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

Characterization and Therapeutic Effect of a pH Stimuli Responsive Polymeric Nanoformulation for Controlled Drug Release

Despite the large number of polymeric nanodelivery systems that have been recently developed, there is still room for improvement in terms of therapeutic efficiency. Most reported nanodevices for controlled release are based on drug encapsulation, which can lead to undesired drug leakage with a consequent reduction in efficacy and an increase in systemic toxicity. Herein, we present a strategy for covalent drug conjugation to the nanodevice to overcome this drawback. In particular, we characterize and evaluate an effective therapeutic polymeric PEGylated nanosystem for controlled pH-sensitive drug release on a breast cancer (MDA-MB-231) and two lung cancer (A549 and H520) cell lines. A significant reduction in the required drug dose to reach its half maximal inhibitory concentration (IC50 value) was achieved by conjugation of the drug to the nanoparticles, which leads to an improvement in the therapeutic index by increasing the efficiency. The genotoxic effect of this nanodevice in cancer cells was confirmed by nucleus histone H2AX specific immunostaining. In summary, we successfully characterized and validated a pH responsive therapeutic polymeric nanodevice in vitro for controlled anticancer drug release.Spanish Ministry of Economy and Competitiveness (MINECO) BIO2016-80519Health Institute Carlos III (ISCIII) DTS18/00121Andalusian Regional Government PAIDI-TC-PVT-PSETC-2.

Selective Anticancer Therapy Based on a HA-CD44 Interaction Inhibitor Loaded on Polymeric Nanoparticles

This research was funded by the Consejeria de Economia, Conocimiento, Empresas y Universidad of the Junta de Andalucia (grant number Excellence Research Project P18-RT-1679) and the Research Results Transfer Office (OTRI) of the University of Granada (grant number PR/17/006 project). This work was partially supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO), grant number PID2019.110987RB.I00; the Health Institute Carlos III (ISCIII), grant number DTS18/00121 the Junta de Andalucia-FEDER, Ministry of Economy, Knowledge, Companies, and University (FEDER 2018: ref. B-FQM-475-UGR18, PAIDI2020: ref. PT18-TP-4160); and the Andalusian Regional Government, grant number PAIDI-TC-PVT-PSETC-2.0. C.D. thanks HECBioSim, the UK High End Computing Consortium for Biomolecular Simulation (hecbiosim.ac.uk), which is supported by the EPSRC (EP/L000253/1) for awarding computing time in Jade, a UK Tier-2 resource. B.R.-R. gratefully acknowledges funding from the European Union's Horizon 2020 Research and Innovation Program under Marie Sklodowska-Curie Grant Agreement no. 754446 and UGR Research and Knowledge Transfer Fund-Athenea3i. J.M.E.-R. thanks the Spanish Ministry of Education for PhD funding (scholarship FPU 16/02061). V.C.-C. thanks the Andalusian Regional Government for her postdoctoral fellowship (POSTDOC_21_00118).Hyaluronic acid (HA), through its interactions with the cluster of differentiation 44 (CD44), acts as a potent modulator of the tumor microenvironment, creating a wide range of extracellular stimuli for tumor growth, angiogenesis, invasion, and metastasis. An innovative antitumor treatment strategy based on the development of a nanodevice for selective release of an inhibitor of the HACD44 interaction is presented. Computational analysis was performed to evaluate the interaction of the designed tetrahydroisoquinoline-ketone derivative (JE22) with CD44 binding site. Cell viability, efficiency, and selectivity of drug release under acidic conditions together with CD44 binding capacity, effect on cell migration, and apoptotic activity were successfully evaluated. Remarkably, the conjugation of this CD44 inhibitor to the nanodevice generated a reduction of the dosis required to achieve a significant therapeutic effect.Junta de Andalucia P18-RT-1679Research Results Transfer Office (OTRI) of the University of Granada PR/17/006Spanish Government PID2019.110987RB.I00Health Institute Carlos III (ISCIII) DTS18/00121Junta de Andalucia-FEDER, Ministry of Economy, Knowledge, Companies, and University (FEDER) B-FQM-475-UGR18 PT18-TP-4160Andalusian Regional Government POSTDOC_21_00118UK Research & Innovation (UKRI)Engineering & Physical Sciences Research Council (EPSRC) EP/L000253/1European Union's Horizon 2020 Research and Innovation Program under Marie Sklodowska-Curie Grant 754446UGR Research and Knowledge Transfer Fund-Athenea3iSpanish Government FPU 16/0206

Polymeric Carriers for Biomedical and Nanomedicine Application

This book focuses on the design of polymeric delivery systems for biomedical and nanomedicine applications as well as on understanding how such biomaterials interact in the physiological environment. The reader will find an encompassing view on the state-of-the-art of polymeric carriers, showing how current research deals with new stimuli-responsive systems for cancer therapies and biomedical challenges, namely overcoming the skin barrier. The published papers cover topics ranging from novel production methods and insights on hybrid polymers to applications as diverse as nanoparticles, hydrogels and microneedles for antifungal skin therapy, peptide and siRNA delivery, enhanced skin absorption of bioactive molecules, and anticancer therapy. The book comprises one review paper and nine research papers

Bifunctional chemical tools for the conditional control of targeted protein degradation

The ubiquitin-proteasome system (UPS) is a key pathway involved in protein homeostasis via the regulation of intracellular protein levels. The application of proximity-induced biology to the UPS has given birth to proteolysis targeting chimeras (PROTACs), which are bifunctional molecules able to co-opt an E3 ligase for the ubiquitination and proteasome-dependent degradation of a selected protein. Targeted protein degradation (TPD) with PROTACs is now established as a disruptive modality both in chemical biology and drug discovery as it allows the efficient knockdown of an intracellular protein with a small molecule in a catalytic manner. With their unique mode of action and modular synthesis, PROTACs are being developed as therapeutics for various human diseases. In order to enable discrete control over PROTACs function, our first investigation used light as a precision tool for the spatiotemporal activation of caged and photoswitchable degraders. The design, synthesis, photochemistry, and cellular activity of novel light-activated PROTACs is reported. Furthermore, with the ambition to apply such tools in vivo, non-conventional light sources were explored to activate the degraders and overcome the limitations of ultraviolet and visible light. In a second approach, variation in intracellular oxygen concentration was used as a means to selectively activate PROTACs. Incorporation of a bioreductive unit on a degrader followed by testing in a hypoxic environment is presented. In summary, this thesis describes our investigation towards the conditional control of TPD with newly designed bifunctional degraders which may help better study and tackle disease-relevant proteins.Open Acces

Drug "Clicking" on Cell-Penetrating Fluorescent Nanoparticles for In Cellulo Chemical Proteomics

This paper introduces a novel method for identifying molecular targets of drugs by conjugating azide-labeled drugs with alkyne-tagged cell-penetrating fluorescent nanoparticles using click chemistry. A modified Baeyer test for alkynes monitors the Cu(I)-catalyzed azide–alkyne cycloaddition reaction, ensuring drug presence on solid support. Dasatinib-conjugated nanoparticles effectively inhibit the SRC protein target in vitro and demonstrate intracellular colocalization