title: Antitumor Activity of a Novel Sphingosine-1-Phosphate 2 Antagonist, AB1, in Neuroblastoma.
abstract: The bioactive lipid sphingosine-1-phosphate (S1P) and its receptors (S1P1-5) play critical roles in many pathologic processes, including cancer. The S1P axis has become a bona fide therapeutic target in cancer. JTE-013 [N-​(2,​6-​dichloro-​4-​pyridinyl)-​2-​[1,​3-​dimethyl-​4-​(1-​methylethyl)-​1H-​pyrazolo[3,​4-​b]pyridin-​6-​yl]-​hydrazinecarboxamide], a known S1P2 antagonist, suffers from instability in vivo. Structurally modified, more potent, and stable S1P2 inhibitors would be desirable pharmacological tools. One of the JTE-013 derivatives, AB1 [N-(1H-4-isopropyl-1-allyl-3-methylpyrazolo[3,4-b]pyridine-6-yl)-amino-N'-(2,6-dichloropyridine-4-yl) urea], exhibited improved S1P2 antagonism compared with JTE-013. Intravenous pharmacokinetics indicated enhanced stability or slower clearance of AB1 in vivo. Migration assays in glioblastoma showed that AB1 was slightly more effective than JTE-013 in blocking S1P2-mediated inhibition of cell migration. Functional studies in the neuroblastoma (NB) cell line SK-N-AS showed that AB1 displayed potency at least equivalent to JTE-013 in affecting signaling molecules downstream of S1P2. Similarly, AB1 inhibition of the growth of SK-N-AS tumor xenografts was improved compared with JTE-013. Cell viability assays excluded that this enhanced AB1 effect is caused by inhibition of cancer cell survival. Both JTE-013 and AB1 trended to inhibit (C-C motif) ligand 2 expression and were able to significantly inhibit subsequent tumor-associated macrophage infiltration in NB xenografts. Interestingly, AB1 was more effective than JTE-013 in inhibiting the expression of the profibrotic mediator connective tissue growth factor. The terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling assay and cleaved caspase-3 detection further demonstrated that apoptosis was increased in AB1-treated NB xenografts compared with JTE-013. Overall, the modification of JTE-013 to produce the AB1 compound improved potency, intravenous pharmacokinetics, cellular activity, and antitumor activity in NB and may have enhanced clinical and experimental applicability.
authors.affiliation: Center for Vascular Biology, University of Connecticut Health Center, Farmington, Connecticut (M.-H.L., L.H.S., F.F.); Arroyo Biosciences, LLC, Princeton, New Jersey (R.S.); Department of Urology and Surgery, Connecticut Children's Medical Center, Hartford, Connecticut (M.H., F.F.); TCG Life Sciences Limited, Hinjewadi, Pune, India (S.J.); Chem-Master International Inc., Stony Brook, New York (E.S.); and Department of Pathology and Laboratory Medicine, Center for Vascular Biology, Weill Medical College of Cornell University, New York, New York (T.H.).
authors.forename: Mei-Hong
authors.initials: MH
authors.lastname: Li
authors.affiliation: Center for Vascular Biology, University of Connecticut Health Center, Farmington, Connecticut (M.-H.L., L.H.S., F.F.); Arroyo Biosciences, LLC, Princeton, New Jersey (R.S.); Department of Urology and Surgery, Connecticut Children's Medical Center, Hartford, Connecticut (M.H., F.F.); TCG Life Sciences Limited, Hinjewadi, Pune, India (S.J.); Chem-Master International Inc., Stony Brook, New York (E.S.); and Department of Pathology and Laboratory Medicine, Center for Vascular Biology, Weill Medical College of Cornell University, New York, New York (T.H.).
authors.forename: Rolf
authors.initials: R
authors.lastname: Swenson
authors.affiliation: Center for Vascular Biology, University of Connecticut Health Center, Farmington, Connecticut (M.-H.L., L.H.S., F.F.); Arroyo Biosciences, LLC, Princeton, New Jersey (R.S.); Department of Urology and Surgery, Connecticut Children's Medical Center, Hartford, Connecticut (M.H., F.F.); TCG Life Sciences Limited, Hinjewadi, Pune, India (S.J.); Chem-Master International Inc., Stony Brook, New York (E.S.); and Department of Pathology and Laboratory Medicine, Center for Vascular Biology, Weill Medical College of Cornell University, New York, New York (T.H.).
authors.forename: Miriam
authors.initials: M
authors.lastname: Harel
authors.affiliation: Center for Vascular Biology, University of Connecticut Health Center, Farmington, Connecticut (M.-H.L., L.H.S., F.F.); Arroyo Biosciences, LLC, Princeton, New Jersey (R.S.); Department of Urology and Surgery, Connecticut Children's Medical Center, Hartford, Connecticut (M.H., F.F.); TCG Life Sciences Limited, Hinjewadi, Pune, India (S.J.); Chem-Master International Inc., Stony Brook, New York (E.S.); and Department of Pathology and Laboratory Medicine, Center for Vascular Biology, Weill Medical College of Cornell University, New York, New York (T.H.).
authors.forename: Sampa
authors.initials: S
authors.lastname: Jana
authors.affiliation: Center for Vascular Biology, University of Connecticut Health Center, Farmington, Connecticut (M.-H.L., L.H.S., F.F.); Arroyo Biosciences, LLC, Princeton, New Jersey (R.S.); Department of Urology and Surgery, Connecticut Children's Medical Center, Hartford, Connecticut (M.H., F.F.); TCG Life Sciences Limited, Hinjewadi, Pune, India (S.J.); Chem-Master International Inc., Stony Brook, New York (E.S.); and Department of Pathology and Laboratory Medicine, Center for Vascular Biology, Weill Medical College of Cornell University, New York, New York (T.H.).
authors.forename: Erik
authors.initials: E
authors.lastname: Stolarzewicz
authors.affiliation: Center for Vascular Biology, University of Connecticut Health Center, Farmington, Connecticut (M.-H.L., L.H.S., F.F.); Arroyo Biosciences, LLC, Princeton, New Jersey (R.S.); Department of Urology and Surgery, Connecticut Children's Medical Center, Hartford, Connecticut (M.H., F.F.); TCG Life Sciences Limited, Hinjewadi, Pune, India (S.J.); Chem-Master International Inc., Stony Brook, New York (E.S.); and Department of Pathology and Laboratory Medicine, Center for Vascular Biology, Weill Medical College of Cornell University, New York, New York (T.H.).
authors.forename: Timothy
authors.initials: T
authors.lastname: Hla
authors.affiliation: Center for Vascular Biology, University of Connecticut Health Center, Farmington, Connecticut (M.-H.L., L.H.S., F.F.); Arroyo Biosciences, LLC, Princeton, New Jersey (R.S.); Department of Urology and Surgery, Connecticut Children's Medical Center, Hartford, Connecticut (M.H., F.F.); TCG Life Sciences Limited, Hinjewadi, Pune, India (S.J.); Chem-Master International Inc., Stony Brook, New York (E.S.); and Department of Pathology and Laboratory Medicine, Center for Vascular Biology, Weill Medical College of Cornell University, New York, New York (T.H.).
authors.forename: Linda H
authors.initials: LH
authors.lastname: Shapiro
authors.affiliation: Center for Vascular Biology, University of Connecticut Health Center, Farmington, Connecticut (M.-H.L., L.H.S., F.F.); Arroyo Biosciences, LLC, Princeton, New Jersey (R.S.); Department of Urology and Surgery, Connecticut Children's Medical Center, Hartford, Connecticut (M.H., F.F.); TCG Life Sciences Limited, Hinjewadi, Pune, India (S.J.); Chem-Master International Inc., Stony Brook, New York (E.S.); and Department of Pathology and Laboratory Medicine, Center for Vascular Biology, Weill Medical College of Cornell University, New York, New York (T.H.) fferrer@connecticutchildrens.org.
authors.forename: Fernando
authors.initials: F
authors.lastname: Ferrer
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002699:Chlorambucil / Q000031:analogs & derivatives* / Q000494:pharmacology; D055513:Connective Tissue Growth Factor / Q000378:metabolism; D005260:Female; D064593:Heterografts; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009447:Neuroblastoma / Q000188:drug therapy* / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D049349:Receptors, Lysosphingolipid / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000081025:Sphingosine-1-Phosphate Receptors
chemical_list: D000970:Antineoplastic Agents; C471998:JTE 013; D011720:Pyrazoles; D011725:Pyridines; D049349:Receptors, Lysosphingolipid; C477522:S1PR2 protein, human; D000081025:Sphingosine-1-Phosphate Receptors; C501107:chlorambucil-N-(2-(4-(4-bis(2-chloroethyl)aminophenyl)butyryl)aminoethyl)-5-(4-amidinophenyl)-2-furancarboxamide hydrochloride; D055513:Connective Tissue Growth Factor; D002699:Chlorambucil; D053148:Caspase 3