Peptides; Proto-Oncogene Proteins p21(ras); Galectin 1; HRAS protein, human; Humans; Proto-Oncogene Proteins p21(ras)/chemistry; Galectin 1/chemistry; Galectin 1/genetics; Protein Binding; Signal Transduction; Peptides/chemistry; Medicine (miscellaneous); Biochemistry, Genetics and Molecular Biology (all)
Abstract :
[en] Hyperactive Ras signalling is found in most cancers. Ras proteins are only active in membrane nanoclusters, which are therefore potential drug targets. We previously showed that the nanocluster scaffold galectin-1 (Gal1) enhances H-Ras nanoclustering via direct interaction with the Ras binding domain (RBD) of Raf. Here, we establish that the B-Raf preference of Gal1 emerges from the divergence of the Raf RBDs at their proposed Gal1-binding interface. We then identify the L5UR peptide, which disrupts this interaction by binding with low micromolar affinity to the B- and C-Raf-RBDs. Its 23-mer core fragment is sufficient to interfere with H-Ras nanoclustering, modulate Ras-signalling and moderately reduce cell viability. These latter two phenotypic effects may also emerge from the ability of L5UR to broadly engage with several RBD- and RA-domain containing Ras interactors. The L5UR-peptide core fragment is a starting point for the development of more specific reagents against Ras-nanoclustering and -interactors.
MANOHARAN, Ganesh Babu ✱; University of Luxembourg > Faculty of Science, Technology and Medicine > Department of Life Sciences and Medicine > Team Daniel ABANKWA
PAVIC, Karolina ; University of Luxembourg > Faculty of Science, Technology and Medicine (FSTM) > Department of Life Sciences and Medicine (DLSM)
Yeste-Vázquez, Alejandro; Department of Chemistry and Pharmaceutical Sciences, VU University Amsterdam, Amsterdam, The Netherlands ; Amsterdam Institute of Molecular and Life Sciences (AIMMS), VU University Amsterdam, Amsterdam, The Netherlands
Knuuttila, Matias ; Turku Bioscience Centre, University of Turku and Åbo Akademi University, 20520, Turku, Finland
Arora, Neha; Department of Integrative Biology and Pharmacology, McGovern Medical School, UT Health, Houston, TX, 77030, USA
Zhou, Yong ; Department of Integrative Biology and Pharmacology, McGovern Medical School, UT Health, Houston, TX, 77030, USA
Härmä, Harri; Chemistry of Drug Development, Department of Chemistry, University of Turku, 20500, Turku, Finland
GAIGNEAUX, Anthoula ; University of Luxembourg > Faculty of Science, Technology and Medicine (FSTM) > Department of Life Sciences and Medicine (DLSM)
Grossmann, Tom N ; Department of Chemistry and Pharmaceutical Sciences, VU University Amsterdam, Amsterdam, The Netherlands ; Amsterdam Institute of Molecular and Life Sciences (AIMMS), VU University Amsterdam, Amsterdam, The Netherlands
ABANKWA, Daniel ; University of Luxembourg ; Turku Bioscience Centre, University of Turku and Åbo Akademi University, 20520, Turku, Finland. daniel.abankwa@uni.lu
✱ These authors have contributed equally to this work.
External co-authors :
yes
Language :
English
Title :
Identification of an H-Ras nanocluster disrupting peptide.
C.L. Steffen P. Kaya E. Schaffner-Reckinger D. Abankwa Eliminating oncogenic RAS: back to the future at the drawing board Biochem. Soc. Trans. 2023 51 447 456 1:CAS:528:DC%2BB3sXkvFWqsbs%3D 36688434 9987992
S.R. Punekar V. Velcheti B.G. Neel K.K. Wong The current state of the art and future trends in RAS-targeted cancer therapies Nat. Rev. Clin. Oncol. 2022 19 637 655 1:CAS:528:DC%2BB38XisVGmurjN 36028717 9412785
D.K. Simanshu D.V. Nissley F. McCormick RAS proteins and their regulators in human disease Cell 2017 170 17 33 1:CAS:528:DC%2BC2sXhtFSqsLfP 28666118 5555610
J. Spiegel P.M. Cromm G. Zimmermann T.N. Grossmann H. Waldmann Small-molecule modulation of Ras signaling Nat. Chem. Biol. 2014 10 613 622 1:CAS:528:DC%2BC2cXpslGisbw%3D 24929527
M.J. Smith Defining bone fide effectors of RAS GTPases Bioessays 2023 45 37401638
Kiel C., Matallanas D. & Kolch W. The ins and outs of RAS effector complexes. Biomolecules11, 236 (2021).
D.K. Simanshu D.K. Morrison A structure is worth a thousand words: new insights for RAS and RAF regulation Cancer Discov. 2022 12 899 912 1:CAS:528:DC%2BB38Xht1Git73J 35046094 8983508
H. Lavoie M. Therrien Regulation of RAF protein kinases in ERK signalling Nat. Rev. Mol. Cell Biol. 2015 16 281 298 1:CAS:528:DC%2BC2MXntFCitbk%3D 25907612
J.A. Martinez Fiesco D.E. Durrant D.K. Morrison P. Zhang Structural insights into the BRAF monomer-to-dimer transition mediated by RAS binding Nat. Commun. 2022 13 1:CAS:528:DC%2BB38XhvFGlsrw%3D 35078985 8789793
T. Rajakulendran M. Sahmi M. Lefrancois F. Sicheri M. Therrien A dimerization-dependent mechanism drives RAF catalytic activation Nature 2009 461 542 545 1:CAS:528:DC%2BD1MXhtVOmu7fI 19727074
Abankwa D., Gorfe A. A. Mechanisms of Ras membrane organization and signaling: Ras rocks again. Biomolecules10, 1522 (2020).
T. Tian et al. Plasma membrane nanoswitches generate high-fidelity Ras signal transduction Nat. Cell Biol. 2007 9 905 914 1:CAS:528:DC%2BD2sXosVSltLo%3D 17618274
S. Sarkar-Banerjee et al. Spatiotemporal analysis of K-Ras plasma membrane interactions reveals multiple high order homo-oligomeric complexes J. Am. Chem. Soc. 2017 139 13466 13475 1:CAS:528:DC%2BC2sXhsVejs7jE 28863262 5663506
K.J. Cho et al. Raf inhibitors target ras spatiotemporal dynamics Curr. Biol. 2012 22 945 955 1:CAS:528:DC%2BC38Xms12msLY%3D 22560614
T. Jin et al. RAF inhibitors promote RAS-RAF interaction by allosterically disrupting RAF autoinhibition Nat. Commun. 2017 8 29084939 5662619
M. Holderfield M.M. Deuker F. McCormick M. McMahon Targeting RAF kinases for cancer therapy: BRAF-mutated melanoma and beyond Nat. Rev. Cancer 2014 14 455 467 1:CAS:528:DC%2BC2cXhtVamur3M 24957944 4250230
K. Pavic R. Chippalkatti D. Abankwa Drug targeting opportunities en route to Ras nanoclusters Adv. Cancer Res. 2022 153 63 99 1:CAS:528:DC%2BB3sXjt1amsro%3D 35101236
B. Rotblat et al. H-Ras nanocluster stability regulates the magnitude of MAPK signal output PLoS ONE 2010 5 e11991 20700538 2916832
I.A. Prior C. Muncke R.G. Parton J.F. Hancock Direct visualization of Ras proteins in spatially distinct cell surface microdomains J. Cell Biol. 2003 160 165 170 1:CAS:528:DC%2BD3sXntVamtg%3D%3D 12527752 2172642
G. Elad-Sfadia R. Haklai E. Ballan H.J. Gabius Y. Kloog Galectin-1 augments Ras activation and diverts Ras signals to Raf-1 at the expense of phosphoinositide 3-kinase J. Biol. Chem. 2002 277 37169 37175 1:CAS:528:DC%2BD38XnsVaqtbs%3D 12149263
A.V. Timoshenko Towards molecular mechanisms regulating the expression of galectins in cancer cells under microenvironmental stress conditions Cell Mol. Life Sci. 2015 72 4327 4340 1:CAS:528:DC%2BC2MXht12mt7%2FM 26245305 11113283
G.A. Rabinovich Galectin-1 as a potential cancer target Br. J. Cancer 2005 92 1188 1192 1:CAS:528:DC%2BD2MXivFWltLw%3D 15785741 2361964
Johannes L., Jacob R. & Leffler H. Galectins at a glance. J. Cell Sci. 131, jcs208884 (2018).
L. Belanis S.J. Plowman B. Rotblat J.F. Hancock Y. Kloog Galectin-1 is a novel structural component and a major regulator of h-ras nanoclusters Mol. Biol. Cell 2008 19 1404 1414 1:CAS:528:DC%2BD1cXlslemt7Y%3D 18234837 2291398
T. Mejuch H. van Hattum G. Triola M. Jaiswal H. Waldmann Specificity of lipoprotein chaperones for the characteristic lipidated structural motifs of their cognate lipoproteins Chembiochem 2015 16 2460 2465 1:CAS:528:DC%2BC2MXhslWks7bF 26503308
B. Lakshman et al. Quantitative biophysical analysis defines key components modulating recruitment of the GTPase KRAS to the plasma membrane J. Biol. Chem. 2019 294 2193 2207 1:CAS:528:DC%2BC1MXis1ehsLo%3D 30559287
O. Blazevits et al. Galectin-1 dimers can scaffold Raf-effectors to increase H-ras nanoclustering Sci. Rep. 2016 6 1:CAS:528:DC%2BC28Xmt1egtLw%3D 27087647 4834570
C. Guzman et al. The efficacy of Raf kinase recruitment to the GTPase H-ras depends on H-ras membrane conformer-specific nanoclustering J. Biol. Chem. 2014 289 9519 9533 1:CAS:528:DC%2BC2cXls1ahur0%3D 24569991 3975003
M. Cho R.D. Cummings Galectin-1, a beta-galactoside-binding lectin in Chinese hamster ovary cells. I. Physical and chemical characterization J. Biol. Chem. 1995 270 5198 5206 1:CAS:528:DyaK2MXksVSltL4%3D 7890630
E. Siljamaki D. Abankwa SPRED1 interferes with K-ras but Not H-ras membrane anchorage and signaling Mol. Cell Biol. 2016 36 2612 2625 1:CAS:528:DC%2BC2sXht1Gqsr8%3D 27503857 5038144
J. Stegmayr et al. Extracellular and intracellular small-molecule galectin-3 inhibitors Sci. Rep. 2019 9 30778105 6379368
Chan, Y. C. et al. Dissecting the structure-activity relationship of galectin-ligand interactions. Int. J. Mol. Sci. 19, 392 (2018).
S. Marullo M. Bouvier Resonance energy transfer approaches in molecular pharmacology and beyond Trends Pharm. Sci. 2007 28 362 365 1:CAS:528:DC%2BD2sXotlWltLw%3D 17629577
Manoharan G. B., Laurini C., Bottone S., Ben Fredj N. & Abankwa D. K. K-Ras binds calmodulin-related Centrin1 with potential implications for K-Ras driven cancer cell stemness. Cancers (Basel)15, 3087 (2023).
R. Rock et al. BRAF inhibitors promote intermediate BRAF(V600E) conformations and binary interactions with activated RAS Sci. Adv. 2019 5 31453322 6693913
L. Elantak et al. Structural basis for galectin-1-dependent pre-B cell receptor (pre-BCR) activation J. Biol. Chem. 2012 287 44703 44713 1:CAS:528:DC%2BC3sXjvVem 23124203 3531785
R.P. Dings et al. Antitumor agent calixarene 0118 targets human galectin-1 as an allosteric inhibitor of carbohydrate binding J. Med. Chem. 2012 55 5121 5129 1:CAS:528:DC%2BC38XmvV2ms74%3D 22575017 4242090
L. Astorgues-Xerri et al. OTX008, a selective small-molecule inhibitor of galectin-1, downregulates cancer cell proliferation, invasion and tumour angiogenesis Eur. J. Cancer 2014 50 2463 2477 1:CAS:528:DC%2BC2cXhtFyht7%2FE 25042151
R.J. Brandwijk et al. Cloning an artificial gene encoding angiostatic anginex: from designed peptide to functional recombinant protein Biochem. Biophys. Res. Commun. 2005 333 1261 1268 1:CAS:528:DC%2BD2MXlvVensrk%3D 15979575
V.L. Thijssen et al. Galectin-1 is essential in tumor angiogenesis and is a target for antiangiogenesis therapy Proc. Natl Acad. Sci. USA 2006 103 15975 15980 1:CAS:528:DC%2BD28XhtFyltr7O 17043243 1635112
I.M. Posada et al. ASPP2 is a novel pan-RAS nanocluster scaffold PLoS ONE 2016 11 e0159677 27437940 4954646
I.M.D. Posada et al. Rapalogs can promote cancer cell stemness in vitro in a Galectin-1 and H-ras-dependent manner Oncotarget 2017 8 44550 44566 28562352 5546501
Y. Wang et al. ASPP1 and ASPP2 bind active RAS, potentiate RAS signalling and enhance p53 activity in cancer cells Cell Death Differ. 2013 20 525 534 1:CAS:528:DC%2BC3sXjvVaqsr0%3D 23392125 3595493
Dhanaraman, T. et al. RASSF effectors couple diverse RAS subfamily GTPases to the Hippo pathway. Sci. Signal. 13, eabb4778 (2020).
T. Jauset M.E. Beaulieu Bioactive cell penetrating peptides and proteins in cancer: a bright future ahead Curr. Opin. Pharm. 2019 47 133 140 1:CAS:528:DC%2BC1MXnvV2nt7g%3D
L. Dietrich et al. Cell permeable stapled peptide inhibitor of Wnt signaling that targets beta-catenin protein-protein interactions Cell Chem. Biol. 2017 24 958 968.e955 1:CAS:528:DC%2BC2sXht1CksrjK 28757184
E. Vives P. Brodin B. Lebleu A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus J. Biol. Chem. 1997 272 16010 16017 1:CAS:528:DyaK2sXktFGnsb4%3D 9188504
H. Adihou et al. A protein tertiary structure mimetic modulator of the Hippo signalling pathway Nat. Commun. 2020 11 33110077 7591920
Siddiqui F. A., Vukic V., Salminen T. A., Abankwa D. Elaiophylin is a potent Hsp90/ Cdc37 protein interface inhibitor with K-Ras nanocluster selectivity. Biomolecules11, 836 (2021).
Siddiqui, F. A. et al. Novel small molecule Hsp90/Cdc37 interface inhibitors indirectly target K-Ras-signaling. Cancers (Basel)13, 927 (2021).
R. Shalom-Feuerstein et al. K-ras nanoclustering is subverted by overexpression of the scaffold protein galectin-3 Cancer Res. 2008 68 6608 6616 1:CAS:528:DC%2BD1cXpslKnu7o%3D 18701484 2587079
E.M. Terrell et al. Distinct binding preferences between Ras and Raf family members and the impact on oncogenic Ras signaling Mol. Cell 2019 76 872 884.e875 1:CAS:528:DC%2BC1MXhvFKjsr%2FF 31606273 7001861
P.M. Cromm et al. Protease-resistant and cell-permeable double-stapled peptides targeting the Rab8a GTPase ACS Chem. Biol. 2016 11 2375 2382 1:CAS:528:DC%2BC28XhtVCjtrzJ 27336832
M. Pelay-Gimeno A. Glas O. Koch T.N. Grossmann Structure-based design of inhibitors of protein-protein interactions: mimicking peptide binding epitopes Angew. Chem. Int. Ed. Engl. 2015 54 8896 8927 1:CAS:528:DC%2BC2MXhtVOmu7rM 26119925 4557054
R. Spencer-Smith et al. Inhibition of RAS function through targeting an allosteric regulatory site Nat. Chem. Biol. 2017 13 62 68 1:CAS:528:DC%2BC28XhvVSntLnP 27820802
I.A. Prior F.E. Hood J.L. Hartley The frequency of Ras mutations in cancer Cancer Res. 2020 80 2969 2974 1:CAS:528:DC%2BB3cXhvVegsrvM 32209560 7367715
B. Burtness et al. Pembrolizumab alone or with chemotherapy versus cetuximab with chemotherapy for recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-048): a randomised, open-label, phase 3 study Lancet 2019 394 1915 1928 1:CAS:528:DC%2BC1MXitV2ms7jM 31679945
A.L. Ho et al. Tipifarnib in head and neck squamous cell carcinoma with HRAS mutations J. Clin. Oncol. 2021 39 1856 1864 1:CAS:528:DC%2BB38Xhslals7Y%3D 33750196 8189627
R.J. Brandwijk et al. Anti-angiogenesis and anti-tumor activity of recombinant anginex Biochem. Biophys. Res. Commun. 2006 349 1073 1078 1:CAS:528:DC%2BD28XpvFCqtbo%3D 16970922
S. Okutachi et al. A covalent calmodulin inhibitor as a tool to study cellular mechanisms of K-Ras-driven stemness Front. Cell Dev. Biol. 2021 9 34307350 8296985
G.B. Manoharan S. Okutachi D. Abankwa Potential of phenothiazines to synergistically block calmodulin and reactivate PP2A in cancer cells PLoS ONE 2022 17 e0268635 1:CAS:528:DC%2BB38XhvFGhsLjP 35617282 9135253
M. Wendt et al. Bicyclic beta-sheet mimetics that target the transcriptional coactivator beta-catenin and inhibit Wnt signaling Angew. Chem. Int. Ed. Engl. 2021 60 13937 13944 1:CAS:528:DC%2BB3MXhtVCktLbF 33783110 8252567
F.M. Paulussen et al. Covalent proteomimetic inhibitor of the bacterial FtsQB divisome complex J. Am. Chem. Soc. 2022 144 15303 15313 1:CAS:528:DC%2BB38XitVKkt7vN 35945166 9413201
A. Kuepper et al. Constrained peptides mimic a viral suppressor of RNA silencing Nucleic Acids Res. 2021 49 12622 12633 1:CAS:528:DC%2BB38XhtFaksb4%3D 34871435 8682738
G.B. Manoharan K. Kopra V. Eskonen H. Harma D. Abankwa High-throughput amenable fluorescence-assays to screen for calmodulin-inhibitors Anal. Biochem. 2019 572 25 32 1:CAS:528:DC%2BC1MXks1Snsb0%3D 30825429
H. Sinijarv et al. Binding assay for characterization of protein kinase inhibitors possessing sub-picomolar to sub-millimolar affinity Anal. Biochem. 2017 531 67 77 1:CAS:528:DC%2BC2sXptFCkur8%3D 28527909
H. Harma et al. A new simple cell-based homogeneous time-resolved fluorescence QRET technique for receptor-ligand interaction screening J. Biomol. Screen 2009 14 936 943 1:CAS:528:DC%2BD1MXhtlSjtLnN 19684287
K. Kopra H. Harma Quenching resonance energy transfer (QRET): a single-label technique for inhibitor screening and interaction studies N. Biotechnol. 2015 32 575 580 1:CAS:528:DC%2BC2MXjs1Gkurw%3D 25721971
K. Kopra et al. A homogeneous quenching resonance energy transfer assay for the kinetic analysis of the GTPase nucleotide exchange reaction Anal. Bioanal. Chem. 2014 406 4147 4156 1:CAS:528:DC%2BC2cXmslKjt78%3D 24760397
C. Guzman C. Oetken-Lindholm D. Abankwa Automated high-throughput fluorescence lifetime imaging microscopy to detect protein-protein interactions J. Lab Autom. 2016 21 238 245 1:CAS:528:DC%2BC2sXlvVyisb0%3D 26384400
G. Babu Manoharan C. Guzman A.K. Najumudeen D. Abankwa Detection of Ras nanoclustering-dependent homo-FRET using fluorescence anisotropy measurements Eur. J. Cell Biol. 2023 102 1:CAS:528:DC%2BB3sXnslChu7g%3D 37058825
Potdar, S. et al. Breeze 2.0: an interactive web-tool for visual analysis and comparison of drug response data. Nucleic Acids Res. 51(W1) W57–W61 (2023).
