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G-quadruplexes (G4s) are non-canonical DNA secondary structures. The identification of selective tools to probe individual G4s over the ∼700 000 found in the human genome is key to unravel the biological significance of specific G4s. We took inspiration from a crystal structure of the bovine DHX36 helicase bound to the G4 formed in the promoter region of the oncogene c-MYC to identify a short peptide that preferentially binds MYC G4 with nM affinity over a small panel of parallel and non-parallel G4s tested.

Thanks to its biocompatibility, versatility and programmable interactions, DNA has been proposed as a building block for functional, stimuli-responsive frameworks with applications in biosensing, tissue engineering and drug delivery. Of particular importance for in vivo applications is the possibility of making such nano-materials responsive to physiological stimuli. Here we demonstrate how combining noncanonical DNA G-quadruplex (G4) structures with amphiphilic DNA constructs yields nanostructures, which we termed "Quad-Stars", capable of assembling into responsive hydrogel particles via a straightforward, enzyme-free, one-pot reaction. The embedded G4 structures allow one to trigger and control the assembly/disassembly in a reversible fashion by adding or removing K+ ions. Furthermore, the hydrogel aggregates can be photodisassembled upon near-UV irradiation in the presence of a porphyrin photosensitiser. The combined reversibility of assembly, responsiveness and cargo-loading capabilities of the hydrophobic moieties make Quad-Stars a promising candidate for biosensors and responsive drug delivery carriers.

Thanks to its biocompatibility, versatility and programmable interactions, DNA has been proposed as a building block for functional, stimuli-responsive frameworks with applications in biosensing, tissue engineering and drug delivery. Of particular importance for in vivo applications is the possibility of making such nano-materials responsive to physiological stimuli. Here we demonstrate how combining noncanonical DNA G-quadruplex (G4) structures with amphiphilic DNA constructs yields nanostructures, which we termed "Quad-Stars", capable of assembling into responsive hydrogel particles via a straightforward, enzyme-free, one-pot reaction. The embedded G4 structures allow one to trigger and control the assembly/disassembly in a reversible fashion by adding or removing K+ ions. Furthermore, the frameworks can be rendered responsive to near-UV light through the addition of a porphyrin photosensitiser. The combined reversibility of assembly, responsiveness and cargo-loading capabilities of the hydrophobic moieties make Quad-Stars a promising candidate for biosensors and responsive drug delivery carriers.

Background and purpose The TRPC1, TRPC4, and TRPC5 proteins form homotetrameric or heterotetrameric, calcium-permeable cation channels that are involved in various disease states. Recent research has yielded specific and potent xanthine-based TRPC1/4/5 inhibitors. Here, we investigated the possibility of xanthine-based activators of these channels. Experimental approach An analogue of the TRPC1/4/5 inhibitor Pico145, AM237, was synthesized and its activity was investigated using HEK cells overexpressing TRPC4, TRPC5, TRPC4-C1, TRPC5-C1, TRPC1:C4 or TRPC1:C5 channels, and in A498 cells expressing native TRPC1:C4 channels. TRPC1/4/5 channel activities were assayed by measuring intracellular concentration of Ca2+ ([Ca2+ ]i ) and by patch-clamp electrophysiology. Selectivity of AM237 was tested against TRPC3, TRPC6, TRPV4, or TRPM2 channels. Key results AM237 potently activated TRPC5:C5 channels (EC50 15-20 nM in [Ca2+ ]i assay) and potentiated their activation by sphingosine-1-phosphate but suppressed activation evoked by (-)-englerin A (EA). In patch-clamp studies, AM237 activated TRPC5:C5 channels, with greater effect at positive voltages, but with lower efficacy than EA. Pico145 competitively inhibited AM237-induced TRPC5:C5 activation. AM237 did not activate TRPC4:C4, TRPC4-C1, TRPC5-C1, TRPC1:C5, and TRPC1:C4 channels, or native TRPC1:C4 channels in A498 cells, but potently inhibited EA-dependent activation of these channels with IC50 values ranging from 0.9 to 7 nM. AM237 (300 nM) did not activate or inhibit TRPC3, TRPC6, TRPV4, or TRPM2 channels. Conclusions and implications This study suggests the possibility for selective activation of TRPC5 channels by xanthine derivatives and supports the general principle that xanthine-based compounds can activate, potentiate, or inhibit these channels depending on subunit composition.

Cellular energy metabolism is fundamental for all biological functions. Cellular proliferation requires extensive metabolic reprogramming and has a high energy demand. The Kv1.3 voltage-gated potassium channel drives cellular proliferation. Kv1.3 channels localise to mitochondria. Using high-resolution respirometry, we show Kv1.3 channels increase oxidative phosphorylation, independently of redox balance, mitochondrial membrane potential or calcium signalling. Kv1.3-induced respiration increased reactive oxygen species production. Reducing reactive oxygen concentrations inhibited Kv1.3-induced proliferation. Selective Kv1.3 mutation identified that channel-induced respiration required an intact voltage sensor and C-terminal ERK1/2 phosphorylation site, but is channel pore independent. We show Kv1.3 channels regulate respiration through a non-conducting mechanism to generate reactive oxygen species which drive proliferation. This study identifies a Kv1.3-mediated mechanism underlying the metabolic regulation of proliferation, which may provide a therapeutic target for diseases characterised by dysfunctional proliferation and cell growth.

TRPC1/4/5 cation channels are emerging drug targets for the treatment of, amongst others, central nervous system (CNS) disorders, kidney disease, and cardiovascular and metabolic disease. Various small-molecule TRPC1/4/5 modulators have been reported, including highly potent xanthine derivatives that distinguish between specific TRPC1/4/5 tetramers. However, tools to profile ligand engagement by TRPC1/4/5 channels in live cells are lacking. Here, we report a set of potent xanthine-based photoaffinity probes that functionally mimic the xanthines Pico145 and AM237. Using these probes, we have developed a photoaffinity labelling protocol for TRPC5 channels, providing the first method for the quantitative assessment of binding interactions of TRPC5 with small molecules in cells. This method could be important for drug discovery efforts targeting the xanthine/lipid binding site of TRPC1/4/5 channels.

TRPC1/4/5 cation channels are emerging drug targets for the treatment of, amongst others, central nervous system (CNS) disorders, kidney disease, and cardiovascular and metabolic disease. Various small-molecule TRPC1/4/5 modulators have been reported, including highly potent xanthine derivatives that can distinguish between specific TRPC1/4/5 tetramers. However, there is a paucity of information about their binding mode, which limits the ability to develop them further as chemical probes of specific TRPC1/4/5 channels for use in fundamental biological studies and drug discovery programmes. Here, we report the development of a set of potent xanthine-based photoaffinity probes that functionally mimic the xanthines Pico145 and AM237, respectively. Using these probes, we have developed a quantitative photoaffinity labelling protocol for TRPC5 channels. Our results provide the first direct evidence that xanthines modulate TRPC5 channels through a direct binding interaction with TRPC5 protein, and the first quantitative method for the assessment of binding interactions of TRPC5 and small molecules. Our method may allow the study of the mode-of-action of other TRPC1/4/5 modulators, and the identification of small molecule binding sites of TRPC1/4/5 channels.

TRPC1/4/5 cation channels are emerging drug targets for the treatment of, amongst others, central nervous system (CNS) disorders, kidney disease, and cardiovascular and metabolic disease. Various small-molecule TRPC1/4/5 modulators have been reported, including highly potent xanthine derivatives that can distinguish between specific TRPC1/4/5 tetramers. However, there is a paucity of information about their binding mode, which limits the ability to develop them further as chemical probes of specific TRPC1/4/5 channels for use in fundamental biological studies and drug discovery programmes. Here, we report the development of a set of potent xanthine-based photoaffinity probes that functionally mimic the xanthines Pico145 and AM237, respectively. Using these probes, we have developed a quantitative photoaffinity labelling protocol for TRPC5 channels. Our results provide the first direct evidence that xanthines modulate TRPC5 channels through a direct binding interaction with TRPC5 protein, and the first quantitative method for the assessment of binding interactions of TRPC5 and small molecules. Our method may allow the study of the mode-of-action of other TRPC1/4/5 modulators, and the identification of small molecule binding sites of TRPC1/4/5 channels.

G-quadruplexes are nucleic acids secondary structures that can be formed in guanine-rich sequences. More than 30 years ago, their formation was first observed in telomeric DNA. Since then, a number of other sequences capable of forming G-quadruplex structures have been described and increasing evidence supporting their formation in the context of living cells has been accumulated. To fully underpin the biological significance of G-quadruplexes and their potential as therapeutic targets, several chemical-biology tools and methods have been developed to map and visualise these nucleic acids secondary structures in human cells. In this review, we critically present the most relevant methods developed to investigate G-quadruplex prevalence in human cells and to study their biological functions, presenting the next key chemical-biology challenges that need to be addressed to fully unravel G-quadruplex mediated biology and their therapeutic potential.

Quinone methides (QMs) are transient reactive species that can be efficiently generated from stable precursors under a variety of biocompatible conditions. Due to their electrophilic nature, QMs have been widely explored as cross-linking agents of DNA and proteins under physiological conditions. However, QMs also have a diene character and can irreversibly react via Diels-Alder reaction with electron-rich dienophiles. This particular reactivity has been recently exploited to label biomolecules with fluorophores in living cells. QMs are characterised by two unique properties that make them ideal candidates for chemical biology applications: i) they can be efficiently generated in situ from very stable precursors by means of bio-orthogonal protocols ii) they are reversible cross-linking agents, making them suitable for “catch and release” target-enrichment experiments. Nevertheless, there are only few examples reported to date that truly take advantage of QMs unique chemistry in the context of chemical-biology assay development. In this review, we will examine the most relevant examples that illustrate the benefit of using QMs for chemical biology purposes and we will anticipate novel approaches to further their applications in biologically relevant contexts.

Proteins of the TRPC family can form many homo- and heterotetrameric cation channels permeable to Na+, K+ and Ca2+. In this review, we focus on channels formed by the isoforms TRPC1, TRPC4 and TRPC5. We review evidence for the formation of different TRPC1/4/5 tetramers, give an overview of recently developed small-molecule TRPC1/4/5 activators and inhibitors, highlight examples of biological roles of TRPC1/4/5 channels in different tissues and pathologies, and discuss how high-quality chemical probes of TRPC1/4/5 modulators can be used to understand the involvement of TRPC1/4/5 channels in physiological and pathophysiological processes.

A self‐sorting network comprising six different hydrogen bonding‐motifs is presented. Detailed analysis of the pairwise molecular recognition behavior of each hydrogen‐bonding motif by NMR spectroscopy, single‐crystal studies, and DFT allowed the construction of cascades exploiting integrative and non‐integrative narcissistic and social self‐sorted product distributions. Multiple distinct pathways within the network could be created depending on the presence or absence of different components. More information can be found in the Full Paper by C. Fonseca Guerra, A. J. Wilson, et al. on page 785-795.

Funder: Imperial College London, Thanks to its biocompatibility, versatility, and programmable interactions, DNA has been proposed as a building block for functional, stimuli-responsive frameworks with applications in biosensing, tissue engineering, and drug delivery. Of particular importance for in vivo applications is the possibility of making such nanomaterials responsive to physiological stimuli. Here, we demonstrate how combining noncanonical DNA G-quadruplex (G4) structures with amphiphilic DNA constructs yields nanostructures, which we termed "Quad-Stars", capable of assembling into responsive hydrogel particles via a straightforward, enzyme-free, one-pot reaction. The embedded G4 structures allow one to trigger and control the assembly/disassembly in a reversible fashion by adding or removing K+ ions. Furthermore, the hydrogel aggregates can be photo-disassembled upon near-UV irradiation in the presence of a porphyrin photosensitizer. The combined reversibility of assembly, responsiveness, and cargo-loading capabilities of the hydrophobic moieties make Quad-Stars a promising candidate for biosensors and responsive drug delivery carriers.

Background and Purpose The TRPC5 proteins assemble to create calcium‐permeable, non‐selective, cationic channels. We sought novel modulators of these channels through studies of natural products. Experimental Approach Intracellular calcium measurements and patch clamp recordings were made from cell lines. Compounds were generated by synthetic chemistry. Key Results Through a screen of natural products used in traditional Chinese medicines, the flavonol galangin was identified as an inhibitor of lanthanide‐evoked calcium entry in TRPC5 overexpressing HEK 293 cells (IC50 0.45 μM). Galangin also inhibited lanthanide‐evoked TRPC5‐mediated current in whole‐cell and outside‐out patch recordings. In differentiated 3T3‐L1 cells, it inhibited constitutive and lanthanide‐evoked calcium entry through endogenous TRPC5‐containing channels. The related natural flavonols, kaempferol and quercetin were less potent inhibitors of TRPC5. Myricetin and luteolin lacked effect, and apigenin was a stimulator. Based on structure–activity relationship studies with natural and synthetic flavonols, we designed 3,5,7‐trihydroxy‐2‐(2‐bromophenyl)‐4H‐chromen‐4‐one (AM12), which inhibited lanthanide‐evoked TRPC5 activity with an IC50 of 0.28 μM. AM12 also inhibited TRPC5 activity evoked by the agonist (−)‐Englerin A and was effective in excised outside‐out membrane patches, suggesting a relatively direct effect. It inhibited TRPC4 channels similarly, but its inhibitory effect on TRPC1–TRPC5 heteromeric channels was weaker. Conclusions and Implications The data suggest that galangin (a natural product from the ginger family) is a TRPC5 inhibitor and that other natural and synthetic flavonoids contain antagonist or agonist capabilities at TRPC5 and closely related channels depending on the substitution patterns of both the chromone core and the phenyl ring.