Tubulin-binding Agents Research Articles

PATH-13. MOLECULAR AND BIOLOGY CORRELATIVE RESULTS FROM THE PHASE 1B TRIAL OF UNESBULIN (PTC596) WITH RADIOTHERAPY IN CHILDREN NEWLY-DIAGNOSED WITH DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) AND HIGH-GRADE GLIOMA (HGG): A REPORT FROM THE COLLABORATIVE NETWORK FOR NEURO-ONCOLOGY CLINICAL TRIALS (CONNECT)

Abstract BACKGROUND Within the CONNECT1702 trial of unesbulin (tubulin-binding agent causing BMI-1 modulation) with radiotherapy in patients with DIPG/HGG, tumor tissue was analyzed at diagnosis and/or autopsy for biology correlatives, enabling comprehensive, longitudinal molecular characterization. METHODS Whole genome sequencing, RNA-sequencing, and Western blot analyses were performed on tumor, normal brain, and peripheral blood mononuclear cells (PBMCs). RESULTS Tumor tissue from diagnosis (n=11) and/or autopsy (n=7) was analyzed for 16 patients (DIPG=9; HGG=7). Western blot analyses demonstrated 1) BMI-1 overexpression in baseline tumor compared to normal brain and 2) primary on-target effect of BMI1-modulation causing M-phase mitotic arrest when comparing pre- and post-treated PBMCs (increased H3S10-phosphorylation). Among 9 DIPG patients, alterations in the following genes were identified: H3-3A (H3.3) (n=5), H3C2 (H3.1) (n=2, both with co-occurring ACVR1 and PIK3CA mutations), TP53 (n=5), ATRX (n=2), and PPM1D (n=2). Tumors from two longer DIPG survivors were H3.1-, ACVR1-mutant (overall survival [OS]=26 months) and MET-amplified (OS=18 months); one patient with H3.3K27M-DIPG had germline MSH6 mutation (Lynch syndrome) (OS=12.6 months). Among 7 HGG patients, alterations in the following genes were identified: H3-3A (n=5), TP53 (n=3), NF1 (n=3), PDGFRA (n=1), EGFR (n=1), and PIK3CA (n=1). A BRAFV600E mutation was observed in the thalamic HGG patient (H3.3K27M) with greatest progression-free survival (>23 months). Early leptomeningeal metastases occurred in one DIPG patient with BCOR alteration and one HGG patient, with MYCN amplification identified in the spinal metastasis, but not intracranial disease. From gene set enrichment analyses, pathways enriched in patients with longer survival included telomerase regulation and ATP synthesis, whereas pathways enriched in patients with shorter survival included interneuron development, neuron semaphorin signaling, and gamma aminobutyric signaling. CONCLUSIONS Comprehensive molecular profiling of diagnostic and post-mortem tumor tissue provides valuable early biological insight, elucidating potential genomic biomarkers predictive of outcome and response to M-phase alteration and BMI-1 modulation in DIPG/HGG.

HURP binding to the vinca domain of β-tubulin accounts for cancer drug resistance

Vinca alkaloids, a class of tubulin-binding agent, are widely used in treating cancer, yet the emerging resistance compromises their efficacy. Hepatoma up-regulated protein (HURP), a microtubule-associated protein displaying heightened expression across various cancer types, reduces cancer cells’ sensitivity to vinca-alkaloid drugs upon overexpression. However, the molecular basis behind this drug resistance remains unknown. Here we discover a tubulin-binding domain within HURP, and establish its role in regulating microtubule growth. Cryo-EM analysis reveals interactions between HURP’s tubulin-binding domain and the vinca domain on β-tubulin -- the site targeted by vinca alkaloid drugs. Importantly, HURP competes directly with vinorelbine, a vinca alkaloid-based chemotherapeutic agent, countering microtubule growth defects caused by vinorelbine both in vitro and in vivo. Our findings elucidate a mechanism driving drug resistance in HURP-overexpressing cancer cells and emphasize HURP tubulin-binding domain’s role in mitotic spindle assembly. This underscores its potential as a therapeutic target to improve cancer treatment.

Interaction of norsecurinine-type monomeric and dimeric alkaloids with α-tubulin: a molecular docking study

Aim: New microtubule-targeting agents are needed to improve cancer treatment. The recent characterization of the anticancer alkaloid securinine as a tubulin-binding agent prompted us to explore the interaction of related monomeric and dimeric analogues with tubulin. The interaction between the α/β-tubulin dimer and alkaloids fluevirines A–F and flueggenines A–I, isolated from the bush Flueggea virosa (Roxb. ex Willd.) Royle, was investigated using molecular docking. Methods: Two molecular models were initially compared for the binding of securinine to α/β-tubulin. The pironetin-binding site model (5FNV) was selected for the subsequent docking analysis with all compounds. Empirical energies of interaction (ΔE) were measured and compared. Results: Fluevirine A has been identified as a potent tubulin binder. This dimeric alkaloid formed more stable complexes with tubulin than the monomeric counterparts, such as fluevirines B–D. The bis-indole derivative fluevirine E also provided more stable complexes than (nor)securinine. The study was extended to the dimeric alkaloids flueggenines A–I and three compounds were identified as potential tubulin binders: the polycyclic product flueggenine B, the norsecurinine-indole hybrid flueggenine E, and the norsecurinine dimer flueggenine I. This later compound proved to be well adapted to fit into the pironetin site of tubulin, extending its two norsecurinine units between the colchicine-binding area and the pironetin site, in close proximity to the pironetin-reactive cysteine-316 residue. Structure-binding relationships were delineated. Conclusions: The study identifies the dimeric alkaloids fluevirine A and flueggenine I as potential α-tubulin binding agents. For the first time, dimeric alkaloids including two C-C connected norsecurinine units are characterized as tubulin ligands. The study contributes to a better understanding of the mechanism of action of Flueggea alkaloids and should help the design of anticancer analogues targeting the pironetin site of α-tubulin.

Retraction: Integrating mechanisms of response and resistance against the tubulin binding agent Eribulin in preclinical models of osteosarcoma.

Retraction: Integrating mechanisms of response and resistance against the tubulin binding agent Eribulin in preclinical models of osteosarcoma.

Debio 1562M, a Next Generation Antibody Drug Conjugate (ADC) Targeting CD37 for AML and MDS Treatment

CD37 is a trans-membrane protein specifically expressed on normal hematopoietic tissues such as B cells, neutrophils and macrophages, and has demonstrated roles in adhesion, cell migration (de Winde et al, 2018; Wee et al, 2015), survival and apoptotic signals (Lapalombella et al, 2012). Increased expression of CD37 has been observed in various hematological cancers such as lymphomas, Chronic Lymphocytic Leukemia (CLL) and Acute Myeloid Leukemia (AML) (Beckwith et al, 2014; Deckert et al, 2013; Pereira et al, 2015; Yan et al, 2021). Higher expression of CD37 was also associated with poor patient outcome in AML (Zhang et al, 2020; Larkin et al, ASH 2018). Debio 1562M is a second-generation novel ADC directed against CD37 and including Debiopharm's proprietary Multilink cleavable linker-payload technology. Multilink improves manufacturing to achieve homogenous high drug antibody ratio while retaining excellent solubility as well as favorable biologic characteristics with preserved internalization kinetics on target cells. Debio 1562M is conjugated to 8 molecules of a tubulin-binding agent and despite cleavable linker that ensure effective intracellular delivery of toxic payload, we show sustained plasma stability in vitro and in vivo with no premature extracellular release. In this work, we report Debio 1562M specificity and potent antitumor activity in multiple AML models both in vitro and in vivo. First, in our in vitro cytotoxicity assay, Debio 1562M displayed nano- to picomolar anti-proliferative activity in both AML cell lines and primary samples. Out of 30 primary samples tested, 20 were sensitive to Debio 1562M with IC50 ranging from 1 to 10nM, this regardless of their mutation status, subtype or medical history. Interestingly, we observed that AML cells have high internalization capability and therefore relatively low levels of surface CD37 is sufficient for cytotoxic activity. The specificity of Debio 1562M was demonstrated by using 3 different CD37 knock out AML cell lines which were generated by CRISPR-Cas9 technology. Parental and CD37 KO cells were treated for 72h with a dose range of Debio 1562M and viability was measured (Figure 1). While Debio 1562M has an IC50 below 1nM in all 3 parental cell lines, its activity is reduced by more than 2 log in the KO cell lines. In vivo, in 3 different AML cell line xenograft mouse models, a single injection of Debio 1562M at 5mg/kg induced complete tumor regression and all mice remained alive at study termination, more than 60 days after treatment, except one mouse in one model. In a patient derived xenograft (PDX) model, the same dose of Debio 1562M significantly reduced tumor burden in blood and bone marrow with sustained activity 42 days after treatment (37% and 98% of blasts in blood and bone marrow respectively in control animals versus 0% and 3% in treated mice, pvalue<0.001). Furthermore, we evaluated the activity of Debio 1562M over venetoclax and azacytidine combination in the MV4-11 Luciferase xenografted mouse model. NCG mice were inoculated with 1x10 7 cells in the tail vein and randomization in groups of 8 mice was performed 14 days later. Mice received either one administration of Debio 1562M at 5mg/kg or a combination of venetoclax (QDx14 100mg/kg) and azacitidine (QDx5 3,5mg/kg). Complete regression of the tumor was observed in all mice after Debio 1562M administration and all mice survived at study termination, 75 days after treatment while none survived after 56 days when treated with venetoclax and azacitidine combination (Figure 2). The toxicology profile of Debio 1562M in mice showed expected clinical signs related to the payload's off target toxicity but with a significant safety margin. Interestingly, no peripheral neuropathy symptoms were detected, as commonly observed for this class of payload. Additionally, Multilink TM-payload construct alone was not associated with any signs of toxicity. On-target toxicity profile couldn't be evaluated as Debio 1562M isn't cross reactive with mouse CD37, however 1 st generation CD37 ADC has already demonstrated safety and tolerability in Phase 1 and 2 clinical trials.Overall, our data confirm the relevance of CD37 targeting for AML treatment. The excellent antitumor activity and tolerability of Debio 1562M in several pre-clinical models now pave the way for upcoming clinical development.

Novel Tetrazole Derivatives Targeting Tubulin Endowed with Antiproliferative Activity against Glioblastoma Cells.

Increasing awareness of the structure of microtubules has made tubulin a relevant target for the research of novel chemotherapies. Furthermore, the particularly high sensitivity of glioblastoma multiforme (GBM) cells to microtubule disruption could open new doors in the search for new anti-GBM treatments. However, the difficulties in developing potent anti-tubulin drugs endowed with improved pharmacokinetic properties necessitates the expansion of medicinal chemistry campaigns. The application of an ensemble pharmacophore screening methodology helped to optimize this process, leading to the development of a new tetrazole-based tubulin inhibitor. Considering this scaffold, we have synthesized a new family of tetrazole derivatives that achieved remarkable antimitotic effects against a broad panel of cancer cells, especially against GBM cells, showing high selectivity in comparison with non-tumor cells. The compounds also exerted high aqueous solubility and were demonstrated to not be substrates of efflux pumps, thus overcoming the main limitations that are usually associated with tubulin binding agents. Tubulin polymerization assays, immunofluorescence experiments, and flow cytometry studies demonstrated that the compounds target tubulin and arrest cells at the G2/M phase followed by induction of apoptosis. The docking experiments agreed with the proposed interactions at the colchicine site and explained the structure-activity relationships.

Mechanism-based peripheral blood biomarkers to evaluate anticancer effects of the plinabulin/docetaxel combination (plin/doc) vs. doc alone in second/third line EGFR wild-type (WT) NSCLC.

e21017 Background: Pretreatment neutrophil to lymphocyte (L) (NLR), platelet to L (PLR), and L to monocyte (LMR) levels are established prognostic biomarkers for advanced NSCLC. Plinabulin (Plin) is a well-tolerated, differentiated tubulin-binding agent being developed for neutropenia prevention as a monotherapy and in combination, and for improving the efficacy of anticancer agents. DUBLIN-3 (NCT02504489) was a randomized, single-blinded (pts only), active-controlled Ph3 study in 2nd/3rd line stage IIIB/IV, EGFR WT NSCLC with a measurable lung lesion (RECIST 1.1). Pts (N = 559) were randomized 1:1 to Plin/Doc or Doc (21-day (D) cycle (C)). Doc (75 mg/m2) on D1 and Plin (30 mg/m2) on D1/D8 were given by IV infusion with steroid premedication (8 mg PO BID x 3D). Differential WBCs and platelet counts were collected at baseline (pre-steroid), D1, and D8. We analyzed the posttreatment effects of Plin on NLR, PLR, and LMR. Methods: All evaluable Plin/Doc vs Doc pts were included. NLR/PLR/LMR ratios and their respective change from baseline (cNLR/cPLR/cLMR) were obtained from blood draws taken at predose, C1D8, and/or C2D8. For intra/inter-arm overall survival (OS) stratification, baseline median NLR (3.2), PLR (177), and LMR (3.5) values were selected as thresholds. By examining responders & long-term survivors in both arms, posttreatment NLR/LMR and cNLR/cLMR thresholds were established for C1D8 and C2D8. Finally, PFS/OS benefits were stratified to determine the statistical significances of Plin/Doc vs Doc. Results: In agreement with literature, baseline threshold levels for NLR = 3.2, PLR = 177, LMR = 3.5 were statistically significantly predictive of OS in both the Plin/Doc and Doc arms (p values ranging from 0.05 to 0.0001). Plin prevents Doc-induced neutropenia, and post-Doc neutrophil counts were higher on D8 with Plin/Doc vs Plin (p < 0.001). Despite Doc-induced myelosuppression, Plin/Doc responding pts maintained similar NLR/LMR values as baseline at C1D8 and C2D8. After a single Plin dose at C1D8, lower NLR (cutoff = 3.0; 1.2x median value of all pts) and higher LMR (cutoff = 4.5; median value of all pts) thresholds independently predicted PFS/OS benefits of Plin/Doc vs Doc. Further, a higher cNLR threshold (cutoff = 0.25) at C2D8, best predicted Plin/Doc vs Doc in PFS and OS (p < 0.0001 and p = 0.0035 respectively). Conclusions: Bone marrow suppression remains a common serious risk of classical/non-classical cancer drugs. Under these conditions, the addition of Plin offers rapid protection of GMP lineage cells which can be captured by simple blood cell counts. Incorporation of blood-based biomarkers may help enrich NSCLC pts with better sensitivity to Plin/Doc combination than Doc alone. Clinical trial information: NCT02504489 .

Abstract 1545: Targeting anticancer agents to the tumor microenvironment with cathepsin B cleavable drug-linker conjugates of phosphatidylserine-binding proteins

Abstract In healthy cells, the membrane lipid phosphatidylserine (PS) is confined to the inner leaflet of the plasma membrane but in cancer cells and in the tumor vasculature PS is prevalent in the outer leaflet where it is highly immunosuppressive. In this study engineered proteins, such as Bavituximab, a chimeric monoclonal antibody that binds to PS via the abundant serum protein β2-glycoprotein 1, and constructs containing the PS binding domain of β2-glycoprotein 1 (betabodies), are conjugated to anticancer drug-linkers designed to be cleavable by cathepsin B. Cathepsin B is a cysteine protease that is abundant in the lysosomes of normal cells but is upregulated and trafficked to the cell surface and is secreted into the extracellular parenchyma in solid tumors, including breast and pancreatic cancer. A series of drug-linker constructs were synthesized that incorporate unique small-molecule payloads with dual mechanism of action, functioning as both potent tubulin-binding antimitotic agents and vascular disrupting agents. The payloads include benzosuberene-based aniline KGP156 and its phenolic congener KGP18, dihydronaphthalene-based KGP05, and indole-based OXi8006. These drug-linker constructs were reacted with N-acetyl-L-cysteine (NAC) and evaluated for cathepsin B cleavage, and liberation of payload after spontaneous 1,6-fragmentation and decarboxylation. The drug linker construct, NAC-succinimidocaproyl-L-valine-L citrulline-p-aminobenzyl carbamate derivatized with aminobenzosuberene, KGP156, was cleaved by cathepsin B (260 nM) with release of payload at the rate of 12.7 μM/min and by cathepsin L (600 nM) at a rate of 11. 5 μM/min. Other drug-linker constructs in this series demonstrated slower release of payload upon cleavage with cathepsin B or cathepsin L. The drug-linker construct incorporating aminodihydronaphthalene-based KGP05 released KGP05 at a rate of 2.4 μM/min with a comparable rate upon exposure to cathepsin L. Payload release from drug-linker constructs was not observed, over 24 h in the absence of enzyme. Drug-linker constructs without the Val-Cit dipeptide were not cleaved by cathepsin B. The most efficient cathepsin B drug-linker substrate, Mc-Val-Cit-PABC-KGP156, was conjugated to Baxituximab to form the corresponding antibody-drug conjugate (ADC). Baxituximab was first reacted with 2-iminothiolane, a thiolating agent that reacts with accessible lysine residues. Reaction with Ellman’s reagent revealed an -SH/antibody ratio of 3.5. Conjugation of thiolated Bavituximab with Mc-Val-Cit-PABC-KGP156 gave a drug/antibody ratio (DAR) of 3.6/1 after cleavage by cathepsin B and determination of released KGP156 by LC-MS. These studies revealed 1) distinct differences in cleavage rates for drug-linkers, 2) cleavage by cathepsin L, and 3) efficient cathepsin B mediated release of payload from the ADC. Citation Format: Yuling Deng, Jacob W. Ford, Jenny M. VanNatta, Casey J. Maguire, Deboprosad Mondal, Natalie Z. Phinney, Rolf A. Brekken, Kevin G. Pinney, Mary Lynn Trawick. Targeting anticancer agents to the tumor microenvironment with cathepsin B cleavable drug-linker conjugates of phosphatidylserine-binding proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1545.

Noscapine-Amino Acid Conjugates Suppress the Progression of Cancer Cells.

Lung cancer is the leading cause of cancer deaths globally; 1 in 16 people are diagnosed with lung cancer in their lifetime. Microtubules, a critical cytoskeletal assembly, have an essential role in cell division. Interference with the microtubule assembly leads to genetic instability during mitosis and cancer cell death. Currently, available antimitotic drugs such as vincas and taxanes are limited due to side effects such as alopecia, myelosuppression, and drug resistance. Noscapine, an opium alkaloid, is a tubulin-binding agent and can alter the microtubule assembly, causing cancer cell death. Amino acids are fundamental building blocks for protein synthesis, making them essential for the biosynthesis of cancer cells. However, the ability of amino acids in drug transportation has yet to be exploited in developing noscapine analogues as a potential drug candidate for cancer. Hence, in the present study, we have explored the ninth position of noscapine by introducing a hydroxymethylene group using the Blanc reaction and further coupled it with a series of amino acids to construct five target conjugates in good yields. The synthesized amino acid conjugate molecules were biologically evaluated against the A549 lung cancer cell line, among which the noscapine-tryptophan conjugate showed IC50 = 32 μM, as compared to noscapine alone (IC50 = 73 μM). Morphological changes in cancer cells, cell cycle arrest in the G1 phase, and ethidium bromide/acridine orange staining indicated promising anticancer properties. Molecular docking confirmed strong binding to tubulin, with a score of -41.47 kJ/mol with all 3D coordinates and significant involvement of molecular forces, including the hydrogen bonds and hydrophobic interactions. Molecular dynamics simulations demonstrated a stable binding of noscapine-tryptophan conjugate for a prolonged time (100 ns) with the involvement of free energy through the reaction coordinates analyses, solving the bioavailability of parent noscapine to the body.

From seaside to bedside: Current evidence and future perspectives in the treatment of breast cancer using marine compounds.

To date, only few marine natural compounds have been proved to be active in breast cancer (BC). The main marine-derived drugs that have been studied for the treatment of BC are tubulin-binding agents (eribulin and plocabulin), DNA-targeting agents (cytarabine and minor groove binders—trabectedin and lurbinectedin) and Antibody-Drug Conjugates (ADCs). Notably, eribulin is the only approved cytotoxic drug for the treatment of advanced BC (ABC), while cytarabine has a limited indication in case of leptomeningeal diffusion of the disease. Also plocabulin showed limited activity in ABC but further research is needed to define its ultimate potential role. The available clinical data for both trabectedin and lurbinectedin are of particular interest in the treatment of BRCA-mutated tumours and HR deficient disease, probably due to a possible immune-mediated mechanism of action. One of the most innovative therapeutic options for the treatment of BC, particularly in TNBC and HER2-positive BC, are ADCs. Some of the ADCs were developed using a specific marine-derived cytotoxic molecule as payload called auristatin. Among these, clinical data are available on ladiratuzumab vedotin and glembatumumab vedotin in TNBC, and on disitamab vedotin and ALT-P7 in HER2-positive patients. A deeper knowledge of the mechanism of action and of the potential predictive factors for response to marine-derived drugs is important for their rational and effective use, alone or in combination. In this narrative review, we discuss the role of marine-derived drugs for the treatment of BC, although most of them are not approved, and the opportunities that could arise from the potential treasure trove of the sea for novel BC therapeutics.

Selective Delivery of Clinically Approved Tubulin Binding Agents through Covalent Conjugation to an Active Targeting Moiety.

The efficacy and tolerability of tubulin binding agents are hampered by their low specificity for cancer cells like most clinically used anticancer agents. To improve specificity, tubulin binding agents have been covalently conjugated to agents that target cancer cells to give actively targeted drug conjugates. These conjugates are designed to increase uptake of the drug by cancer cells while having limited uptake by normal cells, thereby improving efficacy and tolerability. Approaches used include an attachment to small molecules, polysaccharides, peptides, proteins, and antibodies that exploit the overexpression of receptors for these substances. Antibody targeted strategies have been the most successful to date, with six such examples having gained clinical approval. Many other conjugate types, especially those targeting the folate receptor, have shown promising efficacy and toxicity profiles in pre-clinical models and in early-stage clinical studies. Presented herein is a discussion of the success or otherwise of the recent strategies used to form these actively targeted conjugates.

Demonstrating Tumor Vascular Disrupting Activity of the Small-Molecule Dihydronaphthalene Tubulin-Binding Agent OXi6196 as a Potential Therapeutic for Cancer Treatment.

Simple SummaryVascular-disrupting agents promise significant therapeutic efficacy against solid tumors by selectively damaging tumor-associated vasculature. Following administration of the phosphate prodrug OXi6197, dynamic bioluminescence imaging revealed dose-responsive vascular shutdown in MDA-MB-231 human breast tumor xenografts and for the first time murine RENCA kidney tumors. Rapid vascular disruption was observed, which continued over 24 h. Histology showed vascular congestion, hemorrhage and necrosis. Twice-weekly doses of OXi6197 caused a significant growth delay in MDA-MB-231 breast tumors and many RENCA kidney tumors growing orthotopically in mice.The vascular disrupting activity of a promising tubulin-binding agent (OXi6196) was demonstrated in mice in MDA-MB-231 human breast tumor xenografts growing orthotopically in mammary fat pad and syngeneic RENCA kidney tumors growing orthotopically in the kidney. To enhance water solubility, OXi6196, was derivatized as its corresponding phosphate prodrug salt OXi6197, facilitating effective delivery. OXi6197 is stable in water, but rapidly releases OXi6196 in the presence of alkaline phosphatase. At low nanomolar concentrations OXi6196 caused G2/M cell cycle arrest and apoptosis in MDA-MB-231 breast cancer cells and monolayers of rapidly growing HUVECs underwent concentration-dependent changes in their morphology. Loss of the microtubule structure and increased bundling of filamentous actin into stress fibers followed by cell collapse, rounding and blebbing was observed. OXi6196 (100 nM) disrupted capillary-like endothelial networks pre-established with HUVECs on Matrigel®. When prodrug OXi6197 was administered to mice bearing orthotopic MDA-MB-231-luc tumors, dynamic bioluminescence imaging (BLI) revealed dose-dependent vascular shutdown with >80% signal loss within 2 h at doses ≥30 mg/kg and >90% shutdown after 6 h for doses ≥35 mg/kg, which remained depressed by at least 70% after 24 h. Twice weekly treatment with prodrug OXi6197 (20 mg/kg) caused a significant tumor growth delay, but no overall survival benefit. Similar efficacy was observed for the first time in orthotopic RENCA-luc tumors, which showed massive hemorrhage and necrosis after 24 h. Twice weekly dosing with prodrug OXi6197 (35 mg/kg) caused tumor growth delay in most orthotopic RENCA tumors. Immunohistochemistry revealed extensive necrosis, though with surviving peripheral tissues. These results demonstrate effective vascular disruption at doses comparable to the most effective vascular-disrupting agents (VDAs) suggesting opportunities for further development.

Computational-Based Discovery of the Anti-Cancer Activities of Pyrrole-Based Compounds Targeting the Colchicine-Binding Site of Tubulin.

Despite the tubulin-binding agents (TBAs) that are widely used in the clinic for cancer therapy, tumor resistance to TBAs (both inherited and acquired) significantly impairs their effectiveness, thereby decreasing overall survival (OS) and progression-free survival (PFS) rates, especially for the patients with metastatic, recurrent, and unresectable forms of the disease. Therefore, the development of novel effective drugs interfering with the microtubules’ dynamic state remains a big challenge in current oncology. We report here about the novel ethyl 2-amino-1-(furan-2-carboxamido)-5-(2-aryl/tert-butyl-2-oxoethylidene)-4-oxo-4,5-dihydro-1H-pyrrole-3-carboxylates (EAPCs) exhibiting potent anti-cancer activities against the breast and lung cancer cell lines in vitro. This was due to their ability to inhibit tubulin polymerization and induce cell cycle arrest in M-phase. As an outcome, the EAPC-treated cancer cells exhibited a significant increase in apoptosis, which was evidenced by the expression of cleaved forms of PARP, caspase-3, and increased numbers of Annexin-V-positive cells. By using the in silico molecular modeling methods (e.g., induced-fit docking, binding metadynamics, and unbiased molecular dynamics), we found that EAPC-67 and -70 preferentially bind to the colchicine-binding site of tubulin. Lastly, we have shown that the EAPCs indicated above and colchicine utilizes a similar molecular mechanism to inhibit tubulin polymerization via targeting the T7 loop in the β-chain of tubulin, thereby preventing the conformational changes in the tubulin dimers required for their polymerization. Collectively, we identified the novel and potent TBAs that bind to the colchicine-binding site and disrupt the microtubule network. As a result of these events, the compounds induced a robust cell cycle arrest in M-phase and exhibited potent pro-apoptotic activities against the epithelial cancer cell lines in vitro.

Antitumor Activity of Tubulin-Binding Agent MPC-6827 on Different Types of Cancer Cell Lines.

In this study, the antitumor effects of tubulin-binding agent MPC-6827 on HeLa, MCF-7 and A549 cell lines originated from cervix carcinoma, metastatic breast adenocarcinoma and adenocarcinomic human alveolar basal epithelial cells respectively were determined. Cell index, BrdU labelling index, mitotic index and apoptotic index were evaluated in experiments. In cell index experiment 2 nM, 4 nM, 6 nM, 8 nM, 10 nM MPC-6827 applied to three cell lines. These parameters showed that 4 nM was the optimum concentration for HeLa and A549 cells, while 2 nM was the optimum concentration for MCF-7 cells. The use of optimum concentrations for each cell line has shown that while there was a significant decrease in mitotic index, BrdU labelling index, there was a significant increase in apoptotic index.

Antitumoral Effect of Plocabulin in High Grade Serous Ovarian Carcinoma Cell Line Models

Ovarian cancer (OC) is a life-threatening tumor and the deadliest among gynecological cancers in developed countries. First line treatment with a carboplatin/paclitaxel regime is initially effective in the majority of patients, but most advanced OC will recur and develop drug resistance. Therefore, the identification of alternative therapies is needed. In this study, we employed a panel of high-grade serous ovarian cancer (HGSOC) cell lines, in monolayer and three-dimensional cell cultures. We evaluated the effects of a novel tubulin-binding agent, plocabulin, on proliferation, cell cycle, migration and invasion. We have also tested combinations of plocabulin with several drugs currently used in OC in clinical practice. Our results show a potent antitumor activity of plocabulin, inhibiting proliferation, disrupting microtubule network, and decreasing their migration and invasion capabilities. We did not observe any synergistic combination of plocabulin with cisplatin, doxorubicin, gemcitabine or trabectedin. In conclusion, plocabulin has a potent antitumoral effect in HGSOC cell lines that warrants further clinical investigation.

Abstract P2-13-33: Preclinical development of DHES0815A: A HER2-directed antibody-drug conjugate comprised of a reduced potency PBD dimer linked to a domain I binding HER2 antibody

Abstract Background: Many HER2-directed antibody-drug conjugates (ADCs) in early development employ trastuzumab linked to tubulin binding agents as the cytotoxic drug. DHES0815A is an ADC consisting of a THIOMABTM humanized IgG1 anti-HER2 monoclonal antibody (hu7C2), which binds domain 1 of the HER2 ECD, conjugated via a disulfide linker to PBD-monoamide (MA), a DNA mono-alkylating agent. The reduced potency of the PBD-MA payload compared to PBD dimers and the stability of the conjugation site and linker were designed to improve tolerability, whereas the binding of hu7C2 to a HER2 epitope distinct from trastuzumab and pertuzumab allows combination with existing HER2 therapies. . Methods: Modifications at one imine in the PBD (pyrrolobenzodiazepine) dimer produced mono-alkylating PBDs that were assessed for DNA binding, permeability and cell potency. Each modified PBD was conjugated to hu7C2 LC K149C via a disulfide linker and assessed in HER2-positive models in vitro and in vivo. Safety studies were performed in female and male cynomolgus monkey treated every 3 weeks X 5, with a 7-week follow-up. Assessments included hematology, clinical chemistry, anti-drug antibody, standard clinical, neurological and ophthalmic exams and histopathology. Results: Our goal was design of a reduced potency PBD dimer ADC to achieve dosing in the linear PK range for optimal exposure, efficacy and safety. PBD dimers contain 2 reactive imines that bis-alkylate and crosslink DNA. Mono-alkylating PBD dimers were assessed in DNA binding, potency and permeability assays to select the optimal mono-alkylator, PBD-MA, for further investigation. hu7C2-disulfide-PBD-MA, or DHES0815A, was potent across HER2-positive breast and gastric cancer models in vitro and in vivo, with little effect on HER2-negative tumors or normal primary cells in vitro. DHES0815A was more potent than T-DM1 in HER2-positive and HER2-low breast and gastric cancer PDX models and demonstrated combination activity with standard of care agents T-DM1 or docetaxel. Mechanistically, treatment of HER2-positive cells with DHES0815A resulted in late S-phase cell cycle arrest; induction of DNA damage markers γH2AX, phospho-p53, phospho-CHK2, induction of apoptosis, and bystander activity. Pharmacokinetic analysis showed dose-proportional PK, with linear biphasic PK >/= 4mg/kg in cynomolgus monkey. Safety studies in cynomolgus monkey treated with 4, 8 or 12 mg/kg demonstrated that findings were non-adverse, monitorable and manageable. All doses were tolerated with few clinical observations and minimal to mild clinical pathology changes. Target organs were skin, bone marrow, lung and eye. The HNSTD (highest non-severely toxic dose) was 12 mg/kg. This dose enabled a starting dose of 0.6 mg/kg for phase 1 testing. Conclusions: DHES0815A is a HER2-directed ADC comprised of a domain I binding antibody linked to a reduced potency PBD dimer, PBD-MA, via a reducible disulfide linker. The antibody does not interfere with trastuzumab or pertuzumab binding, allowing combination with existing HER2 therapies. The reduced potency payload allows dosing in the linear PK range, supporting our hypothesis. DHES0815A demonstrated dose-dependent anti-tumor efficacy in multiple HER2+ breast and gastric cancer models, and showed efficacy in HER2-low models, where T-DM1 was inactive. Enhanced anti-tumor activity was observed combining DHES0815A with T-DM1, trastuzumab/pertuzumab or docetaxel. Studies in cynomolgus monkey demonstrated a safety profile compatible with initiating a phase 1 clinical trial. The phase 1 data will be presented in a separate presentation. Citation Format: Gail Lewis Phillips, Guangmin Li, Jun Guo, Shang-Fan Yu, Genee Lee, Donglu Zhang, Peter Dragovich, Thomas Pillow, Binqing Wei, M. Violet Lee, Ola Saad, Shab Masih, Douglas Leipold, Lisa Crocker, Melissa Schutten. Preclinical development of DHES0815A: A HER2-directed antibody-drug conjugate comprised of a reduced potency PBD dimer linked to a domain I binding HER2 antibody [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-33.

Polarity protein Par3 sensitizes breast cancer to paclitaxel by promoting cell cycle arrest.

Paclitaxel, belongs to tubulin-binding agents (TBAs), shows a great efficacy against breast cancer via stabilizing microtubules. Drug resistance limits its clinical application. Here we aimed to explore a role of Polarity protein Par3 in improving paclitaxel effectiveness. Breast cancer specimens from 45 patients were collected to study the relationship between Par3 expression and paclitaxel efficacy. The Kaplan-Meier method was used for survival analysis. Cell viability was measured in breast cancer cells (SK-BR-3 and T-47D) with Par3 over-expression or knockdown. The flow cytometry assays were performed to measure cell apoptosis and cell cycle. BrdU incorporation assay and Hoechst 33,258 staining were performed to measure cell proliferation and cell apoptosis, respectively. Immunofluorescence was used to detect microtubule structures. Par3 expression was associated with good response of paclitaxel in breast cancer patients. Consistently, Par3 over-expression significantly sensitized breast cancer cells to paclitaxel by promoting cell apoptosis and reducing cell proliferation. In Par3 overexpressing cells upon paclitaxel treatment, we observed intensified cell cycle arrests at metaphase. Further exploration showed that Par3 over-expression stabilized microtubules of breast cancer cells in response to paclitaxel and resists to microtubules instability induced by nocodazole, a microtubule-depolymerizing agent. Par3 facilitates polymeric forms of tubulin and stabilizes microtubule structure, which aggravates paclitaxel-induced delay at the metaphase-anaphase transition, leading to proliferation inhibition and apoptosis of breast cancer cells. Par3 has a potential role in sensitizing breast cancer cells to paclitaxel, which may provide a more precise assessment of individual treatment and novel therapeutic targets.

Monitoring the Disruptive Effects of Tubulin-Binding Agents on Cellular Microtubules.

Tubulin-binding agents are an important class of chemotherapeutic agents. This chapter describes detailed protocols to examine the effects of tubulin-binding agents on cellular microtubules. The methods can be utilized for the screening of novel chemotherapeutic agents targeting microtubules. These assays can also be extended to study the effects of various proteins on the stability of microtubules. We have described five assays, which together provides qualitative and quantitative information about the effects of tubulin-binding agents on microtubule stability and dynamics. The key steps and crucial information regarding different steps have been included along with the theory of each of the assays.

Development of a proteochemometric-based support vector machine model for predicting bioactive molecules of tubulin receptors.

Microtubules are receiving enormous interest in drug discovery due to the important roles they play in cellular functions. Targeting tubulin polymerization presents an excellent opportunity for the development of anti-tubulin drugs. Drug resistance and high toxicity of currently used tubulin-binding agents have necessitated the pursuit of novel drug candidates with increased therapeutic potency. The design of novel drug candidates can be achieved using efficient computational techniques to support existing efforts. Proteochemometric (PCM) modeling is a computational technique that can be employed to elucidate the bioactivity relations between related targets and multiple ligands. We have developed a PCM-based Support Vector Machine (SVM) approach for predicting the bioactivity between tubulin receptors and small, drug-like molecules. The bioactivity datasets used for training the SVM algorithm were obtained from the Binding DB database. The SVM-based PCM model yielded a good overall predictive performance with an area under the curve (AUC) of 87%, Matthews correlation coefficient (MCC) of 72%, overall accuracy of 93%, and a classification error of 7%. The algorithm allows the prediction of the likelihood of new interactions based on confidence scores between the query datasets, comprising ligands in SMILES format and protein sequences of tubulin targets. The algorithm has been implemented as a web server known as TubPred, accessible via http://35.167.90.225:5000/ .

Imaging-Guided Evaluation of the Novel Small-Molecule Benzosuberene Tubulin-Binding Agent KGP265 as a Potential Therapeutic Agent for Cancer Treatment.

Simple SummaryVascular-disrupting agents promise significant therapeutic efficacy against solid tumors by selectively damaging tumor-associated vasculature. Dynamic BLI and oxygen-enhanced multispectral optoacoustic tomography (OE-MSOT) were used to compare vascular shutdown following administration of KGP265. BLI signal and vascular oxygenation response (ΔsO2) to a gas breathing challenge were both significantly reduced within 2 h indicating vascular disruption, which continued over 24 h. Twice-weekly doses of KGP265 caused a significant growth delay in MDA-MB-231 human breast tumor xenografts and 4T1 syngeneic breast tumors growing orthotopically in mice.The selective disruption of tumor-associated vasculature represents an attractive therapeutic approach. We have undertaken the first in vivo evaluation of KGP265, a water-soluble prodrug of a benzosuberene-based tubulin-binding agent, and found promising vascular-disrupting activity in three distinct tumor types. Dose escalation in orthotopic MDA-MB-231-luc breast tumor xenografts in mice indicated that higher doses produced more effective vascular shutdown, as revealed by dynamic bioluminescence imaging (BLI). In syngeneic orthotopic 4T1-luc breast and RENCA-luc kidney tumors, dynamic BLI and oxygen enhanced multispectral optoacoustic tomography (OE-MSOT) were used to compare vascular shutdown following the administration of KGP265 (7.5 mg/kg). The BLI signal and vascular oxygenation response (ΔsO2) to a gas breathing challenge were both significantly reduced within 2 h, indicating vascular disruption, which continued over 24 h. A correlative histology confirmed increased necrosis and hemorrhage. Twice-weekly doses of KGP265 caused significant growth delay in both MDA-MB-231 and 4T1 breast tumors, with no obvious systemic toxicity. A combination with carboplatin produced significantly greater tumor growth delay than carboplatin alone, though significant carboplatin-associated toxicity was observed (whole-body weight loss). KGP265 was found to be effective at low concentrations, generating long-term vascular shutdown and tumor growth delay, thus providing strong rationale for further development, particularly in combination therapies.