Drug ‘clicking’ on cell-penetrating fluorescent nanoparticles for in cellulo chemical proteomics

Chemical proteomics approaches are widely used to identify molecular targets of existing or novel drugs. This manuscript describes the development of a straightforward approach to conjugate azide-labeled drugs via click chemistry to alkyne-tagged cell-penetrating fluorescent nanoparticles as a novel tool to study target engagement and/or identification inside living cells. A modification of the Baeyer test for alkynes allows monitoring the Cu­(I)-catalyzed azide–alkyne cycloaddition (CuAAC) reaction, guaranteeing the presence of the drug on the solid support. As a proof of concept, the conjugation of the promiscuous kinase inhibitor dasatinib to Cy5-labeled nanoparticles is presented. Dasatinib-decorated fluorescent nanoparticles efficiently inhibited its protein target SRC in vitro, entered cancer cells, and colocalized with SRC in cellulo

Isomeric O-methyl cannabidiolquinones with dual BACH1/NRF2 activity

Oxidative stress and inflammation in the brain are two key hallmarks of neurodegenerative diseases (NDs) such as Alzheimer's, Parkinson's, Huntington's and multiple sclerosis. The axis NRF2-BACH1 has anti-inflammatory and anti-oxidant properties that could be exploited pharmacologically to obtain neuroprotective effects. Activation of NRF2 or inhibition of BACH1 are, individually, promising therapeutic approaches for NDs. Compounds with dual activity as NRF2 activators and BACH1 inhibitors, could therefore potentially provide a more robust antioxidant and anti-inflammatory effects, with an overall better neuroprotective outcome. The phytocannabinoid cannabidiol (CBD) inhibits BACH1 but lacks significant NRF2 activating properties. Based on this scaffold, we have developed a novel CBD derivative that is highly effective at both inhibiting BACH1 and activating NRF2. This new CBD derivative provides neuroprotection in cell models of relevance to Huntington's disease, setting the basis for further developments in vivo

An effective polymeric nanocarrier that allows for active targeting and selective drug delivery in cell coculture systems

In this manuscript, we report the development of a versatile, robust, and stable targeting nanocarrier for active delivery. This nanocarrier is based on bifunctionalized polymeric nanoparticles conjugated to a monoclonal antibody that allows for active targeting of either (i) a fluorophore for tracking or (ii) a drug for monitoring specific cell responses. This nanodevice can efficiently discriminate between cells in coculture based on the expression levels of cell surface receptors. As a proof of concept, we have demonstrated efficient delivery using a broadly established cell surface receptor as the target, the epidermal growth factor receptor (EGFR), which is overexpressed in several types of cancers. Additionally, a second validation of this nanodevice was successfully carried out using another cell surface receptor as the target, the cluster of differentiation 147 (CD147). Our results suggest that this versatile nanocarrier can be expanded to other cell receptors and bioactive cargoes, offering remarkable discrimination efficiency between cells with different expression levels of a specific marker. This work supports the ability of nanoplatforms to boost and improve the progress towards personalized medicine.Health Institute Carlos III (ISCIII) DTS18/00121Junta de Andalucia-FEDER, Ministry of Economy, Knowledge, Companies and University (University of Granada) B-FQM-475-UGR18 PT18-TP-1490 AT17_6096- OTRISpanish State Research Agency RED2018-102469-TFundación Benéfica Anticáncer San Francisco Javier y Santa CándidaSpanish Ministry of Economy and Competitiveness PTQ-16- 08597Spanish Government FPU 16/0206

Tracking cell proliferation using a nanotechnology based approach

yesTo develop an efficient nanotechnology fluorescence-based method to track cell proliferation to avoid the limitations of current cell-labeling dyes. Material & methods: Synthesis, PEGylation, bifunctionalization and labeling with a fluorophore (Cy5) of 200 nm polystyrene nanoparticles (NPs) were performed. These NPs were characterized and assessed for in vitro long-term monitoring of cell proliferation. Results: The optimization and validation of this method to track long-term cell proliferation assays have been achieved with high reproducibility, without cell cycle disruption. This method has been successfully applied in several adherent and suspension cells including hard-to-transfect cells and isolated human primary lymphocytes. Conclusion: A novel approach to track efficiently cellular proliferation by flow cytometry using fluorescence labeled NPs has been successfully developed

Synthesis of 6,8,9 poly-substituted purine analogue libraries as pro-apoptotic inducers of human leukemic lymphocytes and DAPK-1 inhibitors

A 18-member library of 6,8,9-poly-substituted purines was prepared from pyrimidines, primary alcohols, and N,N-dimethylamides under basic conditions via a novel one-pot synthetic pathway controlled by amide sizes and the novel analogues were tested against two leukemia cell lines: Jurkat (acute T cell leukemia) and K562 (chronic erythroleukemia) cells. Compounds having a benzoxy group at C6 position of the aromatic ring exhibited antiproliferative activity in Jurkat cells whereas all compounds induced a lower effect on K562 cells. Analysis of cell cycle, Annexin-V staining, and cleavage of initiator caspases assays showed that the active purine analogues induce cell death by apoptosis. Based on these results, a new purine derivative was synthesized, 6-benzyloxy-9-tert-butyl-8-phenyl-9H-purine (6d), which displayed the highest activity of the series against Jurkat cell lines. Finally, 33P-radiolabeled kinase assays using 96 recombinant human kinases known to be involved in apoptotic events were performed. Just one of the kinases tested, DAPK-1, was inhibited 50% or more by the phenotypic hits at 10 μM, suggesting that the inhibition of this target could be responsible for the induction of cell death by apoptosis. In agreement with the phenotypic results, the most active antiproliferative agent, 6d, displayed also the lowest IC50 value against recombinant DAPK1 (2.5 μM), further supporting the potential role of this protein on the observed functional response. DAPK-1 inhibition led by 6d together with its pro-apoptotic properties against the Jurkat line makes it an interesting candidate to further investigate the role of DAPK1 kinase in triggering apoptosis in cancer cells, a role which is attracting recent interest.JJDM thanks the Spanish Ministerio de Economia y Competitividad for funding a Ramon y Cajal Fellowship. AUB thanks Medical Research Council UK – Institute of Genetics and Molecular Medicine for funding an Academic Fellowship. We acknowledge the generous supply of rIL-2 provided by the National Institutes of Health AIDS Reagent Program. IJM is grateful for the support provided to his laboratory by grant 12UDG01-ATF from Sparks, the children's medical charity, London, UK

New therapeutic approaches for human African trypanosomiasis

Proyecto FP7 Health-2007-2.3.4-1 "Nanotryp" (223048).Tesis Univ. Granada. Departamento de Bioquímica y Biología Molecular III e Inmunologí

New therapeutic approaches for human African trypanosomiasis

Proyecto FP7 Health-2007-2.3.4-1 "Nanotryp" (223048).Tesis Univ. Granada. Departamento de Bioquímica y Biología Molecular III e Inmunologí

Novel therapy based on camelid nanobodies

Nanobodies (Nbs) are small antibody fragments derived from camelid heavy chain antibodies through recombinant gene technology. Their exceptional physicochemical properties, possibility of humanization and unique antigen recognition properties make them excellent candidates for targeted delivery of biologically active components. Several different therapeutic approaches based on the novel camelid Nbs have been developed to treat a wide range of diseases ranging from immune, bone, blood and neurological disorders; infectious diseases and cancer. This review provides a comprehensive overview of the current state of the use of camelid-derived Nbs as novel therapeutic agents against multiple diseases

The dimerization of Delta(9)-tetrahydrocannabinolic acid A (THCA-A)

The renewed interest in dimeric salicylates as broad-spectrum anti-inflammatory and antidiabetic agents provided a rationale to investigate the dimerization of the substituted salicylate Delta(9)-tetrahydrocannabinolic acid (THCA-A, 3a) as a strategy to solve its instability to decarboxylation and to generate analogues and/or pro-drugs of this native pre-cannabinoid. Activation of the carboxylic group with the DCC-HOBt-DMAP protocol afforded a high yield of the OBt ester 4, that was next converted into the highly crystalline di-depsidic dimer 5 upon treatment with DMAP. The mono-depsidic dimer 6 was also formed when the reaction was carried out with partially decarboxylated THCA-A samples. The structure of the depsidic dimers was established by spectroscopic methods and by aminolysis of 5 into the pre-cannabinoid amide 7. Both dimers showed excellent shelf stability and did not generate significant amounts of Delta(9)-THC upon heating. However, only the didepsidic dimer 5 activated PPAR-gamma, the major target of pre-cannabinoids, but strong binding to serum proteins abolished this activity, also shielding it from the action of esterases. (C) 2019 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V

Discovery of senolytics using machine learning

Abstract Cellular senescence is a stress response involved in ageing and diverse disease processes including cancer, type-2 diabetes, osteoarthritis and viral infection. Despite growing interest in targeted elimination of senescent cells, only few senolytics are known due to the lack of well-characterised molecular targets. Here, we report the discovery of three senolytics using cost-effective machine learning algorithms trained solely on published data. We computationally screened various chemical libraries and validated the senolytic action of ginkgetin, periplocin and oleandrin in human cell lines under various modalities of senescence. The compounds have potency comparable to known senolytics, and we show that oleandrin has improved potency over its target as compared to best-in-class alternatives. Our approach led to several hundred-fold reduction in drug screening costs and demonstrates that artificial intelligence can take maximum advantage of small and heterogeneous drug screening data, paving the way for new open science approaches to early-stage drug discovery