Molecular Docking Studies of α-Pyrone Derivatives isolated from Alternaria phragmospora as CRM1 Inhibitors

Some α-Pyrone derivatives isolated from Alternaria phragmospora fungus showed promising anti leukemic activities, while others were inactive. CRM1/XPO1 (chromosome region maintenance 1 protein, also called exportin1 or PO1 in humans) has been chosen as a target for antileukemic molecular docking study for those compounds to understand their modes of interaction and structure activity relationships. The results showed that two (2 and 4), out of six, natural α-Pyrone derivatives exhibited well-established interactions with the amino acids of the receptor, which was in agreement with the experimental anti-leukemic results of these compounds. Moreover, twenty hypothetical chemically modified α-Pyrone derivatives (7-27) have been designed. Compounds 7, 8, 22 and 24 showed more efficient docking properties than the previously considered natural compounds.


INTRODUCTION:
Endophytic fungi can be considered as an untapped reservoir for biologically active secondary metabolites belonging to different chemical classes such as alkaloids (1), steroids(2), terpenoids (3), pyranones(4), quinones (5), isochromenes(6) α-pyrones (7,8) and benzopyran derivatives (9) Molecular docking of a drug molecule with a receptor (target) gives important information about drug receptor interaction and commonly used to find out the binding orientation of drug candidates to their protein targets in order to predict the affinity and activity (10).
Computer-Aided Drug Design (CADD) is a specialized discipline that uses computational methods to stimulate drug-protein interaction (11).
The major nuclear exporter protein CRM1 (chromosome region maintenance 1 protein, also called exportin1 or XPO1 in humans) is one of seven exportins mediate the transport of 220 proteins and several mRNAs. Interestingly, CRM1 is the sole nuclear exporter of the major tumour suppressor and growth regulatory Proteins. (12) CRM1/XPO1 has been found to be a target in chronic lymphocytic leukemia and the selective CRM1/XPO1 inhibitors have been showed efficacy with an acceptable therapeutic index (13).
The natural product Leptomycin B (LMB) act as an efficient selective inhibitor of nuclear export mediated by the chromosomal region maintenance 1 protein (CRM1). (14) .
Keeping in view the therapeutic importance of α-pyrone derivatives as antitumor (15), (16), and in continuation of our work on separation of biologically active α-pyrone derivatives, we herein report the molecular docking of six previously reported α-pyrone derivatives. These compounds were screened for their antileukemic activity before (8). The molecular docking study of all the compounds has been done for the better understanding of the drug-receptor interaction. Moreover, we suggested hypothetical chemical modifications for the examined compounds to identify their antileukemic activity. This step may be an important step in the way to discover new antileukemic agents.

Ligand preparation
Ligand and the designed compounds 2D structures were sketched using ChemBioDraw Ultra 14.0 and saved in .sdf format. Sdf file opened, 3D structures are protonated and energy minimized by applying CHARMM force fields for charge, and MMFF94 force field for partial charge, then prepared for docking by optimization the parameters.

Protein preparation
The 3D crystal structure of CRM1/XPO1 was downloaded from the protein data bank. Before docking, water molecules were removed from protein file. Crystallographic disorders and unfilled valence atoms were corrected using alternate conformations and valence monitor options. Protein was subjected to energy minimization by applying CHARMM (Chemistry at HARvard Macromolecular Mechanics) force fields for charge, and MMFF94 (Merck Molecular force field) force field for partial charge. Inflexibility of structure is obtained by creating fixed atom constraint.

Docking studies
Molecular docking was performed using Discovery the Dock ligands (CDOCKER) protocol which is an implementation of the CDOCKER algorithm. CDOCKER is a grid-based molecular docking method that employs CHARMm-based molecular dynamics (MD) scheme to dock ligands into a receptor binding site. The receptor is held rigid while the ligands are allowed to flex during the refinement.

Result and discussion
The compounds were docked against CRM1 protein (PDB code: 3M1I, resolution: 2.00A) using discovery studio 2.5 to evaluate the free energy (ΔG) and mode of binding with the active site of CRM1 protein.

Docking of reference ligands
The proposed binding mode of Guanosine-5'-triphosphate revealed an affinity value of  LMB contains a β-hydroxy-ketone moiety formed a hydrogen bond with Ile42, a terminal carboxylate formed two hydrogen bonds with Lys25 and Asp20 and an α,β-unsaturated δlactone moiety binded to the hydrophobic groove of CRM1 which formed of Lys25, Lys125, Lys154, Glu38, and Phe37 (14) (Fig. 3).

Docking of the isolated compounds (1-6)
Six α-Pyrone derivatives (1-6) isolated from Alternaria phragmospora fungus have been docked with CRM1. The obtained results of the free energy of binding (ΔG) explained that only two compounds had moderate binding affinity toward the receptor and the computed values reflected the overall trend (Table 1) These data found to be in agreement with the published antileukemic data for compounds (1-

6) before(8).
The weak hydrogen bond interaction of separated compounds (1-6) may be due to the absence of hydrophilic tail in the chemical structure. This explanation drove us to suggest hypothetical chemical modifications for the separated compounds and identify their modes of interactions with the target protein and hence antileukemic activity.

Docking of suggested hypothetical compounds (7-27)
All compounds possessed the required binding energy to dock itself with the binding pocket of CRM1 ranging from -41.97 to -65.82 kcal/mol. (Table 1) The proposed binding mode of compound 8 (affinity value of -61.76 kcal/mol and three hydrogen bonds) (Fig. 4B) where the oxygen atom of pyrone moiety formed a hydrogen bond with Lys125 with a distance of 2.45 A ˚, the sulfur atom formed another hydrogen bond with Gly24 with a distance of 2.39 A ˚, the methyamino group of side chain formed a hydrogen bond withThr26 a distance of 2.23 A ˚ and the αpyrone moiety occupied the hydrophobic pocket formed by Lys25, Lys125, Lys154, Glu38, and Phe37 (Fig. 6B).
The proposed binding mode of compound 22 (affinity value of -60.33 kcal/mol and two hydrogen bonds) (Fig. 4C) where sulfur atom of methylthiazolidin-4-one moiety formed a hydrogen bond with Gly24 with a distance of 2.16 A˚, the carbonyl group of methylthiazolidin-4-one moiety formed another hydrogen bond with Thr44 with a distance of 1.96 A˚ and the α -pyrone moiety occupied the hydrophobic pocket formed by Lys25, Lys125, Lys154, Glu38, and Phe37 (Fig. 6C).
The proposed binding mode of compound 24 (affinity value of -65.82kcal/mol and two hydrogen bonds) (Fig. 4D) where sulfur atom of methylthiazolidin-4-one moiety formed a hydrogen bond with Gly24 with a distance of 2.27 A˚, the carbonyl group of methylthiazolidin-4-one moiety formed another hydrogen bond with Thr44 with a distance of 2.23 A˚ and the α -pyrone moiety occupied the hydrophobic pocket formed by Lys25, Lys125, Lys154, Glu38, and Phe37 (Fig. 6A).
The chemical modification by insertion of hydrophilic tail such as thiosemicarbazide and methylthiazolidin-4-one moieties may be responsible for the increased binding free energy.
The docking results of the suggested hypothetical compounds may lead to discovery a new promising antileukemic drug.  maintenance 1 protein (CRM1/XPO1) (PDB ID: 3M1I) was used to find the best inhibitors by studying the interaction between 3M1I protein receptor and the isolated compounds. Based on the docking score, compound 2 and 4 showed moderate CDOCKER energy. These data found to be in agreement with the published antileukemic data for compounds (1-6) before (8).
Suggested hypothetical chemical modifications for the isolated compounds has been proceeded and resulted in obtaining compounds