Biology subjects

Bon, R. S.

Publications and source records attributed to Bon, R. S..

7 recordsLinked to original sources

bioRxiv2025

(-)-Englerin A binds a conserved lipid site of TRPC5 and exposes a Met-aromatic motif in channel activation

TRPC4/5 cation channels are polymodal cellular sensors that play key roles in signal transduction/integration and have been implicated in various human pathologies, including anxiety, pain and cardiometabolic disease1-3. The plant natural product (-)-englerin A (EA)4 is a potent, selective TRPC4/5 agonist5,6 that has transformed fundamental and translational research on TRPC4/5 channels. However, the structural basis of interactions between EA and TRPC4/5 proteins has remained elusive, limiting our ability to fully understand and exploit mechanisms of TRPC4/5 channel activation by this intriguing natural product. Here, we present nine high-resolution cryo-EM structures (2.4-3.2 [A]) of human TRPC5 - representing different states and ligand occupancies - which show that EA binds to a conserved lipid binding site between transmembrane domains of adjacent TRPC5 subunits. Our structural models are consistent with the effects of mutagenesis of nearby residues on EAs potency, efficacy and activation kinetics, and allow us to rationalise competitive inhibition by other TRPC4/5 modulators as well as EAs selectivity profile within the TRPC family. Comparison of structures containing various TRPC5:EA stoichiometries revealed key structural and molecular determinants of EA-mediated TRPC5 activation - most notably the aromatic interaction network around Phe520 - underscoring the critical function of Met-aromatic motifs in ion channel structure and function. Binding of EA causes conformational changes of nearby amino acid residues, resulting in rearrangement of the pore helices into a pre-open state. Collectively, we provide structural insight into the mode-of-action of the most widely used TRPC4/5 agonist, which will underpin fundamental TRPC4/5 channel research as well as ongoing drug discovery programmes.

molecular biology↗

bioRxiv2024

Ideal efficacy photoswitches for TRPC4/5 channels harness high potency for spatiotemporally-resolved control of TRPC function in live tissues

Directly probing the endogenous biological roles of target proteins with high spatial and temporal resolution, as non-invasively and reproducibly as possible, is a shared conceptual goal for research across many fields, as well as for targeted therapies. Here we describe the rational conceptual design and test-case practical implementation of a photopharmacological paradigm to empower high-performance photomodulation studies in vivo. TRPC4/5 ion channels are involved in many spatiotemporally resolved circuits, from pain and anxiety, to reproductive signaling, digestion, and obesity. To unpick their biology requires spatiotemporally precise tools, which were lacking. We developed "ideal efficacy photoswitch" ligands to control their diverse functions in situ. These E{leftrightarrows}Z-photoswitchable ligands bias TRPC[4]/5 channel activity with exquisite photocontrol, from strong agonism under 360 nm, to low agonism at 385 nm, to strong antagonism at 410-460 nm. Cryo-EM structures of both TRPC4 and TRPC5 with both Z-agonists and E-antagonists support the rationale for efficacy switching through competitive E/Z isomer binding. Crucially, since the E/Z ratio is exclusively determined by the light wavelength applied, their channel photocontrol is exclusively wavelength-dependent, yet drug-concentration-independent: so is reproducible from cell culture to >millimetre-depth tissues. Indeed, we were able to photocontrol both direct and downstream TRPC4/5 biology in cell lines or primary cells in culture, from calcium flux, to primary neuron excitability and adrenaline release; and even in tissues, photoswitching small intestine motility and peristalsis. The TRPC4/5 ligands we develop will thus unlock a range of high-precision investigations in TRP biology. More broadly, we propose that the success of this efficacy photoswitch program, from concept to tissue level translation, is mainly a consequence of how biology has evolved proteins for efficacy control. We therefore foresee that a variety of functionally responsive protein targets, not only sensory and signaling ion channels and receptors, will be amenable to similarly high-performance photocontrol even in vivo, if a new generation of reagent development adopts this paradigm of ideal efficacy photoswitching. Table of Contents Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/602451v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1f8e527org.highwire.dtl.DTLVardef@18c72d0org.highwire.dtl.DTLVardef@1c5d648org.highwire.dtl.DTLVardef@177203d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Pico145 inhibits TRPC4-mediated mICAT and postprandial small intestinal motility

Background & AimsIn intestinal smooth muscle cells, receptor-operated TRPC4 are responsible for the majority of muscarinic receptor cation current (mICAT), which initiates cholinergic excitation-contraction coupling. Our aim was to examine the effects of the TRPC4 inhibitor Pico145 on mICAT and Ca2+ signalling in mouse ileal myocytes, and on intestinal motility. MethodsIleal myocytes freshly isolated from two month-old male BALB/c mice were used for patch-clamp recordings of whole-cell currents and for intracellular Ca2+ imaging using Fura-2. Functional assessment of Pico145s effects was carried out by standard in vitro tensiometry, ex vivo video recordings and in vivo postprandial intestinal transit measurements using carmine red. ResultsCarbachol (50 {micro}M)-induced mICAT was strongly inhibited by Pico145 starting from 1 pM. The IC50 value for the inhibitory effect of Pico145 on this current evoked by intracellularly applied GTP{gamma}S (200 {micro}M), and thus lacking desensitisation, was found to be 3.1 pM, while carbachol-induced intracellular Ca2+ rises were inhibited with IC50 of 2.7 pM. In contrast, the current activated by direct TRPC4 agonist (-)-englerin A was less sensitive to the action of Pico145 that caused only [~]43% current inhibition at 100 pM. The inhibitory effect developed rather slowly and it was potentiated by membrane depolarisation. In functional assays, Pico145 produced concentration-dependent suppression of both spontaneous and carbachol-evoked intestinal smooth muscle contractions and delayed postprandial intestinal transit. ConclusionsPico145 is a potent GI-active small-molecule which completely inhibits mICAT at picomolar concentrations and which is as effective as trpc4 gene deficiency in in vivo intestinal motility tests.

pharmacology and toxicology↗

bioRxiv2021

Suppression of aminoglycoside-induced premature termination codon readthrough by the TRP channel inhibitor AC1903

Nonsense mutations, which occur in ~11% of patients with genetic disorders, introduce premature termination codons (PTCs) that lead to truncated proteins and promote nonsense-mediated mRNA decay. Aminoglycosides such as gentamicin and G418 permit PTC readthrough and so may address this problem. However, their effects are variable between patients, making clinical use of aminoglycosides challenging. In this study, we addressed the hypothesis that TRP non-selective cation channels contribute to the variable effect of aminoglycosides by controlling their cellular uptake. To attempt to identify the channel type involved, we tested AC1903, a 2-aminobenzimidazole derivative recently reported to selectively inhibit TRPC5 cation channels. AC1903 consistently suppressed G418 uptake and G418-induced PTC readthrough in the DMS-114 cell line and patient-derived JEB01 keratinocytes. In an effort to validate the suggested role of TRPC5, we tested an independent and more potent inhibitor called Pico145, which affects channels containing TRPC1, TRPC4 and TRPC5 but not other TRPCs or other channels. Unexpectedly, Pico145 was completely without effect, suggesting that AC1903 may work through other or additional targets. Consistent with this suggestion, AC1903 inhibited multiple TRPC channels including homomeric TRPC3, TRPC4, TRPC5, TRPC6 as well as concatemeric TRPC4-C1 and TRPC5-C1 channels, all with low micromolar IC50 values. It also inhibited TRPV4 channels but had weak or no effects on TRPV1 and no effect on another non-selective cation channel, PIEZO1. Overall, our study reveals a suppressor of aminoglycoside-mediated PTC readthrough (i.e., AC1903) but suggests that this compound has previously unrecognised effects. These effects require further investigation to determine the molecular mechanism by which AC1903 suppresses aminoglycoside uptake and PTC readthrough.

pharmacology and toxicology↗

bioRxiv2020

Need for speed: Examining protein behaviour during cryoEM grid preparation at different timescales

A host of new technologies are under development to improve the quality and reproducibility of cryoEM grid preparation. Here we have systematically investigated the preparation of three macromolecular complexes using three different vitrification devices (Vitrobot, chameleon and a time-resolved cryoEM device) on various timescales, including grids made within 6 ms, (the fastest reported to date), to interrogate particle behaviour at the air-water interface for different timepoints. Results demonstrate that different macromolecular complexes can respond to the thin film environment formed during cryoEM sample preparation in highly variable ways, shedding light on why cryoEM sample preparation can be difficult to optimise. We demonstrate that reducing time between sample application and vitrification is just one tool to improve cryoEM grid quality, but that it is unlikely to be a generic silver bullet for improving the quality of every cryoEM sample preparation.

biochemistry↗

bioRxiv2020

Cryo-EM structures of human TRPC5 reveal interaction of a xanthine-based TRPC1/4/5 inhibitor with a conserved lipid binding site.

TRPC1/4/5 channels are non-specific cation channels implicated in a wide variety of diseases, and TRPC1/4/5 inhibitors have recently entered the first clinical trials. However, fundamental and translational studies require a better understanding of TRPC1/4/5 channel regulation by endogenous and exogenous factors. Although several potent and selective TRPC1/4/5 modulators have been reported, the paucity of mechanistic insights into their modes-of-action remains a barrier to the development of new chemical probes and drug candidates. The xanthine class of modulators includes the most potent and selective TRPC1/4/5 inhibitors described to date, as well as TRPC5 activators. Our previous studies suggest that xanthines interact with a, so far, elusive pocket of TRPC1/4/5 channels that is essential to channel gating. Targeting this pocket may be a promising strategy for TRPC1/4/5 drug discovery. Here we report the first structure of a small molecule-bound TRPC1/4/5 channel - human TRPC5 in complex with the xanthine Pico145 - to 3.0 [A]. We found that Pico145 binds to a conserved lipid binding site of TRPC5, where it displaces a bound phospholipid. Our findings explain the mode-of-action of xanthine-based TRPC1/4/5 modulators, and suggest a structural basis for TRPC1/4/5 modulation by endogenous factors such as (phospho)lipids and Zn2+ ions. These studies lay the foundations for the structure-based design of new generations of TRPC1/4/5 modulators.

molecular biology↗

bioRxiv2017

The small molecule KHS101 induces bioenergetic dysfunction in glioblastoma cells through inhibition of mitochondrial HSPD1

Pharmacological inhibition of uncontrolled cell growth with small molecule inhibitors is a potential strategy against glioblastoma multiforme (GBM), the most malignant primary brain cancer. Phenotypic profiling of the neurogenic small molecule KHS101 revealed the chemical induction of lethal cellular degradation in molecularly-diverse GBM cells, independent of their tumor subtype, whereas non-cancerous brain cells remained viable. Mechanism-of-action (MOA) studies showed that KHS101 specifically bound and inhibited the mitochondrial chaperone HSPD1. In GBM but not non-cancerous brain cells, KHS101 elicited the aggregation of an enzymatic network that regulates energy metabolism. Compromised glycolysis and oxidative phosphorylation (OXPHOS) resulted in the metabolic energy depletion in KHS101-treated GBM cells. Consistently, KHS101 induced key mitochondrial unfolded protein response factor DDIT3 in vitro and in vivo, and significantly reduced intracranial GBM xenograft tumor growth upon systemic administration, without discernible side effects. These findings suggest targeting of HSPD1-dependent oncometabolic pathways as an anti-GBM therapy.

cancer biology↗