Biology subjects

Simmons, K. J.

Publications and source records attributed to Simmons, K. J..

10 recordsLinked to original sources

bioRxiv2025

Large-Scale Structure-Based Virtual Screening Identifies Diverse KNa1.1 (KCNT1) Potassium Channel Inhibitors

Severe drug-resistant childhood epilepsy is caused by KCNT1 gain-of-function genetic variants, resulting in increased KNa1.1 channel activity. KCNT1-associated epilepsy is thought to affect around 1 in 300,000 births worldwide. Current treatment for KCNT1 epilepsy only provides mild symptomatic relief and uses a cocktail of experimental medications which must be personalised for the individual and are often poorly tolerated. Critically, with many patients, no therapeutic benefit is achieved. We sought to address this by using large-scale virtual screening to accelerate the development of a molecule which binds directly to KCNT1 to supress overactivity. We purchased a total of 71 compounds and using a combination of fluorescent thallium flux assays and patch clamp electrophysiology, identified a series of eight structurally diverse, novel inhibitors of the KNa1.1 channel with potency in the low micromolar range. These provide potential starting points for further development of drugs to treat KCNT1-associated epilepsy. HighlightsO_LIWe have discovered a range of structurally distinct inhibitors of the KCNT1 ion channel using large-scale virtual screening C_LIO_LIThese compound exhibit selectivity for the KCNT1 channel over other related ion channels C_LIO_LIThese compounds could provide starting points for new treatments for KCNT1 related Epilepsy. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/679465v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@481813org.highwire.dtl.DTLVardef@12494dforg.highwire.dtl.DTLVardef@a9f787org.highwire.dtl.DTLVardef@b9f4b9_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2025

Identification of new KCNT1-epilepsy drugs by in silico, cell and Drosophila modelling.

ObjectiveHyperactive KCNT1 potassium channels, caused by gain-of-function mutations, are associated with a range of epilepsy disorders. Patients typically experience drug-resistant seizures and in cases with infantile onset, developmental regression can follow. KCNT1-related disorders include epilepsy of infancy with migrating focal seizures and sleep related hypermotor epilepsy. There are currently no effective treatments for KCNT1-epilepsies, but suppressing over-active channels poses a potential strategy. MethodsUsing KCNT1 channel structural data, we in silico screened a library of known drugs for those predicted to block the channel pore to reduce the current amplitude and inhibit channel activity. ResultsEight known drugs were investigated in vitro for their effects on patient-specific mutant KCNT1 channels, with four drugs showing significant reduction of K+ current amplitudes. The action of the four drugs was then analyzed in vivo and two were found to reduce the seizure phenotype in humanized Drosophila KCNT1-epilepsy models. InterpretationThis study identified two known drugs, antrafenine and nelfinavir mesylate, that reduce KCNT1 channel activity and reduce seizure activity in whole animals, suggesting their potential use as new treatments for KCNT1-epilepsy. The sequential in silico, in vitro and in vivo mechanism-based drug selection strategy used here may have broader application for other human disorders where a disease mechanism has been identified.

pharmacology and toxicology↗

bioRxiv2025

Targeting Tenascin-C-Toll-like Receptor 4 signalling with Adhiron-derived small molecules - a viable strategy for reducing fibrosis in Systemic Sclerosis

Tissue fibrosis is a hallmark of systemic sclerosis (SSc) and results from the excessive production and deposition of collagen and other extracellular matrix proteins by resident fibroblasts. This excessive connective tissue accumulation leads to tissue disruption and subsequent dysfunction in the skin, lungs and other internal organs. Recent studies highlight a role for the matricellular protein Tenascin C in SSc, whereby its stimulation of Toll-like receptor 4 triggers self-sustaining fibroblast activation and ensuing fibrosis. We have utilised Adhiron guided ligand discovery to generate small molecules that target the fibrinogen-like globe domain of Tenascin C, a region involved in Toll-like receptor 4 activation and have demonstrated a reduction in the profibrotic phenotype of human dermal fibroblasts. These studies may facilitate the development of effective targeted therapy for fibrosis in SSc and other fibrotic diseases. Moreover, it highlights the utility of Adhiron guided ligand discovery to generate small molecule inhibitors to selectively modulate proteins. HighlightsO_LIAdhiron screening was undertaken to identify binders to the FBG domain of Tenascin C and the crystal structure of the FBG-Adhiron binding was resolved. C_LIO_LIAdhiron guided ligand discovery was used to generate small molecule inhibitors that selectively target the FBG domain of Tenascin C. C_LIO_LISPR was used to show binding of these molecules to the FBG domain of Tenascin C C_LIO_LIThe small molecule inhibitors generated attenuated TGF{beta}-mediated fibrosis in dermal fibroblasts C_LI

physiology↗

bioRxiv2025

Adhirons are efficient tools to guide antiviral ligand discovery

Adhirons are small, antibody-like proteins expressed in bacteria that bind to biological targets with high affinity. Since their discovery, Adhirons have been isolated against over 350 targets, and they can be used for many research applications. However, their therapeutic application is at present in its early stages, hence the interest in developing novel small molecules to mimic their effect. We developed a pipeline for small molecule discovery where Adhirons act as starting templates in a ligand-based virtual screening workflow. For proof of concept, we used Adhirons against two viral systems: Crimean-Congo Haemorrhagic Fever Virus and Influenza A Virus. In both cases, a hit was identified from the mimics using biophysical and cell-based assays, highlighting how Adhirons, commercial libraries and structural approaches can be combined to accelerate small molecule inhibitor discovery of viruses.

cell biology↗

bioRxiv2024

Small molecule modulation of insulin receptor-insulin like growth factor-1 receptor heterodimers in human endothelial cells

ObjectivesThe insulin receptor (IR) and insulin like growth factor-1 receptor (IGF-1R) are heterodimers consisting of two extracellular -subunits and two transmembrane {beta} -subunits. Insulin {beta} and insulin like growth factor-1 {beta} hemi-receptors can heterodimerize to form hybrids composed of one IR {beta} and one IGF-1R {beta}. The function of hybrids in the endothelium is unclear. We sought insight by developing a small molecule capable of reducing hybrid formation in endothelial cells. MethodsWe performed a high-throughput small molecule screening, based on a homology model of hybrid structure. Endothelial cells were studied using western blotting and qPCR to determine the effects of small molecules that reduced hybrid formation. ResultsOur studies unveil a first-in-class quinoline-containing heterocyclic small molecule that reduces hybrids by >50% in human umbilical vein endothelial cells (HUVECs) with no effects on insulin or insulin like growth factor-1 receptors. This small molecule reduced expression of the negative regulatory p85 subunit of phosphatidylinositol 3-kinase, increased basal phosphorylation of the downstream target Akt and enhanced insulin/insulin-like growth factor-1 and shear stress-induced serine phosphorylation of Akt. In primary saphenous vein endothelial cells (SVEC) from patients with type 2 diabetes mellitus undergoing coronary artery bypass (CABG) surgery, hybrid receptor expression was greater than in patients without type 2 diabetes mellitus. The small molecule significantly reduced hybrid expression in SVEC from patients with type 2 diabetes mellitus. ConclusionsWe identified a small molecule that decreases the formation of IR: IGF-1R hybrid receptors in human endothelial cells, without significant impact on the overall expression of IR or IGF-1R. In HUVECs, reduction of IR: IGF-1R hybrid receptors leads to an increase in insulin-induced serine phosphorylation of the critical downstream signalling kinase, Akt. The underpinning mechanism appears, at least in part to involve the attenuation of the adverse effect of IR: IGF-1R hybrid receptors on PI3-kinase signalling. HighlightsO_LIWe have discovered a small molecule (HI) that inhibits insulin receptor/IGF-1 receptor hybrid formation. C_LIO_LIHI reveals previously unrecognised actions of insulin receptor/IGF-1 receptor hybrids distinct to insulin and IGF-1 receptors in endothelial cells. C_LIO_LITreatment of endothelial cells with HI enhances activity of the downstream signalling kinase Akt due to inhibitory regulation via PI3-K. C_LI

cell biology↗

bioRxiv2024

Using site-directed mutagenesis to further the understanding of insulin receptor-insulin like growth factor-1 receptor heterodimer structure.

Type 2 diabetes is characterised by the disruption of insulin and insulin-like growth factor (IGF) signalling. The key hubs of these signalling cascades - the Insulin receptor (IR) and Insulin-like growth factor 1 receptor (IGF1R) - are known to form functional IR-IGF1R hybrid receptors which are insulin resistant. However, the mechanisms underpinning IR-IGF1R hybrid formation are not fully understood, hindering the ability to modulate this for future therapies targeting this receptor. To pinpoint suitable sites for intervention, computational hotspot prediction was utilised to identify promising epitopes for targeting with point mutagenesis. Specific IGF1R point mutations F450A, R391A and D555A show reduced affinity of the hybrid receptor in a BRET based donor-saturation assay, confirming hybrid formation could be modulated at this interface. These data provide the basis for rational design of more effective hybrid receptor modulators, supporting the prospect of identifying a small molecule that specifically interacts with this target.

molecular biology↗

bioRxiv2021

A small molecule reveals role of insulin receptor-insulin like growth factor-1 receptor heterodimers

The insulin receptor and insulin like growth factor-1 receptor are heterodimers consisting of two extracellular -subunits and two transmembrane {beta}-subunits. IR {beta} and IGF1R {beta} hemi-receptors can heterodimerize to form hybrids composed of one IR {beta} and one IGF1R {beta}. Widely distributed in mammalian tissues, in contrast to IR and IGF1R the physiological function of hybrids is unclear. To identify tool compounds that inhibit hybrid formation we performed a high-throughput small molecule screen based on a homology model of hybrid structure. Our studies unveil a first in class quinoline-containing heterocyclic small molecule that reduces hybrids by >50% in human umbilical vein endothelial cells with no effect on IR or IGF1R expression. Downstream of IR and IGF1R our small molecule led to reduced expression of the negative regulatory p85 subunit of phosphatidylinositol 3-kinase, an increase in phosphorylation of its downstream target Akt and enhanced insulin and shear-induced phosphorylation of Akt. We show that hybrids have a role in human endothelial cell physiology distinct from IR and IGF1R.

cell biology↗

bioRxiv2020

Identification of novel Rab46 effector proteins

Rab46 is a novel Ca2+- sensing Rab GTPase shown to have important functions in endothelial and immune cells. The presence of functional Ca2+- binding, coiled-coil and Rab domains suggest that Rab46 will be important for coupling rapid responses to signalling in many cell types. The molecular mechanisms underlying Rab46 function are currently unknown. Here we provide the first resource for studying Rab46 interacting proteins. Using liquid chromatography mass spectrometry (LC-MS/MS) to identify affinity purified proteins that bind to constitutively active GFP-Rab46 or inactive GFP-Rab46 expressed in endothelial cells, we have revealed 922 possible interacting proteins. Further comparative analyses between nucleotide-locked Rab46 proteins enriched this dataset to confidently identify 29 effector proteins. Importantly, through biochemical and imaging approaches we have validated two potential effector proteins; dynein and the Na2+/ K+ ATPase subunit alpha 1 (ATP1α1). Hence, our use of affinity purification and LC-MS/MS to identify Rab46 neighbouring proteins provides a valuable resource for detecting Rab46 effector proteins and analysing Rab46 functions.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsLC-MS/MSliquid chromatography mass spectrometryGEFsliquid chromatography mass spectrometrGAPsGTPase activating proteinsWPBsWeibel-Palade bodiesECsendothelial cellsMTOCmicrotubule organising centreFDRfalse discovery rateDHCdynein heavy chainATP1α1Na2+/ K+ ATPase subunit α1View Full Text

cell biology↗

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↗

bioRxiv2019

Site-directed M2 proton channel inhibitors enable synergistic combination therapy for rimantadine-resistant pandemic influenza

Pandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable.\n\nRational in silico design and in vitro screens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic \"swine\" H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance.

microbiology↗