AACR 2019 Proceedings: Abstracts 1-2748

CANCER CHEMISTRY

Novel Small Molecules for Cancer Therapy

#1

A small molecule inhibits Nrf2 expression and activity in cancer cells.

Di Zhang, Corbin Livingston, Thomas Dexheimer, Edmund Ellsworth, Aaron Odom, Karen Liby. Michigan State Univ., East Lansing, MI.

Activation of the Nrf2-Keap1-ARE pathway is a master defense mechanism protecting against oxidative and electrophilic stress. Nrf2 was traditionally considered cancer-preventing, as Nrf2 deficiency enhances susceptibility to carcinogens in a variety of mouse models. However, increasing evidence suggests a tumor-promoting role for Nrf2 in established tumors: gain of function mutations in NFE2L2, which activates the Nrf2 pathway, or loss of function mutations in its negative regulator, Keap1, have been found most frequently in lung cancer. Constitutive activation of the Nrf2 pathway is associated with chemoresistance and poor survival. Nrf2 inhibitors, therefore, become potential novel drugs for cancer treatment and needed tool compounds to study the Nrf2 pathway in cancer. To identify Nrf2 inhibitors, we conducted a high throughput screen with a diverse set of small molecules (~6500 compounds). 890 compounds were identified as hits, and a funnel strategy was applied to filter through the hits and select lead compounds. Although other known Nrf2 inhibitors were identified, a potentially novel compound, MSU225, was chosen as a lead for further evaluation. MSU225 downregulated tBHQ (tert-butyl hydroquinone)-induced Nrf2 transcriptional activity in a luciferase assay without inducing cell death. When A549 human lung cancer cells, in which Keap1 is mutated inducing constitutive activation of Nrf2, were treated with 5 µM MSU225, the mRNA expression of HMOX1, NQO1, GST1A1, GCLC, GCLM, and UGT1A6, all downstream targets of Nrf2, were decreased. Moreover, MSU225 significantly (p<0.05) decreased the protein level of Nrf2 in A549 cells. The effect of MSU225 on Nrf2 protein levels was blocked by the proteasome inhibitor MG132, suggesting MSU225 enhances Nrf2 degradation through the proteasome system. In addition, MSU225 inhibited the growth of human lung cancer cells (A549, H460, A427) in soft agar. Cells addicted to Nrf2 were more susceptible to MSU225 for suppression of cell proliferation than cells not addicted to Nrf2. MSU225 also sensitized A549 cells to carboplatin treatment. Our results suggest that MSU225 is a novel Nrf2 inhibitor, and additional in vivo studies will be done to determine if it can be used to treat experimental lung cancer.

#2

Discovery of BAY 2402234 by phenotypic screening: A human Dihydroorotate Dehydrogenase (DHODH) inhibitor in clinical trials for the treatment of myeloid malignancies.

Stefan N. Gradl,¹ Thomas Mueller,¹ Steven Ferrara,² Sherif El Sheikh,³ Andreas Janzer,¹ Han-Jie Zhou,⁴ Anders Friberg,¹ Judith Guenther,¹ Martina Schaefer,¹ Timo Stellfeld,¹ Knut Eis,¹ Michael Kroeber,¹ Duy Nguyen,¹ Claudia Merz,¹ Michael Niehues,¹ Detlef Stoeckigt,¹ Sven Christian,¹ Katja Zimmermann,¹ Pascal Lejeune,¹ Michael Bruening,¹ Hanna Meyer,¹ Vera Puetter,¹ David T. Scadden,⁵ David B. Sykes,⁵ Henrik Seidel,¹ Ashley Eheim,¹ Martin Michels,¹ Andrea Haegebarth,¹ Marcus Bauser¹. ¹Bayer AG, Pharmaceuticals Division, Berlin, Germany; ²Broad Institute of MIT and Harvard, Boston, MA; ³Technical University Cologne, Cologne, Germany; ⁴Essa Pharmaceuticals, San Francisco, CA; ⁵Massachusetts General Hospital, Boston, MA.

DHODH is a key enzyme in the biosynthesis of pyrimidines and recent studies have renewed interest in this old anti-cancer target. Here, we disclose the discovery of 4-triazolosalicylamides as inhibitors of DHODH and their structure activity relationship (SAR). The hit cluster was discovered during a phenotypic high throughput screen (HTS) of 2.5 million compounds where proliferation of H460 lung cancer cells was used as read-out. DHODH was successfully identified as the molecular target by comparing the activity profile of the hits in a panel of cell lines to a set of inhibitors with known pharmacological activity. The hit compounds showed good cellular potency but had undesirable DMPK properties. Interestingly, the compounds are non-ionizable in contrast to many other DHODH inhibitors and show no potency shift from biochemical to cellular assays. Structural modifications lead to compounds with sub-nanomolar potency in cellular assays and increased metabolic stability enabling the proof of concept in vivo xenograft experiments. Further optimization guided by lipophilicity efficiency and identification of metabolic hot spots resulted in molecules with low clearance and improved solubility. BAY 2402234 was selected as the clinical candidate after side by side comparison of a number of promising compounds. It shows great oral bioavailability, target engagement in all preclinical species tested, induces differentiation in AML models, and has excellent activity in a variety of leukemia models. A clinical phase I study has been initiated in patients with myeloid malignancies. (NCT03404726)

#3

Discovery and optimization of small molecule Bax activators for cancer therapy.

Gang Liu,¹ Hyejin Kim,² Chunyong Ding,¹ Zhuo Yu,² Hong Wang,² Haiying Chen,¹ Qiang Shen,² Jia Zhou¹. ¹UT Medical Branch, Galveston, TX; ²UT M. D. Anderson Cancer Center, TX.

Bax, an indispensable executioner protein of B-cell lymphoma 2 (Bcl-2) family, plays a pivotal role in controlling the mitochondrial dysfunction and apoptosis in normal and cancer cells. High throughput screening to combat the nicotine-induced Bax phosphorylation at serine 184 (S184) to activate the proapoptotic function of Bax has revealed compound SMBA1 as a promising Bax direct activator as we previously reported. Initial medicinal chemistry campaign by choosing SMBA1 as a lead compound for breast cancer treatment resulted in the identification of compound CYD-2-11 with low micromolar IC50 values against MDA-MB-231 and MCF-7 as well as in vivo inhibitory effects against MDA-MB-231 xenograft tumor growth. Herein, we report the comprehensive structural optimization based on CYD-2-11 as the lead by introducing various alkylamino side chains to have a deeper access to S184 pocket and replacing carbon atoms with nitrogen as well as reducing the nitro group of 9H-fluorene scaffold to construct diverse derivatives for enhanced drug properties. CYD-4-61 and GL0385 have been discovered as the most potent anti-breast cancer agents with submicromolar IC50 values of 0.34 and 0.33 µM against MDA-MB-231 as well as 0.65 and 0.26 µM against MCF-7, respectively. More importantly, these drug candidates exhibited significant in vivo efficacy suppressing xenograft tumor growth by activating Bax to induce cancer cell apoptosis with the potential to overcome resistance against Adriamycin (ADR) in breast cancer. This work was supported by Breast Cancer Research Program (BCRP) Breakthrough Award BC160038 (to J.Z.) and BC160038P1 (to Q.S.) from the Department of Defense (DoD).

#4

Discovery of novel and highly potent small molecule inhibitors of chemokine receptor CXCR4.

Xiong Fang,¹ Qian Meng,¹ Xiao Fang,¹ Siyu Zhu,¹ Yazi Huang,¹ Jing An,² Yan Xu,¹ Ziwei Huang¹. ¹Tsinghua University, Beijing, China; ²University of California at San Diego, San Diego, CA.

Introduction: CXCR4 is a member of chemokine receptor and G protein coupled receptor (GPCR) families. Its interaction with the chemotactic ligand stromal cell-derived factor 1 (SDF-1 or CXCL12) plays important roles in physiological and pathological processes. Recent studies have showed that the blockade of CXCL12-CXCR4 interaction by small molecules can be used in several clinical applications, including sensitizing cancer cells to chemotherapy, mobilizing hematopoietic stem cells (HSCs) to the blood for HSC collection, and inhibiting the tumor metastasis. To date, AMD3100 (Plerixafor) is the only clinically approved drug used in combination with G-CSF for mobilizing HSCs. Here, we report the discovery and development of a new class of highly potent small molecule CXCR4 inhibitors which can strongly antagonize CXCL12-CXCR4 interaction and mobilizing HSCs. Methods: Based on the crystal structures of CXCR4 showing a major and minor subpockets, we applied a fragment-based combinatorial design (FBCD) strategy to design a new class of hybrid molecules to recognize these two subpockets. A series of designed compounds were chemically synthesized and tested in a panel of biological assays including competitive CXCR4 binding, chemotaxis, calcium mobilization and CXCR4 internalization to characterize their biological activities. Furthermore, site-directed mutagenesis coupled with molecular docking simulation was carried out to determine the binding modes of the most potent compounds. Results: More than 30 compounds were synthesized and characterized. Among them, compound HFX51116 showed the most potent CXCR4 binding activity (IC50 = 12.2 nM), which was more potent than AMD3100 tested in parallel (IC50 = 325.3 nM). Furthermore, HFX51116 displayed remarkable inhibition of calcium mobilization (IC50 = 1.478 nM) and chemotaxis activity (IC50 = 252.7 nM), both of which are better than those of AMD3100. In addition, mutational studies and molecular docking simulation suggested that HFX51116 recognizes the minor subpocket mainly and major subpocket partially. Conclusions: Our findings have demonstrated that HFX51116 is a promising novel lead compound for developing a new class of small molecule therapeutics targeting CXCR4.

#5

Pre-clinical development of CDK9 inhibitor - LS007 for the treatment of acute myeloid leukemia.

Wang Hui,¹ Wang Shudong². ¹Changzhou Lesun Pharmaceuticals Ltd, ChangZhou, China; ²University of South Australia, Adelaide, Australia.

Acute myeloid leukemia (AML) is the most common form of acute leukaemia with an increasing incidence and dismal long-term prognosis with age. The most aggressive subtype, MLL-AML, is characterized by translocations of the mixed-lineage leukaemia gene (MLL) in chromosome band 11q23 and resistance to conventional chemotherapy with a short overall survival of only 9 months. Cyclin-dependent 9 (CDK9) activity is required for MLL-driven oncogenic transcription. Inhibition of CDK9 directly targets the transcriptional machinery required for MLL-fusion proteins (e.g. MLL-AF4, MLL-AF9 and MLL-ENL), thereby leading to apoptosis.

We have developed a highly effective drug candidate - LS007 that inhibited CDK9 with a Ki = 4 nM. It suppressed proliferation and induced apoptosis in a range of AML cell lines and patient samples. LS007 was highly efficacious against several AML cell line xenografts and patient-derived xenografts, and well tolerated in mice after oral administration. In a Molm-13 xenograft model, after 21 days of orally daily dosing at 25 mg/kg and 50 mg/kg LS007

inhibited tumor growth (p < 0.001) with T/C = 44 % and 18 %, respectively. Similarly, in a HL-60 xenograft model, LS007 exhibited a significant tumor growth inhibition at 45 mg/kg and 60 mg/kg daily by oral administration with T/C = 40 % and 20 %, respectively.

The pharmacokinetic profiles of LS007 was further assessed in the cynomolgus monkeys. After oral doses at the dose of 2, 4 and 8 mg/kg, LS007 was detected in the plasma with the maximum concentration (Cmax) from 55.9 to 279.5 ng/mL and the area under the plasma drug concentration-time graph (AUC) up to 1862.7 h·ng/mL. The mean terminal half-life (T 1/2) was around 3 h. LS007 possessed the favorable pharmacokinetic profiles with oral bioavailability F = 56% in the cynomolgus monkey (at dose 4 mg/kg), suggesting its therapeutic potential as an orally bioavailable anti-AML agent. The GLP- toxicological studies of LS007 in the cynomolgus monkey were completed, and the maximum tolerated doses were identified, which were used to estimate the safe starting dose for the Phase I clinical trials.

With its excellent anti-cancer efficacy along with appreciable absorption and safety profiles, LS007 offers a very exciting prospect as a clinical development candidate. The application of Investigational New Drug (IND) has been approved allowing the commencement of clinical trials of LS007 in patients with AML.

#6

Computationally assisted target screening of STING agonist for immunologic therapy.

Grace A. Binder, Christine S. Gambino, Anna Kharitonova, Rainer S. Metcalf, Kenyon G. Daniel, Wayne C. Guida. University of South Florida, Tampa, FL.

Stimulator of interferon genes (STING) is a receptor protein involved in the propagation of innate immune sensing of cytosolic DNA through the production of IFN-β. Mechanistic studies have shown IFN-β production within the tumor microenvironment can result in activation of tumor antigen-specific CD8+ T-cell immunity that can lead to tumor regression [1, 2]. STING activation by STING agonists should result in innate T-cell mediated anti-tumor immunity in the tumor microenvironment and have significant potential as a cancer therapeutic. Conversely, inhibition of STING would lead to a decreased production of IFN-β which could have implications in the treatment of autoimmune disease such as lupus erythematosus.

MD equilibrated crystal structures for human HAQ, REF, and WT alleles were clustered to find optimal conformations for diverse chemical library screening. Novel consensus docking protocols utilizing rigid receptor, induced fit, and quantum polarized ligand docking were applied for quantifying and refining proposed binding mechanisms of STING isoforms. Models for STING agonists and antagonists were developed.

From directed virtual screening, a novel low-molecular-weight organic molecule (GF3-002) that is not based on a cyclic dinucleotide was found as a potential STING activator and is currently under investigation.

GF3-002 and analogs were synthesized and structures were confirmed with LCMS and proton NMR. Both the HAQ and WT alleles, representing 78.3% of the human population were tested against compounds and controls. 2,3-cGAMP and DMSO were used as positive and negative controls, respectively. Microscale thermophoresis and SPR confirmed binding of GF3-002 to WT STING CTD with a kD of 3.2 ± 1.7 μM. IFN-dependent luciferase expression was measured by luminescence in THP-1 monocytic leukemia cells and found an EC50 of 29 ± 1.6 μM, compared to the native ligand 2,3-cGAMP EC50 of 42 ± 4.1 μM. Finally, qPCR was used quantify production of IFN-β via treatment of dendritic cells with GF3-002.

1. Sali, Characterization of a Novel Human-Specific STING Agonist that Elicits Antiviral Activity Against Emerging Alphaviruses. 2017.

2. Corrales, L., et al., Direct activation of STING in the tumor microenvironment leads to potent and systemic tumor regression and immunity. Cell reports, 2015. 11(7): p. 1018-1030.

#7

Discovery of OICR-10268: A potent and selective BCL6 inhibitor.

Iain D. Watson,¹ Methvin Isaac,¹ Brian Wilson,¹ Anh Chau,¹ Justin Morin,¹ Pandiaraju Subramanian,¹ Ahmed Mamai,¹ Babu Joseph,¹ Michael Prakesch,¹ David Uehling,¹ Ayome Abibi,¹ Richard Marcellus,¹ Craig Strathdee,¹ Ratheesh Subramaniam,¹ Brigitte Theriault,¹ Jeffrey Winston,¹ Manuel Chan,¹ Carly Griffin,¹ Herman Cheung,¹ Taira Kiyota,¹ Elijus Undzys,¹ Ahmed Aman,¹ Gennady Poda,¹ Doug Kuntz,² Neil C. Pomroy,² Gil G. Privé,² Rima Al-awar¹. ¹Ontario Inst. for Cancer Research, Toronto, Ontario, Canada; ²University of Toronto, Toronto, Ontario, Canada.

The transcription factor B cell lymphoma 6 (BCL6) is required for the generation of an effective humoral immune response through the development and maintenance of germinal centers (GCs). The inhibition of the protein−protein interaction between BCL6 and its corepressors has been implicated as a therapeutic target for diffuse large B-cell lymphoma (DLBCL), a type of non-Hodgkin’s lymphoma (NHL). Using structure-based drug design, we initiated a program to identify novel BCL6 inhibitors. We identified a high micromolar virtual screening hit which was then optimized for potent biophysical binding and anti-proliferative cellular activity resulting in the identification of OICR-10268, a potent and selective Bcl6 inhibitor.

#8

Targeting DNA-damage response pathway with a novel nano-liposomal ATR inhibitor in solid tumors.

Alexander Koshkaryev, Ozan Alkan, Bolin Geng, Lia Luus, Andreas Raue, Walid Kamoun, Suresh Tipparaju, Dmitri Kirpotin, Daryl Drummond. Merrimack Pharmaceuticals, Cambridge, MA.

Introduction.

Ataxia telangiectasia and Rad3-related (ATR) protein kinases have been identified as a key part of the DNA damage repair processes (DDRP) and cell cycle signaling. The DDRP is known to stimulate DNA repair, promote cell survival and, consequently, can diminish the therapeutic effect of existing DNA-damaging chemotherapy agents and ionizing radiation (IR) therapy. Therefore, there is a need for the development of potent and selective therapies to deliver ATR inhibitors for the treatment of cancer, as part of rational combination regimens with DNA damaging therapies. In this work, we develop and characterize a novel inhibitor of ATR kinase (BG129) and its nano-liposomal formulation (nLs-BG129).

Methods. In vitro activity of novel ATR inhibitors (BG129) was tested in a broad panel of cancer cell lines as a monotherapy or in combination with DNA-damaging chemotherapeutics (carboplatin, gemcitabine, and SN-38). On-target effects of BG129 in vitro were evaluated by Western-Blot to demonstrate dose-dependent inhibition of pChk1 and pRPA2, and a corresponding increase in ɣH2AX. The ATR inhibitor was encapsulated into unilamellar liposomal vesicles formed from hydrogenated soy phosphatidylcholine (HSPC), cholesterol, and methoxy-poly(ethylene glycol)-1,2-distearoyl-sn-glyceryl (PEG2000-DSG) at a 3:2:0.15 molar ratio and loaded stably into liposomes employing a triethylammonium sucroseoctasulfategradient. Liposomes were characterized by size and drug loading efficiency. Pharmacokinetic (PK) properties of nLs-BG129 were evaluated in mice. The antitumor activity of nLs- BG129 in combination with nano-liposomal irinotecan (nal-IRI) was tested in vivo in ovarian and various lung cancer xenograft models.

Results.

BG129 synergized with SN-38, gemcitabine, and carboplatin in vitro in cell-based assays. On-target dose-dependent inhibition of pChk1 and pRPA2 and a corresponding increase in ɣH2AX were observed in cells, following treatment with BG129. Nano-liposomal BG129 showed dramatically improved pharmacokinetics (AUC = 2835 μg/ml*h), compared to either free BG129 or VE-822/M6620/Berzosertib (AUC = 18.18 μg/ml*h). nLs-BG129 also enhanced antitumor activity of nal-IRI in multiple ovarian and SCLC and NSCLC xenograft models, when given in combination and was well tolerated.

Conclusions. Here we introduced a novel nano-liposomal formulation of an ATRi with good in vivo stability and prolonged clearance, with the ability to enhance the anti-tumor activity of DNA-damaging chemotherapeutics in vivo. The nano-liposomal formulation of ATRi can potentially sensitize tumor responses to DNA-damaging agents and improve their activity and tolerability in cancer patients when compared to unencapsulated and fast clearing ATR inhibitors, such as those currently in the clinic.

#9

Effect of C8-side chain on the cytotoxicity and NF-kB inhibitory capacity of pyrrolobenzodiazepines.

David B. Corcoran,¹ Thomas Lewis,² Kazi S. Nahar,¹ Christopher Fegan,² Chris Pepper,³ David E. Thurston,¹ Khondaker Miraz Rahman¹. ¹King's College London, London, United Kingdom; ²University of Cardiff, Cardiff, United Kingdom; ³University of Sussex, Brighton, United Kingdom.

Pyrrolobenzodiazepines (PBDs) have long been of interest in chemotherapeutic research as potential clinical agents. Recently, the conjugation of non-covalently interactive moieties to the C8-position of the PBD structure via a linker of variable length has emerged as a strategy to enhance the DNA sequence specificity of PBDs. This has led to the development of highly cytotoxic agents, which also act as inhibitors of transcription factors such as NF-κB. For example, GWL-78, a C8-linked PBD-Py-Py (Py = pyrrole) conjugate, has been shown to block interaction of the transcription factor NF-Y, and KMR-28-39, a C8-linked PBD-MPB conjugate with an affinity for GC-rich sequences of DNA inhibits activity of the transcription factor NF-κB. As up-regulation of NF-κB has been implicated in the development and progression of several cancer types, this may contribute to the high potency observed for PBD monomers such as KMR-28-39 (i.e., femtomolar IC50 values in some cell lines). In this study, the relationship between the cytotoxicity and transcription factor inhibition of a series of C8-linked PBD hybrid structures was explored. The systematic shortening of the non-covalent element of a C8-linked PBD conjugate, DC-1-92 led to the synthesis of a 19-member library of novel C8-PBD monomers. The critical elements of DC-1-92, which were required to render the molecule cytotoxic, were elucidated by an annexin V assay. The effects of shortening the non-covalent element of the molecule on transcription factor inhibitory capacity were also explored through an ELISA-based measurement of nuclear NF-κB subunit DNA binding following exposure of JJN3 cells to the synthesised molecules. While shortening the non-covalently interactive component of DC-1-92 had less of an effect than expected upon cytotoxicity, the shortening notably reduced the NF-κB inhibitory activity of the shortened analogues. DC-1-92, containing the longest non-covalently interactive C8-element, displayed the most effective inhibition of NF-κB, and effects upon nuclear p65, and RelB levels appeared to diminish in tandem with the shortening of the length of the C8 side chain. These data suggest that the length of the C8 side chain of PBD hybrid structures impacts upon the DNA binding of both canonical and non-canonical NF-κB subunits but this is independent of their cytotoxic effects.

#10

Novel derivatives of anaplastic lymphoma kinase inhibitors: Synthesis, radiolabeling and preliminary biological studies on crizotinib and alectinib analogues.

Mian M. Alauddin, Bhasker Radaram, Yi Rao, Ping Yang, David Piwnica-Worms. UT MD Anderson Cancer Center., Houston, TX.

Anaplastic lymphoma kinase (ALK), an oncogenic receptor tyrosine kinase, has emerged as a therapeutic target in various cancers, including lung cancer. Although several ALK inhibitors have gained regulatory approval, some of them do not cross the blood-brain barrier;¹ furthermore, non-invasive indicators of target engagement or biomarkers predicting response to these agents in vivo are lacking. To overcome these limitations, fluorinated analogues of the ALK inhibitors crizotinib and alectinib (fluoroethyl crizotinib [FECr] and fluoroethyl alectinib [FEAl]) were synthesised and radiolabeled. We report the radiosynthesis and preliminary biological results on these compounds, fluoroethyl crizotinib (¹⁹F/¹⁸F-FECr) and fluoroethyl alectinib (¹⁹F/¹⁸F-FEAl). Briefly, ¹⁹F/¹⁸F-FECr and ¹⁹F/¹⁸F-FEAl were synthesized as previously reported.² ¹⁹F/¹⁸F-fluoroethyl tosylate was prepared, purified and coupled with crizotinib or alectinib. The products were purified by flash column chromatography to yield ¹⁹F/¹⁸F-FECr and ¹⁹F/¹⁸F-FEAl. Alternatively, precursor compounds were synthesized, directly fluorinated with ¹⁹F/¹⁸F-fluoride, and purified to yield ¹⁹F/¹⁸F-FECr and ¹⁹F/¹⁸F-FEAl. In vitro cytotoxicity assays were performed in lung cancer ALK-positive H2228 and ALK-negative H441 cells using non-radioactive compounds. In vivo biodistribution and PET/CT imaging studies were performed on tumor-free nude mice using ¹⁸F-labeled compounds. Chemically, the first method produced ¹⁸F-FECr in 20-24% yield (n=8), molar activity of 37 GBq/μmol, and >99% purity. The second method produced ¹⁸F-FECr and ¹⁸F-FEAl in 40% (n=6) and 20% yields, respectively. In in vitro cytotoxicity assays with ALK-positive H2228 cells, FECr showed an IC50 = 7.5 μM and FEAl an IC50 = 40 nM, similar to their cognate parental compound, confirming that the modification was not detrimental to drug potency. Positron emission tomographic (PET) imaging in tumor-free mice showed significant brain uptake of the ¹⁸F-labeled analogues at 10 min post-injection (6.5 %/ID/g, ¹⁸F-FECr; 8.2 %ID/g, ¹⁸F-FEAl). In conclusion, novel fluoroethyl-modified crizotinib and alectinib and their ¹⁸F-labeled analogues were synthesized with good yields, high purity, and high specific activity. PET and biodistribution results suggest that these fluorinated analogues may penetrate the blood-brain barrier and be potential targeted drugs for treatment of lung cancer that has metastasised to brain. References: 1. Kodama T, et al. Cancer Chemother Pharmacol 2014, 74, 1023-1028. 2. Perera et al. J. Labelled Com Radiopharm. 2016, 59 103-108.

#11

Novel dual inhibitors of LSD1-HDAC6/8 for treatment of cancer.

Dhanalakshmi Sivanandhan, Sridharan Rajagopal, Sreekala Nair, Chandru Gajendran, Dimpy Ghosh, P Nagaraj, Subramanyam J. Tantry, Purushottam Dewang, Mahanandeesha S. Hallur, Kannan Murugan, Srinatha K. C, Damodara Kuntrapaku, M Dilipkumar, R Sharma, S Meghashree, Durga Prasanna Kumar, Suraj P. Ingle, Mohd Zainuddin, A B. Vinod, Sriram Rajagopal. Jubilant Biosys, Bangalore, India.

Introduction: Lysine Specific Demethylase 1 (LSD1) is a flavin adenine dinucleotide (FAD)-dependent amine oxidase that has been reported to be over-expressed in many malignant tumors. Down-regulation of LSD1 has been shown to effectively treat cancers by inducing re-expression of aberrantly silenced genes. Studies have shown that LSD1 may contribute to acute myelogenous leukemia pathogenesis by inhibiting the normal pro-differentiative function of ATRA, paving the way for new combinatorial therapies for AML. Similarly, HDAC isoform selective inhibitors are beginning to be explored as less toxic alternatives to panHDAC inhibitors in select cancers. Further, combined inhibition of LSD1 and HDAC has been shown to be more efficacious in inhibiting multiple cancers. Here, we show that JBI-295, a dual inhibitor targeting both LSD1 and HDAC6/8 shows stronger efficacy without enhancing systemic toxicity, in a subset of AML and JAK-dependent myeloproliferative cancer.

Methods: Computational chemistry approaches were used to design LSD1 specific and LSD1-HDAC dual inhibitors. To assess in vitro LSD1 potency, TR-FRET assay was used. For assessing in vitro HDAC activity fluorescence based HDAC6 activity assay was performed. Western blotting was used to assess biomarkers of LSD1 and HDAC inhibition. Alamar blue cytotoxicity assay was used to assess cell proliferation.

Results: Several compounds from this series show strong in vitro potency against LSD1 with more than excellent selectivity against MAOs. JBI-295 one of the lead dual molecules, showed strong LSD1 potency (IC50 of 0.07 μM) and isoform selective HDAC6/8 activity (IC50 of 0.006 and 0.08 µM on HDAC6 and HDAC8, respectively), with about 100 fold selectivity against other HDAC isoforms. JBI-295 showed strong anti-proliferative activity on leukaemia and multiple myeloma cell lines. In cell based and in vivo target engagement studies there was a concomitant increase in CD11b, CD86 and GFI1b and tubulin acetylation levels. JBI-295 was more efficacious in inhibiting the growth of leukemia HEL92.1.7 xenograft by oral administration when compared to IP administration of ACY-1215, a HDAC6 selective inhibitor. Stronger tumor growth inhibition was also observed in melanoma A375 xenograft model as compared to inhibitors with single activity. In addition, JBI-295 showed single agent activity in a syngeneic murine colon cancer model CT26 and also resulted in stronger tumor growth inhibition when combined with anti-PDL1 antibody.

Conclusion: The data obtained demonstrate that dual LSD1-HDAC6/8 inhibitors could serve as novel, effective therapeutic agents for treatment of select subset of cancer. Advanced efficacy and toxicology studies with JBI-295 are in progress.

#12

A novel Hsp90 machine inhibitor combining chemotherapy and immunotherapy.

Ahmed Chadli,¹ Nada Eisa,¹ Vincent Crowley,² Sumin Lu,¹ Yasmeen Jilani,¹ Hasan Korkaya,¹ Brian Blagg³. ¹Augusta Univ. Cancer Ctr., Augusta, GA; ²The University of Kansas, Lawrence, KS; ³University of Notre Dame, Notre Dame, IN.

Molecular chaperones are guardians of the proteome and of cellular homeostasis. Dysregulation in the function of the Hsp90 chaperoning machine in particular has been implicated in metabolic, oncological, neurodegenerative, and cardiovascular diseases. Targeting Hsp90 has been shown to have a combinatorial impact on dysfunctional circuitries that underlie human cancers. Thus, several inhibitors targeting the N-terminal ATP-binding pocket of Hsp90 are undergoing clinical trials as anticancer agents. They inactivate the ATPase activity of the chaperone, causing proteasomal degradation of its client oncogenic proteins. Although these inhibitors provided a proof-of-principal that Hsp90 is a valuable therapeutic target, they exhibit modest activity in the clinic, in part because they concomitantly induce a heat shock response (HSR), which turns on pro-survival pathways. Consequently, new mechanisms to inhibit the Hsp90 chaperone machine that do not induce the heat shock response are needed.

To address this, we have developed a unique high throughput screen (HTS) based on the progesterone receptor (PR) and the five purified chaperones required for folding steroid receptors, i.e., Hsp90, Hsp70, Hsp40, Hsp70/Hsp90 organizing protein (Hop), and p23. During our preliminary screening of natural products, we discovered that the compound AD07 kills cancer cells through inhibition of the Hsp90 machine without a significant induction of HSR. In addition, AD07 induces a powerful T cell-mediated immune response, resulting in highly efficient tumor killing in a syngeneic mouse model and long-term memory against the primary tumor. At the molecular level, AD07 reduces the protein levels of two key mediators of tumor-induced immune tolerance: programmed cell death ligand-1 (PDL-1) and indolamine 2,3 dioxygenase (IDO). We therefore propose that AD07 is a promising anti-tumor agent targeting the Hsp90 machine through a novel mechanism of action involving cancer cell toxicity, which increases its immunogenicity, and modulation of the tumor microenvironment to reduce immunotolerance.

#13

Structure elucidation, metabolism, and drug interaction potential of ACP-5862, an active, major, circulating metabolite of acalabrutinib.

Terry Podoll,¹ Paul G. Pearson,² Jerry Evarts,³ Tim Ingallinera,³ Hao Sun,⁴ Stephen Byard,⁵ Adrian J. Fretland,⁶ J. Greg Slatter³. ¹IV-PO, LLC, Seattle, WA; ²Pearson Pharma Partners, Westlake Village, WA; ³Acerta Pharma, a member of the AstraZeneca group, South San Francisco, CA; ⁴Covance Laboratories, Madison, WI; ⁵Arcinova, Alnwick, United Kingdom; ⁶Oncology DMPK, IMED Biotech Unit, AstraZeneca, Boston, MA.

Acalabrutinib (Calquence®) is a potent, selective, orally administered, covalent inhibitor of Bruton tyrosine kinase (BTK) that received accelerated approval for relapsed/refractory mantle cell lymphoma from the US FDA in October 2017. Profiling of acalabrutinib metabolites in human plasma revealed a late-eluting, +16 Da metabolite circulating at concentrations higher than parent drug. Metabolite regiochemistry could not be determined by mass spectrometry. In vitro metabolism and preparative HPLC was used to generate a pure sample of the metabolite for structural characterization by NMR. Confirmatory chemical synthesis revealed a pyrrolidine ring-opened ketone. The structure of the metabolite, designated ACP-5862, and a smaller -2 Da peak, identified as dehydropyrrolidine, M25, inferred a common carbinolamide intermediate in their genesis. Both metabolites retained the butynamide electrophile responsible for the inactivation of BTK. In vitro studies on the inhibition of BTK and related Tec and Src kinases revealed that ACP-5862 was active against BTK with similar selectivity and potency to acalabrutinib (Kaptein et al, 2019) This work then investigated the in vitro metabolism and drug transport features of acalabrutinib, and the metabolite ACP-5862, to establish the potential for clinical drug-drug interactions (DDI) via CYPs, UGTs and drug transporters. CYP reaction phenotyping indicated CYP3A4 was responsible for both the formation and further metabolism of ACP-5862. Km and Vmax values for the formation of ACP-5862 using rCYP3A4 were 2.78 μM and 4.13 pmol/pmol CYP/min, respectively. The in vitro intrinsic clearance of ACP-5862 was 23.6 μL/min/mg. Acalabrutinib weakly inhibited CYP2C8, CYP2C9 and CYP3A4 in vitro, and ACP-5862 weakly inhibited CYP2C9 and CYP2C19, with no inhibition of CYP1A2, CYP2B6, or CYP2D6. Similarly, UGT1A1, UGT2B7, and aldehyde oxidase were not inhibited. Neither parent or ACP-5862 strongly induced CYP1A2, CYP2B6, or CYP3A4 mRNA. Acalabrutinib and ACP-5862 were substrates of MDR1 and BCRP in vitro, but were not substrates of OATP1B1 or OATP1B3. Acalabrutinib was not a substrate of OAT1, OAT3, and OCT2. Based on static PK model calculations, acalabrutinib may cause a modest increase in exposure to coadministered BCRP substrates by inhibition of intestinal BCRP, but with no inhibition of BCRP at the systemic level. The PK of substrates of MDR1, MATE1, MATE2-K, OATP1B1, OATP1B3, OAT1, OAT3, and OCT2 are not likely to be altered by acalabrutinib or ACP-5862. These data were combined with clinical DDI data (Izumi et al, 2017) to simulate DDI in the presence of CYP3A inhibitors and inducers. PBPK models confirmed that acalabrutinib and ACP-5862 were not likely to perpetrate CYP2C8 or CYP3A4 mediated drug interactions (Zhou et al., 2019). Overall, acalabrutinib and major metabolite, ACP-5862 have a favorable drug interaction profile.

#14

A new orally available drug for the treatment of metastatic cancers.

Kun Zhou,¹ Jae Eun Cheong,² Michaela Zaffagni,¹ Yingjie Xu,¹ Lijun Sun,² Bruce Zetter¹. ¹Boston Children's Hospital, Boston, MA; ²Beth Israel Deaconess Medical Center, Boston, MA.

Metastatic cancers account for the great majority of cancer deaths. We conducted a screen to indetify known drugs that selectively inhibit the viability of metastatic tumor cells. Results of the screen identified members of the FDA-approved benzimidazole methylcarbamate family (e.g. mebendazole (MBZ) and albendazole (ALB)) as potential therapies for metastatic disease. Earlier work also supports a role for this chemical family in the potential treatment of activity in multiple cancers, but progress has been stalled by their poor water solubility and poor bioavailability for systemic delivery to disseminated tumors. We therefore synthesized a novel compound (OBD9) containing the scaffold of MBZ coupled to an oxetane group to enhance aqueous solubility to 361 μM. OBD9 demonstrates significant cytotoxicity toward a variety of cancer cell types including colon, lung, and prostate cancers (IC50: 0.9-2 μM). In a mouse xenograft model of human A549 lung cancer cell line OBD9 dramatically inhibited the growth of established tumors at 30 or 90 μM without noticeable toxicity. In a mouse xenograft model using highly aggressive PCMLN3 prostate cancer cells, oral administration of OBD9 at 30 mg/kg also significantly repressed growth of established tumors with no visible toxicity.

We have studied the working mechanisms behind OBD9 and found that the Wnt signaling pathway is suppressed by OBD9 treatment. Both qPCR and Western blot data suggest that the beta-catenin/TCF complex in the Wnt signaling pathway are inhibited by OBD9. Among the downstream targets of Wnt signaling pathway, the oncogene cMyc is also significantly decreased in different cancers including colon, lung and prostate cancer. Overall, our in vitro and in vivo data suggest that OBD9 potentially represents a novel therapeutic option for multiple cancers especially metastatic cancer through targeting Wnt signaling pathway and the downstream oncogene cMyc.

#15

Targeting vismodegib-resistant medulloblastoma using novel hedgehog pathway inhibitor and miR-29b mimic.

Virender Kumar, Bharti Sethi, Vinod Kumar, Timothy R. Mcguire, Don W. Coulter, Ram I. Mahato. University of Nebraska Medical Center, Omaha, NE.

The purpose of this study to develop targeted liposomes of MDB5 and miR-29b combination for medulloblastoma (MB) treatment. Due to high heterogeneity and diverse genetic make-up, MB treatment is a challenge. Aberrant activation of hedgehog (Hh) pathway regulates cell growth, cancer stem cell (CSC) proliferation, and tumorigenicity in sonic Hh subgroup of MB. Further, Hh signaling regulates yes-associated protein 1 (YAP1) and glutaminolysis for energy need of tumor cells. GDC-0449 a smoothened receptors (SMO) antagonist treats (SHH)-MB initially, but resistance develops after repeated administration. Cholesterol potentiates Hh pathway independent of SHH by binding directly to the SMO. On the other hand, our RNA-seq and miRNA profiling confirmed downregulation of tumor suppressor miR-29b-3p in human MB samples. miR-29b control genes of apoptosis; p53, MDM2, AKT2, and cholesterol biosynthesis including HMGCR and SREBP1. We recently synthesized a potent GDC-0449 analog MDB5 (SMO inhibitors), which binds strongly to various Smo-mutants compared to GDC-0449. We found that a combination of MDB5 and miR-29b can treat MB synergistically. When we modified miR-29b backbone with 2-O’-methoxyethyl phosphorothioate (OMe-PS-miR-29b-3p), it showed enhanced stability in 50% serum. We designed a cationic lipid MDC2 for liposomal delivery of MDB5 and miR-29b1 simultaneously. Liposomes were prepared using film hydration technique and characterized for particle size and zeta potential, drug loading and release profile, miRNA complexation, stability, and transfection efficiency. These liposomes incorporated 88% MDB5 when the drug to phospholipid molar ratio was kept at 5%. Complete complexation was observed at N/P ratio of 3:1. These liposomes when loaded with green fluorescent protein (GFP) siRNA, resulted in 70% of signal silencing into DAOY MB cells (Shh driven). Liposomal MDB5 had higher cytotoxicity in DAOY cells compared to GDC-0449. Further, MDB5 and OMe-PS-miR-29b loaded liposomes resulted in higher, cytotoxicity and % of apoptosis, G0-G1 phase arrest, and decreased target gene expression in DAOY cells. Combination of drugs to simultaneously target multiple pathways related to Hh, glutaminolysis, cholesterol biosynthesis, and apoptosis has the potential to treat MB. Successful completion of this study will provide a nanomedicine for treating SHH-MB and other brain tumors.

#16

3,3'-diindolylmethane induces cytoglobin expression and synergizes with poly ADP ribose polymerase inhibitor PJ34 to inhibit triple negative breast cancer cell growth.

Jonathan V. Wooten,¹ Nicole Mavingire,¹ Leah Rowland,¹ Jason Matthews,² Eileen Brantley¹. ¹Loma Linda University, Loma Linda, CA; ²University of Oslo, Oslo, Norway.

Triple negative breast cancer (TNBC), characterized by tumors that lack expression of the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), carries a poor prognosis. While recently approved poly ADP ribose polymerase (PARP) inhibitor olaparib shows favorable activity in patients with TNBC harboring tumors deficient in DNA repair enzyme BRCA1, patients with TNBC possessing BRCA1-proficient tumors are largely unresponsive to olaparib. Emerging evidence suggests that small molecules that activate aryl hydrocarbon receptor (AhR) signaling have the capacity to confer anticancer actions. 3,3′-diindolylmethane (DIM), an AhR ligand, is the major metabolite of indole-3-carbinol (I3C) found in cruciferous vegetables such as broccoli. Patients often consume natural product-derived AhR ligands while undergoing chemotherapy. It is therefore crucial to enhance our understanding of nutraceutical-pharmaceutical interactions. We previously demonstrated the ability of synthetic AhR ligands to induce the expression of putative tumor suppressor cytoglobin (CYGB) and inhibit Akt signaling in breast cancer cells. Since PARP inhibitor PJ34 has been shown to synergize with Akt inhibitors we hypothesize that DIM induces CYGB expression and synergizes with PJ34 to inhibit TNBC cell growth. Using the Alamar Blue assay, we found that DIM substantially enhanced the sensitivity of TNBC BRCA1 proficient cells to PJ34, though knockdown of CYGB did not significantly influence the responsiveness of cells to DIM. Chromatin immunoprecipitation followed by next-generation sequencing data revealed that DIM promoted the binding of AhR to the CYGB promoter in breast cancer cells. Finally, DIM induced CYGB gene expression in TNBC cells as determined by quantitative PCR. Taken together, our data suggest that DIM upregulates CYGB in TNBC cells and synergizes with PJ34 to confer anticancer actions in TNBC. Our data provide a rationale for incorporating the use of natural product-derived AhR ligands as a strategy to enhance PARP inhibitor efficacy in patients with TNBC that have BRCA1-proficient tumors.

#17

Novel STAT3 inhibitors identified by Structure-Based Virtual Ligand Screening incorporating SH2 domain flexibility.

Ren Kong,¹ Uddalak Bharadwaj,² Thomas Kris Eckols,² Moses Kasembeli,² Mikhail Kolosov,² Anh Tran,² Oluwatomilona I. Ifelayo,² Hong Zhao,³ Stephen T. Wong,³ David J. Tweardy². ¹Jiangsu University of Technology, Changzhou, China; ²UT MD Anderson Cancer Ctr., Houston, TX; ³Houston Methodist Research Institute, Weill Cornell Medicine, Houston, TX.

Efforts to develop STAT3 inhibitors have focused on its SH2 domain, starting with short phosphotyrosylated peptides with STAT3 binding motifs, e.g. pY905LPQTV within gp130. Despite binding to STAT3 with high affinity, issues regarding stability, bioavailability, and membrane permeability of these peptides, as well as peptidomimetics such as CJ-887, have limited their further clinical development and led to increased interest in small-molecule inhibitors. Some small molecule STAT3 inhibitors have been identified using structure-based virtual ligand screening (SB-VLS). While having favorable drug-like properties, most suffer from weak binding affinities, possibly due to the high flexibility of the target domain, especially within the region involved in pY-peptide binding. We conducted molecular dynamic (MD) simulations of the SH2 domain in a complex with CJ-887, with a focus on ligand-induced protein conformation changes that increase binding affinity. We used an averaged structure from this MD trajectory as induced-active site receptor model for SB-VLS of 110,000 compounds in the SPEC database. Screening was followed by re-docking, re-scoring of the top 30% of hits and selection for compounds that directly interact with pY+0 binding pocket residues R609-S613, resulting in 110 initial hits, which were then tested for STAT3 targeting using a panel of cellular and biochemical assays. 24 out of 110 compounds could inhibit G-CSF-stimulated pY-STAT3 by > 50% at 10 µM with 9 compounds inhibiting by > 99%; 8/24 compounds (SPEC-29, 8, 93, 98, 106, 57, 101 and 85) inhibited STAT3-binding to EGFR pY-peptide (SPR) by 29-71% at 10 µM; 5 of these (SPEC-29, 8, 93, 98, and 106) had pY-STAT3 IC50s ranging from 2.7-19.0 µM; 3 compounds (SPEC-29, 8, and 93) potently inhibited growth of pSTAT3-high MDA-MB-468 (IC50 = 2.4-12.1 µM) and MDA-MB-231 (IC50s = 3.0-12.2 µM) cells and the growth-inhibitory abilities correlated to abilities to inhibit pY-STAT3. Thus, we used a dynamic ligand-STAT3, bound-fit docking model to screen libraries that successfully identified two hits with potent activity against STAT3 both of which comply with all of Lipinski’s rules of five for drug-likeness. Importantly, while most STAT3 inhibitors previously identified have negatively charged moieties, which mediate their binding to the pY+0 binding pocket but limit cell penetration, the two hits identified are uncharged and likely will serve as superior candidates for future hit-to-lead development leading to an effective direct small-molecule STAT3 inhibitor. Furthermore, the ability of the technique employed here for identifying neutral hits may also be useful in designing inhibitors targeting SH2 domains within other oncogenic targets.

#18

Doubled lifespan in APCMin/+ mice by targeting MTAP.

Ross S. Firestone, Mu Feng, Vern L. Schramm. Albert Einstein College of Medicine, Bronx, NY.

Humans with Familial Adenomatous Polyposis (FAP) have increased risk of colon cancer. Mice with the APCMin/+ mutation provide an animal model of FAP. Interruption of epigenetic control by preventing salvage of S-adenosylmethionine from 5′-methylthioadenosine (MTA) was tested as a potential FAP therapy. 5′-Methylthioadenosine phosphorylase (MTAP) catalyzes the phosphorolysis of MTA to adenine and methylthioribose-1-phosphate for methionine recycling. The effect of MTDIA, a transition state analogue of MTAP, was tested as an oral agent in APCMin/+ mice. Survival analysis with 10, 20 and 30 mg/kg/day oral doses of MTDIA showed a 2-fold increase in mouse lifespan at the optimal 20 mg/kg/day dose. Metabolomic analysis (mouse liver) showed significant changes in metabolites relevant to methionine metabolism. The >4-fold increase in MTA, confirmed the physiologic target. Histologic analysis of intestinal tissue revealed slowed tumor progression in treated mice. RNAseq and Western blots provide additional information about the consequences of MTAP inhibition.

#19

Design and synthesis of biaryl indolyl benzamides as HDAC1-selective inhibitors via a fragment-based lead generation approach.

Ahmed T. Negmeldin,¹ Mary Kay H. Pflum². ¹Gulf Medical University, Ajman, United Arab Emirates; ²Wayne State Univeristy, Detroit, MI.

Histone deacetylase (HDAC) proteins are epigenetic regulatory enzymes that deacetylate protein substrates, leading to subsequent changes in cell function. HDAC proteins are implicated in cancers and several HDAC inhibitors have been approved by the FDA as anti-cancer drugs. Unfortunately, most of the FDA-approved drugs non-selectively inhibit most of the HDAC isoforms, which may lead to side effects in the clinic and limit their use on the bench top. Isoform-selective HDAC inhibitors may decrease or eliminate the side effects associated with non-selective inhibitors treatment. In addition, isoform-selective HDAC inhibitors can be used as molecular tools to study HDAC-related cancer biology. In this work, several biaryl indolyl benzamide compounds were designed and synthesized based on a fragment-based lead generation approach as potential HDAC1-selective inhibitors. The design was based on combining two different fragments from known HDAC1/2 and HDAC1/3 selective inhibitors. These fragments can discriminate against both HDAC2 and HDAC3 and possibly impart selectivity towards HDAC1. Docking studies were performed to validate the design of the inhibitors and to understand the binding of different isoform-selective inhibitors to HDAC1, HDAC2, and HDAC3. Potency and isoform selectivity of the new compounds were assessed via in vitro screening with different HDAC isoforms. All analogs showed modest selectivity for HDAC1 over HDAC2. Bnz-3 was identified as the best compound in terms of potency and selectivity. In particular, Bnz-3 displayed roughly 10- to 100-fold higher potency than the other compounds, with an IC50 value of 548 nM with HDAC1. In terms of selectivity, Bnz-3 showed about 10.6-fold selectivity for HDAC1 over HDAC2, with almost no inhibition of either HDAC3 or HDAC6. The new biaryl indolyl benzamide HDAC inhibitors represent potential lead compounds that can help to further develop more selective HDAC1 inhibitors.

#20

Pharmacology and metabolism of GLL398, an oral selective estrogen receptor degrader for endocrine therapy of breast cancer.

Shanchun Guo, Changde Zhang, Madhu Mottamal, Ahamed Hossain, Jiawang Liu, Jiawang Liu, Guangdi Wang. Xavier University of Louisiana, New Orleans, LA.

Selective estrogen receptor degrader (SERD) has proven clinically effective in treating advanced or metastatic breast cancer since the approval of fulvestrant by FDA in 2002. Recent expansion of indications as a first line monotherapy and as combination therapy with CDK4/6 inhibitors further demonstrates its clinical utility as an efficacious breast cancer endocrine regimen. However, the poor pharmacokinetic properties of fulvestrant and its injection-only administration route has driven continued efforts to develop orally bioavailability SERD that could potentially improve clinical response to SERD treatment. GLL398, a boron-modified GW5638 analog, showed superior oral bioavailability while retaining both antiestrogenic activity and ER degrading efficacy at a potency level comparable to the more active metabolite of GW5638, GW7604. Here we report further studies on the pharmacology, pharmacokinetics, and metabolism of GLL398. Consistent with GLL398’s robust activities in breast cancer cells that are tamoxifen resistant or express constitutively active, mutant ESR1 (Y537S), it was found to bind the mutant ERY537S at a high affinity. Molecular modeling of the binding mode of GLL398 to ER also found its molecular interactions consistent with the experimentally determined high binding affinity towards WT ER and ERY537S. To test if the superior oral bioavailability can be translated to potent efficacy in vivo, mice bearing MCF-7 derived xenograft breast tumors and patient derived xenograft (PDX) tumors harboring ERY537S were treated with GLL398 which potently inhibited tumor growth in mice. Finally the metabolism of GLL398 was also investigated to elucidate the biotransformation of the drug and its potential contribution to the superior oral bioavailability over GW7604.

#21

Generation and characterization of potent analogues of ERX-11.

Suryavathi Viswanadhapalli,¹ Mengxing Li,¹ Shi-Hong Ma,² Gangadhara Reddy Sareddy,¹ Tae-Kyung Lee,³ Mei Zhou,¹ Yiliao Liu,¹ Xihui Liu,² Dede N. Bahun-Wilson,² Kara Kassees,³ Jung-Mo Ahn,³ Ganesh V. Raj,² Ratna K. Vadlamudi¹. ¹UT Health Science Ctr., San Antonio, TX; ²UT Southwestern, Dallas, TX; ³UT Dallas, TX.

Breast cancer (BC) is the most common malignancy in women. Approximately 70% BC are Estrogen receptor (ER) positive, However, most patients develop resistance to current therapies and progress to incurable metastases. We had earlier reported a novel therapeutic agent, ERX-11, that modulates estrogen receptor coregulator interactions. For lead optimization, we designed, synthesized, and tested over 500 analogs of ERX-11 in multiple models of BC.

Methods: In vitro activity was tested using cell titer glo, MTT, and apoptosis assays. The utility of the ERX analogs in treating therapy resistant ER-positive BC was evaluated using models with acquired resistance (Tamoxifen, Letrozole), and engineered models that express ER mutations. Xenografts, patient derived xenografts (PDX), and patient derived xenograft explants (PDEx) were used for testing the utility of ERX analogs.

Results: Our screening studies identified several ERX analogs with potent activity against BC cells. Subtle changes in the ERX analogs appear to have significant ramifications on both their potency against ER-positive BC cell lines and against other tumors types. Some analogs like ERX-41 were more potent than ERX-11 in their ability to block the proliferation of multiple ER-positive BC cell lines (IC50 ranging from 50-200 nM). Other analogs like ERX-208 showed similar activity as ERX-11 against ER-positive BC cell lines but had potent activity (IC50 ranging from 50 -100 nM) against ovarian cancer cell lines. Through iterative changes, we have identified lead compounds with significant activity against other cancers, including in gliomas, ovarian and pancreatic cancers. Although all these compounds were designed to better target the ligand binding pocket of ER, CRISPR-Cas9 based KO screening revealed that these active compounds do not all target ER and appear to target other proteins, including other nuclear receptors. Some compounds for example have activity in ER-negative BC. In several xenograft and models, including pancreatic cancer and ER-negative BC, the activity of the compounds have been confirmed by oral administration of the ERX analogs in vivo. We have also validated the activity of these analogs using PDX and PDEX models.

Conclusions: From our studies to develop a more potent ERX-11 lead analog, we have identified multiple analogs with activities against multiple cancers. While the intended target of these analogs was ER, our library of analogs has potent activity against both ER-positive and ER-negative tumors. In collaboration, we are pursuing further leads in multiple cancers to further delineate the mechanism of action of these various analogs.

#22

Design and synthesis of substituted tetrahydroisoquinoline derivatives as anti-angiogenic and anti-breast cancer agents.

Kinfe Ken Redda,¹ Madhavi Gangapuram,² Suresh Eyuinni¹. ¹Florida A&M Univ., Tallahassee, FL; ²Florida A&M University, Tallahassee, FL.

Angiogenesis is the formation of new blood vessels and plays a critical role in the development, invasion and metastasis of breast cancer pathogenesis. Vascular Endothelial Growth Factor (VEGF) is one of the important angiogenic factors secreted by tumor cells. Overexpression of VEGF significantly increases tumor growth and angiogenesis in a murine model of breast cancer. Inhibition of VGEF pathway by limiting new blood vessels formation has proven to be a valuable approach in reducing tumor growth. During the last two decades, diversified small molecules were evaluated and developed as anti-angiogenic agents. Trabectedin (ET-743; Yondelis) is a tetrahydroisoquinoline alkaloid derived from a Caribbean tunicate Ecteinascidia turbinata and has a potent in vitro and in vivo antitumor activity, including prostate and breast cancers. Trabectedin significantly inhibited the mRNA and protein expression levels of VEGF in all breast cancer cells tested. Previously, we developed a series of tetrahydroisoquinoline (THIQs) derivatives and their in-vitro anti-proliferative activities were tested against MCF-7, MDA-MB-231 human breast cancer cell lines and Ishikawa human endometrial adenocarcinoma cell lines. These THIQ derivatives have the same core structure as that of Trabectedin alkaloid and are considered potential anti-angiogenic agents. Novel substituted THIQ derivatives were designed and synthesized in our laboratory. N-amination of substituted isoquinolines by the aminating agent, O-mesytelenesulfonylhydroxylamine and treated with acyl chlorides led to the formation of ylides. The ylides were reduced using sodium borohydride to yield the desired substituted tetrahydroisoquinolines in moderate to good yields. The synthesized compounds were characterized using NMR and elemental analysis. Of the synthesized compounds, fifteen compounds were screened for anti-angiogenesis activity in the Eli Lilly’s Open Innovation Drug Discovery Program (OIDD). Five compounds exhibited greater than 50% inhibition of the tube area at 10µM in the primary single point assay. These five compounds were further screened at different concentrations to obtain dose response curves and IC50 Values. Compound 4-ethyl-N-(3,4-dihydroisoquinolin-2(1H)-yl) benzamide showed an IC50 Value of 1.72 µM.

This research was supported by the National Center for Research Resources and the National Institute of Minority Health and Health Disparities of the National Institutes of Health through Grant Number 8 G12MD007582-28.

#23

Small molecule targeting of gankyrin reduces cell migration and proliferation in certain cancers.

Pamela Farrales, Aaron Muth. St. John's University, Queens, NY.

Introduction: Gankyrin is an oncoprotein involved in regulating numerous pathways important to cell growth, signaling, proliferation and death. The overexpression of gankyrin and subsequent increased protein-protein interactions have been identified as key to increased proliferation in a variety of cancers. Consequently, small molecules that bind to and disrupt these protein-protein interactions are promising therapeutic strategies for the treatment of certain cancers. The first small molecule binder of gankyrin, cjoc42, was recently developed and exhibited efficacy in inhibiting the gankyrin-26S proteasome interaction. The aim of this work is to determine the effects of cjoc42 and its derivatives on cell migration and proliferation in gankyrin-overexpressing cancers.

Methods: Wound healing and CyQuant proliferation assays were used to determine the ability of cjoc42 to inhibit migration and proliferation in the gankyrin-overexpressing cell lines A549, MDA-MB231 and Huh6 cells. A protein thermal shift assay was used to screen new cjoc42 derivatives for their ability to bind gankyrin. Western blot experiments will determine if binding of cjoc42 and its derivatives to gankyrin is the mechanism of action in cells.

Results: Cjoc42 demonstrated an ability to inhibit the migratory and proliferative activity of A549 and MDA-MB231 cells. While only a modest inhibition of migration was observed, cjoc42 exhibited a significant antiproliferative effect against these cell lines. Utilization of a protein thermal shift assay yielded cjoc42 derivatives with more potent gankyrin-binding ability. These compounds will be evaluated in-cellulo for their ability to inhibit cell migration and proliferation. Western blot experiments will confirm that gankyrin-binding of cjoc42 attributes to the anti-migration and anti-proliferative effects observed.

Conclusions: These studies show that cjoc42 directly inhibits cell migration and proliferation by inhibiting the interaction between gankyrin and the 26S proteasome. Consequently, this work provides background for the development of small molecule inhibitors as a gankyrin-mediated therapy for the treatment of lung, breast and liver cancers.

#24

Novel STAT3 & HDAC inhibitors that selectively target triple-negative breast cancer cell.

Bocheng Wu. Georgia Institution of Technology, Atlanta, GA.

Signal transducer and activator of transcription 3 (STAT3) is an oncogenetic transcriptional factor which can modulate cancer cells apoptosis. Stimulation of cancer cells by cytokines such as IL-6, INF-a, etc., can induce phosphorylation of STAT3, resulting in the translocation of p-STAT3 into the nucleus where it facilitates the expression of anti-apoptosis genes. This p-STAT3 anti-apoptotic activity is one drug resistance mechanism developed by cancer cells against chemotherapies including histone deacetylase inhibitors (HDACi). Pyrimethamine has been reported to inhibit STAT3 phosphorylation, mRNA transcriptional level, and STAT3-dependent cell viabilities. In this presentation, we disclosed novel pyrimethamine-based bifunctional STAT3-HDAC inhibitors as potential therapeutic agents for p-STAT3-dependent cancers. We will describe the design, synthesis and biological activities of STAT3-HDAC inhibitors. We will show that these bifunctional agents inhibit HDAC deacetylase activity and STAT3 phosphorylation. A subset of these compounds is more cytotoxic to MDA-MB-231, a triple-negative breast cancer cell line that is highly dependent on STAT3.

#25

Identifying new inhibitors of ABCG2 by high-throughput screening.

Shoji Kokubo, Shinobu Ohnuma, Hideaki Karasawa, Akihiro Yamamura, Hideyuki Suzuki, Megumi Murakami, Norihiko Sugisawa, Keigo Kanehara, Keisuke Sato, Takanori Ishida, Takashi Kamei, Takeshi Naitoh, Michiaki Unno. Tohoku University Graduate School of Medicine, Sendai, Japan.

Background: ABCG2, also known as breast cancer resistance protein (BCRP), is a member of the ATP-binding cassette (ABC)-family of membrane transport proteins associated with multidrug resistance (MDR). No ABCG2 inhibitor has shown clinical significance yet.

Aim: To identify novel inhibitors of ABCG2 by high-throughput screening.

Material & Methods: After higher expression of ABCG2 in HCT-116/BCRP cells was confirmed by flow cytometry (FACSVerse) using 5D3 antibody, high-throughput fluorescent cell-based assay was performed with the original chemical library of Tohoku University containing about 6000 compounds using ABCG2-specific fluorescent substrate, Pheophorbide a (PhA) in HCT-116/BCRP cells overexpressing ABCG2. Ko143, a potent ABCG2 inhibitor, was used as positive control. Fluorescence intensity in the cells was read by a fluorescence plate reader (SpectraMax M2e) in bottom read mode, with 395 nm excitation, 670 nm emission. To validate the results, flow cytometry assay was performed with 10 µM PhA in HCT-116/BCRP cells. The fluorescence intensity was analyzed by flow cytometry (FACSVerse).

Results: Fluorescenct cell-based assay with high-throughput screening could detect 23 candidate compounds, which showed higher fluorescence intensity as positive control Ko143 showed. Then, these 23 compounds were validated with flow cytometry assays. The flow cytometry assays revealed that 16 compounds increased fluorescence intensity of PhA in HCT-116/BCRP cells in dose dependent manner, suggesting that these candidate compounds inhibited transport function of ABCG2. Among 16 compounds, 6 compounds showed further inhibitory effect for transport function of ABCG2 as equivalent to that of Ko143.

Conclusion: These 6 compounds may provide a basis for further investigation of ABCG2 function.

#26

Development of potent lead compounds in multiple tumor types.

Ganesh V. Raj,¹ Xihui Liu,¹ Dede Ekoue,¹ Jung-Mo Ahn,² Ratna Vadlamudi³. ¹UT Southwestern Medical Ctr., Dallas, TX; ²UT Dallas, Dallas, TX; ³UT Health Sciences Center at San Antonio, San Antonio, TX.

Background: We had earlier reported a novel therapeutic agent, ERX-11, that modulates estrogen receptor coregulator interactions. For lead optimization, we designed, synthesized and tested over 500 analogs of ERX-11 in multiple models of BC. We also tested this library of compounds for activity against other cancer cell lines both in vitro and in vivo and against primary tumors ex vivo.

Methods: In vitro activity was tested using Cell titer glo, MTT, and apoptosis assays.The utility of the ERX analogs in treating therapy resistant ER-positive BC was evaluated using models with acquired resistance (Tamoxifen, Letrozole), and engineered models that express ER mutations. Xenografts were used for testing the utility of ERX analogs in vivo. Primary patient tumor derived explants were used for ex vivo testing of ERX analogs.

Results: Our screening studies identified several ERX analogs with potent activity against BC cells. Subtle changes in the ERX analogs appear to have significant ramifications on both their potency against ER+ BC cell lines and against other tumors types. Some analogs like ERX-41 were more potent than ERX-11 in their ability to block the proliferation of multiple ER-positive BC cell lines (IC50 ranging from 20-200nM). Other analogs like ERX-208 showed similar activity as ERX-11 against ER-positive BC cell lines (IC50 ranging from 100-500nM) but had potent activity against ovarian cancer cell lines. Through iterative changes, we have identified leads compounds with significant activity against other cancers, including in gliomas, ovarian and pancreatic cancers. Although all these compounds were designed to better target the ligand binding pocket of ER, these active compounds do not all target ER and appear to target other proteins, including other nuclear receptors. Some compounds for example have activity in ER-negative breast cancers. In several xenograft models, including pancreatic cancer and ER-negative, the activity of the compounds have been confirmed by oral administration of the ERX analog in vivo. We have also validated the activity of these analogs in patient-derived explants cultured ex vivoin multiple tumor types.

Conclusions: From our studies to develop a more potent ERX-11 lead analog, we have identified multiple analogs with distinct activities against multiple cancers. While the intended target of these analogs was ER, our library of analogs has potent activity against both ER-positive and ER-negative tumors. In collaboration, we are pursuing further leads in multiple cancers to further delineate the mechanism of action of these various analogs.

#27

Identification and characterization of a novel dual FLT3/microtubule polymerization inhibitor through cell-based screening.

Haleema Sadia Malik,¹ Aishah Bilal,¹ Rahim Ullah,¹ Maheen Iqbal,¹ Rahman Shah Saleem,¹ Hidayat Hussain,² Amir Faisal¹. ¹Lahore University of Management Sciences, Lahore, Pakistan; ²Leibniz Institute of Plant Biochemistry, Halle, Germany.

Activating mutations in FLT3 receptor tyrosine kinase are found in one-third of acute myeloid leukemia (AML) patients and are associated with disease relapse and bad prognosis. Majority of these mutations are Internal tandem duplications (ITD) in the juxtamembrane domain of FLT3 and have been validated as a therapeutic target. Clinical success of selective inhibitors targeting oncogenic FLT3, however, has been limited due to the acquisition of drug resistance. Here we report the identification of a dual FLT3/microtubule polymerization inhibitor, HH-IA-208, through cell-based screenings for the differential killing of leukemia cell lines expressing FLT3-ITD (MV411) or BCR-ABL (K562) using an MTS proliferation assay and for mitotic inducers using a dot-blot assay for histone H3 phosphorylation in HCT116 cell line. HH-IA-208, a chalcone derivative, inhibits FLT3 in vitro and treatment of FLT3-ITD+ cell lines with HH-IA-208 results in reduced FLT3 signaling. Similarly, HH-IA-208 inhibits tubulin polymerization in a biochemical assay and in cells that results in a mitotic arrest. HH-IA-208 is selectively more potent in leukemia cell lines expressing FLT3-ITD and treatment for 24 and 48 hours with the inhibitor results in apoptotic cell death. Furthermore, HH-IA-208 is able to overcome TKD mutation-mediated acquired resistance to FLT3 inhibitors in a MOLM13 cell line expressing FLT3-ITD with the D835Y mutation. HH-IA-208, therefore, is a promising lead for the discovery of dual target FLT3 inhibitors that can overcome resistance to selective FLT3 inhibitors.

#28

Dihydrolipoyl dehydrogenase is a key molecule inducing ferroptotic cell death.

Jihee Kim, Daiha Shin, Jaewang Lee, Jong-Lyel Roh. Asan Medical Center, Unv. of Ulsan College of Medicine, Seoul, Republic of Korea.

Ferroptosis is iron-dependent, non-apoptotic cell death from lipid peroxidation. Cystine-glutamate transporter (xCT) and glutathione peroxidase 4 (GPX4) are recognized as the key regulators of ferroptosis. Glutathione depletion via cystine deficiency or inhibition of xCT or GPX4 accumulates cellular lipid peroxidation and ferrous iron, resulting in ferroptosis. Glutamine is an amino acid essential for growth of cancer cells as well as induction of ferroptotic cell death via the production of alpha-ketoglutarate (αKG) from glutaminolysis. However, it is still unclear whether αKG or its relevant metabolic process is engaged in ferroptotic cell death. Therefore, we examined the role of αKG dehydrogenase (αKGDH) in induction of ferroptosis in head and neck cancer (HNC) cells. Cystine-deficient conditioned medium or xCT inhibition with sulfasalazine induced typical ferroptotic cell death, which was prevented by the inhibition of glutaminolysis or αKG production. Next, we focused the dihydrolipoyl dehydrogenase (DLD) and dihydrolipoyl succinyltransferase (DLST), consisting of the E3 and E2 components of αKGDH respectively. Ferroptosis was significantly prevented by genetic inhibition of the DLD but not the DLST, which was rescued by re-insertion of the DLD gene. Cellular reactive oxygen, lipid peroxidation, and ferrous iron accumulation were induced by the increased function of αKGDH, that is mainly the DLD, when αKG was converted to succinate in the tricarboxylic acid cycle. Sulfasalazine did not significantly suppress the in vivo growth of the shDLD-transfected HNC cells but vector control transplanted in nude mice. Taken together, our data support that the DLD is a key molecule inducing ferroptosis in cancer cells.

TUMOR BIOLOGY

3D Models

#29

Activity of trastuzumab emtansine (T-DM1) in 3D cell culture.

Jean Z. Boyer,¹ Gail Lewis Phillips,² Hiro Nitta,¹ Karl Garsha,¹ Eric May,¹ Brittany Admire,¹ Robert Kraft,¹ Megan Peccarelli,¹ Andre Zamorano,¹ Scott Gill,¹ Eslie Dennis,¹ Liz Vela,¹ Penny Towne¹. ¹Ventana Medical Systems, Inc. Roche Tissue Diagnostics, Oro Valley, AZ; ²Genentech, Inc, CA.

Introduction: Cell spheroids/aggregates generated from three dimensional (3D) cell culture methods are similar to real tumors in terms of tissue morphology, biology, and gene expression. We performed a unique 3D cell culture drug efficacy study with Trastuzumab emtansine (T-DM1) across a number of breast cancer cell lines that were previously investigated in 2D cell culture (Lewis Phillips, et al, 2008). We obtained significantly different results for some cell lines grown as 3D spheroids/aggregates when compared to those grown as 2D cultures.

Methodology: We performed 3D cell culture and produced FFPE blocks (using novel methods) from 3D spheroids/aggregates to determine HER2 (IHC) protein expression levels for five cell lines: SK-BR-3; BT-474; MDA-MB-361; MDA-MB-175 and MCF-7. We also performed HER2 gene-protein assay (HER2 GPA) to determine HER2 gene amplification along with IHC protein expression status in four cell lines: SK-BR-3; BT-474; MDA-MB-361 and MDA-MB-175. Drug (T-DM1) activity testing using CellTiter™-Glo 3D cell viability assay was performed on 3D cell spheroids/aggregates for comparison with 2D cells. Images were obtained of T-DM1 internalization in BT-474 cells and spheroids using pHrodo™ iFL Human IgG Labeling Reagent.

Results: In