Computational evaluation of natural compounds as potential inhibitors of human PEPCK-M: an alternative for lung cancer therapy

Figures & data

Figure 1 Schematic overview of the computer-aided drug design approach used in the identification of HsPEPCK-M potential inhibitors.

Abbreviations: u_HsPEPCK-M, modeled protein without ligands; GTP_HsPEPCK-M, modeled protein complexed with GTP and OAA; GDP_HsPEPCK-M, modeled protein complexed with GDP and PEP.

Figure 2 Set of PEPCK substrates (1–2), PEPCK-C inhibitors (3–9), and PEPCK-M inhibitor (10) used in the docking study. The molecular weight is in g/mol.

Figure 3 The three-dimensional structures of HsPEPCK-M models and the templates. (A) The structural superposition of GTP_HsPEPCK-M (green), the prepared template HsPEPCK-C with GTP (PDB ID 1KHE – grey) and GgPEPCK-M (PDB ID 2FAH – pink). The active site poses of (B) the model GTP_HsPEPCK-M complexed with GTP and OAA (C) GDP_HsPEPCK-M complexed with GDP and PEP and (D) u_HsPEPCK-M without ligands. All ligands are represented as ball and sticks, and residues’ sidechains are represented as sticks. The two manganese ions are represented as purple spheres. Protein structural figures were generated using Pymol 2.1.0.

Figure 4 Structure-based pharmacophore hypothesis (PH). Important pharmacophoric features in the active site of HsPEPCK-C in the 1KHF structure are represented in (A) PH1 and (B) PH2. In the insets are the schematic representations of PH1 and PH2 with the distances between features in angstroms (Å). Pharmacophore features are colored according to their characteristic: red=negative ionic, blue=positive ionic, yellow=H-bond acceptor and white=H-bond donor.

Table 1 Area under the curve and enrichment factors for decoy dockings

Models	DUD-E				DecoyFinder
	AUC	EF2%	EF5%	EF10%	AUC	EF2%	EF5%	EF10%
u_HsPEPCK-M	0.85	4.6	3.7	3.6	0.99	40	20	10
GTP_HsPEPCK-M	0.93	8.8	9.2	6.3	0.98	32	20	9.9
GDP_HsPEPCK-M	0.9	4.4	9.1	5.5	0.99	37	20	10

Abbreviations: AUC, area under the curve; EF%, enrichment factor with respect to N% sample size.

Table 2 Docking Scores of the positive controls and the ten best scoring compounds for HsPEPCK-M in all configurations

Description	GTP_HsPEPCK-M		GDP_HsPEPCK-M		u_HsPEPCK-M
	Compounds	DS	Compounds	DS	Compounds	DS
Substrates	PEP	−11.86	PEP	−10.884	PEP	−11.548
	OAA	−10.591	OAA	−9.47	OAA	−10.229
Known inhibitors	(6) 3-phosphonopropionic acid	−12.369	(7) 1,2-ethanediphosphonic acid	−11.342	(10) 3-mercaptopicolinic acid	−10.365
	(3) oxalic acid	−11.44	(6) 3-phosphonopropionic acid	−10.929	(6) 3-phosphonopropionic acid	−10
	(10) 3-mercaptopicolinic acid	−10.75	(4) phosphonoformic acid	−10.581	(7) 1,2-ethanediphosphonic acid	−9.975
	(7) 1,2-ethanediphosphonic acid	−9.765	(3) oxalic acid	−10.538	(5) phosphoglycolic acid	−9.842
	(5) phosphoglycolic acid	−9.03	(5) phosphoglycolic acid	−9.778	(9) 3-sulfopropanoic acid	−9.762
	(9) 3-sulfopropanoic acid	−8.996	(9) 3-sulfopropanoic acid	−9.733	(4) phosphonoformic acid	−9.468
	(8) sulfoacetic acid	−8.993	(10) 3-mercaptopicolinic acid	−9.197	(3) oxalic acid	−8.521
	(4) phosphonoformic acid	−8.756	(8) sulfoacetic acid	−9.131	(8) sulfoacetic acid	−8.456
Screened compounds	ZINC00895296	−15.93	ZINC00895535	−14.31	ZINC30730321	−15.59
	ZINC00901201	−15.46	ZINC05783661	−14.29	ZINC02571340	−14.86
	ZINC01661435	−14.98	ZINC08551223	−14.2	ZINC08216889	−14.26
	ZINC13545827	−14.98	ZINC00388311	−13.88	ZINC01529331	−14.25
	ZINC01656421	−14.63	ZINC01656424	−13.81	ZINC04028702	−14.15
	ZINC02578809	−14.57	ZINC08216889	−13.8	ZINC13545320	−14.1
	ZINC02572383	−14.56	ZINC01656423	−13.77	ZINC19639050	−14.06
	ZINC00136659	−14.38	ZINC25722529	−13.72	ZINC12502195	−13.7
	ZINC00901335	−14.27	ZINC01854170	−13.67	ZINC04521349	−13.62
	ZINC13339553	−14.26	ZINC13513475	−13.64	ZINC02576963	−13.59

Abbreviations: DS, Docking Score; PEP, phosphoenolpyruvate; OAA, oxaloacetate.

Figure 5 Chemical classification and distribution of compounds on classes based on ClassyFire software. (A) Chemical classes of the HsPEPCK-M potential inhibitors. (B) Chemical superclasses of the HsPEPCK-M potential inhibitors. (C) The chemical superclasses and distribution of the compounds on 13 clusters obtained in the clusterization procedure.

Figure 6 Heat map and dendrogram for the clusters of the 779 potential HsPEPCK-M inhibitors. The selected cut-off point generated 13 groups – an illustrative chemical structure is presented for each cluster (1–13). The heat map represents the docking score for (A) GTP_HsPEPCK-M, (B) GDP_HsPEPCK-M and (C) u_HsPEPCK-M. The best scores are depicted in dark blue while the worst are in light green.

Figure 7 Binding mode of the hydroxycitrate (ZINC01656421) and tartaric acid (ZINC895296) in GTP_HsPEPCK-M active site obtained with the molecular docking assay. (A) and (C) are the 3D binding modes of hydroxycitrate and tartaric acid, respectively. The inhibitors are represented as ball and sticks, the protein represented in light green, and the intermolecular interactions are represented as gray dashes; (B) and (D) are the 2D interactions diagram of molecular docking results of hydroxycitrate and tartaric acid, respectively. The 2D residues are illustrated as negatively charged (red), positively charged (purple), polar (blue), hydrophobic (green). The hydrogen bonds are represented as purple arrows and salt bridges are represented as blue/red lines. The 3D and 2D images were generated using Pymol 2.1.0 and Maestro, respectively.

Figure 8 Binding mode of the danshensu (ZINC00895535) and 2-(2-Phenylhydrazono)malonic acid (ZINC02571340) in GDP_HsPEPCK-M and u_HsPEPCK-M active site, respectively, obtained with the molecular docking assay. (A) and (C) are the 3D binding modes of danshensu and 2-(2-Phenylhydrazono)malonic acid, respectively. The inhibitors are represented as ball and sticks, the protein represented in light purple (GDP_HsPEPCK-M) and light orange (u_HsPEPCK-M), and the intermolecular interactions are represented as gray dashes; (B) and (D) are the 2D interactions diagram of molecular docking results of danshensu and 2-(2-Phenylhydrazono)malonic acid, respectively. The 2D residues are illustrated as negatively charged (red), positively charged (purple), polar (blue), hydrophobic (green). The hydrogen bonds are represented as purple arrows and salt bridges are represented as blue/red lines. The 3D and 2D images were generated using Pymol 2.1.0 and Maestro, respectively.

Table 3 ADME properties, PAINS filters for PEPCK known inhibitors and selected putative HsPEPCK-M inhibitors

Compounds	MW	logP	HBD	HBA	LV	OB VEBER	OB EGAN	PAINS Filter
(6) 3-phosphonopropionic acid	154.06	−2.1	3	5	0	Good	Good	Accepted
(10) 3-mercaptopicolinic acid	155.17	0.86	1	3	0	Good	Good	Warning
(7) 1,2-ethanediphosphonic acid	190.03	−3.43	4	6	0	Good	Good	Accepted
(5) phosphoglycolic acid	156.03	−1.96	3	6	0	Good	Good	Accepted
(9) 3-sulfopropanoic acid	154.14	−1.39	2	5	0	Good	Good	Accepted
(8) sulfoacetic acid	140.12	−1.29	2	5	0	Good	Good	Accepted
(4) phosphonoformic acid	126.01	−1.74	3	5	0	Good	Good	Accepted
ZINC2571340	208.17	2.89	3	6	0	Good	Good	Accepted
ZINC0895535	198.17	0.15	4	5	0	Good	Good	Accepted
ZINC1656421	208.12	−2.56	5	8	0	Good	Good	Accepted

Notes: Observation: tartaric acid (ZINC895296) and oxalic acid (CID 971) were defined as a salt and were not evaluated by FAFDrugs. Also, 3-mercaptopicolinic acid has tPSA slightly lower than 75 Ų raising a warning in the Pfizer 3/75 Rule.

Abbreviations: MW, molecular weight; LogP, octanol/water partition coefficient; HBD, hydrogen bond donor; HBA, hydrogen bond acceptor; LV, Lipinski Violation; OB, oral bioavailability; PAINS, pan-assay interference compounds; DS, docking score; H bonds, hydrogen bonds; tPSA, topological polar surface area.