Cellular Thermal Shift Assay Research Articles

A novel α,β-unsaturated ketone inhibits leukemia cell growth as PARP1 inhibitor.

Leukemia is a malignant disease of the hematopoietic system, in which clonal leukemia cells accumulate and inhibit normal hematopoiesis in the bone marrow and other hematopoietic tissues as a result of uncontrolled proliferation and impaired apoptosis, among other mechanisms. In this study, the anti-leukemic effect of a compound (SGP-17-S) extracted from Chloranthus multistachys, a plant with anti-inflammatory, antibacterial and anti-tumor effects, was evaluated. The effect of SGP-17-S on the viability of leukemic cell was demonstrated by MTT assay, cell cycle, and apoptosis were assessed by flow cytometry using PI staining and Annexin V/PI double staining. Combinations of network pharmacology and cellular thermal shift assay (CETSA) with western blot were used to validate agents that act on leukemia targets. The results showed that SGP-17-S inhibited the growth of leukemia cells in a time- and dose-dependent manner. SGP-17-S blocked HEL cells in the G2 phase, induced apoptosis, decreased Bcl-2 and caspase-8 protein expression, and increased Bax and caspase-3 expression. In addition, CETSA revealed that PARP1 is an important target gene for the inhibition of HEL cell growth, and SGP-17-S exerted its action on leukemia cells by targeting PARP1. Therefore, this study might provide new solutions and ideas for the treatment of leukemia.

Synthesis, Anti-Inflammatory Activities, and Molecular Docking Study of Novel Pyxinol Derivatives as Inhibitors of NF-κB Activation

Pyxinol, an active metabolite of ginsenosides in human hepatocytes, exhibits various pharmacological activities. Here, a series of C-3 modified pyxinol derivatives was designed and virtually screened by molecular docking with the key inflammation-related proteins of the nuclear factor kappa B (NF-κB) pathway. Some of the novel derivatives were synthesized to assess their effects in inhibiting the production of nitric oxide (NO) and mitochondrial reactive oxygen species (MtROS) in lipopolysaccharide-triggered RAW264.7 cells. Derivative 2c exhibited the highest NO and MtROS inhibitory activities with low cytotoxicity. Furthermore, 2c decreased the protein levels of interleukin 1β, tumor necrosis factor α, inducible nitric oxide synthase, and cyclooxygenase 2 and suppressed the activation of NF-κB signaling. Cellular thermal shift assays indicated that 2c could directly bind with p65 and p50 in situ. Molecular docking revealed that 2c’s binding to the p65–p50 heterodimer and p50 homodimer was close to their DNA binding sites. In summary, pyxinol derivatives possess potential for development as NF-κB inhibitors.

Discovery of ganoderic acid A (GAA) PROTACs as MDM2 protein degraders for the treatment of breast cancer

Breast cancer is one of the most common female malignant tumors, with triple-negative breast cancer (TNBC) being the most specific, highly invasive, metastatic and associated with a poor prognosis. Our previous study showed that the natural product ganoderic acid A (GAA) has a certain affinity for MDM2. In this study, two series of novel GAA PROTACs C1–C10 and V1–V10 were designed and synthesized for the treatment of breast cancer. The antitumor activity of these compounds was evaluated against four human tumor cell lines (MCF-7, MDA-MB-231, SJSA-1, and HepG2). Among them, V9 and V10 showed stronger anti-proliferative effects against breast cancer cells, and V10 showed the best selectivity in MDA-MB-231 cells (TNBC), which was 5-fold higher than that of the lead compound GAA. Preliminary structure-activity analysis revealed that V-series GAA PROTACs had better effects than C-series, and the introduction of 2O–4O PEG linkers could significantly improve the antitumor activity. Molecular docking, surface plasmon resonance (SPR), cellular thermal shift assay (CETSA), and Western blot researches showed that both V9 and V10 could bind with MDM2, and degrade the protein through the ubiquitin-proteasome system. Molecular dynamics simulation (MD) revealed that V10 is a bifunctional molecule that can bind to von Hippel-Lindau (VHL) at one end and target MDM2 at the other. In addition, V10 promoted the upregulation of p21 in p53-mutant MDA-MB-231 cells, and induced apoptosis via down-regulation of the bcl-2/bax ratio and the expression of cyclin B1. Finally, in vivo experiments showed that, V10 also exhibited good tumor inhibitory activity in xenografted TNBC zebrafish models, with an inhibition rate of 27.2% at 50 μg/mL. In conclusion, our results suggested that V10 has anti-tumor effects on p53-mutant breast cancer in vitro and in vivo, and may be used as a novel lead compound for the future development of TNBC.

Gastrodin Attenuates Colitis and Prevents Tumorigenesis in Mice by Interrupting TLR4/MD2/NF-ΚB Signaling Transduction.

Chronic inflammation is one of the causative factors for tumorigenesis. Gastrodin is a main active ingredient isolated from Gastrodia elata Blume, a famous medicinal herb with a long edible history. This study aimed to explore the effects of gastrodin on colitis-associated carcinogenesis (CRC) in mice and to elucidate its potential molecular mechanisms. Balb/c mice were induced with azoxymethane (AOM) and dextran sulfate sodium (DSS) for 12 weeks. Gastrodin (50 mg/kg) was administered via oral gavage three times per week until the end of the experiment. Disease indexes, including body weight, bloody diarrhea, colon length, histopathological score, and tumor size, were measured. Tumor cell proliferation was evaluated by BrdU incorporation assay and tumor cell cytotoxicity was assessed by cell counting kit (CCK-8). The expression levels of toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-ΚB) signaling molecules, NF-ΚB luciferase, and pro-inflammatory cytokines were determined by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), immunoblotting, immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), or reporter gene assays. The binding affinity between gastrodin and myeloid differentiation protein-2 (MD2) was analyzed by molecular docking and cellular thermal shift assay (CETSA). Gastrodin administration was demonstrated to mitigate various CRC-related symptoms in mice, including weight loss, diarrhea, and tissue abnormalities. Notably, gastrodin suppressed tumor cell growth during colitis- associated tumorigenesis, resulting in fewer and smaller adenomas in the colon. Unlike irinotecan, a broadspectrum antitumor drug, gastrodin did not exhibit apparent cytotoxicity in various colorectal adenocarcinoma cell lines. Additionally, gastrodin downregulated TLR4/NF-ΚB signaling molecules and pro-inflammatory mediators in mice and macrophages. Molecular docking and CETSA experiments suggested that gastrodin binds to the MD2 protein, potentially interfering with the recognition of lipopolysaccharide (LPS) by TLR4, leading to NF-ΚB pathway inhibition. This study provides evidence for the first time that gastrodin attenuated colitis and prevented colitisrelated carcinogenesis in mice, at least partially, by diminishing tumor-promoting cytokines through the interruption of TLR4/MD2/NF-ΚB signaling transduction.

Development of (2-(Benzyloxy)phenyl)methanamine Derivatives as Potent and Selective Inhibitors of CARM1 for the Treatment of Melanoma.

Coactivator-associated arginine methyltransferase 1 (CARM1), an important member of type I protein arginine methyltransferases (PRMTs), has emerged as a promising therapeutic target for various cancer types. In our previous study, we have identified a series of type I PRMT inhibitors, among which ZL-28-6 (6) exhibited increased activity against CARM1 while displaying decreased potency against other type I PRMTs. In this work, we conducted chemical modifications on compound 6, resulting in a series of (2-(benzyloxy)phenyl)methanamine derivatives as potent inhibitors of CARM1. Among them, compound 17e displayed remarkable potency and selectivity for CARM1 (IC50 = 2 ± 1 nM), along with notable antiproliferative effects against melanoma cell lines. Cellular thermal shift assay and western blot experiments confirmed that compound 6 effectively targets CARM1 within cells. Furthermore, compound 17e displayed good antitumor efficacy in a melanoma xenograft model, indicating that this compound warrants further investigation as a potential anticancer agent.

Water-soluble 4-(dimethylaminomethyl)heliomycin exerts greater antitumor effects than parental heliomycin by targeting the tNOX-SIRT1 axis and apoptosis in oral cancer cells.

The antibiotic heliomycin (resistomycin), which is generated from Streptomyces resistomycificus, has multiple activities, including anticancer effects. Heliomycin was first described in the 1960s, but its clinical applications have been hindered by extremely low solubility. A series of 4-aminomethyl derivatives of heliomycin were synthesized to increase water solubility; studies showed that they had anti-proliferative effects, but the drug targets remained unknown. In this study, we conducted cellular thermal shift assays (CETSA) and molecular docking simulations to identify and validate that heliomycin and its water-soluble derivative, 4-(dimethylaminomethyl)heliomycin (designated compound 4-dmH) engaged and targeted with sirtuin-1 (SIRT1) in p53-functional SAS and p53-mutated HSC-3 oral cancer cells. We further addressed the cellular outcome of SIRT1 inhibition by these compounds and found that, in addition to SIRT1, the water-soluble 4-dmH preferentially targeted a tumor-associated NADH oxidase (tNOX, ENOX2). The direct binding of 4-dmH to tNOX decreased the oxidation of NADH to NAD+ which diminished NAD+-dependent SIRT1 deacetylase activity, ultimately inducing apoptosis and significant cytotoxicity in both cell types, as opposed to the parental heliomycin-induced autophagy. We also observed that tNOX and SIRT1 were both upregulated in tumor tissues of oral cancer patients compared to adjacent normal tissues, suggesting their clinical relevance. Finally, the better therapeutic efficacy of 4-dmH was confirmed in tumor-bearing mice, which showed greater tNOX and SIRT1 downregulation and tumor volume reduction when treated with 4-dmH compared to heliomycin. Taken together, our in vitro and in vivo findings suggest that the multifaceted properties of water-soluble 4-dmH enable it to offer superior antitumor value compared to parental heliomycin, and indicated that it functions through targeting the tNOX-NAD+-SIRT1 axis to induce apoptosis in oral cancer cells.

Flavokawain B inhibits NF-κB inflammatory signaling pathway activation in inflammatory bowel disease by targeting TLR2

Inflammatory bowel disease (IBD) is characterized by recurrent inflammatory reactions in the intestinal mucosa, including ulcerative colitis (UC) and Crohn's disease (CD). The expression of Toll-like receptor 2 (TLR2) has been observed to increase during the progression of IBD. Flavokawain B (FKB), a natural chalcone with potent anti-inflammatory activity, exerts its effects through inhibition of the NF-κB pathway. In this study, we aimed to investigate the effects and mechanisms of FKB targeting TLR2 in IBD. C57BL/6 J mice were treated with 2.5% dextran sulfate sodium (DSS) for 7 days, with administration of FKB or TLR2 inhibitor C29 starting on day 2 to establish the model of IBD. In vitro, bone marrow-derived macrophages (BMDMs) were stimulated with the TLR2 agonist Pam3CSK4 to explore the therapeutic effect of FKB and its pharmacological mechanism. Compared with the model group, the FKB-treated group showed significant reductions in colitis-related injuries in the IBD mouse model, including weight gain, increased colon length and reduced inflammation. FKB decreased the formation of TLR2-MyD88 complex by targeting TLR2, leading to suppression of downstream NF-κB signaling pathway. Similar therapeutic effects were observed in the C29-treated group. Additionally, in vitro data suggested that FKB exerted its anti-inflammatory effect by targeting TLR2 and inhibiting Pam3CSK4-induced activation of the NF-κB pathway. The anti-inflammatory effects of FKB were demonstrated through drug affinity responsive target stability assay and cellular thermal shift assay, revealing its binding affinity to TLR2. By inhibiting the activation of the TLR2/NF-κB signaling pathway, FKB effectively prevented DSS-induced IBD and exhibited promising potential as a therapeutic candidate for IBD treatment.

Costunolide ameliorates MNNG-induced chronic atrophic gastritis through inhibiting oxidative stress and DNA damage via activation of Nrf2

BackgroundChronic atrophic gastritis (CAG) is a chronic digestive disease. Modern research has revealed substantial evidence indicating that the progression of CAG is closely linked to the occurrence of oxidative stress-induced DNA damage and apoptosis in the gastric mucosa. Additionally, research has indicated that Costunolide (COS), the primary active compound found in Aucklandiae Radix, a traditional herb, exhibits antioxidant properties. Nevertheless, the therapeutic potential of COS in treating CAG and its molecular targets have not yet been determined. PurposeThe objective of this research was to explore the potential gastric mucosal protective effects and mechanisms of COS against N-Methyl-N´-nitro-N-nitrosoguanidine (MNNG)-induced CAG. MethodsFirstly, the MNNG-induced rat CAG model was established in vivo. Occurrence of CAG was detected through macroscopic examination of the stomachs and H&E staining. Additionally, we assessed oxidative stress, DNA damage, and apoptosis using biochemical detection, Western blot, immunohistochemistry and immunofluorescence. Then, an in vitro model was developed to induce MNNG-induced damage in GES-1 cells, and the occurrence of cell damage was determined by Hoechst 33342 staining and flow cytometry. Finally, the key targets of COS for the treatment of CAG were identified through molecular docking, cellular thermal shift assay (CETSA), and inhibitor ML385. ResultsIn vivo studies demonstrated that COS promotes the expression of Nrf2 in gastric tissues. This led to an increased expression of SOD, GSH, HO-1, while reducing the production of MDA. Furthermore, COS inhibited DNA damage and apoptosis by suppressing the expression of γH2AX and PARP1 in gastric tissues. In vitro studies showed that COS effectively reversed apoptosis induced by MNNG in GES-1 cells. Additionally, COS interacted with Nrf2 to promote its expression. Furthermore, the expression levels of SOD, GSH, and HO-1 were augmented, while the generation of ROS and MDA was diminished. ConclusionsOur results indicate that COS exhibits therapeutic effects on CAG through the promotion of Nrf2 expression and inhibition of oxidative stress and DNA damage. Therefore, COS has the potential to provide new drugs for the treatment of CAG.

CBC-1 as a Cynanbungeigenin C derivative inhibits the growth of colorectal cancer through targeting Hedgehog pathway component GLI 1

Colorectal cancer (CRC) is one of the most common gastrointestinal cancers that results in death in worldwide. The Hedgehog (HH) signalling pathway regulates the initiation and progression of CRC. Inhibiting the HH pathway has been presented as a potential treatment strategy in recent years. Cynanbungeigenin C (CBC) is a new type of C21 steroid that has been previously reported for the treatment of medulloblastoma. However, its further investigation was limited by its poor water solubility. In this study, six new CBC derivatives were synthesized through the structural modification of CBC, and four of them showed better water solubility than CBC. Moreover, their antiproliferative activities on CRC were evaluated. It was found that CBC-1 presented the best inhibitory effect on three types of CRC cell lines, and this effect was superior to that of CBC. Mechanistically, CBC-1 inhibited the proliferation of CRC cells through regulation of mRNA and proteins of the HH pathway according to qRT–PCR and Western blotting analysis. Furthermore, Cellular Thermal Shift Assay results indicated that CBC-1 regulated this signalling pathway by targeting glioma‑associated oncogene (GLI 1).In addition, cell apoptosis was induced increasingly by transfection with GLI 1 siRNA or treatment with CBC-1 to downregulate GLI 1. Last, the in vivo results demonstrated that CBC-1 significantly reduced tumour size and downregulated GLI 1 in CRC. Therefore, this study suggests that CBC-1, a new GLI 1 inhibitor derived from natural products, may be developed as a potential antitumour candidate for CRC treatment.

Gigantol ameliorates DSS-induced colitis via suppressing β2 integrin mediated adhesion and chemotaxis of macrophage

Ethnopharmacological relevanceDendrobium, recognized as “Shihu” in traditional Chinese medicine, holds a rich history of medicinal utilization documented in the Chinese Pharmacopoeia. Ancient texts like “Shen Nong Ben Cao Jing” extol Dendrobium's virtues as a superior herbal medicine fortifying “Yin” and invigorating the five viscera. Dendrobium is extensively employed for the treatment of gastrointestinal inflammatory disorders, showcasing significant therapeutic efficacy, particularly against ulcerative colitis (UC), within the realm of Chinese ethnopharmacology. Dendrobium plays crucial pharmacological roles due to its rich content of polysaccharides, alkaloids, phenanthrenes, and bibenzyls. Gigantol, a prominent bibenzyl compound, stands out as one of the most vital active constituents within Dendrobium, the gigantol content of Dendrobium leaves can reach approximately 4.79 μg/g. Its significance lies in being recognized as a noteworthy anti-inflammatory compound derived from Dendrobium. Aim of the studyGiven the pivotal role of gigantol as a primary active substance in Dendrobium, the therapeutic potential of gigantol for gastrointestinal diseases remains enigmatic. Our present investigation aimed to evaluate the therapeutic effects of gigantol on dextran sulfate sodium (DSS)-induced colitis and reveal its potential mechanism in countering UC activity. Materials and methodsThe protective efficacy of gigantol against colitis was assessed by examining the histopathological changes and conducting biochemical analyses of colon from DSS-challenged mice. Assessments focused on gigantol's impact on improving the intestinal epithelial barrier and its anti-inflammatory effects in colonic tissues of colitis mice. Investigative techniques included the exploration of the macrophage inflammatory signaling pathway via qPCR and Western blot analyses. In vitro studies scrutinized macrophage adhesion, migration, and chemotaxis utilizing transwell and Zigmond chambers. Furthermore, F-actin and Rac1 activation assays detailed cellular cytoskeletal remodeling. The potential therapeutic target of gigantol was identified and validated through protein binding analysis, competitive enzyme-linked immunosorbent assay (ELISA), cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) assay. The binding sites between gigantol and its target were predicted via molecular docking. ResultsGigantol ameliorated symptoms of DSS-induced colitis, rectified damage to the intestinal barrier, and suppressed the production of pro-inflammatory cytokines in colonic tissues. Intriguingly, gigantol significantly curtailed NF-κB signaling activation in the colons of DSS-induced colitis mice. Notably, gigantol impaired the β2 integrin-dependent adhesion and migratory capacity of RAW264.7 cells. Moreover, gigantol notably influenced the cytoskeleton remodeling of RAW264.7 cells by suppressing Vav1 phosphorylation and Rac1 activation. Mechanistically, gigantol interacted with β2 integrin, subsequently diminishing binding affinity with intercellular adhesion molecule-1 (ICAM-1). ConclusionsIn conclusion, these findings elucidate that gigantol ameliorates DSS-induced colitis by antagonizing β2 integrin-mediated macrophage adhesion, migration, and chemotaxis, thus it may impede macrophage recruitment and infiltration into colonic tissues. This study suggests that gigantol shows promise as a viable candidate for clinical colitis therapy.

Isoalantolactone exerts anti-melanoma effects via inhibiting PI3K/AKT/mTOR and STAT3 signaling in cell and mouse models.

Although the anti-cancer activity of isoalantolactone (IATL) has been extensively studied, the anti-melanoma effects of IATL are still unknown. Here, we have investigated the anti-melanoma effects and mechanism of action of IATL. MTT and crystal violet staining assays were performed to detect the inhibitory effect of IATL on melanoma cell viability. Apoptosis and cell cycle arrest induced by IATL were examined using flow cytometry. The molecular mechanism of IATL was explored by Western blotting, confocal microscope analysis, molecular docking, and cellular thermal shift assay (CETSA). A B16F10 allograft mouse model was constructed to determine the anti-melanoma effects of IATL in vivo. The results showed that IATL exerted anti-melanoma effects in vitro and in vivo. IATL induced cytoprotective autophagy in melanoma cells by inhibiting the PI3K/AKT/mTOR signaling. Moreover, IATL inhibited STAT3 activation both in melanoma cells and allograft tumors not only by binding to the SH2 domain of STAT3 but also by suppressing the activity of its upstream kinase Src. These findings demonstrate that IATL exerts anti-melanoma effects via inhibiting the STAT3 and PI3K/AKT/mTOR signaling pathways, and provides a pharmacological basis for developing IATL as a novel phytotherapeutic agent for treating melanoma clinically.

Abstract 1848: The Cellular Thermal Shift assay and its applications in Target ID, MoA determination and biomarker discovery

Abstract With the Cellular Thermal Shift Assay (CETSA) celebrating its first decade since the PoC publication, the method has gained much interest in the basic science field as well as become an industry standard in applied drug discovery setting. In the last few years it has become apparent that in addition to providing information about drug-protein interaction, CETSA based thermal profiling of cells can help describe additional aspects of cell biology as it gives insight into the activating and rewiring of protein-protein interaction networks inside the cell upon stimuli. In the work presented here, we have used CETSA coupled to high resolution mass spectrometric readout to profile the response patterns of about 8000 proteins to several hundred molecular probes and marketed drugs. The goal has been to provide a map of wanted and unwanted effects following treatment and to correlate it to alternative readouts, by for example cellular imaging and cell death assays. We have applied the CETSA protocol for close to five hundred compounds, many in several cell-lines (HepG2, U87MG and K562). The experimental setup includes the step of treating the cells with a certain compound at a fixed concentration. After incubation for an hour, we heated the cells to a range of temperatures, followed by a lysis protocol and pooling of the samples. After this, the samples have been centrifuged to separate the soluble from insoluble fraction. The soluble fraction has then been prepared for MS acquisition using TMT based multiplexing. Typically, this allowed us to monitor changes in thermal stability among more than 8,000 proteins, as a consequence of compound treatment. This data reveals specific compound “fingerprints” that can be used to decipher mode of action and identify biomarkers. Despite the short incubation time, proteins with compound-induced thermal stability shifts are not only targets/off-targets (direct binders), but also downstream, and sometimes upstream, pathway members and general cellular responses. All data combined makes the basis of the Target Engagement Atlas. Here we can see clusters of compounds, sharing similar protein binding profiles. Among the most prominent ones are mTOR inhibitors, NSAIDs, as well as tubulin binders. Alternatively, one can use this resource to reveal clusters of “pharmacologically” associated protein networks. We show that pharmacological perturbation allows identification of clusters representing tight protein complexes (for example ribosomes), known metabolic pathways (folate biosynthesis), molecular functions (kinases), as well as networks of proteins linked only by ligand binding specificity. CETSA MS allows for unbiased, cellular, and molecular profiling of compound effects. Together with data from clinical settings, as well as in vivo/in vitro end point assays it is possible to correlate the CETSA patterns to wanted but also unwanted responses. Citation Format: Tomas Friman, Alexey Chernobrovkin, Tuomas Tolvanen, Erin Gilson, Stina Lundgren, Victoria Brehmer, Daniel Martinez Molina. The Cellular Thermal Shift assay and its applications in Target ID, MoA determination and biomarker 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 1848.

Abstract 3096: CETSA Navigate HT: A new paradigm for high-throughput screening

Abstract The CEllular Thermal Shift Assay (CETSA) technology is a powerful way to assess target engagement across various matrixes. By the introduction of the high-throughput format (CETSA Navigate HT), this technology offers a reliable high-throughput screening method in natural cellular environments that can successfully be applied in compound screening for hit identification and confirmation already during lead generation. Due to its broad applicability to native, full length protein targets in the live cellular environment, without the need for expensive and time-consuming protein and/or compound labelling efforts, CETSA Navigate HT may be regarded as a new paradigm for high-throughput screening and actives generation. As a case study to demonstrate the applicability of CETSA Navigate HT in hit identification, we have applied the technology for primary screening of a 12000 compound library for CDK4, a protein targeted in several cancers. Hits identified from this screen are privileged from the onset by binding to the target in a physiologically relevant cellular matrix and in a binding-mode agnostic way. This is in contrast to traditional screening methods targeting activity or in vitro interactions where cellular activity is addressed in later stages. Data from follow up studies using functional assays will be presented as well as mass spectrometry based CETSA selectivity profiling and comparison to commercially available de novo CDK4/6 inhibitors.  Citation Format: Helena Almqvist, Victoria Brehmer, Laurence Arnold, Tomas Friman, Stina Lundgren, Daniel Martinez Molina. CETSA Navigate HT: A new paradigm for high-throughput screening [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 3096.

Abstract 646: Proteome wide target deconvolution of pharmacologically active natural products by CETSA® coupled to mass spectrometry

Abstract Nature has provided a large plethora of different molecules that in the hands of man has been ascribed to a vast range of pharmacological actions. In some cases, pharmacology is clearly connected to a phenotype e.g., Golgi apparatus dissolution by Brefeldin A, whereas in other cases the proposed phenotypes are many and disparate e.g., Quercetin. Many claims are made around natural compounds as having various positive therapeutic effects on cancer e.g., curcumin and quercetin. Intriguingly, claims about therapeutic effects of various natural compounds keep on surfacing, and we wanted to address this issue by performing target deconvolution of a selection of natural compounds in Hepatic cancer cells: HepG2. We took advantage of the CETSA® (CEllular Thermal Shift Assay) method coupled to Mass spectrometry detection in order to deconvolute the mode of action of seven highly cited natural compounds that comprised both well annotated compounds with an established mode of action: Brefeldin A and Wortmannin; and compounds with a less well established mode of action: Carnosol, Curcumin, Resveratrol, Rhoifolin, and Quercetin. CETSA is a method that allows detection of compound-induced thermal shifts of proteins in intact living cells as well as in cell lysates. Subsequently, a thermal shift of a protein in the presence of a compound then implicates a direct or in the case of intact cells, also an indirect interaction between the protein in question and compound. As CETSA can be performed in intact living cells we could detect proteins that were potential direct binders as well as proteins that were part of the downstream signaling events or part of the phenotypic response. The Natural compounds were assessed in a screening format in which three bio-replicates were analyzed at a single concentration, 30 µM. Good proteome coverage was obtained in the HepG2 cells and all compounds tested induced thermal shifts of proteins at 30 µM. Most of the compounds displayed many protein shifts of both suggested direct targets and proteins presumed to be part of a phenotypic response, as well as novel targets/off-targets. Curcumin and Rhoifolin were the exceptions, which only had a few shifted proteins. Brefeldin A, Quercetin, and Wortmannin engaged proteins associated to their annotated mode of action pathways, whereas Carnosol, Curcumin, and Resveratrol was less specific in their responses with regards to shifted proteins i.e., no clear signaling pathway was determined. In this study we use CETSA coupled to MS detection to delineate mode of action of natural compounds, which shed light on why they could be implicated to have a role in tumor biology. We also found that compounds with a well-established mode of action give a clearer target engagement profile compared to less annotated compounds. Citation Format: Tomas Friman, Tuomas Tolvanen, Erin Gilson, Alexey Chernobrovkin, Stina Lundgren, Victoria Brehmer, Daniel Martinez Molina. Proteome wide target deconvolution of pharmacologically active natural products by CETSA® coupled to mass spectrometry [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 646.

Abstract 7148: Small molecule inhibitor of FEN1 nuclease utilizing a novel metal-binding pharmacophore synergizes with inhibitors of USP1, PARP, PARG and ATR

Abstract Flap endonuclease 1 (FEN1) is a structure-specific metallonuclease that cleaves 5’ DNA flaps during replication and repair. FEN1 is an attractive target for development of anticancer therapeutics because it is overexpressed in many tumor types and has a large number of synthetic lethality partners including genes in Homologous Recombination (HR) pathway (Mengwasser et al., 2019; Guo et al., 2020). Utilizing fragment-based drug discovery approach we identified a novel metal-binding pharmacophore that binds to the two magnesium ions in the FEN1 active site. Further elaboration using fragment growth strategies resulted in highly potent and selective inhibitors. The current lead (BSM-1516) is ~65-fold more potent against FEN1 than its related enzyme Exonuclease 1 (Exo1) in biochemical assays (IC50 of 7 nM and 460 nM, respectively), an improvement of more than an order of magnitude in selectivity compared to earlier efforts. FEN1 target engagement in live cells was validated by cellular thermal shift assay (CETSA EC50 of 24 nM). Inhibition of FEN1 led to its increased association with chromatin in S-phase cells and recruitment of PARP1 enzyme. In clonogenic assay, BRCA2-deficient DLD1 cells were ~15-fold more sensitive to FEN1 inhibition than their isogenic BRCA2-wild-type counterparts (EC50 of 350 nM and 5 µM, respectively), confirming the increased susceptibility of HR deficient cancer cells to FEN1 inhibition. Treatment of BRCA2-deficient but not wild-type DLD1 cells with BSM-1516 resulted in cell cycle arrest and dose-dependent decrease of S-phase BrdU incorporation into DNA. Cell cycle arrest of BRCA2-deficient DLD1 cells was accompanied by DNA damage signaling and by accumulation of chromatin-bound RPA32, a marker of ssDNA. To explore synthetic lethal interactions of FEN1 with other DNA repair genes we performed FEN1-inhibitor-anchored CRISPR screen. This analysis revealed that in addition to HR pathway inactivation, genomic perturbations in EXO1, USP1 and PARP1 genes sensitized cells to FEN1 inhibition. Synergistic relationships of BSM-1516 and its combination potential were further explored in viability studies with a panel of DDR inhibitors (n=25) in BRCA2-proficient and deficient cell lines. Strong synergy was identified with multiple drug classes that included inhibitors of USP1 (KSQ-4279), PARP (Olaparib, Niraparib, Talazoparib, AZD5305), PARG (PDD 00017273) and ATR (AZD6738, VE-822, Elimusertib). In vitro ADME assays and in vivo PK studies showed that BSM-1516 has properties suitable for in vivo testing, either as a single agent or in combination with synergistic DDR inhibitors, an investigation that is currently underway. Citation Format: Jason Munguia, Sanjay Agarwalla, Elaine Guo, Dave Martin, Junhua Fan, Dave Lonergan, David Puerta, Zachary Zimmerman, Konstantin Taganov. Small molecule inhibitor of FEN1 nuclease utilizing a novel metal-binding pharmacophore synergizes with inhibitors of USP1, PARP, PARG and ATR [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 7148.

Abstract 5787: Structure-function mapping of DHODH shows deviations in protein structure

Abstract Dihydroorotate dehydrogenase (DHODH) is a CoQ-dependent enzyme that converts dihydroorotate to orotate in the de novo pyrimidine biosynthesis pathway. Multiple structures of DHODH in complex with various small molecule inhibitors have been solved that indicate that DHODH inhibitors bind in the CoQ binding tunnel of DHODH and act as competitive inhibitors of CoQ. We performed saturating mutagenesis screens to identify mutations in DHODH that confer resistance to brequinar and BAY2402234, two highly potent DHODH inhibitors. As expected, many of the resistance mutations localized to the drug/CoQ-binding tunnel of the enzyme. Among the top-scoring drug-binding-domain mutants in both the brequinar and BAY2402234 screens were substitutions A58H and A58T. In silico analysis predicted that A58H should impair DHODH binding to both brequinar and BAY2402234. However, A58T was predicted to only impair binding of DHODH to brequinar. We expressed the mutants in a DHODH inhibitor sensitive cell line and confirmed that they both confer resistance to brequinar and BAY2402234, and we then assessed the ability of the mutants to bind drug by cellular thermal shift assay (CETSA). We found that, as predicted, DHODH A58H did not bind brequinar or BAY2402234. However, to our surprise, we detected binding of both brequinar and BAY2402234 to DHODH A58T. We generated recombinant DHODH A58T and confirmed that the purified protein was enzymatically active and strongly drug-resistant by DCIP assay and then assessed binding of the mutant protein to brequinar and BAY2402234 by thermal shift assay (TSA). Consistent with our CETSA results, recombinant DHODH A58T bound both brequinar and BAY2402234 in vitro. These results suggested that DHODH A58T can simultaneously bind drug and CoQ. However, previously reported DHODH structures indicated that the drug/CoQ binding tunnel is quite narrow and rigid. To determine whether the structure of catalytically-active DHODH is more flexible than inhibitor-bound catalytically-inactive DHODH, we co-crystallized wild-type DHODH with decylubiquinone (DCU), a CoQ analog that can act as a DHODH co-substrate in vitro. We found that the structure of DCU-bound DHODH differs from previously published DHODH structures in two important ways: (1) there appears to be a second point of ingress into the active site of DHODH next to the CoQ tunnel that may be able to accommodate a highly non-polar molecule such as CoQ/DCU and (2) the ‘flap’ covering the DHO/orotate ingress/egress region of DHODH, which is ‘closed’ in inhibitor-bound DHODH, is ‘open’ in DCU-bound DHODH. We are currently working to solve the crystal structure of DHODH A58T bound to inhibitor and DCU to ask if DCU can access the active site of DHODH through an alternative hydrophobic tunnel and if DHODH A58T remains in the ‘open’ configuration when bound to drug. Citation Format: Ruchika Pokhriyal, Laura Evans, Hyuk-Soo Seo, Jillian K. O'Neil, Betty Rouaisnel, Atanas Kamburov, Judith Guenther, Stefan Gradl, Haribabu Arthanari, Sven Christian, Xiaoping Yang, Sirano Dhe-Paganon, Julie-Aurore Losman. Structure-function mapping of DHODH shows deviations in protein structure [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 5787.

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 3097: CETSA HT: Employing rapid high-throughput target engagement technology in primary screening of P53

Abstract The confirmation of target engagement for candidate drugs is a fundamental requisite in drug discovery. A significant portion of projects fail to reach the clinic due to lack of efficacy or failure to show lead candidates interacting with the intended target in a more complex environment. The utility of using the CETSA® (CEllular Thermal Shift Assay) technology to measure target engagement in intact cells is increasingly exploited in high throughput screening, where examples demonstrate low false positive rates in screens of up to 500K compounds. The p53 tumor suppressor protein, a crucial guardian against cancer, functions as a transcription factor that mediates cancer suppression by regulating genes involved in a wide range of cellular processes such as DNA repair, cell cycle and apoptosis. Unfortunately, frequent mutations in the TP53 gene hinder p53's tumor suppressor role in human cancers, thus designating it as a promising target for anticancer therapy. However, attempts to restore p53 function for therapeutic purposes have not yet advanced to clinical approval, highlighting the complexities in leveraging p53 for cancer treatment. Its undruggable nature, attributed to the absence of accessible deep pockets, lack of enzymatic activity, and its intracellular location, further complicates targeted interventions with both low and high molecular weight drugs. Here, we demonstrate the analysis of a high-throughput plate based CETSA® HT screen on the challenging target p53, performed using AlphaLISA® SureFire® Ultra™ technology and our newly developed high-throughput screening platform. Hits identified from this screen are privileged from the onset by binding to the target in a physiologically relevant cellular matrix and in a binding-mode agnostic way. This is in contrast to traditional screening methods targeting activity or in vitro interaction where cellular activity is addressed in later stages. This assay cascade highlights a rapid target-to-hit lead generation strategy placing the emphasis on finding chemical matter that interacts with endogenous target in living cells first, followed by analysis of hit material activity. The ‘shortcut’ to chemical matter active in living cells represents significant potential cost savings in comparison to assay development time on recombinantly produced proteins and/or modified cell lines for traditional HTS screening approaches that still rely on target engagement assessment downstream. Citation Format: Merve Kacal, Tomas Friman, Laurence Arnold, Victoria Brehmer, Daniel Martinez Molina, Stina Lundgren. CETSA HT: Employing rapid high-throughput target engagement technology in primary screening of P53 [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 3097.

Abstract 211: Applying cellular thermal shift assays to uncover novel regulators of epithelial to mesenchymal transition in triple negative breast cancer

Abstract Metastatic Breast Cancer is accountable for over half a million deaths worldwide each year, the majority of which are from the triple negative breast cancer (TNBC) subtype, known to be highly aggressive, metastatic and resistant to targeted therapy. Metastasis is driven by the ability of cancer cells to undergo epithelial to mesenchymal transition (EMT), a cell state transition involving a gain of stemness, cell motility and chemoresistance. EMT has been well characterized using a range of techniques based upon quantitative proteomics and transcription profiling, yet targeting EMT to prevent tumor metastasis and chemoresistance is an obstacle that remains to be fully overcome. Using a cellular thermal shift assay coupled with mass spectrometry (CETSA-MS), we have measured changes in protein stability upon induction of EMT and have uncovered key enzymes in the citric acid (TCA) cycle that are stabilized during EMT of human mammary epithelial cells. Such enzymes are responsible for the conversion of TCA cycle intermediates required for a recently discovered post-translational modification. In TNBC cells, we find that loss of these enzymes causes not only global changes to crucial post-translational modification of mitochondrial proteins, but very specific alterations in several cytoskeletal proteins, leading to altered EMT status as well as reduced tumor growth and initiation both in vitro and in vivo. This demonstrates the importance of TCA cycle-controlled post-translational modification of cytoskeletal proteins in tumorigenesis and establishes basis for further investigation into their role in EMT and metastasis of breast cancer. Through pharmacological inhibition and immunofluorescence imaging, we discovered regulators of post-translational modifications that may be viable drug targets to overcome chemoresistance and relapse in TNBC patients with limited and inefficacious options. Citation Format: Jessica A. Lidster, Ser Yue Loo, Zhengwei Wu, Lingyun Dai, Seow Qi Ng, Nayana Prabhu, Dennis Kappei, Wai Leong Tam. Applying cellular thermal shift assays to uncover novel regulators of epithelial to mesenchymal transition in triple negative breast cancer [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 211.

Abstract 4723: Target engagement sheds light on difference in drug efficacy in breast cancer cell lines

Abstract Breast cancer is a serious disease that affects millions of women worldwide, and 700,000 people succumb to the disease annually. Breast cancers are divided into several functional subgroups based on molecular characteristics and growth pattern: Luminal A and B, HER2 positive (HER2+) and triple negative. The subgroups are important from a clinical perspective as they partially guide treatment strategies. In this study we wanted to investigate the difference in target engagement with four drugs indicated for treatment of different types of breast cancer: Alpelisib, Neratinib, Vinorelbine, and Docetaxel. Five different human breast cancer cell lines were included, representing Luminal A (MCF-7, T-47D), HER2+ (SK-BR-3, BT-474), and triple negative (BT-20). We used CETSA® (CEllular Thermal Shift Assay) coupled to mass spectrometry detection to assess proteome wide changes in protein thermal stability in intact breast cancer cell lines. CETSA is unique as a target engagement detection modality as it allows for target engagement assessment in intact cells with unaltered drug molecules. Also, as biology is intact in live cells it is possible to follow compound induced downstream signaling or phenotypic events that result in changed thermal stability of proteins. In addition to assessing protein thermal stability with CETSA, which is a short treatment of 1 h, we also subjected cells in culture to prolonged drug exposures, ≤72 h, in order to measure viability. This was done to correlate early changes in protein thermal stability to the viability phenotype that manifested later. In general, responses both in protein thermal stability and cell viability was heterogenous among the cell lines. The most homogenous response in viability was observed for the Phosphatidyl Inositol 3’ kinase inhibitor Alpelisib, which reduced viability to 20 - 40% in all cell lines. In contrast, partial resistance was shown to the other drugs by at least one of the cell lines. For Alpelisib, the amount and type of proteins that were thermally shifted correlated with the effect on viability. The covalent HER2 inhibitor Neratinib was most effective in inhibiting viability in HER2+ cells and proteins of the HER2 pathway were thermally shifted in these cells, which was not observed for the other cell lines. Viability of the other cell lines were also affected by Neratinib, but only at higher concentrations. Both microtubule inhibitors induced thermal stabilization of tubulins, but that did not always translate into efficacy in the viability assay. However, sensitive cell lines showed thermal shifts of tubulins at lower concentrations of compound. In oncology drug development it is important that effects on viability are driven by on-target efficacy and not by off-target effects. Here we show the benefit of connecting knowledge of target engagement and phenotypic readouts; what targets are engaged, at which concentrations, and what is the corresponding phenotype. Citation Format: Tomas Friman, Tuomas Tolvanen, Merve Kacal, Victoria Brehmer, Stina Lundgren, Laurence Arnold, Daniel Martinez Molina. Target engagement sheds light on difference in drug efficacy in breast cancer cell lines [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 4723.