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Fragment-based drug discovery (FBDD) is now established as a complementary approach to high-throughput screening (HTS). Contrary to HTS, where large libraries of drug-like molecules are screened, FBDD screens involve smaller and less complex molecules which, despite a low affinity to protein targets, display more 'atom-efficient' binding interactions than larger molecules. Fragment hits can, therefore, serve as a more efficient start point for subsequent optimisation, particularly for hard-to-drug targets. Since the number of possible molecules increases exponentially with molecular size, small fragment libraries allow for a proportionately greater coverage of their respective 'chemical space' compared with larger HTS libraries comprising larger molecules. However, good library design is essential to ensure optimal chemical and pharmacophore diversity, molecular complexity, and physicochemical characteristics. In this review, we describe our views on fragment library design, and on what constitutes a good fragment from a medicinal and computational chemistry perspective. We highlight emerging chemical and computational technologies in FBDD and discuss strategies for optimising fragment hits. The impact of novel FBDD approaches is already being felt, with the recent approval of the covalent KRAS

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.

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.

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 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.

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.

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.

The protein structure guided design of a series of pyrazolo[1,5-a]pyrimidines with high potency for human cyclin-dependent kinase 2 (CDK2) is described. Some examples were shown to inhibit the growth of human colon tumour cells, were equipotent for CDK1 and were selective against GSK-3β and other kinases.