BACKGROUND AND PURPOSETransient receptor potential cation channel subfamily M member 7 (TRPM7) is a bifunctional protein comprising a TRP ion channel segment linked to an a-type protein kinase domain. TRPM7 is essential for proliferation and cell growth. Up-regulation of TRPM7 function is involved in anoxic neuronal death, cardiac fibrosis and tumour cell proliferation. The goal of this work was to identify non-toxic inhibitors of the TRPM7 channel and to assess the effect of blocking endogenous TRPM7 currents on the phenotype of living cells.
EXPERIMENTAL APPROACHWe developed an aequorin bioluminescence-based assay of TRPM7 channel activity and performed a hypothesis-driven screen for inhibitors of the channel. The candidates identified were further assessed electrophysiologically and in cell biological experiments.
KEY RESULTSTRPM7 currents were inhibited by modulators of small conductance Ca 2+ -activated K + channels (KCa2.1-2.3; SK) channels, including the antimalarial plant alkaloid quinine, CyPPA, dequalinium, NS8593, SKA31 and UCL 1684. The most potent compound NS8593 (IC50 1.6 mM) specifically targeted TRPM7 as compared with other TRP channels, interfered with Mg 2+ -dependent regulation of TRPM7 channel and inhibited the motility of cultured cells. NS8593 exhibited full and reversible block of native TRPM7-like currents in HEK 293 cells, freshly isolated smooth muscle cells, primary podocytes and ventricular myocytes.
CONCLUSIONS AND IMPLICATIONSThis study reveals a tight overlap in the pharmacological profiles of TRPM7 and KCa2.1-2.3 channels. NS8593 acts as a negative gating modulator of TRPM7 and is well-suited to study functional features and cellular roles of endogenous TRPM7.
Abbreviations
Although there are a large number of studies available for the evaluation of the therapeutic efficacy of targeted polymeric nanoparticles, little is known about the critical attributes that can further influence their uptake into target cells. In this study, varying cRGD ligand densities (0− 100% surface functionalization) were combined with different poly(ethylene glycol) (PEG) spacer lengths (2/3.5/5 kDa), and the specific receptor binding of targeted core−shell structured poly(lactic-co-glycolic acid)/poly(lactic acid)−PEG nanoparticles was evaluated using α v β 3 integrin-overexpressing U87MG glioblastoma cells. Nanoparticles with 100% surface functionalization and short PEG2k linkers displayed a high propensity to form colloidal clusters, allowing for the cooperative binding to integrin receptors on the cellular membrane. In contrast, the high flexibility of longer PEG chains enhanced the chance of ligand entanglement and shrouding, decreasing the number of ligand−receptor binding events. As a result, the combination of short PEG2k linkers and a high cRGD surface modification synergistically increased the uptake of nanoparticles into target cells. Even though to date, the nanoparticle size and its degree of functionalization are considered to be the major determinants for controlling the uptake efficiency of targeted colloids, these results strongly suggest that the role of the linker length should be carefully taken into consideration for the design of targeted drug delivery formulations to maximize the therapeutic efficacy and minimize adverse side effects.
Formation of a protein corona can severely compromise the performance of functionalized polymeric nanoparticles by changing their identity and integrity.
Poor target cell specificity is currently a major shortcoming of nanoparticles (NPs) used for biomedical applications. It causes significant material loss to off-target sites and poor availability at the intended delivery site. To overcome this limitation, we designed particles that identify cells in a virus-like manner. As a blueprint, we chose a mechanism typical of influenza A virus particles in which ectoenzymatic hemagglutinin activation by target cells is a mandatory prerequisite for binding to a secondary target structure that finally confirms cell identity and allows for uptake of the virus. We developed NPs that probe mesangial cells for the presence of angiotensin-converting enzyme on their surface using angiotensin I (Ang-I) as a proligand. This initial interaction enzymatically transforms Ang-I to a secondary ligand angiotensin II (Ang-II) that has the potential to bind in a second stage to Ang-II type-1 receptor (AT1R). The presence of the receptor confirms the target cell identity and triggers NP uptake via endocytosis. Our virus-mimetic NPs showed outstanding target-cell affinity with picomolar avidities and were able to selectively identify these cells in the presence of 90% off-target cells that carried only the AT1R. Our results demonstrate that the design of virus-mimetic cell interactive NPs is a valuable strategy to enhance NP specificity for therapeutic and diagnostic applications. Our set of primary and secondary targets is particularly suited for the identification of mesangial cells that play a pivotal role in diabetic nephropathy, one of the leading causes of renal failure, for which currently no treatment exists.
Amine-modified four- and eight-armed poloxamines were prepared and subsequently functionalized with maleimide or furyl groups. Aqueous solutions of these polymers exhibited an immediate gelation at a temperature above 37 °C. Concomitantly, Diels-Alder reactions gradually cross-linked and cured the gels. Different ratios between four- and eight-armed macromonomers were used to tune hydrogel stability and mechanical properties. In this way, hydrogel stability could be precisely controlled in the range of 14 to 329 days. Controlled release of the model antibody bevacizumab was achieved over a period of 7, 21, and 115 days. Release profiles were triphasic with a low burst; approximately 87% of the released antibody was intact and displayed functional binding. The hydrogels presented in this study are degradable, nontoxic, rapidly gelling, stable, and provide controlled antibody release. They can be tailored to match the demands of various applications and present an attractive platform for antibody delivery.
Viral
infection patterns often rely on precisely coordinated sequences of
distinct ligand–receptor interactions, leading in many cases
to an outstanding target cell specificity. A successful mimicry of
viral targeting strategies to create more site-specific nanoparticles
(NPs) would therefore require particle–cell interactions to
also be adequately controllable. In the present study, hetero-multivalent
block-copolymer NPs present their attached ligands in a sterically
controlled manner to create a sequential NP–cell interaction
similar to the cell infiltration strategy of human adenovirus type
2. Targeting renal mesangial cells, particles therefore initially
bind angiotensin II receptor type 1 (AT1r) on the cell surface via
a structurally flexible AT1r antagonist. After a mandatory spatial
approach, particle endocytosis is realized via binding of immobile
αVβ3 integrins with a previously
concealed secondary ligand, thereby creating a stepwise particle–cell
interplay of primary NP attachment and subsequent uptake. Manufactured
adenovirus-mimetic NPs show great avidity for both target motifs in vitro, leading to a substantial binding as well as subsequent
cell uptake into target mesangial cells. Additionally, steric shielding
of secondary ligand visibility leads to a highly controllable, sequential
ligand–receptor interaction, whereby hetero-functional NPs
activate mesangial cell surface integrins only after a successful
prior binding to the AT1r. This stepwise cell identification significantly
enhances mesangial cell specificity in co-culture assays with different
off-target cells. Additionally, described NPs display excellent in vivo robustness by efficiently accumulating in the mesangium
upon injection, thereby opening new paths for possible drug delivery
applications.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.