Tubulin Polymerization Research Articles

Structure-activity relationship study of new carbazole sulfonamide derivatives as anticancer agents with dual-target mechanism

A series of novel carbazole sulfonamide derivatives were synthesized and evaluated for antiproliferative activity. Among them, compounds 7 and 15 showed strong potency (IC50 values of 0.81–31.19 nM) against five different cancer cells including multidrug-resistant MCF7/ADR cells. Compound 15 displayed a high cancer cell selectivity (IC50(L02)/average IC50: SI = 7.7). The l-valine prodrug 7a and the phosphate prodrug 15a exerted rohust in vivo antitumor efficacies and accepted safety prolifes. Further mechanism studies revealed that 7 and 15 directly bind to the colchicine site in tubulin to block tubulin polymerization, promote microtubule fragmentation at the cellular level, and induce apoptosis with G2/M cell cycle arrest. These compounds also inhibit HEMC-1 cells migration and vascular tube formation. Additionally, compound 7 displayed a selective inhibition of Topo I. Collectively, these studies suggest that 7 and 15 represents a promising new generation of tubulin inhibitors for cancer treatment.

Synthesis and Selective Anticancer Activity Evaluation of 2-phenylacrylonitrile Derivatives as Tubulin Inhibitors.

This study aimed at synthesizing 13 series of novel derivatives with 2-phenylacrylonitrile, evaluating antitumor activity both in vivo and in vitro, and obtaining novel tubulin inhibitors. The 13 series of 2-phenylacrylonitrile derivatives were synthesized by Knoevenagel condensation and the anti-proliferative activities were determined by MTT assay. The cell cycle and apoptosis were analyzed by flow cytometer. Quantitative cell migration was performed using 24-well Boyden chambers. The proteins were detected by western blotting. in vitro kinetics of microtubule assembly was measured using ELISA kit for Human β-tubulin (TUBB). Molecular docking was done by Discovery Studio (DS) 2017 Client online tool. Among the derivatives, compound 1g2a possessed strong inhibitory activity against HCT116 (IC50 = 5.9 nM) and BEL-7402 (IC50 = 7.8 nM) cells. Compound 1g2a exhibited better selective antiproliferative activities and specificities than all the positive control drugs, including taxol. Compound 1g2a inhibited proliferation of HCT116 and BEL-7402 cells by arresting them in the G2/M phase of the cell cycle, inhibited the migration of HCT116 and BEL-7402 cells and the formation of cell colonies. Compound 1g2a showed excellent tubulin polymerization inhibitory activity on HCT116 and BEL-7402 cells. The results of molecular docking analyses showed that 1g2a may inhibit tubulin to exert anticancer effects. Compound 1g2a shows outstanding antitumor activity both in vivo and in vitro and has the potential to be further developed into a highly effective antitumor agent with little toxicity to normal tissues.

Design, synthesis, and biological evaluation of novel 2,3-Di-O-Aryl/Alkyl sulfonate derivatives of l-ascorbic acid: Efficient access to novel anticancer agents via in vitro screening, tubulin polymerization inhibition, molecular docking study and ADME predictions

A series of novel l-ascorbic acid derivatives bearing aryl and alkyl sulfonate substituents were synthesized and characterized. In vitro anticancer evaluation against MCF-7 (breast) and A-549 (lung) cancer cell lines revealed potent activity for most of the compounds, with 2b being equipotent to the standard drug colchicine against MCF-7 (IC50 = 0.04 μM). Notably, compound 2b displayed 89-fold selectivity for MCF-7 breast cancer over MCF-10A normal breast cells. Derivatives with two sulfonate groups (2a-g, 3a-g) exhibited superior potency over those with one sulfonate (4a-c,5g, 6b). Compounds 2b and 2c potently inhibited tubulin polymerization in A-549 cancer cells (73.12 % and 62.09 % inhibition, respectively), substantiating their anticancer potential through microtubule disruption. Molecular docking studies showed higher binding scores and affinities for these compounds at the colchicine-binding site of α, β-tubulin compared to colchicine itself. In-silico ADMET predictions indicated favourable drug-like properties, with 2b exhibiting the highest binding affinity. These sulfonate derivatives of l-ascorbic acid represents promising lead scaffolds for anticancer drug development.

Oxindoline Containing Thiazolidine‐4‐one Tethered Triazoles Act as Antimitotic Agents by Targeting Microtubule Dynamics

AbstractOxindoline containing thiazolidine‐4‐ones linked triazole hybrids (24 a–l) series were synthesized by multistep synthesis and tested for their cytotoxic activity against four human cancer cell lines. Significantly higher susceptibility was observed in liver (HepG2) cancer cells. Among them conjugates 24 c and 24 e exhibited promising cytotoxicity with IC50 values of 1.64 μM, and 2.07 μM respectively when compared to the standard 5‐fluorouracil (IC50=20.8 μM) against the HepG2 cell line. Later on, these compounds changed the morphology of HepG2 cells and significantly inhibited colony formation in HepG2 in a dose‐dependent manner. The scratch assay divulged that these compounds decreased wound size and inhibited cell migration. Interestingly, they induced the cell cycle arrest at the S phase. Moreover, the DCFDA assay indicated ROS production proportionate to compound dose and AO/Eb staining revealed increased apoptotic cell count with higher doses. The tubulin polymerization assay reveals that they inhibited the microtubules and acted as antimitotic agents. In‐silico results of these conjugates also support their tubulin inhibitory properties. Interestingly, these compounds have adequate ADME‐toxicity parameters. Based on free energy calculation results, it was observed that these inhibitors influence the conformational flexibility of the target tubulin protein.

Design, synthesis, and biological evaluation of novel benzimidazole derivatives as anti-cervical cancer agents through PI3K/Akt/mTOR pathway and tubulin inhibition

Phosphatidylinositol 3-kinase (PI3K) is one of the most attractive therapeutic targets for cervical cancer treatment. In this study, we designed and synthesized a series of benzimidazole derivatives and evaluated their anti-cervical cancer activity. Compound 4r exhibited strong antiproliferative activity in different cervical cancer cell lines HeLa, SiHa and Ca Ski, and relative lower cytotoxicity to normal hepatic and renal cell lines LO2 and HEK-293t (IC50 values were at 21.08 μM and 23.96 μM respectively). Its IC50 value was at 3.38 μM to the SiHa cells. Further mechanistic studies revealed that 4r induced apoptosis, arrested cell cycle in G2/M phase, suppressed PI3K/Akt/mTOR pathway and inhibit the polymerization of tubulin. Molecular docking study suggested that 4r formed key H-bonds action with PI3Kα (PDB ID:8EXU) and tubulin (PDB ID:1SA0). Zebrafish acute toxicity experiments showed that high concentrations of 4r did not cause death or malformation of zebrafish embryos. All these results demonstrated that 4r would be a promising lead candidate for further development of novel PI3K and tubulin dual inhibitors in cervical cancer treatment.

Synthesis of Estrone Heterodimers and Evaluation of Their In Vitro Antiproliferative Activity.

Directed structural modifications of natural products offer excellent opportunities to develop selectively acting drug candidates. Natural product hybrids represent a particular compound group. The components of hybrids constructed from different molecular entities may result in synergic action with diminished side effects. Steroidal homo- or heterodimers deserve special attention owing to their potentially high anticancer effect. Inspired by our recently described antiproliferative core-modified estrone derivatives, here, we combined them into heterodimers via Cu(I)-catalyzed azide-alkyne cycloaddition reactions. The two trans-16-azido-3-(O-benzyl)-17-hydroxy-13α-estrone derivatives were reacted with 3-O-propargyl-D-secoestrone alcohol or oxime. The antiproliferative activities of the four newly synthesized dimers were evaluated against a panel of human adherent gynecological cancer cell lines (cervical: Hela, SiHa, C33A; breast: MCF-7, T47D, MDA-MB-231, MDA-MB-361; ovarian: A2780). One heterodimer (12) exerted substantial antiproliferative activity against all investigated cell lines in the submicromolar or low micromolar range. A pronounced proapoptotic effect was observed by fluorescent double staining and flow cytometry on three cervical cell lines. Additionally, cell cycle blockade in the G2/M phase was detected, which might be a consequence of the effect of the dimer on tubulin polymerization. Computational calculations on the taxoid binding site of tubulin revealed potential binding of both steroidal building blocks, mainly with hydrophobic interactions and water bridges.

Modification of the phenyl ring B of phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates by pyridinyl moiety leads to novel antimitotics targeting the colchicine-binding site

A series of 8 novel pyridinyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PYRIB-SOs) were designed, prepared and evaluated for their mechanism of action. PYRIB-SOs were found to have antiproliferative activity in the nanomolar to submicromolar range on several breast cancer cell lines. Moreover, subsequent biofunctional assays indicated that the most potent PYRIB-SOs 1–3 act as antimitotics binding to the colchicine-binding site (C-BS) of α, β-tubulin and that they arrest the cell cycle progression in the G2/M phase. Microtubule immunofluorescence and tubulin polymerisation assay confirm that they disrupt the cytoskeleton through inhibition of tubulin polymerisation as observed with microtubule-destabilising agents. They also show good overall theoretical physicochemical, pharmacokinetic and druglike properties. Overall, these results show that PYRIB-SOs is a new family of promising antimitotics to be further studied in vivo for biopharmaceutical and pharmacodynamic evaluations.

Aluminum-Catalyzed Intramolecular Vinylation of Arenes by Vinyl Cations.

This study addresses the challenges associated with vinyl cation generation, a process that traditionally requires quite specific counterions. Described herein is a novel intramolecular vinylation of arenes catalyzed by aluminum(III) chloride, utilizing practical conditions and readily available vinyl triflates derived from 2-aceto-3-arylpropionates. Comprehensive experimental data support diverse carbocycle synthesis, exemplified by indenes and higher analogues. Control experiments verify the applicability of the vinylation protocol, and synthetic applications showcase a potent tubulin polymerization inhibitor with anticancer properties. Density functional theory computations reveal a Lewis-acid-driven mechanism involving triflate moiety abstraction to generate a reactive vinyl cation.

Design and Synthesis of Aspirin-chalcone Mimic Conjugates as Potential Anticancer Agents.

Extensive research has been conducted on aspirin, a widely recognized NSAID medication, regarding its potential as an anticancer agent. Studies have revealed its ability to trigger cell death in different types of cancer cells. A set of aspirin-chalcone mimic conjugates 5a-k and 6a-d utilizing the freshly prepared acid chloride of aspirin moiety has been designed and synthesized. To evaluate the newly developed compounds, the NCI 60- cell line panel was employed to assess their anti-proliferative properties. Subsequently, cell cycle analysis was conducted along with an examination of the compounds' impact on the levels of p53, Bax, Bcl-2, active caspase- 3, and their inhibition mechanism of tubulin polymerization. Derivative 6c displayed the best anticancer activity among the tested series while 6d was the best against breast cancer MDA-MB-468, therefore both of them were selected for the 5-dose stage, however, targeting MDA-MB-468, PI-flow cytometry of compound 6d proved the triggered cell growth arrest at the G1/S phase avoiding the mitotic cycle in MDA-MB-468 cells. Similarly, the upregulation of oncogenic parameters such as caspase-3, p53, and Bax/Bcl-2, along with the inhibition of PARP-1 enzyme level, was observed with compound 6d. This compound also exhibited a significant ability to induce apoptosis and disrupt the intracellular microtubule network through a promising activity as a tubulin polymerization inhibitor with IC50 = 1.065 ± 0.024 ng/ml. Furthermore, to examine the manner in which compound 6d binds to the active pocket of the tubulin polymerization enzyme, a molecular docking study was conducted. The study indicated that compound 6d could be a powerful microtubule-destabilizing agent. Therefore, further research on 6d could be worthwhile.

Dual molecule targeting HDAC6 leads to intratumoral CD4+ cytotoxic lymphocytes recruitment through MHC-II upregulation on lung cancer cells

BackgroundDespite the current therapeutic treatments including surgery, chemotherapy, radiotherapy and more recently immunotherapy, the mortality rate of lung cancer stays high. Regarding lung cancer, epigenetic modifications altering cell cycle, angiogenesis...

Synthesis of Morpholine Linked Indole-1,2,3-triazole Hybrids as In Vitro Tubulin Polymerization Inhibiting Agents

Synthesis of Morpholine Linked Indole-1,2,3-triazole Hybrids as In Vitro Tubulin Polymerization Inhibiting Agents

Exploring the potential of Ziziphus nummularia and luteolin-7-O-glucoside as tubulin inhibitors in cancer therapy and survival

Cancer is responsible for approximately 10 million deaths worldwide, with 70% of the deaths occurring in low- and middle-income countries; as such safer and more effective anti-cancer drugs are required. Therefore, the potential benefits of Ziziphus nummularia and Ziziphus spina-christi as sources of anti-cancer agents were investigated. Z. nummularia and Z. spina-christi extracts were prepared using chloroform, ethanol, ethyl acetate, and water. The extracts’ anti-cancer properties were determined using the MTT Cell Viability Assay in four cancer cell lines: breast (KAIMRC2 and MDA-MB-231), colorectal (HCT8), and liver (HepG2). The ApoTox-Glo Triplex Assay and high-content imaging (HCI)-Apoptosis Assay were used to assess KAIMRC2 and HCT8 cells further. In addition, KAIMRC2 cells were tested for microtubule staining, and AKT/mTOR protein expression was determined by western blot analysis. Liquid chromatography-mass spectrometry (LC–MS) was performed to identify the secondary metabolites in the ethanol and ethyl acetate extracts, followed by in silico techniques to predict molecular targets and interactions, safety, and pharmacokinetic profile for identified metabolites. Out of the eight extracts, the ethanolic extract of Z. nummularia, exhibited the most potent activity against KAIMRC2 cells with an IC50 value of 29.2 μg/ml. Cancer cell treatment with the ethanolic extract of Z. nummularia resulted in a dose-dependent decrease in cell viability with increased apoptosis and cytotoxic effects. Microtubule staining showed a disrupted microtubular network. The ethanolic extract treatment of KAIMRC2 cells led to upregulated expression of pAKT and pmTOR. In silico studies predicted luteolin-7-O-glucoside to be a ligand for tubulin with the highest docking score (− 7.686) and similar binding interactions relative to the native ligand. Further computational analysis of the metabolites showed acceptable pharmacokinetic and safety profiles, although ethanolic extract metabolites were predicted to have cardiotoxic effects. Ethanolic extraction is optimal for solubilizing active anticancer metabolites from Z. nummularia, which may act by causing M-phase arrest via inhibition of tubulin polymerization. Luteolin-7-O-glucoside is the lead candidate for further research and development as an anti-cancer agent. In addition, this study suggests that herbal treatment could switch on mechanisms of adaptation and survival in cancer cells.

Abstract 2033: Rigosertib promotes anti-tumor activity of cancer cells via CETSA revealed novel targets and activates NLRP3-dependent inflammatory responses

Abstract Background: Rigosertib (ON 01910.Na) is considered a promising anticancer agent due to its ability to inhibit multiple disease signaling pathways, including RAS-MAPK signaling. Rigosertib is in a Phase 2 program in advanced squamous cell carcinoma complicating recessive dystrophic epidermolysis bullosa (RDEB-associated SCC) (NCT03786237, NCT04177498), and a Phase 2 trial in combination with pembrolizumab in patients with metastatic melanoma (NCT05764395). The aim of our study was to further elucidate the mechanism of action and to identify potential novel targets engaged by rigosertib. Methods: A variety of biochemical, molecular and cell biological assays were performed to characterize the binding of rigosertib to previously proposed targets and its effect on RAS-MAPK signaling and tumor cell survival. A mass spectrometry-based Cellular Thermal Shift Assay (CETSA-MS) was conducted to identify potential new protein targets of rigosertib. Time-lapse microscopy was used for detection of ROS levels and tubulin polymerization in rigosertib treated lung cancer cells. Bone Marrow Derived Macrophages (BMDM) collected from C57/BL6 mice and human lung cancer organoids were treated with rigosertib and the cell death was determined by flow cytometry. Results: Comparative CETSA profiling of rigosertib and colchicine, a classical tubulin depolymerizing agent, reveals unique targets of rigosertib in multiple tumor cell types. We identify two ROS-related proteins, ERO1A and NQO2 that potentially contribute to the induction of ROS-dependent JNK activation. Fluorescence live-cell imaging confirms the effect of ERO1A and NQO2 on rigosertib induced ROS generation. NEK7, a relevant protein for microtubule organization, is another potential target for rigosertib. NEK7 knock-down reveals reduced microtubule depolymerization in rigosertib treated lung cancer cells. While we confirm that rigosertib might affect microtubules at higher concentrations, the main cellular target(s) responsible for the induction of stress and JNK-mediated inhibition of RAS-RAF-MEK signaling needs to be further characterized. Rigosertib affects the tumor immune environment by activating NLRP3-dependent inflammatory responses and Caspase-1 to trigger IL-1β and IL-18 secretion in THP1 cells, as well as in human cancer organoids, and in wild-type, Casp1/11−/−, Asc−/− and Nlrp3−/− BMDMs. Conclusion: These findings indicate that rigosertib may represent an effective compound for inhibition of RAS-MAPK signaling through ROS-mediated JNK activation. The rigosertib-dependent mechanism for NLRP3 activation suggests a reprogramming of the tumor immune environment which may contribute to the synergistic effect seen with immune checkpoint inhibitors pre-clinically and clinically. Clinical trials in several difficult-to-treat cancers continue. Citation Format: Petros Kechagioglou, Camille Dupont, Hajime Yurugi, Alexey Chernobrovkin, Rossana Romero, Rebecca Tweedell, Thirumala-Devi Kanneganti, Stephen Cosenza, Steven M. Fruchtman, Krishnaraj Rajalingam. Rigosertib promotes anti-tumor activity of cancer cells via CETSA revealed novel targets and activates NLRP3-dependent inflammatory responses [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 2033.

Abstract 4701: A novel microtubule disruptor exerts broad anticancer efficacy with a tolerable safety profile

Abstract Initial evaluation of the prenylated hydroxy-stilbene isolated from bee propolis, AUS_001, in the NCI 60 human cell line anticancer screen showed significant growth inhibitory effects with the most robust being observed in leukemia, central nervous system and breast cancer cell lines. The aim of the current study was to further assess the potency of AUS_001 in vitro and investigate its safety profile and mechanism of action. A commercially available cell-based profiling screen (OncoPanel®) identified significant AUS_001-induced growth inhibition in 273 out of 280 cancer cell lines with a concentration causing 50% cell growth inhibition in the range of 0.021-0.94 µM, while non-neoplastic cells required an average of 20x higher concentration to generate similar efficacy. These findings indicate that AUS_001 exerts a high degree of potency across 30 cancer types with a selectivity for cancer versus normal cells. Maximum tolerated dose studies in mice illustrated that orally administered AUS_001 is well tolerated at doses 4 times greater than those that conferred antitumor efficacy, with no myelosuppression or other overt toxicities observed. Predictive toxicology and safety screening indicated that AUS_001 exerts a strong safety profile as evidenced by absence of mutagenic potential in the Ames test using up to 100 µM drug doses and low hERG-blocking liability with a half-maximal inhibitory concentration of 65 µM. Notably, AUS_001 is a poor substrate of the drug efflux transporter, P-glycoprotein 1 (Pgp), as it does not inhibit drug-stimulated Pgp ATPase activity and retains efficacy in high Pgp-expressing models in vitro. Subsequently, the effects of AUS_001 were explored in the ToxTracker® assay, consisting of a panel of mammalian stem cell lines that contain fluorescent reporters for the exploration of DNA damage, oxidative stress, and protein damage using flow cytometry. Robust activation of the Rtkn genotoxicity reporter under low cytotoxicity conditions, in combination with accumulation of cells in G2/M phases of the cell cycle within 4 h and an increase in aneuploid cells after 24 h of drug treatment, suggested an aneugenic mode of action, typically caused by tubulin-targeting agents or mitotic kinase inhibitors. Biochemical tubulin polymerization assays then revealed that AUS_001 acts as a direct tubulin destabilizing agent and monitoring microtubule dynamics in GFP-tubulin reporter cells provided evidence that AUS_001 blocks microtubule assembly. Our studies have elaborated the mechanism of AUS_001 as an inhibitor of tubulin polymerization. The favorable safety profile of AUS_001, along with its ability to circumvent Pgp-mediated multidrug resistance, provides potential for efficacy against multiple cancers where microtubule destabilization is proven to be an effective target. Citation Format: Herman Lelie, Inger Brandsma, Giel Hendriks, Lee R. Cavedine, Brogan A. Epkins, Steven M. Garner, Andrew J. Cook, Muthukrishnan Renganathan, Yong Zhao, Alastair J. King, Peter Y.W. Chan, April Risinger, Marina Koutsioumpa. A novel microtubule disruptor exerts broad anticancer efficacy with a tolerable safety profile [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 4701.

Abstract 507: HLS-22001 induces G2/M cell cycle arrest and apoptotic cell death via tubulin depolymerization in colon cancer

Abstract Colorectal cancer (CRC) is one of the main malignant tumors and among the leading cause of death in cancer patients worldwide considering that recurrence and chemotherapeutic drug resistance are highly common in the advanced stage of CRC. Mutations in KRAS and BRAF occur frequently in colorectal cancers, and abnormal cell cycles caused by these mutations affect tumor progression and metastasis. Accordingly, inhibition of cell cycle has been a potential target for colorectal cancers and therefore, we have developed a new lead compound, HLS-22001. Our studies confirmed that HLS-22001 inhibited the proliferation of several cancer cells and significantly induced G2/M arrest and apoptosis of colon cancer cells and chemotherapy resistant colon cancer cells. The docking model studies showed that HLS-22001 was expected to bind to the colchicine binding site of beta-tubulin, and in fact, as a tubulin inhibitor, it had the effect of inhibiting tubulin dimerization and polymerization in the microtubule dynamics. Consistently, in xenograft mouse model, oral administration of HLS-22001 significantly suppressed tumor weight and volume without acute toxicity in mice. Collectively, these results suggest further development of HLS-22001 as a novel and promising oral drug for colorectal cancer. Citation Format: Chi-hyun Ahn, Gyu-beom Jang, Chunhwa Kim, Junho Lee, Taewon Kim, Youngeun Yang, Eunji Cho, Minchae Kim, Yun-tai Kim, Yong-Hae Han, Heebum Kang. HLS-22001 induces G2/M cell cycle arrest and apoptotic cell death via tubulin depolymerization in colon 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 507.

Abstract 513: Intra-tubular damage is targeted by maytansinoids and rescued by NF1: Revisiting mechanism and biomarkers of an established ADC payload

Abstract There is great interest in the identification of biomarkers to guide development of antibody-drug conjugates (ADC). We previously showed that loss of Neurofibromatosis 1 (NF1), a gene frequently mutated across cancers, enhances the activity of DM1, the maytansinoid payload of T-DM1, through a novel function in regulating microtubule (MT) dynamics. Maytansinoids are puzzlingly more effective in cells (in the nanomolar range) vs in vitro (in the micromolar range). Since maytansinoids bind at the interface between tubulin dimers, they are thought to only bind soluble tubulin dimers or MT ends, which would suggest very few binding sites available for pharmacological interaction in vivo, at odds with data. Here we investigated the interaction of DM1 with NF1 and MTs, using cellular and reductionist in vitro systems. To measure in vivo MT dynamics, we transiently transfected the MT end-binding protein EB3-GFP and reconstructed MT trajectories by live-cell imaging. Upon DM1 treatment, KO cells showed a highly significant reduction in MT speed, demonstrating a direct role for NF1 on MT dynamics in cells. In turbidity-based tubulin polymerization assays, recombinant NF1 greatly accelerated polymerization, and completely rescued DM1-induced inhibition. Visual inspection of fluorescent MTs showed that NF1 induced significant MT bundling, a defining feature of many MT-associated proteins, which generates signal indistinguishable from true MT polymerization in turbidity assays. To follow the dynamics of individual microtubules, we applied Total Internal Reflection (TIRF) microscopy on glass-immobilized MTs. As expected, polymerization in the presence of NF1 led to a significant increase in MT dynamics (elongation speed, rescue and catastrophe rate). Expectedly, DM1 led to significant reduction in the fraction of elongating MTs and speed, but these defects were completely or partially rescued by NF1. Importantly, DM1 did not only lead to MT shortening (as proposed by the current model), but also to clear and frequent MT fracturing, indicating that the drug is not only engaging MT ends but also intra-tubular binding sites. This is consistent with recent models of MT formation which incorporate the frequent presence of areas of discontinuity or damage induced by mechanical stress, exposing intra-tubular DM1 binding sites. Interestingly, adding NF1 to DM1-treated MTs generated areas of de novo intra-tubular tubulin insertion, coincident with damaged sites, suggesting an entirely novel role for NF1 in MT repair.In conclusion, we provide evidence for a model in which maytansinoids bind not only to soluble tubulin dimers and MT ends, but also to intra-tubular damaged sites. Thus, the number of binding sites in cells would be proportional to MT damage, suggesting a mechanism for differential efficacy across tumor types and a potential avenue for combinatorial drug development. Citation Format: Eleonora Messuti, Bruno Achutti Duso, Alessia Castiglioni, Giulia Tini, Emanuele Bonetti, Giuseppe Ciossani, Silvia Monzani, Daria Khuntsariya, Zdeněk Lánský, Marcus Braun, Luigi Scietti, Luca Mazzarella. Intra-tubular damage is targeted by maytansinoids and rescued by NF1: Revisiting mechanism and biomarkers of an established ADC payload [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 513.

Abstract 7140: A new series of flavonoid derivatives with anti-cancer activity includes SDN207, SND210, SND524, and SND562 as potent microtubule-targeting agents

Abstract Background: Plant-derived compounds, including flavonoids, can be potent anti-cancer agents, but low bioavailability, lack of specificity, and complexity in their synthesis can hamper their clinical development. Here, we present seven novel flavonoid derivatives, and test them for their anti-tumor activity in various models derived from hematological cancers and solid tumors. Methods: MTT assessed anti-proliferative activity after 72h of exposure to increasing concentrations of drugs. Tubulin Polymerization Assay was performed with 3-fold dilutions (15-0.02 μM), compared to paclitaxel (1-3 μM) or vinblastine (3 μM) as references. The OncolinesProfiler analysis was used to compare IC50 fingerprints determined on the Oncolines cell line panel to the IC50 fingerprints of 213 pre-profiled reference drugs. Results: SDN207, SND210, SND216, SND462, SND504, SND524, and SND562 were designed and synthesized using a flavonoid polyphenol scaffold selectively modified to markedly increase cytotoxicity towards cancer cells. All seven compounds showed dose-dependent anti-proliferative activity across 15 lymphoma cell lines derived from germinal center B-cell-like (GCB) diffuse large B cell lymphoma (DLBCL) (n=6), activated B cell-like (ABC) DLBCL (n=2), mantle cell lymphoma (n=4), and marginal zone lymphoma (MZL) (n=3). Activity in the nM range was seen with five compounds: SDN207 (median IC50 1.6 nM; 95%CI, 1.3-3.2), SND210 (1.4 nM; 95%CI, 0.9-3.3), SND216 (19.5 nM; 95%CI, 10-38 nM), SND562 (28.9 nM; 95%CI, 15.4-57.7), and SND524 (46.7 nM; 95%CI, 32-133.3). Both other compounds had a median IC50 of 2 μΜ (SND462, 95%CI, 1.1-4.5 μM; SND504, 95%CI, 0.5-3.9 μM). There were no differences based on lymphoma histology. The anti-tumor activity was maintained in MZL cell lines with acquired resistance to BTK and PI3K inhibitors (n=3). Six compounds were also assessed for their anti-tumor activity in models other than lymphomas (SDN207, n=31; SND210, n=20; SND462, n=4; SND504, n=4; SND524, n=9; SND562, n=35), confirming the higher activity for SDN207, SND210, SND562, and SND524. Regarding the mechanism of action, up to now, a tubulin polymerization assay was performed for three compounds. Microtubule depolymerization (SND207), and microtubule polymerization (SND524, SND562) were observed at concentrations as low as 20 nM. The OncolinesProfiler identified the tubulin-targeting agents vinblastine, vincristine, paclitaxel, and docetaxel among the top reference compounds most correlated with SND207 and SND210 (i.e., Pearson correlation > 0.5). Conclusions: Novel compounds developed using plant-derived skeletons have shown in vitro anti-tumor activity in lymphomas, leukemias, and solid tumors, including models of resistance to targeted agents. Microtubule targeting appeared as a possible mechanism of action for at least four of them. Citation Format: Alberto J. Arribas, Eleonora Cannas, Eugenio Gaudio, Paulina Biniecka, Dan Stoicescu, Francesco Bertoni. A new series of flavonoid derivatives with anti-cancer activity includes SDN207, SND210, SND524, and SND562 as potent microtubule-targeting agents [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 7140.

Abstract 7139: Anti-cancer effects of benzimidazole derivative BNZ-111 on paclitaxel-resistant ovarian cancer

Abstract Ovarian cancer, a leading cause of cancer-related deaths in women, remains a formidable challenge, especially in the context of platinum-resistant disease. This study investigated the potential of the benzimidazole derivative BNZ-111 as a novel treatment strategy for platinum-resistant ovarian cancer. With improved pharmacokinetic parameters, BNZ-111 effectively disrupts tubulin polymerization through hydrogen bonds and hydrophobic interactions. It demonstrated strong cytotoxicity in both chemo-sensitive and chemo-resistant epithelial ovarian cancer cell lines, inducing apoptosis and G2/M cell cycle arrest. In vivo experiments using orthotopic and patient-derived xenograft models showed significant tumor growth inhibition without apparent toxicity to vital organs. Unlike paclitaxel, BNZ-111 proved effective in paclitaxel-resistant cells, potentially by bypassing interaction with MDR1 and modulating β-3 tubulin expression to suppress microtubule dynamics. This study suggests that BNZ-111, with favorable drug-like properties, holds promise as a therapeutic option for platinum-resistant ovarian cancer, addressing a critical clinical need in gynecologic oncology Citation Format: Jeong-Won Lee, Kwangho Lee, Byumseok Koh, Yoo-Young Lee, Duk-Soo Bae. Anti-cancer effects of benzimidazole derivative BNZ-111 on paclitaxel-resistant ovarian 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 7139.

Abstract 7144: BR0L075 (SCB01A), a microtubule inhibitor targeting the colchicine binding site, induces immunogenic cell death

Abstract BPR0L075 (also known as SCB01A in phase II clinical trial) is a novel anti-microtubule drug with potent anti-tumor and anti-angiogenic activity in vitro and in vivo. BPR0L075 inhibits tubulin polymerization (microtubule de-stabilizer) by binding to the colchicine-binding site of tubulin. We previously reported that BPR0L075 overcomes paclitaxel (microtubule stabilizer) resistance and induces vascular-disruption in vitro and in vivo. We observed that breast and ovarian cancer cells treated with BPR0L075 in vitro induced key hallmarks of immunogenic cell death (ICD). In both human and murine, ovarian (OVCAR3, ID8) and breast cancer (MDA-MB-231, 4T1) cell lines, treatment with low nanomolar concentration of BPR0L075 significantly decreased the viability of cells evidenced by SRB assay. Using a known ICD inducer mitoxantrone as a positive control, both flow cytometric analysis and immunofluorescence staining assays demonstrated that BPR0L075 treatment led to a time- and dose-dependent translocation of calreticulin, an endoplasmic reticulum chaperone protein, on the plasma membrane of dying cells which sends an “eat me” signal. The BPR0L75 treatment also led to secretion of High Mobility Group Box 1 (HMGB1), a non-histone chromatin-binding nuclear protein, as well as adenosine triphosphate (ATP) from the cell into the extracellular space using ELISA assay. These damage-associated molecular patterns (DAMPs) are known to lead to antigen-presenting cell recruitment, phagocytosis, maturation of dendritic cells, and chemotaxis. The appearance of the ICD biomarkers (calreticulin exposure, HMGB1 and ATP release) supports BPR0L075 as an ICD-inducing antimitotic agent. Further in vivo studies are warranted to delineate the effect of BPR0L075 on immune activation and recruitment of immune cells to the tumor microenvironment. Citation Format: Ayesha Bano, Xinli Liu. BR0L075 (SCB01A), a microtubule inhibitor targeting the colchicine binding site, induces immunogenic cell death [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 7144.

Genetic imputation of kidney transcriptome, proteome and multi-omics illuminates new blood pressure and hypertension targets

Genetic mechanisms of blood pressure (BP) regulation remain poorly defined. Using kidney-specific epigenomic annotations and 3D genome information we generated and validated gene expression prediction models for the purpose of transcriptome-wide association studies in 700 human kidneys. We identified 889 kidney genes associated with BP of which 399 were prioritised as contributors to BP regulation. Imputation of kidney proteome and microRNAome uncovered 97 renal proteins and 11 miRNAs associated with BP. Integration with plasma proteomics and metabolomics illuminated circulating levels of myo-inositol, 4-guanidinobutanoate and angiotensinogen as downstream effectors of several kidney BP genes (SLC5A11, AGMAT, AGT, respectively). We showed that genetically determined reduction in renal expression may mimic the effects of rare loss-of-function variants on kidney mRNA/protein and lead to an increase in BP (e.g., ENPEP). We demonstrated a strong correlation (r = 0.81) in expression of protein-coding genes between cells harvested from urine and the kidney highlighting a diagnostic potential of urinary cell transcriptomics. We uncovered adenylyl cyclase activators as a repurposing opportunity for hypertension and illustrated examples of BP-elevating effects of anticancer drugs (e.g. tubulin polymerisation inhibitors). Collectively, our studies provide new biological insights into genetic regulation of BP with potential to drive clinical translation in hypertension.