Using Chemistry to Target Neuroblastoma - ACS Chemical

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Using Chemistry to Target Neuroblastoma Jeanne N. Hansen, Xingguo Li, Y. George Zheng, Louis T. Lotta, Abhishek Dedhe, and Nina F. Schor ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00258 • Publication Date (Web): 11 Aug 2017 Downloaded from http://pubs.acs.org on August 13, 2017

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ACS Chemical Neuroscience

Using Chemistry to Target Neuroblastoma

Jeanne N. Hansen1, Xingguo Li1, Y. George Zheng2, Louis T. Lotta, Jr. 1, Abhishek Dedhe1, and Nina F. Schor1 1

2

Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, NY, 14642, U.S.A.

Department of Pharmaceutical and Biochemical Sciences, University of Georgia, Athens, Georgia, 30602, U. S. A.

Keywords: Neuroblastoma; Diamidines; Asymmetric dimethylarginine; Reactive oxygen species; Kidins220 [See Textbox 1.]

Address correspondence to: Nina F. Schor, MD, PhD Department of Pediatrics Golisano Children’s Hospital 601 Elmwood Avenue, Box 777 Rochester, NY 14642 Tel.:

585-275-4673

Fax:

585-273-1079

E-mail:

[email protected] 1 ACS Paragon Plus Environment


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ABSTRACT

Neuroblastoma is a cancer of the neural crest almost exclusively seen in childhood. While children with single, small primary tumors are often cured with surgery alone, the 65% of children with neuroblastoma whose disease has metastasized have less than a 50% chance of surviving five years after diagnosis. Innovative pharmacological strategies are critically needed for these children. Efforts to identify novel targets that afford ablation of neuroblastoma with minimal toxicity to normal tissues are underway. Developing approaches to neuroblastoma include those that target the catecholamine transporter; ubiquitin E3 ligase; the ganglioside, GD2; the retinoic acid receptor; the protein kinases ALK and Aurora; and protein arginine Nmethyltransferases. Here, as examples of the use of chemistry to combat neuroblastoma, we describe targeting of: the protein arginine N-methyltransferases and their role in prolonging the half-life of the neuroblastoma oncoprotein N-Myc; redox signaling in neuroblastoma; and developmentally regulated proteins expressed in primitive neuroblastoma cells but not in mature neural crest elements.

2 ACS Paragon Plus Environment

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Neuroblastoma is a cancer of the cells of the neural crest. It occurs almost exclusively in children. While children with neuroblastoma that is limited to a small primary tumor and infants with neuroblastoma with metastases limited to the liver, bone marrow, and/or skin fare extremely well, the nearly 70% of children with metastatic neuroblastoma at the time of diagnosis have only a 50% chance of living 5 years after diagnosis.1 The search continues for new prognostic markers and therapeutic targets that help identify neuroblastoma early in its course and that enable therapies with maximal efficacy and minimal toxicity. Developing approaches to neuroblastoma include those that target the catecholamine transporter; ubiquitin E3 ligase; the ganglioside, GD2; the retinoic acid receptor; the protein kinases ALK and Aurora; and protein arginine N-methyltransferases.2 Here, to exemplify the use of chemistry to combat neuroblastoma, we propose strategies to target, in turn, the protein arginine N-methyltransferases and their role in prolonging the half-life of the neuroblastoma oncoprotein N-Myc; redox signaling in neuroblastoma; and developmentally regulated proteins expressed in primitive neuroblastoma cells but not in mature neural crest elements. [Place Textbox 1 about here.]

PROTEIN ARGININE METHYLATION AS A TARGET

Protein Arginine N-methyltransferases (PRMTs) PRMTs transfer a methyl group from S-adenosylmethionine (SAM) to arginine residues in proteins. They are divided into three types depending on the stereochemistry of the transferred methyl groups. Type I PRMTs (PRMT1, -3, -4, -6, and -8) form asymmertric dimethylarginines; type II PRMTs (PRMT5 and -9) form symmetric dimethylarginines; and the type III PRMT,



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PRMT7, forms only monomethylarginines. PRMTs are evolutionarily conserved in eukaryotes and expressed in most organs in mammals. Structurally, PRMT is composed of a Rossmann fold domain for SAM binding and a β-barrel domain for substrate binding.3 Beyond this catalytic domain, most PRMTs also contain a variable N-terminal region; for example, the SH3 domain in PRMT2, the zinc finger of PRMT3, the PH domain in CARM1, and the TIM barrel in PRMT5. These unique sequences are likely involved in recruitment of interactive proteins and formation of homo-oligomers, and may also take part in substrate binding.4 Further, Both PRMT7 and PRMT9 contain a pseudo-catalytic domain at their C-terminus. The activity of PRMTs has been implicated in oncogenesis of several kinds of cancer, including breast, colon, prostate and lung cancers and acute myeloid leukemia.5

PRMTs in Normal Neural Crest Development and Neuroblastoma The reaction catalyzed by PRMTs is of importance during development of the nervous system. PRMT1 knockout is embryonic lethal6 and a CNS-specific conditional knockdown (heterozygous gene deletion) results in widespread hypomyelination in the central nervous system.7 In the case of retinoid-induced neuronal differentiation, PRMT1 plays a role in governing the potency of retinoic acid in modulation of transcription at specific “hot spots” on the genome.8 PRMT1 is also highly expressed in normal ganglion cells in the myenteric plexus, and is absent in the colonic mucosa of patients with aganglionic Hirschsprung’s disease, a disorder characterized by congenital absence of ganglion cells in the neural plexuses in the wall of the distal segment of the colon.9 Mouse embryos engineered to carry a null mutation in the Prmt1 gene do not develop beyond day E6.5, but embryonic stem cells from them are both viable and PRMT1-deficient,


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demonstrating only 15% of the native methyltransferase activity and 46% of the native asymmetric NG,NG-dimethylarginine levels, presumably from other type I PRMTs.6 On the other hand, overexpression of PRMT1 is a harbinger of chemotherapeutic resistance and poor prognosis in neuroblastoma.10 Several mechanisms have been implicated in the role of PRMTs in neural crest development. Histone methylation alters the transcriptome of normal and cancer cells. This was first shown for β-globin gene expression, in which PRMT1-mediated dimethyl H4R3 facilitates histone acetylation and enhancer/promoter communications, and leads to the efficient recruitment of transcription preinitiation complexes to active β-globin gene promoters.11 The oncogene MYCN encodes a basic helix-loop-helix oncoprotein, N-Myc, expressed most notably in neuroblastomas. High levels of N-Myc are associated with poor prognosis in neuroblastoma.12 Asymmetric dimethylation by PRMT1 of arginine 65 in the oncoprotein NMyc prolongs the half-life of N-Myc by enhancing dual phosphorylation of N-Myc at tyrosine 58 and serine 62. Only after dephosphorylation at serine 62 is tyrosine 58-phosphorylated N-Myc targeted for ubiquitination and degradation.10 N-Myc is also symmetrically dimethylated and stabilized by PRMT5, but the methylation sites and their functional roles remain to be determined.13 The potential for modulation of N-Myc half-life and consequent elevation of cellular levels of N-Myc by PRMT1 make PRMT1 an attractive potential target for neuroblastoma therapy. [Place Textbox 2 about here.] Methylation by PRMT1 of Smad/1/5/8 facilitates its phosphorylation and participation in antiapoptotic signaling, enhancing the resistance of neuroblastoma cells from oxidative death as occurs after treatment with some chemotherapeutic agents. Induction of oxidative stress in



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neuroblastoma cells using iron decreased the PRMT1 content of the cells and enhanced the chemotherapeutic responsiveness of neuroblastoma cells.14 It has been inferred that downregulation or inhibition of PRMT1 might make neuroblastoma cells more responsive to chemotherapeutic agents.

Inhibitors of PRMT1 as Therapy for Neuroblastoma The realization of the role of PRMT1 in oncogenesis has led to efforts to suppress the activity of PRMT1 towards the conquest of cancer. The uniqueness of the tissue expression profiles and the structures of the active site of each PRMT and, potentially, each relevant substrate thereof hold the promise of enabling specifically targeted pharmacotherapies.15 Type I-selective PRMT inhibitors have been developed and used as anti-parasitic agents, but most of these are believed to be efficacious because they hit other targets in addition to PRMTs.16-18 Perhaps the best understood of these are polyamine transporters15, NMDA receptors17, and ETS family transcription factors18. Recent efforts have focused on enhancing the specificity and potency of these compounds and minimizing off-target effects. Diamidines are one of the best studied classes of type Iselective PRMT inhibitors. These compounds are composed of two terminal benzamidine groups connected through a central aryl or alkyl linker (Fig. 1). The amidine moiety is a structural mimic of the side-chain guanidino group of the PRMT substrate, arginine. Although not yet experimentally confirmed, the amidine group may act as a substrate-competitive functional group for PRMT binding in the active site. Indeed, molecular modeling studies support interaction of the diamidine functionality with the acidic glutamate residues in the catalytic site that are essential for substrate binding. Thus far, several potent diamidine


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compounds have been developed for selective PRMT1 inhibition. These include stilbamidine19, furamidine20, SKLB63921, and decamidine22. For the alkyl chain-linked diamidine inhibitors of PRMT1, the alkyl chain length is critical, with the order of decreasing activity being n=10 > 6 > 5.22 Steric hindrance appears to be an issue, as alkyl chains off of which come bulky constituents are less efficacious than their parent compounds. Substitution of sulfur for the oxygen of the central furan ring does not appear to alter the anti-PRMT activity of diamidines.15,20 We have studied the effects of a number of PRMT1-selective diamidines and related compounds on neuroblastoma cell lines in culture. Our preliminary study indicates that the structure-activity relationships of PRMT inhibitors vis-à-vis growth of neuroblastoma cell cultures (Fig. 1) reflect the structure-activity relationships of these compounds vis-à-vis enzyme activity in cell-free assays. This finding is consistent with the hypothesis that, despite the several known activities of diamidines, their anti-neuroblastoma activity relates, at least in part, to their inhibition of PRMT1. In light of our observation that PRMT1 knockdown or inhibition decreases the cellular halflife of N-Myc10, we have also begun studies of the differential anti-neuroblastoma activity of PRMT1 inhibitors in MYCN-amplified and MYCN-non-amplified cell lines. It is interesting but of unclear significance that alkyl and aromatic diamidines appear in this preliminary study to have opposite differential effects on MYCN-amplified and MYCN-non-amplified neuroblastoma cell lines (Fig. 2). Studies are currently ongoing to examine the in vivo activity and structure-activity relationships of diamidines in neuroblastoma-bearing mice. In addition, others have begun studying inhibition of the binding of N-Myc to Max or Aurora kinase A, both of which also prevent degradation of N-Myc in the cell.23.24



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REDOX SIGNALING AS A TARGET

Fenretinide (4-hydroxyphenyl retinamide; 4-HPR) The occurrence of congenital neuroblastoma and the presence of neuroblastic rests that spontaneously differentiate or undergo apoptosis in normal infants led to the notion that neuroblastoma represents an arrest in the process of differentiation of neural crest cells. This, in turn, led to efforts to treat neuroblastoma by inducing differentiation in these cells. Retinoic acid has been used in this regard and remains a component of some chemotherapeutic regimens for neuroblastoma.1,2 In efforts to improve upon the pharmacodynamic characteristics of retinoic acid, a series of synthetic retinoids have been piloted as well. One, 4-HPR, proved to be quite efficacious in vitro and has been studied with promising results in patients with neuroblastoma.25 This turned out to be surprising, however, because 4-HPR is, at best, a weak ligand for retinoic acid receptors. Furthermore, 4-HPR induces apoptosis, not differentiation, in neuroblastoma cells.26,27 [Place Textbox 3 about here.] The mechanism by which 4-HPR kills neuroblastoma cells is thought to involve accumulation of reactive oxygen species, particularly in the mitochondria, in turn thought to be the result of “leakiness” of mitochondrial Complex II.28 Induction of apoptosis by 4-HPR also involves upregulation of MKK4 and MEKK1, and phosphorylation of MKK3/6.29 This effect is likely not specific to neuroblastoma; fenretinide is known to demonstrate mitochondrial toxicity in many cancer cell types, but appears to be well tolerated in Phase I clinical trials.25 The reason for its apparent cancer selectivity is not known.


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Potentiation of 4-HPR-induced Redox Stress and Apoptosis by the p75 Neurotrophin Receptor (p75NTR) Expression of the p75NTR in neuroblastoma cells enhances apoptosis induction and mitochondrial accumulation of reactive oxygen species by 4-HPR in neuroblastoma cells.30 This enhancement does not involve induction of overexpression or increased activity of Complex II. Also, induction of p75NTR expression does not result in a consistent change across neuroblastoma cell lines in expression of the retinoid binding protein, CRABP-I.31,32 Potentiation by p75NTR of redox stress and apoptosis induction by 4-HPR requires p38MAPK phosphorylation, JNK phosphorylation, caspase 3 activation, Akt cleavage, and a decrease in Akt phosphorylation.29 It is hypothesized that this pro-apoptotic signaling cascade enhances the generation and/or half-life of mitochondrial reactive oxygen species at the level of Complex II.

4-HPR and Potentiation of Its Activity as Therapy for Neuroblastoma Ongoing clinical trials of 4-HPR alone and in combination with other agents have employed modified preparations of 4-HPR aimed at enhancing its bioavailability and prolonging its halflife in vivo.25 Adjunctive agents have been proposed to accomplish this purpose, as well. For example, the antifungal P450 inhibitor, ketoconazole, has been found to increase intratumoral levels and antitumor efficacy of 4-HPR in murine xenograft models of human neuroblastoma.33 The microtubule inhibitor, ABT-751, synergistically enhances reactive oxygen species accumulation in response to 4-HPR treatment in xenograft models.34



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The fragment of p75NTR thought to be responsible for its potentiation of 4-HPR activity is its intracellular domain (p75ICD), the result of sequential cleavage of p75NTR by α- and γsecretases.30,35 In the future, it may be possible to deliver this peptide to neuroblastoma cells or to specifically transfect neuroblastoma cells in vivo with an expression construct for it. Alternatively, pharmacological induction of p75NTR or its downstream effectors specifically in neuroblastoma cells may be possible.

MARKERS OF NEURAL LINEAGE BRANCHES AS TARGETS

Leveraging Tumor-selective Chemistry The long-pursued goal in cancer therapy has been to identify those chemical species uniquely expressed on or in cancer cells so that they can be targeted without toxicity to normal cells. Targeting neuroblastoma using antibodies to GD2, a disialoganglioside most abundant on neuroblastoma cells, has been used in several different approaches to neuroblastoma.36 These antibodies have been rendered radioactive to effect targeted radiation therapy.37 They have been attached to toxins or nanoparticles containing apoptosis-inducing microRNAs.38 Dinutuximab, a chimeric, human-murine, anti-GD2 monoclonal antibody given in combination with granulocytemacrophage colony-stimulating factor, interleukin-2, and 13-cis-retinoic acid, has been shown to increase relative to conventional therapies the 2-year event-free survival of patients with highrisk neuroblastoma that responds at least partially to multi-agent, multimodality therapies.39


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Leveraging Developmentally-regulated Chemistry Because neuroblastoma can be conceptualized as an arrest of the process of differentiation, the hope has also been that one could target those proteins that are expressed by sympathetic neuroblasts or precursor neuronal or Schwann cells and leave mature neural crest-derived elements unscathed. The kinase D-interacting substrate of 220kDa (Kidins220) is a protein involved in neurotrophin and cytokine signaling, vesicle formation and transport, and cytoskeletal regulation. Kidins220 also stabilizes NGF- but not BDNF-induced signaling in neural cells. It is expressed uniquely on immature neural lineage cells. Downregulation of Kidins220 in neuroblastoma cells results in adoption of a Schwann cell-like morphology and coordinate downregulation of neuronal lineage markers, including doublecortin, strathmin, and the light subunit of neurofilament protein. This is of particular interest because as tumors of the neural crest progress from most to least malignant (i.e., from neuroblastoma to ganglioneuroblastoma to ganglioneuroma), doublecortin, strathmin, and Kidins220 expression decreases.40,41 Despite this association of expression profile with level of malignancy, downregulation of Kidins220 did not alter motility, proliferation, or response to conventional chemotherapeutic drugs in neuroblastoma cell lines in culture.41

Homogenizing the Target Pool towards Therapy for Neuroblastoma While it appears that downregulation of Kidins220 does not rid neuroblastoma cells of their proliferative or migratory characteristics, nor does it enhance the efficacy of conventional antineuroblastoma pharmacotherapeutics,41 it does align the morphology and expression profile of the cells in the Schwann cell pathway.40 In their native state, neuroblastomas include neural



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crest stem, neuronal precursor, and epithelioid (Schwann-like) precursor cells. As illustrated in Fig. 3, aligning the cells of a given tumor in one of these lineage types and stages may facilitate targeted therapies against one or more chemical species unique to that particular cell type and stage of differentiation. (This potential strategy is reminiscent of synchronization of cells in the cell cycle before treating them with cell cycle-active agents.) Identification of specific novel targets and the pharmacological agents that might modulate their expression or activity is a potential step towards creation of innovative approaches aimed at the conquest of neuroblastoma.

CONCLUSION

Neuroblastoma is a common and frequently fatal cancer of childhood. New therapeutic approaches to this tumor are needed. Targeted approaches could afford anti-tumor efficacy with minimization of systemic toxicity. Recent and ongoing attempts at such therapeutic approaches include targeting of protein arginine methyltransferases, mechanisms of redox regulation, or developmentally-regulated markers of specific branches of the neural ineage pathways. These innovative approaches to neuroblastoma hold the promise of prevention and cure of this cancer of childhood. Their translation from the tissue culture dish to the clinic will require demonstration of efficacy and delineation of toxicity first in animal models of neuroblastoma and ultimately in human subjects. Such studies are well underway in the TH-MYCN murine neuroblastoma model42 and in human neuroblastoma-derived neurosphere xenografted mice.


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AUTHOR CONTRIBUTIONS

Jeanne N. Hansen, Ph.D. performed all of the studies involving diamidine effects on neuroblastoma cell lines; determined the relationship between neuroblastoma prognosis in children and tumor content of N-Myc PRMT1; and read, edited, and approved all versions of the manuscript before submission.

Xingguo Li, Ph.D. determined the mechanism of the relationship between PRMT1 activity and neuroblastoma cell virulence; conceived of the potential use of PRMT1 inhibition in the treatment of neuroblastoma; directly supervised Dr. Hansen and Mr. Lotta in performance of their studies; and read, edited, and approved all versions of the manuscript before submission.

Y. George Zheng, Ph.D. synthesized the diamidines used in these studies and discussed in this paper; wrote the sections of the paper devoted to describing the relationship between diamidine structure and selective effects on PRMT1 activity; and read, edited, and approved all versions of the manuscript before submission.

Louis T. Lotta, Jr. provided technical assistance for all studies performed by Drs. Li and Schor; and read, edited, and approved all versions of the manuscript before submission.

Abhishek Dedhe provided technical assistance for all studies performed by Dr. Hansen; and read, edited, and approved all versions of the manuscript before submission.



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Nina F. Schor, M.D., Ph.D. conceived of and oversaw all aspects of the studies described; performed the literature review and wrote and compiled the text of this paper; supervised the students who performed the studies described relevant to redox and neural marker targeting, and interpreted the results of these studies; created all figures shown in this paper; and read, edited, and approved the final version of the manuscript before submission.

ACKNOWLEDGEMENTS

The studies for which primary data are presented in this paper were funded by a grant from Crosby’s Fund for Neuroblastoma Pediatric Cancer Research (N.F.S.), the William H. Eilinger Endowment of Golisano Children’s Hospital (N.F.S.), a grant from the Strong Children’s Research Center Small Grants Program (X.L.), and NIH grant R01GM110387 (Y.G.Z.).


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Page 22 of 29

FIGURE LEGENDS

Fig. 1: Relationship between linker structure and LC50 (µM; 48 h after treatment) of diamidines and the related compound, TCE 5003, in six different neuroblastoma cell lines (LAN5, KELLY, SH-EP1, SH-SY5Y, SK-N-AS, and SK-N-BE2C). Data were pooled across all cell lines (i.e., n=6 for each bar) and the mean LC50 is plotted for each compound. The figure shows the result of one preliminary experiment in which varying concentrations of a compound were added to sister culture wells at t = 0. Error bars represent the S.E.M. For alkyl diether-linked diamidines, the number above each bar is the alkyl chain length.

Fig. 2: LC50 (µM; 48 h after treatment) of diamidines with various linker structures and the related compound, TCE 5003, in MYCN-amplified (filled bars; n=3; LAN5, KELLY, SK-NBE2C) and MYCN-non-amplified (open bars; n=3; SH-EP1, SH-SY5Y, SK-N-AS) neuroblastoma cell lines. Data were pooled across all cell lines of similar MYCN expression (i.e., n=3 for each bar) and the mean LC50 is plotted for each compound. The figure shows the result of one preliminary experiment in which varying concentrations of a compound were added to sister culture wells at t = 0. Error bars represent the S.E.M.

Fig. 3: Cartoon depicting heterogeneity of neuroblastoma cell line morphology in the native state; homogeneous Schwann cell-like morphology after downregulation of Kidins220; and the potential of a drug aimed at a Schwann cell protein to induce apoptosis in this homogeneous Schwann cell-like neuroblastoma population.


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TEXTBOXES

Textbox 1:

Neuroblastoma: Neuroblastoma is a cancer of developing nerve cells (called neuroblasts) in the sympathetic (peripheral autonomic) nervous system. It can arise anywhere along the sympathetic chain, including in the chest or abdomen. Diamidines: A group of compounds each of which contains two -C(=NH)-NH2 moieties. Asymmetric dimethylarginine: Arginine with two methyl groups coming off of one of the two nitrogens in the “R” group; (H3C)2-N-C(=NH)-NH-(CH2)3-CH(NH2)-C(=O)-OH Reactive Oxygen Species: Chemically reactive species that contain oxygen. Kidins220: Kinase D-interacting substrate of 220 kDa or ARMS (ankyrin repeat-rich membrane spanning) is a protein that acts as a scaffold for signal transductants. It plays important roles in the immune system and in neurotrophin and ephrin signaling in neurons.



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Textbox 2:

Protein arginine methyltransferase-1 (PRMT1) and Neuroblastoma Oncogenesis

PRMT1-mediated R65 methylation primes the neuroblastoma oncoprotein, MYCN, for phosphorylation at S62, stabilizing MYCN and further priming it for phosphorylation at T58. Dephosphorylation of pS62 then sensitizes MYCN, still phosphorylated at T58, to bind to E3 ligase, which leads to subsequent ubiquitination and degradation.9


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Textbox 3:

All-trans retinoic acid

13-cis retinoic acid

Fenretinide


Fig. 1

45

Page 26 of 29

Aromatic Ring Linkage

40

35

30

--------Alkyl Diether Linkage-------LC50 (µM)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


25

n= 5

6

10

20

15

10

Sulfonyl Linkage (non-diamidine)

5

0 Pentamidine

Hexamidine

Decamidine

TCE5003

ACS Paragon Plus Environment

SKLB639

Furamidine

Fig. 2 Page 27 of 29

60

50

40

LC50 (µM)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


30

20

10

0 Pentamidine

Hexamidine

Decamidine

TCE5003


SKLB639

Furamidine

Fig. 3

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Page 28 of 29

Graphical Table Page 29 of 29 of Contents 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

HN


Linker

H2N

NH NH2

p75NTR

Kidins220

Anti-epithelioid Drug


Using Chemistry to Target Neuroblastoma - ACS Chemical

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