Ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP) family members (ENPP1–7) have been implicated in key biological and pathophysiological processes, including nucleotide and phospholipid signaling, bone mineralization, fibrotic diseases, and tumor-associated immune cell infiltration. ENPPs are single-pass transmembrane ecto-enzymes, with notable exceptions of ENPP2 (Autotaxin) and ENNP6, which are secreted and glycosylphosphatidylinositol (GPI)-anchored, respectively. ENNP1 and ENNP2 are the best characterized and functionally the most interesting members. Here, we review the structural features of ENPP1–7 to understand how they evolved to accommodate specific substrates and mediate different biological activities. ENPPs are defined by a conserved phosphodiesterase (PDE) domain. In ENPP1–3, the PDE domain is flanked by two N-terminal somatomedin B-like domains and a C-terminal inactive nuclease domain that confers structural stability, whereas ENPP4–7 only possess the PDE domain. Structural differences in the substrate-binding site endow each protein with unique characteristics. Thus, ENPP1, ENPP3, ENPP4, and ENPP5 hydrolyze nucleotides, whereas ENPP2, ENPP6, and ENNP7 evolved as phospholipases through adaptions in the catalytic domain. These adaptations explain the different biological and pathophysiological functions of individual members. Understanding the ENPP members as a whole advances our insights into common mechanisms, highlights their functional diversity, and helps to explore new biological roles.

This version is available at https://strathprints.strath.ac.uk/59722/ Strathprints is designed to allow users to access the research output of the University of Strathclyde. Unless otherwise explicitly stated on the manuscript, Copyright © and Moral Rights for the papers on this site are retained by the individual authors and/or other copyright owners. Please check the manuscript for details of any other licences that may have been applied. You may not engage in further distribution of the material for any profitmaking activities or any commercial gain. You may freely distribute both the url (https://strathprints.strath.ac.uk/) and the content of this paper for research or private study, educational, or not-for-profit purposes without prior permission or charge.Any correspondence concerning this service should be sent to the Strathprints administrator: strathprints@strath.ac.ukThe Strathprints institutional repository (https://strathprints.strath.ac.uk) is a digital archive of University of Strathclyde research outputs. It has been developed to disseminate open access research outputs, expose data about those outputs, and enable the management and persistent access to Strathclyde's intellectual output. Rational design of Autotaxin inhibitors by structural evolution of endogenous modulatorsWillem-Jan Keune, † Frances Potjewyd, Tatjana AbstractAutotaxin produces the bioactive lipid lysophosphatidic acid (LPA), and is a drug target of considerable interest for numerous pathologies. We report the expedient, structure-guided evolution of weak physiological allosteric inhibitors (bile salts) into potent competitive Autotaxin inhibitors that do not interact with the catalytic site. Functional data confirms that our lead compound attenuates LPA mediated signalling in cells, and reduces LPA synthesis in vivo, providing a promising natural product derived scaffold for drug discovery.

SUMMARY Autotaxin (ATX; ENPP2 ) produces lysophosphatidic acid (LPA) that regulates multiple biological functions via cognate G protein-coupled receptors LPAR1–6. ATX/LPA promotes tumor cell migration and metastasis via LPAR1 and T cell motility via LPAR2, yet its actions in the tumor immune microenvironment remain unclear. Here, we show that ATX secreted by melanoma cells is chemorepulsive for tumor-infiltrating lymphocytes (TILs) and circulating CD8 + T cells ex vivo , with ATX functioning as an LPA-producing chaperone. Mechanistically, T cell repulsion predominantly involves Gα 12/13 -coupled LPAR6. Upon anti-cancer vaccination of tumor-bearing mice, ATX does not affect the induction of systemic T cell responses but, importantly, suppresses tumor infiltration of cytotoxic CD8 + T cells and thereby impairs tumor regression. Moreover, single-cell data from melanoma tumors are consistent with intratumoral ATX acting as a T cell repellent. These findings highlight an unexpected role for the pro-metastatic ATX-LPAR axis in suppressing CD8 + T cell infiltration to impede anti-tumor immunity, suggesting new therapeutic opportunities.

Autotaxin is a secreted glycoprotein and the only member of the ectonucleotide pyrophosphatase/phosphodiesterase family that converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA). LPA controls key responses such as cell migration, proliferation, and survival, implicating ATX-LPA signalling in various (patho)physiological processes and establishing it as a drug target. ATX structural and functional studies have revealed an orthosteric and an allosteric site, called the "pocket" and the "tunnel," respectively. However, the mechanisms in allosteric modulation of ATX's activity as a lysophospholipase D are unclear. Here, using the physiological LPC substrate, a new fluorescent substrate, and diverse ATX inhibitors, we revisited the kinetics and allosteric regulation of the ATX catalytic cycle, dissecting the different steps and pathways leading to LPC hydrolysis. We found that ATX activity is stimulated by LPA and that LPA activates ATX lysophospholipase D activity by binding to the ATX tunnel. A consolidation of all experimental kinetics data yielded a comprehensive catalytic model supported by molecular modelling simulations, and suggested a positive feedback mechanism that is regulated by the abundance of the LPA products activating hydrolysis of different LPC species. Our results complement and extend the current understanding of ATX hydrolysis in light of the allosteric regulation by ATX-produced LPA species, and have implications for the design and application of both orthosteric and allosteric ATX inhibitors.Autotaxin (ATX or ENPP2) is a secreted glycoprotein and a unique member of the ectonucleotide pyrophosphatase / phosphordiesterase (ENPP) family (1). It is the only ENPP family member with lysophospholipase D (lysoPLD) activity (EC 3.1.4.39), and it is the main enzyme responsible for the hydrolysis of lysophosphatidylcholine (2-acyl-sn-glycero-3-phosphocholine or LPC) to produce the bioactive lipid lysophosphatidic acid (monoacyl-snglycerol-3-phosphate or LPA) (2-4). LPA acts as a ligand for several LPA receptors (LPARs) showing overlapping activities. The ATX-LPA signalling axis is vital for embryonic development and has been implicated in many (patho)physiological processes, which include vascular development (5), cancer metastasis (6), and other human diseases, such as fibrosis (7) and cholestatic pruritus (8). ATX is translated as a preproenzyme that is secreted to plasma upon its proteolytic processing, resulting in its native structural domains (9, 10). Close to the Nterminus, ATX presents two somatomedin B (SMB)-like domains, which are followed by the central catalytic phosphodiesterase (PDE) domain, and an inactive nuclease-like domain. Catalysis occurs in a bimetallic active site presenting two Zn 2+ atoms, and resembles that of other members of the alkaline phosphatase family (11). The catalytic site of ATX is organized in a tripartite binding site (Fig.1), where the active site is followed by a shallow hydrophilic groove that (11)(12)(13), and a tunnel, also called ...

Autotaxin (ATX) is a secreted lysophospholipase D, catalysing the conversion of lysophosphatidylcholine (LPC) to bioactive lysophosphatidic acid (LPA). LPA acts through two families of G protein-coupled receptors (GPCRs) controlling key cellular responses, and it is implicated in many physiological processes and pathologies. ATX, therefore, has been established as an important drug target in the pharmaceutical industry. Structural and biochemical studies of ATX have shown that it has a bimetallic nucleophilic catalytic site, a substrate-binding (orthosteric) hydrophobic pocket that accommodates the lipid alkyl chain, and an allosteric tunnel that can accommodate various steroids and LPA. In this review, first, we revisit what is known about ATX-mediated catalysis, crucially in light of allosteric regulation. Then, we present the known ATX catalysis-independent functions, including binding to cell surface integrins and proteoglycans. Next, we analyse all crystal structures of ATX bound to inhibitors and present them based on the four inhibitor types that are established based on the binding to the orthosteric and/or the allosteric site. Finally, in light of these data we discuss how mechanistic differences might differentially modulate the activity of the ATX-LPA signalling axis, and clinical applications including cancer.

Autotaxin (ATX) is a secreted enzyme responsible for the hydrolysis of lysophosphatidylcholine (LPC) to the bioactive lysophosphatidic acid (LPA) and choline. The ATX-LPA signaling pathway is implicated in cell survival, migration, and proliferation; thus, the inhibition of ATX is a recognized therapeutic target for a number of diseases including fibrotic diseases, cancer, and inflammation, among others. Many of the developed synthetic inhibitors for ATX have resembled the lipid chemotype of the native ligand; however, a small number of inhibitors have been described that deviate from this common scaffold. Herein, we report the structure-activity relationships (SAR) of a previously reported small molecule ATX inhibitor. We show through enzyme kinetics studies that analogues of this chemotype are noncompetitive inhibitors, and by using a crystal structure with ATX we confirm the discrete binding mode.

Autotaxin is a secreted phosphodiesterase that converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA). LPA controls key cellular responses such as migration, proliferation and survival, implicating ATX-LPA signalling in various (patho)physiological processes and establishing it as a drug target. ATX structural and functional studies have revealed an orthosteric and an allosteric site, the "pocket" and the "tunnel". Here, we revisit the kinetics of the ATX catalytic cycle in light of allosteric regulation, dissecting the different steps and pathways that lead to LPC hydrolysis. Consolidating all experimental kinetics data to a comprehensive catalytic model supported by molecular modelling simulations, suggests a positive feedback mechanism, regulated by the abundance of the LPA products activating hydrolysis of different LPC species. Our results complement and extend current understanding of ATX hydrolysis in light of the allosteric regulation by produced LPA species, and have implications for the design and application of orthosteric and allosteric ATX inhibitors.

To improve immunotherapy efficacy, a better understanding of the factors that regulate Tcell migration into tumors is essential. Here we uncover a role for autotaxin (ATX) in this process. ATX (encoded by ENPP2) produces lysophosphatidic acid (LPA) that activates G protein-coupled receptors (LPAR1-6) to regulate multiple (patho)physiological processes, including tumor progression via LPAR1 and lymphocyte homing via LPAR2.Unexpectedly, we find that melanoma cell-secreted ATX is a major chemorepellent for tumor-infiltrating lymphocytes ex vivo through Gα12/13-coupled LPAR6, with ATX functioning as an LPA-producing chaperone. Using an anti-cancer vaccination model, we provide proof-of-concept that secreted ATX opposes tumor infiltration of CD8+ T cells.Additionally, ENPP2 expression in melanoma tumors correlates with reduced CD8+ T-cell infiltration as inferred from single-cell transcriptomics. Hence, by counteracting T-cell infiltration while activating tumor cells via different LPA receptors, the ATX/LPA complex exerts dual actions in the tumor immune microenvironment, which may provide new therapeutic approaches.