Centromere-associated Protein E Inhibitor Research Articles

Abstract 4945: Computational modeling-based discovery of novel anticancer drug candidates targeting centromere-associated protein E

Abstract Purpose: Centromere-associated protein E (CENP-E), a mitotic motor protein, is crucial for cell division. Inhibition of CENP-E can lead to chromosome misalignment and aneuploidy. Our recent studies revealed that the CENP-E inhibitor (CENP-Ei), Compound A, induces aneuploid-mediated cGAS-STING pathway activation in cancer cell lines, which is expected to trigger innate immune response and induce immunological conversion of the tumor microenvironment from cold to hot. However, known CENP-Eis exhibited limited efficacy, and none have gone beyond phase I trials since 2012. The aim of this study is to develop a workflow integrating structure and artificial intelligence-based modeling approaches to accelerate the discovery of novel CENP-Eis with improved efficacy and overcome related challenges. Method: The inhibitor-bound CENP-E structure was modeled using chimeric homology modeling followed by induced fit docking with known CENP-Eis. Structure-based virtual screening (VS) was performed on large compound libraries in Enamine REAL, Synthetically Accessible Virtual Inventory, and MolPort databases. Consensus scoring methods were applied by averaging ranks of individual molecules obtained from VS against different CENP-E conformations. An active learning-enhanced VS pipeline was developed using ATOM Modeling PipeLine (AMPL) to find the best-ranked ligands more efficiently. CENP-E ligand candidates were further filtered based on their safety properties predicted by machine learning models trained with curated databases of adverse drug reactions using AMPL. The success of this study was measured by its ability to reveal novel chemotypes that could modulate CENP-E in kinesin ATPase assays. Result: Conformations of CENP-E’s ligand binding site were predicted for VS using the homology model. Structure-based VS was performed to select high-ranked compounds based on consensus scoring. The active learning-enhanced VS pipeline achieved 75% accuracy in ranking the top 20% of hit compounds. The graph convolutional network model with a ROCAUC, 0.76, was created using AMPL to filter ligand candidates based on their predicted safety properties. Seventy-three potential CENP-E ligands computationally prioritized from the Molport database were experimentally tested using kinesin ATPase assays from which 4 potential inhibitors and 8 potential activators were identified indicating overlapping binding sites of inhibitors and activators. Conclusion: Our integrated workflow is effective in discovering novel CENP-E modulators. The discovery of CENP-E activators provides an opportunity to identify novel therapeutics in hepatocellular carcinoma malignancies known to experience decreased CENP-E activity leading to aneuploidy. The predictions generated by computational models combined with experimental validations could further accelerate anticancer drug discovery. Citation Format: Pinyi Lu, Ryo Kamata, Michael R. Weil, Naomi Ohashi, Akihiro Ohashi, Eric A. Stahlberg. Computational modeling-based discovery of novel anticancer drug candidates targeting centromere-associated protein E [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 4945.

Abstract 411: PI3K-AKT-mTOR signaling pathways play important roles in chromosomal instability-induced innate immune response in cancer cells

Abstract Background: Chromosomal instability (CIN) frequently exerts innate immune response, being expected to sensitize immunotherapy. cGAS-STING pathway is a hub to regulate CIN-mediated innate immune activation. We previously demonstrated that small-molecule inhibitors targeting Centromere-associated Protein-E (CENP-E) profoundly accumulated CIN in cancer cells, leading to activation of cGAS-STING and its related innate immune pathways. In this study, aiming to deeply understand molecular mechanisms of CENP-E inhibitor (CENP-Ei)-induced CIN and innate immune activation, we conducted large-scale chemical screening with ~1,400 kinase inhibitors in CENP-Ei-treated reporter cells for NF-κB and IRF. We also experimentally validated the effects of hit compounds on CENP-Ei-induced innate immune activation. Material and Methods: GSK923295 and Cmpd-A were used as CENP-E inhibitors. HeLa, A549, A549 dual reporter cells were treated with the CENP-Ei at the indicated concentrations, subjected to immunofluorescent, transcriptome, FACS, reporter activity, or gene expression analyses. High-throughput screening (HTS) with ~1,400 kinase library was conducted in A549 dual reporter cells in combinational treatment with GSK923295. Combination effects of the representative hit compounds were confirmed in matrix-combination studies with the CENP-Ei. Results: Treatment with the CENP-Ei, Cmpd-A and GSK923295, induced CIN (e.g., multinucleation) in both A549 and A549 dual reporter cells, which elevated NFkB and IRF reporter activities as well as gene expressions of inflammatory cytokines and chemokines in innate immune pathways. Transcriptome analyses also confirmed that gene ontologies (GO) for innate immune response, such as cytosolic DNA sensing pathway and cytokine-cytokine receptor interaction pathway, were significantly enriched in CENP-Ei-treated CIN cells. Next, we conducted HTS of IRF-reporter activity with ~1,400 kinase library in combination with GSK923295 in A549 dual reporter cells. The HTS revealed that a series of inhibitors targeting PI3K-AKT-mTOR signaling pathways intensively suppressed CENP-Ei-induced IRF activation, which occupied ~40% of hit compounds. The matrix-combination studies confirmed that sapanisertib (TORC1/2 inhibitor) significantly attenuated CENP-Ei-induced IRF activity. Conclusions: CENP-Ei-induced CIN potently activates innate immune response pathways in cancer cells, in which PI3K-AKT-mTOR signaling pathway appears to be involved. We are initiating AI-based drug discovery to develop novel CENP-E inhibitors. Citation Format: Ryo Kamata, Hitoshi Saito, Yumi Hakozaki, Yukie Kashima, Gaku Yamamoto, Tomoko Yamamori Morita, Pinyi Lu, Akihiro Ohashi. PI3K-AKT-mTOR signaling pathways play important roles in chromosomal instability-induced innate immune response in cancer cells [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 411.

Identification of benzo[d]pyrrolo[2,1-b]thiazole derivatives as CENP-E inhibitors

Kinesin centromere-associated protein E (CENP-E) has emerged as a potential target for the development of anticancer drugs due to its involvement in the mitotic progression of the cell cycle. Although several CENP-E inhibitors have been reported, more knowledge of chemical structures and inhibitory mechanisms is necessary for developing CENP-E inhibitors. Here, we describe the identification of new CENP-E inhibitors. Screening of a small-molecule chemical library identified benzo[d]pyrrolo[2,1-b]thiazole derivatives, including 1, as compounds with inhibitory activity against the microtubule-stimulated ATPase of the CENP-E motor domain. Among the mitotic kinesins examined, 1 selectively inhibited the kinesin ATPase activity of CENP-E. In a steady-state ATPase assay, 1 exhibited ATP-competitive behavior, which was different from the CENP-E inhibitor GSK923295. Compound 1 inhibited the proliferation of tumor-derived HeLa and HCT116 cells more efficiently than that of non-cancerous WI-38 cells. The inhibition of cell proliferation was attributed to the ability of 1 to induce apoptotic cell death. The compound showed antimitotic activity, which caused cell cycle arrest at mitosis via interference with proper chromosome alignment. We identified 1 and its derivatives as the lead compounds that target CENP-E, thus providing a new opportunity for the development of anticancer agents targeting kinesins.

First-time-in-human study of GSK923295, a novel antimitotic inhibitor of centromere-associated protein E (CENP-E), in patients with refractory cancer

GSK923295 is an inhibitor of CENP-E, a key cellular protein important in the alignment of chromosomes during mitosis. This was a Phase I, open-label, first-time-in-human, dose-escalation study, to determine the maximum-tolerated dose (MTD), safety, and pharmacokinetics of GSK923295. Adult patients with previously treated solid tumors were enrolled in successive cohorts at GSK923295 doses ranging from 10 to 250mg/m(2). GSK923295 was administered by a 1-h intravenous infusion, once weekly for three consecutive weeks, with treatment cycles repeated every 4 weeks. A total of 39 patients were enrolled. The MTD for GSK923295 was determined to be 190mg/m(2). Observed dose-limiting toxicities (all grade 3) were as follows: fatigue (n=2, 5%), increased AST (n=1, 2.5%), hypokalemia (n=1, 2.5%), and hypoxia (n=1, 2.5%). Across all doses, fatigue was the most commonly reported drug-related adverse event (n=13; 33%). Gastrointestinal toxicities of diarrhea (n=12, 31%), nausea (n=8, 21%), and vomiting (n=7, 18%) were generally mild. Frequency of neutropenia was low (13%). There were two reports of neuropathy and no reports of mucositis or alopecia. GSK923295 exhibited dose-proportional pharmacokinetics from 10 to 250mg/m(2) and did not accumulate upon weekly administration. The mean terminal elimination half-life of GSK923295 was 9-11 h. One patient with urothelial carcinoma experienced a durable partial response at the 250mg/m(2) dose level. The novel CENP-E inhibitor, GSK923295, had dose-proportional pharmacokinetics and a low number of grade 3 or 4 adverse events. The observed incidence of myelosuppression and neuropathy was low. Further investigations may provide a more complete understanding of the potential for GSK923295 as an antiproliferative agent.

Phase I first-in-human study of the centromere-associated protein E (CENP-E) inhibitor GSK923295 in patients with advanced solid tumors (study CPE107602).

3012 Background: CENP-E plays a key role in maintaining and silencing mitotic checkpoint signaling. CENP-E is overexpressed in many tumors relative to surrounding normal tissue. Inhibition of CENP-E protein results in cell cycle arrest in mitosis. GSK923295 is a selective inhibitor of CENP-E with potent and broad spectrum antitumor activity against solid tumors and hematologic malignancies in vitro. We present the maximum-tolerated dose (MTD), dose-limiting toxicities (DLTs), and pharmacokinetics (PK) of this first-in-class CENP-E inhibitor, GSK923295, in patients (pts) with advanced solid tumors. Methods: Adult pts with relapsed or refractory advanced solid tumors received GSK923295 by 1-hour IV infusion on days 1, 8, and 15 of a 28-day cycle. Pts continued therapy until disease progression or toxicity. Doses were escalated using a modified accelerated titration schema. Results: 38 of 39 pts enrolled were evaluable. Doses were escalated from 10 to 250 mg/m2. DLTs included grade 3 AST elevation (n=1/7 at 80 mg/m2), grade 3 hypoxia (n=1/7 at 140 mg/m2), and 3 DLTs at 250 mg/m2 (grade 3 fatigue (n=2) and grade 3 hypokalemia (n=1)). Thus, MTD was determined to be 190 mg/m2. Fatigue was the most common adverse event (AE) (n=21/37), mostly grade 1 (n=16/21), but appeared dose related. Other grade 3 and 4 AEs occurring after cycle 1 were (n=1 each): grade 3 vomiting (105 mg/m2), grade 4 hyponatremia (140 mg/m2), grade 4 AST (140 mg/m2), grade 4 ALT (140 mg/m2), grade 3 total bilirubin (250 mg/m2). Preliminary PK data show that GSK923295 exposure appears dose proportional with low (15%) intrasubject- and moderate (41%) intersubject-variability in body-size normalized systemic clearance. One pt with urothelial carcinoma experienced a partial response (RECIST) after 6 months (mo) of GSK923295 at 250 mg/m2, lasting ≥ 3 mo. Six pts had stable disease lasting ≥ 3 mo across a wide range of doses (20 mg/m2 6 mo, n=1 [liver]; 80 mg/m2 3 mo, n=3 [colon/rectum, pancreas]; 140 mg/m2 5 mo, n=1 [prostate]; 190 mg/m2 4 mo, n=1 [ovary]). Conclusions: The MTD of GSK923295 is 190 mg/m2 administered on days 1, 8, and 15 of a 28-day cycle. Fatigue was the most common AE and was dose limiting. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration GlaxoSmithKline GlaxoSmithKline GlaxoSmithKline GlaxoSmithKline

Pediatric Preclinical Testing Program (PPTP) testing of the CENP-E inhibitor GSK923295A

10015 Background: GSK923295A is a small molecule inhibitor of centromere-associated protein E (CENP-E), a mitotic kinesin that is required for metaphase chromosome alignment and integration of mitotic spindle mechanics with mitotic checkpoint signaling. An initial phase I clinical trial in adults is ongoing. Methods: The PPTP includes a molecularly characterized in vitro panel of cell lines (n = 27) and in vivo panel of xenografts (n = 60) representing most of the common types of childhood solid tumors and childhood acute lymphoblastic leukemia (ALL). GSK923295A was tested in vitro at concentrations from 1.0 nM to 10.0 μM (96 hour exposure) and was tested in vivo using a daily × 3 for 2 weeks schedule, repeated at day 21. GSK923295A was administered IP at a dose of 125 mg/kg. Three measures of antitumor activity were used: 1) an objective response measure modeled after the clinical setting; 2) a time to event measure based on the median event-free survival (EFS); and 3) a treated to control (T/C) tumor volume measure. Results: GSK923295A demonstrated potent in vitro activity against the PPTP cell line panel with a median IC50 of 27 nM (range 12 nM to > 10 μM). 35 of 37 solid tumor xenograft models were evaluable. GSK923295A induced significant differences in EFS distribution compared to controls in 32 of 35 evaluable models. Objective responses were noted in 13 of 35 xenografts, including 9 with maintained complete responses (MCR), 3 with complete response (CR), and 1 with partial response (PR). Three of 5 Ewing sarcoma xenografts achieved MCR or CR, as did 2 of 3 rhabdoid tumor, and 2 of 5 rhabdomyosarcoma models. For the neuroblastoma panel, the best response was progressive disease (PD) with growth delay compared to controls (PD2 response), which was observed in 5 of 6 xenografts. GSK923295A showed activity against the ALL panel, but unexplained toxicity (generally on or after day 21) precluded formal analysis. Conclusions: GSK923295A has substantial in vitro and in vivo activity against the PPTP's preclinical models. The observed high level of preclinical activity for GSK923295A will need to be evaluated in the context of systemic exposures achieved in the xenograft models and those achievable in humans at tolerable doses. (Supported by NCI NO1CM42216) [Table: see text]

Therapeutic potential of mitotic kinesin inhibitors in cancer

Background: Mitotic kinesins are attractive targets in cancer chemotherapy. A large number of small-molecule inhibitors of kinesin spindle protein (KSP) and centromere-associated protein E (CENP-E) have been discovered. Objective: To estimate the therapeutic potential of mitotic kinesin inhibitors. Methods: We comprehensively summarized and analysed the trend among recent patents and conducted a literature review of clinical trials performed since July 2004. Results/conclusion: More than 80 patent applications have been made, spanning a wide range of structural diversity. Five KSP inhibitors and one CENP-E inhibitor have advanced to the early clinical stage, and some of the results for these clinical trials have been disclosed. The therapeutic potential for mitotic kinesin inhibitors is likely to become obvious within the next few years.