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Dual-specificity tyrosine-regulated kinase 1A (DYRK1A) regulates the proliferation and differentiation of neuronal progenitor cells during brain development. Consequently, DYRK1A has attracted interest as a target for the treatment of neurodegenerative diseases, including Alzheimer's disease (AD) and Down's syndrome. Recently, the inhibition of DYRK1A has been investigated as a potential treatment for diabetes, while DYRK1A's role as a mediator in the cell cycle has garnered interest in oncologic indications. Structure-activity relationship (SAR) analysis in combination with high-resolution X-ray crystallography leads to a series of pyrazolo[1,5-b]pyridazine inhibitors with excellent ligand efficiencies, good physicochemical properties, and a high degree of selectivity over the kinome. Compound 11 exhibited good permeability and cellular activity without P-glycoprotein liability, extending the utility of 11 in an in vivo setting. These pyrazolo[1,5-b]pyridazines are a viable lead series in the discovery of new therapies for the treatment of diseases linked to DYRK1A function.

Ectopic expression in T-cell precursors of LIM only protein 2 (LMO2), a key factor in hematopoietic development, has been linked to the onset of T-cell acute lymphoblastic leukaemia (T-ALL). In the T-ALL context, LMO2 drives oncogenic progression through binding to erythroid-specific transcription factor SCL/TAL1 and sequestration of E-protein transcription factors, normally required for T-cell differentiation. A key requirement for the formation of this oncogenic protein-protein interaction (PPI) is the conformational flexibility of LMO2. Here we identify a small molecule inhibitor of the SCL-LMO2 PPI, which hinders the interaction in vitro through direct binding to LMO2. Biophysical analysis demonstrates that this inhibitor acts through a mechanism of conformational modulation of LMO2. Importantly, this work has led to the identification of a small molecule inhibitor of the SCL-LMO2 PPI, which can provide a starting point for the development of new agents for the treatment of T-ALL. These results suggest that similar approaches, based on the modulation of protein conformation by small molecules, might be used for therapeutic targeting of other oncogenic PPIs.

Purpose: There is considerable interest in positive allosteric modulators (PAMs) of the α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) subtype of ionotropic glutamate receptors as therapeutic agents for a range of cognitive and mood disorders. However, the challenge is to increase AMPA receptor (AMPAR) function sufficient to enhance cognitive function but not to the extent that there are mechanism-related pro-convulsant or convulsant side effects. In this present study, we report the preclinical pharmacology data for MDI-222, an AMPAR PAM which enhances cognition but has a much reduced side-effect (i.e. convulsant) liability relative to other molecules of this mechanism. Methods: The pharmacological effects of MDI-222 were characterised in in vitro and in vivo preclinical electrophysiology, efficacy (cognition), side-effect (pro-convulsant/convulsant), tolerability and toxicity assays. Results: We demonstrate that MDI-222 is an AMPAR PAM, since it enhanced AMPAR function in vitro at human (hGluA1-4) and rat (rGluA2) homomeric receptors, and potentiated hetero-oligomeric AMPARs in rat neurons. MDI-222 enhanced electrically evoked AMPAR-mediated synaptic transmission in the anaesthetised rat at 10 mg/kg (administered intravenously) and did not significantly lower the seizure threshold in the pro-convulsant maximal electroshock threshold test (MEST) at any dose tested up to a maximum of 30 mg/kg (administered by oral gavage (p.o.)). MDI-222 reversed a delay-induced deficit in novel object recognition (NOR) in rats with a minimum effective dose (MED) of 0.3 mg/kg (p.o.) following acute administration, which was reduced to 0.1 mg/kg following sub-chronic administration, and improved passive avoidance performance in scopolamine-impaired rats with a MED of 10 mg/kg p.o. On the other hand, MDI-222 was not pro-convulsant in the MEST, resulting in a therapeutic window between plasma concentrations that enhanced cognitive performance and those associated with mechanism-related side effects of ⩾1000-fold. Unfortunately, despite the excellent preclinical profile of this compound, further development had to be halted due to non-mechanism-related issues. Conclusions: We conclude that MDI-222 is an AMPAR PAM which enhances cognitive performance in rats and has a significantly improved safety profile in preclinical species.

Simple Summary Liver metastasis remains the major contributor in colorectal cancer-related death. It has become clear that the unique immune features of liver microenvironment take part in many steps of metastatic cascade, from pre-metastatic niche formation, tumor cell colonization to metastatic tumor establishment. Therefore, better understanding of mechanisms orchestrating the formation of a hospitable hepatic metastatic niche is necessary for the development of effective therapies. This review summarizes the current understandings of the critical role of liver immune microenvironment in metastasis development and provides therapeutic perspective on targeting the metastasis-prone microenvironment. Abstract A drastic difference exists between the 5-year survival rates of colorectal cancer patients with localized cancer and distal organ metastasis. The liver is the most favorable organ for cancer metastases from the colorectum. Beyond the liver-colon anatomic relationship, emerging evidence highlights the impact of liver immune microenvironment on colorectal liver metastasis. Prior to cancer cell dissemination, hepatocytes secrete multiple factors to recruit or activate immune cells and stromal cells in the liver to form a favorable premetastatic niche. The liver-resident cells including Kupffer cells, hepatic stellate cells, and liver-sinusoidal endothelial cells are co-opted by the recruited cells, such as myeloid-derived suppressor cells and tumor-associated macrophages, to establish an immunosuppressive liver microenvironment suitable for tumor cell colonization and outgrowth. Current treatments including radical surgery, systemic therapy, and localized therapy have only achieved good clinical outcomes in a minority of colorectal cancer patients with liver metastasis, which is further hampered by high recurrence rate. Better understanding of the mechanisms governing the metastasis-prone liver immune microenvironment should open new immuno-oncology avenues for liver metastasis intervention.

To identify small molecules that shield mammalian sensory hair cells from the ototoxic side effects of aminoglycoside antibiotics we screened 10,240 compounds, selecting those that protected against neomycin and gentamicin in zebrafish lateral-line hair cells and, when retested in mouse cochlear cultures, prevented gentamicin-induced death of outer hair cells (OHCs). Of 64 compounds that protected zebrafish hair cells, 8 protect OHCs from gentamicin in vitro. These hits share structural features and all block, to varying degrees, the OHC’s mechano-electrical transducer (MET) channel, a known route of aminoglycoside entry into hair cells. Further characterisation of one of the strongest MET-channel blockers, UoS-7692, revealed it additionally protects against kanamycin and tobramycin, and does not abrogate the bactericidal activity of gentamicin. UoS-7692 behaves, like the aminoglycosides, as a permeant blocker of the MET channel, significantly reduces gentamicin-Texas Red loading into OHCs, and preserves lateral-line function in neomycin-treated zebrafish. Trans-tympanic injection of UoS-7692 protects mouse OHCs from furosemide-kanamycin exposure in vivo and partially preserves hearing. The results confirm the hair-cell MET channel as a viable target for the identification of compounds that protect the cochlea from aminoglycosides, and provide a series of hit compounds that will inform the design of future otoprotectants.

RV521 is an orally bioavailable inhibitor of respiratory syncytial virus (RSV) fusion that was identified after a lead optimization process based upon hits that originated from a physical property directed hit profiling exercise at Reviral. This exercise encompassed collaborations with a number of contract organizations with collaborative medicinal chemistry and virology during the optimization phase in addition to those utilized as the compound proceeded through preclinical and clinical evaluation. RV521 exhibited a mean IC50 of 1.2 nM against a panel of RSV A and B laboratory strains and clinical isolates with antiviral efficacy in the Balb/C mouse model of RSV infection. Oral bioavailability in preclinical species ranged from 42 to >100% with evidence of highly efficient penetration into lung tissue. In healthy adult human volunteers experimentally infected with RSV, a potent antiviral effect was observed with a significant reduction in viral load and symptoms compared to placebo.

BLM (Bloom syndrome protein) is a RECQ-family helicase involved in the dissolution of complex DNA structures and repair intermediates. Synthetic lethality analysis implicates BLM as a promising target in a range of cancers with defects in the DNA damage response, however selective small molecule inhibitors of defined mechanism are currently lacking. Here we identify and characterise a specific inhibitor of BLM’s ATPase-coupled DNA helicase activity, by allosteric trapping of a DNA-bound translocation intermediate. Crystallographic structures of BLM-DNA-ADP-inhibitor complexes identify a hitherto unknown interdomain interface, whose opening and closing are integral to translocation of ssDNA, and which provides a highly selective pocket for drug discovery. Comparison with structures of other RECQ helicases provides a model for branch migration of Holliday junctions by BLM.

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter within the central nervous system (CNS) with fast, transsynaptic, and modulatory extrasynaptic effects being mediated by the ionotropic GABA type A receptors (GABAARs). These receptors are of particular interest because they are the molecular target of a number of pharmacological agents, of which the benzodiazepines (BZDs), such as diazepam, are the best described. The anxiolytic, sedating, and myorelaxant effects of BZDs are mediated by separate populations of GABAARs containing either α1, α2, α3, or α5 subunits and the molecular dissection of the pharmacology of BZDs indicates that subtype-selective GABAAR modulators will have novel pharmacological profiles. This is best exemplified by α2/α3-GABAAR positive allosteric modulators (PAMs) and α5-GABAAR negative allosteric modulators (NAMs), which were originally developed as nonsedating anxiolytics and cognition enhancers, respectively. This review aims to summarize the current state of the field of subtype-selective GABAAR modulators acting via the BZD binding site and their potential clinical indications.

Background and Purpose AMPA receptor positive allosteric modulators represent a potential therapeutic strategy to improve cognition in people with schizophrenia. These studies collectively constitute the preclinical pharmacology data package used to build confidence in the pharmacology of this molecule and enable a clinical trial application. Experimental Approach [N-[(2S)-5-(6-fluoro-3-pyridinyl)-2,3-dihydro 1H–inden-2-yl]-2-propanesulfonamide] (UoS12258) was profiled in a number of in vitro and in vivo studies to highlight its suitability as a novel therapeutic agent. Key Results We demonstrated that UoS12258 is a selective, positive allosteric modulator of the AMPA receptor. At rat native hetero-oligomeric AMPA receptors, UoS12258 displayed a minimum effective concentration of approximately 10 nM in vitro and enhanced AMPA receptor-mediated synaptic transmission at an estimated free brain concentration of approximately 15 nM in vivo. UoS12258 reversed a delay-induced deficit in novel object recognition in rats after both acute and sub-chronic dosing. Sub-chronic dosing reduced the minimum effective dose from 0.3 to 0.03 mg·kg−1. UoS12258 was also effective at improving performance in two other cognition models, passive avoidance in scopolamine-impaired rats and water maze learning and retention in aged rats. In side-effect profiling studies, UoS12258 did not produce significant changes in the maximal electroshock threshold test at doses below 10 mg·kg−1. Conclusion and Implications We conclude that UoS12258 is a potent and selective AMPA receptor modulator exhibiting cognition enhancing properties in several rat behavioural models superior to other molecules that have previously entered clinical evaluation.

Aminoglycosides (AGs) are broad-spectrum antibiotics used for the treatment of serious bacterial infections but have use-limiting side effects including irreversible hearing loss. Here, we assessed the otoprotective profile of carvedilol in mouse cochlear cultures and in vivo zebrafish assays and investigated its mechanism of protection which, we found, may be mediated by a block of the hair cell’s mechanoelectrical transducer (MET) channel, the major entry route for the AGs. To understand the full otoprotective potential of carvedilol, a series of 18 analogues were prepared and evaluated for their effect against AG-induced damage as well as their affinity for the MET channel. One derivative was found to confer greater protection than carvedilol itself in cochlear cultures and also to bind more tightly to the MET channel. At higher concentrations, both carvedilol and this derivative were toxic in cochlear cultures but not in zebrafish, suggesting a good therapeutic window under in vivo conditions.

The neurotransmitter glutamate and its receptors have long been of interest to scientists involved in pharmaceutical research since dysfunction of the glutamatergic signalling pathway has been associated with the pathophysiology of\ud several psychiatric and neurological disorders. The research on AMPAR positive allosteric modulators offers opportunities to modulate fast excitatory synaptic transmission and identify new potential therapeutic agents for a range of neurodiseases. The field of AMPAR modulators continues to be a dynamic area of drug discovery with a pronounced diversification of the chemotypes explored in recent\ud years. This article reviews literature published in this area in the last 6 years, focusing on the new core templates, some derived from high-throughput screens, with an emphasis on structure-activity relationships, drug metabolism and pharmacokinetics properties, and pharmacological profiles of these series.

Cyclin-dependent kinase 20 (CDK20), or more commonly referred to as cell cycle-related kinase (CCRK), is the latest member of CDK family with strong linkage to human cancers. Accumulating studies have reported the consistent overexpression of CCRK in cancers arising from brain, colon, liver, lung and ovary. Such aberrant up-regulation of CCRK is clinically significant as it correlates with tumor staging, shorter patient survival and poor prognosis. Intriguingly, the signalling molecules perturbed by CCRK are divergent and cancer-specific, including the cell cycle regulators CDK2, cyclin D1, cyclin E and RB in glioblastoma, ovarian carcinoma and colorectal cancer, and KEAP1-NRF2 cytoprotective pathway in lung cancer. In hepatocellular carcinoma (HCC), CCRK mediates virus-host interaction to promote hepatitis B virus-associated tumorigenesis. Further mechanistic analyses reveal that CCRK orchestrates a self-reinforcing circuitry comprising of AR, GSK3β, β-catenin, AKT, EZH2, and NF-κB signalling for transcriptional and epigenetic regulation of oncogenes and tumor suppressor genes. Notably, EZH2 and NF-κB in this circuit have been recently shown to induce IL-6 production to facilitate tumor immune evasion. Concordantly, in a hepatoma preclinical model, ablation of Ccrk disrupts the immunosuppressive tumor microenvironment and enhances the therapeutic efficacy of immune checkpoint blockade via potentiation of anti-tumor T cell responses. In this review, we summarized the multifaceted tumor-intrinsic and -extrinsic functions of CCRK, which represents a novel signalling hub exploitable in cancer immunotherapy.

New tools are required to ensure the adequate control of the neglected tropical disease human African trypanosomiasis. Annual reports of infection have recently fallen to fewer than 5000 cases per year; however, current therapies are hard to administer and have safety concerns and, hence, are far from ideal. Trypanosome alternative oxidase is an exciting target for controlling the infection; it is unique to the parasite, and inhibition of this enzyme with the natural product ascofuranone has shown to clear in vivo infections. We report the synthesis and associated structure activity relationships of inhibitors based upon this natural product with correlation to T. b. brucei growth inhibition in an attempt to generate molecules that possess improved physicochemical properties and potential for use as new treatments for human African trypanosomiasis.

Aminoglycoside antibiotics are used to treat life-threatening bacterial infections but can cause deafness due to hair cell death in the inner ear. Compounds have been described that protect zebrafish lateral line hair cells from aminoglycosides, but few are effective in the cochlea. As the aminoglycosides interact with several ion channels, including the mechanoelectrical transducer (MET) channels by which they can enter hair cells, we screened 160 ion-channel modulators, seeking compounds that protect cochlear outer hair cells (OHCs) from aminoglycoside-induced death in vitro. Using zebrafish, 72 compounds were identified that either reduced loading of the MET-channel blocker FM 1-43FX, decreased Texas red–conjugated neomycin labeling, or reduced neomycin-induced hair cell death. After testing these 72 compounds, and 6 structurally similar compounds that failed in zebrafish, 13 were found that protected against gentamicin-induced death of OHCs in mouse cochlear cultures, 6 of which are permeant blockers of the hair cell MET channel. None of these compounds abrogated aminoglycoside antibacterial efficacy. By selecting those without adverse effects at high concentrations, 5 emerged as leads for developing pharmaceutical otoprotectants to alleviate an increasing clinical problem.

African trypanosomiasis is a parasitic disease affecting 5000 humans and millions of livestock animals in sub-Saharan Africa every year. Current treatments are limited, difficult to administer and often toxic causing long term injury or death in many patients. Trypanosome alternative oxidase is a parasite specific enzyme whose inhibition by the natural product ascofuranone (AF) has been shown to be curative in murine models. Until now synthetic methods to AF analogues have been limited, this has restricted both understanding of the key structural features required for binding and also how this chemotype could be developed to an effective therapeutic agent. The development of 3 amenable novel synthetic routes to ascofuranone-like compounds is described. The SAR generated around the AF chemotype is reported with correlation to the inhibition of T. b. brucei growth and corresponding selectivity in cytotoxic assessment in mammalian HepG2 cell lines. These methods allow access to greater synthetic diversification and have enabled the synthesis of compounds that have and will continue to facilitate further optimisation of the AF chemotype into a drug-like lead., Graphical abstract Image 1, Highlights • Synthesis of ascofuranone like inhibitors of trypanosome alternative oxidase. • Structure activity relationships of trypanosome alternative oxidase inhibitors. • Correlation of trypanosome alternative oxidase inhibition and T. b. brucei growth.

Aminoglycoside antibiotics are widely used for the treatment of life-threatening bacterial infections, but cause permanent hearing loss in a substantial proportion of treated patients. The sensory hair cells of the inner ear are damaged following entry of these antibiotics via the mechano-electrical transducer (MET) channels located at the tips of the hair cell’s stereocilia. d-Tubocurarine (dTC) is a MET channel blocker that reduces the loading of gentamicin-Texas Red (GTTR) into rat cochlear hair cells and protects them from gentamicin treatment. Berbamine is a structurally related alkaloid that reduces GTTR labeling of zebrafish lateral-line hair cells and protects them from aminoglycoside-induced cell death. Both compounds are thought to reduce aminoglycoside entry into hair cells through the MET channels. Here we show that dTC (≥6.25 µM) or berbamine (≥1.55 µM) protect zebrafish hair cells in vivo from neomycin (6.25 µM, 1 h). Protection of zebrafish hair cells against gentamicin (10 µM, 6 h) was provided by ≥25 µM dTC or ≥12.5 µM berbamine. Hair cells in mouse cochlear cultures are protected from longer-term exposure to gentamicin (5 µM, 48 h) by 20 µM berbamine or 25 µM dTC. Berbamine is, however, highly toxic to mouse cochlear hair cells at higher concentrations (≥30 µM) whilst dTC is not. The absence of toxicity in the zebrafish assays prompts caution in extrapolating results from zebrafish neuromasts to mammalian cochlear hair cells. MET current recordings from mouse outer hair cells (OHCs) show that both compounds are permeant open-channel blockers, rapidly and reversibly blocking the MET channel with half-blocking concentrations of 2.2 µM (dTC) and 2.8 µM (berbamine) in the presence of 1.3 mM Ca2+ at −104 mV. Berbamine, but not dTC, also blocks the hair cell’s basolateral K + current, IK,neo, and modeling studies indicate that berbamine permeates the MET channel more readily than dTC. These studies reveal key properties of MET-channel blockers required for the future design of successful otoprotectants.

Glutamate receptors and the associated neurotransmission have been of interest to the pharmaceutical research community for many years since dysfunction of the glutamatergic signaling pathway has been associated with the pathophysiology of several psychiatric and neurological disorders. The research on ligands modulating the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), one subtype of glutamate ionotropic receptor, offers opportunities to modulate fast excitatory synaptic transmission, and to identify new potential therapeutic agents for a range of neurodisease, such as schizophrenia and epilepsy. The field of AMPAR ligands continues to be a dynamic area of drug discovery, with a pronounced diversification of the chemotypes explored as positive allosteric modulators in recent years. This article reviews literature published in this area in the last decade.

MASTL (microtubule-associated serine/threonine kinase-like), commonly known as Greatwall (GWL), is a member of the AGC kinase family and has recently emerged as a novel cancer therapy target. GWL regulates mitotic division via phosphorylation of its known substrates ENSA and ARPP19, which upon phosphorylation act as inhibitors of the PP2A-B55δ phosphatase. Inactivation of PP2A-B55δ induces sustained activity of the CDK1-cyclin B complex, triggering mitotic entry and preventing mitotic collapse during cell division. Towards the end of mitosis, GWL inactivation is required to initiate mitotic exit. Overexpression of GWL in breast cancer cells drives oncogenic properties, such as transformation and invasions. In particular, triple negative breast cancer tumors, which are associated with a poor prognosis, overexpress GWL and are highly sensitive to GWL depletion. In addition, GWL expression has been found to be elevated in oral squamous cell carcinoma and prostate cancer tissues. A number of studies have also indicated that GWL may contribute directly to tumorigenesis, suggesting that modulation of GWL activity with small-molecule inhibitors could afford a potential new anti-cancer therapy. The precise role of GWL in cells remaining largely unexplored, the identification of a pharmacological tool would be useful to further assess the tumor-associated functions of this protein. To that end, we have recently developed and optimized a robust HTRF assay, based on a specific ENSA peptide analogue, and used it to screen kinase focused libraries, made available to us by GlaxoSmithKline and Roche. A 10.2% hit rate was achieved from the screening of the 11,000 compound set at a single concentration of 10 µM. Molecules inducing over 50% inhibition in the single point screen were subsequently tested in concentration-dependent experiments. Analysis of the IC50 generated enabled to identify molecular clusters and potential hits to initiate a medicinal chemistry campaign. A review of the literature on the selectivity of the hits identified indicated that a significant number of these molecules also had affinity for other AGC kinases and that selectivity among the AGC kinase family could be challenging to achieve. Computational studies using an in-house co-crystal structure of GWL in complex with Staurosporine and the published crystal structures of other AGC kinases will be used to design out selectivity and guide the design of inhibitors to obtain a potent chemical probe. Citation Format: Tristan D. Reuillon, Sarah Walker, Darren Le Grand, Simon E. Ward, Ben Wahab, Mohan B. Rajasekaran, Helfrid Hochegger, Antony W. Oliver. Screening of kinase focused libraries for the identification of Greatwall inhibitors [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 4210. doi:10.1158/1538-7445.AM2017-4210

Alzheimer's disease is arguably the largest healthcare issue of our time, with over 45 million people currently diagnosed with dementia worldwide. With the single biggest risk factor being age, this number is only going to increase as our populations' age. Alzheimer's disease pathology is highly complex, but is believed to be principally the result of an inter-play between the toxic proteins beta-amyloid and tau and driven by several genetic and environmental risk factors. Current treatments focus on treating cognitive and behavioral symptoms but have only modest effects and duration of efficacy. To discuss these topics, a 1-day symposium was held in Brussels to look at the progress that the field has made over recent years in attempting to target different aspects and stages of Alzheimer's disease pathology and what lessons have been learned. It also looked at the current biological mechanisms under clinical and preclinical evaluation, together with exploring how new and developing genetic insights into the disease are directing us to novel mechanisms for potential new treatment options and to address the critical question: "Are we making progress towards a disease-modifying treatment for patients?"