Novel Mtorc1 Inhibitors Kill Glioblastoma Stem Cells

pharmaceuticals

Article Novel mTORC1 Inhibitors Kill Glioblastoma Stem Cells

Jose A. Sandoval 1, Alexey Tomilov 1, Sandipan Datta 1, Sonia Allen 1, Robert O’Donnell 2, Thomas Sears 3, Kevin Woolard 3, Dmytro Kovalskyy 4, James M. Angelastro 1 and Gino Cortopassi 1,*

1 Department of Molecular Biosciences, University of California Davis, Davis, CA 95616, USA; [email protected] (J.A.S.); [email protected] (A.T.); [email protected] (S.D.); [email protected] (S.A.); [email protected] (J.M.A.) 2 Department of Internal Medicine, University of California Davis School of Medicine, Sacramento, CA 95817, USA; [email protected] 3 Department of Pathology, Microbiology and Immunology, University of California Davis, Davis, CA 95616, USA; [email protected] (T.S.); [email protected] (K.W.) 4 Department of Biochemistry, University of Texas Health and Science Center at San Antonio, San Antonio, TX, 78229, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-530-754-9665

Received: 6 October 2020; Accepted: 20 November 2020; Published: 24 November 2020

Abstract: Glioblastoma (GBM) is an aggressive tumor of the brain, with an average post-diagnosis survival of 15 months. GBM stem cells (GBMSC) resist the standard-of-care therapy, temozolomide, and are considered a major contributor to tumor resistance. Mammalian target of rapamycin Complex 1 (mTORC1) regulates cell proliferation and has been shown by others to have reduced activity in GBMSC. We recently identified a novel chemical series of human-safe piperazine-based brain-penetrant mTORC1-specific inhibitors. We assayed the piperazine-mTOR binding strength by two biophysical measurements, biolayer interferometry and field-effect biosensing, and these confirmed each other and demonstrated a structure–activity relationship. As mTORC1 is altered in human GBMSC, and as mTORC1 inhibitors have been tested in previous GBM clinical trials, we tested the killing potency of the tightest-binding piperazines and observed that these were potent GBMSC killers. GBMSCs are resistant to the standard-of-care temozolomide therapy, but temozolomide supplemented with tight-binding piperazine meclizine and flunarizine greatly enhanced GBMSC death over temozolomide alone. Lastly, we investigated IDH1-mutated GBMSC mutations that are known to affect mitochondrial and mTORC1 metabolism, and the tight-binding meclizine provoked ‘synthetic lethality’ in IDH1-mutant GBMSCs. In other words, IDH1-mutated GBMSC showed greater sensitivity to the coadministration of temozolomide and meclizine. These data tend to support a novel clinical strategy for GBM, i.e., the co-administration of meclizine or flunarizine as adjuvant therapy in the treatment of GBM and IDH1-mutant GBM.

Keywords: mTOR; mTORC1; glioblastoma; piperazine; meclizine

1. Introduction Glioblastoma multiforme (GBM) is a deadly tumor with a usual post-diagnosis survival of 15 months. One major hypothesis for GBM tumor resistance is that GBM stem cells (GBMSCs) resist standard chemotherapy and, through growth, proliferation, and mutation, cause tumor relapse [1–3]. Several studies have shown that mTORC1 inhibition through rapamycin attenuates cancer stem cell chemotherapy insensitivity [4–6].

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The mechanistic or mammalian target of rapamycin (mTOR) is a highly conserved protein kinase that serves as a central regulator of cell growth and connects cellular metabolism with environmental factors [7]. It is divided into two distinct complexes labelled mTORC1 and mTORC2, both of which are often dysregulated in cancer, including GBM [8–10]. mTORC1 is made up of a regulatory-associated-protein-of-mTOR (RAPTOR), proline-rich AKT substrate 40 kDa (PRAS40), mammalian lethal with Sec-13 protein 8 (mLST8), and DEP-domain TOR-binding protein (DEPTOR) [10]. It is inhibited by rapamycin, which binds to FK506-binding protein that only interacts with mTORC1. Growth factors, energy levels, oxygen levels, and amino acids all modulate mTORC1 activity through their effect on TSC1/2, a GTP-ase-activating protein that acts on Ras-homolog enriched in the brain (Rheb), which interacts with mTORC1. In the active form, mTORC1 promotes cell growth and proliferation mainly by phosphorylating eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) and ribosomal protein S6Kinase (S6K). mTORC1 plays a significant role in autophagy, lipid synthesis, and mitochondrial metabolism and biogenesis, and is altered in many tumor types [11–13]. mTORC1 activity is altered in patient-derived GBMSCs [14], suggesting that inhibition of mTORC1 could be specifically lethal to GBMSCs. Numerous mutations occur to drive GBM tumor progression. One such mutation is an R132H in the isocitrate dehydrogenase 1 (IDH1) protein. This is a gain of function mutation that alters metabolism, increasing the conversion of alpha-ketoglutarate into 2-hydroxyglutarate (D-2HG) [15]. Recently, it was shown that among patients with GBM IDH1 mutations, those patients with the lowest mTORC1 activity survived longest [16]. These authors found that among GBM patients with the IDH1 mutation, the low-mTORC1 group survived about 10-fold longer (200 months) than the high-mTORC1 group (20 months) [16]. Thus, it could be hypothesized that pharmacologically decreasing the mTORC1 activity in IDH1-mutant-bearing GBM patients could be a novel strategy for extending disease-free survival. There are other connections between IDH1 and mTORC1. It was found that the 2HG metabolite produced in IDH1-mutant cells is an mTORC1 stimulator [17]. Others have observed that inhibition of mTORC1 reduces the 2HG metabolite that appears to underlie GBMSC progression [18]. Thus, in the IDH1-mutant > 2HG > mTORC1 > GBMSC progression pathway, a novel mTORC1 inhibitor might potentially slow progression, or be ‘synthetically lethal’ to IDH1-mutant tumors specifically. Previously, we identified four piperazine compounds (cinnarizine, hydroxyzine, meclizine, and flunarizine) as novel mTORC1-specific inhibitors [19]. Two of the piperazine compounds, cinnarizine and flunarizine, have shown promise as radiation sensitizers in numerous mouse tumor types, perhaps through this previously unknown capacity to inhibit mTORC1 [20,21]. Given the finding of altered mTORC1 in patient-derived GBMSCs [14], we hypothesized that piperazine mTORC1 inhibitors might be effective killers of GBMSCs in vitro. Furthermore, because these drugs are known to be brain-penetrant and have low side-effect profiles, we tested the concept that they might be used as adjuvant therapy alongside the chemotherapeutic standard of care in GBM, namely temozolomide. Lastly, we tested the hypothesis that through the metabolic connection between IDH1 mutations and mTORC1, these mTORC1 inhibitors would be synthetically lethal to GBMSCs harboring the IDH1 mutation. Our results tend to support the ideas that piperazine toxicity to GBMSCs is related to their binding to the mTOR protein itself, that the piperazines plus temozolomide combination is much more toxic to GBMSCs than temozolomide alone, and that piperazines appear to have synthetic lethality in GBMSCs in the context of IDH1 mutations.

2. Results

2.1. Piperazine Compounds Dose-Dependently Inhibit mTORC1 Downstream Target Previously, our lab has shown that members of the piperazine drug class are mTORC1-specific inhibitors [19]. In Figure1A, we show that four piperazines dose-dependently inhibit the downstream target of mTORC1, pS6K (Figure1). Hydroxyzine and cinnarizine yield significant inhibition beginning at 0.1 µM and have a maximum effect at 10 µM. Meclizine and flunarizine follow similar trends, Pharmaceuticals 2020, 13, 419 3 of 18

Pharmaceuticals 2020, 13, x FOR PEER REVIEW 3 of 20 with meclizine showing significant inhibition beginning at 0.1 µM and flunarizine at 1 µM. Though 1 µM. Though meclizine does not show a maximum effect at the highest dose, all results are consistent meclizinewith doeseach other not show. In Figure a maximum 1B, we show effect that at the these highest four piperazine dose, all results compounds are consistent at a single with dose each of 10 other. In FigureµM 1inhibitB, we show mTORC1, that these assessed four through piperazine the compounds phosphorylation at a single of its downstreamdose of 10 µ M target inhibit pS6K, mTORC1, in assessedpatient through-derived the glioblastoma phosphorylation stem cells. of its Out downstream of the four piperazine target pS6K, compounds, in patient-derived flunarizine glioblastoma exhibits stem cells.the highest Out ofinhibitory the four potency, piperazine followed compounds, by hydroxyzine, flunarizine meclizine, exhibits and the cinnarizine highest inhibitory, which shows potency, followedan in byhibitory hydroxyzine, trend. meclizine, and cinnarizine, which shows an inhibitory trend.

FigureFigure 1. Conﬁrmation 1. Confirmation of of mTORC1mTORC1 inhibition inhibition by bypiperazine piperazine compounds. compounds. (A) Piperazine (A) Piperazine drugs dose drugs- dose-dependentlydependently inhibit inhibit mTORC1 mTORC1 downstream downstream target target S6Kinase S6Kinase in mousein mouse FL83B FL83B cells. cells. (B) (PiperazineB) Piperazine drugsdrugs inhibit inhibit GBM GBM stem stem cells cells at at a single a single concentration concentration of 10 10 µµM.M. The The amount amount of ofphosphorylated phosphorylated S6Kinase relative to total S6Kinase was measured by ELISA. The bars are the fold change over the S6Kinase relative to total S6Kinase was measured by ELISA. The bars are the fold change over the vehicle control. Error bars are 1 standard deviation. Data shown for meclizine, hydroxyzine, vehicle control. Error bars are 1 standard deviation. Data shown for meclizine, hydroxyzine, cinnarizine, cinnarizine, and flunarizine as indicated. *, **, *** ,**** correspond to p values less than 0.05, 0.001, and ﬂunarizine as indicated. *, **, ***, **** correspond to p values less than 0.05, 0.001, 0.0001, and 0.00001 0.0001, and 0.00001 respectively. n = 3 for each group. p value calculated with Student’s t-test. respectively. n = 3 for each group. p value calculated with Student’s t-test.

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2.2.2.2. Two Two Biophysical Biophysical Assays Assays Confirm Confirm Piperazine Piperazine Interaction Interaction with withHuman Human mTOR mTOR InIn order order to tobetter better-characterize-characterize the thepiperazine piperazine compounds compounds and andmove move forward forward in our in selection our selection of of a a top cancer therapeutic therapeutic candidate, candidate, we we measured measured the the absolute absolute response response of small of small molecules molecules binding binding to tothe the human human mTOR mTOR protein protein with with bio-layer bio-layer interferometry interferometry (BLI). (BLI). As As a a further further confirmation, confirmation, we we plotted plotted the BLI response (pm) against the affinity expressed as the reciprocal of the dissociation the BLI response (pm) against the affinity expressed as the reciprocal of the dissociation constant of the constant of the same interactions as determined by field-effect biosensing (FEB). A linear relationship same interactions as determined by field-effect biosensing (FEB). A linear relationship was observed. was observed. Flunarizine yielded the strongest response and highest affinity to human mTOR Flunarizine yielded the strongest response and highest affinity to human mTOR followed by cinnarizine, followed by cinnarizine, meclizine, hydroxyzine, and cetirizine (Figure 2). Rapamycin is included as meclizine, hydroxyzine, and cetirizine (Figure2). Rapamycin is included as a potent mTORC1 inhibitor; a potent mTORC1 inhibitor; however, rapamycin has a different molecular weight, optical density, andhowever, charge-to rapamycin-mass ratio has that a dilimitsfferent the molecular integrity of weight, comparing optical it to density, the piperazine and charge-to-mass compounds ratioin that theselimits two the biophysical integrity ofassays. comparing it to the piperazine compounds in these two biophysical assays.

Figure 2. Affinity plotted against binding response for members of the piperazine drug class and Figure 2. Affinity plotted against binding response for members of the piperazine drug class and rapamycinrapamycin.. The The Y-axis Y-axis is the is response the response measured measured with withbi-layer bi-layer interferometry. interferometry. Affinity A ffimeasurementsnity measurements generatedgenerated with with field field-e-effectff ectbiosensing biosensing is on is the on X the-axis, X-axis, as indicated. as indicated. The line The is linelinear is linearregression. regression. 2.3. Piperazine Drugs Kill GBMSCs 2.3. Piperazine Drugs Kill GBMSCs The killing potency of four mTORC1-inhibiting piperazine compounds was tested (Figure3). The killing potency of four mTORC1-inhibiting piperazine compounds was tested (Figure 3). KillingKilling was was compared compared to to rapamycin, rapamycin, the the classic classic mTORC1 mTORC1 inhibitor, inhibitor, as well well asas temozolomide,temozolomide, thethe current currentstandard standard of care of for care GBM. for GBM. All four All offour the of tested the tested piperazine piperazine compounds compounds reduced reduced cell viabilitycell viability compared comparedto either to vehicle either control vehicle orcontrol temozolomide. or temozolomide. Meclizine Meclizine and flunarizine and flunarizine were more were toxic more to toxic GBMSC to than GBMSCrapamycin than rapamycin (Figure3). Rapamycin(Figure 3). Rapamycin has been usedhas been in multiple used in multiple GBM clinical GBM trialsclinical [22 trials–25]. [22–25].

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FigureFigure 3.3. PiperazinePiperazine drugsdrugs reducereduce viabilityviability inin thethe patient-derivedpatient-derived GBMSCGBMSC line.line. ExperimentsExperiments werewere completedcompleted used used Trypan Trypan Blue Blue Exclusion. Exclusion. The Thecolumns columns are the are average the average percent viabilitypercent ofviability three biological of three replicatesbiological normalizedreplicates normalized to the vehicle to control.the vehicle Error control. bars are Error the standardbars are the error standard of the mean. error *,of **: thep

2.4. Comparison of GBM Cell Viability after Treatment with Meclizine, Flunarizine, or Rapamycin Meclizine, flunarizine, and rapamycin were tested in GBMSCs at four doses. Meclizine reduced GBMSC viability by nearly 70% at 10 µM. Flunarizine reduced GBMSC viability to a similar extent (Figure 4). Both meclizine and flunarizine were significantly more toxic to GBMSCs than rapamycin, a drug that has been used in several GBM clinical trials [22–25]. The IC50’s for meclizine, flunarizine, and rapamycin were 5.3, 6.8, and 14 µM, respectively (Figure 4).

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2.4. Comparison of GBM Cell Viability after Treatment with Meclizine, Flunarizine, or Rapamycin Meclizine, flunarizine, and rapamycin were tested in GBMSCs at four doses. Meclizine reduced GBMSC viability by nearly 70% at 10 µM. Flunarizine reduced GBMSC viability to a similar extent (Figure4). Both meclizine and flunarizine were significantly more toxic to GBMSCs than rapamycin, a drug that has been used in several GBM clinical trials [22–25]. The IC50’s for meclizine, flunarizine, Pharmaceuticalsand rapamycin 2020 were, 13, x 5.3,FOR 6.8,PEER and REVIEW 14 µ M, respectively (Figure4). 6 of 20

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Figure 4. MeclizineMeclizine and and flunarizine flunarizine dose dose-dependently-dependently kill kill patientpatient-derived-derived GBMSC better than rapamycin. Experiments werewere completedcompleted used used Trypan Trypan Blue Blue Exclusion. Exclusion. Values Values are are cell cell viability viability at 1 atµM 1 (µM6.0) (–6.0) and and 10 µ 10M( µM5.0) (–5.0) of rapamycin,of rapamycin, meclizine, meclizine, and and flunarizine. flunarizine. Error Error bars bars are are the the standard standard error error of − − ofthe the mean. mean. A significantA significant diff differenceerence for for meclizine meclizine v. rapamycin v. rapamycin and and flunarizine flunarizine v. rapamycin v. rapamycin at 10 atµ 10M µMp < 0.05p < 0 was.05 was observed, observed this, this is represented is represented by*. byp -values*. p-values were were calculated calculated using using the two-tailedthe two-tailedt-test. t- test.Lines Lines are nonlinear are nonlinear sigmoidal sigmoidal dose–response dose–response curves curves fitted fitted by the by three-parameterthe three-parameter model. model.n = n6 = for 6 vehicle,for vehicle,n = n4 for= 4 eachfor each concentration concentration rapamycin, rapamycin,n = 6nfor = 6 each for each concentration concentration of meclizine, of meclizine, and n and= 3 n for = 3each for concentrationeach concentration of flunarizine. of flunarizine. The total The numbertotal number of measurements of measurements was 6, was 8, 12, 6, and 8, 12, 6 for and vehicle, 6 for vehicle,rapamycin, rapamycin, meclizine, meclizine, and flunarizine, and flunarizine respectively., respectively. 2.5. Co-Administration of Temozolomide with Meclizine or Flunarizine Potentiates GBMSC Killing 2.5. Co-Administration of Temozolomide with Meclizine or Flunarizine Potentiates GBMSC Killing Temozolomide is the standard-of-care chemotherapeutic agent used in GBM [9]. Temozolomide is Temozolomide is the standard-of-care chemotherapeutic agent used in GBM [9]. Temozolomide used daily for 6 weeks during radiation therapy, and then again after a one-month break, for a further is used daily for 6 weeks during radiation therapy, and then again after a one-month break, for a 6 months, and is given for 5 days per month. further 6 months, and is given for 5 days per month. Because meclizine has a benign safety profile (one tradename is Dramamine) [26–29], we tested the Because meclizine has a benign safety profile (one tradename is Dramamine) [26–29], we tested idea of meclizine as adjuvant therapy, i.e., the ability of piperazine plus temozolomide to kill GBMSCs. the idea of meclizine as adjuvant therapy, i.e., the ability of piperazine plus temozolomide to kill As seen in Figure5, there was a negligible decrease in GBMSC viability at all doses of temozolomide. GBMSCs. As seen in Figure 5, there was a negligible decrease in GBMSC viability at all doses of Even at 100 µM of temozolomide, the cell viability remained above 90%. The four-parameter sigmoidal temozolomide. Even at 100 µM of temozolomide, the cell viability remained above 90%. The four- dose–response model used for nonlinear regression predicted the survival of the cells (Rmin) to be parameter sigmoidal dose–response model used for nonlinear regression predicted the survival of 93.15%. This means that at any pharmacologically relevant concentrations of temozolomide treatment the cells (Rmin) to be 93.15%. This means that at any pharmacologically relevant concentrations of temozolomide treatment alone, there will be no more than a 6.85% reduction in viability of the GBMSC. In other words, the maximum killing potency of temozolomide alone is 6.85% for the GBMSC. Thus, temozolomide alone is not sufficient to combat GBMSC in vitro. Therefore, we tested our top candidates, flunarizine and meclizine, in combination with temozolomide, and observed dose-dependent killing (Figure 5). The tightest mTOR-binding compound, flunarizine, plus temozolomide appeared to be the most potent killing combination. The IC50 of flunarizine + temozolomide was 1 µM and the maximum cell killing potency in this experiment was 82.5%, p = 4.5 × 10–5. For meclizine + temozolomide, the IC50 was 5.7 µM and the maximum killing potency was 66.7%, R2 = 99, p = 7.5 × 10−8. Thus, the maximum killing potency of meclizine in

Pharmaceuticals 2020, 13, 419 7 of 18 alone, there will be no more than a 6.85% reduction in viability of the GBMSC. In other words, the maximum killing potency of temozolomide alone is 6.85% for the GBMSC. Thus, temozolomide alone is not sufficient to combat GBMSC in vitro. Therefore, we tested our top candidates, flunarizine and meclizine, in combination with temozolomide, and observed dose-dependent killing (Figure5). The tightest mTOR-binding compound, flunarizine, plus temozolomide appeared to be the most potent killing combination. The IC50 of flunarizine + temozolomide was 1 µM and the maximum cell killing potency in this experiment was 5 82.5%,Pharmaceuticalsp = 4.5 201020, 1.3 For, x FOR meclizine PEER REVIEW+ temozolomide, the IC was 5.7 µM and the maximum killing7 of 20 × − 50 potency was 66.7%, R2 = 99, p = 7.5 10 8. Thus, the maximum killing potency of meclizine in × − combinationcombination with with temozolomide temozolomide was was 66.7 66.7/6.85%/6.85% = 9.6 = 9.6 times times better better than than TMZ TMZ alone; alone; the the maximum maximum killingkilling potency potency of flunarizineof flunarizine in combinationin combination with with temozolomide temozolomide was was 82.5% 82.5%/6.85%/6.85% = 12 = times12 times better better thanthan TMZ TMZ alone alone (maximum (maximum killing killing potency potency= 100-R = 100/min).-R/min).

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Figure 5. Figure Meclizine5. Meclizine and and flunarizine flunarizine co-administered co-administered with with temozolomide,temozolomide, the current current standard standard of of care care for GBM. Experiments were conducted using Trypan Blue Exclusion in patient-derived GBMsc. for GBM. Experiments were conducted using Trypan Blue Exclusion in patient-derived GBMsc. Experimental points are the average of 3 experiments. Error bars are the standard error of the mean. Experimental points are the average of 3 experiments. Error bars are the standard error of the mean. The curve is a four-parameter-model sigmoidal dose–response curve. The X-axis is drug concentration, The curve is a four-parameter-model sigmoidal dose–response curve. The X-axis is drug while the Y-axis is % viability of GBSC normalized to vehicle control. Best-fitting values for the concentration, while the Y-axis is % viability of GBSC normalized to vehicle control. Best-fitting co-administration of meclizine and temozolomide were: IC50 = 5.7 µM, Rmin = 33.27, R2 = 0.99, values for the co-administration of meclizine and temozolomide were: IC50 = 5.7 µM, Rmin = 33.27, p = 7.5 10 8. Best fitting values for flunarizine and temozolomide were: IC50 = 1.0 µM, Rmin = 17.50, R2 ×= 0.99,− p = 7.5 × 10–8. Best fitting values for flunarizine and temozolomide were: IC50 = 1.0 µM, R2 = 0.99, p = 4.5 10 5. Values for temozolomide alone: IC50 = 3.2 10 5, Rmin = 93.2, R2 = 0.71, Rmin = 17.50, R2× = −0.99, p = 4.5 × 10–5. Values for temozolomide alone:× −IC50 = 3.2 × 10–5, Rmin = 93.2, p-value = 3.5 10 2. n = 4 for vehicle, 3 for each concentration of temozolomide, 3 for each concentration R2 = 0.71,× p-value− = 3.5 × 10–2. n = 4 for vehicle, 3 for each concentration of temozolomide, 3 for each of meclizine + temozolomide, 3 for each concentration flunarizine + temozolomide. The total number of concentration of meclizine + temozolomide, 3 for each concentration flunarizine + temozolomide. The measurements are 3, 12, 12, and 9 for vehicle, temozolomide, meclizine + temozolomide, and flunarizine total number of measurements are 3, 12, 12, and 9 for vehicle, temozolomide, meclizine + + temozolomide, respectively. *, **, ***, and **** correspond to p values less than 0.05, 0.001, 0.0001, temozolomide, and flunarizine + temozolomide, respectively. *, **, ***, and **** correspond to p values and 0.00001 respectively. less than 0.05, 0.001, 0.0001, and 0.00001 respectively.

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2.6. Meclizine Confers Synthetic Lethality on GBM Stem Cell Harboring R132H IDH1 Mutation GBM IDH1 mutations change metabolism and drive mTORC1 activity [17], and patients with GBMs and the IDH1 mutation have low mTORC1 activity and a 10-fold longer survival [16]. Thus, we tested the idea that mTORC1 inhibition via meclizine might cause synthetic lethality in IDH1 mutant cells. Figure6A shows the IDH1 wildtype response to both temozolomide (black line) and meclizine + 10 µM temozolomide (dashed line). No significant difference in cell viability between the vehicle control and highest concentrations of temozolomide was observed. At higher concentrations, there was only about a 7% cell viability reduction observed in temozolomide alone. In experiments with the IDH1 wildtype cells, the co-administration of meclizine and temozolomide significantly decreased cell viability beginning at a 10 µM dose of meclizine. As stated above, the killing potency of meclizine in combination with temozolomide was about 67%, roughly 10 times better than temozolomide alone. Best-fit values for meclizine in combination with temozolomide were IC50: 5.7 µM, (100-Rmin): 66.7%, R2 = 0.99, p = 7.5 10 8, as determined by the four-parameter model. Figure6B shows the × − R132H IDH1 mutant response. The IDH1 mutant appeared to be more sensitive to temozolomide alone than the IDH1 wild type, IC = 3.2 10 6, Rmin = 62.5% R2 = 0.97, p-value = 6.0 10 5; 50 × − × − however, the maximum killing potency was only (100 62.5%) = 38%. While there was a significant − reduction in cell viability at the two highest concentrations of temozolomide which are much higher than achieved in human dosing (30 and 100 µM), temozolomide alone is not very effective for killing GBMSC with the R132H genotype, as all cells survive at any pharmacologically relevant concentration of temozolomide alone. However, a significant reduction in cell viability was observed beginning at 1 µM of meclizine co-administered with 10 µM of temozolomide in the IDH1 mutant GBM stem cell line. Thus, the R1423 IDH1 mutant GBMSC was effectively and dose-dependently killed by the co-administration of meclizine with temozolomide. The IC50 of meclizine in combination with temozolomide was 2 µM, and the maximum killing potency was 95.5%. Therefore, the combination with meclizine was (95%/38%) >2 times more effective at killing the IDH1 GBMSC than temozolomide alone. Thus, the combination of meclizine with temozolomide might be an effective strategy to combat GBMSC, and especially the R132H mutant cells. Confirmatory dose-dependent killing was observed in another pair of IDH1 wt and mutant cell lines; the mutant in this case was a grade III astrocytoma rather than grade IV astrocytoma (Figure A1 in AppendixA). Pharmaceuticals 2020, 13, 419 9 of 18 Pharmaceuticals 2020, 13, x FOR PEER REVIEW 9 of 20

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Log (Conc.) [M] FigureFigure 6. Comparison 6. Comparison of cell of cell viability viability reduction reduction by by temozolomide temozolomide and and by by co co-administration-administration of of meclizinemeclizine and 10 andµM 10 temozolomide µM temozolomide to theto the IDH1 IDH1 wildtype wildtype( A(A),), andand in IDH1 IDH1 R132H R132H mutant mutant (B) ( BGBM) GBM patient-derived stem cells. The X-axis is the concentration of compounds tested. When meclizine was patient-derived stem cells. The X-axis is the concentration of compounds tested. When meclizine co-administered, the concentration of temozolomide was fixed at 10 µM. Experimental points are the was co-administered, the concentration of temozolomide was fixed at 10 µM. Experimental points are average of 3 independent experiments; error bars are the standard error of the mean. The lines are the the average4-parameter of 3 independent-model sigmoidal experiments; dose–response error curves. bars *, are **, **** the p standard < 0.05, 0.01 error, 0.001 of, 0 the.0001 mean., respectively. The lines are theBest 4-parameter-model-fitting values for meclizine sigmoidal + temozolomide dose–response co-administration curves. *, **, in **** wildtypep < 0.05, (A) 0.01,were:0.001, IC50 = 0.0001,5.7 respectively.µM, Rmin Best-fitting = 33.27, R2 values= 0.99, p for= 7.5 meclizine × 10–8. n = 4+ fortemozolomide vehicle, n = 3 for co-administration each concentration inof meclizine wildtype + (A) were:temozolomide. IC50 = 5.7 µM, Best Rmin-fit values= 33.27, for temozolomide R2 = 0.99, palone= 7.5 in wildtype10 8. (nA)= were:4 for IC50 vehicle, = 3.2 ×n 10=–5, 3Rmin for each= × − concentration93.2, R2 = of 0.71 meclizine, p-value =+ 3.5temozolomide. × 10–2. n = 4 for vehicle, Best-fit and values 3 for for each temozolomide concentration of alone temozolomide in wildtype for (A) were:a IC50 total =of 3.212 concentrations10 5, Rmin of =temozolomide.93.2, R2 = 0.71, Best-fitp-value values =for3.5 meclizine10 2 co. -nadministered= 4 for vehicle, in mutant and 3 × − × − for each(B) concentration were: IC50 = 1.9 of µM, temozolomide Rmin = 4.1%, R2 for = a0.99, total p-value of 12 = concentrations 2.0 × 10–6. n = 10 offor temozolomide. vehicle. n = 4 for each Best-fit valuesconcentration for meclizine of co-administered meclizine + temozolomide in mutant for (B ) a were: total of IC50 16 = measurements.1.9 µM, Rmin Best= -4.1%,fit values R2 = for0.99, –6 –5 temozolomide6 in mutant (B) were: IC50 = 3.3 × 10 , Rmin = 62.5%, R2 = 0.982, p–value = 6.1 × 10 . n = p-value = 2.0 10− . n = 10 for vehicle. n = 4 for each concentration of meclizine + temozolomide for 1 for vehicle.× n = 6 for each concentration of temozolomide (24 measurements in total). a total of 16 measurements. Best-fit values for temozolomide in mutant (B) were: IC50 = 3.3 10 6, × − Rmin = 62.5%, R2 = 0.982, p-value = 6.1 10 5. n = 1 for vehicle. n = 6 for each concentration of 3. Discussion × − temozolomide (24 measurements in total).

Pharmaceuticals 2020, 13, 419 10 of 18

3. Discussion We recently demonstrated that a series of piperazine compounds are also mTORC1 inhibitors [19]. mTORC1 activity is a validated clinical target, and the mTORC1 inhibitor rapamycin, everolimus, and temsirolimus have been used in clinical trials of GBM [30–32]. Rapamycin, the most common mTORC1-specific inhibitor, has been approved for numerous clinical trials; however, rapamycin has an extensive side-effect profile [33]. This extensive side-effect profile may limit its effectiveness. Thus, the discovery of a novel class of mTORC1 inhibitors, such as meclizine with a lower toxicity profile than rapamycin, may be a relevant candidate for novel GBM therapy. As seen in AppendixA Figure A2, Rapamycin, through its mTORC1 inhibition, is an effective killer of 2 IDH1 wt GBMSC and of 1 IDH1 mutant GBMSC and another IDH1 mutant grade III astrocytoma stem cell line. In other words, we observe an increase in stem cell death with the increase in mTORC1 inhibition. This observation emphasizes the significance of novel mTORC1 inhibitors with milder safety profiles and similar potency to rapamycin as mTORC1 inhibition has shown benefits ranging from in vitro studies to human clinical trials [34,35]. Cinnarizine and flunarizine were two of the tightest binders to mTORC1, and were toxic to GBMSCs in our assays, having been shown by others to kill lymphomas and myeloma cells as well [36,37]. However, cinnarizine and flunarizine both have a chemical liability in that after prolonged exposure, they can cause iatrogenic parkinsonism [36–38], and are less likely to win FDA approval, even for a very severe cancer. Thus, we tested the potency of our tightest mTORC1-binders with milder side-effect profiles, meclizine, toward killing GBMSC. We also conducted these tests, though less extensive, on flunarizine as it was our tightest binder overall. Both meclizine and flunarizine performed better than rapamycin. If a clinical trial were ever to be carried out using meclizine in GBM, ethical considerations would require it be co-administered alongside the standard-of-care drug temozolomide, as has been done with other mTORC1 clinical trials [24,39]. Meclizine and flunarizine when co-administered with temozolomide greatly increased cytotoxicity, suggesting them as possible adjuvant therapies with a low side-effect profile. Meclizine is an antihistamine prescribed to treat nausea and motion sickness; flunarizine was previously prescribed to treat vertigo and migraines [40,41]. Due to meclizine’s mild safety profile, precedence in human dosing, and similar potency in reducing cell viability when compared to flunarizine and rapamycin, we propose meclizine as a potential GBM therapeutic agent. Additionally, meclizine has been well documented to cross the blood–brain barrier, an essential characteristic for treating GBM [42]. Nonetheless, we do not diminish the potential of flunarizine as a potential therapeutic agent as well. Further experiments in mouse models of GBM are necessary before implementing meclizine, or any other piperazine compound, as a cancer therapeutic. However, as safety and pharmacological studies have been extensively conducted, the timeline to clinical trial should be more straightforward than for a new chemical entity. Additionally, we observed synthetic lethality when comparing the cell viability response of IDH1 wildtype GBMSCs and those bearing the R132H IDH1 mutation. Mutant cells have more 2-hydroxyglutarate, which mediates energy depletion and activated AMPK, which in turn, represses mTOR activity [43]. Seeing as alpha-ketoglutarate levels are reduced, indicating lower nutrient levels, a further decrease in mTORC1 through direct inhibition may be sufficient to remarkably reduce cell viability and proliferation when compared to IDH1 wildtype GBM stem cells. Elevated 2-HG levels also inhibit the DNA repair enzyme ALKBH and the DNA damage response proteins KDM4A/B[44,45]. Temozolomide, the standard-of-care chemotherapeutic agent for treating GBM, is a DNA alkylating agent, meaning that it targets GBM tumors by damaging DNA [46]. Clinically, IDH1 mutations are associated with a better response to temozolomide treatment, a fact supported by our temozolomide dose–response experiment in IDH1 mutant GBM cells (Figure6B) [ 43,47]. The combination of an increased sensitivity to temozolomide and compound inhibition of mTORC1 by two means provide a molecular basis for IDH1 R132H synthetic lethality to the described treatment. Pharmaceuticals 2020, 13, 419 11 of 18

The need for additional cell lines is recognized as a limitation of the study. We have conducted less extensive studies on a subsequent IDH1 WT GBMSC labeled “GB SC2” in the appendix figures. In addition, we also conducted tests on an IDH1 mutant Grade III astrocytoma patient-derived cell line in the appendix figures. Though comparisons between Grade III astrocytoma and GBM, which is a grade IV astrocytoma, are not fair, results from the astrocytoma cell line are still interesting as Grade III astrocytoma develop into Glioblastoma. However, these experiments do confirm the benefit of the co-administration of meclizine with temozolomide in vitro and support their use as adjuvant therapy for GBM. In other words, meclizine co-administered with temozolomide killed more than temozolomide alone in three patient-derived GBM stem cell lines and in one grade III astrocytoma patient-derived cell line. Nonetheless, more primary cells harboring the IDH1 R132H mutation, either patient-derived or WT-derived, are necessary to sufficiently demonstrate that IDH1 mutant cells are more sensitive to mTORC1 inhibition than wildtype cells.

4. Materials and Methods

4.1. Cell Lines and Cell Culture The Mus musculus (mouse) normal hepatocyte liver cell line (FL83B) was purchased from American Type Culture Collection (Manassas, VA, USA). FL83B cells were cultured in Dulbecco’s modified Eagle’s medium/Ham’s F-12 50/50 Mix with l-glutamine and 15 mM HEPES and 10% fetal bovine serum (FBS), both from Corning (Fremont, CA, USA). The cell line was grown at the standard 37 ◦C and 5% CO2 conditions. All drug treatment was performed in the absence of pen/strep and FBS. The GBMSC lines used were patient-derived from recurring tumors and gifted to us by Kevin Woolard DVM, PhD from the University of California Davis. The cell lines were named 0827 and 0923. 0827 is the only IDH1 WT cell line used in the main figures in the manuscript and is referred to as IDH1 wildtype in Figure6. 923 is used only in appendix and is referred to as “IDH1 WT” in AppendixA Figure A1 and “GB SC 2” in AppendixA Figure A2. The 0827 and 923 GBM stem cells were cultured in Neurobasal A minimal media with 1X N-2 supplement, 1X B-27 supplement, 1X GlutaMAX, and (50 units/mL of penicillin/50 µg/mL of streptomycin) from Gibco (Gaithersburg, MD, USA), and human EGF and FGF-154 from Shenandoah Biotechnology (Warwick, PA, USA). The IDH1 mutant cell line was named 905 and will be referred to as IDH1 mutant or R132H IDH1 mutant. It is the only IDH1 mutant cell line used in the main figures. The IDH1 mutant Grade III astrocytoma was named BT 142 and is only used in the appendix figures. It is referred to as “IDH1 mutant” in AppendixA Figure A1 and “IDH1 mt 2” in AppendixA Figure A2. These cells were cultured in the same media as the IDH1 wildtype with the addition of 1 mL of 10 µg/mL PDGF. Cells used did not exceed passage 15. Patient-derived glioma stem cells were generated at the National Cancer Institute, National Institutes of Health (Bethesda, MD). This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research and approved by the Institutional Review Board [48,49]. The stemness profile of IDH1 wildtype cells are included as a appendix figure (Figure A3). GBM-differentiated cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and pen/strep.

4.2. Compounds All chemicals (DMSO, insulin, penicillin/streptomycin) were purchased from Sigma-Aldrich, unless indicated otherwise. Rapamycin was purchased from EMD Millipore (Billerica, MA, USA). Pharmaceuticals 2020, 13, 419 12 of 18

4.3. Bi-Layer Interferometry Proteins we puriﬁed from HEK T293 cells. Terminally biotinylated mTOR protein and the GFP as a nonbinding control were produced using the following plasmids, for mTOR—EX Mm31144-M48, and for GFP—EX-EGFP-M48 GeneCopoeia (Rockville, MD, USA), and loaded onto sets of Octet RED 384 SSA biosensors (Pall ForteBio LLC., Menlo Park, CA, USA) to a density of 12 nm followed by the blocking of nonoccupied streptavidin residues of biosensors with 200 u biotin. Sensors were tested against indicated concentrations of compounds using the Octet RED 384 BLI instrument in BLI Kinetic Buﬀer (Pall ForteBio LLC., Menlo Park, CA, USA) containing 1% BSA. Parameters: Baseline 20 s, association 30 s, dissociation 40 s. Real-time binding sensorgrams were recorded and analyzed using Octet BLI software 8.1 (Pall ForteBio LLC., Menlo Park, CA, USA) and Excel.

4.4. Field-Effect Biosensing The human 6His::mTOR was purified from HEK T293 cells using plasmid Ex-E1870-M01-GS GeneCopoeia (Rockville, MD, USA) and loaded onto the Ni-NTA chip Nanomedical Diagnostics (San Diego, CA, USA); compounds were tested at 10 µM concentration in triplicates; kon, koff, and KD were determined using Nanomedical Diagnostics Agile Plus Version 4.2.2.20754 software, Nanomedical Diagnostics (San Diego, CA, USA). The association time was 5 min, the dissociation time was 10 min, and the baseline was 15 min.

4.5. Cell Viability Assay 0827 IDH1 WT GBM, 923 IDH1 WT GBM, 905 IDH1 mutant GBM, and BT142 gIII astrocytoma stem cells were seeded, 300,000 cells/well, in 12-well cell culture plates and allowed to grow for 24 h. Cells were treated with vehicle (0.1% DMSO) or compounds dissolved in DMSO for 48 h before being assayed. Cells in media plus drug were then removed and centrifuged at 1200 rpm for 3 min. Media were aspirated oﬀ, and 0.5 mL of 0.05% trypsin was added to each well, incubated for 3 min, and then neutralized with serum containing media. Samples were re-suspended and pipetted thoroughly to create single cell suspensions for analysis. We utilized the Vi-CELLTM Cell Viability Analyzer from Beckman Coulter (Indianapolis, IN, USA) to evaluate cell viability and survival of both GBM stem cells after drug treatment.

4.6. pS6K ELISA The PathScan® Phospho-S6Kinase (Thr389) Sandwich ELISA and PathScan® Total S6K Sandwich ELISA Kits were purchased from Cell Signaling Technologies (Danvers, MA). In addition, 200,000 cells/well were seeded onto 24-well plates and allowed to grow for 48 h; the media were changed to DMEM-F12 without FBS for the FL83B and standard Neurobasal for the Stem Cells, and cells were incubated for another 18 to 20 h. Cells were treated for two hours with tested compounds at indicated concentrations; the media were aspirated, and plates were washed once with ice-cold PBS. Cells were lysed with 200 µL of lysis buﬀer; lysates were transferred to a 96-well plate for ELISA. ELISA was performed according to manufacturer’s instructions.

4.7. Co-Immunoprecipitation The HEK cells were co-transfected with N-terminally His-tagged Human mTOR Gene Copoeia Cat# EX-Mm31144-M48) and C-terminally Avi-tagged Human RAPTOR (Gene Copoeia Cat# EX-E0449-M62). The IP was performed with Ni-NTA magnetic beads (Thermo Fisher, Cat# 88831). Westerns were developed using IRDye® 680RD Streptavidin (LiCor Biosciences, Cat# 926-68079) and anti-mTOR antibody, before the protein isolation cells were pre-treated with a 10 µM concentration of indicated drugs for 2 h. The bands intensities were quantiﬁed using Image Studio Software (Licor Biosciences Lincoln, Nebraska). Pharmaceuticals 2020, 13, 419 13 of 18

4.8. Western Blot GBM cells were lysed using Cell Lysis buffer from Cell Signaling Technologies (Danvers, MA, USA) with 1X Halt™ Protease and Phosphatase Inhibitor Cocktail from ThermoFisher Scientific (Waltham, MA). Nitrocellulose membranes were incubated overnight with the following antibodies diluted in blocking buffer: 1:5000 dilution of anti-actin antibody, mouse monoclonal from Sigma-Aldrich, and 1:1000 Sox2 Rabbit mAb 1:1000; Nestin (Rat-401) Mouse mAb, β3-Tubulin (D71G9) XP® Rabbit mAb, and GFAP (D1F4Q) XP® Rabbit mAb all from Cell Signaling Technologies (Danvers, MA). Membranes were then incubated with the following secondary antibody pair: IRDye 680CW and IRDye 800CW-coupled secondary antibodies from LI-COR Biosciences (Lincoln, NE, USA) at 1:20,000 dilution. Western Blots were then visualized with the Odyssey infrared imager and software from LI-COR Biosciences (Lincoln, NE) according to the manufacturer’s instructions.

4.9. Data Analysis Graphpad Prism 9.0 and Microsoft Excel were used for the statistical analysis. The three- or four-parameter sigmoidal dose–response model was used for ﬁtting analysis (as indicated in legend). Signiﬁcance was assigned as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. All p-values were generated through Student’s t-test.

Author Contributions: Verification of mTORC1 inhibition—S.A.; B.L.I. and F.E.B.—A.T.; Single-Dose Cell Viability Experiment—S.A. and J.A.S. Multiple-Dose Cell Viability and IDH1 Comparison—J.A.S.; Consultation—A.T., S.D., R.O., J.M.A.; AppendixA Figure A1—J.A.S.; AppendixA Figure A2—J.A.S. AppendixA Figure A3—T.S., K.W.; AppendixA Figure A4—A.T.; AppendixA Figure A5—D.K. Overseeing entire project—G.C. All authors have read and agreed to the published version of the manuscript. Funding: This research received support from P30CA093373 to UCDMC. Presented work was supported by unrestricted funds and foundation awards. Pharmaceuticals 2020, 13, x FOR PEER REVIEW 14 of 20 Conflicts of Interest: The authors declare no conflict of interest. Appendix A Appendix A

Figure A1. Cont.

Figure A1. Coadministration of temozolomide and meclizine is more toxic than temozolomide alone. (A) Temozolomide and meclizine co-administered with temozolomide, the current standard of care, in a subsequent GBM IDH1 wildtype cell line and a (B) IDH1 mutant World Health Organization (WHO)-grade III astrocytoma. Experiments were conducted using Trypan Blue Exclusion in patient-derived GBMsc. Experimental points are an average of 3 experiments. Error bars are the standard error of the mean. The curve is a 3-parameter-model sigmoidal dose–response curve. The X-axis is drug concentration, while the Y-axis is % viability of GBMsc normalized to vehicle control. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Pharmaceuticals 2020, 13, x FOR PEER REVIEW 14 of 20

Appendix A

Pharmaceuticals 2020, 13, 419 14 of 18

FigureFigure A1. Coadministration A1. Coadministration of temozolomide of temozolomide and and meclizine meclizine is is more more toxic toxic than than temozolomide temozolomide alone. (alone.A) Temozolomide (A) Temozolomide and meclizineand meclizine co-administered co-administered with with temozolomide, temozolomide, the the current current standard standard of care,of in care, a subsequent in a subsequent GBM GBM IDH1 IDH1 wildtype wildtype cell cell line line and and a a ((BB)) IDH1IDH1 mutant mutant World World Health Health OrganizationOrganization (WHO)-grade (WHO)- IIIgrade astrocytoma. III astrocytoma. Experiments Experiments were conductedwere conducted using Trypan using TrypanBlue Exclusion Blue in patient-derivedExclusion in patient GBMsc.-derived Experimental GBMsc. Experimental points are points an average are an average of 3 experiments. of 3 experiments. Error Error bars bars are the standardare the errorstandard of theerror mean. of the mean. The curve The curve is a is 3-parameter-model a 3-parameter-model sigmoidal sigmoidal dose dose–response–response curve. curve. The X-axis is drug concentration, while the Y-axis is % viability of GBMsc normalized to vehicle PharmaceuticalsThe X-axis 2020, 1 is3,drug x FOR concentration, PEER REVIEW while the Y-axis is % viability of GBMsc normalized to vehicle control.15 of 20 * p < 0.05,control. ** p < * p0.01, < 0.05, *** **p p< <0.001, 0.01, *** **** p

FigureFigure A2. A2. TheThe mTORC1 mTORC1 inhibitor inhibitor rapamycin rapamycin is dose-dependently is dose-dependently toxic to toxic GBMSCs. to GBMSCs (A) Rapamycin,. (A) Rapamycin,also known alsomTORC1-speciﬁc known mTORC1-specific inhibitor, inhibitor, dosed dosed at increasing at increasing dosages dosage in 2s in patient-derived 2 patient-derived IDH1 IDH1wildtype wildtype GBMsc GBMsc and and in( Bin) ( 2B IDH1) 2 IDH1 mutant mutant cell cell lines, lines, with with IDH1 IDH1 mt 1 mt being 1 being a patient-derived a patient-derived GBMsc GBMscand and IDH1 IDH1 mt mt 2 a 2 patient-deriveda patient-derived WHO-grade WHO-grade III III astrocytoma,astrocytoma, which which is is known known to todevelop develop into into glioblastomaglioblastoma (WHO (WHO grade grade IV astrocytoma). astrocytoma). Experiments Experiments were were conducted conducted using using Trypan Trypan Blue Exclusion Blue Exclusionin patient-derived in patient-derived GBMsc. GBMsc. Experimental Experimental points points are anare averagean average of 3of experiments. 3 experiments. Error Error bars bars are arethe the standardstandard error of of the the me mean.an. The The curve curve is isa 4 a-parameter 4-parameter-model-model sigmoidal sigmoidal dose dose–response–response curve. curve. TheThe X-axis X-axis is is drug drug concentration concentration,, while while the Y-axisY-axis is is % % viability viability of GBMscof GBMsc normalized normalized to vehicle to vehicle control. control.

Figure A3. GBMSCs have appropriate stemness markers. Western Blot showing stemness of cells used in manuscript. 0827 and 0923 refer to IDH1 wt 1 and IDH1 wt 2, respectively. NBE refers to the cells in the “stem” state, and “Diff” refers to the cells after ten days of culture in media containing 10% FBS. Cells were in the “NBE” or “stem” state for all experiments presented in the manuscript. Molecular markers for stemness included are sox2 and Nestin. Markers of differentiation are TUJ-1 and GFAP. Actin served as the loading control.

Pharmaceuticals 2020, 13, x FOR PEER REVIEW 15 of 20

Figure A2. The mTORC1 inhibitor rapamycin is dose-dependently toxic to GBMSCs. (A) Rapamycin, also known mTORC1-specific inhibitor, dosed at increasing dosages in 2 patient-derived IDH1 wildtype GBMsc and in (B) 2 IDH1 mutant cell lines, with IDH1 mt 1 being a patient-derived GBMsc and IDH1 mt 2 a patient-derived WHO-grade III astrocytoma, which is known to develop into glioblastoma (WHO grade IV astrocytoma). Experiments were conducted using Trypan Blue Exclusion in patient-derived GBMsc. Experimental points are an average of 3 experiments. Error bars are the standard error of the mean. The curve is a 4-parameter-model sigmoidal dose–response curve. Pharmaceuticals 2020The, 13 X,- 419axis is drug concentration, while the Y-axis is % viability of GBMsc normalized to vehicle 15 of 18 control.

Figure A3. GBMSCs have appropriate stemness markers. Western Blot showing stemness of cells Figure A3. GBMSCs have appropriate stemness markers. Western Blot showing stemness of cells used used in manuscript. 0827 and 0923 refer to IDH1 wt 1 and IDH1 wt 2, respectively. NBE refers to the in manuscript.cells 0827in the and“stem” 0923 state, refer and to“Diff” IDH1 refers wt to 1the and cells IDH1 after ten wt days 2, respectively. of culture in media NBE containing refers to the cells in the “stem” state,10% FBS. and Cell “Dis wereﬀ” in refers the “NBE” to the or “stem” cells after state for ten all days experiments of culture presented in media in the manuscript. containing 10% FBS. Cells were inMolecular the “NBE” markers or for “stem” stemness state included for allare sox2 experiments and Nestin. presentedMarkers of differentiation in the manuscript. are TUJ-1 Molecular and GFAP. Actin served as the loading control. markers for stemness included are sox2 and Nestin. Markers of diﬀerentiation are TUJ-1 and GFAP. Pharmaceuticals 2020, 13, x FOR PEER REVIEW 16 of 20 Actin served as the loading control.

Vehicle Vehicle Vehicle Meclizine Cinnarizine Nebivolol Cetrizine B. 10µM 10µM 10µM 10µM

RAPTOR

mTOR

Figure A4. Piperazine molecules significantly block the association of Raptor with mTOR by co- Figure A4. Piperazine molecules significantly block the association of Raptor with mTOR by immunoprecipitation. Co-immunoprecipitation showing mTOR and RAPTOR, component of co-immunoprecipitation.mTORC1 complex. Experiment Co-immunoprecipitation shows reduced loading of showing RAPTOR onto mTOR mTOR and when RAPTOR, treated with component of mTORC1m complex.eclizine, cinnarizine, Experiment and nebivolol shows at 10 reduced µM. This provides loading a potential of RAPTOR mechanism onto for mTORC1 mTOR- when treated with meclizine,specific inhibiti cinnarizine,on by piperazine and compounds nebivolol as atRAPTOR 10 µM. is only This associated provides with mTOR a potential Complex 1. mechanism for (A) The quantification of the gel, (B) bars are means and SD of 2 independent experiments. p-value mTORC1-specificcalculated using inhibition two-tailed by t-test. piperazine Meclizine, compoundscinnarizine, and asnebivolol RAPTOR were significantly is only associated inhibiting with mTOR Complex 1.the( associationA) The quantification of mTOR with RAPTOR, of the gel,but not (B )cetirizine. bars are Alpha means was andunder SD 5%. of * and 2 independent ** refer to p- experiments. p-value calculatedvalues less than using 0.05 and two-tailed 0.01 respectively.t-test. Meclizine, cinnarizine, and nebivolol were significantly inhibiting the association of mTOR with RAPTOR, but not cetirizine. Alpha was under 5%. * and ** refer to p-values less than 0.05 and 0.01 respectively.

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Pharmaceuticals 2020, 13, x FOR PEER REVIEW 17 of 20

FigureFigure A5. AA5. A pose pose of of meclizine meclizine within the the kinase kinase domain domain of mTOR. of mTOR.

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