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bioRxiv preprint doi: https://doi.org/10.1101/2020.10.12.336677; this version posted October 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Brain-Restricted mTOR Inhibition with Binary Pharmacology 2 3 Ziyang Zhang1, Qiwen Fan2,3, Xujun Luo2,3, Kevin J. Lou1, William A. Weiss2,3,4,5, Kevan 4 M. Shokat1,* 5 6 1 Department of Cellular and Molecular Pharmacology and Howard Hughes Medical 7 Institute, University of California, San Francisco, California 8 2 Helen Diller Family Comprehensive Center, San Francisco, California 9 3 Department of Neurology, University of California, San Francisco, California 10 4 Department of Pediatrics, University of California, San Francisco, California 11 5 Department of Neurological Surgery, University of California, San Francisco, California 12 * Corresponding author. [email protected] (K.M.S.).

1 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.12.336677; this version posted October 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

13 Abstract 14 On-target-off-tissue drug engagement is an important source of adverse effects 15 that constrains the therapeutic window of drug candidates. In diseases of the central 16 , drugs with brain-restricted pharmacology are highly desirable. Here we 17 report a strategy to achieve inhibition of mTOR while sparing mTOR activity elsewhere 18 through the use of a brain-permeable mTOR inhibitor RapaLink-1 and brain-impermeable 19 FKBP12 ligand RapaBlock. We show that this drug combination mitigates the systemic 20 effects of mTOR inhibitors but retains the efficacy of RapaLink-1 in 21 xenografts. We further present a general method to design cell-permeable, FKBP12- 22 dependent inhibitors from known drug scaffolds. These inhibitors are sensitive to 23 deactivation by RapaBlock enabling the brain-restricted inhibition of their respective 24 kinase targets.

2 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.12.336677; this version posted October 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

25 Administration of a drug often leads to systemic pharmacological 26 effects that contribute to both efficacy and and define its therapeutic index. While 27 off-target effects may be mitigated by chemical modifications that improve the specificity 28 of the drug, on-target-off-tissue toxicity represents a unique challenge that requires 29 precise control over tissue partitioning. On-target-off-tissue affect commonly 30 used drugs such as (myopathy caused by HMG CoA reductase inhibition in 1 31 ) and first-generation antihistamines (drowsiness caused by H1 receptor 32 blockade in the brain)2 and can sometimes preclude the safe usage of otherwise effective 33 therapeutic agents. Chemical inhibitors of the mechanistic target of rapamycin (mTOR) 34 provide a case in point. Although mTOR inhibitors have shown efficacy in treating a 35 number of central nervous system (CNS) diseases including complex 36 (TSC)3,4, glioblastoma (GBM)5,6, and alcohol use disorder (AUD)7–9, systemic mTOR 37 inhibition is associated with a variety of dose-limiting adverse effects: immune 38 suppression, metabolic disorders, and growth inhibition in children10,11. If the 39 pharmacological effects of these mTOR inhibitors could be confined to the CNS, their 40 therapeutic window could be substantially widened. Here we present a chemical strategy 41 that allows brain-specific mTOR inhibition through the combination of two 42 pharmacological agents – a brain-permeable mTOR inhibitor (RapaLink-1) whose 43 function requires the intracellular FK506-binding protein 12 (FKBP12) and a brain- 44 impermeant ligand of FKBP12 (RapaBlock). When used in a glioblastoma xenograft 45 model, this drug combination drove tumor regression without detectable systemic toxicity. 46 We further demonstrate that this strategy can be adapted to achieve brain-specific 47 inhibition of other kinase targets by developing a method to convert known kinase 48 inhibitors into FKBP12-dependent formats. 49 Therapeutic targeting of mTOR kinase can be achieved both allosterically and 50 orthosterically. First-generation mTOR inhibitors rapamycin and its analogs (rapalogs) 51 bind to the FK506 rapamycin binding (FRB) domain of mTOR as a complex with the 52 intracellular protein FKBP12, resulting in substrate-dependent allosteric inhibition of 53 mTOR complex 112–14. Second-generation mTOR kinase inhibitors (TORKi) directly bind 54 in the ATP pocket of mTOR and inhibit the activity of both mTOR complex 1 and complex 55 215–17. The third generation mTOR inhibitor RapaLink-1 is a bitopic ligand that

3 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.12.336677; this version posted October 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

56 simultaneously engages the ATP pocket and an allosteric pocket (the FRB domain) of 57 mTOR, achieving potent and durable inhibition of its kinase activity18. Despite its large 58 molecular weight (1784 Da), RapaLink-1 is cell- and brain-permeant and has shown 59 enhanced in vivo efficacy in driving glioblastoma regression compared to earlier mTOR 60 inhibitors6. 61 Because RapaLink-1 contains a TOR kinase inhibitor (TORKi) moiety (Fig. 1a, grey 62 shades) that may directly bind in the ATP pocket, we wondered whether the interaction 63 of RapaLink-1 with FKBP12 is essential for its inhibition of mTOR. We performed 64 kinase assay with purified mTOR protein and found that RapaLink-1 exhibited identical 65 IC50 values as MLN0128 (the TORKi portion of RapaLink-1) and that of 10 µM 66 FKBP12 had no effect on its activity (Fig. 1b). Surprisingly, when we tested RapaLink-1 67 in cells, we observed a strong dependency on FKBP12 for mTOR inhibition. In 68 (K562/dCas9-KRAB cells), CRISPRi-mediated knockdown of FKBP12 with two distinct 69 single guide RNAs (sgRNAs) drastically impeded its cellular activity (Fig. 1c). This effect 70 was even more pronounced when we used FK506 (a high affinity natural ligand of 71 FKBP12) to pharmacologically block the ligand of FKBP12. Similar FKBP12- 72 dependence of RapaLink-1 was observed when we monitored mTOR signaling by 73 Western Blot (Fig. 1d, see also ref18) – whereas 10 nM RapaLink-1 reduced P-S6 74 (S240/244) and P-4EBP(T37/46) to almost undetectable levels, combination of 10 nM 75 RapaLink-1 and 10 µM FK506 showed no effect on either marker. 76 These results highlight that while FKBP12 is not essential for RapaLink-1 to bind 77 and inhibit the of mTOR in vitro, it is required for cellular activity perhaps 78 serving as an intracellular sink for RapaLink-1 to accumulate in the cell. We probed this 79 possibility using a structural analog of RapaLink-1, where the TORKi moiety had been 80 replaced with tetramethylrhodamine (RapaTAMRA, Extended Data Fig. 1). This 81 fluorescent analog of RapaLink-1 allowed us to quantify intracellular compound 82 concentration by flow cytometry. Consistent with previous findings with RapaLink-118 and 83 other FKBP-binding compounds19,20, RapaTAMRA showed high cellular retention (10-fold 84 increase in median fluorescence intensity) even after extensive washout, but this effect 85 was diminished by FKBP12 knockdown (Extended Data Fig. 1c). While other factors may

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86 contribute, we believe that FKBP12-mediated cellular partitioning is partly responsible for 87 the exceptional potency of RapaLink-1. 88 We next considered whether the dependence of RapaLink-1 on FKBP12 could be 89 harnessed to achieve CNS-restricted mTOR inhibition. Specifically, selective blockade 90 of FKBP12 in peripheral tissues with a potent, brain-impermeant small molecule ligand 91 would allow RapaLink to accumulate in the brain but not peripheral tissues, resulting in 92 brain-specific inhibition (Fig. 2a). To identify a candidate FKBP12 ligand with the suitable 93 permeability profile (i.e. cell-permeable but BBB-impermeable), we considered known 94 natural and synthetic high-affinity FKBP12 ligands (e.g., FK50621,22, rapamycin23,24, 95 SLF/Shield-125,26), but all these compounds readily cross the -brain barrier (BBB). 96 Therefore, we synthesized a panel of derivatives of SLF and FK506 where polar 97 substituents (particularly donors and acceptors) had been attached to the 98 solvent exposed parts of these two molecules, a design principle directly against the 99 empirical rules for designing BBB-permeable drugs27–30 (Extended Data Fig. 2). In 100 addition, modification of the C21-allyl of FK506 has the added advantage that it abolishes 101 the binding to calcineurin31, the natural target of FK506, whose inhibition causes 102 suppression of Nuclear factor of activated T-cells (NFAT) signaling and hence 103 (Fig. 2b). The majority of Shield-1 and FK506 derivatives we 104 synthesized maintained potent FKBP12 binding (Extended Data Fig . 2), as measured by 105 a competition fluorescence polarization assay32. We then screened these compounds 106 (10 µM) in a cell-based assay, evaluating whether they can protect mTOR from inhibition 107 by RapaLink-1 (10 nM) by monitoring phospho-S6 level by Western Blot. While none of 108 the SLF derivatives were found to be effective, most FK506 analogs attenuated the 109 potency of RapaLink-1 (rescuing phospho-S6 levels), with some of them completely 110 blocking RapaLink-1 from inhibiting mTOR (Extended Data Fig. 2). Four of the most 111 effective RapaLink-1 blocking compounds were subjected to an additional in vivo screen, 112 where mTOR signaling was separately analyzed in the brain and skeletal muscle tissues 113 of mice treated with a combination of RapaLink-1 and the candidate compound (1 mg/kg 114 and 40 mg/kg, respectively). A pyridine N-oxide derivative of FK506 protected peripheral 115 tissues from RapaLink-1 but allowed potent inhibition of mTOR in the brain (Extended

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116 Data Fig. 3). We therefore chose this compound for our further study and refer to it as 117 “RapaBlock”.

118 RapaBlock and FK506 bind to FKBP12 with comparable affinity (Fig. 2d, Ki’s: 3.1 119 nM and 1.7 nM, respectively), but unlike FK506, RapaBlock does not exhibit any inhibitory 120 activity for (Fig. 2e). In cultured cells, RapaBlock does not affect mTOR 121 signaling by itself (up to 10 µM) but attenuates the pharmacological effects of RapaLink- 122 1 in a dose-dependent fashion (Fig. 3a). Interestingly, RapaBlock appears more effective 123 at blocking rapamycin, restoring phospho-S6 signal to the same level as untreated cells 124 at 100:1 stoichiometry (Fig. 3d). Because rapamycin and rapalogs exert 125 immunosuppressive effects by inhibiting mTOR activity and thus suppressing cell 126 proliferation in response to costimulatory and -mediated signals33,34, we asked 127 whether RapaBlock can prevent RapaLink-1-mediated immunosuppression. We 128 stimulated human peripheral mononuclear blood cells (PBMC) with anti-CD3 and anti- 129 CD28 in the presence of different concentrations of RapaLink-1 and 130 RapaBlock and assessed after 5 days. Whereas RapaLink-1 potently 131 inhibited PBMC proliferation at nanomolar concentrations, addition of RapaBlock

132 abolished this effect, shifting the IC50 by more than 100-fold (Fig. 3b). Though mTOR 133 inhibition does not directly control cytokine production, we observed higher cumulative IL- 134 2 release in cells co-treated with RapaLink-1 and RapaBlock compared to cells treated 135 with RapaLink-1 alone, presumably as a result of greater cell proliferation (Fig. 3c). 136 Consistent with our observation of mTOR signaling, RapaBlock appeared more effective 137 at protecting PBMC from rapamycin-mediated proliferation inhibition (Figs. 3e, 3f). 138 Together, these data demonstrate that by competitively binding to intracellular FKBP12, 139 RapaBlock renders RapaLink-1 and rapamycin incapable of inhibiting mTOR, and in so 140 doing diminishes the immunosuppressive effects of these two drugs. 141 To examine whether the combination of RapaLink-1 and RapaBlock allows brain- 142 specific inhibition of mTOR in vivo, we treated healthy BALB/cnu/nu mice with RapaLink-1 143 (1 mg/kg) or a combination of RapaLink-1 (1 mg/kg) and RapaBlock (40 mg/kg), 144 stimulated mTOR activity with (250 mU) after 4 h or 24 h, and analyzed dissected 145 tissues by immunoblot (Fig. 4a). RapaLink-1, when used as a single agent, potently 146 inhibited mTOR signaling in both skeletal muscle and brain tissues, as revealed by the

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147 reduced of S6 and 4EBP1. The combination of RapaLink-1 and 148 RapaBlock, however, exhibited remarkable tissue-specific effects: while mTOR activity in 149 the brain was inhibited at a comparable level to mice treated with RapaLink-1 only, mTOR 150 activity in skeletal muscle was not affected. 151 RapaLink-1 has been shown to be efficacious in orthotopic mouse models of 152 glioblastoma but inhibition of mTOR in the periphery does not contribute to efficacy. We 153 asked whether our combination regimen, lacking the ability to inhibit mTOR activity in 154 peripheral tissues, could retain the efficacy of RapaLink-1 in treating glioblastoma. We 155 established orthotopic intracranial xenografts of U87MG cells expressing firefly luciferase 156 in nude mice and treated these mice with i.p. injections of RapaLink-1 (1 mg/kg), 157 RapaBlock (40 mg/kg), or a combination of both every 5 days. All treatments were well 158 tolerated, and no significant changes of body weight were observed (Fig. 4b). RapaLink- 159 1, both as a single-agent or in combination with RapaBlock, significantly suppressed 160 tumor growth and improved survival, whereas RapaBlock alone had no significant effect 161 on either. 162 Having established a binary therapeutic approach to achieve brain-specific 163 inhibition of mTOR, we wondered whether the same strategy could be adapted to other 164 drugs for which brain-restricted pharmacology would be desirable. One critical challenge 165 is that for this approach to be generalizable, the “active” component (e.g. RapaLink-1) 166 must be dependent on the availability FKBP12. Drugs with this property are rare, few if 167 any besides rapamycin and FK506 are known. Our earlier investigation of RapaLink-1 168 (Fig. 1) and RapaTAMRA (Extended Data Fig. 1) led us to hypothesize that other 169 bifunctional molecules consisting of a FKBP12-binding moiety and a kinase inhibitor 170 moiety could be conditionally active and amenable to modulation with RapaBlock. 171 We first tested our hypothesis with GNE7915, a potent and specific inhibitor of 172 -rich repeat kinase 2 (LRRK2) a pre-clinical agent under investigation for the 173 treatment of Parkinson’s disease35–37. As gain-of-function of LRRK2 are 174 strongly associated with hereditary and sporadic forms of Parkinson’s diseases, LRRK2 175 kinase inhibition has been pursued as a potential therapeutic strategy38,39. However, 176 recent studies demonstrating that systemic LRRK2 inhibition with small molecule 177 inhibitors induced cytoplasmic vacuolation of type II pneumocytes suggests a potential

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178 safety liability for these compounds40,41. We synthesized FK-GNE7915 by chemically 179 linking the pharmacophores of FK506 and GNE7915 with a piperazine group (Fig. 5a). 180 In in vitro LRRK2 kinase assays, FK-GNE7915 was an inferior inhibitor to GNE7915 (Fig. 181 5c, IC50s of 107 nM and 3.0 nM, respectively), though its activity was potentiated by

182 including 10 µM FKBP12 in the assay (IC50: 21 nM). However, in a cellular assay where 183 we quantified phospho-LRRK2 (S935) levels as a marker for LRRK2 inhibition, FK- 184 GNE7915 was more potent than the parent compound GNE7915 by more than 10-fold 185 (Fig 5d, IC50s of 6.7 nM and 81 nM, respectively). The juxtaposition of these two results 186 suggested a role of the FK506 moiety in the enhanced cellular potency of FK-GNE7915. 187 We reasoned that the high-affinity FK506-FKBP12 interaction can promote the 188 intracellular accumulation of FK-GNE7915 similar to the case of RapaLink-1 and 189 confirmed this using a bifunctional fluorescent probe FK-TAMRA (Extended Data Fig. 4). 190 This feature allowed us to program LRRK2 inhibition by controlling the availability of 191 FKBP12: whereas 100 nM FK-GNE7915 treatment reduced phosphor-LRRK2 (S935) 192 level to 15% of basal level, the combination of 100 nM FK-GNE7915 and 1 µM RapaBlock 193 had no effect on LRRK2 activity (Fig. 5e). 194 The successful conversion of GNE7915 into a FKBP-dependent LRRK2 inhibitor 195 by simply linking it to an FK506 fragment prompted us to evaluate the generality of this 196 strategy. We explored a number of kinase inhibitors that are being investigated for CNS- 197 diseases: (Src family kinase inhibitor, glioblastoma)42–44, (EGFR/HER2 198 inhibitor, glioblastoma)45,46, and prostetin (MAP4K4 inhibitor, amyotrophic lateral sclerosis 199 and Alzheimer’s disease)47. In all three cases, chemically linking the kinase inhibitor to 200 FK506 yielded bifunctional molecules that retained the kinase inhibitory activities of the 201 parent molecules (Extended Data Figs. 5-7). For FK-dasatinib, we also examined its 202 target specificity using both biochemical (Invitrogen SelectScreen Kinase Profiling) and 203 live-cell kinase profiling48 and observed an identical spectrum of kinase targets as 204 dasatinib with the exception of DDR2, a known off-target of dasatinib (Extended Data Fig. 205 5). Despite their large molecular weights, these bifunctional molecules are active in cells 206 with comparable potencies to their parent compounds and sensitive to deactivation by 207 RapaBlock. While these examples represent a limited set of kinase inhibitors with

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208 potential CNS disease indications, it is conceivable more drugs can be similarly 209 configured with programmable pharmacology without losing cellular potency. 210 211 Conclusion 212 Exploiting the unique functional dependence of rapamycin analogs on FKBP12, we 213 have developed an approach to achieve brain-specific mTOR inhibition through the 214 simultaneous administration of a potent mTOR inhibitor (RapaLink-1) and a cell- 215 permeable, BBB-impermeable ligand of FKBP12 (RapaBlock). Tissue-restricted mTOR 216 inhibition enabled by this binary pharmacology strategy reduces the toxicity in peripheral 217 tissues but maintains therapeutic benefits of RapaLink-1 in glioblastoma xenograft 218 models. On the basis of these findings, it seems reasonable to anticipate that the same 219 drug combination may be of broader value in other CNS diseases driven by dysregulated 220 mTOR activity such as Alcohol Use Disorder. 221 The applicability of our approach extends beyond mTOR inhibition. We show that 222 chemically linking ATP-site kinase inhibitors to FK506 through solvent-exposed groups 223 leads to a new class of cell-permeable kinase inhibitors whose activity depends on the 224 abundant endogenous protein FKBP12. These inhibitors are characterized by their ability 225 to mediate the formation of a ternary complex of the drug, the target kinase and FKBP12, 226 as well as their amenability to activity modulation by RapaBlock. Further in vivo studies 227 and medicinal chemistry are necessary to assess and optimize these compounds on a 228 case-by-case basis, but our initial investigations show that it is feasible to attain brain- 229 selective kinase inhibition of LRRK2 using a FKBP-dependent kinase inhibitor (FK- 230 GNE7915) and RapaBlock. 231 We have investigated the mechanism by which RapaBlock controls the cellular 232 activity of RapaLink-1 as well as other FKBP-dependent kinase inhibitors. Our current 233 data has revealed at least two roles of cellular FKBP12 in the function of RapaLink-1 (and 234 other FKBP-binding compounds). First, we have shown that FKBP12 serves as a 235 reservoir to retain and accumulate RapaLink-1 inside the cell, achieving exceptional 236 cellular concentration. Second, for most FKBP-dependent kinase inhibitors we have 237 investigated, FKBP12 improves their potency in cell-free assays. Though we have not 238 obtained direct evidence, we hypothesize that in aqueous solutions, a bifunctional

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239 compound built with flexible linkers between hydrophobic pharmacophores will mainly 240 adopt a binding-incompetent conformation to minimize hydration penalty (“hydrophobic 241 collapse”), and binding of FKBP12 will expose the inhibitor moiety to enable target 242 inhibition. RapaBlock impedes both of these processes by occupying the ligand-binding 243 site of FKBP12. While further investigation is clearly necessary to elucidate how these 244 high molecular weight compounds enter cells and whether FKBP12 participates in the 245 binding interaction with the target protein, we believe this system provides a generalizable 246 approach for programmable kinase inhibition. 247 Combining two pharmaceutical agents to achieve tissue-selective therapeutic 248 effects has been previously employed in drugs approved for clinical use or in development. 249 Examples include Levodopa/Carbidopa (BBB-permeable dopamine precursor/BBB- 250 impermeable dopa decarboxylase inhibitor) for Parkinson’s Disease49, conjugated 251 estrogens/bazedoxifene (BBB-permeable estrogen/BBB-impermeable estrogen receptor 252 modulator) for post-menopausal hot flashes and osteoporosis50, and 253 donepezil/solifenacin (BBB-permeable acetylcholinesterase inhibitor/BBB-impermeable 254 anticholinergic) for Alzheimer’s disease51. Our approach differs from these precedents in 255 that it does not involve two drugs with counteracting effects on the same target or pathway; 256 instead, RapaBlock controls tissue-specificity by directly attenuating the activity of the 257 kinase inhibitor. The present system therefore has the advantage of being adaptable for 258 a multitude of targets, only requiring an invariant RapaBlock molecule and a FKBP12- 259 dependent inhibitor that can be readily designed based on the structures of FK506 and 260 lead compounds. While we have focused on protein in this study, it seems 261 reasonable to expect that the approach is also applicable to other classes of therapeutic 262 targets, such as and histone modification . 263 264 Acknowledgements 265 We thank Douglas Wassarman for discussions. Z.Z. is a Damon Runyon Fellow 266 supported by the Damon Runyon Foundation (DRG-2281-17). 267 K.M.S, Q.W.F. and W.A.W. acknowledge NIH 1R01CA221969. K.M.S, and W.A.W. 268 acknowledge the Samuel Waxman Cancer Research Foundation. W.A.W. acknowledges 269 NIH U01CA217864 and Cancer Research UK A28592. K.M.S. acknowledges the Michael

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270 J. Fox Foundation P0536220, The Mark Foundation for Cancer Research and the Howard 271 Hughes Medical Institute. 272 273 Author Contributions 274 Z.Z., Q.F., W.A.W and K.M.S. conceived the project, designed and analyzed the 275 experiments, and wrote the manuscript. Z.Z., Q.F., X.L. and K.J.L. performed the 276 laboratory experiments. W.A.W. supervised the in vivo experiments. 277 278 Supplementary Information is available for this paper. 279 280 Author Information 281 Correspondence and requests for materials should be addressed to K.M.S. 282 ([email protected]).

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14 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.12.336677; this version posted October 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. ) a b 100 FKBP12 Binding mTOR FRB Domain Binding mTOR ATP Site Binding FKBP12 Binding MLN128

CH3 CH3 OCH3 OCH3 CH3 MLN128+FKBP Activity (%

H2N Calcineurin Rapa H O H Binding 50 O OCH3 O O Rapa+FKBP H3C CH3 N H3C H3C OH OH RL-1 O HO O O O RL-1+FKBP OCH3 NH2 O

OH Residual Kinase 0 N H CH3 N N H N 10-3 10-2 10-1 100 101 102 O O N CH3 N CH3 [Drug] (nM) O O O CH3 CH3 c

H H ) 100 ) No sgRNA O NH GAL4-4 sgRNA H3CO H3CO aibility (% O O OH V 50 FKBP12 sgRNA-1 O N 8 FKBP12 sgRNA-2 N N No sgRNA + 10 M FK506 (Normalized to DMSO

RapaLink-1 (RL-1) FK506 Relative Cell 0 10-3 10-2 10-1 100 101 102 [RapaLink-1] (nM)

d e MCF7 RapaLink-1 Only FKBP12 knockdown RapaLink-1 + FK506 (1:1000)

10 nM RL-1 – – + + 10 µM FK506 – + – +

P-Akt [S473] FRB

P-S6 [S240/244]

P-4EBP1 [T37/46] FKBP12 mTOR mTOR Akt mTOR FK506 S6 +FKBP12

4EBP1 mTOR mTOR mTOR 410 inhibited not inhibited not inhibited 411 Figure 1. RapaLink-1 is a potent mTOR inhibitor that requires FKBP12 for its 412 cellular activity. a, Chemical structures of RapaLink-1 and FK506. b, Inhibition of mTOR 413 activity by MLN128, RapaLink-1 (RL-1) or Rapamycin in the presence or absence of 10 414 µM FKBP12 in vitro kinase assay. c, K562-dCas9-KRAB cells transduced with GAL4-4 415 (control) or FKBP12-targeting sgRNAs were treated with RapaLink-1 and cell proliferation 416 was assessed after 72 h. In the last listed condition, cells were transduced with sgRNA 417 but treated with RapaLink-1 in the presence of 10 µM FK506. d, Immunoblot analysis of 418 mTOR signaling in MCF7 cells treated with DMSO, RapaLink-1, FK506, or a combination 419 of RapaLink-1 and FK506. e, Schematics of the proposed working model. Grey circles 420 indicate cellular membranes.

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a b c OCH3 OCH3 CH3 Brain BBB Periphery H O H3C H3C OH O O O N+ FKBP12 FK506 OH O– N H RapaLink-1 O CH3 O CH3 FRB domain FRB domain H C21 allyl:

calcineurin binding H3CO FKBP12 OH

RapaBlock mTOR mTOR FKBP12 inhibited RapaBlock active d e 120 120 )

110 100 ) 100 FKBP12 Ki NFAT Activation IC50 80 90 FK506 1.7 ± 0.2 nM FK506 0.094 ± 0.008 nM 60 80 RapaBlock 3.1 ± 0.4 nM RapaBlock >10000 nM 70 40 Activation (DMSO% 60 T Polarization (mP

A 20 F FKBP12 Fluorescence 50 N 40 0 10-1 100 101 102 10-3 10-2 10-1 100 101 102 103 104 421 Concentration [nM] Concentration [nM] 422 423 Figure 2. RapaBlock is a potent, non-immunosuppressive FKBP12 ligand. a, a 424 proposed model to achieve brain-specific mTOR inhibition through the combination of an 425 FKBP12-dependent mTOR inhibitor (RapaLink-1) and a brain impermeable FKBP12 426 ligand (RapaBlock). b, a FK506–FKBP12 co-crystal structure (PDB: 1FKJ) showing that 427 the C21 allyl group is solvent exposed and its modification may lead to abolished 428 calcineurin binding. c, chemical structures RapaBlock. Blue shaded areas indicate the 429 FKBP12-binding moiety. d, competition fluorescence polarization assay using 430 fluorescein-labeled rapamycin as the tracer compound (n = 3). e, Jurkat cells expressing 431 a luciferase under the control of NFAT response element were stimulated 432 with phorbol myristate acetate and ionomycin in the presence of various concentrations 433 of compounds (n = 3).

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a RapaLink-1(nM) 0 10 10 10 10 0 b c RapaBlock(nM) 0 0 100 1000 10000 10000

P-Akt[S473] 8000 ) ) P-S6 [S240/244] n 100 6000 P-4EBP1 [T37/46] 4000 Total Akt 50 Total S6 2000 Relative Proliferatio Secreted IL-2 (pg/mL (Normalized to DMSO, % Total 4EBP1 0 0 10-2 10-1 100 101 102 10-2 10-1 100 101 102 GAPDH [RapaLink-1] (nM) [RapaLink-1] (nM)

FKBP12 No RapaBlock +10 M RapaBlock

d Rapamycin (nM) 0 10 10 10 10 0 e f RapaBlock(nM) 0 0 100 1000 10000 10000

P-Akt[S473] 6000 ) )

P-S6 [S240/244] n

100 4000 P-4EBP1 [T37/46]

Total Akt 50 2000 Total S6 Relative Proliferatio Secreted IL-2 (pg/mL (Normalized to DMSO, % Total 4EBP1 0 0 10-2 10-1 100 101 102 10-2 10-1 100 101 102 GAPDH [Rapamycin] (nM) [Rapamycin] (nM) 434 FKBP12 No RapaBlock +10 M RapaBlock 435 Figure 3. RapaBlock protects cells from mTOR inhibition by RapaLink-1 and 436 Rapamycin. a, d, MCF7 cells were treated with a combination of RapaLink-1 and 437 RapaBlock (a), or Rapamycin and RapaBlock (d) for 4 h, then phosphorylation of mTOR 438 substrates were analyzed by immunoblotting. Results shown are representative of three 439 independent experiments. b, e, Human PBMCs were stimulated with anti-CD3 and anti- 440 CD28 in the presence of varying amounts of RapaLink-1 and RapaBlock (b) or rapamycin 441 and RapaBlock (e), and cell proliferation was measured after 120 h. c, f, Interleukin-2 442 secretion in the culture supernatant of PBMCs in c and f was quantified by sandwich 443 ELISA.

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a b Muscle Brain 26 1010 (Hr) 0 0.25 4 4 4 24 24 24 0 0.25 4 4 4 24 24 24 l 100

l n.s. Insulin (250 mU) + + + + + + + + + + + + + + 109 RapaBlock (40 mg/kg) + + + + + + + + *** ) 24 n.s. RapaLink-1 (1mg/kg) + + + + + + + + n.s. 108 + 50 eight(g + n.s.

p-RPS6 (S235/236) W *** 22

RPS6 (tumor size) 107

Probability of Surviva + p-4EBP1 (T37/46) Bioluminscence signa n.s. ** ** 4EBP1 106 0 20 0 10 20 30 40 0 20 40 60 0 10 20 30 40 GAPDH Days Days Days RapaLink-1 (1 mg/kg) + + + + Vehicle RapaLink-1 (1 mg/kg) Rapablock (40 mg/kg) 444 +RapaBlock (40 mg/kg) 445 Figure 4. Cotreatment with RapaLink-1 and RapaBlock allows brain-specific 446 inhibition of mTOR. a, Mice were treated intraperitoneally with a single dose of 447 RapaLink-1 (1 mg/kg), RapaBlock (40 mg/kg), or a combination of both. mTOR activity 448 was stimulated with insulin (250 mU) 15 minutes before tissue dissection. Whole brain 449 and skeletal muscle were analyzed by immunoblot (n = 3 except for the following: no- 450 insulin group, n = 1; insulin-only group, n = 2. Results are shown for one animal from each 451 group). b, Mice bearing luciferase-expressing orthotopic glioblastoma xenografts 452 (U87MG-Luc) were randomized to four different groups (n = 7) and treated 453 intraperitoneally every 5 days: (1) vehicle; (2) RapaBlock (40 mg/kg); (3) RapaLink-1 (1 454 mg/kg); (4) RapaLink-1 (1 mg/kg) and RapaBlock (40 mg/kg). Tumor size was monitored 455 every 5 days using bioluminescence imaging. Statistical tests were performed for each 456 treatment group versus vehicle-treated group: unpaired Student’s t-test (tumor size on 457 day 20); log rank test (survival). n.s., not significant; **, p£0.01; ***, p£0.001.

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a b OCH3 OCH3 CH3 no inhibition H F N N H O H3C H3C N OH O CF 3 O O – FKBP12 O O HN OH N H FKBP12 O potent inhibition CH3 LRRK2 O O CH3 GNE7915 H N +RapaBlock

O H3CO OH N FK-GNE7915 FKBP12 no inhibition N = FK506 C21 Fragment

c d e ) ) )

100 100 100 Activity (%

50 50 50 Cellular p-LRRK S935 Cellu;ar p-LRRK S935 (Normalized to DMSO, % (Normalized to DMSO, % Residual Kinase 0 0 0 -1 0 1 2 3 4 10-1 100 101 102 103 100 nM 100 nM 10 10 10 10 10 10 DMSO [Drug] (nM) Conc (nM) GNE7915 FK-GNE7915

GNE7915 GNE7915 + 10 µM FKBP12 GNE7915 No RrapaBlock 458 FK-GNE7915 FK-GNE7915+ 10 µM FKBP12 FK-GNE7915 +1 µM RapaBlock 459 Figure 5. Programmable kinase inhibition with FKBP-dependent kinase inhibitors 460 and RapaBlock. a, Structures of GNE7915 and FK-GNE7915. b, Proposed working 461 model for FKBP-dependent kinase inhibitors. c, kinase inhibition in the absence or 462 presence of supplemented 10 µM recombinant FKBP12 protein. Data is the average of 463 two replicates. d, e, RAW264.7 cells were treated with GNE7915, FK-GNE7915, and/or 464 RapaBlock and phospho-LRRK2 (S935) was analyzed by time-resolved FRET using 465 epitopically orthogonal antibodies for LRRK2 and p-LRRK2 (S935). See Supplementary 466 Information for experimental details.

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467 468 Extended Data Figure 1. FKBP12-Rapamycin interaction contributes to cellular 469 accumulation of a fluorescent analog of RapaLink-1. a, Illustration of the flow 470 cytometry-based assay to assess cellular accumulation of TAMRA compounds. b, 471 Structure of a fluorescent probe Rapa-TAMRA. c, FKBP12 knockdown decreases 472 cellular retention of Rapa-TAMRA, but not TAMRA-PEG8-N3. See Supplementary 473 Information for gating strategy.

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a H H H N N O N 2 + N H2N H2N O O

Kd (FKBP12) Rapamycin RapaLink-1 SLF 01-025 01-038 01-040 Compound [nM] P-S6 P-S6 H H CH3 O H N H3C N N N N O H N SLF 23 0.56 0.05 HO 2 O O CH O 3 O 01-025 95 0.13 ND 01-038 9.4 0.22 ND 01-041 01-043 01-044 01-059 01-040 21 0.89 ND O NH 01-041 38 0.07 ND O HO H H H N H2N N N N 01-043 15 0.89 ND N H2N N O HO H H O O 01-044 13 0.64 ND N CH3 HO O 01-059 14 0.13 ND H CH3 O H3C O O 01-060A 01-060B 01-065 01-070 01-060A 46 0.08 ND OCH3 01-060B 75 0.24 ND H O H N H H 01-065 38 0.30 ND N N N OCH3 N HO H N N O 2 01-070 21 0.11 ND H C O 3 O 01-072 13 0.78 ND 01-083 13 0.08 ND 01-072 01-083 02-014 02-032 02-014 64 0.55 ND

O- 02-032 36 0.06 ND H H H N H N N N N+ N Cl HS 02-033 10 0.00 ND O O O O 02-055 64 0.02 ND 02-096 6.0 0.40 ND 02-033 02-055 03-083 03-087 03-077 44 0.91 ND 03-083 31 0.85 ND H H H N O N N H2N O N 3 O H2N 03-084 19 0.44 ND O O O 03-087 29 0.76 0.03

02-096 03-077 03-084

Kd (FKBP12) Rapamycin RapaLink-1 b Compound N N NH2 S [nM] P-S6 P-S6

05-011 FK506 0.8 0.99 0.71 FK506 05-050 05-084 05-011 0.7 0.76 0.52 CH3 HO N 05-012 0.4 0.36 0.03 S CH 3 05-013 1.4 0.93 0.61 OH H3CO 05-013 CH3 05-020 1.5 0.63 0.03 O N N 05-026 3.7 0.92 1.07 O 05-012 05-051 05-085 OH NH2 N S 05-027 1.1 0.89 0.03 O O CO H O O 2 05-028 2.5 0.94 0.02 HO – H3C H3C O O– 05-037 05-037 8.7 ND 0.03 O OH N+ N+ 05-050 3.6 ND 0.70 H O N N 05-051 1.2 ND 0.65 OCH3 OCH3 CH3 S 05-060 0.6 ND 0.09 05-020 05-060 05-086 O 05-026 05-061 0.2 ND 0.01 05-064 0.2 ND 0.60 H N N H 05-084 3.3 ND ND N + S N N O N N+ H 05-085 1.5 ND 0.56 O– – O O 05-086 1.6 ND 0.44 05-064 05-061 05-092 05-028 05-092 2.5 ND 1.02 474 475 Extended Data Figure 2. Structures polar FKBP12 ligands synthesized (a, SLF 476 derivatives; b, FK506 derivatives). Listed in the table are their affinity to recombinant 477 FKBP12 (fluorescence polarization assay) and their efficacy of blocking mTOR inhibition 478 by rapamycin or RapaLink-1 (assessed by western blot analysis of p-S6 level after 479 treatment of MCF7 cells with a combination of 10 nM Rapamycin/Rapalink + 10 µM 480 candidate compound for 24 h. P-S6 level is quantified as fraction of DMSO control). ND, 481 not determined.

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a +RapaLink-1 (10 nM) – – FK506 (µM) 05-026 (µM) 05-037 (µM) 06-039 (µM) 06-041 (µM) – – 0.1 1 10 0.1 1 10 0.1 1 10 0.1 1 10 0.1 1 10 P-S6 [S240/244] Total S6

P-4EBP1 [T37/46]

Total 4EBP1 GAPDH b Muscle

Insulin (250 mU) + + + + + + + + + + + + + + + + + + + + RapaBlock (05-026) + + + + RapaBlock (05-037) + + + + RapaBlock (06-039) + + + + RapaBlock (06-041) + + + + RapaLink-1 + + + + + + + + + + p-RPS6 (S235/236) RPS6 p-4EBP1 (T37/46) 4EBP1 GAPDH

Brain

Insulin (250 mU) + + + + + + + + + + + + + + + + + + + + RapaBlock (05-026) + + + + RapaBlock (05-037) + + + + RapaBlock (06-039) + + + + RapaBlock (06-041) + + + + RapaLink-1 + + + + + + + + + + p-RPS6 (S235/236) RPS6 p-4EBP1 (T37/46) 4EBP1 482 GAPDH 483 Extended Data Figure 3. Four candidate RapaBlock molecules were evaluated in 484 cells (a) and in vivo (b).

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a b DMSO 1 nM FK-TAMRA 100 nM FK-TAMRA 0.0% 0.1% 0.1% 0.2% 50.5% 49.5%

NegCtrl sg

50.2% 49.7% 50.2% 49.6% 0.0% 0.0%

0.1% 0.1% 0.1% 0.1% 50.1% 47.4%

OCH3 OCH3 CH3 FKBP12 sg1

H O H3C H3C O OH 49.6% 50.3% 49.4% 50.4% 0.1% 2.4% – O O O2C N O N 0.0% 0.0% 0.2% 0.1% 48.8% 50.5% OH N N H O O O CH 3 FKBP12 sg2 O CH3 H N+

48.8% 51.1% 48.8% 51.0% 0.0% 0.6% H CO TAMRA 3 sgRNA- sgRNA+ sgRNA- sgRNA+ sgRNA- sgRNA+ OH

FK-TAMRA DMSO 1 nM FK-TAMRA 100 nM FK-TAMRA 105 l

signa 104 1.00 0.17 0.23 A sgRNA-

AMR sgRNA+ T 103 1.01 1.01 1.01 1.00 0.94 0.95 Median 102

g 1 2 g 1 2 g 1 2

NegCtrl s NegCtrl s NegCtrl s 485 FKBP12 sg FKBP12 sg FKBP12 sg FKBP12 sg FKBP12 sg FKBP12 sg 486 Extended Data Figure 4. (Related to Extended Data Figure 1) FKBP12-FK506 487 interaction contributes to cellular accumulation of a fluorescence analog of FK506. 488 a, Structure of a fluorescent probe FK-TAMRA. b, FKBP12 knockdown decreases 489 cellular retention of FK-TAMRA. See Supplementary Information for gating strategy.

23 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.12.336677; this version posted October 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

a OCH3 OCH3 CH3 b

H O H3C H3C N OH O O H O no inhibition N N S O Cl OH N N N N NH H O CH CH3 – FKBP12 3 H C 3 O CH3 FKBP12 H

HO Dasatinib H3CO +RapaBlock OH O FK-Dasatinib potent inhibition no inhibition = FK506 C21 Fragment

c d SRC CSK DDR2 K562 ) ) )

100 100 100 )

1.0 Activity (% Activity (% Activity (%

50 50 50 0.5 Relative Growth (Normalized to DMSO 0 0 0 Inhibition of Kinase Inhibition of Kinase Inhibition of Kinase 10-2 10-1 100 101 102 103 10-2 10-1 100 101 102 103 10-2 10-1 100 101 102 103 10-4 10-3 10-2 10-1 100 101 102 103 [Drug] (nM) [Drug] (nM) [Drug] (nM) [Drug] (nM)

Dasatinib Dasatinib + 10 µM FKBP12 FK-Dasatinib FK-Dasatinib + 10 µM FKBP12 Dasatinib Dasatinib + 10 µM RapaBlock FK-Dasatinib FK-Dasatinib + 10 µM RapaBlock

e f

– – + + – – Dasatinib (100 nM) FK-Dasatinib (nM) Drug Dasatinib (100 nM) FK-Dasatinib (100 nM) FK506-Dasa (100 nM) – – – – + + – – RapaBlock (10 µM) – + – + – + 0 1 3 10 30 100 Time after washout (h) 0 1 2 3 6 12 24 0 1 2 3 6 12 24

180 – 180 – 130 – 130 – 180 – 130 – 100 – 100 – 100 – 70 – 70 – 70 –

IB: pTyr 55 – 55 – 55 – IB: pTyr 40 – 40 – 40 –

35 – 35 – 35 – 25 – 25 – 25 – 15 – 15 – 15 –

COX IV COX IV

g h ] 2

1 100 Kinase activity inhibited P 100

B >70% by both Dasatinib and 1:

K PRKCA F

] CSK Inhibition% Inhibition% M ABL1, BLK, BMX, BTK, CSF1R, EPHA2,

µ Name [Dasatinib] [FK-Dasatinib] 0 EPHA4, EPHA5, EPHA8, EPHB1, EPHB2,

1 80 LCK EPHB4, FGR, FRK, FYN, HCK, LCK, LYN A, 80 + LYN B, PEAK1, SRC, SRC N1, YES1 M FK-Dasa ABL1 100 100 n 0

0 YES1 100 97 1 FK-Dasa [

60 60 ) MAPK14 M GAK 100 98 % n

(

0 CAMK2G n BLK 100 100 1 o [ Kinase activity inhibited i

) t >70% by Dasatinib and <70% by 1: i SIK2 100 100 %

b PRKDC i (

40 ROCK1 MTOR

h 40 n

n NEK7 ABL2 100 100 o i

i DDR1, EPHA1, EPHA3, EPHB3, FYN A, PKMYT1 t e i EIF2AK4 s

b RIPK2, SIK1, SNF1LK2, SRMS, TNK2, TXK SRC 100 100 a i n h i n K I CSK 94 90 20 e 20 CDK7 CAMK1D

s STK35

a LCK 92 86

n FER PAK1 i

K CHEK2 JAK1 0 0

0 20 40 60 80 100 0 20 40 60 80 100 490 Kinase Inhibit ion (%) [10 nM Dasat inib + 10 µM FKBP12] Kinase inhibit ion (%) [100 nM Dasat inib] 491 Extended Data Figure 5. FK-Dasatinib is a FKBP12-dependent Src family kinase 492 inhibitor with long cellular retention time. a, Structures of Dasatinib and FK-Dasatinib. 493 b, Proposed working model for FKBP-dependent kinase inhibitors. c, Inhibition of Src, 494 Csk and DDR2 kinases by Dasatinib and FK-Dasatinib in the absence or presence of 495 supplemented 10 µM recombinant FKBP12 protein. Data is the average of two replicates.

24 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.12.336677; this version posted October 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

496 d, Inhibition of K562 cell proliferation by Dasatinib and FK-Dasatinib, in the presence or 497 absence of 10 µM RapaBlock. Data is the average of three replicates. e, Jurkat cells were 498 stimulated with anti-CD3 (OKT3) in the presence of dasatinib, FK-dasatinib, 499 and/or RapaBlock and analyzed by immunoblot. f, Jurkat cells were pulse-treated with 500 Dasatinib (100 nM) or FK-Dasatinib (100 nM) for 1 h, then drugs were washed out and 501 cells were incubated in drug-free media for various amounts of time, stimulated with anti- 502 CD3 antibody (OKT3) for 5 min and analyzed by immunoblot. g, inhibition of 476 purified 503 kinases by Dasatinib (10 nM) or FK-Dasatinib (10 nM) in the presence of 10 µM 504 recombinant FKBP12 protein. h, kinase profiling of Dasatinib (100 nM Dasatinib) or FK- 505 Dasatinib (100 nM) in Jurkat cells using the covalent occupancy probe XO44. See 506 Supplementary Information for experimental details.

25 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.12.336677; this version posted October 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

a b

O F

HN Cl EGFR HER2 ) ) 120 120 O N 100 100 N Activity (% Activity (% 80 80

60 60 H H3C N 40 40 S Lapatinib O O 20 20

0 0 Inhibition of Kinase Inhibition of Kinase -2 -1 0 1 2 3 O 10-2 10-1 100 101 102 103 10 10 10 10 10 10 [Drug] (nM) [Drug] (nM) N FK-Lapatinib N Lapatinib Lapatinib+10 µM FKBP12 FK-Lapatinib FK-Lapatinib+10 µM FKBP12

= FK506 C21 Fragment

c d Drug (1 µM) DMSO Lap FK-Lap

RapaBlock (10 µM) – + – + – +

P-HER2 (Y1221/1222) SK-BR-3

120 P-HER3 (Y1289) ) ) 100 P-AKT (S473) aibility (%

V 80 P-ERK (T202/Y204) 60

(Normalized to DMSO 40 HER2 Relative Cell

100 101 102 103 104 HER3 [Drug] (nM) AKT Lapatinib Lapatinib+10 µM RapaBlock ERK FK-Lapatinib FK-Lapatinib+10 µM RapaBlock GAPDH 507 508 Extended Data Figure 6. FK-Lapatinib is a FKBP12-dependent EGFR/HER2 kinase 509 inhibitor. a, Chemical structures of Lapatinib and FK-Lapatinib. b, Inhibition of EGFR 510 and HER2 kinase activity by Lapatinib and FK-Lapatinib in the absence or presence of 511 supplemented 10 µM recombinant FKBP12 protein. Data is the average of two replicates. 512 c, Inhibition of proliferation of SK-BR3 cells by lapatinib and FK-lapatinib in the presence 513 of absence of 10 µM RapaBlock. Data is the average of three replicates. d, SK-BR3 cells 514 were treated with Lapatinib, FK-Lapatinib, and/or RapaBlock for 1 h and analyzed by 515 immunoblot.

26 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.12.336677; this version posted October 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

a b

H N N

MAP4K4 (HGK)

) 120 Cl 100 Prostetin Activity (%

80 Prostetin + 10 µM FKBP12 60 HO FK-Prostetin N Prostetin 40 N FK-Prostetin + 10 µM FKBP12 20

0 O Inhibition of Kinase 10-3 10-2 10-1 100 101 102 N FK-Prostetin [Drug] (nM) N 516 = FK506 C21 Fragment 517 Extended Data Figure 7. FK-Prostetin is a FKBP12-dependent MAP4K4(HGK) 518 kinase inhibitor. a, Chemical structures of Prostetin and FK-Prostetin. b, Inhibition of 519 MAP4K4(HGK) kinase activity by Lapatinib and FK-Lapatinib in the absence or presence 520 of supplemented 10 µM recombinant FKBP12 protein. Data is the average of two 521 replicates.

27 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.12.336677; this version posted October 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Days 0 5 10 15 20 25 Vehicle RapaBlock RapaLink-1

522 RapaLink-1 +RapaBlock 523 Extended Data Figure 8. Exemplary bioluminescence images of mice bearing 524 intracranial glioblastoma xenografts. U87MG cells expressing firefly luciferase were 525 injected intracranially into BALB/cnu/nu mice. After tumor establishment, mice were sorted 526 into four groups and treated by i.p. injections every 5 days of vehicle, RapaBlock (1 mg/kg), 527 RapaBlock (40 mg/kg), or a combination of RapaLink-1 and RapaBlock (1 mg/kg and 40 528 mg/kg, respectively). Bioluminescence imaging of tumor-bearing mice was obtained at 529 days shown (day 0 was start of treatment), using identical imaging conditions.

28