inhibitor, which potently sensitizes KEAP1-mutated cells/tumors to anticancer agents, with little effects on the WT-KEAP1. Our work demonstrated it is feasible to restore the NRF2-inhibitory function of KEAP1 mutants, by targeting the mutants with compounds to repair the disrupted KEAP1 mutant/NRF2 interactions. The KEAP1 mutant-selectivity is critical, as NRF2 is a master regulator of cellular defense response in WT-KEAP1 normal tissues. Citation Format: Xiaohong Tian, Tahar Aboulkassim, Qiang Liu, Mark Hancock, Jian Hui Wu, Gerald Batist. Discovery of a smart molecular glue: A small-molecular compound selectively degrades Nrf2 in KEAP1-mutated tumor cells, by restoring the broken KEAP1 mutant/Nrf2 interaction [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 4507.

Abstract 677: Discovery of a first-in-class allosteric inhibitor of the small GTPase Rac1

Abstract The Rac1 signaling pathway is known to be hyperactivated in numerous solid tumors. We developed a Rac1 AlphaLISA assay that is amenable to high-throughput screening (HTS) to screen large libraries of small molecules. Based on molecular modeling of a putative allosteric binding site for several of the hits, we optimized a series of compounds that lead to RP-102124. RP-102124 engages with Rac1 in both biochemical and cellular target engagement assays. RP-102124 has greater potency than previously reported Rac1 inhibitors in biochemical assays, with potent inhibition of phenotypic characteristics of Rac signaling such as cell migration and cytoskeletal rearrangements. RP-102124 is a Racmulti inhibitor with activity against oncogenic Rac1b. RP-102124 is orally bioavailable and shows dose dependent inhibition of tumor growth in xenograft models at doses that are well-tolerated. RP-102124 is a first-in-class allosteric Racmulti inhibitor that shows promising therapeutic potential. Citation Format: Robert Silverman, Alan Cooper, Ronodip Kar, Mayuri V. Raman, Ramu Ravirala, Mubarak Hasan Shah, Sandeep Mahankali, Erik T. Goka. Discovery of a first-in-class allosteric inhibitor of the small GTPase Rac1 [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 677.

Abstract 2655: Unexpected role of the NuRD component, Chd4, in Foxp3+ Treg cells and its relevance to tumor immunotherapy

Abstract We are investigating basic questions like why do Foxp3+ T-regulatory (Treg) cells have at least 3 nuclear complexes involving Hdac1 and Hdac2 (CoREST, Sin3, NuRD), and how can the inhibitory effects of Foxp3+ Tregs on host anti-tumor immune responses be targeted therapeutically? Here, we report on the immune functions of chromodomain-helicase-DNA-binding protein 4 (Chd4), a key enzyme of the nucleosome remodeling and deacetylase complex (NuRD) complex. TCGA analysis showed increased Chd4 expression and Foxp3+ Tregs within lung tumors, leading us to assess the effects of conditional gene deletion within Foxp3+ murine Tregs. Chd4 knockout led to systemic autoimmunity and death of mice within 3 weeks of life and RNAseq studies showed Chd4 loss led to derepression of ~1400 genes and repression of &amp;gt;700 further genes resulting in a Treg signature markedly different from conditional deletion of Hdac1, Hdac2 or Mbd2 in Treg cells. Chd4 is an intrinsically disordered therapeutic target without a defined structure outside its physiological cell environment. To date, no specific inhibitors targeting Chd4 have been developed. We have identified Ch41, a novel and potent inhibitor of Chd4, using a novel cellular target engagement platform. Transcriptomic and mass-spec investigations showed that Ch41 impaired thymic Treg function and iTreg development, led to decreased CNS2 demethylation within the Foxp3 locus, and significantly impaired growth of lung tumors and hepatocellular carcinomas in immunocompetent but not in immunodeficient C57BL/6 mice (p&amp;lt;0.01). Following Chd4 inhibitor therapy, intratumoral conventional T cells had increased activation and cytokine production (IL-2, IFN-g) compared to intratumoral T cells in control tumor-bearing mice but, perhaps unexpectedly, treated mice did not show histologic or biochemical evidence of autoimmunity. We conclude that while tumor associated Tregs have unique properties that include their increased suppressive activity compared to non-tumor associated Treg cells, they are also more susceptible than the latter and conventional T cells to newly developed therapeutic interventions, including targeting of the Chd4 chromodomain of the NuRD complex. Citation Format: Wayne W. Hancock, Liqing Wang, Martina Minisini, Eros di Giorgio, Ivan Babic, Elmar Nurmemmedov. Unexpected role of the NuRD component, Chd4, in Foxp3+ Treg cells and its relevance to tumor immunotherapy [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 2655.

Abstract 3098: CETSA for navigating your chemistry and exploring the biology of protein degraders

Abstract Protein degradation is a non-classical modality with the potential to tackle therapeutically interesting proteins, previously deemed undruggable. In this area, CETSA is a powerful technology for identifying new binders, assessing target engagement, and investigating the mechanism of action of the degraders in the intact cell environment - without the need to modify your compound, the proteins or the cellular environment. CETSA can provide insights into the binding of the degrader to both the E3 ligase and the protein of interest (POI), as well as follow the downstream cellular and molecular effects. Here we will exemplify how CETSA can be applied in various stages of drug discovery to provide biologically relevant data to guide the design and optimization of new degraders. For example, CETSA can provide valuable information of the cell permeability and the in-cell performance of the degrader, which is important for understanding how the degrader enters cells and reaches its target proteins. By combining data from CETSA with data from degradation readout, in our case quantitative proteomics, it is possible to correlate cellular target engagement potencies with degradation efficiency and importantly, also identify any drug:protein binding that does not result in degradation By using the proteome-wide CETSA Explore format, the selectivity of the degrader and the downstream protein consequences of the elimination of the target protein could be studied. Citation Format: Victoria Brehmer, Stina Lundgren, Tomas Friman, Daniele Amadio, Alexey Chernobrovkin, Daniel Martinez Molina. CETSA for navigating your chemistry and exploring the biology of protein degraders [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 3098.

Abstract 6011: Pharmacological inhibition of PMS2 increases tumor mutational burden, induces microsatellite instability and elicits immune mediated rejection in vivo

Abstract Tumors carrying defects in DNA mismatch repair (MMR-d) display high tumor mutational burden (TMB) and increased tumor neoantigen levels. MMR-d leads to accumulation of single nucleotide variants (SNVs) and insertions or deletions (indels) amongst repetitive DNA sequences generating microsatellite instability (MSI). Cancers that harbor &amp;gt;40% microsatellite variations are described as MSI-high (MSI-H). These tumors display unique clinical features including better prognosis, increased immune infiltration and remarkable response to immune checkpoint blockade (ICB) therapy. Colorectal cancer (CRC) patients with MMR-d often respond to therapies based on immune checkpoint blockade, whilst those with mismatch repair proficient (MMR-p) tumors do not. Therefore, identifying strategies to enhance the efficacy of immunotherapeutic treatments and transform this prevalent subgroup of CRCs from immunologically cold to hot remains an urgent and an unmet clinical need. We discovered NP1867 a first-in-class, potent, selective, covalent small molecule inhibitor of the DNA Mismatch Repair protein PMS2. We show by biochemical, biophysical, and protein-ligand X-ray crystallographic methods that NP1867 binds irreversibly to the ATP binding site of the N-terminal ATPase domain of PMS2. NP1867 displays potent binding to PMS2 in a NanoBRET cellular target engagement assay and inhibits MMR activity in mechanistic and functional cell-based assays of MMR-dependent DNA repair. Continuous treatment of murine CRC cell line CT26 (MMR-p) with NP1867 increases TMB, specifically enriches gained mutations with typical MMR-d related COSMIC mutational signatures, and elicits MSI-H status in a time-dependent manner, demonstrating for the first time that inhibition of PMS2 with a small molecule phenocopies the inactivation of MMR observed in patients. Remarkably, inoculation of immune-competent mice with CT26 cells pre-treated with NP1867 elicits tumor regressions in anti-PD1-treated animals, recapitulating the unique clinical features of MMR-d/MSI-H tumors. Conversely, no response is observed in immune-competent animals inoculated with control-treated cells. The extent of in vivo response correlates with the duration of in vitro NP1867 treatment and with the MSI-H status of treated CT26 cells. In conclusion, small molecule NP1867 functionally inhibits DNA MMR by targeting PMS2, enriches gained mutations with MMR-d COSMIC signatures, elicits MSI-H status, and enhances immune surveillance in preclinical models. These findings pave the way for the development of orally bioavailable compounds suitable for long-term dosing in animal models. Citation Format: Eleonora Piumatti, Alexia Hervieu, Philippe Riou, Julian Blagg, Adam Peall, Sam Weeks, Maria T. Rodríguez-Plata, Giuseppe Rospo, Sasi Arunachalam, Bettina Meier, Paige Tongue, Tessa McLaren, Kalpesh Parmar, Pradip Patel, David Clark, Gareth Langley, Charles Nichols, Benoît Rousseau, Paul Winship, Matthew Baker, Martin Drysdale, Giovanni Germano, Alberto Bardelli. Pharmacological inhibition of PMS2 increases tumor mutational burden, induces microsatellite instability and elicits immune mediated rejection in vivo [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 6011.

Abstract 5730: Targeting the cell cycle with AZD8421, a potent and highly selective CDK2 inhibitor

Abstract Cyclin-dependent Kinase 2 (CDK2) is a Ser/Thr kinase activated via interaction with its cyclin partners CCNE and CCNA, driving G1/S progression of the cell cycle. CDK2 inhibition has the potential to address multiple resistance mechanisms to CDK4/6 inhibitors in breast cancer. In addition, CCNE1 is frequently amplified and over-expressed in cancers including ovarian, uterine, breast, gastric and others, with pre-clinical data linking CDK2 inhibition sensitivity to high CCNE1. The CDK2 inhibitor field has historically suffered from high attrition in Phase I due to off-target toxicity. AZD8421 is a uniquely selective CDK2 inhibitor, that offers potential to show improved therapeutic index and combinability. As well as achieving CDK family selectivity in cells versus key off-targets (CDK1, CDK4/6, CDK9), AZD8421 had no significant kinase inhibition outside the CDK family. Only 7 distinct targets showed greater than 50% inhibition in a panel of 403 kinases when screened with 1µM of AZD8421. In nanoBRET assays measuring cellular target engagement, AZD8421 had an IC50 against CDK2 of 9nM with selectivity over CDK1, CDK4 and CDK6. In vitro cellular assays demonstrated that AZD8421 inhibits endogenous phospho-substrate at 58nM with &amp;gt;327-fold selectivity over CDK9 phospho-substrate pSer2 of RNAPII (&amp;gt;19.2 µM). In a CCNE1 amplified cell line, AZD8421 potently inhibited cell proliferation (69nM, OVCAR3), correlated with inhibition of pRB, arrest in G1/S phase of the cell cycle and induction of senescence, compared to a CCNE1 non-amplified cell line (SKOV3) with IC50 of 2.05 µM. In vitro combination assays in CDK4/6 inhibitor resistant breast cancer cell lines showed combination benefit with AZD8421 plus approved CDK4/6 inhibitors. In vivo, AZD8421 potently suppressed phosphorylation of Rb, and demonstrated robust monotherapy and CDK4/6i combination activity in breast and ovarian in vivo models. In vivo PD marker suppression and efficacy was demonstrated in CDK4/6 inhibitor resistant breast PDXs in combination with palbociclib (CDK4/6i). AZD8421 showed robust monotherapy activity in a CCNE1 amplified ovarian model OVCAR3 with regressions seen with monotherapy and in combination with palbociclib. Monotherapy activity and combination benefit in vivo with standard of care agents was also observed in ovarian PDX models with elevated levels of CCNE1. AZD8421 is a potent and highly selective CDK2 inhibitor, with double digit nM potency in cells, as well as suitable physical and pharmacokinetic properties for progression into the clinic. Citation Format: Christopher R. Denz, Michael Grondine, Jun Fan, Jessie Hao-Ru Hsu, Anne Jackson, James Robinson, Grace Guo, Wen Li, Yanjun Wang, Maryann San Martin, Laura Prickett, Jeffrey Johannes, Avipsa Ghosh, Dhivya Sudhan, Christina Vasalou, Ryan Richards, Kevin Beaumont, Matt Peters, Lisa Drew, Stephen Fawell, Frederick W. Goldberg. Targeting the cell cycle with AZD8421, a potent and highly selective CDK2 inhibitor [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 5730.

Abstract 5976: R9: A novel NuRD complex inhibitor for targeted therapy in triple negative breast cancer (TNBC)

Abstract Triple Negative Breast Cancer (TNBC) is a highly aggressive breast cancer subtype, disproportionately affecting young and African American populations. TNBC constitutes up to 15% of breast cancers that lack effective targeted therapy. The Nucleosome Remodeling and Deacetylase (NuRD) complex, a pivotal chromatin remodeling entity, plays a crucial role in cancer development and progression. Specifically, the NuRD complex associates with SALL4 in cancer cells, contributing to the silencing of tumor-suppressor genes like PTEN. In this study, we discover R9, a novel small-molecule nucleotide analog, a first-in-class NuRD inhibitor. Utilizing computational and cellular methods, R9 demonstrated selective engagement with RBBP4, a key NuRD complex subunit. Cellular target engagement technology has been utilized to verify direct interaction of R9 with NuRD complex as well as to guide medicinal chemistry efforts. Mass spectrometry thermal proteome profiling (TPP) affirmed RBBP4 as the primary target of R9 with minimal off-target effects. In vitro studies reveal R9 displaying robust toxicity in TNBC cells, while sparing normal tissue derived cells. Encouragingly, R9 effectively impeded TNBC tumors growth in mice without inducing toxicity. Mechanistically, we demonstrate R9 induces DNA damage in sensitive TNBC cells, and the sensitivity correlates to DNA damage-vulnerable cancer cells, indicating DNA damage is a major effector of NuRD complex inhibition in TNBCs. We further demonstrate that R9 induces DNA damage through a c-MYC dependent manner. Ongoing studies aim to elucidate key transcription factors involved in NuRD complex inhibition and the role of MYC and DNA damage in response to NuRD complex inhibition. This research positions R9 as a promising candidate for targeted therapy in TNBC, offering potential insights into the intricate molecular pathways involved in TNBC tumor progression. Citation Format: Kun Zhang, Sheeba Jacob, Mikhail Dozmorov, Ivan Babic, Elmar Nurmemmedov, Anthony C. Faber. R9: A novel NuRD complex inhibitor for targeted therapy in triple negative breast cancer (TNBC) [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 5976.

Abstract 5782: Probing monomer - dimer and conformational transitions of PLK1 using catalytic and polo-box domain inhibitors

Abstract Polo Like Kinase 1 (PLK1), a sought-after target for drug discovery, is an essential mitotic kinase consisting of a catalytic domain (KD) and a polobox domain (PBD), the latter of which plays roles in substrate recognition, subcellular localization and further acting as an autoinhibitory regulatory domain. PLK1 can exist either in an inhibited closed conformation or an open active state allowing interaction with and phosphorylation of substrates. PLK1 has also been shown to be regulated by interchange between monomeric and dimeric forms through binding to the Bora protein and by activation loop phosphorylation. We have described PBD-binding molecules termed abbapolins that inhibit cellular phosphorylation of a PLK1 substrate and induce loss of intracellular PLK1. In addition, due to their engagement of the cryptic pocket (ligand induced) of the PBD, we proposed that abbapolins would interfere with formation of PLK1 dimers. Here we provide evidence further supporting the ability of abbapolins to block PLK1 cellular dimerization and that this is related to their mechanism of anti-proliferative activity. In a separate study, using a cellular thermal shift assay, KD inhibitors were shown to decrease soluble PLK1, indicating that catalytic-site binding results in less stable PLK1 suggestive of an open conformation. We have extended these observations using a sensitive readout of cellular target engagement employing a Microtag overexpressed PLK1. In this format, we observe heat-induced destabilization of PLK1 upon nanomolar engagement by two lead KD inhibitors, BI2536 and onvansertib, currently being clinically evaluated. Furthermore, cyclapolin 9, a low molecular weight kinase inhibitor that, unlike the other two KD inhibitors does not protrude from the active site, did not induce significant destabilization. We have also carried out experiments using a fluorescently labeled version of BI2536 to probe the conformational effects of PBD binding upon the KD. These data solidify the observations that relief of autoinhibited PLK1 is induced by certain KD binders and highlight the conformational perturbations from KD versus PBD binding. These observations have implications for the development of ATP-competitive PLK1 inhibitors because catalytic inhibitors may conversely promote PLK1 non-catalytic functions, which may explain their lack of clinical efficacy to date. Furthermore, the demonstration that the abbapolins are mechanistically unique in terms of blocking the dimerization of PLK through the PBD provides impetus for their development as next generation PLK1 inhibitors. Citation Format: Mourad Sanhaji, Monika Raab, George Merhej, Chintada Nageswara Rao, Ivan Babic, Elmar Nurmemmedov, Klaus Strebhardt, MIchael Wyatt, Campbell McInnes. Probing monomer - dimer and conformational transitions of PLK1 using catalytic and polo-box domain inhibitors [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 5782.

Abstract 3092: MICRO-TAG: A novel fluorescence-based real-time cellular target engagement platform for drug discovery

Abstract Success of drug discovery programs can be amplified by adopting emerging technologies that can interrogate drug targets within their physiological context. Cell target engagement (CTE) presents a combined biological &amp; biophysical method, which provides quantitative evaluation of target-drug interaction within the native environment of the cell. The currently available luminescence-based CTE methods are limited to measurement of end-point signal and poor resolution, thereby limiting their mechanistic and stoichiometric insight. We have developed MICRO-TAG, a novel fluorescence-based CTE platform, which relies on complementation of split-RNAse A. It enables interrogation of target engagement without interfering with folding, localization and function of target proteins - all within the physiological milieu of the cell. Uniquely, this new platform allows for quantitation and monitoring of CTE in real- time in the cell. It enables extraction of important parameters from target-drug interaction: target thermal profile and temperature of aggregation (Tagg), time required to reach equilibrium (teq), apparent association rate (Kon) and physiologically relevant drug:target binding affinity (KD). We provide data demonstrating potential and utility of MICRO-TAG platform on drug targets, such as KRAS, MTH1, EGFR, and UBE2N. The platform demonstrates relevance for discovery of novel drug candidates targeting challenging drug targets such as transcription factors. This highly scalable and quantitative novel method for assessing drug-target interaction within cells can enhance drug discovery programs, and offers potential to rapidly identify clinically relevant hit compounds for challenging drug targets. Citation Format: Ivan Babic, Nikolas Bryan, Claire Cunningham, Avery Sampson, Daniel Starczynowski, Elmar Nurmemmedov. MICRO-TAG: A novel fluorescence-based real-time cellular target engagement platform for drug discovery [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 3092.