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Increased GBM cell invasion in vivo is confirmed by a human specific antibody against HLA and measurement of tumour volume by multi-slice T2 MR imaging

GFP E2 cells irradiated with 2Gy in an ex vivo brain slice motility assay

Supplemental Figure 1. Kinome selectivity profiles for BDP8900 and BDP9066. Supplemental Figure 2. Cancer cell line sensitivity to BDP8900 and BDP9066. Supplemental Figure 3. MRCKα phosphorylations compared with MRCKβ. Supplemental Figure 4. MRCKα, MRCKβ and MRCKα pS1003 antibody validation. Supplemental Figure 5. MRCK and DMPK expression in MDA MB 231 D3H2 LN human breast cancer cells, SCC12 human squamous cell carcinoma cells, and human skeletal muscle. Supplemental Table 1. Crystallographic data and model statistics. Supplemental Table 2: BDP8900 and BDP9066 selectivity in vitro (in-house determinations). Supplemental Table 3. Kinase selectivity screen for BDP8900 and BDP9066. Supplemental Table 4. BDP8900 selectivity in vitro (out-sourced determinations). Supplemental Table 5. BDP9066 selectivity in vitro (out-sourced determinations). Supplemental Table 6. Cancer cell line EC50 values for BDP8900 and BDP9066. Supplemental Table 7. MRCKα phosphorylation sites. Supplemental Table 8. MRCKα phosphorylation on S1003.

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.

Subconfluent migration assays in the presence of 100nM BDP-9066

Summary of the properties of BDP-9066

Subconfluent migration assay in the presence of DMSO

Antibodies used for western blotting

untreated GFP E2 cells in an ex vivo brain slice motility assay

Supplemental figure legends

Ex vivo migration assay in the presence of BDP-9066.

Supplementary video figure legend

The myotonic dystrophy–related Cdc42-binding kinases MRCKα and MRCKβ 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.

Subconfluent migration assays in the presence of 1000nM BDP-9066

We recently reported a deep learning model to facilitate fragment library design, which is critical for efficient hit identification. However, our model was implemented in Python. We have now created an implementation in the KNIME graphical pipelining environment which we hope will allow experimentation by users with limited programming knowledge.

Fragment-based hit identification (FBHI) allows proportionately greater coverage of chemical space using fewer molecules than traditional high-throughput screening approaches. However, effectively exploiting this advantage is highly dependent on the library design. Solubility, stability, chemical complexity, chemical/shape diversity, and synthetic tractability for fragment elaboration are all critical aspects, and molecule design remains a time-consuming task for computational and medicinal chemists. Artificial neural networks have attracted considerable attention in automated de novo design applications and could also prove useful for fragment library design. Chemical autoencoders are neural networks consisting of encoder and decoder parts, which respectively compress and decompress molecular representations. The decoder is applied to samples drawn from the space of compressed representations to generate novel molecules that can be scored for properties of interest. Here, we report an autoencoder model using a recurrent neural network architecture, which was trained using 486,565 fragments curated from commercial sources, to simultaneously reconstruct both SMILES and chemical fingerprints. To explore its utility in fragment design, we applied transfer learning to the fingerprint decoder layers to train a classifier using 66 frequent hitter fragments identified from our screening campaigns. Using a particle swarm optimization sampling approach, we compare the performance of this "dual" model to an architecture encoding SMILES only. The dual model produced valid SMILES with improved features, considering a range of properties including aromatic ring counts, heavy atom count, synthetic accessibility, and a new fragment complexity score we term Feature Complexity (FeCo). Additionally, we demonstrate that generative performance is further enhanced by use of a simple syntax-correction procedure during training, in which invalid and undesirable SMILES are spiked into the training set. Finally, we used the syntax-corrected model to generate a library of novel candidate privileged fragments.

RAS is a major anticancer drug target which requires membrane localization to activate downstream signal transduction. The direct inhibition of RAS has proven to be challenging. Here, we present a novel strategy for targeting RAS by stabilizing its interaction with the prenyl-binding protein PDE6D and disrupting its localization. Using rationally designed RAS point mutations, we were able to stabilize the RAS:PDE6D complex by increasing the affinity of RAS for PDE6D, which resulted in the redirection of RAS to the cytoplasm and the primary cilium and inhibition of oncogenic RAS/ERK signaling. We developed an SPR fragment screening and identified fragments that bind at the KRAS:PDE6D interface, as shown through cocrystal structures. Finally, we show that the stoichiometric ratios of KRAS:PDE6D vary in different cell lines, suggesting that the impact of this strategy might be cell-type-dependent. This study forms the foundation from which a potential anticancer small-molecule RAS:PDE6D complex stabilizer could be developed. ispartof: JOURNAL OF MEDICINAL CHEMISTRY vol:65 issue:3 pages:1898-1914 ispartof: location:United States status: published

Graphical abstract, 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.

Additive manufacturing often has higher environmental impacts per part than traditional manufacturing at scale, but new materials can enable more sustainable 3D printing. This study developed and tested novel materials for paste extrusion printing, and tested materials invented by others. Testing compared their whole-system environmental impacts to standard ABS extrusion, measured by life cycle assessment (LCA); testing also assessed material strength, printability, and cost. Materials were chosen for low print energy (chemical bonding, not melting), low toxicity, and circular life cycle (biodegradable, ideally sourced from waste biomaterial). Printing energy was reduced 75% (from 160 to 40 Wh/part), and embodied impacts of materials were reduced 82% (from 6.6 to 1.2 ReCiPe Endpoint H millipoints/part). Overall impacts per part were reduced 78% (from 27 to 6 ReCiPe Endpoint H millipoints/part), including embodied impacts of the printer itself, in a maximum utilization scenario. Results were also compared to previous studies of seven different 3D printers of various types. More than ten material recipes were tested, and pecan shell flour with sodium silicate showed the best print quality. Strength and print quality did not approach ABS, but material cost was cut by 50%. Thus, while further development is required, some materials show promise for greener additive manufacturing.

The myotonic dystrophy–related Cdc42-binding kinases MRCKα and MRCKβ 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.

In a hybrid vehicle the speeds at which the transmission shifts between gears has a large impact on energy consumption. Dynamic Programming (DP) can be used to find the optimal transmission gear for a given torque and speed. Neuroevolution of Augmenting Topologies (NEAT) is then utilized to find the shift lines which allow the transmission to be at the most energy efficient point in an implementable way. Through this strategy, an improvement of 7% was achieved compared to the traditional approach.

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.

The current technological trend in transportation is a move toward cleaner energy and one of the fastest growing technologies in this field is the energy storage system (ESS)-regularly comprised of batteries. This trend can be seen in battery (BEV) and hybrid-electric vehicles (HEV). Efficiency of energy storage system (ESS) operation is absolutely crucial to electrified vehicles as batteries are still only capable of delivering a relatively limited amount of power before reaching a discharged state. Minimizing power losses within the electrical system is central to the challenge of developing electric vehicles or plug-in HEV's (PHEV) which can reach extended ranges and create broad consumer appeal. The purpose of our research will be to optimize the operation of the HV/LV electrical system by reducing power loss through control of the DC/DC buck converter duty ratio. A variable DC/DC voltage output will allow the system to use power more efficiently by online matching of the system's real-time power needs. This will be achieved through simulation by a model predictive control (MPC) based power management strategy. Hardware in the loop testing can then be accomplished.

Gene synthesis services have largely superseded traditional PCR methods for the generation of cDNAs destined for bacterial expression vectors. This, in turn, has increased the application of codon-optimized cDNAs where codons rarely used by Escherchia coli are replaced with common synonymous codons to accelerate translation of the target. A markedly accelerated rate of expression often results in a significant uplift in the levels of target protein but a substantial proportion of the enhanced yield can partition to the insoluble fraction rendering a significant portion of the gains unavailable for native purification. We have assessed several expression attenuation strategies for their utility in the manipulation of the soluble fraction towards higher levels of soluble target recovery from codon optimized systems. Using a set of human small GTPases as a case study, we compare the degeneration of the T7 promoter sequence, the use of alternative translational start codons and the manipulation of synonymous codon usage. Degeneration of both the T7 promoter and the translational start codon merely depressed overall expression and did not increase the percentage of product recovered in native purification of the soluble fraction. However, the selective introduction of rare non-optimal codons back into the codon-optimized sequence resulted in significantly elevated recovery of soluble targets. We propose that slowing the rate of extension during translation using a small number of rare codons allows more time for the co-translational folding of the nascent polypeptide. This increases the proportion of the target recovered in the soluble fraction by immobilized metal affinity chromatography (IMAC). Thus, a "de-optimization" of codon-optimized cDNAs, to attenuate or pause the translation process, may prove a useful strategy for improved recombinant protein production.

Whilst fragment-based screening has found significant utility in aiding the discovery of high quality hits against a range of targets, the use of this technology in the protein-protein interaction inhibitor field is very much in its infancy. This review aims to highlight the key technologies used to identify fragment hits, such as NMR, SPR, X-ray crystallography and biochemical screening, the fragment-based protein-protein interaction case studies reported to date and, more importantly, the potential of this methodology in unearthing high quality hit molecules in this critical area of drug discovery. In addition, we also discuss some of the key aspects of fragment library design, the composition of a high quality library and suggest ways in which future, more structurally diverse fragments which occupy different regions of chemical space to the vast majority of current fragment libraries may be selected.

Myotonic dystrophy kinase-related CDC42-binding kinases MRCKα and MRCKβ regulate actin-myosin contractility and are members of the AGC family of serine/threonine kinases. MRCKs are closely related to the Rho-regulated ROCK kinases, which is reflected in their shared abilities to phosphorylate a similar set of substrates, including myosin II light chain proteins (MLC) and myosin phosphatase target subunit 1 (MYPT1). However, MRCK and ROCK may phosphorylate these substrates at different subcellular localisations leading to diverging effects on cell invasion and migration. Glioblastoma (GBM) is an aggressive and incurable primary brain tumor. Patients are treated with surgery, radiotherapy and chemotherapy but prognosis remains poor, with a median survival of 15 months. Complete surgical resection of GBM is difficult because these tumours are frequently invasive. Recent research has indicated that, while radiotherapy extends life expectancy of patients, it can provoke a more infiltrative phenotype in those GBM cells that survive treatment. We have developed potent and selective small molecule MRCK inhibitors exemplified by BDP-9066, which inhibits pMLC in a selective cellular assay with an EC50 28.3 nM, whilst displaying high selectivity over ROCK1 where the EC50 10.6 µM. This compound, and others in the series, have been used in numerous cellular assay systems to demonstrate that MRCK inhibition suppresses the invasion and migration of cancer cells including, breast, skin SCC and glioblastoma. We have used BDP-9066 to demonstrate that MRCK inhibition is effective at perturbing radiation-induced migration of GBM cells in culture at concentrations that block phosphorylation of MLC. Using a clinically relevant intracranial mouse model that recapitulates key histological features of the disease, we have shown that BDP-9066 inhibits the invasion of G7 GBM cells into the contralateral hemisphere of the brain. These experiments highlight the potential utility of MRCK inhibition in the treatment of GBM in patients. Citation Format: Heather J. McKinnon, Joanna Birch, Laura McDonald, Mairi Sime, Daniel Croft, Diane Crighton, Martin Drysdale, Lesley Gilmore, Christopher Gray, Jennifer Konczal, Duncan McArthur, Patricia McConnell, Mokdad Mezna, Alexander Schuettelkopf, Karen Strathdee, Justin Bower, Michael F. Olson, Anthony J. Chalmers. A novel small molecule inhibitor of MRCK shows utility in blocking radiation induced invasion of glioblastoma cells in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1933.

[reaction: see text] The synthesis of dimethyl sulfomycinamate, the acidic methanolysis product of the sulfomycin family of thiopeptide antibiotics, from methyl 2-oxo-4-(trimethylsilyl)but-3-ynoate is achieved in a 2,3,6-trisubstituted pyridine synthesis that proceeds with total regiocontrol in 13 steps by the Bohlmann-Rahtz heteroannulation of a 1-(oxazol-4-yl)enamine or in 12 steps and 9% yield by three-component cyclocondensation with N-[3-oxo-3-(oxazol-4-yl)propanoyl]serine and ammonia in ethanol.

The targeting of RNA for the design of novel anti-viral compounds represents an area of vast potential. We have used NMR and computational methods to model the interaction of a series of synthetic inhibitors of the in vitro RNA binding activities of a peptide derived from the transcriptional activator protein, Tat, from human immunodeficiency virus type 1. Inhibition has been measured through the monitering of fluorescence resonance energy transfer between fluorescently labeled peptide and RNA components. A series of compounds containing a bi-aryl heterocycle as one of the three substituents on a benzylic scaffold, induce a novel, inactive TAR conformation by stacking between base-pairs at the site of a three-base bulge within TAR. The development of this series resulted in an enhancement in potency (with Ki < 100 nM in an in vitro assay) and the removal of problematic guanidinium moieties. Ligands from this series can act as inhibitors of Tat-induced transcription in a cell-free system. This study validates the drug design strategy of using a ligand to target the RNA receptor in a non-functional conformation.

[reaction: see text] Dimethyl sulfomycinamate, the oxazole-thiazole-pyridine product generated in the methanolysis of the thiopeptide antibiotic sulfomycin I, is prepared in 13 steps and 8% overall yield by the Bohlmann-Rahtz heteroannulation of 1-(oxazol-4-yl)enamines and methyl 4-(trimethylsilyl)-2-oxobut-3-ynoate.

New facile experimental procedures for the preparation of 2,3,4,6-tetrasubstituted pyridines in a single synthetic step have been developed. Thus, an enamino ester and alkynone react by Michael addition–cyclodehydration in a heterocyclisation process that is catalysed by acetic acid, Amberlyst 15 ion exchange resin, zinc(II) bromide or ytterbium(III) triflate. The new one-step Bronsted or Lewis acid-catalysed Bohlmann–Rahtz reaction is a simple, direct and highly expedient method for the synthesis of pyridines that proceeds at a lower reaction temperature and avoids the need to isolate reaction intermediates.

The actin-bundling protein fascin 1 is markedly overexpressed in a range of invasive tumors and is believed to play a critical role in cancer cell metastasis. Targeting fascin is however very challenging owing to its mechanism of protein-protein interaction and a lack of knowledge regarding the crucial actin-binding sites. By combining a fragment-based approach, biophysical assay screening and X-ray crystallography, we have been able to identify and optimize novel fascin 1 inhibitors that show nanomolar affinity in biochemical binding and bundling assays. We now show the development of functional cell assays in which the lead fascin inhibitor compounds have been tested. For the first time we disclose the structures of the most advanced compounds and show their effects in 2D and 3D cell culture. Fascin 1 binds and cross-links filamentous actin (F-actin) into parallel bundles that are used in the formation of dynamic cellular protrusions (such as lamellipodia and filopodia) used during cell migration, and in the formation of invadopodia used by tumor cell lines to degrade the tumor extracellular matrix (ECM). Whereas fascin 1 expression is low or absent in normal epithelia, its expression is dramatically increased in a variety of invasive tumor types including colon, lung, ovarian and pancreas, with increased expression being an independent prognostic indicator of poor clinical outcome in the most aggressive and metastatic tumor types. Knockdown of fascin expression has been shown to reduce tumor cell invasion both in vitro and in vivo, thereby highlighting fascin as an important drug discovery target. Small molecule inhibitors of fascin 1 were identified by screening our fragment library (~1000 compounds) using surface plasmon resonance (SPR). X-ray co-crystallography with these hit compounds showed four distinct ligand binding sites within the fascin protein. Optimized site 2 binders induce a substantial conformational change, with the deeply enclosed pocket between fascin domains 1 and 2 opening a channel that accesses the surface of the protein. A virtual screen identified the compound BDP-00010834, which binds in this pocket with an SPR Kd of 29.7µM and also inhibits (IC50=50µM) the functional activity of fascin measured in an F-actin bundling assay. Structure-based optimization in combination with X-ray co-crystallography has enabled the generation of compounds with more than 300-fold increase in both binding affinity and functional activity over this screening hit. Our current best-in-series compounds include BDP-00013544 (Kd=29nM, IC50=185nM). These lead compounds have now been tested in a number of cell based invasion assays including both 2D and 3D cultures. Citation Format: Daniel Croft, Stuart Francis, Justin Bower, Andrea Gohlke, Gillian Goodwin, Christopher Gray, Jennifer Konczal, Sophie Macconnachie, Patricia McConnell, Laura McDonald, Heather McKinnon, Mokdad Mezna, Charles Parry, Nikki Paul, Angelo Pugliese, Alexander Schuettelkopf, Laura Machesky, Martin Drysdale. Identifying small molecule inhibitors of Fascin 1 using fragment-based drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-228. doi:10.1158/1538-7445.AM2017-LB-228

Background:\ud The myotonic dystrophy kinase-related CDC42-binding kinases MRCKα and MRCKβ 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.\ud \ud Results:\ud 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 MRCKβ kinase domain in complex with BDP5290 revealed how this ligand interacts with the nucleotide binding pocket. BDP5290 demonstrated marked selectivity for MRCKβ 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.\ud \ud Conclusions:\ud 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.

A derivative of (Ia) with a methyl-substituent at the terminal double bond yields no cycloadduct under the presented conditions.

The synthesis of silaheterocycles through the first examples of an intramolecular silene Diels-Alder reaction is described.

The identification of high-quality hits during the early phases of drug discovery is essential if projects are to have a realistic chance of progressing into clinical development and delivering marketed drugs. As the pharmaceutical industry goes through unprecedented change, there are increasing opportunities to collaborate via pre-competitive networks to marshal multifunctional resources and knowledge to drive impactful, innovative science. The 3D Fragment Consortium is developing fragment-screening libraries with enhanced 3D characteristics and evaluating their effect on the quality of fragment-based hit identification (FBHI) projects.

A novel N-aryl piperazine-1-carboxamide series of human CCR2 chemokine receptor antagonists was discovered. Early analogues were potent at CCR2 but also inhibited the hERG cardiac ion channel. Structural modifications which decreased lipophilicity and basicity resulted in the identification of a sub-series with an improved margin over hERG. The pharmacological and pharmacokinetic properties of the lead compound from this series, N-(3,4-dichlorophenyl)-4-[(2R)-4-isopropylpiperazine-2-carbonyl]piperazine-1-carboxamide, are described.

Fascin 1 is an actin-bundling protein that is dramatically overexpressed in a variety of invasive tumors and thought to have a critical role in cancer cell metastasis. However, as a drug target it is highly challenging due to its mechanism of protein-protein interaction and the lack of knowledge around the critical actin-binding sites. Using a fragment-based approach, biophysical assay screening and X-ray crystallography, we have been able to identify and optimize novel fascin 1 inhibitors. Furthermore we have developed robust and reproducible biochemical binding and bundling assays which have allowed us to develop ligands with submicromolar affinity. Fascin 1 cross-links filamentous actin (F-actin) into parallel bundles that are involved in the formation of dynamic cellular protrusions (such as lamellipodia and filopodia) used during cell migration. It also contributes to the formation of actin-rich finger-like protrusions, termed invadopodia, that tumor cell lines use to degrade the tumor extracellular matrix (ECM). Fascin 1 expression is low or absent in normal epithelia but is dramatically increased in a variety of tumor types including bladder, colon, lung, ovarian and pancreas. Its increased expression is associated with the most aggressive and metastatic tumor types and has been shown to be an independent prognostic indicator of poor clinical outcome. Fascin knockdown has been shown to reduce tumor cell invasion both in vitro and in vivo, suggesting fascin as a valid target for small molecule inhibitors. We have utilized a fragment-based screening approach incorporating surface plasmon resonance (SPR) to identify small molecules that bind to fascin 1. Our fragment library (∼1000 compounds) yielded 53 hit compounds that were confirmed by dose-response. Using these hits, X-ray co-crystallography identified four independent ligand binding sites within fascin. We have concentrated our efforts on one site, a deeply enclosed pocket between fascin domains 1 and 2. A number of fragment hits and commercial analogues were found to bind in this pocket and open a channel towards the surface of the protein. Subsequent virtual screening studies identified BDP-00010834, a compound which binds in this pocket with an SPR Kd of 29.7μM. Moreover, BDP-00010834 shows activity (IC50 = 50μM) in a functional biochemical screening assay that measures fascin-mediated F-actin bundling. Structure-based optimization together with X-ray co-crystallography has generated compounds with a 25- to 50-fold increase in both binding affinity and functional activity over this screening hit. Best-in-series compounds include BDP-00011163 (Kd = 1.2μM, IC50 = 0.8μM) and BDP-00011165 (Kd = 0.6μM, IC50 = 1.1μM). Citation Format: Daniel Croft, Stuart Francis, Alexander Schuettelkopf, Charles Parry, Gillian Goodwin, Angelo Pugliese, Laura McDonald, Maeve Clarke, Andrew Pannifer, Jen Lonsdale, Christopher Gray, Justin Bower, Heather McKinnon, Laura Machesky, Martin Drysdale. A fragment-based approach towards the identification of small molecule inhibitors of fascin 1. [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 A113.