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Discovery of Small Molecule WWP2 Ubiquitin Ligase Inhibitors

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Discovery of Small Molecule WWP2 Ubiquitin Ligase Inhibitors DOI:10.1002/chem.201804169 Communication & Biochemistry Discovery of Small Molecule WWP2Ubiquitin Ligase Inhibitors Jessica E. Watt,[a] Gregory R. Hughes,[a, c] Samuel Walpole,[b] Serena Monaco,[b] G. Richard Stephenson,[c] Philip C. BulmanPage,[c] AndrewM.Hemmings,[a, c] Jesus Angulo,*[b] and AndrewChantry*[a] There have been anumber of recent attempts to develop Abstract: We have screened small molecule libraries spe- HECT ligase inhibitors, and more specifically targeting the cificallyfor inhibitors that target WWP2, an E3 ubiquitin Nedd4sub-family.Heclin wasidentified from abicyclic peptide ligase associated with tumouroutgrowth and spread.Se- libraryscreen and can selectively inhibit Smurf2, Nedd4 and lected hits demonstrateddose-dependentWWP2inhibi- WWP1byinducingaconformational change that causes oxida- tion, low micromolar IC50 values, and inhibition of PTEN tion of the activesite cysteine.[12] Chlomipramineemergedasa substrate-specific ubiquitination. Binding to WWP2 was candidate ITCH ligase inhibitor based on an enzymatic assay- confirmed by ligand-based NMR spectroscopy.Further- basedhigh throughput screen and was shown to block both more, we used acombinationofSTD NMR, the recently ITCH auto-ubiquitination and substrate-specific p73 ubiquitina- developed DEEP-STD NMR approach, and docking calcula- tion, althoughIC50 values are in the low millimolarrange.[13] tions, to propose for the first time an NMR-validated 3D Using acovalent tethering method, anew class of Nedd4 molecular model of aWWP2-inhibitor complex. These first ligase inhibitors were identified and showntoreact with a generation WWP2 inhibitors provide amolecular frame- non-catalytic cysteinetohinderubiquitin binding to this exo- work for informing organic synthetic approaches to im- site and subsequent enzymatic processivity.[14] Additional at- prove activity and selectivity. tempts to discover Nedd4 inhibitors have utilisedinsilico modelling approaches supported by thermal shift binding ex- periments and revealed that derivatives of indole-3-carbinol There are approximately 600 E3 ligases in the human genome, may also bind to the processivity exosite in the Nedd4 catalytic and they can be sub-divided into RING type and HECT domain HECT domain and inhibit substrate-specific ubiquitination ligases.[1] Within the HECT E3s is asmall group (9 in total) re- linkedtooncogenic signalling events.[15,16] ferred to as the Nedd4superfamily,which includes the WWP1, Here, we focused our efforts on developing small molecule WWP2, Nedd4, ITCH and Smurf2Ubligases.[2] The 3D crystal inhibitors of the WWP2 ubiquitin ligase, an enzymethat can structureshave been resolved for the HECT domains within cause ubiquitin-dependent degradation of specific tumour Nedd4,[3] WWP1,[4] and WWP2.[5,6] Accumulating evidence sug- suppressor proteins that are commonly lost in many types of gests that the Nedd4 ligases, and notably WWP1 and WWP2, cancer,namely Smad transcriptionfactors, PTEN, and Oct4. To are frequently mis-expressed in cancer.[7–9] Theiroverexpression that aim, we have used high-throughput screening to identify in animalmodels causesincreased tumour outgrowth,[10] and hits from the NCI DiversitySet V and NCI Approved Oncology Set they also selectively control key oncogenic signaling events V smallmolecule libraries, we have confirmed binding to linked to both cancerinitiation and spread.[11] WWP2byNMR spectroscopy,and used acombination of STD NMR,DiffErential EPitope mapping (DEEP)-STD NMR and dock- [a] J. E. Watt, G. R. Hughes, Dr.A.M.Hemmings,Dr. A. Chantry ing calculations to generate for the first time an NMR-validated SchoolofBiological Sciences 3D molecular model of aWWP2-inhibitor complex. To discover University of East Anglia, Norwich NR4 7TJ (UK) E-mail:[email protected] first generation WWP2ubiquitin ligase inhibitors we used a96- [b] S. Walpole,S.Monaco, Dr.J.Angulo well plate format high-throughput screen (HTS) using recombi- SchoolofPharmacy nant proteinsand auto-ubiquitination as areadout for WWP2 University of East Anglia, Norwich NR4 7TJ (UK) activity (Figure 1a). E-mail:[email protected] Initially,todevelopand optimise the HTS assay full-length [c] G. R. Hughes, Dr.G.R.Stephenson, Prof. P. C. Bulman Page, WWP2(WWP2-FL) expressed as aGST fusionprotein in bacte- Dr.A.M.Hemmings SchoolofChemistry ria was attached onto glutathione-coated plates and then incu- University of East Anglia, Norwich NR4 7TJ (UK) batedwith recombinantE1, E2 enzymes and Flag-tagged ubiq- Supporting information and the ORCID identification number(s) for the au- uitin (Supporting InformationFigure S1a). The specificity for thor(s) of this article can be found under: WWP2auto-ubiquitinationwas confirmed by detection of Ub- https://doi.org/10.1002/chem.201804169. Flag conjugated WWP2 in the presence of all assay compo- 2018 The Authors. Published by Wiley-VCH Verlag GmbH&Co. KGaA. nentsand confirmed using acatalytically inactive WWP2-FLC838A This is an open access article under the terms of the Creative Commons At- tributionLicense, whichpermits use, distributionand reproduction in any mutant (Supporting Information FigureS1b). Next,weused medium, provided the original work is properly cited. this assay to screen the NCI Diversity Set V and NCI Approved Chem. Eur.J.2018, 24,17677 –17680 17677 2018 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Communication Table 1. Summary of selected hits from WWP2 inhibitorscreen and IC50 values. Data for STD-NMR andCPMG are included,and (?)indicates in- conclusive data due to poorsolubility and/or low signal/noise ratios. Cpd./NSC Structure IC50 (mm)STD-NMRCPMG 1/2805 0.38 33 7/228155 0.84 12/44750 0.85 ?? 19/228150 1.29 ?? Figure 1. High throughput screenfor WWP2 inhibitors. (a) HTS assays for NCI Diversity Set Vwere carried out using 10 mm compound at 0.1%DMSO in single shot with internal controls for 100 %activity (DMSO) and 0% activi- ty (E3 only) on each individual plate. All assays were normalised using 100 % 20/288387 2.30 33 and 0% controls and had athreshold of 70%asindicated by the yellow line. 0.5 <Z’<0.9 for all single shot assays. (b) Auto-ubiquitinationassay on 24 Diversity set Vhits at 10 mm and 0.1%DMSOwith athreshold set at 20% as indicated by the yellow line. hibitors were affecting this ubiquitin priming step earlier within in the auto-ubiquitination cascade. We therefore devel- Oncology Set V small molecule libraries consisting of 1,593 and oped and optimised ubiquitination assays to measureUbcH7 450 compounds, respectively.The initial screens were run as (E2) and Uba1 (E1) activities. For UbcH7, we used asimilar single shot assays at aconcentration of 10 mm and 0.1% plate assay approachasused to measureE3activity but used DMSO,withZ’ values over 0.5 suggestingthat these plate bacterially His-tagged UbcH7 and bound this protein onto assays were robust. The controls foreach library screen nickel-coated 96-well plates. Once the assay was optimised to showednosignificant difference betweenthe buffer and E3 obtain significant 100%and 0% activity controls, compounds only controlsand no significant effect was observed in the were tested at the same initial concentrations as used in the presence of DMSO (Supporting Information Figure S1c). Results GST-WWP2-FL screening, and no significant decrease in activity were normalised using the high and low controlstocalculate was observed for all compounds in the His-E2 ubiquitination the percentage residual activity compared to the DMSO con- assay (Supporting Information Figure S3a). To assesscom- trol. Athreshold of 70 %(> 2SDfrom the DMSO control mean) pound effects on E1 activity,weused adifferent gel-based ap- was used to determine which compounds would be selected proach duetodifficulties in developing robustand reproduci- for further testing. Weidentified 24 initial hits from the NCI Di- ble plate assays. RecombinantHis-Uba1 and ubiquitin undergo versity Set Vscreen, but no hits emerged from the NCI Oncolo- E1-mediated transthiolation when incubated together andthe gy set (Figure 1a and Supporting Information Figure S1d). Po- formation of highermolecular weight mono-Ub-E1 conjugates tential hits emerging from the single shot assays were then was clearly apparent when separated by SDS-PAGE and stained tested in triplicate under the same assayconditions and at with Coomassie Blue. In the presence of the WWP2 lead inhibi- 10 mm compound concentration, and 17 of the initial 24 hits tory compounds we found no discernible impact on E1 ubiqui- demonstrated significant decrease in WWP2 activity (Fig- tin conjugation (Supporting Information Figure S3b). Hitcom- ure 1b). These were then further ranked more stringently pounds were next tested against Nedd4 and WWP1 to find out based on <20 %residual activity thresholds, and furtherde-se- whether they were capable of inhibiting otherHECT E3 ligases lected based on dose-dependent WWP2 inhibition (Supporting in the same family as WWP2. GST-tagged Nedd4and His- Information Figure S2). In total, 10 potentialhit inhibitors with tagged WWP1 were immobilisedonto glutathione coated and varied structural compositions were identified andthe majority nickel coated plates, respectively.Auto-ubiquitination levels of these gave IC50 values in the low micromolar range (Sup- were determined using HRP-conjugated anti-Flagantibody and porting Information Table S1), and five of these inhibitors had TMB substrate. Both Nedd4and WWP1 produced asignificant IC50 values below 3 mm (Table 1). increaseinODwhen in the presence of E1, E2, and
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