The bile acid phospholipid conjugate ursodeoxycholate lysophoshatidylethanolamide acts by binding to calcium independent membrane phospholipase A₂ type beta

Introduction

The actual challenge in hepatology is the therapy of non-alcoholic steatohepatitis (NASH). It presents as steatosis hepatitis with features of inflammation. NASH is the most common liver disease in the world. It develops to cirrhosis with its complications, including hepatocellular carcinoma. Several indicative laboratory parameters such as γ-glutamyl-transferase (GGT) and alanine aminotransferase (ALT) are elevated in this disease. However, a therapy is not available. Predisposing conditions are metabolic syndrome, diabetes and hypercholesterolemia. However, the pathogenesis is still unclear, but a key feature is the increased ratio of lysophosphatidylcholine (LPC) to phosphatidylcholine (PC), suggesting a higher activity of phospholipase A₂ (PLA₂) (1). The PLA₂ family of phospholipases is classified into five distinct groups, namely the secretory (sPLA₂), cytosolic (cPLA₂), Ca²⁺-independent (iPLA₂), lysosomal (LPLA₂), and platelet-activating factor (PAF) acetylhydrolases (2). This complex family of enzymes plays a significant role in inflammation by triggering the synthesis of regulatory lipid molecules, which are produced and activated at the site of inflammation. Each member of this hydrolase enzyme family serves a distinct role by generating active lipid metabolites that promote inflammatory diseases including hyperlipidemia, obesity, and diabetes. In particular, the cPLA₂s composed of different isoforms (PLA2α, PLA2β, and PLA2γ) participate in inflammation by hydrolyzing the ester bond at the sn-2 position of phospholipids to produce non-esterified fatty acids and lysophospholipids. In contrast, most sPLA₂s contain a conserved calcium-binding loop and strongly dependent on Ca²⁺ concentrations for their catalytic activity (2). Members of the iPLA₂ family have significant functions in transmembrane signal transduction, while the lysosomal PLA₂ (known as PLA2G15) are involved in the catabolism of pulmonary surfactant and play a role in host defense and in the processing of lipid antigens for presentation by CD1 proteins (3). The three types of PAF acetylhydrolases, i.e., PAF-AH(I), PAF-AH(II), and PAF-AH(III) mediate the inactivation of the PAF by deacetylation of the acetyl group located at the sn-2 position of the glycerol backbone (4,5).

The PC breakdown product LPC is metabolically active for generation of phosphorylated C-Jun-N-terminal kinase 1 (pJNK1) (6). This key player accelerates cellular fatty acid influx (7) and induces inflammation and fibrosis. All downstream features of JNK1 signaling are well established (8). Nevertheless, the generation of LPC as precipitating event remains unresolved. Interestingly, hepatic fatty acid uptake is mediated by a heterotetrameric protein arrangement consisting of the fatty acid binding protein of plasma membranes (FABP_PM) (9), the cluster of differentiation (CD36) (10), caveolin-1 (11), and calcium-independent membrane PLA₂ type beta (iPLA₂β) also known as group 6 iPLA2s (7). The complex is localized within detergent-resistant membrane platforms of the plasma membrane (DRM-PM) (7), representing specialized lipid domains constituting raft structures which mediate cellular communication (12). The iPLA₂β is the prominent player in cellular PC hydrolysis (13). Within the fatty acid uptake complex, iPLA₂β serves as the constitutive protein maintaining the required structural stability (14). It may not be directly involved in the fatty acid influx process, but it holds contact to intracellular metabolism for uptake regulation. It remains to be established, whether it may simply respond to cytosolic LPC concentration with feedback inhibition of its enzymatic activity or a more complex regulation takes place as previously proposed (15).

The bile acid phospholipid conjugate, ursodeoxycholate-lysophosphatidylethanolamide (UDCA-LPE) inhibits iPLA₂β and suppresses pro-inflammatory LPC generation in a dose-dependent mode (7). According to the described dual function of iPLA₂β as member of the fatty acid uptake complex and its intrinsic capability to generate LPC, UDCA-LPE is a potent inhibitor also of cellular fatty acid influx. Analysis of the uptake kinetics revealed a reduction of V_max, which is compatible with fading of available active transport sites (7). Consequently, it reversed liver steatosis, inflammation, and fibrosis in animal studies, which are all critical features observed in NASH (16). Even TNF-α-induced acute liver failure could be prevented, if the drug was given concomitantly (17,18). Thus, UDCA-LPE could serve as hepatoprotective, anti-inflammatory drug for the liver (19). The open question remains, whether iPLA₂β interacts with UDCA-LPE.

In the present study, we used in silico techniques for predicting potential docking sites of UDCA-LPE in iPLA₂β. Our computational approach revealed a clear affinity of UDCA-LPE for iPLA₂β with a region between Phe84 and Leu125 that should have the largest affinity for UDCA-LPE. Noteworthy, the proposed region is nearly identical with those that were determined for binding of pyrrophenone to the evolutionarily conserved PLA₂α. Hence, the proposed direct binding of UDCA-LPE to iPLA₂β might explain the efficacy of UDCA-LPE in preventing hepatic fatty acid uptake.

We present the following article in accordance with the handout on scientific writing and checklist of scientific manuscript components (available at https://amj.amegroups.com/article/view/10.21037/amj-21-10/rc).

Methods

General strategy used for molecular docking analysis

In order to analyze the binding energy and the docking pose of UDCA-LPE on iPLA₂β protein from Chinese hamster (PDB ID: 6AUN), molecular docking was performed as described previously (20). Pyrrophenone (21) was used as positive control compound. The pyrrolidine derivative pyrrophenone is a cell-permeable highly potent and reversible inhibitor of the catalytic domain (CAT) cPLA₂α through formation of a hemiketal between its ketone carbonyl and the active site serine of the enzyme based on the ability of polarized ketones to form stable hemiketals with serine proteases and esterases (22). For molecular docking analysis, this component is ideally suited, because its molecular mass is similar to UDCA-LPE. We used the following strategy for docking analysis. At first, unbiased blind docking was performed by covering the whole protein surface. The binding site of pyrrophenone was determined, and defined docking was performed by covering the binding site of pyrrophenone found after blind docking analysis. Then, we used the same setting for UDCA-LPE and identified potential interaction regions. In each case, the binding energy and docking pose of UDCA-LPE was compared with those of pyrrophenone.

Performance of docking analysis

For docking analysis, we used AutoDock 4.2, representing software with a graphical user interface offering a variety of search algorithms to explore a given docking problem (23). This automated docking algorithm is widely used for prediction of biomolecular complexes in structure/function analysis and in molecular design (23). It allows fully flexible modelling of specific portions of protein, in a similar manner as the ligand. In the algorithms, semi-empirical free energy force fields are used to predict the binding free energies of small molecules such as pyrrophenone or UDCA-LPE to supposed macromolecular target structures such as iPLA₂β (23). The molecular graphics program Visual Molecular Dynamics (VMD), which is designed for the display and analysis of molecular assemblies (24), was used to visualize the docking poses.

Statistical analysis of docking prediction

In our analysis, the docking parameters were set to 250 runs and 2,500,000 energy evaluations for each cycle and three independent runs were performed by using AutoDock 4.2 algorithm (23). Average values of binding energies and standard deviations (SDs) were calculated and mentioned at the corresponding table.

Sequence alignments

Human, mouse, rat and Chinese hamster iPLA₂β proteins were aligned using the Clustal Omega program (25). Sequence information from respective proteins were taken from the GenBank (Homo sapiens: NP_001336795.1; Mus musculus: NP_001185954; Rattus norvegicus: NP_001257725.1; Cricetulus griseus: 6AUN_B).

Remarks on scientific writing

We present the following article in accordance with the handout on scientific writing and checklist of scientific manuscript components according to the forms provided by the Teaching Issues and Experiments in Ecology (TIEE), which is a peer-reviewed web-based collection of ecological educational materials. The respective form can be found at: https://tiee.esa.org/vol/v4/experiments/habitat_shifts/pdf/Appendix3.pdf.

Results

iPLA₂β, an evolutionarily conserved protein

The 85-kD cytosolic calcium-independent PLA2 (iPLA₂β, also known as PLA2G6A or PNPLA9) was originally isolated from Chinese hamster ovary (CHO) cells (26). The full-length protein encodes an evolutionarily highly conserved 752-amino acid protein with one lipase motif (GXS465XG) and eight residue ankyrin repeats (Figure 1). It shares about 90.56% to 98.54% identity between human, Chinese hamster, mouse, and rat (Table 1).

Figure 1 Multiple sequence alignment of iPLA₂β. Human, mouse, rat and Chinese hamster iPLA₂β proteins were aligned using the Clustal Omega program. Amino acid residues fully conserved in all four sequences are indicated by asterisks (*) below the bottom line. Colons (:) indicate conservation between groups of strongly similar properties, while periods (.) indicate conservation between groups of weakly similar properties. Amino acid positions of the proposed UDCA-LPE binding site are marked in reversed black type. Positions of ankyrin repeats (❶–❽) and lipase domain boxed in grey are indicated. UDCA-LPE, ursodeoxycholate-lysophosphatidylethanolamide; iPLA₂β, calcium-independent membrane phospholipase A₂ type beta.

The enzyme belongs to a diverse group of enzymes catalyzing hydrolysis of the sn-2 substituent form glycerophospholipid substrates to yield a free fatty acid and a 2-lysophopholipid. There is now ample evidence that alterations in iPLA₂β function is involved the pathogenesis of many diseases including cardiovascular disease, cancer, and muscular dystrophy (27). In addition, there is evidence that iPLA₂β is involved in the pathogenesis of diabetes and non-alcoholic steatohepatitis (7,28). Therefore, iPLA₂β inhibitors are potential good drug candidates to interfere with respective diseases. In line with this assumption, the selective inhibition of iPLA₂β with a fluoroketone inhibitor (i.e., FKGK18) showed beneficial effects in models of autoimmune type 1 diabetes (29). We have recently shown that UDCA-LPE disintegrated the lipid backbone of raft plasma membrane domains by the removal of iPLA₂β (14). In the mentioned study, we discussed that UDCA-LPE potentially exerted its effects by acting as an iPLA₂β inhibitor. However, providing experimental proof for this hypothesis is complex, because interaction studies are mainly aggravated by the low solubility of UDCA-LPE and the fact that the CAT of the enzyme forms a tight dimer that is not easily accessible (27).

Prediction of interactions of iPLA₂β UDCA-LPE

A well-established potent inhibitor for cPLA₂α is pyrrophenone. This non-peptide and low-molecular weight inhibitor is structurally a 1,2,4-trisubstituted pyrrolidine that strongly inhibits the esterase and lysophospholipase activities of cellular PLA₂α (30). Although structurally different, the overall size as measured by its molecular mass is similar to UDCA-LPE (Figure 2).

Figure 2 Structure of pyrrophenone and ursodeoxycholic-lysophosphatidylethanolamide (UDCA-LPE). The average mass (849.96 Da) of pyrrophenone (CAS no. 341973-06-6) with a molecular formula of C₄₉H₃₇F₂N₃O₅S₂ is similar to the coupled bile acid-phospholipid conjugate UDCA-LPE composed out of ursodeoxycholic acid (UDCA, CAS no. 128-13-2, C₂₄H₄₀O₄, M_r =392.56) and lysophosphatiylethanolamine composed of an ethanolamine head group and glycerophosphoric acid with various fatty acids located in sn-1 position (LPE, CAS 95046-40-5, M_r =~479). UDCA-LPE, ursodeoxycholate-lysophosphatidylethanolamide.

To get an impression of a possible binding of UDCA-LPE to iPLA₂β, we performed a molecular docking approach using the pyrrophenone as a positive control compound. In an unbiased “blind docking” attempt, we found that potential interacting residues within iPLA₂β for pyrrophenone are located between amino acid position Gln86 and Glu128. Upon performing the same setting for UDCA-LPE, we identified a region between Pro500 and Val678 that might have binding capacity. However, in a defined docking approach, in which potential pyrrophenone interacting residues were considered, we identified a region between Phe84 and Leu125 that should have the largest affinity for UDCA-LPE. The proposed region is nearly identical with those that were determined for pyrrophenone. The results for the blind and defined docking runs are summarized in Table 2.

It is remarkable that multiple sequence alignment for human, Chinese hamster, mouse, and rat iPLA₂β protein revealed that the residues in the UDCA-LPE proposed docking site were all conserved for 10 out of 14 residues (cf. Figure 1) except for position His85 in humans, for position Tyr87 in humans and rats, for position Leu92 in humans, and for position Ser121 in humans. As shown in Figure 3, UDCA-LPE bound in close proximity to the pyrrophenone binding site but with lower affinity (−7.9±0.6 vs. −14.3±0.6 kcal/mol). Leu91 was forming a hydrogen bond with UDCA-LPE whereas Ser88, Ser111 and Trp120 were forming a hydrogen bond with pyrrophenone. Ten residues were commonly interacting with UDCA-LPE and pyrrophenone.

Figure 3 Docking poses of UDCA-LPE (yellow) and pyrrophenone (red) on iPLA₂β (PDB ID: 6AUN) after defined docking analysis. Bold labeled residues are forming hydrogen bonds. Coloring of the protein was applied according to the secondary structure; turn (green), β-sheet (yellow), α-helix (violet), coil (white). UDCA-LPE, ursodeoxycholate-lysophosphatidylethanolamide; iPLA₂β, calcium-independent membrane phospholipase A₂ type beta; LBE, ligand-binding energy.

Discussion

The question addressed in this paper was, how UDCA-LPE exerts its inhibitory effect on fatty acid influx as well as inflammation of the liver. One option is based on its detergent effect on DRM-PM leading to dissolution of these platforms as prerequisite for constitution of the fatty acid uptake complex. In vitro experiments with isolated DRM-PM indeed showed that UDCA-LPE (50 µM) resulted in a 63.13%±7.40% reduction of phospholipids and an 81.94%±8.30% reduction of cholesterol in relation to mg total protein (14). The ratio of phospholipids to cholesterol changed from 2:1 to 4:1, resembling those of non-DRM fractions (31). Thus, iPLA₂β could lose contact to DRM-PM and distribute to other cell compartments.

The other option is that iPLA₂β is primarily and specifically removed from the membrane fatty acid uptake complex. This is followed by removal of the other members of the fatty acid uptake complex (7,14). Moreover, if PC remains bound to iPLA₂β even if iPLA₂β is displaced from DRM-PMs and the enzymatic activity is silenced after UDCA-LPE exposure, it is likely that the DRM-PM lipid platform is consequently dissolved (14).

In favor of the iPLA₂β removal option is the observation that the same phenomenon was observed with other phospholipase inhibitors, which are not considered as detergents: methyl arachidonyl fluorophosphonate (MAFP) and bromoenol lactone (BEL) (7). Therefore, the next question, which had to be answered, is whether UDCA-LPE binds to iPLA₂β. Recently the structure of iPLA₂β was resolved, and it was suggested that this protein forms a stable dimer, in which the active sites of the dimer are wide open providing sufficient space for phospholipids to access the catalytic centers (27). Moreover, the enzymatic activity of the dimer can be allosterically inhibited by calmodulin altering the confirmation of the dimerization interface (27). The question arises, whether dimerization is obligatory for its enzymatic function and whether monomeric iPLA₂β within the heterotetrameric fatty acid uptake complex serves for the same activation purpose. One could argue that the subunit of iPLA₂β communicates via ankyrin repeats with other proteins (15). Indeed, mutations lacking the ankyrin repeats of iPLA₂β retain their CAT, but are catalytically inactive (15).

In this study, we demonstrated that UDCA-LPE bound with high affinity to iPLA₂β. Since the UDCA-LPE binding energy was lower than −7 kcal/mol, it reflects binding affinity. According to the literature, −7 kcal/mol can be considered as threshold value reflecting strong interaction with the protein of interest (32-34). Based on theoretical considerations and respective modeling systems, the applied molecular docking approach is a valuable tool to provide an estimate of the location and binding energy of a ligand toward a target protein. In contrast, a direct experimental evaluation of UDCA-LPE binding is prone to artifacts, because iPLA₂β is embedded in DRM-PM platforms and the required purification disturbs its structure and, thus, its function.

Next, it has to be addressed, whether UDCA-LPE leads to allosteric inhibition of iPLA₂β via conformational change, in a comparable manner as reported for MAFP and BEL (15). As shown in Figure 3, UDCA-LPE binding is proposed outside the catalytic side of the enzyme, which is compatible with a concomitant structural change. If the requirements for binding to the enzyme are defined, other small molecules can be designed for optimal clinical use. Bioavailability, effectiveness, metabolic disposition, adverse events and toxicity are the relevant evaluation criteria.

These findings are of fundamental importance and may have direct therapeutic implications. UDCA-LPE is a potent inhibitor of cellular fatty acid influx and a direct inhibitor of iPLA₂β activity. As such, compounds with similar structure to UDCA-LPE but overall higher solubility might be an effective therapeutic mean for NASH therapy. It will be interesting to follow up, how the drug-likeness of these compounds could be translated into novel therapies.

Our study has some limitations. For docking analysis, we used a template consisting of pyrrophenone and cPLA₂α for determination of binding sites of UDCA-LPE in iPLA₂β. Although both proteins belong to the phospholipase superfamily, it is obvious that they have defined functions that might predict different structures. However, sophisticated structure analysis has shown that the fold of the core secondary structure elements in the CAT of iPLA₂β resembles that of cPLA₂α CAT (27), which suggests that cPLA2α is a good template protein. Likewise, the cPLA₂α inhibitor pyrrophenone (CAS no. 341973-06-6) has a molecular weight of ~850 and molecular formula C₄₉H₃₇F₂N₃O₅S₂, while the composite of ursodeoxycholic acid (CAS no. 128-13-2; molecular weight ~393, C₂₄H₄₀O₄) and lysophosphatidylethanolamine (CAS no. 95046-40-5; molecular weight ~479; C₇H₁₅NO₇P⁻) have a different molecular structure and, thus, mobility that might interfere with the dynamics in binding to its predicted target. Moreover, the water solubility of pyrrophenone and UDCA-LPE may vary, which per se might influence the binding to their target sites. UDCA-LPE has limited solubility and the cell-permeable inhibitor pyrrophenone is only sparingly soluble in aqueous buffers too (35,36). This suggests that the solubility in our assay may affect both compound-enzyme interactions. Unfortunately, the low solubility of UDCA-LPE hindered us to document the binding of UDCA-LPE to iPLA₂β, because UDCA-LPE formed micelles in water, which prevented effective testing of the proposed interaction by techniques such as surface plasmon resonance, microscale thermophoresis, or isothermal titration calorimetry.

In case an optional iPLA₂β inhibitor is found, the perspective for development of an anti-inflammatory, antisteatotic and antifibrotic drug for NASH and other inflammatory liver diseases is opened.

Acknowledgments

Funding: None.

Footnote

Provenance and Peer Review: This article was commissioned by the editorial office, AME Medical Journal for the series “Liver Diseases: Symptoms, Causes and Novel Therapies”. The article has undergone external peer review.

Reporting Checklist: The authors have completed the handout on scientific writing and checklist of scientific manuscript components according to the forms provided by the Teaching Issues and Experiments in Ecology (TIEE), which is a peer-reviewed web-based collection of ecological educational materials. Available at https://amj.amegroups.com/article/view/10.21037/amj-21-10/rc

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Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://amj.amegroups.com/article/view/10.21037/amj-21-10/coif). The series “Liver Diseases: Symptoms, Causes and Novel Therapies” was commissioned by the editorial office without any funding or sponsorship. WS and RW served as unpaid Guest Editors of the series. The authors have no other conflicts of interest to declare.

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