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

Stein, Adam J.; Bain, Gretchen; Prodanovich, Pat; Santini, Angelina M.; Darlington, Janice; Stelzer, Nina M. P.; Sidhu, Ranjinder S.; Schaub, Jeffrey M.; Goulet, Lance; Lonergan, Dave; Calderon, Imelda; Evans, Jilly F.; Hutchinson, John H.

doi:10.1124/mol.115.100404

Cited by 53 publications

(73 citation statements)

References 35 publications

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“…The structural characterization of the ATX tripartite site (Fig.1A-B) has created a remarkable potential for selective inhibitor design (14)(15)(16)) that includes lipid-based inhibitors (17), DNA aptamers (18) and small molecules. The latter can be classified in four distinct types depending on their binding modes to the tripartite site (19) (Fig.1C).…”

Section: Introductionmentioning

confidence: 99%

Lysophosphatidic acid produced by autotaxin acts as an allosteric modulator of its catalytic efficiency

Salgado-Polo

Fish

Matsoukas

et al. 2018

Journal of Biological Chemistry

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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 ...

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Section: Introductionmentioning

confidence: 99%

Lysophosphatidic acid produced by autotaxin acts as an allosteric modulator of its catalytic efficiency

Salgado-Polo

Fish

Matsoukas

et al. 2018

Journal of Biological Chemistry

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“…3, Figure 1). By contrast, a recent series of indole-based ATX inhibitors 7 and the lead compound of an independent discovery program 8 are reported to occupy the hydrophobic pocket and tunnel, without interacting with the hydrophilic groove or the active site, acting as allosteric inhibitors, similar to natural bile salts.…”

Section: Introductionmentioning

confidence: 99%

Rational Design of Autotaxin Inhibitors by Structural Evolution of Endogenous Modulators

et al. 2017

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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.

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“…It is worth noting that the currently available human ATX structures (PDB ID: 4ZG6, 4ZG7, 4ZG9, and 4ZGA) were published after this modeling was developed. [30] However, the mouse and rat crystal structures were not without limitations. For example, the rat ATX structure was missing portions of the backbone distant from the active site and hydrophobic regions, whereas the mouse structure contained several sidechains truncated to alanine (methyl) due to unresolved atomic positions (K59, E67, K104, R162, R244, R246, F274, N398, L458, K462, R549, Q559, R602, E642, and K666).…”

Section: Resultsmentioning

confidence: 99%

“…The recently published crystal structures of ATX, enzyme inhibition kinetics, and molecular docking studies show distinct binding regions for small molecule ATX inhibitors. [26–30] Small molecule, non-lipid inhibitors are divided between their interactions with either the polar active site[30,53–55] and the distant hydrophobic domain. [28–30,53,57]…”

Section: Discussionmentioning

confidence: 99%

“…[22–25] Recent crystallized structures of ATX are useful tools from which to develop structure-based pharmacophores. [26–30] Crystal structures of mouse, rat, and human ATX all are composed of three main domains, including a catalytic domain, which contains a polar active site, a hydrophobic tunnel, and a hydrophobic pocket (figure 1). The prevalence of non-polar amino acid sidechains in the hydrophobic tunnel of ATX might lead to a structure-based pharmacophore that contains predominantly non-specific hydrophobic features, capable of finding compounds which fit into the ATX hydrophobic pocket but may also bind to other receptors.…”

Section: Introductionmentioning

confidence: 99%

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Discovery and synthetic optimization of a novel scaffold for hydrophobic tunnel-targeted autotaxin inhibition

Ragle

Palanisamy

Joe

et al. 2016

Bioorganic & Medicinal Chemistry

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Autotaxin (ATX) is a ubiquitous ectoenzyme that hydrolyzes lysophosphatidylcholine (LPC) to form the bioactive lipid mediator lysophosphatidic acid (LPA). LPA activates specific G-protein coupled receptors to elicit downstream effects leading to cellular motility, survival, and invasion. Through these pathways, upregulation of ATX is linked to diseases such as cancer and cardiovascular disease. Recent crystal structures confirm that the catalytic domain of ATX contains multiple binding regions including a polar active site, hydrophobic tunnel, and a hydrophobic pocket. This finding is consistent with the promiscuous nature of ATX hydrolysis of multiple and diverse substrates and prior investigations of inhibitor impacts on ATX enzyme kinetics. The current study used virtual screening methods to guide experimental identification and characterization of inhibitors targeting the hydrophobic region of ATX. An initially discovered inhibitor, GRI392104 (IC50 4 μM) was used as a lead for synthetic optimization. In total twelve newly synthesized inhibitors of ATX were more potent than GRI392104 and were selective for ATX as they had no effect on other LPC-specific NPP family members or on LPA1–5 GPCR.

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

Cited by 53 publications

References 35 publications

Lysophosphatidic acid produced by autotaxin acts as an allosteric modulator of its catalytic efficiency

Lysophosphatidic acid produced by autotaxin acts as an allosteric modulator of its catalytic efficiency

Rational Design of Autotaxin Inhibitors by Structural Evolution of Endogenous Modulators

Discovery and synthetic optimization of a novel scaffold for hydrophobic tunnel-targeted autotaxin inhibition

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