cancers Review The Structural Binding Mode of the Four Autotaxin Inhibitor Types that Differentially Affect Catalytic and Non-Catalytic Functions Fernando Salgado-Polo and Anastassis Perrakis * Oncode Institute and Division of Biochemistry, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands; [email protected] * Correspondence: [email protected] Received: 10 September 2019; Accepted: 8 October 2019; Published: 16 October 2019 Abstract: 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. Keywords: lysophosphatidic acid; autotaxin; inhibitor; allosteric; orthosteric; lipid chaperone; signalling; GPCR 1. Introduction Lysophosphatidic acid (1- or 2-acyl-sn-glycero-3-phosphate or LPA) is a bioactive lipid found in many body fluids and involved in many physiological and pathological processes. Historically, LPA had been identified as a growth factor in serum that could induce motility in fibroblasts and cancer cells through G protein-coupled receptors (GPCRs) [1,2]. Subsequent research identified specific LPA GPCRs (LPA1–6), which have distinct expression patterns [3]. Deregulation of the LPA signalling axis has been linked to different diseases, such liver disease [4], fibrosis [5], pruritus [6], multiple sclerosis [7], inflammation, and cancer [8,9]. The LPA receptors are classified into distinct families: the endothelial cell differentiation gene (EDG) (LPA1–3) and non-EDG (LPA4–6) families. The crystallographic structures of LPA1 and LPA6 have provided the field with key mechanistic indications with respect to their ligand binding mode [10,11]. Namely, a structural comparison between the LPA1 and LPA6 substrate-binding sites has indicated contrasting LPA binding modes from the extracellular milieu or the plasma membrane, respectively. The former is consistent with a model by which LPA is carried by a lipid chaperone, such as albumin, to bind to the flexible N-terminal domain of LPA1 and deliver LPA specifically [10], whereas the latter would not require lipid-carrying molecules [12,13]. Cancers 2019, 11, 1577; doi:10.3390/cancers11101577 www.mdpi.com/journal/cancers Cancers 2019, 11, 1577 2 of 17 Cancers 2019, 11, x 2 of 17 Since LPA can can promote promote a a plethora plethora of of downstream downstream signalling signalling events, events, both both its production its production and anddegradation degradation are tightly are tightly regulated, regulated, resulting resulting in an estimated in an estimated half‐life half-life of approximately of approximately three minutes three minutesin circulation in circulation [14–16]. Such [14– 16a short]. Such‐lived a short-lived existence is existence due to its is fast due degradation to its fast degradation by three membrane by three‐ membrane-boundbound lipid phosphate lipid phosphatephosphatases phosphatases (LPPs), which (LPPs), cleave which the cleave LPA thephosphate LPA phosphate group and group release and releasesignalling signalling-inactive‐inactive monoacylglycerol monoacylglycerol [17,18]. Contrary [17,18]. Contrary to this, LPA to this, production LPA production originates originates from the fromfollowing the following two sources: two phosphatidic sources: phosphatidic acid hydrolysis acid by hydrolysis PLA1/2 and by from PLA the1/2 and enzymatic from the conversion enzymatic of conversionlysophosphatidylcholine of lysophosphatidylcholine (1‐ or 2‐acyl (1-‐sn or‐glycero 2-acyl-sn‐3-glycero-3-phosphocholine‐phosphocholine or LPC) or to LPC) LPA to LPAby bya alysophospholipase lysophospholipase D D (lysoPLD) (lysoPLD) that that has has been been established established to to be be Autotaxin Autotaxin (ATX) (ATX) (Figure (Figure 11))[ [19,20].19,20]. ATX-catalysedATX‐catalysed production production constitutes constitutes the the main main physiological physiological source source of extracellular of extracellular LPA, and LPA, therefore and ATXtherefore has been ATX widely has been studied widely as studied a target as for a drugtarget development for drug development [14,21,22]. [14,21,22]. Figure 1.1. Distinct modes for lysophosphatidic acid (LPA) binding to itsits cognatecognate GG protein-coupledprotein‐coupled receptorsreceptors (GPCRs)(GPCRs) proposed based on theirtheir crystallographiccrystallographic structures.structures. Autotaxin (ATX) isis thethe main producerproducer ofof LPA,LPA, whichwhich cancan thenthen bindbind toto thethe extracellularlyextracellularly openopen lipid-bindinglipid‐binding pocketpocket ofof LPALPA1–31–3, potentiallypotentially assisted assisted by by lipid lipid chaperones, chaperones, or di orff usediffuse laterally laterally towards towards the membrane-open the membrane ligand-binding‐open ligand‐ sitebinding of LPA site4–6 of. LPA4–6. ATX isis the the only only member member of the of ectonucleotidethe ectonucleotide pyrophosphatase pyrophosphatase/phosphodiesterase/phosphodiesterase family (ENPP)family that(ENPP) presents that presents lysoPLD lysoPLD activity (EC activity 3.1.4.39) (EC over3.1.4.39) lysophospholipids over lysophospholipids [22]. ATX [22]. is first ATX translated is first astranslated a pre-proenyme as a pre‐ thatproenyme undergoes that twoundergoes proteolytic two processing proteolytic steps, processing resulting steps, in a resulting mature, glycosylated in a mature, andglycosylated secreted formand [23secreted]. The determinationform [23]. The of thedetermination structure of ATXof the by X-raystructure crystallography of ATX by enabled X‐ray thecrystallography determination enabled of its domain the determination organization, of i.e.,its thedomain two N-terminalorganization, somatomedin i.e., the two B N (SMB)-like‐terminal domainssomatomedin are followed B (SMB) by‐like a central domains catalytic are followed phosphodiesterase by a central (PDE) catalytic domain, phosphodiesterase which is adjacent (PDE) to an inactivedomain, nuclease-likewhich is adjacent domain. to Substratean inactive hydrolysis nuclease‐ requireslike domain. a bimetallic Substrate active hydrolysis site containing requires two a Znbimetallic2+ ions active and a site threonine containing nucleophile, two Zn2+ ions which and act a threonine in an associative nucleophile, two-step which in-line act in displacementan associative catalytictwo‐step mechanism in‐line displacement [24]. catalytic mechanism [24]. Structural studiesstudies have have also also established established that that ATX ATX has a has unique a unique tripartite tripartite binding binding site. The site. catalytic The bimetalliccatalytic bimetallic site is next site to is a next hydrophilic to a hydrophilic shallow grooveshallow that groove accommodates that accommodates the hydrophilic the hydrophilic glycerol moietyglycerol of moiety lipid substrates. of lipid substrates. This groove This is connectedgroove is connected by a T-junction by a T to‐junction a hydrophobic to a hydrophobic pocket where pocket acyl chainswhere canacyl bind, chains and can a tunnel bind, (oftenand a calledtunnel the (often “hydrophobic called the channel”)“hydrophobic that leads channel”) to the that other leads side to of the PDEother domain side of [ 25the]. PDE It is noteworthy domain [25]. that It theis noteworthy tunnel (or channel) that the is tunnel only partially (or channel) hydrophobic is only inpartially nature andhydrophobic has hydrophilic in nature patches, and has unlike hydrophilic the pocket patches, (Figure unlike2). The the tunnel pocket binds (Figure steroid 2). The molecules tunnel binds [ 26], assteroid well asmolecules the LPA [26], product as well [27, 28as], the which LPA results product in a[27,28], modulation which of results catalytic in a e ffimodulationciency. Thus, of catalytic we refer toefficiency. the tunnel Thus, as the we allostericrefer to the site, tunnel while as we the refer allosteric to the site, substrate-binding, while we refer to hydrophilic the substrate groove‐binding, and thehydrophilic hydrophobic groove pocket, and the as the hydrophobic orthosteric pocket, site (Figure as the3). orthosteric site (Figure 3). Cancers 2019, 11, 1577 3 of 17 CancersCancers 2019 2019, ,11 11, ,x x 33 ofof 1717 FigureFigure 2. 2.2. HydrophobicityHydrophobicity comparisoncomparison betweenbetween between thethe pocketpocket and and the the tunnel. tunnel.tunnel. SideSide Side sectionssections