Hits of a high-throughput screen identify the hydrophobic pocket of autotaxin/lysophospholipase D as an inhibitory surface

James I. Fells, Sue Lee, Yuko Fujiwara, Derek D. Norman, Keng Gat Lim, Ryoko Tsukahara, Jianxiong Liu, Renukadevi Patil, Duane Miller, R. Jason Kirby, Sandra Nelson, William Seibel, Ruben Papoian, Abby L. Parrill, Daniel L. Baker, Robert Bittman, Gabor Tigyi

Research output: Contribution to journalArticle

22 Citations (Scopus)

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 IC 50 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 59-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 targetbinding site for inhibitors of enzymatic activity.

Original languageEnglish (US)
Pages (from-to)415-424
Number of pages10
JournalMolecular Pharmacology
Volume84
Issue number3
DOIs
StatePublished - Sep 1 2013

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Fluorescent Dyes
Phosphoric Diester Hydrolases
nucleotide pyrophosphatase
Melanoma
Lysophospholipase
Lysosphingolipid Receptors
Thymidine Monophosphate
Neoplasm Metastasis
Lysophosphatidylcholines
Neuralgia
Enzymes
Drug Discovery
Pruritus
Inbred C57BL Mouse
Mutagenesis
Catalytic Domain
Neoplasms
Carcinogenesis
Hydrolysis
Animal Models

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Pharmacology

Cite this

Hits of a high-throughput screen identify the hydrophobic pocket of autotaxin/lysophospholipase D as an inhibitory surface. / Fells, James I.; Lee, Sue; Fujiwara, Yuko; Norman, Derek D.; Lim, Keng Gat; Tsukahara, Ryoko; Liu, Jianxiong; Patil, Renukadevi; Miller, Duane; Kirby, R. Jason; Nelson, Sandra; Seibel, William; Papoian, Ruben; Parrill, Abby L.; Baker, Daniel L.; Bittman, Robert; Tigyi, Gabor.

In: Molecular Pharmacology, Vol. 84, No. 3, 01.09.2013, p. 415-424.

Research output: Contribution to journalArticle

Fells, JI, Lee, S, Fujiwara, Y, Norman, DD, Lim, KG, Tsukahara, R, Liu, J, Patil, R, Miller, D, Kirby, RJ, Nelson, S, Seibel, W, Papoian, R, Parrill, AL, Baker, DL, Bittman, R & Tigyi, G 2013, 'Hits of a high-throughput screen identify the hydrophobic pocket of autotaxin/lysophospholipase D as an inhibitory surface', Molecular Pharmacology, vol. 84, no. 3, pp. 415-424. https://doi.org/10.1124/mol.113.087080
Fells, James I. ; Lee, Sue ; Fujiwara, Yuko ; Norman, Derek D. ; Lim, Keng Gat ; Tsukahara, Ryoko ; Liu, Jianxiong ; Patil, Renukadevi ; Miller, Duane ; Kirby, R. Jason ; Nelson, Sandra ; Seibel, William ; Papoian, Ruben ; Parrill, Abby L. ; Baker, Daniel L. ; Bittman, Robert ; Tigyi, Gabor. / Hits of a high-throughput screen identify the hydrophobic pocket of autotaxin/lysophospholipase D as an inhibitory surface. In: Molecular Pharmacology. 2013 ; Vol. 84, No. 3. pp. 415-424.
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AU - Nelson, Sandra

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N2 - 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 IC 50 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 59-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 targetbinding site for inhibitors of enzymatic activity.

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