Oxygenated Polyketides from Plakinastrella mamillaris as a New Chemotype of PXR Agonists

Further purification of the apolar extracts of the sponge Plakinastrella mamillaris, afforded a new oxygenated polyketide named gracilioether K, together with the previously isolated gracilioethers E–G and gracilioethers I and J. The structure of the new compound has been elucidated by extensive NMR (1H and 13C, COSY, HSQC, HMBC, and ROESY) and ESI-MS analysis. With the exception of gracilioether F, all compounds are endowed with potent pregnane-X-receptor (PXR) agonistic activity and therefore represent a new chemotype of potential anti-inflammatory leads. Docking calculations suggested theoretical binding modes of the identified compounds, compatible with an agonistic activity on hPXR, and clarified the molecular basis of their biological activities.


Introduction
Pregnane-X-receptor (PXR; NR1I2), a member of the nuclear receptor superfamily, primarily expressed in the liver and intestine [1], has a major role in the induction of genes involved in drug and xenobiotic transport and metabolism [2], thus preventing the accumulation of toxic chemicals in the body. In addition to its role as a xeno-sensor, PXR, through a complex, yet fully deciphered crosstalk with other nuclear receptors [3], such constitutive androstane receptor (CAR), farnesoid-X-receptor (FXR), liver-X-receptor (LXR), vitamin D receptor (VDR), peroxisome proliferator-activated receptors (PPARs), estrogen receptor (ER), glucocorticoid receptor (GR), COUP transcription factor I (COUP-TFI), and II, plays a relevant physiological role in the regulation of bilirubin clearance [4], bile acid detoxification [5][6][7], lipid, steroid [8], and glucose metabolism [9]. Moreover, recent studies demonstrated mutual inhibition between PXR and nuclear factor (NF)-κB signaling pathway, providing a potential molecular mechanism that links xenobiotic metabolism and inflammation [10,11]. These potential roles of PXR in disease pathogenesis suggest PXR as a novel target for drug discovery.
In recent years, several natural products from herbal medicine and marine sources were disclosed for their potentials to act as modulators of PXR [12][13][14]. In particular, studies from our laboratories disclosed some sterols [15][16][17][18] from the marine sponge Theonella swinhoei as a new class of PXR agonists that exerted potent immunomodulatory activity. Extensive in vivo and in vitro studies on solomonsterol A confirmed the therapeutic potential of PXR agonists in the treatment of inflammatory bowel disease (IBD) [19]. Further characterization of natural PXR agonists, and their biological activities, may hold the promise of developing novel PXR modulators for the treatment of metabolic diseases.
A recent report [20] described ginkgolides, terpene trilactones present in Ginkgo biloba extract, as a new chemotype for ligand activation of hPXR.
In connection with our interest toward the discovery of new ligands for nuclear receptors, we were intrigued by the structural resemblance between the polycyclic "cage-like" backbone of ginkgolides with the oxygenated polycyclic structure of gracilioethers E, F, G, I, and J (1-5, Figure 1), polyketides recently isolated by our group [21]. Therefore docking studies were performed to assess the accommodation of gracilioethers in the ligand binding domain (LBD) of hPXR, and to highlight the pharmacophoric parts able to establish weak interactions with the key residues of the investigated target.
In order to re-obtain the gracilioethers for pharmacological evaluations on the pure components of the apolar extracts of the sponge Plakinastrella mamillaris, a new oxygenated polyketide, named gracilioether K (6), was also isolated.
In the present study we reported the results of the docking analysis and the pharmacological evaluation indicating that, with the exception of gracilioether F (2), all compounds are endowed with potent PXR agonistic activity.
The structural characterization of the new polyketide derivative, gracilioether K, is also reported.

Results and Discussion
The starting hypothesis of considering gracilioethers 1-5, as characterized by structural requirements compatible with an agonistic activity on hPXR, was investigated by means of molecular docking experiments. As previously described [22][23][24], a putative PXR ligand should be able to interact with polar residues in the LBD. In this context, Ekins et al. proposed several agonist pharmacophoric models, characterized by the presence of a hydrogen bond acceptor and/or donor, and hydrophobes [25]. In particular Ser247 and other polar residues (namely Cys284, Gln285, His407) have been reported as key amino acids involved in hydrogen bond/polar interactions with the ligand counterpart.
From a structural point of view, gracilioethers 1-5 are characterized by a well defined region with a high density of oxygens, potentially able to establish hydrogen bonds and polar interactions, and by a spatially distinct hydrophobic part. Starting from these assumptions, we have envisaged a binding mode, in which a part of these molecules is involved in polar interactions and/or hydrogen bonds with the key residues in the hPXR LBD, while the remaining part stabilizes the complex by van der Waals interactions ( Figure 2). For each investigated compound, the docking analysis of the different binding poses was then conducted using a distance criterion, filtering and considering the number of poses interacting with key residues, Ser247, Cys284, Gln285, and His407.
Firstly, this analysis highlighted the ability of the investigated compounds to interact with the Ser247 [24] via polar interactions or hydrogen bonds in a high number of docking poses. In particular, for gracilioethers 1, 3, and 5 we found these fundamental interactions in more than 55% of the 50 docking poses saved for each compound. The significant number of polar groups in a defined part of their structures determines the stabilization of these putative complexes in the hPXR LBD through further hydrogen bonds or polar interactions (Gln285, Cys284, and His407), while the hydrophobic components are involved in van der Waals contacts (namely Leu240, Phe251, Phe281, Ile414).
If compared to compounds 1, 3, and 5, gracilioethers 2 and 4 are characterized by the absence of the unsaturated side chain at C-3, determining a minor number of interactions with the above reported key residues. Interestingly, a smaller percentage of poses interacting with Ser247 were obtained (30% for Gracilioether I (4)

Isolation of Gracilioethers and Structural Characterization of Gracilioether K (6)
Prompted by good docking results, a re-isolation procedure was performed on the CHCl 3 extract of the sponge Plakinastrella mamillaris. MPLC followed by HPLC purification afforded 1-5 in reasonable amounts for further pharmacological tests and the new oxygenated polyketide named gracilioether K (6).
Gracilioether K (6)  Analysis of the 1 H NMR and HSQC spectra revealed the presence of three ethyls, one methoxyl, one methyl, four methines (including one oxymethine), and two methylenes ( Table 1). The 13 C NMR spectrum further showed the presence of one acyl carbon at δ C 171.1, two oxygenated quaternary carbons at δ C 93.8 and 96.7, and two ketalic carbons at δ C 110.49 and 110.51 (Table 1). A detailed analysis of 2D NMR data indicated a carbon framework for gracilioether K (6) very similar to that of gracilioether A [26]. The most significant difference is the loss, in gracilioether K, of two sp 2 carbons (δ H/C 4.86/86.8 and δ C 174.6), assigned respectively at C-2 and C-3 in gracilioether A [26].
Analysis of COSY data allowed to the identify five spin systems: Two ethyl groups linked to quaternary carbons, a CH(5)CH(9)CH(8)(CH 2 CH 3 )CH 2 (7), a CH 3 (12)CH(11)OH, and one isolated CH 2 (2) (δ H 2.63 and 2.71) spin systems (Figure 3a and Table 1). The HMBC correlations H 3 -14/C-4 and H 3 -16/C-6 connected the two ethyl units to C-4 and C-6 quaternary carbons, respectively. The hydroxyethyl group, CH 3 (12)CH(11)OH, was connected to the ketalic quaternary carbon C-10 (δ C 110.51) on the basis of HMBC correlations H 3 -12/C-10 and H-11/C-10. The isolated AB spin system (δ H 2.63 and 2.71) was connected to the acyl group at δ C 171.1 and to the ketal carbon at δ C 110.49 on the basis of HMBC correlations H-2/C-1 and H-2/C-3. A methyl ester group was easily inferred by the diagnostic long-range correlation between the methoxy protons at δ H 3.62 and the acyl carbonyl at δ C 171.1. Several diagnostic HMBC correlations (Figure 3a and Table 1) allowed us to secure the presence of the hexahydro-cyclopentanfurane system common to all gracilioethers, in which an ether bridge connecting C-3 and C-6 was placed by analogy with other gracilioethers and chemical shift considerations. Although no diagnostic heteronuclear long-range correlations were found to prove the C-9/C-10 linkage, the attachment of C-10/C-12 subunit to C-9 was supposed by analogy with gracilioether A, and confirmed by the strong dipolar couplings between H 3 -12/H-9 and H 3 -18 ( Figure 3b). Finally, two ether bridges between C-3 and C-10 and between C-4 and C-10 were assigned to satisfy the degrees of unsaturation and the molecular formula and to fulfil the functionalization degree of C-3, C-4, and C-10, as determined by 13 C NMR chemical shifts value analysis.
The relative configuration of the tetracyclic system in 6 was established from ROESY correlations (Table 1) 19 19 a ) b) The analysis of the ROESY spectrum gave also information on the stereochemistry at C-3. In particular ROESY correlations H-2/H 2 -13 and H 3 -16 indicated that the carbomethoxy methyl substituent at C-3 and the ethyl group at C-4 were cis-oriented.
The stereochemical homology observed for all members of the gracilioethers family previously isolated from the sponge Plakinastrella mamillaris [21,26] suggested a common biogenetic origin. By analogy with well known biosynthetic pathway to PGE 2 , endoperoxy rearrangement in gracilioether A could afford hydroxyketone 7 that, through hemiacetal ring closure followed by 1,4 nucleophilic addition of the C-10 oxygen group to the unsaturated ester moiety, generated the complex polycyclic system of gracilioether K (Figure 4). Therefore, from this biogenetic relationship, gracilioether K is likely to retain the same absolute configuration of all stereogenic centers of the hexahydro-cyclopentanfurane system as in gracilioether A [26].

Pharmacological Evaluation
All compounds of this small series were challenged in a reporter gene assay using HepG2 cells, a human hepatoma cell line. As shown in Figure 6, all compounds, with the exception of gracilioether F (2), were PXR agonists without effect when administered in combination with rifaximin. However, when HepG2 cells transfected with FXR vectors were treated with gracilioethers 1-6, alone or in the presence of chenodeoxycholic acid (CDCA) 10 μM, no significant agonistic or antagonistic activities were observed, with the exception for the compound 4, which revealed a slight antagonistic activity on FXR (Figure 7).   The lyophilized material (171 g) was extracted with methanol (3 × 1.5 L) at room temperature and the crude methanolic extract (40 g) was subjected to a modified Kupchan's partitioning procedure as follows. The methanol extract was dissolved in a mixture of MeOH/H 2 O containing 10% H 2 O and partitioned against n-hexane to give 17.3 g of the crude extract. The water content (% v/v) of the MeOH extract was adjusted to 30% and partitioned against CHCl 3 to give 16.6 g of the crude extract.

Computation Details
The chemical structures of the compounds were built and processed with Macromodel 8.5 (Schrödinger, LLC, New York, NY, USA, 2003). Conformational search and Molecular Dynamics calculations were performed on a 4× AMD Opteron SixCore 2.4 GHz using Macromodel 8.5.
Conformational search simulations were performed using the OPLS_2005 force field. The Monte Carlo multiple minimum (MCMM) method (10 5 steps) was used to allow a full exploration of the conformational space. Molecular Dynamics simulations were performed at 450 K and with a simulation time of 10 ns, after an equilibration phase of 1 ns. A constant dielectric term, mimicking the presence of the solvent, was used to reduce artifacts. Finally, the optimization (Conjugate Gradient, 0.0005 Å convergence threshold) of the structures was applied to identify the three-dimensional starting models for the subsequent steps of docking calculations.

Docking Calculation
Docking calculations were performed using the Autodock-Vina software [28]. In the configuration files of the crystallographic structure of hPXR (PDB code: 1M13) we specified the coordinate values for the targets, focusing the grid on the site of presumable pharmacological interest. In particular a grid box size of 30 × 36 × 32 and centered at 14.282 (x), 74.983 (y), and 0.974 (z) was used, with spacing of 1.0 Å between the grid points. The exhaustiveness value was set to 64, saving 50 conformations as maximum number of binding modes, and choosing 3.0 kcal/mol as maximum energy difference between the best and worst binding pose calculated. For all the investigated compounds, all open-chain bonds were treated as active torsional bonds. Docking results were analyzed with Autodock Tools 1.4.5. Illustrations of the 3D models were generated using VMD software [29].

Conclusions
The demonstration that gracilioethers, with the exception of gracilioether F, are PXR agonists, devoid of a significant activity on the bile acid sensor FXR, might have pharmacological and therapeutic relevance. Indeed, PXR agonists are being increasingly investigated for their ability to reduce inflammation in gastrointestinal tract and for their potential application in the treatment of a variety of human disorders including altered bone homeostasis, liver steatosis, and liver and lung fibrosis. PXR has, additionally, a multi-factorial impact on cancer, either by directly affecting cell proliferation and apoptosis, or by inducing chemotherapy resistance in colon, breast, prostate, and endometrial cancer, and in osteosarcoma, suggesting that identification of novel PXR ligands, agonists and antagonists, might have relevance for the treatment of these disorders. This study reaffirms sponges of Plakinastrella genus as an invaluable source of nuclear receptors ligands [30] endowed with unusual chemical scaffolds and useful for discovery of new pharmacological leads in several human diseases.