title: Hits of a high-throughput screen identify the hydrophobic pocket of autotaxin/lysophospholipase D as an inhibitory surface.
abstract: Autotaxin (ATX), a lysophospholipase D, plays an important role in cancer invasion, metastasis, tumor progression, tumorigenesis, neuropathic pain, fibrotic diseases, cholestatic pruritus, lymphocyte homing, and thrombotic diseases by producing the lipid mediator lysophosphatidic acid (LPA). A high-throughput screen of ATX inhibition using the lysophosphatidylcholine-like substrate fluorogenic substrate 3 (FS-3) and ∼10,000 compounds from the University of Cincinnati Drug Discovery Center identified several small-molecule inhibitors with IC₅₀ vales ranging from nanomolar to low micromolar. The pharmacology of the three most potent compounds: 918013 (1; 2,4-dichloro-N-(3-fluorophenyl)-5-(4-morpholinylsulfonyl) benzamide), 931126 (2; 4-oxo-4-{2-[(5-phenoxy-1H-indol-2-yl)carbonyl]hydrazino}-N-(4-phenylbutan-2-yl)butanamide), and 966791 (3; N-(2,6-dimethylphenyl)-2-[N-(2-furylmethyl)(4-(1,2,3,4-tetraazolyl)phenyl)carbonylamino]-2-(4-hydroxy-3-methoxyphenyl) acetamide), were further characterized in enzyme, cellular, and whole animal models. Compounds 1 and 2 were competitive inhibitors of ATX-mediated hydrolysis of the lysophospholipase substrate FS-3. In contrast, compound 3 was a competitive inhibitor of both FS-3 and the phosphodiesterase substrate p-nitrophenyl thymidine 5'-monophosphate. Computational docking and mutagenesis suggested that compounds 1 and 2 target the hydrophobic pocket, thereby blocking access to the active site of ATX. The potencies of compounds 1-3 were comparable to each other in each of the assays. All of these compounds significantly reduced invasion of A2058 human melanoma cells in vitro and the colonization of lung metastases by B16-F10 murine melanoma cells in C57BL/6 mice. The compounds had no agonist or antagonist effects on select LPA or sphingosine 1-phosphate receptors, nor did they inhibit nucleotide pyrophosphatase/phosphodiesterase (NPP) enzymes NPP6 and NPP7. These results identify the molecular surface of the hydrophobic pocket of ATX as a target-binding site for inhibitors of enzymatic activity.
authors.affiliation: Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA.
authors.forename: James I
authors.initials: JI
authors.lastname: Fells
authors.forename: Sue Chin
authors.initials: SC
authors.lastname: Lee
authors.forename: Yuko
authors.initials: Y
authors.lastname: Fujiwara
authors.forename: Derek D
authors.initials: DD
authors.lastname: Norman
authors.forename: Keng Gat
authors.initials: KG
authors.lastname: Lim
authors.forename: Ryoko
authors.initials: R
authors.lastname: Tsukahara
authors.forename: Jianxiong
authors.initials: J
authors.lastname: Liu
authors.forename: Renukadevi
authors.initials: R
authors.lastname: Patil
authors.forename: Duane D
authors.initials: DD
authors.lastname: Miller
authors.forename: R Jason
authors.initials: RJ
authors.lastname: Kirby
authors.forename: Sandra
authors.initials: S
authors.lastname: Nelson
authors.forename: William
authors.initials: W
authors.lastname: Seibel
authors.forename: Ruben
authors.initials: R
authors.lastname: Papoian
authors.forename: Abby L
authors.initials: AL
authors.lastname: Parrill
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Baker
authors.forename: Robert
authors.initials: R
authors.lastname: Bittman
authors.forename: Gabor
authors.initials: G
authors.lastname: Tigyi
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry* / Q000494:pharmacology; D001549:Benzamides / Q000737:chemistry* / Q000494:pharmacology; D044482:Benzeneacetamides / Q000737:chemistry* / Q000494:pharmacology; D045744:Cell Line, Tumor; D004354:Drug Screening Assays, Antitumor; D057166:High-Throughput Screening Assays; D006801:Humans; D006834:Hydrazines / Q000737:chemistry* / Q000494:pharmacology; D057927:Hydrophobic and Hydrophilic Interactions; D007211:Indoles / Q000737:chemistry* / Q000494:pharmacology; D008175:Lung Neoplasms / Q000188:drug therapy / Q000473:pathology / Q000556:secondary; D008546:Melanoma, Experimental / Q000188:drug therapy / Q000556:secondary; D051379:Mice; D008810:Mice, Inbred C57BL; D062105:Molecular Docking Simulation; D009154:Mutation; D009361:Neoplasm Invasiveness; D010726:Phosphodiesterase Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013449:Sulfonamides / Q000737:chemistry* / Q000494:pharmacology; D013777:Tetrazoles / Q000737:chemistry* / Q000494:pharmacology; D000099189:Lysophospholipase D
chemical_list: D000970:Antineoplastic Agents; D001549:Benzamides; D044482:Benzeneacetamides; D006834:Hydrazines; D007211:Indoles; D010726:Phosphodiesterase Inhibitors; D010727:Phosphoric Diester Hydrolases; D013449:Sulfonamides; D013777:Tetrazoles; D000099189:Lysophospholipase D; C584433:2,4-dichloro-N-(3-fluorophenyl)-5-(4-morpholinylsulfonyl)benzamide; C584434:4-oxo-4-(2-((5-phenoxy-1H-indol-2-yl)carbonyl)hydrazino)-N-(4-phenylbutan-2-yl)butanamide; C584435:N-(2,6-dimethylphenyl)-2-(N-(2-furylmethyl)(4-(1,2,3,4-tetraazolyl)phenyl)carbonylamino)-2-(4-hydroxy-3-methoxyphenyl)acetamide