Article Published: 24 May 2023 A small-molecule PI3Kα activator for cardioprotection and neuroregeneration Grace Q. Gong  ORCID: orcid.org/0000-0002-7609-04661, Benoit Bilanges  ORCID:

orcid.org/0000-0003-4400-37161, Ben Allsop2, Glenn R. Masson  ORCID: orcid.org/0000-0002-1386-47193,4, Victoria Roberton  ORCID: orcid.org/0000-0002-0404-69845, Trevor Askwith2, Sally

Oxenford2, Ralitsa R. Madsen1, Sarah E. Conduit  ORCID: orcid.org/0000-0002-5075-88511, Dom Bellini3, Martina Fitzek6, Matt Collier6, Osman Najam7, Zhenhe He7, Ben Wahab8, Stephen H.

McLaughlin  ORCID: orcid.org/0000-0001-9135-62533, A. W. Edith Chan9, Isabella Feierberg10, Andrew Madin11, Daniele Morelli1, Amandeep Bhamra12, Vanesa Vinciauskaite  ORCID:

Stephens  ORCID: orcid.org/0000-0002-2771-348713, Phillip T. Hawkins  ORCID: orcid.org/0000-0002-6979-046413, Matilda Katan  ORCID: orcid.org/0000-0001-9992-837515, Derek M. Yellon7 na1,

Sean M. Davidson  ORCID: orcid.org/0000-0001-5182-49807 na1, David M. Smith  ORCID: orcid.org/0000-0001-6831-280X16 na1, James B. Phillips  ORCID: orcid.org/0000-0001-8117-30745 na1, Richard

Angell2,8 na1, Roger L. Williams  ORCID: orcid.org/0000-0001-7754-42073 na1 & …Bart Vanhaesebroeck  ORCID: orcid.org/0000-0002-7074-36731 Show authors Nature volume 618, pages 159–168

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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 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.

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

options: Log in Learn about institutional subscriptions Read our FAQs Contact customer support Fig. 1: Biochemical mechanism of PI3Kα activation by 1938.Fig. 2: Structural mechanism of PI3Kα

activation by 1938.Fig. 3: 1938 activates PI3Kα signalling in cells.Fig. 4: Phosphoproteomics analysis of PI3Kα-WT and PI3Kα-KO MEFs.Fig. 5: 1938 induces biological responses in cultured

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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.

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 PRIDE partner repository77 with the dataset identifier PXD037721.

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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. (1

80348)) 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.

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

AuthorsGrace Q. GongView author publications You can also search for this author inPubMed Google Scholar

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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.

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.

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.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

1 Additional biochemical data on 1938.

a, Determination of Kd 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 Kd = 36 ± 5 µM. b, Determination of Kd 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

Kd = 16 ± 2 µM. Kd 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).

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).

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.

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.

selectivity profile of 1938 (1 µM) on 133 protein kinases and 7 lipid kinases.

visualised as a kinome tree using KinMap.

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.

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.

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.

neuro-regeneration e

xperiments.

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.

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 Tables 2a,b and 3, Supplementary Figs. 1 and 2, and legends for Supplementary Figs. 1 and 2 and for Supplementary Videos 1–4.

Table 1

HDX data and analysis.

1938 Thermo Fisher SSBK-Adapta screen.

1938 Thermo Fisher SSBK-LanthaScreen binding.

1938 Thermo Fisher SSBK-Z′-LYTE screen.

MEF phosphoproteomics analysis of 1938 and insulin signalling.

MEF phosphoproteomics sites represented in PhosphoSite.

Geometry of 1938 checked against crystallographic database (CSD) using MOGUL.

Mechanisms of activation by 1938. See Supplementary Information file for the full legend.

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

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

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

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About this articleCite this article Gong, G.Q., Bilanges, B., Allsop, B. et al. A small-molecule PI3Kα activator for cardioprotection and neuroregeneration. Nature 618, 159–168 (2023).

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Received: 17 September 2021

Accepted: 17 March 2023

Published: 24 May 2023

Issue Date: 01 June 2023

DOI: https://doi.org/10.1038/s41586-023-05972-2

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