The success of new therapies hinges on our ability to understand their molecular and cellular mechanisms of action. We modified BET bromodomain inhibitors, an epigenetic-based therapy, to create functionally conserved compounds that are amenable to click chemistry and can be used as molecular probes in vitro and in vivo. We used click proteomics and click sequencing to explore the gene regulatory function of BRD4 (bromodomain containing protein 4) and the transcriptional changes induced by BET inhibitors. In our studies of mouse models of acute leukemia, we used high-resolution microscopy and flow cytometry to highlight the heterogeneity of drug activity within tumor cells located in different tissue compartments. We also demonstrate the differential distribution and effects of BET inhibitors in normal and malignant cells in vivo. This study provides a potential framework for the preclinical assessment of a wide range of drugs.

Background and Purpose: Chronic spontaneous urticaria presents as a heterogeneous syndrome characterised by wheals, angioedema, or both for greater than 6 weeks. Spleen tyrosine kinase mediates allergen-induced mast cell degranulation via the IgE signalling pathway, a central component of wheal formation and inflammation. In this study, we investigated the effects of perfused or topically administered GSK2646264 on IgE-mediated histamine release from mast cells in an ex vivo human skin model. Experimental Approach: Using a novel SkiP device, ex vivo human skin from mastectomy surgeries was challenged with anti-IgE, complement 5a (C5a), and buffer to induce histamine release from skin mast cells. Histamine was collected via microdialysis fibres and measured fluorometrically. GSK2646264 was delivered via perfusion either using microdialysis fibres or topically in a cream. Drug concentrations in the skin were measured by LC-MS, and a pharmacokinetic/ pharmacodynamic (PK/PD) relationship developed.Key Results: Perfused GSK2646264 significantly inhibited anti-IgE (but not C5a)induced histamine release in a concentration-dependent manner. The 0.5, 1, and 3% cream delivered GSK2646264 to the dermis above the IC 90 and dosedependently attenuated anti-IgE-induced histamine release.Conclusions and Implications: GSK2646264 administered topically or direct to the dermis blocked histamine release from in situ skin mast cells. A PK/PD relationship curve suggests that dermal concentrations above 6.8 μM should lead to approximately 90% inhibition of histamine release from skin mast cells following activation of the Fc fragment of IgE receptor 1a, implicating a potential use for the compound in skin mast cell diseases such as urticaria.

A simple method for the analysis of peptides based on mass spectrometry has been applied to the study of interactions between human bleomycin hydrolase (hBH) or neprilysin (NEP; EC 3.4.24.11) and peptides Ac-HHQKLVFFAG-NH2, Ac-SEVNLDAEFG-NH2 and Ac-GGVVIATVIG-NH2, three substrates cleaved by α, β and γ-secretase, respectively. These proteases are involved in the catabolism of amyloid protein precursor (APP), a protein implicated in Alzheimer's disease. The results indicate that hBH does not cleave the three substrates tested. Conversely, NEP cleaves Ac-HHQKLVFFAG-NH2 at multiple sites and Ac-SEVNLDAEFG-NH2 at only one position, whereas no cleavage was observed with Ac-GGVVIATVIG-NH2. The fragments generated by cleavage with NEP were clearly identified without any further analysis by tandem mass spectrometry. The implication of the role of NEP in the Alzheimer detoxification process is discussed. This technique provides useful information on peptide cleavage points and could be applied easily to a mixture of peptides for the determination of amino acid sequences preferred by any protease.