Biology subjects

Scott, J. W.

Publications and source records attributed to Scott, J. W..

11 recordsLinked to original sources

bioRxiv2026

Fine tuning energy metabolism in skeletal muscle: Discovery of a novel autoinhibitory mechanism in the N-terminal extension of AMPKγ3

AMP-activated protein kinase (AMPK) regulates metabolism in response to metabolic stress that includes stimulating glucose uptake in skeletal muscle independently of the canonical insulin signalling pathway, positioning it as an attractive therapeutic target for insulin resistance and type 2 diabetes mellitus (T2DM). AMPK is an {beta}{gamma} heterotrimer, with multiple isoforms for each subunit enabling the formation of 12 different complexes with distinct tissue expression profiles. Among these, the 2{beta}2{gamma}3 complex is predominantly expressed in skeletal muscle, the major site of glucose disposal and a highly desirable therapeutic target for T2DM. Here, we characterise the functional role of a unique, 182 residue N-terminal extension (NTE) within {gamma}3 subunit. Deletion of the {gamma}3-NTE from 2{beta}2{gamma}3 complex increases basal AMPK activity without affecting activation by AMP or pharmacological AMPK activators, demonstrating the {gamma}3-NTE performs an autoinhibitory function. Using complementary biophysical techniques, including hydrogen-deuterium exchange-mass spectrometry, surface plasmon resonance, chemical crosslinking and co-pulldowns, we identified a 39-residue sequence in the {gamma}3-NTE (residues 129-168), that directly interacts with the C-helix of the AMPK kinase domain small lobe, a key regulatory element in many protein kinases. Using AlphaFold3, we probe the interaction predicted to take place between a {gamma}3-NTE -helix ({gamma}3-iHelix; [~]T142-E154) and the C-helix in the 2{beta}2{gamma}3 complex. These findings provide the groundwork for developing novel T2DM therapies that target AMPK activation selectively in skeletal muscle involving reversal of the {gamma}3 autoinhibition.

biochemistry↗

bioRxiv2026

Fanconi Anaemia E3 Ligase complex activity is regulated by a druggable metabolite binding site in FANCX

The Fanconi anaemia (FA) DNA repair pathway is an emerging target for precision cancer therapy. Using a high-throughput FANCD2-monoubiquitination assay, we identified a class of small molecules, including MSG010, that inhibit the FA E3 ligase complex in vitro. Because these molecules, and the metabolite, palmitoyl-CoA, are known to engage allosteric drug and metabolite (ADaM) binding site in AMP-activated kinase (AMPK), we hypothesised that a similar pocket exists within the FA complex. Supporting this, long-chain, but not short-chain, fatty acyl-CoA molecules inhibited the FA E3 ligase complex activity, and sequence analysis revealed similarity between the AMPK ADaM site and a WD40 repeat in the FA subunit FANCX. Targeted mutagenesis of this FANCX region disrupted E3 ligase activity or abolished inhibition by MSG010, suggesting the presence of an ADaM-like site in FANCX. Moreover, MSG010 preferentially killed BRCA1-deficient cells in vitro. These findings identify a putative small-molecule binding site in the FA pathway that may be developed further to test for exploitation as anticancer therapeutics.

cancer biology↗

bioRxiv2025

SONIC: A Benchmarking Paradigm for Brain-Computer Interfaces

Brain-computer interfaces (BCIs) can restore function for individuals with neuro-logical disorders and have the potential to transform the way people interact with digital systems. However, the development of advanced BCI applications, such as fluent speech synthesis, is dependent on the underlying information transfer capacity of the physical neural interface employed. A significant barrier to progress has been the lack of standardized, application-agnostic methods for benchmarking BCI system performance prior to clinical trials. Here, we introduce SONIC, a novel preclinical benchmarking paradigm designed to evaluate the information transfer rate (ITR) of a BCI system. This paradigm treats the brain and BCI as a noisy communication channel, where information is sent into the brain via precisely controlled sensory stimuli and read out by the neural interface. We implemented this paradigm in an ovine model by presenting rapid sequences of pure tones while recording neural activity from the primary auditory cortex with the Paradromics Connexus(C) BCI, a fully implanted system utilizing high-density intracortical micro-electrode arrays with wireless power and data transmission. A convolutional neural network was used to decode tones based on neural features. Our results demonstrate an achieved ITR of over 200 bits per second (bps), which is the highest reported BCI ITR to date. For reference, this rate exceeds the linguistic information content of human speech. This ITR is achieved with a total neural interface, filtering, and data aggregation delay of 56 milliseconds. Further analysis demonstrated that ITR remains high (> 100 bps) for the lowest total delay tested (11 ms), supporting the needs of latency-sensitive applications (e.g., direct speech synthesis). This work establishes a new benchmark for BCI performance and demonstrates that the Connexus BCI possesses the bandwidth necessary to support highly advanced applications. This benchmark provides a robust framework for preclinical BCI evaluation, enabling principled system design optimization to accelerate the translation of next-generation neurotechnology.

bioengineering↗

bioRxiv2025

Mechanism and cellular actions of the potent AMPK inhibitor BAY-3827

Inhibition of AMP-activated protein kinase (AMPK) is under increasing investigation for its therapeutic potential in many diseases, including certain cancers. However, existing AMPK- inhibitors available as tool compounds are largely limited to compound C/dorsomorphin and SBI-0206965, both of which suffer from poor selectivity and off-target effects. Here we describe the structure-based molecular insights and cellular actions of a recently identified potent AMPK inhibitor, BAY-3827. Kinase selectivity profiling and sequence analyses of kinases that are highly or weakly inhibited by BAY-3827 uncovered key conserved residues involved in its inhibitory mechanism. A 2.5 [A] co-crystal structure of the AMPK kinase domain (KD)-BAY-3827 complex and comparison with known KD-inhibitor structures, revealed an overlapping site in the ATP-binding pocket and an C helix-out conformation. A distinct feature of the BAY-3827-bound state is the formation of a disulfide bridge between the D helix Cys106 and the activation loop residue Cys174. This bridge appears to stabilize the activation loop such that Asn162 repositions the DFG motif Phe158 toward the C-terminal kinase lobe, displacing His137 and disrupting the regulatory spine, thereby promoting an inactive state. In hepatocytes, 2.5-5 M BAY-3827, but not the structurally resembling inactive BAY-974, fully blocked AMPK activator (MK-8722)-mediated phosphorylation of ACC1 and corresponding inhibition of lipogenesis. Unbiased transcriptome analysis in MK- 8722-treated wild-type and AMPK-null hepatocytes revealed that 5 M BAY-3827 downregulated >30% of MK-8722-stimulated AMPK-dependent genes. Based on its greater selectivity and potency substantiated by comprehensive structural and cellular investigations, BAY-3827 is a powerful tool to delineate AMPK functions. One-sentence summaryWe provide the mechanism of action of the potent and selective AMPK inhibitor BAY-3827, which blocks AMPK-dependent cellular functions.

biochemistry↗

bioRxiv2024

PSKH1 kinase activity is differentially modulated via allosteric binding of Ca2+ sensor proteins

Protein Serine Kinase H1 (PSKH1) was recently identified as a crucial factor in kidney development and is overexpressed in prostate, lung and kidney cancers. However, little is known about PSKH1 regulatory mechanisms, leading to its classification as a "dark" kinase. Here, we used biochemistry and mass spectrometry to define PSKH1s consensus substrate motif, protein interactors, and how interactors, including Ca2+ sensor proteins, promote or suppress activity. Intriguingly, despite the absence of a canonical Calmodulin binding motif, Ca2+-Calmodulin activated PSKH1 while, in contrast, the ER-resident Ca2+ sensor of the CREC family, Reticulocalbin-3, suppressed PSKH1 catalytic activity. In addition to antagonistic regulation of the PSKH1 kinase domain by Ca2+ sensing proteins, we identified UNC119B as a protein interactor that activates PSKH1 via direct engagement of the kinase domain. Our findings identify complementary allosteric mechanisms by which regulatory proteins tune PSKH1s catalytic activity, and raise the possibility that different Ca2+ sensors may act more broadly to tune kinase activities by detecting and decoding extremes of intracellular Ca2+ concentrations.

biochemistry↗

bioRxiv2024

Unconventional binding of Calmodulin to CHK2 kinase inhibits catalytic activity

Calmodulin (CaM) serves an essential role in eukaryotic cells as a Ca2+ sensor. Ca2+ binding leads to conformation changes in CaM that enable engagement of a repertoire of enzymes and the regulation of their catalytic activities. Classically, Ca2+-CaM binds to an inhibitory pseudosubstrate sequence C-terminal to the kinase domain in members of the Ca2+-CaM dependent protein kinase (CAMK) family, and relieves inhibition to promote catalytic activity. Here, we report an unexpected mechanism by which CaM can bind CHK2 kinase to inhibit its kinase activity. Using biochemical, biophysical, structural mass spectrometry, and cellular approaches, we identify a direct interaction of Ca2+-CaM with the CHK2 kinase domain that suppresses CHK2 catalytic activity in vitro and is crucial for cell proliferation in human cells following DNA damage. Our findings add direct suppression of kinase activity to the repertoire of CaMs functions, complementing the paradigmatic mechanism of promoting kinase activity through autoinhibitory domain sequestration.

biochemistry↗

bioRxiv2022

SGC-CAMKK2-1: A chemical probe for CAMKK2

The serine/threonine protein kinase calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) plays critical roles in a range of biological processes. Despite its importance, only a handful of inhibitors of CAMKK2 have been disclosed. Having a selective small molecule tool to interrogate this kinase will help demonstrate that CAMKK2 inhibition can be therapeutically beneficial. Herein, we disclose SGC-CAMKK2-1, a selective chemical probe that targets CAMKK2.

cancer biology↗

bioRxiv2022

Systemic ablation of Camkk2 impairs metastatic colonization and improves insulin sensitivity in TRAMP mice: Evidence for cancer cell-extrinsic CAMKK2 functions in prostate cancer

Despite early studies linking calcium-calmodulin protein kinase kinase 2 (CAMKK2) to prostate cancer cell migration and invasion, the role of CAMKK2 in metastasis in vivo remains unclear. Moreover, while CAMKK2 is known to regulate systemic metabolism, whether CAMKK2s effects on whole body metabolism would impact prostate cancer progression and/or related comorbidities is not known. Here, we demonstrate that germline ablation of Camkk2 slows, but does not stop, primary prostate tumorigenesis in the TRansgenic Adenocarcinoma Mouse Prostate (TRAMP) genetic mouse model. Consistent with prior epidemiological reports supporting a link between obesity and prostate cancer aggressiveness, TRAMP mice fed a high-fat diet exhibited a pronounced increase in the colonization of lung metastases. We demonstrated that this effect on metastatic spread was dependent on CAMKK2. Notably, diet-induced lung metastases exhibited a highly aggressive neuroendocrine phenotype. Concurrently, Camkk2 deletion improved insulin sensitivity in the same mice. Histological analyses revealed that cancer cells were smaller in the TRAMP;Camkk2-/- mice compared to TRAMP;Camkk2+/+ controls. Given the differences in circulating insulin levels, a known regulator of cell growth, we hypothesized that systemic CAMKK2 could promote prostate cancer cell growth and disease progression in part through cancer cell-extrinsic mechanisms. Accordingly, host deletion of Camkk2 impaired the growth of syngeneic murine prostate tumors in vivo, confirming nonautonomous roles for CAMKK2 in prostate cancer. Cancer cell size and mTOR signaling was diminished in tumors propagated in Camkk2-null mice. Together, these data indicate that, in addition to cancer cell-intrinsic roles, CAMKK2 promotes prostate cancer progression via tumor-extrinsic mechanisms. Further, we propose that CAMKK2 inhibition may also help combat common metabolic comorbidities in men with advanced prostate cancer.

cancer biology↗

bioRxiv2021

Novel torin1-sensitive phosphorylation sites on the metabolic regulator AMPK revealed by label-free mass spectrometry

AMPK and mTORC1 are nutrient-sensitive protein kinases that form a fundamental negative feedback loop that governs cell growth and proliferation. AMPK is an {beta}{gamma} heterotrimer that is directly phosphorylated by mTORC1 on 2S345 to suppress AMPK activity and promote cell proliferation under nutrient stress conditions. Using mass spectrometry, we generated precise phosphorylation profiles of all 12 AMPK complexes expressed in proliferating human cells. Of the 18 phosphorylation sites detected, seven were sensitive to pharmacological mTORC1 inhibition, including four in the AMPK {gamma}2 isoform NH2-terminal domain and 2S377 which is located in the nucleotide-sensing motif. In particular, {beta}1S182 and {beta}2S184 were found to be mTORC1 substrates in vitro and near-maximally or substantially phosphorylated under cellular growth conditions. {beta}S182 phosphorylation was elevated in 1-containing complexes, relative to 2, an effect partly attributable to the non-conserved -subunit serine/threonine-rich loop. While mutation of {beta}1S182 to a non-phosphorylatable Ala had no effect on basal and ligand-stimulated AMPK activity, {beta}2-S184A mutation increased nuclear AMPK activity and enhanced cell proliferation under nutrient stress. We conclude that mTORC1 governs the nuclear activity of AMPK to regulate transcription factors involved in metabolism and cell survival during nutrient shortage.

biochemistry↗

bioRxiv2021

Compound- and fiber type-selective requirement of AMPKγ3 for insulin-independent glucose uptake in skeletal muscle

Objective: The metabolic master-switch AMP-activated protein kinase (AMPK) mediates insulin-independent glucose uptake in muscle and regulates the metabolic activity of brown and beige adipose tissue (BAT). The regulatory AMPK{gamma}3 isoform is uniquely expressed in skeletal muscle and also potentially in BAT. Here, we investigated the role that AMPK{gamma}3 plays in mediating skeletal muscle glucose uptake and whole-body glucose clearance in response to small-molecule activators that act on AMPK via distinct mechanisms. We also assessed if {gamma}3 plays a role in adipose thermogenesis and browning. Methods: Global AMPK{gamma}3 knockout (KO) mice were generated. A systematic whole-body, tissue and molecular phenotyping linked to glucose homeostasis was performed in {gamma}3 KO and wild type (WT) mice. Glucose uptake in glycolytic and oxidative skeletal muscle ex vivo, as well as blood glucose clearance in response to small molecule AMPK activators that target nucleotide-binding domain of {gamma} subunit (AICAR) and allosteric drug and metabolite (ADaM) site located at the interface of the and {beta} subunit (991, MK-8722) were assessed. Oxygen consumption, thermography, and molecular phenotyping with a {beta}3-adrenergic receptor agonist (CL-316,243) treatment were performed to assess BAT thermogenesis, characteristics and function. Results: Genetic ablation of {gamma}3 did not affect body weight, body composition, physical activity, and parameters associated with glucose homeostasis under chow or high fat diet. {gamma}3 deficiency had no effect on fiber-type composition, mitochondrial content and components, or insulin-stimulated glucose uptake in skeletal muscle. Glycolytic muscles in {gamma}3 KO mice showed a partial loss of AMPK2 activity, which was associated with reduced levels of AMPK2 and {beta}2 subunit isoforms. Notably, {gamma}3 deficiency resulted in a selective loss of AICAR-, but not MK-8722-induced blood glucose-lowering in vivo and glucose uptake specifically in glycolytic muscle ex vivo. We detected {gamma}3 in BAT and found that it preferentially interacts with 2 and {beta}2. We observed no differences in oxygen consumption, thermogenesis, morphology of BAT and inguinal white adipose tissue (iWAT), or markers of BAT activity between WT and {gamma}3 KO mice. Conclusions: These results demonstrate that {gamma}3 plays a key role in mediating AICAR- but not ADaM site binding drug-stimulated blood glucose clearance and glucose uptake specifically in glycolytic skeletal muscle. We also showed that {gamma}3 is dispensable for thermogenesis and browning of iWAT.

physiology↗

bioRxiv2020

In depth analysis of kinase cross screening data to identify CAMKK2 inhibitory scaffolds

The calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) plays a central role in many cell signaling pathways. CAMKK2 activates CAMK1, CAMK4, AMPK, and AKT leading to numerous physiological responses. Deregulation of CAMKK2 is linked to several diseases, suggesting utility of CAMKK2 inhibitors for oncological, metabolic and inflammatory indications. In this work we review the role of CAMKK2 in biology and disease. Through analysis of literature and public databases we have identified starting points for CAMKK2 inhibitor medicinal chemistry campaigns. These starting points provide an opportunity for the development of selective CAMKK2 inhibitors and will lead to tools that delineate the roles of this kinase in disease biology.

cancer biology↗