Structure-based design, synthesis and biological evaluation of a novel series of isoquinolone and pyrazolo[4,3-c]pyridine inhibitors of fascin 1 as potential anti-metastatic agents.

Fascin is an actin binding and bundling protein that is not expressed in normal epithelial tissues but overexpressed in a variety of invasive epithelial tumors. It has a critical role in cancer cell metastasis by promoting cell migration and invasion. Here we report the crystal structures of fascin in complex with a series of novel and potent inhibitors. Structure-based elaboration of these compounds enabled the development of a series with nanomolar affinities for fascin, good physicochemical properties and the ability to inhibit fascin-mediated bundling of filamentous actin. These compounds provide promising starting points for fascin-targeted anti-metastatic therapies.

A fully automated procedure for the parallel, multidimensional purification and nucleotide loading of the human GTPases KRas, Rac1 and RalB.

Small GTPases regulate many key cellular processes and their role in human disease validates many proteins in this class as desirable targets for therapeutic intervention. Reliable recombinant production of GTPases, often in the active GTP loaded state, is a prerequisite for the prosecution of drug discovery efforts. The preparation of these active forms can be complex and often constricts the supply to the reagent intensive techniques used in structure base drug discovery. We have established a fully automated, multidimensional protein purification strategy for the parallel production of the catalytic G-domains of KRas, Rac1 and RalB GTPases in the active form. This method incorporates a four step chromatography purification with TEV protease-mediated affinity tag cleavage and a conditioning step that achieves the activation of the GTPase by exchanging GDP for the non-hydrolyzable GTP analogue GMPPnP. We also demonstrate that an automated method is efficient at loading of KRas with mantGDP for application in a SOS1 catalysed fluorescent nucleotide exchange assay. In comparison to more conventional manual workflows the automated method offers marked advantages in method run time and operator workload. This reduces the bottleneck in protein production while generating products that are highly purified and effectively loaded with nucleotide analogues.

A novel small-molecule MRCK inhibitor blocks cancer cell invasion.

BACKGROUND: The myotonic dystrophy kinase-related CDC42-binding kinases MRCKα and MRCKß regulate actin-myosin contractility and have been implicated in cancer metastasis. Along with the related ROCK1 and ROCK2 kinases, the MRCK proteins initiate signalling events that lead to contractile force generation which powers cancer cell motility and invasion. A potential strategy for cancer therapy is to reduce metastasis by blocking MRCK activity, either alone or in combination with ROCK inhibition. However, to date no potent small molecule inhibitors have been developed with selectivity towards MRCK. RESULTS: Screening a kinase-focused small molecule chemical library resulted in the identification of compounds with inhibitory activity towards MRCK. Medicinal chemistry combined with in vitro enzyme profiling led to the discovery of 4-chloro-1-(4-piperidyl)-N-[5-(2-pyridyl)-1H-pyrazol-4-yl]pyrazole-3-carboxamide (BDP00005290; abbreviated as BDP5290) as a potent MRCK inhibitor. X-ray crystallography of the MRCKß kinase domain in complex with BDP5290 revealed how this ligand interacts with the nucleotide binding pocket. BDP5290 demonstrated marked selectivity for MRCKß over ROCK1 or ROCK2 for inhibition of myosin II light chain (MLC) phosphorylation in cells. While BDP5290 was able to block MLC phosphorylation at both cytoplasmic actin stress fibres and peripheral cortical actin bundles, the ROCK selective inhibitor Y27632 primarily reduced MLC phosphorylation on stress fibres. BDP5290 was also more effective at reducing MDA-MB-231 breast cancer cell invasion through Matrigel than Y27632. Finally, the ability of human SCC12 squamous cell carcinoma cells to invade a three-dimensional collagen matrix was strongly inhibited by 2 µM BDP5290 but not the identical concentration of Y27632, despite equivalent inhibition of MLC phosphorylation. CONCLUSIONS: BDP5290 is a potent MRCK inhibitor with activity in cells, resulting in reduced MLC phosphorylation, cell motility and tumour cell invasion. The discovery of this compound will enable further investigations into the biological activities of MRCK proteins and their contributions to cancer progression.

5-Deazaflavin derivatives as inhibitors of p53 ubiquitination by HDM2.

Based on previous reports of certain 5-deazaflavin derivatives being capable of activating the tumour suppressor p53 in cancer cells through inhibition of the p53-specific ubiquitin E3 ligase HDM2, we have conducted an structure-activity relationship (SAR) analysis through systematic modification of the 5-deazaflavin template. This analysis shows that HDM2-inhibitory activity depends on a combination of factors. The most active compounds (e.g., 15) contain a trifluoromethyl or chloro substituent at the deazaflavin C9 position and this activity depends to a large extent on the presence of at least one additional halogen or methyl substituent of the phenyl group at N10. Our SAR results, in combination with the HDM2 RING domain receptor recognition model we present, form the basis for the design of drug-like and potent activators of p53 for potential cancer therapy.

Small molecules that bind the Mdm2 RING stabilize and activate p53.

p53 is a tumor suppressor that responds to a variety of stresses such as oncogenes and DNA damage by activating its transcriptional targets to allow repair or elimination of damaged cells. In the absence of stress signals, p53 needs to be kept in check and this is achieved by the E3 ligase MDM2. For tumors that retain wild-type p53, therapeutic strategies aimed at removing the inhibitory activity of MDM2 on p53 are under development and to date have focused on drugs that prevent the binding of p53 to MDM2. Here, we report the analysis of a group of synthetic analogs derived from 5-deazaflavin compounds previously identified in a screen as inhibitors of MDM2 autoubiquitination. Using measurement of surface plasmon resonance, we demonstrated that active 5-deazaflavin analogs bind to the MDM2 RING, whereas inactive compounds show no binding. In cellular assays, these active MDM2 RING binding compounds inhibited the ubiquitination of p53, stabilized p53, led to increased expression of p53 targets and caused corresponding cell cycle effects. Deazaflavin analogs therefore function to activate p53 through a novel mechanism, by inhibiting the E3 ligase activity of MDM2 in a manner that involves binding to the MDM2 RING.

The Meisenheimer Complex as a Paradigm in Drug Discovery: Reversible Covalent Inhibition through C67 of the ATP Binding Site of PLK1.

The polo kinase family are important oncology targets that act in regulating entry into and progression through mitosis. Structure-guided discovery of a new class of inhibitors of Polo-like kinase 1 (PLK1) catalytic activity that interact with Cys67 of the ATP binding site is described. Compounds containing the benzothiazole N-oxide scaffold not only bind covalently to this residue, but are reversible inhibitors through the formation of Meisenheimer complexes. This mechanism of kinase inhibition results in compounds that can target PLK1 with high selectivity, while avoiding issues with irreversible covalent binding and interaction with other thiol-containing molecules in the cell. Due to renewed interest in covalent drugs and the plethora of potential drug targets, these represent prototypes for the design of kinase inhibitory compounds that achieve high specificity through covalent interaction and yet still bind reversibly to the ATP cleft, a strategy that could be applied to avoid issues with conventional covalent binders.

Discovery of Potent and Selective MRCK Inhibitors with Therapeutic Effect on Skin Cancer.

The myotonic dystrophy-related Cdc42-binding kinases MRCKα and MRCKß contribute to the regulation of actin-myosin cytoskeleton organization and dynamics, acting in concert with the Rho-associated coiled-coil kinases ROCK1 and ROCK2. The absence of highly potent and selective MRCK inhibitors has resulted in relatively little knowledge of the potential roles of these kinases in cancer. Here, we report the discovery of the azaindole compounds BDP8900 and BDP9066 as potent and selective MRCK inhibitors that reduce substrate phosphorylation, leading to morphologic changes in cancer cells along with inhibition of their motility and invasive character. In over 750 human cancer cell lines tested, BDP8900 and BDP9066 displayed consistent antiproliferative effects with greatest activity in hematologic cancer cells. Mass spectrometry identified MRCKα S1003 as an autophosphorylation site, enabling development of a phosphorylation-sensitive antibody tool to report on MRCKα status in tumor specimens. In a two-stage chemical carcinogenesis model of murine squamous cell carcinoma, topical treatments reduced MRCKα S1003 autophosphorylation and skin papilloma outgrowth. In parallel work, we validated a phospho-selective antibody with the capability to monitor drug pharmacodynamics. Taken together, our findings establish an important oncogenic role for MRCK in cancer, and they offer an initial preclinical proof of concept for MRCK inhibition as a valid therapeutic strategy.Significance: The development of selective small-molecule inhibitors of the Cdc42-binding MRCK kinases reveals their essential roles in cancer cell viability, migration, and invasive character. Cancer Res; 78(8); 2096-114. ©2018 AACR.

Colorectal Tumors Require NUAK1 for Protection from Oxidative Stress.

Exploiting oxidative stress has recently emerged as a plausible strategy for treatment of human cancer, and antioxidant defenses are implicated in resistance to chemotherapy and radiotherapy. Targeted suppression of antioxidant defenses could thus broadly improve therapeutic outcomes. Here, we identify the AMPK-related kinase NUAK1 as a key component of the antioxidant stress response pathway and reveal a specific requirement for this role of NUAK1 in colorectal cancer. We show that NUAK1 is activated by oxidative stress and that this activation is required to facilitate nuclear import of the antioxidant master regulator NRF2: Activation of NUAK1 coordinates PP1ß inhibition with AKT activation in order to suppress GSK3ß-dependent inhibition of NRF2 nuclear import. Deletion of NUAK1 suppresses formation of colorectal tumors, whereas acute depletion of NUAK1 induces regression of preexisting autochthonous tumors. Importantly, elevated expression of NUAK1 in human colorectal cancer is associated with more aggressive disease and reduced overall survival.Significance: This work identifies NUAK1 as a key facilitator of the adaptive antioxidant response that is associated with aggressive disease and worse outcome in human colorectal cancer. Our data suggest that transient NUAK1 inhibition may provide a safe and effective means for treatment of human colorectal cancer via disruption of intrinsic antioxidant defenses. Cancer Discov; 8(5); 632-47. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 517.

A Novel Small-Molecule Inhibitor of MRCK Prevents Radiation-Driven Invasion in Glioblastoma.

Glioblastoma (GBM) is an aggressive and incurable primary brain tumor that causes severe neurologic, cognitive, and psychologic symptoms. Symptoms are caused and exacerbated by the infiltrative properties of GBM cells, which enable them to pervade the healthy brain and disrupt normal function. Recent research has indicated that although radiotherapy (RT) remains the most effective component of multimodality therapy for patients with GBM, it can provoke a more infiltrative phenotype in GBM cells that survive treatment. Here, we demonstrate an essential role of the actin-myosin regulatory kinase myotonic dystrophy kinase-related CDC42-binding kinase (MRCK) in mediating the proinvasive effects of radiation. MRCK-mediated invasion occurred via downstream signaling to effector molecules MYPT1 and MLC2. MRCK was activated by clinically relevant doses per fraction of radiation, and this activation was concomitant with an increase in GBM cell motility and invasion. Furthermore, ablation of MRCK activity either by RNAi or by inhibition with the novel small-molecule inhibitor BDP-9066 prevented radiation-driven increases in motility both in vitro and in a clinically relevant orthotopic xenograft model of GBM. Crucially, treatment with BDP-9066 in combination with RT significantly increased survival in this model and markedly reduced infiltration of the contralateral cerebral hemisphere.Significance: An effective new strategy for the treatment of glioblastoma uses a novel, anti-invasive chemotherapeutic to prevent infiltration of the normal brain by glioblastoma cells.Cancer Res; 78(22); 6509-22. ©2018 AACR.

Catch the kinase conformer.

Protein kinases exist in inactive and active states, but little attention has been paid to which state is or should be the target in drug discovery efforts. In this issue of Chemistry & Biology, Okram et al. tackle this issue and show that inhibitors can be designed specifically to bind to inactive Abl.