ABSTRACT Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development1,2,3,4,5. 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

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. Access through your institution Buy or subscribe This is a preview of subscription content,

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SpringerLink * Instant access to full article PDF Buy now Prices may be subject to local taxes which are calculated during checkout ADDITIONAL ACCESS OPTIONS: * Log in * Learn about

institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS DISCOVERY OF SMALL-MOLECULE ACTIVATORS OF NICOTINAMIDE PHOSPHORIBOSYLTRANSFERASE

(NAMPT) AND THEIR PRECLINICAL NEUROPROTECTIVE ACTIVITY Article 22 April 2022 REPURPOSING CANCER DRUGS IDENTIFIES KENPAULLONE WHICH AMELIORATES PATHOLOGIC PAIN IN PRECLINICAL MODELS VIA

NORMALIZATION OF INHIBITORY NEUROTRANSMISSION Article Open access 27 October 2021 PROTEIN KINASES IN NEURODEGENERATIVE DISEASES: CURRENT UNDERSTANDINGS AND IMPLICATIONS FOR DRUG DISCOVERY

Article Open access 07 May 2025 DATA AVAILABILITY All raw images for the TIRF microscopy experiments are provided at the Open Science Framework (https://osf.io/gzxfm/). MS data (raw and

processed data) have been deposited into the ProteomeXchange Consortium through the PRIDE partner repository77 with the dataset identifier PXD037721. The MS proteomics data have been

deposited into the ProteomeXchange Consortium through the PRIDE partner repository with the dataset identifier PXD027993. Crystallography data have been deposited into the PDB91

(https://www.rcsb.org/) with the following identifiers: 8BFU (apo p110α)[8OW2 (p110α/1938 complex); 7PG5 (apo p110α/p85α); and 7PG6 (BYL719–p110α/p85α). Protein structures used for analysis

are available from the PDB database (4JPS, 4ZOP and 4OVV). Protein sequences (_PIK3CA_, _PIK3CB_ and _PIK3CD_) were obtained from the UniProt database (https://www.uniprot.org/). The other

data that support the findings in this study are available from the corresponding author upon request. Source data are provided with this paper. CODE AVAILABILITY All macros and R analysis

scripts for the TIRF microscopy experiments are provided at the Open Science Framework (https://osf.io/gzxfm/). MS scripts have been deposited into the ProteomeXchange Consortium through the

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(2000). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS We thank D. Petrovic, A. Davis, G. Pairaudeau, S. Cosulich and R. Knoll (AstraZeneca)

for advice and support; C. Boshoff, D. Linch, B. Williams, D. Miller, N. McNally and A. Holmes (UCL/UCLH) for support during the early stages of this work; O. Perisic for help with cloning

and protein expression (MRC-LMB); S. Arjun and P. Golforoush (The Hatter Cardiovascular Institute) for help with the analysis of the cardioprotection experiments; Y. Posor (UCL) and G. 

Hammond (University of Pittsburgh) for help and advice with TIRF microscopy; F. Gorrec (MRC-LMB) for advice on crystallization; N. Vasan (Columbia University) for the gift of the

Flag–_PIK3R1_ plasmid; J. Shi (MRC-LMB) for help with insect cell culture; M. Schimpl (AstraZeneca) for help with Mogul geometry analysis; M. Graupera (Barcelona), E. Aksoy (London), L. 

Foukas (London) and K. Okkenhaug (Cambridge) for extensive feedback on manuscript; J. Kinghorn and the other team members of the UCL Translational Research Office, A. Sullivan and V. 

Dominguez for general support; D. Leisi, R. Colman and S. Garcia Gomez (UCLB) for business support; staff at the Diamond Light Source (DLS-UK) and EMBL-Hamburg (PETRA III/DESY, Germany) for

beamtime (proposals mx23583 and mx28677 – DLS; and mx647- EMBL); the staff of beamlines i03 (DLS), i04 (DLS), i24 (DLS) and P13 (EMBL-Hamburg) for assistance with crystal testing and data

collection; and staff at the Diamond-CCP4 Data Collection and Structure Solution Workshop 2022 for training. This research was funded in part by the Wellcome Trust and UKRI (BBRSRC and MRC).

For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. Grant funding details are as

follows: UK NIHR UCLH Biomedical Research Centre (to B.V. (High Impact Experimental Medicine Initiative BRC80a/HI/TE/5995 and BRC80b/HI/TE/5995), to B.V. and R.A. (BRC504/CV/BV/101320,

BRC732/JK/101400 and RCF309/BVH/101440/2017) and to the UCL Drug Discovery Group (BRC247/HI/DM/101440 and BRC454/HI/JK/104360)); MRC (to R.L.W. (MC_U105184308); to B.V. and R.A. (UCL

Proximity to Discovery Fund (MC_PC_15063, MC_PC_16087 and MC_PC_17202); MRC Confidence in Concept (MC_PC_16063 and MC_PC_18063); to G.R.M. and V.V. (MRC iCase Studentship (MR/R01579/1)); to

G.R.M. and R.L.W. (Blue Sky collaboration MRC Laboratory of Molecular Biology and AstraZeneca (BSF33)); the Rosetrees Trust (to V.R. (Rosetrees Trust Seedcorn 2020 (100049)) and J.B.P. (UCL

Rosetrees Stoneygate Prize 2018; M827)); Cancer Research UK (to B.V. (C23338/A29269 and C23338/A25722)); the BBSRC (to R.A. and B.V. (BBSRC Global Challenges Research Fund Impact

Acceleration Account GCRF-IAA), to G.R.M. (BBSRC Capital Equipment Grant BB/V019635/1), and to L.R.S., P.T.H. and K.E.A. (BBSRC Institute Strategic Programme Grant BB/PO13384/1)); the

British Heart Foundation (to S.M.D., D.M.Y., B.V. and R.A. (PG/18/44/33790)); the Fidelity Foundation (to T.D.B. and M.K. (180348)) and the UCL Enterprise HEIF Knowledge Exchange and

Innovation Fund (to R.A. (KEI2017-05-18)). The UCL Drug Discovery Group received additional support from the Wellcome Trust (Institutional Strategic Support Fund; awarded to UCL

(105604/Z/14/Z and 204841/Z/16/Z), with subaward to the Drug Discovery Group (ISSF2/H17RCO/033 and ISSF3/H17RCO/006)). A.B. is supported by a CRUK Cancer Immunotherapy Network Accelerator

(CITA) Award (C33499/A20265) and a CRUK UCL Centre award (C416/A25145). S.S. and the UCL Cancer Institute Translational Technology Platforms are supported by a CRUK UCL Centre Award

(C416/A25145). Personal fellowships were from EU Marie Skłodowska-Curie (to G.Q.G. (contract number 839032) and S.E.C. (contract number 838559)) and the Wellcome Trust (to R.R.M.

(220464/Z/20/Z)). G.R.M. was supported by the AstraZeneca/LMB Blue Sky Initiative (MC-A024-5PF9G to R.L.W.) and a Henslow Research Fellowship from The Cambridge Philosophical Society and St

Catharine’s College, Cambridge, UK. AUTHOR INFORMATION Author notes * These authors jointly supervised this work: Derek M. Yellon, Sean M. Davidson, David M. Smith, James B. Phillips,

Richard Angell, Roger L. Williams AUTHORS AND AFFILIATIONS * Cell Signalling, Cancer Institute, University College London, London, UK Grace Q. Gong, Benoit Bilanges, Ralitsa R. Madsen, Sarah

E. Conduit, Daniele Morelli, Elena Lopez-Guadamillas, Maria A. Whitehead & Bart Vanhaesebroeck * Drug Discovery Group, Translational Research Office, University College London, London,

UK Ben Allsop, Trevor Askwith, Sally Oxenford, Alice Hooper, Chandni Patel & Richard Angell * Medical Research Council Laboratory of Molecular Biology, Cambridge, UK Glenn R. Masson, Dom

Bellini, Stephen H. McLaughlin & Roger L. Williams * Division of Cellular Medicine, School of Medicine, University of Dundee, Dundee, UK Glenn R. Masson & Vanesa Vinciauskaite * UCL

Centre for Nerve Engineering, UCL School of Pharmacy, University College London, London, UK Victoria Roberton, Matthew Wilcox & James B. Phillips * Hit Discovery, Discovery Sciences,

R&D, AstraZeneca, Alderley Park, Macclesfield, UK Martina Fitzek & Matt Collier * The Hatter Cardiovascular Institute, University College London, London, UK Osman Najam, Zhenhe He, 

Derek M. Yellon & Sean M. Davidson * Medicines Discovery Institute, School of Biosciences, Cardiff University, Cardiff, UK Ben Wahab & Richard Angell * Wolfson Institute for

Biomedical Research, University College London, London, UK A. W. Edith Chan * Molecular AI, Discovery Sciences, R&D, AstraZeneca, Waltham, MA, USA Isabella Feierberg * Hit Discovery,

Discovery Sciences, R&D, AstraZeneca, Cambridge, UK Andrew Madin * Proteomics Research Translational Technology Platform, Cancer Institute, University College London, London, UK Amandeep

Bhamra & Silvia Surinova * Signalling Programme, Babraham Institute, Cambridge, UK Karen E. Anderson, Len R. Stephens & Phillip T. Hawkins * Institute of Structural and Molecular

Biology, Birkbeck College, London, UK Nikos Pinotsis * Institute of Structural and Molecular Biology, Division of Biosciences, University College London, London, UK Tom D. Bunney & 

Matilda Katan * Emerging Innovations, Discovery Sciences, R&D, AstraZeneca, Cambridge, UK David M. Smith Authors * Grace Q. Gong View author publications You can also search for this

author inPubMed Google Scholar * Benoit Bilanges View author publications You can also search for this author inPubMed Google Scholar * Ben Allsop View author publications You can also

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search for this author inPubMed Google Scholar CONTRIBUTIONS B.V. provided the initial study conceptualization, with input from R.A., R.L.W., D.M.S., S.M.D. and D.M.Y. B.V. took the lead in

writing the manuscript, with major input from G.Q.G., B.B., B.A., V.R., T.A., R.R.M., S.E.C., S.M.D., J.B.P. and R.L.W., with other authors contributing to manuscript editing and

finalization. G.Q.G., B.B., B.A., G.R.M., V.R., T.A., S.O., R.R.M., S.E.C., D.B., O.N., Z.H., B.W., S.H.M., A.W.E.C., V.V., K.E.A., N.P., E.L.-G. and J.B.P. designed and performed

experiments and data analyses supporting the study. M.F., M.C., I.F. and A.M. supported the HCS and drug modelling studies. D.M., A.B., S.S., M.W., A.H., C.P. and T.D.B. performed

experiments and analysis. M.A.W. and M.K. provided general support. B.V., R.A., J.B.P. and R.L.W. supervised the study, with input from D.M.S., D.M.Y., S.M.D., L.R.S. and P.T.H. D.M.Y.,

S.M.D., D.M.S., J.B.P., R.A. and R.L.W. are joint senior authors. CORRESPONDING AUTHOR Correspondence to Bart Vanhaesebroeck. ETHICS DECLARATIONS COMPETING INTERESTS B.V. is a consultant for

iOnctura, Venthera, Pharming and Olema Pharmaceuticals, and has received speaker fees from Gilead. M.F., M.C., I.F., A.M. and D.M.S. are or were employees and shareholders in AstraZeneca at

the time of the work done. J.B.P. is co-Founder and Chief Scientific Officer of the UCL spin-out company Glialign. A patent application WO2023041905, with relevance to this work has been

filed by UCL Business Ltd, covering ‘Aminopyridines as activators of PI 3 kinase’ that lists B.A., T.A, S.O., E.A.W.C., H.E., D.M.Y., R.A, R.L.W. and B.V. as inventors. The other authors

declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION _Nature_ thanks John Burke, Arvin (C.) Dar and Takehiko Sasaki for their contribution to the peer review of this work.

Peer reviewer reports are available. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional

affiliations. EXTENDED DATA FIGURES AND TABLES EXTENDED DATA FIG. 1 ADDITIONAL BIOCHEMICAL DATA ON 1938. A, Determination of _K_d for the dissociation of 1938 from p110α/p85α by surface

plasmon resonance (SPR). SPR equilibrium response titration of 1938 binding to immobilized p110α/p85α, yielding a dissociation constant _K_d = 36 ± 5 µM. B, Determination of _K_d for the

dissociation of 1938 from p110α/p85α by differential scanning fluorimetry (DSF). The first derivatives of the fluorescence change of p110α/p85α upon thermal denaturation at the stated 1938

concentrations _(left panel)_ were used to plot the melting temperature (Tm) _(right panel)_. Fits to data gave a _K_d = 16 ± 2 µM. _K_d shown as mean ± SD (n = 3 independent experiments).

Representative experiment is shown. C, Effect of 1938 on the IC50 of BYL719 for PI3Kα. Data shown as mean ± SEM (n = 3 independent experiments). D, Activation of class IA PI3K isoforms by a

concentration range of pY using the ADP-Glo assay. Data shown as mean ± SEM (n = 3 independent experiments). EXTENDED DATA FIG. 2 ADDITIONAL DATA ON HDX-MS AND CRYSTALLOGRAPHY. Structural

changes induced by BYL719 (A_)_, or 1938 in combination with BYL719 (B), assessed by HDX-MS in full-length p110α/p85α, highlighted on the structure of p110α (gray)/niSH2-p85α (green) (PDB:

4ZOP). Selection threshhold for significant peptides: a-b difference ≥2.5%, Da difference ≥0.25, p-value <0.05 (unpaired t-test). C, Peptide uptake from HDX-MS. A selection of peptides

(peptides 848-859, 532-551, 1002-1013 and 1006-1016 are from p110α, peptide 555-570 is from p85α) exhibiting significant differences in solvent exchange rates on the addition of 1938 (red),

BYL719 (green), both (purple) or neither (blue). Data presented here is from one of three biological replicates Five time points were measured in triplicate. Each point is the mean of one

biological repeat. D, Omit map of ligand 1938 (mFo-DFc) calculated at +/− 3 σ using phenix.polder. E, 1938 bound to p110α shown in multiple orientations. F, Two possible orientations shown 

for the 1938 ligand (magenta and yellow sticks) in the p110α crystal structure, both fit the Sigma-weighted density map (blue; 2mFo-DFc) equally well. Yellow dashes show predicted hydrogen

bonds.G, Effect of 1938 on catalytic activity of p110α proteins with mutations in the 1938-binding pocket. Data shown as mean ± SEM (n = 4 independent experiments). EXTENDED DATA FIG. 3

ADDITIONAL DATA ON 1938-DRIVEN SIGNALLING. A, MEFs were stimulated at different time points with 1938 (5 µM) or for 2 min with PDGF (20 ng/ml), followed by lipid extraction and PtdIns(3,4)P2

measurement by mass spectrometry. B, MEFs were stimulated for 2 min with 1938 (30 µM) or PDGF (0.5 or 1 ng/ml), followed by lipid extraction and PtdIns(3,4)P2 measurement by mass

spectrometry. (A,B) n = independent experiments, shown in figure. Error bars represent SD. C, Control TIRF microscopy data from DMSO-treated HeLa cells expressing the PtdIns(3,4,5)P3 or the

PtdIns(3,4)P2 reporter. HeLa cells expressing the EGFP-tagged PIP3 reporter PH-ARNO-I303Ex2 (ARNO) _(black lines)_ or the PtdIns(3,4)P2 reporter mCherry-cPH-TAPP1x3 _(blue lines_) were

stimulated with DMSO as indicated. Overlay plots (mean ± SEM) were generated by scaling to minimum and maximum values of the normalised fluorescence intensity for each time point (Fn(t)).

PtdIns(3,4,5)3 reporter data are representative of 2 experiments and 16 single cells. PtdIns(3,4)P2 reporter data are representative of 4 experiments and 28 single cells. Individual

measurements were acquired every 2 min. D, pAKTS473 induction by 1938 in PI3Kα-KO MEFs transiently transfected with p110α-WT or p110α-mutants. Blot representative of n = 2 experiments. E,

Time course analysis of 1938-induced pAKTS473 in A549 by 1938+BYL719 or a saturating insulin concentration. Blot representative of n = 3 experiments. F, Time course analysis of 1938-induced

pAKTS473 and pS6S240/244 in MCF10A cells in the presence or absence of BYL719. Shown is a representative blot of n = 2 independent experiments. G, Time course analysis of insulin- or

1938-induced PI3K/AKT/mTORC1 signalling in A549, n = 2 experiments. EXTENDED DATA FIG. 4 _IN VITRO_ SELECTIVITY PROFILE OF 1938 (1 ΜM) ON 133 PROTEIN KINASES AND 7 LIPID KINASES. VISUALISED

AS A WATERFALL PLOT. In the waterfall plot, the protein and lipid kinases are labelled in black and red, respectively, with the dashed line delineating 25% of kinase inhibition. EXTENDED

DATA FIG. 5 _IN VITRO_ SELECTIVITY PROFILE OF 1938 (1 ΜM) ON 133 PROTEIN KINASES AND 7 LIPID KINASES. visualised as a kinome tree using KinMap. EXTENDED DATA FIG. 6 EFFECT OF 1938 ON _IN

VITRO_ KINASE ACTIVITY OF THE PI3K-RELATED KINASES ATM AND MTORC1 (MTOR/RAPTOR/LST8 COMPLEX). The kinases were incubated at 30 °C for 30 min (ATM) or 3 h (mTORC1), with or without 200 µM

1938 in the presence of their respective substrates (GST-p53 for ATM and 4E-BP1 for mTORC1), followed by analysis and quantification as described in Methods. The positive control for ATM was

inclusion of the MRN complex (Mre11-Rad50-Nbs1), known to activate ATM, in the kinase reaction. The positive control for mTORC1 was the use of a triple amount of mTORC1 complex in the

kinase reaction. Data show individual experiments (n = 3), error bars represent mean ± SD. EXTENDED DATA FIG. 7 PHOSPHOPROTEOMICS EXPERIMENTAL SET-UP AND CONTROL DATA. A, Experimental design

and workflow of phosphoproteomics experiment. PI3Kα-WT and PI3Kα-KO MEFs were serum-starved overnight, stimulated with DMSO, 1938 (5 µM) or insulin (100 nM) for 15 min or 4 h and processed

for phosphoproteomics analysis. 10,611 phosphosites fom 3,093 proteins were analysed by MSstats, the majority of which were pSer and pThr residues. B, Validation of phosphoproteomics

conditions. PI3Kα-WT and PI3Kα-KO MEFs were serum-starved overnight and stimulated with DMSO, 1938 (5 µM) or insulin (100 nM) for 15 min or 4 h as indicated. Lysates were immunoblotted with

antibodies to pAKTS473, pAKTT308, total AKT, pPRAS40/AKT1S1T247, pS6RPS240/244, S6RP or GAPDH. Samples were from a representative phosphoproteomics experiment. Representative of n = 2

independent experiments. C, Volcano plot of phosphosites differentially regulated by 1938 (5 µM) relative to DMSO in PI3Kα-WT MEFs. Note, these data are reproduced, enlarged and labelled

from Fig. 4b. Red, upregulated phosphosites, Green, downregulated phospho-sites. Boxed phosphosites have been previously reported to be regulated by PI3K signalling (PhosphoSitePlus). D,

Insulin stimulation induces phosphorylation of expected PI3K targets in PI3Kα-WT MEFs. Volcano plot of Log2(fold change) _versus_ -log10(adjusted p-value) for phosphosites differentially

regulated by (right) 15 min or (left) 4 h 100 nM insulin treatment in PI3Kα-WT MEFs relative to DMSO-treated cells. E, High experimental reproducibility of phosphoproteomics experiment.

Quantified phosphopeptides were analysed within the model-based statistical framework MSstats. Data were log2 transformed, quantile normalised, and a linear mixed-effects model was fitted to

the data. The group comparison function was employed to test for differential abundance between conditions. p-values were adjusted to control the FDR using the Benjamini-Hochberg procedure.

Multi-scatter plot of the Log2(intensity) of signals obtained from each replicate against the Log2(intensity) of the same sample from all other replicates. Numbers indicate the Pearson

correlation coefficient for each pair. EXTENDED DATA FIG. 8 ADDITIONAL DATA RELATED TO THE FUNCTIONAL ACTIVITIES OF 1938 IN CULTURED CELLS, TISSUES AND ORGANISMS. A, Time-dependent

dose-response of MEFs to 1938 as measured by CellTiter-Glo®. PI3Kα-WT and PI3Kα-KO MEFs were serum starved for 4 h, followed by stimulation with a dose range of 1938 in serum-free media for

the indicated time points. Cellular metabolic activity was assessed by measurement of cellular ATP content by CellTiter-Glo®. Luminescence normalised to DMSO-only as 100%. Data shown from 2

individual experiments. B, MEFs were serum-starved overnight, followed by 24 h stimulation in serum-free medium with 1938+BYL719, insulin, or culture medium containing 10% FBS, followed by

measurement of cell number (crystal violet staining). Data show 2 independent experiments. C, _Ex vivo_ perfused Langendorff rat heart model. Generation of pAKTS473 in ischaemic hearts

treated with vehicle, 1938 or insulin upon reperfusion. Rat hearts were perfused for 10 min for stabilization, followed by 45 min global ischaemia and then reperfused for 2 h. During the

first 15 min of reperfusion, the buffer contained either vehicle (0.1% DMSO), 1938 (5 µM) or insulin (1 µM). After 2 h, all hearts were freeze-clamped and frozen in liquid nitrogen followed

by tissue extraction in RIPA buffer, SDS-PAGE and immunoblotting with the indicated antibodies. The quantification for this blot is shown in Fig. 5d. Statistics: 1-way ANOVA with Tukey’s

post test. Each lane contains the extract of an individual heart: vehicle (n = 5), 1938 (n = 6) or insulin (n = 2). D, _In vivo_ perfused mouse heart model. Left panel, area at risk in

vehicle- and 1938-treated hearts. Mice were subjected to 40 min coronary artery ligation followed by 2 h reperfusion. 15 min prior to reperfusion, 50 µl of DMSO or 10 mg/kg 1938 in DMSO, was

administered i.v. Following reperfusion, the hearts were then excised, perfused with Evans Blue, sliced and stained with tetrazolium chloride, prior to blinded assessment of infarct size as

a percentage of the total ischaemic “area at risk” (AAR) (this is shown in Fig. 5e). The AAR in each heart is indicated as a % of the total area of the left ventricular (LV) myocardium.

Since there was no significant difference in AAR between the two groups (P = 0.86), this control measurement demonstrates experimental consistency in suture positioning etc. Statistics:

Student’s unpaired 2-sided t-test, data shown as mean±SEM. Right panel, generation of pAKTS473 in ischaemic hearts treated with vehicle or 1938 upon reperfusion. 50 µl of DMSO vehicle or 10 

mg/kg 1938 in DMSO was injected i.v. into anaesthetized and intubated mice. After 15 min, the chest was opened, the heart removed and immediately freeze-clamped in liquid nitrogen followed

by tissue extraction in RIPA buffer, SDS-PAGE and immunoblotting with the indicated antibodies. Each lane contains the extract of an individual heart of mice treated with vehicle (n = 4) or

1938 (n = 4). The quantification for this blot is shown in Fig. 5e, right panel. EXTENDED DATA FIG. 9 ADDITIONAL AND CONTROL STUDIES FOR NEURO-REGENERATION EXPERIMENTS. A, _Top panel_;

Control experiment to test the biological activity of 1938 post-freezing. An aliquot of 100 μM 1938 stock solution in dH2O and vehicle was defrosted and tested for induction of pAKTS473 by

15 min treatment of A549 cells, using insulin (1 μM) or 1938 (10 µM from control stocks in DMSO) as positive controls. _Bottom panel_; pAKTS473 induction in exposed sciatic nerves, injected

with vehicle (autoclaved H2O) or 1938 (from stocks in autoclaved H2O) or bathed in a solution of vehicle or 1938. After 30 min, the nerves were washed and processed for analysis as described

in Materials and Methods. Cell extracts of MCF7 breast cancer cells stimulated for 15 min with 5 µM 1938 or vehicle (DMSO) were loaded on the gels as positive controls. n = 1 experiment. B,

Representative immunohistochemistry images of a transverse section through the distal common peroneal rat nerve, showing ChAT- and neurofilament-positive axons with tissue architecture

typical of normal tissue. Scale bar = 50 µm. C, Representative immunohistochemistry images of rat TA muscle, showing a α-BTX-stained post-synaptic neuromuscular structure with associated

neurofilament-positive neurons. Scale bar = 20 µm. n = 5 animals. EXTENDED DATA FIG. 10 ADDITIONAL DATA FOR METHODOLOGY. _Left panel_, Sanger sequencing of the genomic _PIK3CA_ locus of A549

cell clones subjected to CRISPR/Cas9 gene-targeting. _Lower traces:_ reference genomic _PIK3CA_ sequence (wild-type), with the crispr RNA sequence underlined. _Top traces:_ DNA sequence of

CRISPR/Cas9 gene-targeted or control-edited A549 clones. The _PIK3CA-_KO clone 12 shows a +1 bp insertion (arrow), leading to frameshift and the generation of 2 consecutive premature

stop-codons (asterisk) immediately downstream of the +1 bp insertion. Note that the first stop-codon occurs 80 bp upstream from the 3′ exon-exon junction and will therefore result in

nonsense-mediated decay of the mRNA. The _PIK3CA-_WT clone 9 shows wild-type genomic DNA sequence. _Right panel_, Western blot for p110α using antibody CST#4255. SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION Supplementary Tables 2a,b and 3, Supplementary Figs. 1 and 2, and legends for Supplementary Figs. 1 and 2 and for Supplementary Videos 1–4. REPORTING SUMMARY PEER

REVIEW FILE SUPPLEMENTARY TABLE 1 HDX data and analysis. SUPPLEMENTARY TABLE 4 1938 Thermo Fisher SSBK-Adapta screen. SUPPLEMENTARY TABLE 5 1938 Thermo Fisher SSBK-LanthaScreen binding.

SUPPLEMENTARY TABLE 6 1938 Thermo Fisher SSBK-Z′-LYTE screen. SUPPLEMENTARY TABLE 7 MEF phosphoproteomics analysis of 1938 and insulin signalling. SUPPLEMENTARY TABLE 8 MEF phosphoproteomics

sites represented in PhosphoSite. SUPPLEMENTARY TABLE 9 Geometry of 1938 checked against crystallographic database (CSD) using MOGUL. SUPPLEMENTARY VIDEO 1 Mechanisms of activation by 1938.

See Supplementary Information file for the full legend. SUPPLEMENTARY VIDEO 2 Representative TIRF microscopy time-lapse videos of WT A549 cells treated with vehicle. See Supplementary

Information file for the full legend. SUPPLEMENTARY VIDEO 3 Representative TIRF microscopy time-lapse videos of WT A549 cells treated with 1938. See Supplementary Information file for the

full legend. SUPPLEMENTARY VIDEO 4 Representative TIRF microscopy time-lapse videos of KO A549 cells treated with 1938. See Supplementary Information file for the full legend. SOURCE DATA

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Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Gong, G.Q., Bilanges, B., Allsop, B. _et al._ A small-molecule PI3Kα activator for cardioprotection and neuroregeneration.

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