Mark Charles

A Novel Small-Molecule Inhibitor of Protein Kinase D Blocks Pancreatic Cancer Growth In vitro and In vivo

Protein kinase D (PKD) family members are increasingly implicated in multiple normal and abnormal biological functions, including signaling pathways that promote mitogenesis in pancreatic cancer. However, nothing is known about the effects of targeting PKD in pancreatic cancer. Our PKD inhibitor discovery program identified CRT0066101 as a specific inhibitor of all PKD isoforms. The aim of our study was to determine the effects of CRT0066101 in pancreatic cancer. Initially, we showed that autophosphorylated PKD1 and PKD2 (activated PKD1/2) are significantly upregulated in pancreatic cancer and that PKD1/2 are expressed in multiple pancreatic cancer cell lines. Using Panc-1 as a model system, we showed that CRT0066101 reduced bromodeoxyuridine incorporation; increased apoptosis; blocked neurotensin-induced PKD1/2 activation; reduced neurotensin-induced, PKD-mediated Hsp27 phosphorylation; attenuated PKD1-mediated NF-κB activation; and abrogated the expression of NF-κB-dependent proliferative and prosurvival proteins. We showed that CRT0066101 given orally (80 mg/kg/d) for 24 days significantly abrogated pancreatic cancer growth in Panc-1 subcutaneous xenograft model. Activated PKD1/2 expression in the treated tumor explants was significantly inhibited with peak tumor concentration (12 μmol/L) of CRT0066101 achieved within 2 hours after oral administration. Further, we showed that CRT0066101 given orally (80 mg/kg/d) for 21 days in Panc-1 orthotopic model potently blocked tumor growth in vivo. CRT0066101 significantly reduced Ki-67–positive proliferation index (P < 0.01), increased terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling–positive apoptotic cells (P < 0.05), and abrogated the expression of NF-κB–dependent proteins including cyclin D1, survivin, and cIAP-1. Our results show for the first time that a PKD-specific small-molecule inhibitor CRT0066101 blocks pancreatic cancer growth in vivo and show that PKD is a novel therapeutic target in pancreatic cancer. Mol Cancer Ther; 9(5); 1136–46. ©2010 AACR.

Characterization of the biological effects of a novel protein kinase D inhibitor in endothelial cells.

VEGF (vascular endothelial growth factor) plays an essential role in angiogenesis during development and in disease largely mediated by signalling events initiated by binding of VEGF to its receptor, VEGFR2 (VEGF receptor 2)/KDR (kinase insert domain receptor). Recent studies indicate that VEGF activates PKD (protein kinase D) in endothelial cells to regulate a variety of cellular functions, including signalling events, proliferation, migration and angiogenesis. To better understand the role of PKD in VEGF-mediated endothelial function, we characterized the effects of a novel pyrazine benzamide PKD inhibitor CRT5 in HUVECs (human umbilical vein endothelial cells). The activity of the isoforms PKD1 and PKD2 were blocked by this inhibitor as indicated by reduced phosphorylation, at Ser916 and Ser876 respectively, after VEGF stimulation. The VEGF-induced phosphorylation of three PKD substrates, histone deacetylase 5, CREB (cAMP-response-element-binding protein) and HSP27 (heat-shock protein 27) at Ser82, was also inhibited by CRT5. In contrast, CRT6, an inactive analogue of CRT5, had no effect on PKD or HSP27 Ser82 phosphorylation. Furthermore, phosphorylation of HSP27 at Ser78, which occurs solely via the p38 MAPK (mitogen-activated protein kinase) pathway, was also unaffected by CRT5. In vitro kinase assays show that CRT5 did not significantly inhibit several PKC isoforms expressed in endothelial cells. CRT5 also decreased VEGF-induced endothelial migration, proliferation and tubulogenesis, similar to effects seen when the cells were transfected with PKD siRNA (small interfering RNA). CRT5, a novel specific PKD inhibitor, will greatly facilitate the study of the role of PKD signalling mechanisms in angiogenesis.

Identification of 3-aminothieno[2,3-b]pyridine-2-carboxamides and 4-aminobenzothieno[3,2-d]pyrimidines as LIMK1 inhibitors

A high throughput chemical screening campaign has led to the identification of 3-aminobenzo[b]thiophene-2-carboxamides as LIMK1 inhibitors. Evolution of bicyclic hits to the tricyclic 4-aminobenzothieno[3,2-d]pyrimidine, using a traditional medicinal chemistry SAR guided approach, resulted in a significant increase in potency. Further elaboration has seen the 7-phenyl-4-aminobenzothieno[3,2-d]pyrimidine emerge as a LIMK1 inhibitor lead candidate.

LIM kinase inhibitors disrupt mitotic microtubule organization and impair tumor cell proliferation

The actin and microtubule cytoskeletons are critically important for cancer cell proliferation, and drugs that target microtubules are widely-used cancer therapies. However, their utility is compromised by toxicities due to dose and exposure. To overcome these issues, we characterized how inhibition of the actin and microtubule cytoskeleton regulatory LIM kinases could be used in drug combinations to increase efficacy. A previously-described LIMK inhibitor (LIMKi) induced dose-dependent microtubule alterations that resulted in significant mitotic defects, and increased the cytotoxic potency of microtubule polymerization inhibitors. By combining LIMKi with 366 compounds from the GSK Published Kinase Inhibitor Set, effective combinations were identified with kinase inhibitors including EGFR, p38 and Raf. These findings encouraged a drug discovery effort that led to development of CRT0105446 and CRT0105950, which potently block LIMK1 and LIMK2 activity in vitro, and inhibit cofilin phosphorylation and increase αTubulin acetylation in cells. CRT0105446 and CRT0105950 were screened against 656 cancer cell lines, and rhabdomyosarcoma, neuroblastoma and kidney cancer cells were identified as significantly sensitive to both LIMK inhibitors. These large-scale screens have identified effective LIMK inhibitor drug combinations and sensitive cancer types. In addition, the LIMK inhibitory compounds CRT0105446 and CRT0105950 will enable further development of LIMK-targeted cancer therapy.

Discovery, Development, and SAR of Aminothiazoles as LIMK Inhibitors with Cellular Anti-Invasive Properties

As part of a program to develop a small molecule inhibitor of LIMK, a series of aminothiazole inhibitors were discovered by high throughput screening. Scaffold hopping and subsequent SAR directed development led to a series of low nanomolar inhibitors of LIMK1 and LIMK2 that also inhibited the direct biomarker p-cofilin in cells and inhibited the invasion of MDA MB-231-luc cells in a matrigel inverse invasion assay.

INVESTIGATION OF THE ROLE OF PROTEIN KINASE D IN HUMAN RHINOVIRUS REPLICATION

ABSTRACT Picornavirus replication is known to cause extensive remodeling of Golgi and endoplasmic reticulum membranes, and a number of the host proteins involved in the viral replication complex have been identified, including oxysterol binding protein (OSBP) and phosphatidylinositol 4-kinase III beta (PI4KB). Since both OSBP and PI4KB are substrates for protein kinase D (PKD) and PKD is known to be involved in the control of Golgi membrane vesicular and lipid transport, we hypothesized that PKD played a role in viral replication. We present multiple lines of evidence in support of this hypothesis. First, infection of HeLa cells with human rhinovirus (HRV) induced the phosphorylation of PKD. Second, PKD inhibitors reduced HRV genome replication, protein expression, and titers in a concentration-dependent fashion and also blocked the replication of poliovirus (PV) and foot-and-mouth disease virus (FMDV) in a variety of cells. Third, HRV replication was significantly reduced in HeLa cells overexpressing wild-type and mutant forms of PKD1. Fourth, HRV genome replication was reduced in HAP1 cells in which the PKD1 gene was knocked out by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. Although we have not identified the molecular mechanism through which PKD regulates viral replication, our data suggest that this is not due to enhanced interferon signaling or an inhibition of clathrin-mediated endocytosis, and PKD inhibitors do not need to be present during viral uptake. Our data show for the first time that targeting PKD with small molecules can inhibit the replication of HRV, PV, and FMDV, and therefore, PKD may represent a novel antiviral target for drug discovery. IMPORTANCE Picornaviruses remain an important family of human and animal pathogens for which we have a very limited arsenal of antiviral agents. HRV is the causative agent of the common cold, which in itself is a relatively trivial infection; however, in asthma and chronic obstructive pulmonary disease (COPD) patients, this virus is a major cause of exacerbations resulting in an increased use of medication, worsening symptoms, and, frequently, hospital admission. Thus, HRV represents a substantial health care and economic burden for which there are no approved therapies. We sought to identify a novel host target as a potential anti-HRV therapy. HRV infection induces the phosphorylation of PKD, and inhibitors of this kinase effectively block HRV replication at an early stage of the viral life cycle. Moreover, PKD inhibitors also block PV and FMDV replication. This is the first description that PKD may represent a target for antiviral drug discovery.

Abstract LB-C20: Lead generation and optimisation of a series of novel glutaminase (GLS) inhibitors

Glutamine is an essential nutrient for cancer cells and is used to support cancer cell growth. Glutamine is converted to glutamate by the enzyme glutaminase, which is then converted into alpha-ketoglutarate, a metabolic intermediate within the TCA cycle which serves as a precursor for the biosynthesis of ATP, NADPH, fatty acids, glutathione, nucleic acids and amino acids. As such, we regarded glutaminolysis as a key pathway for therapeutic intervention. At the outset of the project there were several reported inhibitors of glutaminase known in the literature; however, they possess undesirable drug like properties. We describe here our efforts to discover novel inhibitors of glutaminase with a promising development profile. From a high throughput screen of approximately 900,000 compounds in the AstraZeneca collection, we were able to identify three series of glutaminase inhibitors. One of these series has been optimised to deliver cell potent, selective and orally bioavailable inhibitors of glutaminase 1. Citation Format: Mark D. Charles, Ceri Cairnduff, Joanna Brookfield, Verity Sabin, Tennyson Ekwuru, Neil Jones, Attilla Ting, James Smith, Willem Nissink, Ray Finlay, Steve Powell, Susan Critchlow, Richard Ward, Matthew Wood, Linette Ruston, Jon Winter, Ian Hollingsworth. Lead generation and optimisation of a series of novel glutaminase (GLS) inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C20.

Abstract C210: LIM kinase small molecule inhibitors block tumor cell invasion

The invasive potential of carcinomas greatly contributes to their ability to metastasize, a process which is estimated to cause 90% of all human cancer deaths. The LIM kinase (LIMK) family of Ser/Thr kinases sit at a hub of signaling pathways downstream of the Rho family of GTPases. Functionally, LIMK is directly involved in regulating the activity of cofilin, a family of proteins which modulate cell movement through reorganization of the actin cytoskeleton network. LIMK is up‐regulated in a number of invasive cancer cell lines and metastatic breast and prostate tumors, whilst an increase in LIMK activity has been shown to cause increased cellular invasion in multiple model systems. We demonstrate that LIMK inhibition by siRNA reduces the invasive capacity of MDA‐MB‐231‐Luc breast cancer cells in an in vitro matrigel invasion assay and reduces fibroblast lead collective invasion in a co‐culture organotypic model. Potent, selective, small molecule LIMK inhibitors have been identified that inhibit LIMK in vitro with sub‐nanomolar activity in biochemical assays and low micromolar activity in cells as measured by cofilin phosphorylation. Treatment of MDA‐MB‐231‐Luc cells or tumor associated fibroblasts with these small molecule inhibitors decreased cell invasion in vitro with minimal cellular toxicity. Collectively, these data further validate LIMK as an anti‐invasive therapeutic target in tumor cells and demonstrate the potential utility of LIMK inhibitors in metastatic disease. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C210.

Abstract B261: A novel small molecule inhibitor of protein kinase D blocks pancreatic cancer growth in vivo

Background: Protein kinase D (PKD) is a novel family of serine‐threonine kinase with diverse biological functions including cell proliferation and growth. Pancreatic Cancer (PaCa) is a devastating disease with few therapeutic options. We showed earlier that PKD signaling pathways promote mitogenesis in multiple PaCa cell lines. However, nothing is known about targeting biological functions of PKD in PaCa. Our PKD inhibitor discovery program yielded CRT0066101 that specifically blocks PKD activation. Aim: The aim of our study was to determine the effects of CRT0066101 in PaCa, both in vitro and in vivo. Methods and Results: Our immunohistochemical analysis showed that activated PKD (pS916PKD1) is significantly upregulated in PaCa as compared to normal ducts (91% vs 22%; p Conclusion: Our results demonstrate for the first time that the PKD‐specific small molecule inhibitor CRT0066101 blocks PaCa growth both in vitro and in vivo. Thus, PKD is a novel therapeutic target in PaCa. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B261.