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 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.

Structural Basis for Inhibition of Human Autotaxin by Four Potent Compounds with Distinct Modes of Binding

“…However, unlike the competitive inhibitor HA155, which coordinates to zinc (PDB ID 2XRG), PAT-078 does not extend far enough to interact with the active site zincs, and thus is unable to inhibit cleavage of the nucleotidelike BNPP substrate. In support of this hypothesis, Fells et al (2013) have identified compounds that competitively inhibit cleavage of LPC-like substrates (LPC, FS-3), but also do not inhibit cleavage of nucleotide substrates. Molecular docking studies using these compounds identified a number of key interactions with ATX residues, including Leu214, Ala218, Phe275, and Tyr307, that correspond well with the ATX residues identified in this study (Fells et al, 2013).…”

“…In support of this hypothesis, Fells et al (2013) have identified compounds that competitively inhibit cleavage of LPC-like substrates (LPC, FS-3), but also do not inhibit cleavage of nucleotide substrates. Molecular docking studies using these compounds identified a number of key interactions with ATX residues, including Leu214, Ala218, Phe275, and Tyr307, that correspond well with the ATX residues identified in this study (Fells et al, 2013). Further, the authors predicted that these compounds bind in the hydrophobic pocket away from the zinc ions in a similar manner to PAT-078.…”

Autotaxin: structure-function and signaling

“…Binding of ATX ␣ to cellsurface HSPGs, via its polybasic insertion loop, represents an additional isoform-specifi c mechanism for spatially and temporally restricted LPA production. Precisely how ATX activity is regulated, what the structural determinants of and nucleotide products bound to ATX and ENPP1 suggest that many additional cores can be envisaged in the further development of inhibitors [see also ( 81 )]. The ATX inhibitors structurally characterized until now share common characteristics and explore the hydrophobic pocket with fl uoro-benzene and chloro-benzene moieties.…”

Thematic Review Series: Phospholipases: Central Role in Lipid Signaling and Disease Autotaxin, a lysophospholipase D with pleomorphic effects in oncogenesis and cancer progression

“…Further, additional analysis is needed to determine whether other pathway alterations may serve compensatory roles in the small set of patients with ENPP2 deletions. Together, these results indicate that the ATX/LPA signaling axis may be dysregulated in specifi c cancer subtypes, and that patients could potentially benefi t from targeted therapies blocking ATX/ LPA signaling Due to the pleiotropic connection of ATX in cancer pathology, considerable effort has been made to generate small molecule inhibitors that target its enzymatic activity ( 120,(164)(165)(166)(167)(168)(169)(170)(171)(172)(173). Using sensitive fl uorescence probes, such as TG-mTMP, Kawaguchi et al ( 174 ) identifi ed several novel ATX inhibitor scaffolds and solved the crystal structures of ATX-compound complexes at high resolution (1.75-1.95 Å).…”