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Supporting Information for:

Phenotypic Screening Identifies Protein Synthesis Inhibitors as H- Ras-Nanocluster Increasing Tumor Growth Inducers

Arafath K. Najumudeen1, Itziar M. D. Posada1, Benoit Lectez1, Yong Zhou2, Sebastian K. -J. Landor1, 3, Adyary Fallarero4, Pia Vuorela4, John Hancock2, Daniel Abankwa*1

Author affiliations 1 Turku Centre for Biotechnology, Åbo Akademi University, Tykistökatu 6B, 20520 Turku, Finland 2 University of Texas Health Science Center at Houston, Medical School, Houston, Texas 77030, United States 3 Department of Cell and Molecular Biology (CMB), Karolinska Institutet, SE- 171 77 Stockholm, Sweden 4 Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland

* e-mail: [email protected]

Page 1 of 23 Supporting experimental procedures

β -galactosidase enzymatic assay BHK and HEK cells were seeded on 12-well plates and transfected with pCMV-β– Gal plasmid at 150 or 50 ng/mL, respectively, using jetPRIME reagent. A negative control sample was included (untransfected) in order to follow the endogenous β–gal activity of the cell. Cells were subsequently incubated with the compounds for 16 h at the following concentrations: CHX at 0.18 or 10 µM, anisomycin and harringtonine at 10 µM. The cells were next lysed with PBS buffer containing 1% Triton-X-100 and 100 µg/mL PMSF and samples were centrifuged (15,000 rpm, 5 min, 4 °C) to remove cell debris. 10-to-20 µl of cell lysate was then incubated with 100 µl reaction buffer containing ortho-nitrophenyl-β-galactoside (0.6 mg/mL ONPG in 0.1 M sodium phosphate buffer, 1 mM MgCl2 and 45 mM β-mercaptoethanol) and β–galactosidase activity was followed spectrophotometrically at λ= 420 nm on a Synergy™ H1 Multi- Mode Reader (BioTek, VT, USA). For β–gal activity normalization, the absorbance was normalized with protein amount, measured by the bicinchoninic acid assay (Thermo-Fischer Scientific).

Western Blot For the protein synthesis inhibition assay (Supporting Information Figure 2a), BHK cells were treated for 24 h with CHX at 0.18 or 10 µM, and anisomycin and harringtonine at 10 µM. Cell lysates were run on an SDS-PAGE and electroblotted, and membranes were immunolabeled with anti-p53 antibody (Cell Signaling Technology, cat. no. 9282) or β-Actin which was used as loading control, and membranes were developed as described on methods section. For the signaling assay (Supporting Information Figure 3b) BHK cells were treated with anisomycin and harringtonine at 2 µM for 24 h.

FLIM-FRET For the FLIM experiments on Supporting Information Figure 3a, HEK cells were seeded on a 6-well plate with glass coverslips, and transfection was done after 24 h using jetPRIME (Polyplus) transfection reagent with pmGFP-H-rasG12V alone or together with pmCherry-H-rasG12V (pmGFP/pmCherry plasmids at 1:3 ratio, 2 µg total plasmid). 24 h after transfections, cells were treated for 24 h with either control

Page 2 of 23 (DMSO 0.05% (v/v)) or 0.18 µM of CHX or 2 µM of anisomycin or harringtonine and incubated for additional 24 h. Cells were fixed in 4% PFA/PBS for 20 min and then washed in PBS, and coverslips with cells were then mounted with Mowiol 4-88 (Sigma Aldrich, cat. no. 81381) on glass slides.

Cell proliferation assays For MDA-MB-231 cell proliferation assay, cells were seeded in 96-well plates and treated after 24 h with either control (DMSO 0.05% (v/v)) or 10 µM CHX and incubated for 72 h. DMSO concentration in the final samples was kept under 0.05% (v/v). Cell proliferation was measured every 24 h as change in fluorescence intensity using alamarBlue (Invitrogen, #DAL1100). Each treatment condition comprised of at least five replicate wells and was repeated three times. The fluorescence intensities were expressed as change in cell proliferation relative to the control. Data analysis was performed using GraphPad Prism.

Page 3 of 23 a 0.6 positive deviators R2 = 0.87 0.55 Anisomycin CHX 0.5 Harringtonine Emetine 2HCl

(HEK) control Actinomycin D

max 0.50 0.4 Echinomycin Diacetoxyscirpenol

0.3

FRET E FRET 0.45 0.2 control 0.2 0.3 0.4 0.5 0.6 0.5 0.6

FRET Emax (BHK) FRET Emax (BHK)

negative deviators control 0.4 (HEK) max Tubercidin 0.3 Lapachone, b DHTS Mevastatin FRET E FRET A-23187 2-Hydroxy acetophenone 0.2 0.2 0.3 0.4 0.5 FRET E (BHK) max Supporting Information Figure 1. Cross-validation of hits in HEK cells.

(a) Correlation analysis between Emax values from HEK and BHK cells expressing H- ras-NANOPS. Cells were treated for 24 h with 10 µM of hits from the screen. Dots represent average Emax in HEK and BHK cells from three independent experiments. Block lines indicate the line fit with 95% confidence interval band. Dotted lines mark the hit selection thresholds; red - increase in Emax and blue - decrease in Emax from the average of the control (green). Right, zoomed from (a) showing the positive deviators marked in red. Left, zoomed from (a) showing the negative deviators marked in blue.

Page 4 of 23 M M μ a μ M μ M μ

controlCHX 0.18CHX 10Anisomycin Harringtonine 10 10 1 0.12 0 0 0 p53

β-actin

b c

100 ) ns ) 100

****

50 **** 50 -gal activity (% -gal activity (%

0 0 l d M M M M d l µ µ µ µ ro M M M M µ µ µ µ 10 ont sfectecontro 10 c 10 10 n X in X in tra CH CH n CHX 0.18 myc myc u o untransfecte CHX 0.18 o s s ni ni A A Harringtonine 10 Harringtonine 10

Supporting Information Figure 2. PSI treatment inhibits protein translation.

(a) p53 (t1/2= 10 min) expression level was measured under PSI treatment in BHK cells. Cells were treated with the compounds for 24 h and cell lysates were immunoblotted against p53 and β-Actin (n=1). (b, c) β-galactosidase enzymatic activity was measured under PSI treatment. BHK cells (b) and HEK cells (c) were transfected with pCMV-β-gal and subsequently incubated with the following compounds for 16 h: CHX (at 0.18 and 10 µM) anisomycin (10 µM) and harringtonine (10 µM). Cell lysates were incubated with ONPG and β-gal activity was followed spectrophotometrically (mean ± SEM, n≥2). Loss of β-gal activity was related to loss of protein translation. Statistical significance between control and treated sample was determined using one-way ANOVA test (ns, not significant; ****, p<0.0001).

Page 5 of 23 a b control AnisomycinHarringtonine control 246 1 7.3* 8.7* + Anisomycin 2 μM 112 ppErk1/2

+ Harringtonine 2 μM 139 **** Erk1/2 + CHX 0.18 μM 171 1 2.8* 2.5* 10 15 20 25 pAkt apparent FRET efficiency (%) Akt β-actin

Supporting Information Figure 3. PSIs increase H-ras nanoclustering and Erk- Akt signaling. (a) Nanoclustering-FRET analysis in HEK cells co-expressing mGFP- and mCherry- tagged H-rasG12V. Cells were treated for 24 hours with either DMSO control or the indicated protein synthesis inhibitors. The apparent FRET efficiency (mean ± SEM, n=3) was calculated from FLIM data. Statistical significance between control and treated cells were examined using one-way ANOVA tests (****, p<0.0001). The numbers in the bars indicate the number of analyzed cells. (b) Representative western blots for BHK cells treated for 24 h with either DMSO control or 2 µM anisomycin and harringtonine. Numbers indicate the ratio of the phosphorylated Erk and Akt relative to total Erk and Akt, respectively. β-Actin was used as a loading control (mean ± SEM, n=3). Statistical significance was determined using Student’s t-test (*, p<0.05).

Page 6 of 23

a 6 ) control CHX 10 μM 4

2 cell proliferation

(relative to control 0 0 24 48 72 time (h)

Supporting Information Figure 4: CHX inhibits normal proliferation of MDA- MB-231. Proliferation of MDA-MB-231 cells treated for 72 h with either DMSO or CHX at 10 µM. The graph shows mean fold change compared to DMSO control at each time point ± SEM (n=6).

Page 7 of 23 Supporting Information Table 1: List of compounds included in the commercially available Enzo® Screen-Well® Natural Product Library (Enzo Life Sciences Inc., USA) and the in-house diverse natural compounds collection obtained from different commercial suppliers. ID codes of the compounds for each library are indicated with their corresponding molecular weight (MW).

ENZO codes Compound Name MW 1 Acivicin 178.6 2 Actinomycin D 1255.5 3 Anisomycin 265.3 4 Antibiotic A-23187 523.6 5 Aristolochic acid A 341.3 6 Artesunate 384.4 7 Australine HCl 225.5 8 Baicalein 270.2 9 Betulinic acid 456.7 10 Bilobalide 326.3 11 Brefeldin A 280.4 12 Bromocriptine mesylate 750.7 13 C2 Phytoceramide 359.6 14 C6 Ceramide 397.7 15 Caffeic Acid 180.2 16 Camptothecin 348.4 17 Cantharidin 196.2 18 CAPE 284.3 19 305.4 20 Castanospermine 189.2 21 Cerulenin 223.3 22 Cevadine 591.7 23 Chaetomellic acid A 370.4 24 Chelerythrine Cl 383.8 25 Chromomycin A3 1183.3 26 250.3 27 Colchicine 399.4 28 Coumermycin A1 1110.1 29 Curcumin 368.4 30 Cycloheximide 281.1 31 Cyclopamine 411.6 32 Cyclopiazonic acid 336.4 33 Cycloserine, L- 102.1 34 Cyclosporin A 1202.6 35 Cytochalasin B 479.6 36 Cytochalasin D 507.6 37 Cytochalasin E 495.6 38 Daidzein 254.2 39 Daunorubicin HCl 564 40 Decoyinine 279.2 41 Deguelin 394.3 42 Deoxyphorbol 13-acetate, 12- 390.5 43 Deoxyphorbol 13-phenylacetate 20-acetate, 12- 508.6 44 Dihydroergocristine mesylate 707.8 45 Domoic acid 311.3 46 Doxorubicin HCl 580 47 E6 Berbamine 757.8 48 E-64 357.4 49 E-64-C 314.4 50 E-64-D 342.4

Page 8 of 23 51 Ebelactone B 352.5 52 Ellipticine 246.3 53 Embelin 294.4 54 , (±)- 208.7 55 Epigallocatechin gallate 458.4 56 Etoposide 588.6 57 Forskolin 410.5 58 Fumagillin 458.6 59 705.8 60 Galanthamine HBr 368.3 61 Gambogic acid 628.8 62 Genistein 270.2 63 Geranylgeranoic acid 304.5 64 Gingerol 294.4 65 Ginkgolide B 424.4 66 326.4 67 Gossypol 518.6 68 Grayanotoxin III 414.5 69 Himbacine 345.5 70 Huperzine A, (-)- 242.3 71 Hydroxycamptothecin, 10- 364.4 72 Hypericin 504.4 73 Indirubin 262.3 74 Ingenol 3,20-dibenzoate 556.7 75 Isotetrandrine 622.8 76 Jervine 425.6 77 Kainic acid 231.3 78 Kavain (+/-) 230.3 79 Kenpaulone 327.2 80 Lapachone, b - 242.4 81 Lincomycin 406.5 82 Lycorine HCl 323.8 83 Mevastatin 390.5 84 3-B-Indoleacrylic acid 187.2 85 Mimosine, L- 198.2 86 Mithramycin A 1085.2 87 Monensin Na 692.9 88 Mycophenolic acid 320.3 89 Myriocin 401.5 90 Neomycin sulfate 908.9 91 Nigericin Na 747 92 Oligomycin A 791.1 93 Ouabain (-)- 584.7 94 Parthenolide 248.3 95 Perillic acid 166.2 96 Phloretin 274.2 97 Phorbol 12,13-dibutyrate 504.6 98 Phorbol 12,13-dibutyrate, 4-a - 504.6 99 Phorbol 12-myristate 13-acetate 616.8 100 Phorbol 12-myristate 13-acetate, 4-a - 616.8 101 Phytosphingosine 317.5 102 Piceatannol 244.3 103 Prostaglandin A1 336.5 104 Prostaglandin B1 336.5 105 Prostaglandin E1 354.5 106 Prostaglandin E2 352.5

Page 9 of 23 107 Prostaglandin F2a 354.5 108 Kahweol acetate 356.5 109 Quisqualic acid 189.1 110 Radicicol 364.8 111 Rapamycin 914.2 112 Rauwolscine 390.9 113 Resveratrol 228.2 114 Retinoic acid (all trans) 300.4 115 Retinoic acid, 13-cis- 300.4 116 Retinoic acid, 9-cis- 300.4 117 Rifampicin 823 118 Rosmarinic acid 360.3 119 Rotenone 394.4 120 Rottlerin 516.6 121 Ryanodine 493.5 122 Shikonin 288.3 123 Spectinomycin sulfate 430.4 124 Swainsonine 173.2 125 Tanshinone IIA 294.3 126 Taxol 853.9 127 Tetrandrine 622.8 128 Thapsigargin 652.8 129 Tomatidine 415.6 130 Troleandomycin 814 131 Tunicamycin B 830.9 132 Ursolic acid 456.7 133 Valinomycin 1111.4 134 Aconitine 645.7 135 Veratridine 673.8 136 sulfate 909.1 137 Vincristine sulfate 923 138 Vinpocetine 350.5 139 Wedelolactone 314.3 140 Wortmannin 428.4 141 Apigenin 270.2 142 Arecoline HBr 236.1 143 Atropine sulfate 694.8 144 Berbamine 2HCl 681.6 145 Bicuculline, (+)- 367.4 146 Bufalin 236.4 147 Brucine n-oxide 394.5 148 Butein 272.2 149 Catalpol 362.4 150 Chrysine 254.2 151 Desoxypeganine HCl 244.7 152 Veratramine 409.6 153 Emodin 270.2 154 Gramine 174 155 Harmaline HCl 250.7 156 Harmine HCl 248.7 157 Hyoscyamine 289 158 Ivermectin 875.1 159 Luteolin 286.2 160 Melatonin 232.3 161 Morin 338.2 162 Myricetin 318.2 163 Naringenin 272.2 164 Nicotine, (-)- 162.2

Page 10 of 23 165 Nonactin 736.9 166 Penicillamine, L- 149.2 167 Picrotoxinin 292.3 168 Pilocarpine HCl 244.7 169 Quassin 388.4 170 Quercetin 338.3 171 Quinidine HCl 378.9 172 Quinine HCl 396.9 173 Robinetine 302.2 174 Menadione 172.2 175 Strychnine HCl 370.9 176 Tryptanthrin 248.2 177 Yohimbine HCl 390.9 178 Eburnamonine, (-)- 294.4 179 Lysergol 254.3 180 Monocrotaline 325.4 181 Oxytetracycline, a -apo- 442.3 182 Pseudopelletierin HCl 189.6 183 Salsolinol HBr 260.1 184 Sitosterol, b - 414.7 185 324.3 186 Trimethylpsoralen, 4,5',8- 228.2 187 442.5 188 Emetine 2HCl 553.5 189 Kaempferol 286 190 Kanamycin 582.6 191 Celastrol 450.6 192 Taxifolin (+) 304.3 193 Theobromine 180.1 194 Baccatin III 586.6 195 Carminic acid 492.4 196 Cotinine, (-)- 176.2 197 Austricin 262 198 Condelphine 449 199 Delcorine 479 200 Deltaline 523.6 201 Diacetylkorseveriline 515 202 Dubinidine 275 203 Eudesmine 386 204 Feroline 358.4 205 Fillalbin 291 206 Graveoline 279 207 Heliotrine 313.1 208 Hernandezine 652 209 Heteratisine 391 210 Imperialine 429.6 211 Karakoline 377 212 Lapidine 334 213 Lapiferine 394 214 Nitrarine 2HCl 380 215 Norfluorocurarine 292 216 Peganole 188 217 Pinocembrin 256.2 218 Protopine HCl 389.6 219 Remerine HCl 315.6 220 Sevedindione 427 221 Skimmianine 259

Page 11 of 23 222 Songorine 357 223 Trichodesmine 353 224 Tschimganidin 388.5 225 Tschimganine 304 226 Ungerine nitrate 392 227 Genistin 432.4 228 Laudanosine methiodide 499.4 229 Apigenin-7-O-glucoside 432.5 230 Bavachinin A 338.4 231 Decylubiquinone 322.4 232 Convolvamine HCl 341.8 233 Daidzin 416.3 234 Datiscetin 286.2 235 Deacetylcolchicine, N-formyl- 385.4 236 Oridonin 364.4 237 Eriocitrin 596.5 238 Eriodictyol 288.2 239 Eriodictyol-7-O-glucoside 450.4 240 Homobutein 286.2 241 Homoeriodictyol (-) 302.2 242 Homoorientin 448.3 243 Hydroxyflavone, 7- 238.2 244 Isorhamnetine-3-O-glucoside 478.4 245 Isorhoifoline 578.5 246 Isosakuranetin 286.2 247 Isovitexin 432.4 248 Dihydromethysticin 276.3 249 (Beta, Beta-Dimethylacryl) Shikonin 370.4 250 Kaempferol-7-neohesperidoside 594.5 251 Luteolin-3',7-di-o-glucoside 610.5 252 Flavokawain B 284.3 253 Marein 450.4 254 Maritimein 448.3 255 Picropodophyllin 414.4 256 Myricitrin 464.4 257 Naringenin-7-O-glucoside 434.4 258 Narirutin 580.6 259 Picrotin 310.3 260 Plumbagin 188.2 261 Ketopinic acid 182.2 262 Scopolomine N-butylbromide 440.4 263 Rhamnetine 316.2 264 Rhoifolin 578.5 265 Sanguinarine 332.3 266 Saponarin 594.5 267 Manool 290.5 268 Citreoviridin 402.5 269 Sinensetine 372 270 Sulfuretine 270.2 271 Atropine-N-oxide 341.8 272 Tamarixetine 316.2 273 Tetrahydroalstonine 352.4 274 Diacetoxyscirpenol 366.4 275 Vitexin-2"-O-rhamnoside 587.5 276 Laudanosoline HBr 436.3 277 Bromolaudanosine, (±)-6'- 436.3 278 Andrographolide 350.5 279 Ajmaline 326.4

Page 12 of 23 280 Chelidonine, (+)- 353.4 281 Dihydroxyflavone, 6,7- 254.2 282 Fisetin 286.2 283 Harmalol HCl 272.7 284 Harmol HCl 270.7 285 Isorhamnetine-3-O-rutinoside 624.5 286 Isoscopoletine 192.1 287 Methoxyflavone, 5- 252.2 288 Pratol 268.2 289 Syringetine-3-O-glucoside 508.4 290 Conessine 356.6 291 Sarsasapogenin 416.6 292 Strophanthidin acetate 446.5 293 sulfate 428.5 294 Ferutinin 358.5 295 Piperine 285.3 296 Quercitrin 448.4 297 Scopolamine N-oxide HBr, (-)- 400.3 298 Shikimic Acid 174.2 299 Stachydrine HCl 179.6 300 Ochratoxin A 403.8 301 Patulin 154.1 302 318.4 303 Isorhamnetine 316.2 304 Abscisic acid, (±)- 264.3 305 Rifamycin SV-NA 719.8 306 Aloe-emodine 270.2 307 Antimycin A1 548.6 308 Asarinin, (-)- 354.4 309 Aucubin 346.3 310 Deoxyshikonin 272.3 311 Boldine 327.3 312 Caryophyllene oxide 220.4 313 Catechin hydrate, (+)- 290.3 314 Cinchonidine, (-)- 294.4 315 Cinchonine, (+)- 294.4 316 Cotininecarboxylic acid, trans-4- 220.2 317 Demissidine 399.6 318 Dipterocarpol 442.7 319 Dehydrocostus Lactone 230.3 320 Friedelin 426.7 321 Indole-3-butyric acid 203.2 322 Gibberellic acid, (+)- 346.4 323 Gitoxigenin 390.5 324 Harmane HCl 218.7 325 Hydroxytropinone, 6- 155.2 326 Isocorydine HCl 377.9 327 Isoreserpine, (-)- 608.7 328 Leucomisine 246 329 Methylergonovine 455.5 330 Corydaline 369.4 331 Muscarine Cl, (+)- 209.7 332 Nalidixic acid 232.2 333 Narasin 765 334 Noreleagnine 172.2 335 Norharmane 168.2 336 Palmatine Cl 405.8 337 Peruvoside 548.7

Page 13 of 23 338 Physostigmine 275.4 339 6-Acetamido-6-deoxy-castanospermine 230.3 340 Podocarpic acid 274.4 341 Retrorsine 351.4 342 Rhapontin 420.4 343 Sclareolide, (3aR)-(+)- 250.4 344 Streptonigrin 506.5 345 Tetrahydropapaverine 379.9 346 Ingenol 348.4 347 Syrosingopine 666.7 348 Visnagin 230.2 349 Wogonin 284.2 350 Zearalanol, b - 322.4 351 4-Methylumbelliferone 176.2 352 Echinomycin 1101.3 353 Ellagic acid 302.2 354 Epicatechin, (-)- 290.3 355 Puromycin 471.5 356 Glycyrrhetinic acid, 18-b - 470.7 357 Griseofulvin, (+)- 352.8 358 Isoquercitrine 464.3 359 Kinetin 215.2 360 Lasalocid A Na 612.8 361 Vanillylacetone 194.2 362 Sclareol 308.5 363 Trigonelline HCl 173.6 364 Tubercidin 266.3 365 Usnic acid, (+)- 344.3 366 Vitexin 432.3 367 Acacetine 284.2 368 Capreomycin sulfate 750.8 369 Carnitine Cl, (±)- 197.6 370 Cephradine 349.4 371 Vasicine 188.2 372 Homatropine HBr 356.2 373 Hydrastine, D-b - 383.4 374 Khellin 260.3 375 Lobeline HCl 373.9 376 Osthole 244.3 377 Tetrahydrolipstatin 495.8 378 Neohesperidin 610.5 379 Noscapine, (±)- 413.4 380 Oleanolic acid 456.7 381 Papaverine HCl 375.8 382 Phlorizine 472.4 383 Protoveratrine B 809.9 384 Reserpine 608.7 385 Salinomycin 751 386 Xanthotoxin 216.2 387 Scopoletin 192.2 388 Digitoxin 764.9 389 Solanine, a - 868.1 390 Solasodine 413.6 391 Tropine 141.2 392 D-Tubocurarine-chloride 681.7 393 Myristicin 192.2

Page 14 of 23 394 Vincamine 354.5 395 Anabasine HCl 162.2 396 Cephaeline HBr 466.6 397 Dicoumarol 336.3 398 Artemisinin 282.3 399 Asiatic acid 488.7 400 Auraptene 298.4 401 Vulpinic acid 322.3 402 Berberine HCl 371.8 403 Bergenin 346.3 404 Biochanin A 284.3 405 Bulleyaconitine A 643.8 406 Cafestol 316.4 407 Cafestol acetate 358.5 408 Zerumbone 218.3 409 Catharanthine base 336.4 410 Cepharanthine 606.7 411 Cryptotanshinone 296.4 412 Dehydrokawain, 5,6- 228.2 413 Demethylepipodophyllotoxin, 4'- 400.4 414 Mitomycin C 334.3 415 Methysticin 274.3 416 Thymoquinone 164.2 417 Dihydrotanshinone 278.3 418 Azomycin 113.1 419 Diosmetine 300.2 420 Diosmin 608.5 421 Ecdysone 464.6 422 Ecdysone, b- 480.6 423 Euphorbiasteroid 552.7 424 Flavokawain A 314.3 425 Lupinine 169.3 426 Formononetin 268.2 427 Ginkgolide A 408.4 428 Harringtonine 531.6 429 Hesperetine 302.2 430 Hesperidine 610.5 431 Honokiol 266.3 432 Hypocrellin A 546.5 433 Hypocrellin B 528.5 434 Lagochiline 356.5 435 Lappaconitine 665.6 436 Limonin 470.5 437 Madecassic acid 504.7 438 Magnolol 266.3 439 Matrine 248.4 440 Minocycline 493.9 441 Naringin 580.5 442 Indole-3-acetic acid 175.2 443 Oxocafestol, 16- 284.4 444 Oxokahweol, 16- 282.4 445 Panaxadiol 460.7 446 Panaxatriol 476.7 447 GERI-BP002-A 340.5 448 Pimaricin 665.7 449 Podophylotoxin 414.4 450 Rubescensin A 364.4 451 Rutaecarpine 287.3

Page 15 of 23 452 Rutin 664.5 453 Salsolidine 207.3 454 Salsoline 193.2 455 Santonin 246.3 456 Schisandrin A, R(+)- 416.5 457 Schisandrin B, S(-)- 400.5 458 Schisantherin A 536.6 459 Securinine 217.3 460 Sedanolide 194.1 461 Silybine 482.4 462 Silymarin 482.4 463 Sinomenine 329.4 464 Solanesol 631.1 465 Vindoline 456.5 466 Vinorelbine base 778.9 467 Yangonin 258.3 468 Bergapten 216.2 469 Betulin 442.7 470 Corynanthine 354.4 471 Cytisine, (-)- 190.2 472 Spartein sulfate (-)- 422.5 473 Brassinin 236.4 474 Dihydrorobinetin 304.2 475 Flavanomarein 450.2 476 Lavendustin B 365.4 477 Evodiamine 303.3 478 Oxyacanthine sulfate 704.8 479 Galangine 270.2 480 Lavendustin A 381.4 481 Verruculogen 513.6 482 Gelsemine HCl 358.8 483 Hydrocotarnine HBr 302.1 484 Senecionine 335.1 485 Bis demethoxycurcumin 308.3 486 Dihydrolysergol, 9,10- 256.3 487 Amphotericin B 924.1 488 Amygdalin 457.4 489 Anisodamine 386.3 490 Aphidicolin 338.5 491 Arbutin 272.3 492 Sclerotiorin 390.8 493 Bleomycin 1415.6 494 Chartreusin 640.6 495 Chlorogenic acid 354.3 496 Geraldol 300.2 497 Coumestrol 268.2 498 Diindolylmethane 246.3 499 Ferulic acid 194.2 500 Bakuchiol 256.4 501 Hirsutine 368.5 502 Indole-3-carbinol 147.2

Page 16 of 23 In-house diverse natural compounds collection:

NP codes Compound Name MW NP1 Acacetin (5,7-dihydroxy-4′-methoxyflavone) 284.3 NP2 3-(a-Acetonylbenzyl)-4-hydroxycoumarin 308.3 NP3 Angelicin 186.2 NP4 Apigenin (5,7-Dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) 270.2 NP5 Apigenin-7-glucoside 432.38 NP6 L-ascorbic acid 176.1 NP7 Benzoic acid 122.1 NP8 3-Benzoylbenzo(F)coumarin 300.3 NP9 3-(2-Benzoxazolyl)umbelliferone 279.3 NP10 Bergapten, 5-Methoxypsoralen 216.2 NP11 Betulin 442.7 NP12 Brucinum 430.5 NP13 Butylated hydroxyanisole 180.2 NP14 Butylated hydroxytoluene 220.4 NP15 Caffeic acid 180.2 NP16 Camptothecin 348.4 NP17 Capsaicin 305.4 NP18 (+/-)-Catechin 290.3 NP19 Chrysin (5,7-dihydroxyflavone) 254.3 NP20 m-Coumaric acid 164.2 NP21 Coumarin 146.1 NP22 Coumarin 102 255.3 NP23 Coumarin 106 281.4 NP24 Coumarin 153 302.2 NP25 Coumarin 30 347.4 NP26 Coumarin 7 333.4 NP27 Daidzein 254.2 NP28 Daphnetin (7,8-Dihydroxycoumarin) 178.2 NP29 6,8-Dibromocoumarin-3-carboxylic acid 348.0 NP30 7-Diethylamino-3-thenoylcoumarin 327.4 NP31 5,7-dihydroxy-4-methylcoumarin 192.2 NP32 6,7-dihydroxy-4-methylcoumarin 192.2 NP33 7,8-dihydroxy-6-methoxy coumarin (fraxetin) 208.2 NP34 5,8-dihydroxypsoralen 218.2 NP35 6,7-dimethoxycoumarin (scoparone) 206.2 NP36 6,7-dimethoxy-8-hydroxycoumarin (fraxidin) 222.2 NP37 Ellagic acid 302.2 NP38 (-)-Epicatechin 290.3 NP39 (-)-Epigallocatechin 306.28 NP40 (-)-Epigallocatechin gallate 458.37 NP41 Ergocalciferol (vitamin D2) 396.7 NP42 Esculetin (6,7-dihydroxycoumarin) 178.2 NP43 Esculin sesquihydrate 367.3 NP44 Eserine 275.4 NP45 198.2 NP46 Farnesol 222.4 NP47 Galantamine 368.3 NP48 170.1 NP49 Gitoxigenin 390.5 NP50 Gossypin (3,5,7,3',4'-Pentahydroxy-8-O-glucosylflavone) 648.5 NP51 Hamamelitannin 484.4 NP52 Herniarin (7-methoxycoumarin) 176.2 NP53 4-Hydroxycoumarin 162.1 NP54 Isoscopoletin (6-hydroxy-7-methoxycoumarin) 192.2

Page 17 of 23 NP55 (2-Hydroxypropyl)-β-cyclodextrin 1380.0 NP56 Hyperoside (quercetin-3-galactoside) 464.38 NP57 Isopropyl gallate 212.2 NP58 Kaempferol-3-glucoside (astragalin) 448.4 NP59 Lauryl gallate 338.5 NP60 Linarin (Acacetin-7-O-rutinoside) 592.55 NP61 Luteolin (2-(3,4-Dihydroxyphenyl)- 5,7-dihydroxy-4-chromenone) 286.3 NP62 Luteolin-3',7-di-O-glucoside 610.53 NP63 Luteolin-7-O-glucoside (cynaroside) 448.4 NP64 Lysergol 254.3 NP65 Methyl -β-cyclodextrin 1303.9 NP66 6-Methylcoumarin 160.2 NP67 4-Methyl daphnetin (4-methyl-7,8-Dihydroxycoumarin) 192.2 NP68 4-Methyl-umbelliferone 176.2 NP69 6-methoxy-4-methylcoumarin 190.2 NP70 7-methoxy-4-methylcoumarin 190.2 NP71 Morin dihydrat 338.3 NP72 Myricetin 318.3 NP73 N-(4-hydroxyphenyl)retinamide 391.6 NP74 Naringenin 272.3 NP75 Naringenin-7-O-glucoside 434.4 NP76 Naringin 580.6 NP77 Nordihydroguaiaretic acid 302.4 NP78 Octyl gallate 282.3 NP79 Palmitic acid 256.4 NP80 Piperidin hydrochloride 121.6 NP81 Piperine 285.3 NP82 Pirenperone 393.5 NP83 n-Propyl gallate 212.2 NP84 (3,4-) 154.1 NP85 Psoralen 186.2 NP86 Quercetagetin (6-Hydroxyquercetin) 318.2 NP87 Quercetin dihydrat krist. 338.3 NP88 Quercitrin dihydrat ?? 484.4 NP89 Resveratrol (3,5,4'-trihydroxy-trans-stilbene) 228.3 NP90 Retinoic acid p-hydroxyanilide 391.6 NP91 Retinol 286.5 NP92 Rhamnetin (7-Methylquercetin) 316.3 NP93 Rosmarinic acid 360.3 NP94 Rotenone 394.4 NP95 Rutin 664.6 NP96 Scopoletin (6-Methoxy-7-hydroxycoumarin) 192.2 NP97 Sebacic acid (1,8-octanedicarboxylic acid) 202.2 NP98 Silybin (silymarin) 482.5 NP99 Sinigrin monohydrat 415.5 NP100 Tannic acid 1701.2 NP101 (+)-Taxifolin 304.3 NP102 (-)-α-Thujone 152.2 NP103 α+β-Thujone 152.2 NP104 Thujone standard mixture 152.2 NP105 Umbelliferone 162.1 NP106 Usnic acid 344.3 NP107 Vanillic acid ((4-hydroxy-3-methoxybenzoic acid) 168.2 NP108 Vitexin (Apigenin-8-C-glucoside) 432.38 NP109 Xanthotoxin (8-Methoxypsoralen) 216.2 NP110 Ethoxyquin 217.3 NP111 2-Hydroxyacetophenone 136.2 NP112 Alpha-naphthoflavone (7,8 benzoflavone) 272.9

Page 18 of 23 NP113 (+)-Catechin 290.3 NP114 Catechol (1,2 hydroxybenzene) 110.1 NP115 o-Coumaric acid 164.2 NP116 3,4-Dihydroxybenzoic acid 154.1 NP117 2,3-Dihydroxybenzoic acid 154.1 NP118 2,4-Dihydroxybenzoic acid 154.1 NP119 2,5-Dihydroxybenzoic acid 154.1 NP120 2,6-Dihydroxybenzoic acid 154.1 NP121 3,5-Dihydroxybenzoic acid 154.1 NP122 2,5-Dimethyl-phenol 122.2 NP123 2,6-Dimethoxyflavone 282.3 NP124 Ferulic acid 194.2 NP125 Flavone 222.3 NP126 Genistein 270.2 NP127 Hesperidin 610.6 NP128 3-hydroxyacetophenone 136.2 NP129 4-Hydroxyacetophenone 136.2 NP130 Khellin 260.2 NP131 184.2 NP132 2'-Methoxy-alpha-naphthoflavone 302.3 NP133 4-Methyl pyrocatechol 124.1 NP134 Phthalic acid (benzene-1,2-dicarboxylic acid) 166.1 NP135 Pyrogallol (benzene-1,2,3-triol) 126.1 (-)-Quinic acid ((1S,3R,4S,5R)-1,3,4,5-tetrahydroxycyclohexanecarboxylic NP136 acid) 192.2 NP137 Resorcin (1,3-Dihydroxybenzene) 110.1 NP138 D(-)-Salicin 286.3 NP139 (2-Hydroxybenzoic acid) 138.1 NP140 Sinapic acid (3,5-Dimethoxy-4-) 224.2 NP141 (4-hydroxy-3,5-dimethoxybenzoic acid) 198.2 NP142 3,4,5-Trimethoxybenzoic acid 212.2 NP143 Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) 250.3 NP144 Thymol (2-isopropyl-5-methylphenol) 150.2 NP145 Vanillin (4-Hydroxy-3-methoxybenzaldehyde) 152.2 NP146 2-Hydroxyphenylacetic acid 152.2 NP147 3-hydroxyphenylacetic acid 152.2 NP148 4-Hydroxyphenylacetic acid 152.2 NP149 Hydroquinone 110.1 NP150 2,4-Dinitrophenylhydrazine 198.1 NP151 5-Dimethylaminonaphthalene-1-sulfonylchloride 269.8 NP152 Isopimpinellin (5,8-Dimethoxypsoralene) 246.2 NP153 Silybin 482.4 NP154 Umbelliferone 162.1 NP155 NA NP156 NA NP157 NA

Page 19 of 23 Supporting Information Table 2: List of primary hits from the ENZO and NP library screen with H-ras-NANOPS using cytometry-FRET. Table shows the list of compounds (with library origin and library IDs) that satisfied the selection threshold of 15% change in the Emax FRET-value of H-ras-NANOPS compared to the average Emax of the plate. Hits that increase FRET Emax ≥15% are marked in red and those that decrease FRET Emax ≥15% are marked blue. Auto fluorescent compounds are highlighted in grey and were removed from further analysis.

No. Library ID Compound 1 ENZO E2 Actinomycin D 2 ENZO E3 Anisomycin 3 ENZO E15 Caffeic Acid 4 ENZO E17 Cantharidin 5 ENZO E30 Cycloheximide 6 ENZO E46 Doxorubicin Hydrochloride 7 ENZO E72 Hypericin 8 ENZO E95 Perillic acid 9 ENZO E188 Emetine dihydrochloride 10 ENZO E274 Diacetoxyscirpenol 11 ENZO E352 Echinomycin 12 ENZO E376 Osthole 13 ENZO E334 Noreleagnine 14 ENZO E447 GERI-BP002-A 15 ENZO E428 Harringtonine 16 ENZO E4 Antibiotic A-23187 17 ENZO E52 Ellipticine 18 ENZO E67 Gossypol 19 ENZO E76 Jervine 20 ENZO E80 Lapachone, b - 21 ENZO E83 Mevastatin 22 ENZO E174 Menadione 23 ENZO E269 Sinensetine 24 ENZO E364 Tubercidin 25 ENZO E310 Deoxyshikonin 26 ENZO E459 Securinine 27 ENZO E494 Chartreusin 28 ENZO E417 Dihydrotanshinone 29 NP NP9 3-(2-Benzoxazolyl) umbelliferone 30 NP NP25 Coumarin 30 31 NP NP33 7,8-dihydroxy-6-methoxy coumarin 32 NP NP26 Coumarin 7 33 NP NP22 Coumarin 102 34 NP NP30 7-Diethylamino-3-thenoyl coumarin 35 NP NP24 Coumarin 153 36 NP NP125 Flavone 37 NP NP110 Ethoxyquin 38 NP NP111 2-Hydroxyacetophenone Page 20 of 23 Supporting Information Table 3: List of cross-validated hits in BHK and HEK cells. Table shows percentage change of FRET Emax value for hit compounds as compared to the respective non-treated control values for H-ras-NANOPS in BHK and HEK cells. Shown are hits satisfying the selection threshold 15% change in FRET Emax for either BHK or HEK cells compared to their respective non-treated control. Hits that increase FRET Emax ≥15% are marked in red and those that decrease FRET Emax ≥- 15% are marked blue. Activity: Known activity of the compounds is indicated and compounds with similar activity are color-coded. Percentage deviation from No. Compound Name control FRET Emax Activity BHK HEK 1 Anisomycin 15.00 25.58 Protein synthesis inhibitor 2 Cycloheximide 24.44 20.93 Protein synthesis inhibitor 3 Emetine dihydrochloride 26.67 18.60 Protein synthesis inhibitor 4 Harringtonine 17.78 18.60 Protein synthesis inhibitor 5 Actinomycin D 20.00 16.28 DNA synthesis inhibitor 6 Diacetoxyscirpenol 22.22 13.95 Protein synthesis inhibitor 7 Tubercidin 2.22 -20.93 DNA chelating agent 8 Dihydrotanshinone -6.67 -37.21 FTase inhibitor 9 Lapachone b- -24.44 -39.53 Topoisomerase inhibitor 10 Antibiotic A -23187 -28.89 -48.84 Calcium ionophore 11 Mevastatin -51.11 -51.16 HMG-CoA inhibitor 12 Dihydroxyacetophenone -11.11 -53.49 -

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Supporting Information Table 4: List of validated protein synthesis inhibitor activities.

Compound Activity -Blocks translation elongation by binding the E-site of the 60S Cycloheximide 1 ribosomal subunit, inhibiting eEF2 mediated translocation -Inhibits the initial elongation phase of translation, preventing aa-tRNA 2 Harringtonine binding to the 60S acceptor site 3 -Abolishes c-Jun N-terminal kinase/p38 activation - Binds 60S ribosomal subunit and blocks peptide bond formation, 4 5 preventing elongation and causing polysome stabilization Anisomycin 4 -Inhibits partially DNA synthesis at high concentration 6, 7 -Activates c-Jun N-terminal kinase /p38 stress pathways 8, 9, 10 -Inhibits translation by binding the 40S subunit-S14 protein Emetine 2 HCl -Reduces the synthesis of rRNA at low conc. and prevents the synthesis 11 of all RNA species at high concentration

- Interferes with the peptidyl-transferase activity: inhibits the peptide Diacetoxyscirpenol chain initiation after the first peptide bond formation, breaking down 12, 13 (Anguidine) polyribosomes

Supporting Information References

(1) Schneider-Poetsch, T., Ju, J., Eyler, D. E., Dang, Y., Bhat, S., Merrick, W. C., Green, R., Shen, B., and Liu, J. O. (2010) Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin. Nat Chem Biol 6, 209–217. (2) Fresno, M., Jiménez, A., and Vázquez, D. (1977) Inhibition of translation in eukaryotic systems by harringtonine. Eur. J. Biochem. 72, 323–330. (3) Ouyang, D. Y., Wang, Y. Y., and Zheng, Y. T. (2005) Activation of c-Jun N- terminal kinases by ribotoxic stresses. Cell. Mol. Immunol. 2, 419–425. (4) Grollman, A. P. (1967) Inhibitors of protein biosynthesis. II. Mode of action of anisomycin. J. Biol. Chem. 242, 3226–3233. (5) Barbacid, M., and Vázquez, D. (1975) Ribosome changes during translation. J. Mol. Biol. 93, 449–463. (6) Cano, E., Hazzalin, C. A., and Mahadevan, L. C. (1994) Anisomycin-activated protein kinases p45 and p55 but not mitogen-activated protein kinases ERK-1 and -2 are implicated in the induction of c-fos and c-jun. Mol. Cell. Biol. 14, 7352–7362. (7) Hazzalin, C. A., Le Panse, R., Cano, E., and Mahadevan, L. C. (1998) Anisomycin selectively desensitizes signalling components involved in stress kinase activation and fos and jun induction. Mol. Cell. Biol. 18, 1844–1854. (8) Jiménez, A., Carrasco, L., and Vázquez, D. (1977) Enzymic and nonenzymic translocation by yeast polysomes. Site of action of a number of inhibitors. Biochemistry 16, 4727–4730. (9) Gupta, R. S., and Siminovitch, L. (1977) The molecular basis of emetine resistance in Chinese hamster ovary cells: alteration in the 40S ribosomal subunit. Cell 10, 61–66.

Page 22 of 23 (10) Rhoads, D. D., and Roufa, D. J. (1985) Emetine resistance of Chinese hamster cells: structures of wild-type and mutant ribosomal protein S14 mRNAs. Mol. Cell. Biol. 5, 1655–1659. (11) Gilead, Z., and Becker, Y. (1971) Effect of emetine on ribonucleic acid biosynthesis in HeLa cells. Eur. J. Biochem. 23, 143–149. (12) Mizuno, S. (1975) Mechanism of inhbition of protein systhesis initiation by diacetoxyscirpenol and fusarenon X in the reticulocyte lysate system. Biochim. Biophys. Acta 383, 207–214. (13) Liao, L. L., Grollman, A. P., and Horwitz, S. B. (1976) Mechanism of action of the 12,13-epoxytrichothecene, anguidine, an inhibitor of protein synthesis. Biochim. Biophys. Acta 454, 273–284.

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