Tony M. Raynham

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.

A high-throughput screen identified 3-(3,4-dihydroisoquinolin-2(1H)-ylsulfonyl)benzoic acid as a novel, highly potent (low nM), and isoform-selective (1500-fold) inhibitor of aldo-keto reductase AKR1C3: a target of interest in both breast and prostate cancer. Crystal structure studies showed that the carboxylate group occupies the oxyanion hole in the enzyme, while the sulfonamide provides the correct twist to allow the dihydroisoquinoline to bind in an adjacent hydrophobic pocket. SAR studies around this lead showed that the positioning of the carboxylate was critical, although it could be substituted by acid isosteres and amides. Small substituents on the dihydroisoquinoline gave improvements in potency. A set of "reverse sulfonamides" showed a 12-fold preference for the R stereoisomer. The compounds showed good cellular potency, as measured by inhibition of AKR1C3 metabolism of a known dinitrobenzamide substrate, with a broad rank order between enzymic and cellular activity, but amide analogues were more effective than predicted by the cellular assay.

Aberrant Notch signalling has been implicated in a variety of tumor types, where it is known to play a role in tumorigenisis and angiogenisis. In particular, recent evidence suggests that the Notch pathway is activated by somatic mutations in a significant proportion of human T cell acute lymphoblastic leukaemias (T-ALLs). Therefore targeting of the Notch pathway represents an attractive avenue for anti-cancer drug discovery. However, apart from the potential use of gamma-secratase inhibitors, there are very few known tractable intervention points along this predominantly protein-protein interaction mediated pathway. The structural characteristics of protein-protein contact surfaces present a huge challenge for small molecule drug discovery as these contact surfaces are generally larger than those involved in protein-small molecule interactions and often lack the grooves and pockets on the protein surface that small molecules efficiently bind to. Recent discoveries in our laboratories indicate that the interaction of the HES transcription factor with the TLE co-repressor could provide a tractable point for small molecule inhibitors that would inhibit the Notch signalling pathway. The interaction between HES/TLE is dependent on a highly conserved contiguous tetra-peptide WRPW motif present in the C-terminus of HES family members which binds to the WD domain of TLE. The binding site on TLE is a potentially druggable hydrophobic recess located at the central pore of a 7-bladed β-propeller structure, and the WRPW motif sits on top of the recess contacting all 7 blades of the -propeller. We have established and validated biochemical fluorescence polarisation (FP) and GST-WRPW pull down assays to identify compounds that disrupt HES/TLE binding in vitro and a GAL4-WRPW reporter luciferase assay was developed as an effective way of monitoring the WRPW dependent interactions between HES and TLE proteins in a cell culture model. Using these assays a small library of peptide derivatives, based around the highly conserved WRPW motif, was screened. SAR from the peptide array in conjunction with structural information from a WRPW peptide bound to TLE1 WD domain was used to construct a 3D pharmacophore model which was applied as a search query for in silico virtual screening. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C126.

Structure-based design was applied to the optimization of a series of 2-(quinazolin-2-yl)phenols to generate potent and selective ATP-competitive inhibitors of the DNA damage response signaling enzyme checkpoint kinase 2 (CHK2). Structure−activity relationships for multiple substituent positions were optimized separately and in combination leading to the 2-(quinazolin-2-yl)phenol 46 (IC₅₀ 3 nM) with good selectivity for CHK2 against CHK1 and a wider panel of kinases and with promising in vitro ADMET properties. Off-target activity at hERG ion channels shown by the core scaffold was successfully reduced by the addition of peripheral polar substitution. In addition to showing mechanistic inhibition of CHK2 in HT29 human colon cancer cells, a concentration dependent radioprotective effect in mouse thymocytes was demonstrated for the potent inhibitor 46 (CCT241533).

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.

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.