Michael Grondine

AZD7762, a novel checkpoint kinase inhibitor, drives checkpoint abrogation and potentiates DNA-targeted therapies

Insights from cell cycle research have led to the hypothesis that tumors may be selectively sensitized to DNA-damaging agents resulting in improved antitumor activity and a wider therapeutic margin. The theory relies on the observation that the majority of tumors are deficient in the G1-DNA damage checkpoint pathway resulting in reliance on S and G2 checkpoints for DNA repair and cell survival. The S and G2 checkpoints are regulated by checkpoint kinase 1, a serine/threonine kinase that is activated in response to DNA damage; thus, inhibition of checkpoint kinase 1 signaling impairs DNA repair and increases tumor cell death. Normal tissues, however, have a functioning G1 checkpoint signaling pathway allowing for DNA repair and cell survival. Here, we describe the preclinical profile of AZD7762, a potent ATP-competitive checkpoint kinase inhibitor in clinical trials. AZD7762 has been profiled extensively in vitro and in vivo in combination with DNA-damaging agents and has been shown to potentiate response in several different settings where inhibition of checkpoint kinase results in the abrogation of DNA damage-induced cell cycle arrest. Dose-dependent potentiation of antitumor activity, when AZD7762 is administered in combination with DNA-damaging agents, has been observed in multiple xenograft models with several DNA-damaging agents, further supporting the potential of checkpoint kinase inhibitors to enhance the efficacy of both conventional chemotherapy and radiotherapy and increase patient response rates in a variety of settings. [Mol Cancer Ther 2008;7(9):2955–66]

AZD1208, a potent and selective pan-Pim kinase inhibitor, demonstrates efficacy in preclinical models of acute myeloid leukemia

Upregulation of Pim kinases is observed in several types of leukemias and lymphomas. Pim-1, -2, and -3 promote cell proliferation and survival downstream of cytokine and growth factor signaling pathways. AZD1208 is a potent, highly selective, and orally available Pim kinase inhibitor that effectively inhibits all three isoforms at <5 nM or <150 nM in enzyme and cell assays, respectively. AZD1208 inhibited the growth of 5 of 14 acute myeloid leukemia (AML) cell lines tested, and sensitivity correlates with Pim-1 expression and STAT5 activation. AZD1208 causes cell cycle arrest and apoptosis in MOLM-16 cells, accompanied by a dose-dependent reduction in phosphorylation of Bcl-2 antagonist of cell death, 4EBP1, p70S6K, and S6, as well as increases in cleaved caspase 3 and p27. Inhibition of p4EBP1 and p-p70S6K and suppression of translation are the most representative effects of Pim inhibition in sensitive AML cell lines. AZD1208 inhibits the growth of MOLM-16 and KG-1a xenograft tumors in vivo with a clear pharmacodynamic-pharmacokinetic relationship. AZD1208 also potently inhibits colony growth and Pim signaling substrates in primary AML cells from bone marrow that are Flt3 wild-type or Flt3 internal tandem duplication mutant. These results underscore the therapeutic potential of Pim kinase inhibition for the treatment of AML.

AZD2014, an Inhibitor of mTORC1 and mTORC2, Is Highly Effective in ER+ Breast Cancer When Administered Using Intermittent or Continuous Schedules

mTOR is an atypical serine threonine kinase involved in regulating major cellular functions, such as nutrients sensing, growth, and proliferation. mTOR is part of the multiprotein complexes mTORC1 and mTORC2, which have been shown to play critical yet functionally distinct roles in the regulation of cellular processes. Current clinical mTOR inhibitors only inhibit the mTORC1 complex and are derivatives of the macrolide rapamycin (rapalogs). Encouraging effects have been observed with rapalogs in estrogen receptor–positive (ER+) breast cancer patients in combination with endocrine therapy, such as aromatase inhibitors. AZD2014 is a small-molecule ATP competitive inhibitor of mTOR that inhibits both mTORC1 and mTORC2 complexes and has a greater inhibitory function against mTORC1 than the clinically approved rapalogs. Here, we demonstrate that AZD2014 has broad antiproliferative effects across multiple cell lines, including ER+ breast models with acquired resistance to hormonal therapy and cell lines with acquired resistance to rapalogs. In vivo, AZD2014 induces dose-dependent tumor growth inhibition in several xenograft and primary explant models. The antitumor activity of AZD2014 is associated with modulation of both mTORC1 and mTORC2 substrates, consistent with its mechanism of action. In combination with fulvestrant, AZD2014 induces tumor regressions when dosed continuously or using intermittent dosing schedules. The ability to dose AZD2014 intermittently, together with its ability to block signaling from both mTORC1 and mTORC2 complexes, makes this compound an ideal candidate for combining with endocrine therapies in the clinic. AZD2014 is currently in phase II clinical trials. Mol Cancer Ther; 14(11); 2508–18. ©2015 AACR.

Discovery of Checkpoint Kinase Inhibitor (S)-5-(3-Fluorophenyl)-N-(piperidin-3-yl)-3-ureidothiophene-2-carboxamide (AZD7762) by Structure-Based Design and Optimization of Thiophenecarboxamide Ureas.

Checkpoint kinases CHK1 and CHK2 are activated in response to DNA damage that results in cell cycle arrest, allowing sufficient time for DNA repair. Agents that lead to abrogation of such checkpoints have potential to increase the efficacy of such compounds as chemo- and radiotherapies. Thiophenecarboxamide ureas (TCUs) were identified as inhibitors of CHK1 by high throughput screening. A structure-based approach is described using crystal structures of JNK1 and CHK1 in complex with 1 and 2 and of the CHK1-3b complex. The ribose binding pocket of CHK1 was targeted to generate inhibitors with excellent cellular potency and selectivity over CDK1and IKKβ, key features lacking from the initial compounds. Optimization of 3b resulted in the identification of a regioisomeric 3-TCU lead 12a. Optimization of 12a led to the discovery of the clinical candidate 4 (AZD7762), which strongly potentiates the efficacy of a variety of DNA-damaging agents in preclinical models.

Discovery and Optimization of a Novel Series of Potent Mutant B-Raf V600E Selective Kinase Inhibitors.

B-Raf represents an attractive target for anticancer therapy and the development of small molecule B-Raf inhibitors has delivered new therapies for metastatic melanoma patients. We have discovered a novel class of small molecules that inhibit mutant B-Raf(V600E) kinase activity both in vitro and in vivo. Investigations into the structure-activity relationships of the series are presented along with efforts to improve upon the cellular potency, solubility, and pharmacokinetic profile. Compounds selectively inhibited B-Raf(V600E) in vitro and showed preferential antiproliferative activity in mutant B-Raf(V600E) cell lines and exhibited selectivity in a kinase panel against other kinases. Examples from this series inhibit growth of a B-Raf(V600E) A375 xenograft in vivo at a well-tolerated dose. In addition, aminoquinazolines described herein were shown to display pERK elevation in nonmutant B-Raf cell lines in vitro.

Sensitivity to PI3K and AKT inhibitors is mediated by divergent molecular mechanisms in subtypes of DLBCL

Activated B-cell-like (ABC) and germinal center B-cell-like diffuse large B-cell lymphoma (DLBCL) represent the 2 major molecular DLBCL subtypes. They are characterized by differences in clinical course and by divergent addiction to oncogenic pathways. To determine activity of novel compounds in these 2 subtypes, we conducted an unbiased pharmacologic in vitro screen. The phosphatidylinositol-3-kinase (PI3K) α/δ (PI3Kα/δ) inhibitor AZD8835 showed marked potency in ABC DLBCL models, whereas the protein kinase B (AKT) inhibitor AZD5363 induced apoptosis in PTEN-deficient DLBCLs irrespective of their molecular subtype. These in vitro results were confirmed in various cell line xenograft and patient-derived xenograft mouse models in vivo. Treatment with AZD8835 induced inhibition of nuclear factor κB signaling, prompting us to combine AZD8835 with the Brutons tyrosine kinase inhibitor ibrutinib. This combination was synergistic and effective both in vitro and in vivo. In contrast, the AKT inhibitor AZD5363 was effective in PTEN-deficient DLBCLs through downregulation of the oncogenic transcription factor MYC. Collectively, our data suggest that patients should be stratified according to their oncogenic dependencies when treated with PI3K and AKT inhibitors.

Synthesis and Evaluation of Triazolones as Checkpoint Kinase 1 Inhibitors.

Checkpoint kinase 1 (Chk1, CHEK1) is a Ser/Thr protein kinase that plays a key role in mediating the cellular response to DNA-damage. Synthesis and evaluation of a previously described class of Chk1 inhibitors, triazoloquinolones/triazolones (TZs) is further described herein. Our investigation of structure-activity relationships led to the identification of potent inhibitors 14c, 14h and 16e. Key challenges included modulation of physicochemical properties and pharmacokinetic (PK) parameters to enable compound testing in a Chk1 specific hollow fiber pharmacodynamic model. In this model, 16e was shown to abrogate topotecan-induced cell cycle arrest in a dose dependent manner. The demonstrated activity of TZs in this model in combination with a chemotherapeutic agent as well as radiotherapy validates this series of Chk1 inhibitors. X-ray crystal structures (PDB code: 2YEX and 2YER) for an initial lead and an optimized analog are also presented.

Abstract 2796: Efficacy and biomarker modulation by AZD1208, a novel, potent and selective pan-Pim kinase inhibitor, in models of acute myeloid leukemia

The Pim serine/threonine kinase family is composed of three highly homologous members; Pim-1, Pim-2 and Pim-3, identified by the ability of the prototype member Pim-1 to drive lymphomagenesis in mice. Upregulation of Pim-1 and Pim-2 is observed in leukemias and lymphomas, including AML, NHL and CLL, highlighting the potential of these kinases as therapeutic targets in these indications. Overexpression of Pim-1 or Pim-3 has also been observed in prostate, pancreatic, gastric, bladder and hepatocellular cancers. Pim kinases are downstream effectors of many cytokine and growth factor signaling pathways and are direct transcriptional targets of STAT transcription factors activated by these pathways. Pims can phosphorylate multiple substrates to mediate cell proliferation and survival. Here we describe the activity of AZD1208, an orally available, potent and highly selective Pim inhibitor that effectively inhibits all three isoforms. AZD1208 inhibits the growth of several AML cell lines and sensitivity correlates with the level of Pim-1 expression, STAT5 activation and presence of protein tyrosine kinase mutation. AZD1208 causes cell cycle arrest and apoptosis in MOLM-16 cells in culture. This is accompanied by a dose-dependent reduction in phosphorylation of BAD, 4EBP1 and p70S6K. AZD1208 suppresses the growth of MOLM-16 and KG-1a xenograft tumors in vivo in a dose proportional manner. In addition, AZD1208 leads to potent inhibition of colony growth of primary AML cells from bone marrow aspirates and downregulates phosphorylation of Pim targets. These results underscore the therapeutic potential of Pim kinase inhibition by AZD1208 for the treatment of AML. They also further support investigation of this inhibitor in other hematological and solid tumor malignancies where PIM signaling may play a role in tumorigenesis and survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2796. doi:1538-7445.AM2012-2796

Generation of stable PDX derived cell lines using conditional reprogramming

Efforts to develop effective cancer therapeutics have been hindered by a lack of clinically predictive preclinical models which recapitulate this complex disease. Patient derived xenograft (PDX) models have emerged as valuable tools for translational research but have several practical limitations including lack of sustained growth in vitro. In this study, we utilized Conditional Reprogramming (CR) cell technology- a novel cell culture system facilitating the generation of stable cultures from patient biopsies- to establish PDX-derived cell lines which maintain the characteristics of the parental PDX tumor. Human lung and ovarian PDX tumors were successfully propagated using CR technology to create stable explant cell lines (CR-PDX). These CR-PDX cell lines maintained parental driver mutations and allele frequency without clonal drift. Purified CR-PDX cell lines were amenable to high throughput chemosensitivity screening and in vitro genetic knockdown studies. Additionally, re-implanted CR-PDX cells proliferated to form tumors that retained the growth kinetics, histology, and drug responses of the parental PDX tumor. CR technology can be used to generate and expand stable cell lines from PDX tumors without compromising fundamental biological properties of the model. It offers the ability to expand PDX cells in vitro for subsequent 2D screening assays as well as for use in vivo to reduce variability, animal usage and study costs. The methods and data detailed here provide a platform to generate physiologically relevant and predictive preclinical models to enhance drug discovery efforts.

Adventures in Scaffold Morphing: Discovery of Fused Ring Heterocyclic Checkpoint Kinase 1 (CHK1) Inhibitors

Checkpoint kinase 1 (CHK1) inhibitors are potential cancer therapeutics that can be utilized for enhancing the efficacy of DNA damaging agents. Multiple small molecule CHK1 inhibitors from different chemical scaffolds have been developed and evaluated in clinical trials in combination with chemotherapeutics and radiation treatment. Scaffold morphing of thiophene carboxamide ureas (TCUs), such as AZD7762 (1) and a related series of triazoloquinolines (TZQs), led to the identification of fused-ring bicyclic CHK1 inhibitors, 7-carboxamide thienopyridines (7-CTPs), and 7-carboxamide indoles. X-ray crystal structures reveal a key intramolecular noncovalent sulfur-oxygen interaction in aligning the hinge-binding carboxamide group to the thienopyridine core in a coplanar fashion. An intramolecular hydrogen bond to an indole NH was also effective in locking the carboxamide in the preferred bound conformation to CHK1. Optimization on the 7-CTP series resulted in the identification of lead compound 44, which displayed respectable drug-like properties and good in vitro and in vivo potency.