Structure-Function Relationship of Autotaxin and the Importance of its Inhibitors. Lexi Tatem

Transcription

1 Structure-Function Relationship of Autotaxin and the Importance of its Inhibitors Lexi Tatem Department of Chemistry and Biochemistry, The University of Arizona, Tucson, Arizona Abstract Word Count: 214 Main Body Word Count: 3043

2 Abstract Autotaxin (ATX) is an enzyme and the major producer of lysophosphatidic acid (LPA) in the human body, a lipid involved in many cell-signaling pathways. ATX is a heterotetramer that contains an active site with a threonine nucleophile surrounded by two zinc ions, three aspartates, and three histidines. A hydrophobic pocket serves to accommodate various alkyl chains of LPA analogs. In humans, LPA signaling causes cellular survival, proliferation, differentiation, and migration. Resultantly, ATX is involved in development of the brain and blood vessels, the mobility of lymphocytes, wound healing, and neuropathic pain. Some of the negative effects associated with the dysfunction of ATX are obesity, various cancers, neuropathic pain, and heart disease. Due to its involvement in a large number of diseases, inhibitors of ATX have high therapeutic potential. As a result, there has been a recent surge in interest of ATX inhibitors. Several types of ATX inhibitors have been identified, including LPA based lipid inhibitors, cyclic phosphatidic acid based inhibitors, and non-lipid small molecule inhibitors. It was shown that LPA based inhibitors are not realistic for therapeutic use because they could be harmful toward LPA receptors, but non-lipid smallmolecular inhibitors have a higher probability for therapeutic use. Further research is needed to analyze the crystal structure of ATX in complex with nonlipid inhibitors.

3 Introduction Autotaxin (ATX) is a lysophospholipase D biological enzyme, or one that catalyzes the production of LPA. It is an extracellular glycoprotein in the ectonucleotide pyrophosphatase/phosphodisterase enzyme family (ENPP), and is also called ENPP2, due to its designation as the second family member. This enzyme family is composed of seven proteins that all have nucleotide pyrophosphatase activity and cleave diester phosphates. The substrates of the ENPP family include nucleoside triphosphates, lysophospholipids, and choline phosphate esters (Hausmann et al., 2012). The hydrolysis of lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA) is catalyzed by ATX, the major producer of LPA in the human body. The general structure of LPC contains a carbonyl group, a hydroxyl group, a phosphate group, a positively charged nitrogen group, and an alkyl chain of variable length. In addition, the alkyl groups can be in either saturated or unsaturated form (Van Meeteren et al., 2005). LPA and choline are the products of hydrolysis. Figure 1 shows the mechanism of the reaction with a threonine nucleophile. Figure 2 shows the multistep reaction scheme of the catalytic cycle of ATX. LPA is an agonist lipid that binds to six specific G-protein coupled receptors and thus, activates many pathways and is involved in a variety of cellular responses. Moreover, LPA signals can cause cellular proliferation, differentiation, and migration. Nishimasu et al. illustrated that ATX is involved in a number of processes in the body including development of the brain and blood

4 vessels, the mobility of lymphocytes, wound healing, and neuropathic pain (Nishimasu et al., 2010). Similarly, the findings of Houben and Moolenaar indicate that ATX-LPA signaling promotes tumor formation, angiogenesis, and experimental metastasis in mice (2011). Due to its involvement in LPA production and the activities described above, irregular expression of ATX is associated with a range of diseases including obesity, various cancers, neuropathic pain, and heart disease. For instance, Kawaguchi et al. showed that overexpression of ATX can be seen in various cancer tissues. Houben and Moolenaar showed that abnormal LPA production or expression could cause the initiation and development of cancer. Additionally, they found that heightened expression of ATX and abnormal expression of LPA receptors are found in several human malignancies, while loss of LPA function has been connected to bladder cancer (2011). As a result, the development of ATX inhibitors would have a vast amount of therapeutic potential, and particularly, could serve as a solution for an anticancer drug. In an attempt to develop effective inhibitors, researchers have recently begun to analyze the crystal structure of ATX with various molecules that have shown inhibition activity. Three types of ATX inhibitors that have been found thus far include LPA based lipid inhibitors, cyclic phosphatidic acid (CPA) based inhibitors, and non-lipid small-molecular inhibitors.

5 Main body Basic Structure of ATX The work of Nishimasu et al. produced crystal structures, which showed that ATX is a dense, multi-domain protein. Two of the domains in ATX are located on the amino-terminus side of the protein and are somatomedin-b-like complexes called SMB1 and SMB2. Somatomedin B is a small peptide that is rich in the amino acid cysteine and is found in many proteins. In addition, there is a nuclease-like domain on the carboxy-terminus of the protein called NUC and a catalytic phosphodiesterase domain in the center called PDE (Nishimasu et al., 2010). Figure 3 shows the protein ATX with each of the four domains highlighted. The two SMB regions have a low sequence identity, at 15%, but are very similar with respect to their structural conformation. There is a disulfide-bonded region conserved in all three of these SMB forms, indicating that its presence is important to the function of the protein. The C-terminal NUC domain contains a mixed α/β fold and is binds various ions including calcium, sodium, and potassium ions. These ions are bound and stabilized with different residues. Calcium is coordinated by Asp735, Asn737, Asn739, Asp743, Leu741, and a water molecule. Met671, Asp668, Tyr665, and three water molecules stabilize the potassium ion. Finally, Asp797, Ser800, Ser803, and three water molecules coordinate the sodium ion. The work of Nishimasu et al. shows that the calcium ion is bound very tightly by a hand-like motif, and is not involved in the production of LPA, but is involved in oligodendrocyte development. Oligodendrocytes are a type of glial cell involved

6 in support and insulation of the axons. Calcium and sodium are bound near the boundary between the NUC and PDE domains, and thus are thought to participate in the interactions between the two domains. In addition, water molecules located in-between these two domains form a stabilizing hydrogen bonding network (Nishimasu et al., 2010). The catalytic PDE domain contains an α/β fold in the core. The active site of ATX is in the PDE domain and contains a threonine nucleophile surrounded by two zinc ions, three aspartates and three histidines (Figure 4). There is a hydrophobic pocket (Figure 5) in the PDE domain that serves to accommodate the alkyl chain of LPA, and a tunnel formed by the SMB1 and PDE domains that connects the hydrophobic pocket to the catalytic site (Hausmann et al., 2012). This hydrophobic channel is thought to act as a second LPA binding site (Nishimasu et al., 2010). Nishimasu et al. identifies two linker regions that serve as additional support for the entire heterotetramer. The L1 linker region is located between the SMB2 domain and the PDE domain, but only interacts with the PDE domain. Contrarily, the L2 linker region is located between the PDE and NUC domains and interacts with both. The L1 and L2 linker regions are depicted in Figure 3. Different Forms of ATX There are three isoforms of ATX that are produced via alternative splicing. These three forms are referred to as ATXα, ATXβ, and ATXγ. ATXα was the first identified form of ATX and has a 52-amino acid polybasic insert in the PDE

7 domain (Hausmann et al., 2012). ATXγ is a form generally found in the central nervous system and has a 22-amino acid insertion (Hausmann et al., 2012). ATXβ is the most studied and recognized form of the enzyme and is identical to lysophospholipase D. This is the isoform that this paper focuses on. Structure of ENPP family The ENPP family of enzymes can be split into two groups. The first is comprised of ENPP1, ENPP2 (ATX), and ENPP3. This group has the multidomain structure made up of two N-terminal SMB domains, a central PDE domain, and the C-terminal NUC domain (Figure 3). The second group contains ENPP4 through ENPP7, all of which contain only the PDE domain. However, the structure of the active site of all ENPP family members is conserved (Figure 4). This indicates that its structure is essential to the function of the protein. The seven members of the ENPP family differ in their physiological functions. This is a result of the fact that they bind different substrates. However, it is important to note that the actual molecular feature that causes substrate specificity among the ENPP family is not known. ATX is unique in the fact that it is the only ENPP protein that is secreted. All other members of ENPP are transmembrane proteins (Hausmann et al., 2012). Moreover, all members of ENPP aside from ATX have an insertion loop and therefore do not promote the hydrolysis of LPC to LPA.

8 Structure-Function Relationship There are several structural characteristics that help to stabilize the ATX heterotetramer and assist in its function. These features contain both hydrophilic and hydrophobic interactions. They include a lasso loop region, an N-linked glycan chain, and a disulfide bond (Hausmann et al., 2012). The glycan chain and disulfide bond are shown in Figure 3. The loop structure is around fifty amino acids in length, is connected to the PDE domain, and wraps around the carbonyl-terminus NUC domain. The N- linked glycan chain occurs at Asn524, while the disulfide bond is between Cys413 and Cys805 and connects the PDE and NUC domains. It has been shown by Jansen et al. in 2007 and 2009 that the N-linked glycan chain and the disulfide linkage are important to the catalytic activity of the enzyme. Both the SMB1 and SMB2 domains are small, rich in the amino acid cysteine, and help assist in binding of ATX to trans-membrane receptors on cells. Therefore, these domains are important with respect to the release of LPA once it is hydrolyzed. Additionally, Hausmann et al. described that there is evidence that catalytic activity may be improved through interaction between SMB2 and the integrin, or transmembrane receptors (2012). Another significant characteristic of ATX structure that assists in function is the hydrophobic pocket on the PDE domain, which is involved in both binding of the substrate and binding of inhibitors (Nishimasu et al., 2010). The hydrophobic pocket is lined with nonpolar amino acid residues such as isoleucine, leucine, alanine, methionine, and phenylalanine, shown in Figure 5.

9 These nonpolar residues are able to stabilize the lipid tail of various LPA molecules through hydrophobic interactions. In addition, the pocket is formed though the deletion of 18 amino acids, which are present in all other ENPP family members. This deletion is unique to ATX, which suggests that it is the only enzyme in the ENPP family with a hydrophobic pocket (Hausmann et al., 2012). LPA analogs LPA is a feedback inhibitor of ATX, and the hydrophobic pocket on the PDE domain is able to bind to different forms of LPA in unique conformations. Nishimasu et al. analyzed ATX-LPA complexes with LPAs of various acyl chain lengths and saturations, and found that the optimal acyl chain length, with respect to the hydrophobic pocket, is 14 molecules. However, longer chain lengths can be stabilized if there are unsaturated bonds in the acyl chain. This is because the unsaturated bonds create sharp turns. Moreover, Hausmann found that LPA with unsaturated alkyl chains bind in a bent conformation, while those with saturated chains bind in a more linear conformation (Hausmann 2012). In addition, the analysis by Nishimasu et al. illustrated the importance of hydrogen bonds, van der Waals interactions, and hydrophobic interactions in these complexes (Nishimasu et al., 2010). One oxygen molecule on the phosphate group of LPA is coordinated by the divalent ion, while another forms hydrogen bonds with asparagine and threonine residues. The third oxygen atom on the phosphate group forms hydrogen bonds with lysine, asparagine, and

10 aspartic acid. These three amino acid residues are conserved in the ENPP family, which illustrates their importance for function (Nishimasu et al., 2010). Variety of ATX Inhibitors ATX has recently taken on an emerging role in disease. Therefore, inhibitors of ATX could potentially be therapeutic. The work of East et al. focused on analyzing LPA analogs as ATX inhibitors because LPA is a feedback inhibitor of ATX. The authors found that electron density in the pyridine region of an ATX inhibitor influences its inhibition potential (East et al., 2010). However, in 2013 Kawaguchi et al. reported that LPA based inhibitors are not realistic for therapeutic use because they could have strong negative effects on LPA receptors. In addition, they have harmful or non-complementary properties such as intestinal absorption, water solubility, and metabolic stability (Kawaguchi et al., 2013). Another class of inhibitors called non-lipid small molecule inhibitors is an attractive option for therapeutic use. It has been found that non-lipid inhibitors show good inhibition activity within an organism. Additionally, Hausmann et al. found that one inhibitor that the hydrophobic pocket stabilizes is HA155 (2012). This boronate-based molecule targets the threonine nucleophile. Kawaguchi argues that there is a need for such inhibitors to be optimized based on crystal structures of ATX in complex with various compounds. Kawaguchi et al. developed a fluorescence probe to analyze the effectiveness of various inhibitors. Using this probe, they solved the crystal

11 structures of various ATX complexes with potential inhibitors. The crystal structure of ATX with compound 10 (PDB: 3WAV) showed that compound 10 binds to the active site of ATX, and is therefore a competitive inhibitor. There is a dichlorobenzene segment on compound 10 that is stabilized by the hydrophobic pocket discussed earlier. Hydrogen bonds also help to accommodate this complex. The other inhibitor that Kawaguchi et al. focused on was the hydrophilic compound 16. However, the crystal structure of this molecule showed that compound 16 does not bind to the catalytic pocket (Kawaguchi et al., 2013). Factors of ATX Inhibitory Potential The work of Kawaguchi et al. shows that the most important aspects of inhibitory molecules are their hydrophobicity and molecular size. Using these factors and compound 10 as a guideline, Kawaguchi et al. attempted to find compounds with improved inhibitory activity. Using a benzene moiety, the authors found that the compounds with the most potent inhibitors in this study were 3BoA and 4BoA, while 2BoA had weaker inhibitory activity. Kawaguchi notes that 3BoA and 4Boa have boronic acid further from the substituent than 2BoA. Particularly, 3BoA has boronic acid at the Meta position and 4BoA has boronoic acid at the Para position. Contrarily, 2BoA has boronic acid at the Ortho position (Kawaguchi et al., 2013). See Figure 6 for an illustration of ATX in complex with 2BoA and 4BoA. Kawaguchi et al. utilized crystal structures to show why 2BoA was a weaker inhibitor than 3BoA and 4BoA (PDB: 3WAW, 3WAX, and 3WAY,

12 respectively). The crystal structures of 3BoA and 4BoA show that the inhibitors bind to the active site of ATX and that the oxygen and boron atoms of boronic acid help stabilize the structure by interacting with the zinc ions and threonine residue, respectively (see Figure 6). Contrarily, the crystal structure shows that boronic acid cannot interact with the active site when ATX is in complex with 2BoA, which illustrates why it is not an effective inhibitor (Kawaguchi et al., 2013). Concurrently, the work of St-Coeur et al. shows that a thiazolidinedione compound with a boronic acid moiety is also an effective non-lipid inhibitor (St- Coeur et al., 2013). These results support the findings of Kawaguchi et al. that boronic acid helps to stabilize an inhibitor. Mouse ATX Much of the structural work of ATX has utilized mouse ATX. It is noted by Nishimasu et al. that mouse and human ATX have 93% sequence identity, an extremely high percentage. In addition, the amino acids important to the function of the ATX molecule are strictly conserved and both forms of ATX reacted similarly toward LPC substrates. See Figure 7 for the structural alignment and Figure 8 for the sequence alignment of human and mouse ATX. Due to the high sequence identity, the results found in research based on mouse ATX can be used for human research purposes, and can be used to find inhibitors of human ATX for anticancer drugs (Nishimasu et al., 2010). For

13 example, the article by Kawaguchi et al. utilized mouse ATX to screen various ATX inhibitors. Further studies One area that needs further research is the structure of the PDE domain. While the interactions of the hydrophobic pocket on the catalytic domain are somewhat understood, the exact structure of this domain is not. This is partly due to the fact that the PDE domain is not thermodynamically stable on its own, but is stabilized by interactions with other domains on ATX. Discussion The structure of ATX, a heterotetramer, is somewhat complex and contains various molecular interactions that help stabilize its structure and the binding of substrates. The active site of ATX contains a threonine nucleophile surrounded by two zinc ions, three aspartates, and three histidines. The negative and positive charges on aspartate and histidine, respectively, have the potential to form ionic interactions. These charges further stabilize the active site. Nearby, the hydrophobic pocket serves to accommodate different alkyl chains of LPA analogs. Among all ENPP family members, the structure of the active site is conserved, which indicates that its structure is essential to function. Inhibition of ATX is an area of study that could lead to significant therapeutic treatments, most notably in the realm of cancer, due to the emerging

14 role of ATX in disease. Analysis of ATX crystal structure in complex with inhibitors shows that the most important factors for inhibitory molecules are hydrophobicity and molecular size. Furthermore, the critical size at which an inhibitor binds best to the active site is increased with the presence of unsaturated bonds. Boronic acid mimics the phosphate and helps to accommodate inhibitors in ATX. However, the location of boronic acid affects its ability to interact with the active site. Crystal structures of 2BoA, 3BoA, and 4BoA show that they are located at the Ortho, Meta, and Para positions, respectively. Thus, 3BoA and 4BoA bind to the active site of ATX, while 2BoA does not (Kawaguchi et al., 2013). Researchers continue to use the crystal structure of various forms of ATX, alone and in complex with LPA and other inhibitors, in hopes of finding the most effective inhibitors.

15 References East, J.E., Kennedy, A.J., Tomsig, J.L., De Leon, A.R., Lynch, K.R., and Macdonald, T.L. (2010) Synthesis and structure-activity relationships of tyrosinebased inhibitors of autotaxin (ATX). Bioorg and Med Chem Letters. 20, Hausmann, J., Perrakis, A., and Moolenaar, W.H. (2012) Structure-function relationships of autotaxin a secreted lysophospholipase D. Adv in Biol Regulation. 53, Houben, A.J. and Moolenaar, W.H. (2011) Autotaxin and LPA receptor signaling in cancer. Cancer Metastasis Rev. 30(3-4), Kawaguchi, M., Okabe, T., Okudaira, S., Nishimasu, H., Ishitani, H.K., Nureki, O., Aoki, J., and Nagano, T. (2013) Screening and x-ray crystal structure-based optimization of autotaxin (ENPP2) inhibitors, using a newly developed fluorescence probe. ACS Chem Bio. 8, Mize, C.D., Abbott, A.M., Gacasan, S.B., Parrill, A.L., and Baker, D.L. (2011) Ligand-based autotaxin pharmacophore models reflect structure-based docking results. Nat Inst of Health. 31,

16 Nishimasu, H., Okudaira, S., Hama, K., Mihara, E., Dohmae, N., Inoue, A., Ishitani, R., Takagi, J., Aoki, J., and Nureki, O. (2010) Crystal structure of autotaxin and insight into GPCR activation by lipid mediators. Nature Struc and Molec Bio. 18, North, E.J., Osborne, D.A., Bridson, P.K., Baker, D.L., Parrill, A.L. (2009) Autotaxin structure-activity relationships revealed through lysophosphatidylcholine analogs. Bioorg and Med Chem Letters, 17, Saunders, L.P., Cao, W., Chang, W.C., Albright, R.A., Braddock, D.T., and De La Cruz, E.M. (2011) Kinetic analysis of autotaxin reveals substrate-specific catalytic pathways and a mechanism for lysophosphatidic acid distribution. J Biol Chem, 286(34): St-Coeur, P.D., Ferguson, D., Morin, P.J., and Touaibia, M. (2012) PF-8380 and closely related analogs: synthesis and structure-activity relationship towards autotaxin inhibition and glioma cell viability. Arch Pharm Chem Life Sci. 346, van Meeteren, L.A., Ruurs, P., Christodoulou, E., Goding, J.W., Takakusa, H., Kikuchi, K., Perrakis, A., Nagano, T., and Moolenaar, W.H. (2005) Inhibition of autotaxin by lysophospatidic acid and sphingosine 1-phosphate. J Biol Chem. 280,

17 Figure Legends Figure 1. The Hydrolysis of LPC to LPA. This figure was created with ChemDraw The mechanism for the hydrolysis of LPC to LPA is shown (as one step). The mechanism includes the threonine nucleophile that is present in ATX. The products of the hydrolysis are choline and LPA. Figure 2. The Reaction Scheme. This figure was obtained from Saunders et al. and illustrates the reaction scheme of the catalytic cycle of ATX. C represents choline in the figure. Note that this is a multistep mechanism (2011). Figure 3. The Domains of ATX. Image created in PyMOL using PDB file 2XR9. The figure shows the whole ATX molecule with the SMB1 region colored magenta, the SMB2 region colored orange, the catalytic PDE region colored cyan, and the NUC region colored yellow. The L1 linker region is red and the L2 linker region is green. Additionally, the asparagine in the N linked glycan chain is shown as a salmon-colored stick (near the center of the molecule), and the cysteine residues participating in a disulfide bond are shown as blue sticks (near the bottom). Figure 4. The Active Site of ATX. Image created in PyMOL using PDB file 2XR9. This image shows only the active site of ATX. The zinc ions are colored red, the threonine nucleophile is a blue stick, the three surrounding aspartates are orange lines, and the three surrounding histidines are purple lines. Figure 5. The Hydrophobic Pocket. Images created in PyMOL using PDB file 2XR9. The seven nonpolar amino acid residues that make up the hydrophobic pocket are shown as sticks and highlighted various colors. They are Ile167 (red),

18 Leu216 (orange), Ala217 (yellow), Leu259 (green), Phe273 (blue), Trp275 (purple), and Met512 (hot pink). Figure 6. ATX in Complex with 2BoA and 4BoA. Images created in PyMOL using PDB files 3WAW and 3WAY. ATX bound with 2BoA is aligned with ATX bound with 4BoA. The inhibitor 4BoA is shown as blue sticks and 2BoA is shown as red sticks. Boronic acid is located in the Ortho position in 2BoA, as opposed to the Para position in 4BoA, resulting in weaker inhibitor activity. Figure 7. Human and Mouse ATX. Images created in PyMOL using PDB files 3NKM and 2XR9. Mouse ATX, (cyan) and Human ATX (blue) were structurally aligned, illustrating their extremely high sequence identity. The zinc ions in the active site are highlighted showing their conservation. Figure 8. Sequence Alignment of Human and Mouse ATX. Sequence alignment generated using COBALT. The sequence alignment shows that the two proteins have 93% sequence identity.

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