Targeting DYRK1A/B kinases to modulate p21-cyclin D1-p27 signalling and induce anti-tumour activity in a model of human glioblastoma.

The dual-specificity tyrosine-regulated kinases DYRK1A and DYRK1B play a key role in controlling the quiescence-proliferation switch in cancer cells. Serum reduction of U87MG 2D cultures or multi-cellular tumour spheroids induced a quiescent like state characterized by increased DYRK1B and p27, and decreased pRb and cyclin D1. VER-239353 is a potent, selective inhibitor of the DYRK1A and DYRK1B kinases identified through fragment and structure-guided drug discovery. Inhibition of DYRK1A/B by VER-239353 in quiescent U87MG cells increased pRb, DYRK1B and cyclin D1 but also increased the cell cycle inhibitors p21 and p27. This resulted in exit from G0 but subsequent arrest in G1. DYRK1A/B inhibition reduced the proliferation of U87MG cells in 2D and 3D culture with greater effects observed under reduced serum conditions. Paradoxically, the induced re-expression of cell cycle proteins by DYRK1A/B inhibition further inhibited cell proliferation. Cell growth arrest induced in quiescent cells by DYRK1A/B inhibition was reversible through the addition of growth-promoting factors. DYRK inhibition-induced DNA damage and synergized with a CHK1 inhibitor in the U87MG spheroids. In vivo, DYRK1A/B inhibition-induced tumour stasis in a U87MG tumour xenograft model. These results suggest that further evaluation of VER-239353 as a treatment for glioblastoma is therefore warranted.

Fatty acid amide hydrolase inhibitors. 3: tetra-substituted azetidine ureas with in vivo activity.

We describe here our attempts to optimise the human fatty acid amide hydrolase (FAAH) inhibition and physicochemical properties of our previously reported tetrasubstituted azetidine urea FAAH inhibitor, VER-156084. We describe the SAR of a series of analogues and conclude with the demonstration of in vivo dose-dependant FAAH inhibition in an anandamide-loading study in rats.

Structure-Guided Discovery of Potent and Selective DYRK1A Inhibitors.

The kinase DYRK1A is an attractive target for drug discovery programs due to its implication in multiple diseases. Through a fragment screen, we identified a simple biaryl compound that is bound to the DYRK1A ATP site with very high efficiency, although with limited selectivity. Structure-guided optimization cycles enabled us to convert this fragment hit into potent and selective DYRK1A inhibitors. Exploiting the structural differences in DYRK1A and its close homologue DYRK2, we were able to fine-tune the selectivity of our inhibitors. Our best compounds potently inhibited DYRK1A in the cell culture and in vivo and demonstrated drug-like properties. The inhibition of DYRK1A in vivo translated into dose-dependent tumor growth inhibition in a model of ovarian carcinoma.

Fragment-Derived Selective Inhibitors of Dual-Specificity Kinases DYRK1A and DYRK1B.

The serine/threonine kinase DYRK1A has been implicated in regulation of a variety of cellular processes associated with cancer progression, including cell cycle control, DNA damage repair, protection from apoptosis, cell differentiation, and metastasis. In addition, elevated-level DYRK1A activity has been associated with increased severity of symptoms in Down's syndrome. A selective inhibitor of DYRK1A could therefore be of therapeutic benefit. We have used fragment and structure-based discovery methods to identify a highly selective, well-tolerated, brain-penetrant DYRK1A inhibitor which showed in vivo activity in a tumor model. The inhibitor provides a useful tool compound for further exploration of the effect of DYRK1A inhibition in models of disease.

Discovery and functional evaluation of diverse novel human CB(1) receptor ligands.

Ligand-based virtual screening with a 3D pharmacophore led to the discovery of 30 novel, diverse and drug-like ligands of the human cannabinoid receptor 1 (hCB(1)). The pharmacophore was validated with a hit rate of 16%, binding selectivity versus hCB(2), and expected functional profiles. The discovered compounds provide new tools for exploring cannabinoid pharmacology.

Fatty acid amide hydrolase inhibitors. Surprising selectivity of chiral azetidine ureas.

We report the discovery of a novel, chiral azetidine urea inhibitor of Fatty Acid Amide Hydrolase (FAAH,) and describe the surprising species selectivity of VER-156084 versus rat and human FAAH and also hCB1.

Discovery and optimization of potent and selective functional antagonists of the human adenosine A2B receptor.

We herein report the discovery of a novel class of antagonists of the human adenosine A2B receptor. This low molecular weight scaffold has been optimized to offer derivatives with potential utility for the alleviation of conditions associated with this receptor subtype, such as nociception, diabetes, asthma and COPD. Furthermore, preliminary pharmacokinetic analysis has revealed compounds with profiles suitable for either inhaled or systemic routes of administration.

Antagonists of the human adenosine A2A receptor. Part 1: Discovery and synthesis of thieno[3,2-d]pyrimidine-4-methanone derivatives.

The (-)-(11R,2'S)-enantiomer of the antimalarial drug mefloquine has been found to be a reasonably potent and moderately selective adenosine A(2A) receptor antagonist. Further investigation of this compound has led to the discovery of a series of keto-aryl thieno[3,2-d]pyrimidine derivatives, which are potent and selective antagonists of the adenosine A(2A) receptor. These derivatives show selectivity against the A(1) receptor. Furthermore, some of these compounds have been shown to have in vivo activity in a commonly used model, suggesting the potential for the treatment of Parkinson's disease.

The cannabinoid CB1 receptor inverse agonist, rimonabant, modifies body weight and adiponectin function in diet-induced obese rats as a consequence of reduced food intake.

The cannabinoid CB1 receptor inverse agonist rimonabant induces hypophagia and body weight loss. Reduced body weight may potentially be due to decreased food intake or to direct metabolic effects of drug administration on energy expenditure. This study uses a paired-feeding protocol to quantify the contributions of energy intake to rimonabant-induced body weight loss. Diet-induced obese (DIO) rats were dosed with rimonabant (3, 10 mg/kg PO once daily) and matched with pair-fed controls. Food intake and body weight were measured daily. Blood samples and adipose tissue were collected on day 15 for measurement of plasma adiponectin and adiponectin mRNA levels. DIO rats treated with rimonabant and pair-fed controls showed very similar changes in body weight. Although tolerance developed to the anorectic effect of rimonabant, total food intake was significantly decreased over the 14-day study period and fully accounted for the observed reductions in body weight. Adiponectin mRNA and plasma adiponectin were elevated in vehicle-treated chow-fed animals compared to obese controls, and did not differ between rimonabant-treated and pair-fed animals. The similarities between rimonabant-treated and pair-fed animals in body weight loss and the absence of differences in measures of adiponectin activity between drug-treated and pair-fed animals suggest that the outcomes of this experiment were solely mediated by the drug-induced reduction in food intake.

Pharmacological characterisation of the agonist radioligand binding site of 5-HT(2A), 5-HT(2B) and 5-HT(2C) receptors.

In the present study we compared the affinity of various drugs for the high affinity "agonist-preferring" binding site of human recombinant 5-HT(2A), 5-HT(2B) and 5-HT(2C) receptors stably expressed in monoclonal mammalian cell lines. To ensure that the "agonist-preferring" conformation of the receptor was preferentially labelled in competition binding experiments, saturation analysis was conducted using antagonist and agonist radiolabels at each receptor. Antagonist radiolabels ([3H]-ketanserin for 5-HT(2A) receptor and [3H]-mesulergine for 5-HT(2B) and 5-HT(2C) receptor) bound to a larger population of receptors in each preparation than the corresponding agonist radiolabel ([125I]-DOI for 5-HT(2A) receptor binding and [3H]-5-HT for 5-HT(2B) and 5-HT(2C) receptor binding). Competition experiments were subsequently conducted against appropriate concentrations of the agonist radiolabels bound to the "agonist-preferring" subset of receptors in each preparation. These studies confirmed that there are a number of highly selective antagonists available to investigate 5-HT2 receptor subtype function (for example, MDL 100907, RS-127445 and RS-102221 for 5-HT(2A), 5-HT(2B) and 5-HT(2C) receptors respectively). There remains, however, a lack of highly selective agonists. (-)DOI is potent and moderately selective for 5-HT(2A) receptors, BW723C86 has poor selectivity for human 5-HT(2B) receptors, while Org 37684 and VER-3323 display some selectivity for the 5-HT(2C) receptor. We report for the first time in a single study, the selectivity of numerous serotonergic drugs for 5-HT2 receptors from the same species, in mammalian cell lines and using, exclusively, agonist radiolabels. The results indicate the importance of defining the selectivity of pharmacological tools, which may have been over-estimated in the past, and highlights the need to find more selective agonists to investigate 5-HT2 receptor pharmacology.

Pyrrolo(iso)quinoline derivatives as 5-HT(2C) receptor agonists.

A series of 1-(1-pyrrolo(iso)quinolinyl)-2-propylamines was synthesised and evaluated as 5-HT(2C) receptor agonists for the treatment of obesity. The general methods of synthesis of the precursor indoles are described. The functional efficacy and radioligand binding data for the compounds at 5-HT(2) receptor subtypes are reported. The analogue which showed the highest 5-HT(2C) binding affinity (27, 1.6nM) was found to be successful in reducing food intake in rats.