An image-based screen for secreted proteins involved in breast cancer G0 cell cycle arrest.

Secreted proteins regulate the balance between cellular proliferation and G0 arrest and therefore play important roles in tumour dormancy. Tumour dormancy presents a significant clinical challenge for breast cancer patients, where non-proliferating, G0-arrested cancer cells remain at metastatic sites, below the level of clinical detection, some of which can re-enter proliferation and drive tumour relapse. Knowing which secreted proteins can regulate entry into and exit from G0 allows us to manipulate their signalling to prevent tumour relapse. To identify novel secreted proteins that can promote breast cancer G0 arrest, we performed a secretome-wide, image-based screen for proteins that increase the fraction of cells in G0 arrest. From a secretome library of 1282 purified proteins, we identified 29 candidates that promote G0 arrest in non-transformed and transformed breast epithelial cells. The assay we have developed can be adapted for use in other perturbation screens in other cell types. All datasets have been made available for re-analysis and our candidate proteins are presented for alternative bioinformatic refinement or further experimental follow up.

Compromised chronic efficacy of a glucokinase activator AZD1656 in mouse models for common human GCKR variants.

Glucokinase activators (GKAs) have been developed as blood glucose lowering drugs for type 2 diabetes. Despite good short-term efficacy, several GKAs showed a decline in efficacy chronically during clinical trials. The underlying mechanisms remain incompletely understood. We tested the hypothesis that deficiency in the liver glucokinase regulatory protein (GKRP) as occurs with common human GCKR variants affects chronic GKA efficacy. We used a Gckr-P446L mouse model for the GCKR exonic rs1260326 (P446L) variant and the Gckr-del/wt mouse to model transcriptional deficiency to test for chronic efficacy of the GKA, AZD1656 in GKRP-deficient states. In the Gckr-P446L mouse, the blood glucose lowering efficacy of AZD1656 (3 mg/kg body wt) after 2 weeks was independent of genotype. However after 19 weeks, efficacy was maintained in wild-type but declined in the LL genotype, in conjunction with raised hepatic glucokinase activity and without raised liver lipids. Sustained blood glucose lowering efficacy in wild-type mice was associated with qualitatively similar but more modest changes in the liver transcriptome compared with the P446L genotype, consistent with GKA therapy representing a more modest glucokinase excess than the P446L genotype. Chronic treatment with AZD1656 in the Gckr-del/wt mouse was associated with raised liver triglyceride and hepatocyte microvesicular steatosis. The results show that in mouse models of liver GKRP deficiency in conjunction with functional liver glucokinase excess as occurs in association with common human GCKR variants, GKRP-deficiency predisposes to declining efficacy of the GKA in lowering blood glucose and to GKA induced elevation in liver lipids.

The Retrosplenial Cortical Role in Delayed Spatial Alternation.

The retrosplenial cortex (RSC) plays an important role in spatial cognition. RSC neurons exhibit a variety of spatial firing patterns and lesion studies have found that the RSC is necessary for spatial working memory tasks. However, little is known about how RSC neurons might encode spatial memory during a delay period. In the present study, we trained control rats and rats with excitotoxic lesions of the RSC on spatial alternation task with varying delay durations and in a separate group of rats, we recorded RSC neuronal activity as the rats performed the alternation task. We found that RSC lesions significantly impaired alternation performance, particularly at the longest delay duration. We also found that RSC neurons exhibited reliably different firing patterns throughout the delay periods preceding left and right trials, consistent with a working memory signal. These differential firing patterns were absent during the delay periods preceding errors. We also found that many RSC neurons exhibit a large spike in firing rate leading up to the start of the trial. Many of these trial start responses also differentiated left and right trials, suggesting that they could play a role in priming the 'go left' or 'go right' behavioral responses. Our results suggest that these firing patterns represent critical memory information that underlies the RSC role in spatial working memory.

ProEnd: A Comprehensive Database for Identifying HbYX Motif-Containing Proteins Across the Tree of Life.

The proteasome plays a crucial role in cellular homeostasis by degrading misfolded, damaged, or unnecessary proteins. Understanding the regulatory mechanisms of proteasome activity is vital, particularly the interaction with activators containing the hydrophobic-tyrosine-any amino acid (HbYX) motif. Here, we present ProEnd, a comprehensive database designed to identify and catalog HbYX motif-containing proteins across the tree of life. Using a simple bioinformatics pipeline, we analyzed approximately 73 million proteins from 22,000 reference proteomes in the UniProt/SwissProt database. Our findings reveal the widespread presence of HbYX motifs in diverse organisms, highlighting their evolutionary conservation and functional significance. Notably, we observed an interesting prevalence of these motifs in viral proteomes, suggesting strategic interactions with the host proteasome. As validation two novel HbYX proteins found in this database were tested and found to directly interact with the proteasome. ProEnd's extensive dataset and user-friendly interface enable researchers to explore the potential proteasomal regulator landscape, generating new hypotheses to advance proteasome biology. This resource is set to facilitate the discovery of novel therapeutic targets, enhancing our approach to treating diseases such as neurodegenerative disorders and cancer. Link: http://proend.org/.

Occupancy of the HbYX hydrophobic pocket is sufficient to induce gate opening in the archaeal 20S proteasomes.

Enhancing proteasome function has been a long-standing but challenging target of interest for the potential treatment of neurodegenerative diseases, emphasizing the importance of understanding proteasome activation mechanisms. Most proteasome activator complexes use the C-terminal HbYX motif to bind and trigger gate-opening in the 20S proteasome. This study defines a critical molecular interaction in the HbYX mechanism that triggers gate opening. Here, we focus on the Hb site interaction and find it plays a surprisingly central and crucial role in driving the allosteric conformational changes that induce gate opening in the archaeal 20S. We examined the cryo-EM structure of two mutant archaeal proteasomes, αV24Y T20S and αV24F T20S. These two mutants were engineered to place a bulky aromatic residue in the HbYX hydrophobic pocket and both mutants are highly active, though their mechanisms of activation are undefined. Collectively, our findings indicate that the interaction between the Hb group of the HbYX motif and its corresponding hydrophobic pocket is sufficient to induce gate opening in a mechanistically similar way to the HbYX motif. The involved activation mechanism appears to involve expansion of this hydrophobic binding site affecting the state of the IT switch to triggering gate-opening. Furthermore, we show that the canonical αK66 residue, understood to be critical for proteasome activator binding, plays a key role in stabilizing the open gate, irrespective of activator binding. This study differentiates between the residues in the HbYX motif that support binding interactions ("YX") versus those that allosterically contribute to gate opening (Hb). The insights reported here will guide future drug development efforts, particularly in designing small molecule proteasome activators, by targeting the identified hydrophobic pocket.

Risk Analysis of Respiratory Syncytial Virus Among Infants in the United States by Birth Month.

BACKGROUND: Respiratory syncytial virus (RSV) is a major cause of morbidity and mortality among US infants. A child's calendar birth month determines their age at first exposure(s) to RSV. We estimated birth month-specific risk of medically attended (MA) RSV lower respiratory tract infection (LRTI) among infants during their first RSV season and first year of life (FYOL). METHODS: We analyzed infants born in the USA between July 2016 and February 2020 using three insurance claims databases (two commercial, one Medicaid). We classified infants' first MA RSV LRTI episode by the highest level of care incurred (outpatient, emergency department, or inpatient), employing specific and sensitive diagnostic coding algorithms to define index RSV diagnoses. In our main analysis, we focused on infants' first RSV season. In our secondary analysis, we compared the risk of MA RSV LRTI during infants' first RSV season to that of their FYOL. RESULTS: Infants born from May through September generally had the highest risk of first-season MA RSV LRTI-approximately 6-10% under the specific RSV index diagnosis definition and 16-26% under the sensitive. Infants born between October and December had the highest risk of RSV-related hospitalization during their first season. The proportion of MA RSV LRTI events classified as inpatient ranged from 9% to 54% (specific) and 5% to 33% (sensitive) across birth month and comorbidity group. Through the FYOL, the overall risk of MA RSV LRTI is comparable across birth months within each claims database (6-11% under the specific definition, 17-30% under the sensitive), with additional cases progressing to care at outpatient or ED settings. CONCLUSIONS: Our data support recent national recommendations for the use of nirsevimab in the USA. For infants born at the tail end of an RSV season who do not receive nirsevimab, a dose administered prior to the onset of their second RSV season could reduce the incidence of outpatient- and ED-related events.

A comparison of hippocampal and retrosplenial cortical spatial and contextual firing patterns.

The hippocampus (HPC) and retrosplenial cortex (RSC) are key components of the brain's memory and navigation systems. Lesions of either region produce profound deficits in spatial cognition and HPC neurons exhibit well-known spatial firing patterns (place fields). Recent studies have also identified an array of navigation-related firing patterns in the RSC. However, there has been little work comparing the response properties and information coding mechanisms of these two brain regions. In the present study, we examined the firing patterns of HPC and RSC neurons in two tasks which are commonly used to study spatial cognition in rodents, open field foraging with an environmental context manipulation and continuous T-maze alternation. We found striking similarities in the kinds of spatial and contextual information encoded by these two brain regions. Neurons in both regions carried information about the rat's current spatial location, trajectories and goal locations, and both regions reliably differentiated the contexts. However, we also found several key differences. For example, information about head direction was a prominent component of RSC representations but was only weakly encoded in the HPC. The two regions also used different coding schemes, even when they encoded the same kind of information. As expected, the HPC employed a sparse coding scheme characterized by compact, high contrast place fields, and information about spatial location was the dominant component of HPC representations. RSC firing patterns were more consistent with a distributed coding scheme. Instead of compact place fields, RSC neurons exhibited broad, but reliable, spatial and directional tuning, and they typically carried information about multiple navigational variables. The observed similarities highlight the closely related functions of the HPC and RSC, whereas the differences in information types and coding schemes suggest that these two regions likely make somewhat different contributions to spatial cognition.

Proteasome hyperactivation rewires the proteome enhancing stress resistance, proteostasis, lipid metabolism and ERAD in C. elegans.

Proteasome dysfunction is implicated in the pathogenesis of neurodegenerative diseases and age-related proteinopathies. Using a C. elegans model, we demonstrate that 20S proteasome hyperactivation, facilitated by 20S gate-opening, accelerates the targeting of intrinsically disordered proteins. This leads to increased protein synthesis, extensive rewiring of the proteome and transcriptome, enhanced oxidative stress defense, accelerated lipid metabolism, and peroxisome proliferation. It also promotes ER-associated degradation (ERAD) of aggregation-prone proteins, such as alpha-1 antitrypsin (ATZ) and various lipoproteins. Notably, our results reveal that 20S proteasome hyperactivation suggests a novel role in ERAD with broad implications for proteostasis-related disorders, simultaneously affecting lipid homeostasis and peroxisome proliferation. Furthermore, the enhanced cellular capacity to mitigate proteostasis challenges, alongside unanticipated acceleration of lipid metabolism is expected to contribute to the longevity phenotype of this mutant. Remarkably, the mechanism of longevity induced by 20S gate opening appears unique, independent of known longevity and stress-resistance pathways. These results support the therapeutic potential of 20S proteasome activation in mitigating proteostasis-related disorders broadly and provide new insights into the complex interplay between proteasome activity, cellular health, and aging.

Time Cells in the Retrosplenial Cortex.

The retrosplenial cortex (RSC) is a key component of the brain's memory systems, with anatomical connections to the hippocampus, anterior thalamus, and entorhinal cortex. This circuit has been implicated in episodic memory and many of these structures have been shown to encode temporal information, which is critical for episodic memory. For example, hippocampal time cells reliably fire during specific segments of time during a delay period. Although RSC lesions are known to disrupt temporal memory, time cells have not been observed there. In the present study, we examined the firing patterns of RSC neurons during the intertrial delay period of two behavioral tasks, a blocked alternation task and a cued T-maze task. For the blocked alternation task, rats were required to approach the east or west arm of a plus maze for reward during different blocks of trials. Because the reward locations were not cued, the rat had to remember the goal location for each trial. In the cued T-maze task, the reward location was explicitly cued with a light and the rats simply had to approach the light for reward, so there was no requirement to hold a memory during the intertrial delay. Time cells were prevalent in the blocked alternation task, and most time cells clearly differentiated the east and west trials. We also found that RSC neurons could exhibit off-response time fields, periods of reliably inhibited firing. Time cells were also observed in the cued T-maze, but they were less prevalent and they did not differentiate left and right trials as well as in the blocked alternation task, suggesting that RSC time cells are sensitive to the memory demands of the task. These results suggest that temporal coding is a prominent feature of RSC firing patterns, consistent with an RSC role in episodic memory.

The Stokes-Einstein-Sutherland Equation at the Nanoscale Revisited.

The Stokes-Einstein-Sutherland (SES) equation is at the foundation of statistical physics, relating a particle's diffusion coefficient and size with the fluid viscosity, temperature, and the boundary condition for the particle-solvent interface. It is assumed that it relies on the separation of scales between the particle and the solvent, hence it is expected to break down for diffusive transport on the molecular scale. This assumption is however challenged by a number of experimental studies showing a remarkably small, if any, violation, while simulations systematically report the opposite. To understand these discrepancies, analytical ultracentrifugation experiments are combined with molecular simulations, both performed at unprecedented accuracies, to study the transport of buckminsterfullerene C60 in toluene at infinite dilution. This system is demonstrated to clearly violate the conditions of slow momentum relaxation. Yet, through a linear response to a constant force, the SES equation can be recovered in the long time limit with no more than 4% uncertainty both in experiments and in simulations. This nonetheless requires partial slip on the particle interface, extracted consistently from all the data. These results, thus, resolve a long-standing discussion on the validity and limits of the SES equation at the molecular scale.

Anisotropic molecular diffusion in confinement II: A model for structurally complex particles applied to transport in thin ionic liquid films.

HYPOTHESIS: Diffusion in confinement is an important fundamental problem with significant implications for applications of supported liquid phases. However, resolving the spatially dependent diffusion coefficient, parallel and perpendicular to interfaces, has been a standing issue and for objects of nanometric size, which structurally fluctuate on a similar time scale as they diffuse, no methodology has been established so far. We hypothesise that the complex, coupled dynamics can be captured and analysed by using a model built on the 2-dimensional Smoluchowski equation and systematic coarse-graining. METHODS AND SIMULATIONS: For large, flexible species, a universal approach is offered that does not make any assumptions about the separation of time scales between translation and other degrees of freedom. The method is validated on Molecular Dynamics simulations of bulk systems of a family of ionic liquids with increasing cation sizes where internal degrees of freedom have little to major effects. FINDINGS: After validation on bulk liquids, where we provide an interpretation of two diffusion constants for each species found experimentally, we clearly demonstrate the anisotropic nature of diffusion coefficients at interfaces. Spatial variations in the diffusivities relate to interface-induced structuring of the ionic liquids. Notably, the length scales in strongly confined ionic liquids vary consistently but differently at the solid-liquid and liquid-vapour interfaces.

Austerity, Health and Public Safety in Low-Income Neighborhoods: Grassroots Responses to the Decline of Local Services in Southeast England.

This article uses ethnographic and qualitative research to explore the health implications and social responses of a low-income neighborhood in Southeast England, to more than a decade of austerity policies and declining institutional and welfare support. Findings examine how cuts to public services and welfare programs alongside changes to the area's social structure shape resident's perceptions of health risks and threats. Residents pointed to poor levels of mental health that were exacerbated by financial insecurity, the closure of community facilities and difficulties accessing support and professional help. An increase in social disorder and sense of danger within the vicinity were attributed to changes in the area's social composition and a reduction of policing in the neighborhood, which were an additional cause of anxiety for residents. Many people felt their neighborhood was treated inequitably with regard to law-and-order, health provision and other services designed to address health problems and risks and dangers in their social environment. This institutional vacuum generates unmet health needs facilitating informal practices and methods for managing health, such as through self-provision or using alternative, and more readily available, sources of medical advice and treatment. The demise of older forms of social control and surveillance that ran parallel with closure of the area's communal spaces had been partly compensated by social media usage, while informal methods of policing were a growing presence in the neighborhood in reaction to rising lawlessness and the ineffectiveness of police and local authorities.

Fragment-Based Screening Identifies New Quinazolinone-Based Inositol Hexakisphosphate Kinase (IP6K) Inhibitors.

A high-throughput fragment-based screen has been employed to discover a series of quinazolinone inositol hexakisphosphate kinase (IP6K) inhibitors. IP6Ks have been studied for their role in glucose homeostasis, metabolic disease, fatty liver disease, chronic kidney disease, blood coagulation, neurological development, and psychiatric disease. IP6Ks phosphorylate inositol hexakisphosphate (IP6) to form pyrophosphate 5-diphospho-1,2,3,4,6-pentakisphosphate (IP7). Molecular docking studies and investigation of structure-activity relationships around the quinazolinone core resulted in compounds with submicromolar potency and interesting selectivity for IP6K1 versus the closely related IP6K2 and IP6K3 isoforms.

The glucose transporter 2 regulates CD8+ T cell function via environment sensing.

T cell activation is associated with a profound and rapid metabolic response to meet increased energy demands for cell division, differentiation and development of effector function. Glucose uptake and engagement of the glycolytic pathway are major checkpoints for this event. Here we show that the low-affinity, concentration-dependent glucose transporter 2 (Glut2) regulates the development of CD8+ T cell effector responses in mice by promoting glucose uptake, glycolysis and glucose storage. Expression of Glut2 is modulated by environmental factors including glucose and oxygen availability and extracellular acidification. Glut2 is highly expressed by circulating, recently primed T cells, allowing efficient glucose uptake and storage. In glucose-deprived inflammatory environments, Glut2 becomes downregulated, thus preventing passive loss of intracellular glucose. Mechanistically, Glut2 expression is regulated by a combination of molecular interactions involving hypoxia-inducible factor-1 alpha, galectin-9 and stomatin. Finally, we show that human T cells also rely on this glucose transporter, thus providing a potential target for therapeutic immunomodulation.

Structure, Function, and Allosteric Regulation of the 20S Proteasome by the 11S/PA28 Family of Proteasome Activators.

The proteasome, a complex multi-catalytic protease machinery, orchestrates the protein degradation essential for maintaining cellular homeostasis, and its dysregulation also underlies many different types of diseases. Its function is regulated by many different mechanisms that encompass various factors such as proteasome activators (PAs), adaptor proteins, and post-translational modifications. This review highlights the unique characteristics of proteasomal regulation through the lens of a distinct family of regulators, the 11S, REGs, or PA26/PA28. This ATP-independent family, spanning from amoebas to mammals, exhibits a common architectural structure; yet, their cellular biology and criteria for protein degradation remain mostly elusive. We delve into their evolution and cellular biology, and contrast their structure and function comprehensively, emphasizing the unanswered questions regarding their regulatory mechanisms and broader roles in proteostasis. A deeper understanding of these processes will illuminate the roles of this regulatory family in biology and disease, thus contributing to the advancement of therapeutic strategies.

Block Chemistry for Accurate Modeling of Epoxy Resins.

Accurate molecular modeling of the physical and chemical behavior of highly cross-linked epoxy resins at the atomistic scale is important for the design of new property-optimized materials. However, a systematic approach to parametrizing and characterizing these systems in molecular dynamics is missing. We therefore present a unified scheme to derive atomic charges for amine-based epoxy resins, in agreement with the AMBER force field, based on defining reactive fragments─blocks─building the network. The approach is applicable to all stages of curing from pure liquid to gelation to fully cured glass. We utilize this approach to study DGEBA/DDS epoxy systems, incorporating dynamic topology changes into atomistic molecular dynamics simulations of the curing reaction with 127,000 atoms. We study size effects in our simulations and predict the gel point utilizing a rigorous percolation theory to recover accurately the experimental data. Furthermore, we observe excellent agreement between the estimated and the experimentally determined glass transition temperatures as a function of curing rate. Finally, we demonstrate the quality of our model by the prediction of the elastic modulus based on uniaxial tensile tests. The presented scheme paves the way for a broadly consistent approach for modeling and characterizing all amine-based epoxy resins.

High resolution structures define divergent and convergent mechanisms of archaeal proteasome activation.

Considering the link between neurodegenerative diseases and impaired proteasome function, and the neuro-protective impact of enhanced proteasome activity in animal models, it's crucial to understand proteasome activation mechanisms. A hydrophobic-tyrosine-any residue (HbYX) motif on the C-termini of proteasome-activating complexes independently triggers gate-opening of the 20S core particle for protein degradation; however, the causal allosteric mechanism remains unclear. Our study employs a structurally irreducible dipeptide HbYX mimetic to investigate the allosteric mechanism of gate-opening in the archaeal proteasome. High-resolution cryo-EM structures pinpoint vital residues and conformational changes in the proteasome α-subunit implicated in HbYX-dependent activation. Using point mutations, we simulated the HbYX-bound state, providing support for our mechanistic model. We discerned four main mechanistic elements triggering gate-opening: 1) back-loop rearrangement adjacent to K66, 2) intra- and inter- α subunit conformational changes, 3) occupancy of the hydrophobic pocket, and 4) a highly conserved isoleucine-threonine pair in the 20S channel stabilizing the open and closed states, termed the "IT switch." Comparison of different complexes unveiled convergent and divergent mechanism of 20S gate-opening among HbYX-dependent and independent activators. This study delivers a detailed molecular model for HbYX-dependent 20S gate-opening, enabling the development of small molecule proteasome activators that hold promise to treat neurodegenerative diseases.

Minimal mechanistic component of HbYX-dependent proteasome activation that reverses impairment by neurodegenerative-associated oligomers.

The implication of reduced proteasomal function in neurodegenerative diseases combined with studies showing the protective effects of increasing proteasome activity in animal models highlight the need to understand the capacity for proteasome activation by small molecules. The C-terminal HbYX motif is present on many proteasome binding proteins and functions to tether activators to the 20S core particle. Previous studies have shown that peptides with a HbYX motif can autonomously activate 20S gate-opening to allow protein degradation. In this study, through an iterative process of peptide synthesis, we design a HbYX-like dipeptide mimetic that represents only the fundamental components of the HbYX motif. The mimetic robustly induces gate-opening in archaeal, yeast, and mammalian proteasomes. We identify multiple proteasome α subunit residues in the archaeal proteasome involved in HbYX-dependent activation. When stimulated by the mimetic, the mammalian 20S can degrade unfolded proteins such as tau. Findings using our peptide mimetic suggest the HbYX-dependent mechanism requires cooperative binding in at least two intersubunit pockets of the α ring. Most significantly, our peptide mimetic reverses proteasome impairment by neurodegenerative disease-associated oligomers. Collectively, these results validate HbYX-like molecules as having robust potential to stimulate proteasome function, which are potentially useful for treating neurodegenerative diseases.

Anisotropic molecular diffusion in confinement I: Transport of small particles in potential and density gradients.

HYPOTHESIS: Diffusion in confinement is an important fundamental problem with significant implications for applications of supported liquid phases. However, resolving the spatially dependent diffusion coefficient, parallel and perpendicular to interfaces, has been a standing issue. In the vicinity of interfaces, density fluctuations as a consequence of layering locally impose statistical drift, which impedes the analysis of spatially dependent diffusion coefficients even further. We hypothesise, that we can derive a model to spatially resolve interface-perpendicular diffusion coefficients based on local lifetime statistics with an extension to explicitly account for the effect of local drift using the Smoluchowski equation, that allows us to resolve anisotropic and spatially dependent diffusivity landscapes at interfaces. METHODS AND SIMULATIONS: An analytic relation between local crossing times in system slices and diffusivity as well as an explicit term for calculating drift-induced systematic errors is presented. The method is validated on Molecular Dynamics simulations of bulk water and applied to simulations of water in slit pores. FINDINGS: After validation on bulk liquids, we clearly demonstrate the anisotropic nature of diffusion coefficients at interfaces. Significant spatial variations in the diffusivities correlate with interface-induced structuring but cannot be solely attributed to the drift induced by local density fluctuations.

A small-molecule PI3Kα activator for cardioprotection and neuroregeneration.

Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development1-5. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia-reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.