The Bitter Barricading of Prostaglandin Biosynthesis Pathway: Understanding the Molecular Mechanism of Selective Cyclooxygenase-2 Inhibition by Amarogentin, a Secoiridoid Glycoside from <i>Swertia chirayita</i>

<div><p><i>Swertia chirayita</i>, a medicinal herb inhabiting the challenging terrains and high altitudes of the Himalayas, is a rich source of essential phytochemical isolates. Amarogentin, a bitter secoiridoid glycoside from <i>S. chirayita</i>, shows varied activity in several patho-physiological conditions, predominantly in leishmaniasis and carcinogenesis. Experimental analysis has revealed that amarogentin downregulates the cyclooxygenase-2 (COX-2) activity and helps to curtail skin carcinogenesis in mouse models; however, there exists no account on selective inhibition of the inducible cyclooxygenase (COX) isoform by amarogentin. Hence the computer-aided drug discovery methods were used to unravel the COX-2 inhibitory mechanism of amarogentin and to check its selectivity for the inducible isoform over the constitutive one. The generated theoretical models of both isoforms were subjected to molecular docking analysis with amarogentin and twenty-one other Food and Drug Authority (FDA) approved lead molecules. The post-docking binding energy profile of amarogentin was comparable to the binding energy profiles of the FDA approved selective COX-2 inhibitors. Subsequent molecular dynamics simulation analysis delineated the difference in the stability of both complexes, with amarogentin-COX-2 complex being more stable after 40ns simulation. The total binding free energy calculated by MMGBSA for the amarogentin-COX-2 complex was −52.35 KCal/mol against a binding free energy of −8.57 KCal/mol for amarogentin-COX-1 complex, suggesting a possible selective inhibition of the COX-2 protein by the natural inhibitor. Amarogentin achieves this potential selectivity by small, yet significant, structural differences inherent to the binding cavities of the two isoforms. Hypothetically, it might block the entry of the natural substrates in the hydrophobic binding channel of the COX-2, inhibiting the cyclooxygenation step. To sum up briefly, this work highlights the mechanism of the possible selective COX-2 inhibition by amarogentin and endorses the possibility of obtaining efficient, futuristic and targeted therapeutic agents for relieving inflammation and malignancy from this phytochemical source.</p></div

Interaction of amarogentin with COX-1 based on its position at 0ns and 40ns.

<p>(A) Interaction of amarogentin with channel gate forming residues, at 0ns (red) and 40ns (yellow). The movement in the position and angle of the residue, at 0ns (green) and 40ns (cyan), can differentiate their deviations and suggest a movement of amarogentin outside the channel breaching the channel gate. (B) Position of amarogentin at 0ns (red) and at 40ns (yellow) clearly indicates a complete shift in its orientation, in the course of the simulation. The figure was generated using the PyMol molecular visualisation tool.</p

AutoDock binding energy values and H-bond forming residues of the lead molecules.

<p>Docking prediction of high scoring poses of different COX inhibitors and their corresponding binding energy values was compared with the binding free energy of amarogentin. Subsequently, the H-bonding residues were also analysed for the stability of the docking poses.</p

2D representation of the docking pose of amarogentin inside the hydrophobic cavity of COX.

<p>(A) The docking pose of amarogentin inside the binding cavity of COX-1 shows that besides forming several van der Waals interactions, amarogentin forms four H-bonds, two with Arg119 and one with Ser352. (B) A total of five H-bonds were also observed in the docked amarogentin-COX-2 complex, one each with Arg106, Ser516, Tyr371, and Met508. Amarogentin formed van der Waals interaction with a several residues along the hydrophobic channel. (Legend: green spheres represent hydrophobic residues; cyan spheres represent residues with polar side chains; red spheres and purple spheres show negatively and positively charged amino acid residues, respectively; solid red lines denote cation- π interaction; dotted purple lines indicate hydrogen bonds with side-chain atoms, with the direction of arrow denoting the acceptor atom; solid green lines indicate π-π interactions; and the grey spheres surrounding the atoms indicate that the atoms are exposed to solvent). The illustrations have been generated using Schrödinger Maestro open-source visualisation package.</p

RMSD of amarogentin-COX complexes for 40ns time frame.

<p>RMSD of amarogentin-COX-1 complex (black) shows an elevation in the deviation from the initial structure, reaching around 0.3nm for the final 40ns frame. The RMSD of amarogentin-COX-2 complex (red) showed initial deviations but attained stability at 15ns and remained so till the final 40ns time frame with an RMSD of 0.18nm. The plots have been generated using the GRACE plotting tool.</p

RMSD of the backbone of COX-1 and COX-2 modelled proteins after stabilisation.

<p>Protein backbone RMSD of COX-1 model, over a time frame of 15ns (in black), shows stability in the last 5ns time frame, deviating about 0.35nm from the native structure whereas, protein backbone RMSD of COX-2 model, for 10ns (in red), achieved stability after 5ns and maintained till the final 10ns, with an average deviation of about 0.3nm. The plot has been generated using the GRACE plotting tool.</p

Overall Non-bonded Interaction profile of amarogentin with COX-1 and COX-2.

<p>The total interaction profile of amarogentin covers the H-bond interactions, electrostatic interactions, π-π interactions, cation-π interaction, and the van der Waals interactions.</p

2D representation of the binding pose of amarogentin inside the hydrophobic cavity of COX isoforms after 40ns simulation.

<p>(A) Amarogentin formed only one H-bond with Ser352 in the COX-1 hydrophobic cavity. However, atoms which formed H-bonds after docking were present as van der Waal contacts. (B) Amarogentin made a total of six hydrogen bonds and was positioned up in the channel making the pose look more stable after simulation. Legends are same as expressed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090637#pone-0090637-g004" target="_blank">Figure 4</a>. The illustrations have been generated using Schrödinger Maestro open-source visualisation package.</p

Structure of Amarogentin, a secoiridoid glycoside from <i>Swertia chirayita</i>.

<p>Amarogentin consists of three essential subgroups, the iridoid group, the glucose moiety and the biphenyl-triol rings.</p

Movement of amarogentin inside the COX-1 binding cavity.

<p>Amarogentin after 40ns (green) simulation shows a clear movement outside the binding channel (grey) with respect to its initial 0ns frame (purple), indicating that the complex may not be stable in nature. The figure was generated using the PyMol molecular visualisation tool.</p