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Chinese Medicine logoLink to Chinese Medicine
. 2015 Dec 17;10:37. doi: 10.1186/s13020-015-0067-4

In silico target fishing and pharmacological profiling for the isoquinoline alkaloids of Macleayacordata (Bo Luo Hui)

Qifang Lei 1, Haibo Liu 1,, Yong Peng 1, Peigen Xiao 1
PMCID: PMC4683977  PMID: 26691584

Abstract

Background

Some isoquinoline alkaloids from Macleaya cordata (Willd). R. Br. (Bo Luo Hui) exhibited antibacterial, antiparasitic, antitumor, and analgesic effects. The targets of these isoquinoline alkaloids are undefined. This study aims to investigate the compound–target interaction network and potential pharmacological actions of isoquinoline alkaloids of M. cordata by reverse pharmacophore database screening.

Methods

The targets of 26 isoquinoline alkaloids identified from M. cordata were predicted by a pharmacophore-based target fishing approach. Discovery Studio 3.5 and two pharmacophore databases (PharmaDB and HypoDB) were employed for the target profiling. A compound–target interaction network of M. cordata was constructed and analyzed by Cytoscape 3.0.

Results

Thirteen of the 65 predicted targets identified by PharmaDB were confirmed as targets by HypoDB screening. The targets in the interaction network of M. cordata were involved in cancer (31 targets), microorganisms (12 targets), neurodegeneration (10 targets), inflammation and autoimmunity (8 targets), parasitosis (5 targets), injury (4 targets), and pain (3 targets). Dihydrochelerythrine (C6) was found to hit 23 fitting targets. Macrophage migration inhibitory factor (MIF) hits 15 alkaloids (C12, C1116, C1925) was the most promising target related to cancer.

Conclusion

Through in silico target fishing, the anticancer, anti-inflammatory, and analgesic effects of M. cordata were the most significant among many possible activities. The possible anticancer effects were mainly contributed by the isoquinoline alkaloids as active components.

Background

Macleaya cordata (Willd). R. Br. (Bo Luo Hui) (Fig. 1) has been used for the treatment of cancer [1], insect bites [2], and ringworm infection [3] in Mainland China, North America, and Europe. Phytochemical and pharmacological studies demonstrated that the isoquinoline alkaloids derived from M. cordata are its major active components [4]. Thirty isoquinoline alkaloids have been isolated from M. cordata (Fig. 2), including chelerythrine (C12), sanguinarine (C15), sanguidimerine (C17), chelidimerine (C18), berberine (C21), coptisine (C23), allocryptopine (C24, C25), and protopine (C26). These alkaloids exhibited a broad spectrum of biological activities, such as antitumor [58], anti-inflammatory [911], antimicrobial [1214], analgesic [15], and antioxidant [16] activities.

Fig. 1.

Fig. 1

The original plant of Macleaya cordata

Fig. 2.

Fig. 2

The isoquinoline alkaloids of Macleaya cordata

In our previous study [17], we found that M. cordata could be counted not only as one of the richest resources in Mainland China among all species of the tribe Chelidonieae, but also as one of the most promising natural resources for drug discovery. M. cordata has gained the attention of pharmacognosists since early 1990s (Fig. 3). However, its obscure molecular actions have hindered its use in drug development.

Fig. 3.

Fig. 3

The statistics of Pubmed publications on Macleaya cordata between 1972 and 2014

Although protein–ligand docking techniques have been available in virtual drug screening for specific targets, such as tumor necrosis factor α-converting enzyme (TACE) [18], inducible nitric oxide synthase (iNOS) [19], and Janus-activated kinase 2 (JAK2) [20], these docking approaches to virtual screening are often too computationally expensive [21].

This study aims to investigate the compound-target interaction network of isoquinoline alkaloids of M. cordata by reverse pharmacophore database screening technology, and outline its potential action mechanisms.

Methods

Workflow

Figure 4 shows the workflow of this study. The structures and bioactivities of the isoquinoline alkaloids of M. cordata were collected by literature review [17]. The alkaloids were then applied to target fishing with two pharmacophore and target databases, PharmaDB and HypoDB. The hit pharmacophore models were picked out according to the threshold of a predetermined fit value. The results from PharmaDB screening were compared with those from HypoDB screening. After analysis of the hit targets and their associated pathways and diseases, as well as the interactions between the alkaloids and the targets, an action network of M. cordata was constructed. Literature retrieval was simultaneously carried out to verify the findings.

Fig. 4.

Fig. 4

The workflow of this study

Compound collection

The active components of M. cordata were collected from our own database [17] and the literature. All 26 isoquinoline alkaloids of M. cordata and their bioactivities are listed in Table 1. As shown in Fig. 2, the alkaloids were divided into three classes: benzo[c]phenanthridines (Ben, C1–C18), protoberberines (Ber, C19–C23), and protopines (Pro, C24–C26). Based on the replacement of the C-ring, C1–C9 belong to the dihydro-benzo[c]phenanthridines, C10 is a N-demethyl subtype, and C11–C16 are quaternary ammonium bases that share an iminium moiety (C=N+). The remaining two bisbenzo[c]phenanthridines (BisBen, C17–C18) are epimers to one another.

Table 1.

Basic information of the isoquinoline alkaloids in M. cordata

No. Compounds Bioactivities Virtual hitting targets
1 6-Acetonyl-dihydrosanguinarine Anti-bacteria
Insecticidal
MIF; TTR; NQO1
2 6-Acetonyl-dihydrochelerythrine Anti-oxidant
Anti-HIV
HSD1; MIF; PDE4D; NQO1; PLA2s; nAChR 7α; AknH; TtgR
3 6-Methoxy-dihydrochelerythrine Anti-cancer
Anti-parasitic
CAR/RXR; MR; ERα; JNK3; SHBG; AR; 15S-LOX; MMP12; PPARγ; SARS M(pro); Scy D; MAO-A
4 6-Methoxy-dihydrosanguinarine Anti-bacteria
Anti-cancer
Anti-platelet aggregation
MR; ERα; FNR; MAO-A
5 Bocconoline Anti-bacteria
Anti-fungal
Opsin 2; HSD1; CAR/RXR; MD; ERα; JNK3; SHBG; Chk1; AR; 15S-LOX; CDK2; CAMKII; Aurora A; PIM1; MMP12; Tankyrase 2; SARS M(pro); PfENR; FabZ; DHODH; CDPKs; FNR; ENR; Scy D; MAO-A
6 Dihydrochelerythrine Anti-bacteria
Anti-fungal
CAR/RXR; MR; ERα; PPO; TTR; JNK3; SHBG; NQO1; RBP4; 15S-LOX; CK2; PIM1; FabZ; DHODH; SnoaL; FNR; ENR; Scy D; MAO-A; MAO-B; AchE; HIV-1 RT; OSBP
7 Dihydrosanguinarine Anti-bacteria
Anti-fungal
MR; ERα; PPO; SHBG; 15S-LOX; CDK2; CK2; MAO-A; AchE
8 Oxysanguinarine Anti-platelet aggregation PIM1; CK2
9 Oxychelerythrine Cytotoxic CAR/RXR; TTR; JNK3; SHBG; 15S-LOX; CLK1; CK2; PIM1; MMP12; MAPK p38; COMP; FabZ; SonaL; FNR; ENR; MAO-A; MAO-B; AchE; OSBP
10 Norsanguinarine Anti-fungal CK2; NmrA
11 6-ethoxychelerythrine Anti-bacteria
Anti-fungal
MIF; TTR; JNK3; GAPDH; nAChR 7α; FabZ; CAT; LmrR; HS5B Pol
12 Chelerythrine Anti-bacteria
Anti-fungal
Anti-parasitic
Anti-cancer
MIF; TTR; FabZ; HS5B Pol
13 Chelirubine Anti-proliferative MIF; NQO1; GR; ZipA-FtsZ; AknH; opdA
14 Macarpine Cytotoxic
Anti-proliferative
PDE4B; PDE 4B; MIF; TTR; NQO1; PIM1; MAPK p38; GR; ZipA-FtsZ; AknH
15 Sanguinarine Anti-bacteria
Anti-fungal
Anti-parasitic
Anti-cancer
Anti-oxidant
Hepatotoxicity
MIF; nAChR 7α
16 Sanguilutine Anti-proliferative HSD1; MIF; PDE4D; PLA2s; FabZ
17 Sanguidimerine Unreported ATTP
18 Chelidimerine Unreported MDR HIV-1 Protease
19 Chelanthifoline Anti-malarial ALR; ERα; ERβ; MIF; PDK-1; CK2; PIM1; Pi3 Kγ; GR; nAChR 7α; TEM-1; ActR; MAO-B; HIV-1 RT; OSBP
20 Dehydrocicanthifoline Unreported HSD1; MR; PDE4B; PDE4D; PPO; MIF; TTR; JNK3; CRBP-2; MAPK p38; AR; PIM1; ZipA-FtsZ; HS5B Pol; HIV-1 RT
21 Berberine Anti-fungal
Anti-malarial
Anti-cancer
Cytotoxic
Anti-inflammatory
Anti-Alzheimer’s
Anti-fertility
Anti-diabetes
MIF; FabZ; Scy D; AchE
22 Dehydrochelanthifoline Anti-virus ERα; ERβ; MIF; GSK-3β; TTR; CDK2; PLA2s; MAO-B
23 Coptisine Cytotoxic
Anti-diabetes
CYP2D6 inhibition
Anti-oxidative
Anti-spasmodic
MIF
24 α-Allocryptopine Anti-funga
Anti-arrhythmic
HSD1; MIF; HS5B Pol; Scy D; BACE1
25 β-Allocryptopine Anti-parasitic
Anti-hepatic fibrosis
HSD1; MIF; HS5B POl; Scy D; BACE1; CRALBP; PPO; TTR; nAChR 7α
26 Protopine Anti-malarial
Anti-parasitic
Anti-fertility
Anti-spasmodic
NQO1; PfENR; TtgR

Conformation analysis

The structures of all 26 alkaloid candidates were prepared in MOL format, and converted from 2D drawings to 3D models. Their energies were minimized by the software Discovery Studio (DS, v3.5) developed by BIVIA (USA) with the CHARMM force field. A Monte Carlo-based conformational analysis (FAST mode) was performed to generate conformers from the initial conformations. The maximal 255 conformers were allowed with an energy interval of 20 kcal/mol. These alkaloid molecules were rigid, and the number of conformers for each compound was much fewer than 255. Hence, a total of 135 conformers were generated for the 26 isoquinoline alkaloids.

Ligand profiling

A pharmacophore model represented a series of common features of a set of ligands with a special pharmacological target. The features of a pharmacophore model reflected the target–ligand interaction mode. Pharmacophore-based virtual screening was an alternative to docking. By fitting a compound against a panel of pharmacophore models derived from multiple pharmacological targets, the potential targets of the compound can be outlined.

Automated ligand profiling was available in DS 3.5 as the so-called “Ligand Profiler” protocol. The software offered automated pharmacophore-based activity profiling and reporting [22]. In this study, the default parameters of DS 3.5 were used. For each candidate ligand, three or more features were mapped.

Pharmacophore databases

DS 3.5 was equipped with two available pharmacophore databases, i.e., HypoDB [23] and PharmaDB [24]. HypoDB contained about 2500 pharmacophore models derived from protein–ligand 3D complex structures as well as structural data on small bioactive organic molecules. PharmaDB was created from the sc-PDB, a well-accepted data source in structure-based profiling protocols. The sc-PDB was a collection of 3D structures of binding sites found in the Protein Data Bank (PDB). The binding sites were extracted from crystal structures in which a complex between a protein cavity and a small molecule ligand could be identified. PharmaDB consisted of about 68,000 pharmacophores derived from 8000 protein–ligand complexes from the sc-PDB dataset. PharmaDB is a new and updated pharmacophore database developed in collaboration with Prof. Didier Rognan [25, 26]. The target and pharmacophore models from PharmaDB and HypoDB were not entirely consistent. PharmaDB had a larger quantity of targets, while the models in the HypoDB were fewer and described as being experimentally validated. Therefore, in this study, PharmaDB was employed in the target fishing, and HypoDB was used to validate the results.

Regarding PharmaDB, multiple pharmacophores with shape or excluded volume constraints were generated for each protein target. For the pharmacophores with shape constraints, the suffix “-s” was added to the name. In addition, a numerical suffix referred to the ranking of selectivity evaluated by a default algorithm in DS v3.5. In this study, only the best models with “−1” in their names were employed in the ligand profiling [23]. For each pharmacophore database, a classification tree was available, from which the individual models could be selected.

Parameters

In the profiling with PharmaDB, all the pharmacophore models with the shape of the binding pocket were selected for the virtual screening with default settings. The RIGID mode was used as the molecular mapping algorithm. No molecular features were allowed to be missed while mapping these ligands to the pharmacophore models to increase selectivity. The minimal inter-feature distance was set at 0.5 Å. Parallel screening technology for one or more compounds against a multitude of pharmacophore models was available as a Pipeline Pilot protocol. The number of parallel processing procedures was set at 4. The whole calculation was carried on a T5500 workstation (DELL inc., USA).

Binding mode refinement

All the poses of the ligands mapped into the pharmacophore were preserved. A series of target-ligand pairs were selected as emphasis for further examinations. The selection was based upon compatibility with the reported pharmacological activities, as well as traditional usage of M.cordata. A further refinement was carried out in Molecular Operating Environment (MOE) developed by CCG (Canada) to identify the protein–ligand binding modes. Energy minimization was carried out by conjugated gradient minimization with the MMFF94x force field, until an RMSD of 0.1 kcal mol−1 Ǻ−1 was reached.

Network construction

An interaction table between alkaloids and targets was presented as the ligand profiling results. For each target, the name and pathway information were collected from the PDB and KEGG. The diseases related to the targets were collected from the Therapeutic Target Database (TTD; http://bidd.nus.edu.sg/group/cjttd/) [27] and DrugBank (http://www.drugbank.ca/) [28] databases. Compound-Target-Pathway networks were generated by Cytoscape 3.0 (Cytoscape Consortium, USA) [29]. In the networks, nodes represented the compounds, targets, and biological pathways. The edges linking the compound-target and target-pathway represented their relationships and were marked with different types of lines. After the network was built, the basic parameters of the network were computed and analyzed.

Results and discussion

The profiling results are presented in two HTML tables, designated MoleculeFits and PharmacophoreFits. Two descriptors, fit value and shape similarity, were used to measure the fitness of the ligand and pharmacophore. A fit value equal to or greater than 0.3 was used as a heuristic threshold to select targets from the activity profiler. For each pharmacophore model, the classification information of the target can be indicated in a HTML table created by DS 3.5 called as Pharmacophores. Finally, 98 pharmacophore models were mapped. The models belonged to 65 protein targets, and were involved in 60 pathways. A complete list of the 241 target-ligand pairs is shown in Table 2. The name and indication information of the targets are shown in Table 3. The 13 targets verified by HypoDB screening are marked with an asterisk in Table 3.

Table 2.

The results of ligand profiling

Class CMD-ID ph4 Target short name Gene Uniprot-AC Fit value Shape similarity
Ben 1 3cfn TTR TTHY_HUMAN P02766 0.750635 0.508475
Ben 1 1h69 NQO1 NQO1_HUMAN P15559 0.923086 0.536437
Ben 2 3kba Progesterone receptor PRGR_HUMAN P06401 0.334698 0.506897
Ben 2 1xom PDE4D PDE4D_HUMAN Q08499 0.346437 0.527574
Ben 2 2wnj nAChR 7α Q8WSF8_APLCA Q8WSF8 0.43985 0.505495
Ben 2 1h69 NQO1 NQO1_HUMAN P15559 0.928518 0.504604
Ben 3 2oz7 AR ANDR_HUMAN P10275 0.360685 0.500849
Ben 3 2a3i MR MCR_HUMAN P08235 0.375601 0.528195
Ben 3 5std ScyD SCYD_MAGGR P56221 0.418672 0.543119
Ben 3 1l2i ERα ESR1_HUMAN P03372 0.420147 0.542969
Ben 3 1xvp CAR/RXR NR1I3_HUMAN Q14994 0.460385 0.534672
Ben 3 3lmp PPARγ PPARG_HUMAN P37231 0.526039 0.500787
Ben 3 1d2s SHBG SHBG_HUMAN P04278 0.558685 0.563525
Ben 3 2gz7 SARS M(pro) R1AB_CVHSA P0C6X7 0.559512 0.547348
Ben 3 2p0m 15S-LOX LOX15_RABIT P12530 0.639897 0.537344
Ben 3 3f15 MMP12 MMP12_HUMAN P39900 0.725254 0.50503
Ben 3 2bxr MAO-A AOFA_HUMAN P21397 0.799156 0.521008
Ben 3 2o2u JNK3 MK10_HUMAN P53779 0.835637 0.577825
Ben 4 2bgi FNR Q9L6V3_RHOCA Q9L6V3 0.427793 0.516878
Ben 4 1l2i ERα ESR1_HUMAN P03372 0.452535 0.593291
Ben 4 2bxr MAO-A AOFA_HUMAN P21397 0.793632 0.533917
Ben 5 2ol4 PfENR Q9BH77_PLAFA Q9BH77 0.315455 0.518182
Ben 5 3g0u DHODH PYRD_HUMAN Q02127 0.369491 0.508604
Ben 5 2bxr MAO-A AOFA_HUMAN P21397 0.387312 0.544118
Ben 5 2a3i MR MCR_HUMAN P08235 0.387489 0.563771
Ben 5 7std ScyD SCYD_MAGGR P56221 0.422146 0.504744
Ben 5 2uue CDK2 CDK2_HUMAN P24941 0.424268 0.567108
Ben 5 2oz7 AR ANDR_HUMAN P10275 0.426337 0.510961
Ben 5 3coh Aurora-A STK6_HUMAN O14965 0.4511 0.531532
Ben 5 3kr8 Tankyrase 2 TNKS2_HUMAN Q9H2K2 0.464801 0.548729
Ben 5 1d2s SHBG SHBG_HUMAN P04278 0.480784 0.571721
Ben 5 1l2i ERα ESR1_HUMAN P03372 0.493882 0.57529
Ben 5 2wel CAMKII KCC2D_HUMAN Q13557 0.493929 0.516729
Ben 5 3fne ENR INHA_MYCTU P0A5Y6 0.50498 0.546169
Ben 5 1xvp CAR/RXR NR1I3_HUMAN Q14994 0.508683 0.576427
Ben 5 5std ScyD SCYD_MAGGR P56221 0.522671 0.566972
Ben 5 2brg Chk1 CHK1_HUMAN O14757 0.541427 0.51711
Ben 5 3doz FabZ Q5G940_HELPY Q5G940 0.546379 0.507843
Ben 5 2bgi FNR Q9L6V3_RHOCA Q9L6V3 0.553334 0.549296
Ben 5 3fnf ENR INHA_MYCTU P0A5Y6 0.562321 0.511494
Ben 5 3fnh ENR INHA_MYCTU P0A5Y6 0.614823 0.507547
Ben 5 2p0m 15S-LOX LOX15_RABIT P12530 0.673148 0.541414
Ben 5 3dp1 FabZ Q5G940_HELPY Q5G940 0.687924 0.53816
Ben 5 2gz7 SARS M(pro) R1AB_CVHSA P0C6X7 0.694125 0.551789
Ben 5 2o2u JNK3 MK10_HUMAN P53779 0.805153 0.553719
Ben 5 3f15 MMP12 MMP12_HUMAN P39900 0.893862 0.507187
Ben 6 3fj6 DHODH PYRD_HUMAN Q02127 0.341464 0.566038
Ben 6 5std ScyD SCYD_MAGGR P56221 0.364451 0.555556
Ben 6 1d2s SHBG SHBG_HUMAN P04278 0.367805 0.529289
Ben 6 2a3i MR MCR_HUMAN P08235 0.368001 0.559289
Ben 6 1xvp CAR/RXR NR1I3_HUMAN Q14994 0.423023 0.572534
Ben 6 2v60 MAO-B AOFB_HUMAN P27338 0.436774 0.511294
Ben 6 1l2i ERα ESR1_HUMAN P03372 0.451108 0.529175
Ben 6 1rbp RBP4 RET4_HUMAN P02753 0.486949 0.516378
Ben 6 2nsd ENR INHA_MYCTU P0A5Y6 0.509088 0.530738
Ben 6 1kgj TTR TTHY_RAT P02767 0.522255 0.529412
Ben 6 1tv6 HIV-1 TR POL_HV1B1 P03366 0.564494 0.529981
Ben 6 2bgi FNR Q9L6V3_RHOCA Q9L6V3 0.636419 0.536325
Ben 6 2p0m 15S-LOX LOX15_RABIT P12530 0.663082 0.545045
Ben 6 3imu TTR TTHY_HUMAN P02766 0.690803 0.577011
Ben 6 3dp1 FabZ Q5G940_HELPY Q5G940 0.705916 0.565401
Ben 6 2o2u JNK3 MK10_HUMAN P53779 0.705945 0.507463
Ben 6 1h69 NQO1 NQO1_HUMAN P15559 0.755827 0.508911
Ben 6 2bxr MAO-A AOFA_HUMAN P21397 0.795152 0.541573
Ben 6 1sjw SnoaL Q9RN59_STRNO Q9RN59 0.904111 0.661572
Ben 6 2j3q AChE ACES_TORCA P04058 0.992134 0.661327
Ben 7 2x1n CDK2 CDK2_HUMAN P24941 0.329358 0.521253
Ben 7 1d2s SHBG SHBG_HUMAN P04278 0.340019 0.542857
Ben 7 1l2i ERα ESR1_HUMAN P03372 0.465563 0.553846
Ben 7 2j3q AChE ACES_TORCA P04058 0.470546 0.67
Ben 7 2p0m 15S-LOX LOX15_RABIT P12530 0.639874 0.545254
Ben 7 2bxr MAO-A AOFA_HUMAN P21397 0.822509 0.548694
Ben 8 3bgp PIM-1 PIM1_HUMAN P11309 0.659102 0.52193
Ben 9 2wu7 CLK1 CLK3_HUMAN P49761 0.333353 0.541053
Ben 9 1d2s SHBG SHBG_HUMAN P04278 0.40988 0.526096
Ben 9 2nsd ENR INHA_MYCTU P0A5Y6 0.417703 0.522727
Ben 9 1tha TTR TTHY_HUMAN P02766 0.42188 0.505071
Ben 9 1xvp CAR/RXR NR1I3_HUMAN Q14994 0.459386 0.600775
Ben 9 1fbm COMP COMP_RAT P35444 0.540756 0.509542
Ben 9 2p0m 15S-LOX LOX15_RABIT P12530 0.609094 0.548596
Ben 9 2bxr MAO-A AOFA_HUMAN P21397 0.636415 0.524336
Ben 9 3iw7 MAPK p38 MK14_HUMAN Q16539 0.671331 0.532803
Ben 9 1sjw SnoaL Q9RN59_STRNO Q9RN59 0.679871 0.665953
Ben 9 2bgi FNR Q9L6V3_RHOCA Q9L6V3 0.68446 0.509554
Ben 9 3dp1 FabZ Q5G940_HELPY Q5G940 0.723083 0.601732
Ben 9 2v60 MAO-B AOFB_HUMAN P27338 0.818911 0.501006
Ben 9 2o2u JNK3 MK10_HUMAN P53779 0.878824 0.532609
Ben 9 2j3q AChE ACES_TORCA P04058 0.992667 0.679157
Ben 10 2wmd NmrA NMRL1_HUMAN Q9HBL8 0.635677 0.601671
Ben 11 3doz FabZ Q5G940_HELPY Q5G940 0.380298 0.514677
Ben 11 3kvx JNK3 MK10_HUMAN P53779 0.408174 0.518987
Ben 11 2wnj nAChR 7α Q8WSF8_APLCA Q8WSF8 0.408648 0.512476
Ben 11 3doy FabZ Q5G940_HELPY Q5G940 0.456816 0.515444
Ben 11 1k3t GAPDH G3PG_TRYCR P22513 0.56209 0.500931
Ben 11 3lmp PPARγ PPARG_HUMAN P37231 0.648243 0.508527
Ben 11 1qca CAT CAT3_ECOLX P00484 0.780585 0.505747
Ben 11 3f8f LmrR A2RI36_LACLM A2RI36 0.817481 0.51932
Ben 13 1xan GR GSHR_HUMAN P00390 0.573697 0.520833
Ben 13 3kba Progesterone receptor PRGR_HUMAN P06401 0.651629 0.522059
Ben 13 1h69 NQO1 NQO1_HUMAN P15559 0.811055 0.503055
Ben 13 3a3w opdA Q93LD7_RHIRD Q93LD7 0.833715 0.510158
Ben 14 3huc MAPK p38 MK14_HUMAN Q16539 0.345074 0.534091
Ben 14 1xan GR GSHR_HUMAN P00390 0.517848 0.510823
Ben 14 1h69 NQO1 NQO1_HUMAN P15559 0.723867 0.515504
Ben 14 1xom PDE4D PDE4D_HUMAN Q08499 0.745933 0.503704
Ben 14 1xlx PDE4B PDE4B_HUMAN Q07343 0.796555 0.52037
Ben 15 2wnj nAChR 7α Q8WSF8_APLCA Q8WSF8 0.495424 0.509356
Ben 16 3kba Progesterone receptor PRGR_HUMAN P06401 0.342606 0.537671
Ben 16 1xom PDE4D PDE4D_HUMAN Q08499 0.816692 0.539427
BisBen 17 1r5 l ATTP TTPA_HUMAN P49638 0.32356 0.514156
BisBen 18 1rq9 MDR HIV-1 Protease Q5RTL1_9HIV Q5RTL1 0.621229 0.507743
Ber 19 1u3s ERβ ESR2_HUMAN Q92731 0.408794 0.535377
Ber 19 2j3q AChE ACES_TORCA P04058 0.485705 0.596737
Ber 19 3l54 Pi3 Kγ PK3CG_HUMAN P48736 0.498919 0.59589
Ber 19 1pzo TEM-1 BLAT_ECOLX P62593 0.523404 0.526667
Ber 19 2ikg ALR ALDR_HUMAN P15121 0.561704 0.507109
Ber 19 1c1c HIV-1 TR POL_HV1H2 P04585 0.577074 0.542373
Ber 19 2r7b PDK-1 PDPK1_HUMAN O15530 0.587223 0.533049
Ber 19 1yye ERβ ESR2_HUMAN Q92731 0.679767 0.56691
Ber 19 1qkt ERα ESR1_HUMAN P03372 0.681913 0.56351
Ber 19 1xan GR GSHR_HUMAN P00390 0.715506 0.548544
Ber 19 2wnj nAChR 7α Q8WSF8_APLCA Q8WSF8 0.87937 0.501031
Ber 19 3b6c ActR Q53901_STRCO Q53901 0.880577 0.597561
Ber 19 1x78 ERβ ESR2_HUMAN Q92731 0.909059 0.522565
Ber 20 1xm4 PDE4B PDE4B_HUMAN Q07343 0.402388 0.569138
Ber 20 1tha TTR TTHY_HUMAN P02766 0.45615 0.514286
Ber 20 1tv6 HIV-1 TR POL_HV1B1 P03366 0.459002 0.521154
Ber 20 2nw4 AR ANDR_RAT P15207 0.463505 0.541203
Ber 20 1opb CRBP2 RET2_RAT P06768 0.485837 0.534653
Ber 20 2waj JNK3 MK10_HUMAN P53779 0.572085 0.603104
Ber 20 1kgj TTR TTHY_RAT P02767 0.740087 0.56531
Ber 20 1xom PDE4D PDE4D_HUMAN Q08499 0.811727 0.542406
Ber 20 1xlx PDE4B PDE4B_HUMAN Q07343 0.859016 0.51341
Ber 20 3i6d PPO PPOX_BACSU P32397 0.97618 0.570499
Ber 21 5std ScyD SCYD_MAGGR P56221 0.324086 0.505682
Ber 21 2j3q AChE ACES_TORCA P04058 0.992907 0.672209
Ber 22 1di8 CDK2 CDK2_HUMAN P24941 0.406566 0.501094
Ber 22 1u3s ERβ ESR2_HUMAN Q92731 0.661777 0.509434
Pro 24 5std ScyD SCYD_MAGGR P56221 0.538905 0.543636
Pro 24 3ine BACE1 BACE1_HUMAN P56817 0.54561 0.522968
Pro 25 1tyr TTR TTHY_HUMAN P02766 0.356425 0.526412
Pro 25 3inf BACE1 BACE1_HUMAN P56817 0.37118 0.504303
Pro 25 2wnj nAChR 7α Q8WSF8_APLCA Q8WSF8 0.553029 0.533461
Pro 25 3ine BACE1 BACE1_HUMAN P56817 0.597712 0.51259
Pro 25 3hx3 CRALBP RLBP1_HUMAN P12271 0.604625 0.513158
Pro 25 5std ScyD SCYD_MAGGR P56221 0.763034 0.522523
Pro 26 2ow2 PfENR MMP9_HUMAN P14780 0.302479 0.507865
Pro 26 2f1o NQO1 NQO1_HUMAN P15559 0.432407 0.52183

Table 3.

The targets identified

Targets Short name Type Pathway Diseases
Retinaldehyde-binding protein CRALBP Research Retinaldehyde metabolism Retinitis pigmentosa
Rhodopsin Opsin 2 Research Retina metabolism Retinitis pigmentosa
11-Beta-hydroxysteroid dehydrogenase HSD1 Successful Glucocorticoid concentration Diabetes
Osteoporosis
Hepatotoxicity
CAR/RXR heterodimer CAR/RXR Research Triglyceride metabolism Diabetes
Hepatitis
Aldose reductase ALR Successful Glucolipid metabolism Diabetes
Pain
Mineralocorticoid receptors MR Successful Na+/K+ equilibrium Inflammatory, autoimmune disease
Injury
Phosphodiesterase 4B PDE4B Successful AKT/mTOR pathway Cancer
Obesity
Phosphodiesterase 4D PDE4D Successful Intracellular cAMP//CREB signaling Cancer
Alzheimer’s
Protoporphyrinogen oxidase PPO Research Heme biosynthesis Cancer
Parasitosis
Transthyretin TTR Clinic Trial Thyroxine carrier Cancer
Alzheimer’s
Mitogen-activated protein kinase 10 JNK3 Research GbRH/ErbB/MAPK/insulin signaling pathway Cancer
Alzheimer’s
Sex hormone-binding globulin SHBG Research Sex steroids biosynthesis Cancer
NAD(P)H:quinone oxidoreductase NQO1 Research Quinones metabolism Cancer
Cellular retinol binding protein II CRBP2 Research Retinol metabolism Cancer
Estrogen receptor alphaa ERαa Successful Estrogen metabolism
Insulin-like growth factor pathway
Cancer
Alzheimer’s
Injury
Osteoporosis
Alpha-tocopherol (alpha-T) transfer protein ATTP Research α-Tocopherol metabolism Cancer
Human serum retinol binding protein 4 RBP4 Research Retinol metabolism Cancer
Estrogen receptor betaa ERβa Successful Estrogen metabolism
MAPK, PI3K signaling
Cancer
Alzheimer’s
Injury
Checkpoint kinase 1a Chk1a Research DNA damage response Cancer
Androgen receptor AR Successful Hormone metabolism Cancer
Reticulocyte 15S-lipoxygenase 15S-LOX Research Arachidonic acid metabolism Cancer
3-Phosphoinositide-dependent kinase-1a PDK-1a Research Phosphatidylinositol 3 kinase (PI3K) signaling Cancer
Casein kinase 2a CK2a Research Ser/Thr pathway Cancer
Cyclin dependent kinase 2a CDK2a Research Cell cycle Cancer
Calcium/calmodulin dependent protein kinase II delta CAMKII Research NF-κB-mediated inflammatory response
Ca2+-linked signaling
Cancer
Inflammatory, autoimmune disease
Dual-specificity protein kinase 1 CLK1 Research Nuclear redistribution of SR proteins Cancer
Proto-oncogene serine threonine kinasea PIM-1a Research Cell cycle regulation JAK/STAT pathway Cancer
Aurora kinase A Aurora-A Clinical trial Cell cycle arrest Cancer
Matrix metalloproteinases MMP12 Research Cell invasion, metastasis Cancer
Inflammatory, autoimmune disease
Phospholipase A2 PLA2s Successful VEGF/MAPK/GnRH signaling Cancer
Inflammatory, autoimmune disease
Mitogen-Activated Protein Kinases p38 MAPK p38 Clinical trial MAPK signaling Cancer
Pain
Inflammatory, autoimmune disease
Dermatosis
Tankyrase 2 Tankyrase 2 Research Canonical Wnt signaling Cancer
Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma isoform Pi3Kγ Research Cancer migration, invasion
Inositol phosphate metabolism
Cancer
Inflammatory, autoimmune disease
PPARgamma-LBDa PPARγa Research LPS-induced iNOS expression Cancer
Inflammatory, autoimmune disease
Osteoporosis
Cartilage oligomeric matrix protein COMP Research Bone regeneration Autoimmune disease
Injury
Severe acute respiratory syndrome coronavirus (SARS-CoV) main protease (M(pro)) SARS M(pro) Research Virus maturation Virus infection
Glycosomal glyceraldehyde-3-Phosphate Dehydrogenase GAPDH Successful Glyceraldehydes metabolism Parasitosis
Glutathione disulfide oxidoreductase GR Research Glutathione metabolism Parasitosis
Acyl carrier protein reductasea PfENRa Successful Fatty acid biosynthesis Parasitosis
Acetylcholine binding protein alpha7 nAChR 7α Successful Calcium signaling pathway Alzheimer’s
Pain
3R-hydroxyacyl-acyl carrier protein dehydratase FabZ Research Fatty acid biosynthesis Parasitosis
Dihydroorotate dehydrogenase DHODH Successful Pyrimidine metabolism Parasitosis
TEM-1 Beta-Lactamasea TEM-1a Successful Cefotaxime metabolism Bacterial infection
Chloramphenicol acetyltransferase CAT Research Chloramphenicol metabolism Bacterial infection
Polyketide cyclase SnoaL SnoaL Research Nogalamycin biosynthesis Bacterial infection
ZipA attaches FtsZ protein ZipA-FtsZ Research Cell division Bacterial infection
Ferredoxin-NADP+ reductase FNR Successful Redox metabolism Bacterial infection
Polyketide cyclase AknH AknH Research Aclacinomycin biosynthesis Bacterial infection
Enoyl-acyl carrier protein reductase ENR Successful Fatty acid biosynthesis Bacterial infection
Multidrug binding protein TtgR TtgR Research Active extrusion of drug Bacterial infection
NmrA-like family domain NmrA Research Transcriptional repress Fungal infection
Bacterial phosphotriesterase opdA Research Organophosphate metabolism Bacterial infection
Streptomyces coelicolor TetR family protein ActRa ActRa Research Transcriptional repress Bacterial infection
Multidrug binding transcriptional regulator LmrR LmrR Research Autoregulatory mechanism Bacterial infection
Scytalone Dehydratase ScyD Research Fungicide Fungal infection
Human monoamine oxidase A MAO-A Successful Monoamines metabolism Depression
Acetylcholin esterase AChE Successful Glycerophospholipid metabolism Alzheimer’s
Parkinson’s
β-Site amyloid precursor protein cleaving enzyme BACE1 Clinical trial Neuregulin processing Alzheimer’s
Multidrug-resistant HIV-1 proteasea MDR HIV-1 proteasea Successful Self-activation AIDs
HIV-1 reverse transcriptase HIV-1 TR Successful ATP-dependent excision, pyrophosphorolysis AIDs
Oxysterol binding protein OSBP Research Intracellular lipid homeostasis
Signal conduction
Virus infection
Cancer
Rhodopsin Opsin 2 Research Rod photoreceptor Retinitis pigmentosa
Macrophage migration inhibitory factor MIF Clinical trial Phenylalanine, tyrosine metabolism Cancer
Inflammatory, autoimmune disease
Glycogen synthase kinase-3 beta GSK-3β Research Glycogen biosynthesis Cancer
Alzheimer’s
Diabetes
Hepatitis C virus (HCV) polymerase HS5B Pol Successful DNA biosynthesis Virus infection

aThe targets verified by HypoDB screening

Analysis of the interaction network

A topological analysis of the interaction network offered insights into the biologically relevant connectivity patterns, and highly influential compounds or targets. Some Chinese medicines had been investigated by interaction network analysis [3032].

The pharmacological network of M. cordata had three types of nodes (Fig. 5). The 26 alkaloid nodes formed the core of the network, and were surrounded by 65 target nodes. Each target was linked to at least one pathway. A total of 60 pathway nodes constituted the outer layer of the network. Each alkaloid was the center of a star-shaped action net except for the two bisbenzo[c]phenanthridines (BisBen), which were only linked to one target and one pathway, respectively. The alkaloids and targets were strongly interconnected in many-to-many relationships.

Fig. 5.

Fig. 5

The pharmacological network of Macleaya cordata. Hexagon, targets; Rectangle, biopathway; Ellipse, alkaloids (bright green Ben, dark green BisBen, breen Ber, orange Pro)

A general overview of the global topological properties of the network was obtained from the statistical data by the Network Analyzer of Cytoscape. The diameter of the network was 8.0, the centralization was 0.14, and the density was 0.024. The node degree indicated the number of edges linking to other nodes. The highly connected nodes were referred to as the hubs of the network. The degrees of all the alkaloids (Fig. 6a) and important targets (Fig. 6b) were investigated. The compounds with higher degree values, such as C5, C6, C9, C19, and C20, that might participate in more interactions than the other components were the hubs in the network. The target degree values mostly ranged between 2 and 7. The targets with the highest degree values included MIF (16), TTR (11), FabZ* (11), ERα* (10), and MR (10). The targets with higher degree values might be involved in the pharmacological actions of M. cordata.

Fig. 6.

Fig. 6

Degree distribution in the network. a alkaloids, b targets

Interpreting the pharmacological actions

By mining the PubMed and TTD, the targets of M. cordata in the PharmaDB profiling results were annotated with biological functions and clinical indications (Table 3). Furthermore, the targets were classified according to the reported pharmacological activities of M. cordata as follows: microorganism (including bacterial, fungal, and viral) infection (12 targets, with 3 targets verified by HypoDB screening), parasitic disease (5 targets, with 2 targets validated by HypoDB screening), pain (3 targets), cancer (31 targets, with 8 targets confirmed by HypoDB screening), inflammation (8 targets, with 1 target verified by HypoDB screening), and injury (4 targets, with 2 targets fished by HypoDB screening).

Antibacterial activity

The extracts and their purified alkaloids from M. cordata exhibited notable activities against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, Tetracoccus spp., and methicillin-resistant Staphylococcus aureus (MRSA) [12, 33]. In this study, 12 proposed targets were closely related to microorganisms, and seven of them exhibited antibacterial activities (Fig. 7). the key types of alkaloids with antibacterial activity were dihydro-benzo[c]phenanthridine alkaloids and protoberberines.

Fig. 7.

Fig. 7

The compounds mapping of microorganism related targets

Five targets (LmrR, TEM-1*, CAT, FNR, and ActR) were related to multidrug-resistant bacterial strains. LmrR, a multidrug binding transcriptional regulator and the predicted target of C11, was a PadR-related transcriptional repressor that regulated the production of LmrCD, a major multidrug ABC transporter in Lactococcus lactis [34, 35]. TEM-1* (TEM-1 beta-lactamase) fished by C19 was one of the antibiotic-resistance determinants for penicillins, early cephalosporins, and novel drugs from their derivatives [36]. A new drug, Avibactam™, innovated by AstraZeneca is a TEM-1 inhibitor that has already entered phase III clinical development [37]. In addition, chloramphenicol acetyltransferase (CAT), an antibiotic-inactivating enzyme predicted by C11, catalyzed the acetyl-S-CoA-dependent acetylation of chloramphenicol at the 3-hydroxyl group and resulted in chloramphenicol-resistance in bacteria [38]. Ferredoxin-NADP+ reductase (FNR), targeted in silico by C4, C5, C6, and C9, participated in numerous electron transfer reactions, had no homologous enzyme in humans, and was a target for the accumulation of multidrug-resistant microbial strains [39]. The Streptomyces coelicolor TetR family protein ActR* was found by C19. ActR* may mediate timely self-resistance to an endogenously-produced antibiotic. TetR-mediated antibiotic-resistance might have been acquired from an antibiotic-producer organism [40].

Two targets indicating other pathways were involved in the antibacterial activity. The ZipA-FtsZ complex was fished by C13, C14, and C20 (Fig. 8). ZipA was a membrane-anchored protein in E. coli that interacted with FtsZ-mediated bacterial cell division, and was considered a potential target for antibacterial agents [41]. The target ENR catalyzed an essential step in fatty acid biosynthesis. ENR was a target for narrow-spectrum antibacterial drug discovery because of its essential role in metabolism and its sequence conservation across many bacterial species [42].

Fig. 8.

Fig. 8

Three alkaloids mapped to ZipA-FtsZ. Left the crystal structure and pharmacophore of target, right the alkaloids fit to the pharmacophore

Antiparasitic activity

M. cordata showed remarkable effects against Ichthyophthirius multifiliis in grass carp [43] and richadsin [44], as well as against Dactylogyrus intermedius in Carassius auratus [45]. The total alkaloids of M. cordata were able to kill gastrointestinal parasites [46].

In this study, five targets involved in parasitic diseases were predicted. Because of the lack of reported protein–ligand crystal structures for parasitosis, these five targets were not related to the above parasitosis in either humans or other animals. However, the findings suggested the potential of M.cordata to treat other parasitosis, such as malaria, Chagas disease, and Kala-azar. The enoyl-acyl carrier reductase PfENR* fished by two alkaloids (C5 and C26) and the (3R)-hydroxymyristoyl acyl carrier protein dehydratase FabZ* in silico targeted by six alkaloids (C5, C6, C9, C11, C12, and C16) were involved in the fatty acid biosynthesis of Plasmodium falciparum. The antioxidant enzyme GR fished by C13, C14, and C19 was a target for antimalarial drug development [47]. The target glycosomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) found by C11 was a target for the development of novel chemotherapeutic agents for the treatment of Chagas disease [48]. Dihydroorotate dehydrogenase (DHODH) retrieved by C5 and C6 was related to both Leishmania infection and Trypanosoma infection [49].

Analgesic activity

A mixture of the isoquinoline alkaloids from M. cordata exhibited strong analgesic activity towards the pain caused by inflammatory cytokines and direct peripheral nerve stimulation [50]. In this study, three targets related to pain were identified. nAChR7α was abundantly expressed in the central and peripheral nervous systems, and involved in subchronic pain and inflammation [51]. In the profiling results, nAChR7α was picked out by five alkaloids (C2, C11, C15, C19, and C25). MAPK p38 fished by C9, C14, and C20 was involved in the development and maintenance of inflammatory pain [52, 53]. The reductase ALR fished by C19 was a specific target of painful diabetic neuropathy [54, 55]. Inhibitors of ALR relieved pain and improved somatic and autonomic nerve function [56]. In addition, based on the action network, berberines (Ber) such as C19 and C20 may also be involved in the analgesic activity of M. cordata.

Anti-inflammatory activity

Eight targets related to inflammation were identified in this study. Phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit gamma isoform (PI3 Kγ) fished by C19 recruited leukocytes [57]. The proteinase MMP12, also known as macrophage metalloelastase (MME) or macrophage elastase (ME), was identified with three fitted compounds (C3, C5, and C9) in this study. MMP12 mediated neutrophil and macrophage recruitment and T cell polarization [58], and was a potential therapeutic target for asthma [59]. PPARγ* fished by C3 was another inflammation-related target. Some early findings demonstrated the anti-inflammatory effects of PPARγ by activating human or murine monocytes/macrophages and monocyte/macrophage cell lines [60].

MAPK p38 was involved in a signaling cascade controlling cellular responses to inflammatory cytokines, and it was verified for this pathway in murine macrophage RAW264.7 cells that the M. cordata extract increased both the mRNA and protein levels of cytoprotective enzymes including heme oxygenase-1 (HO-1) and thioredoxin 1 via activation of the p38 MAPK/Nrf2 pathway [16]. The kinase calcium/calmodulin-dependent protein kinase II (CAMKII) was a regulator of intracellular Ca2+ levels, which triggered activation of the transcription factor nuclear factor-kappa B (NF-κB) after T-cell receptor stimulation. An inhibitory effect of CAMKII on NF-κB was confirmed [61]. Phospholipase A2 (PLA2s) was a key enzyme in prostaglandin (PG) biosynthesis for discharging arachidonic acid. Selective inhibitors of PLA2s were implicated in inflammation and connected to diverse diseases, such as cancer, ischemia, atherosclerosis, and schizophrenia [62].

The target mineralocorticoid receptor (MR) fished by five compounds (C3, C4, C6, C7, and C20) was activated by mineralocorticoids, such as aldosterone and deoxycorticosterone, as well as by glucocorticoids, like cortisol. Antagonists of MR had cardioprotective and anti-inflammatory effects in vivo via aldosterone-independent mechanisms [63]. Macrophage migration inhibitory factor (MIF) was involved in both innate and adaptive immune responses. Inhibitors of MIF were potential anti-inflammatory agents [64].

Seven of the eight predicted targets were also related to cancer. These dual correlative targets were PI3Kγ, MMP12, PPARγ*, MAPK p38, CAMKII, PLA2s, and MIF. Their matching compounds are shown in Fig. 9, and the benzo[c]phenanthridine (Ben) alkaloids and berberine (Ber) alkaloids were involved in the anti-inflammatory activity.

Fig. 9.

Fig. 9

Alkaloid C11 mapped to GAPDH. Left the crystal structure and pharmacophore of GAPDH, upper right the alkaloid C11 docked into the target, lower right C11 fitting into the pharmacophore and the shape of the pocket

Injury healing activity

In this study, four predicted targets (ERα*, ERβ*, MR, and COMP) were involved in injury repair. Among them, ERα*, ERβ*, and MR were linked with internal injuries, such as brain injury [65], vascular injury [66], and neuronal injury [67]. The other target, cartilage oligomeric matrix protein (COMP), found by C9 was a non-collagenous extracellular matrix protein found predominantly in cartilage, but also in tendon, ligament, and meniscus [68]. COMP was a marker for joint destruction associated with osteoarthritis, rheumatoid arthritis, trauma, and intense activity [69].

Antitumor activity

Both the mixed and single alkaloids of M. cordata strongly inhibited proliferation and induced apoptosis of cancer cells [6, 70]. The anticancer drug Ukrain™ is an isoquinoline type. The major components of Ukrain™ are chelidonine, sanguinarine, chelerythrine, protopine, and allocryptopine. Ukrain™ exerted cytotoxic effects in cancer cells without negative effects on normal cells [71], and had radiosensitization effects on cancer cells, while exerting radioprotective effects on normal cells [72].

In the pharmacological profiling results, almost half of the predicted targets (31 of 65 targets) had a close relationship with cancer, and ten of them (Table 3) successfully entered into clinical trial observations. In total, nine targets related to cancer were fished by more than five compounds. The results revealed promising prospects for M. cordata in antitumor drug research and development. Based on the action network (Fig. 5), possible antitumor molecular mechanisms of M.cordata were analyzed as follows: (1) most possible effective targets and (2) most likely contributing compounds.

The MIF column was particularly tall (Fig. 10) because it was fished by 15 compounds, including all quaternary benzo[c]phenanthridine (Ben) alkaloids (C11–C16), two other benzo[c]phenanthridine (Ben) alkaloids, five protoberberine (Ber) alkaloids, and two protopine (Pro) alkaloids. The discovered pathways of these 15 compounds mainly included NF-κB and ERK signaling pathways [73, 74], Bax/Bcl and caspase-dependent pathway [75], ROS-mediated mitochondrial pathway [76], p38 MAPK/Nrf2 pathway [77], and VEGF-induced Akt phosphorylation pathway [78]. All of these pathways were linked closely with MIF [7984]. However, there have been no experimental reports on to the interactions between MIF and these alkaloids.

Fig. 10.

Fig. 10

The alkaloids mapping of cancer related targets

Both transthyretin (TTR) and proto-oncogene serine threonine kinase* (PIM-1) were found by seven compounds. TTR was a biomarker for lung cancer [85] and pancreatic ductal adenocarcinoma [86], but has not yet been confirmed as a therapeutic target. PIM-1* fished by C5, C6, C8, C9, C14, C19, and C20, and also verified by HypoDB screening, was responsible for cell cycle regulation, antiapoptotic activity, mediation of homing, and migration of receptor tyrosine kinases via the JAK/STAT pathway. PIM-1 was upregulated in many hematological malignancies and solid tumors. Although PIM kinases were described as weak oncogenes, they were heavily targeted for anticancer drug discovery [87]. C12 was partially involved in the JAK/STAT pathway [88].

The benzo[c]phenanthridine (Ben) alkaloids of M. cordata hit cancer-related targets a total of 75 times, compared with 25 times for protoberberines (Ber), five times for protopines (Pro), and one time for bis-benzo[c]phenanthridines (BisBen) (Fig. 11). According to the quantitative determination of alkaloids from M. cordata, the quaternary benzo[c]phenanthridine alkaloids C12, C13, and C15 were the main active components [89]. However, the dihydro-benzo[c]phenanthridines such as C5, C6, and C9 rarely reached the limit of detection (LOD), and hit more targets than the main alkaloids. As the quaternary and dihydro-benzo[c]phenanthridines can be transformed into one another, the dihydro-benzo[c]phenanthridines could be active compounds in vivo. The metabolism of C15 was examined in pig liver microsomes and cytosol by electrospray ionization hybrid ion trap/time-of-flight mass spectrometry, and C7 was one of the main metabolites in liver microsomes and the only metabolite in cytosol [90]. Hence, the issue of whether the dihydro-benzo[c]phenanthridines were the main compounds combining with the targets in vivo requires further investigation.

Fig. 11.

Fig. 11

The hit number of the alkaloids to cancer related targets

Among the 31 cancer-related targets, at least seven (including MIF, PPARγ*, CAMKII, and Pi3Kγ) were involved in the immune system. These immune-associated targets might be crucial to for oncotherapy with M. cordata.

Potential pharmacological activities

According to the pharmacological profiling, some unreported pharmacological performances of M.cordata emerged. In this study, 10 targets linked with neurodegeneration were fished, among which AChE and MAO-B were crucial therapeutic targets in Alzheimer’s disease and Parkinson’s disease [9194].

In addition, antiviral activities, especially anti-HIV, anti-SARS coronavirus, and antifungal activities, were kinds of extensions of the antibacterial function of M. cordata. The possible anti-HIV activity was notable, because HIV-1 reverse transcriptase and multidrug-resistant HIV-1 protease* were particularly related to AIDS [9599]. Meanwhile, the anti-HIV activity was partly confirmed by HypoDB screening. The protein SARS-CoV M(pro) predicted by C3 and C5 was an attractive target for structure-based drug design of anti-SARS drugs owing to its indispensability for the maturation of severe acute respiratory syndrome coronavirus (SARS-CoV) [100]. Another target, HS5B Pol, fished by five alkaloids was a target for anti-HCV therapeutic advances [101]. Inhibitors of HS5B Pol would be a principal option for the treatment of HCV [102]. Meanwhile, scytalone dehydratase and negative transcriptional regulator NmrA were suggested to be physiological targets of new fungicides and the subjects of inhibitor design and optimization [103105].

In this paper, we proposed a very wide range of the promising targets for the isoquinoline alkaloids of M. cordata. Most of the hits are not yet proven by pharmacological experiment.

Conclusion

Through in silicotarget fishing, the anticancer, anti-inflammatory, and analgesic effects of M. cordata were the most significant among many possible activities. The possible anticancer effects were mainly contributed by the isoquinoline alkaloids as active components.

Authors’ contributions

HBL, QFL, PGX and YP conceived and designed the study. HBL, QFL and PGX performed the experiments. HBL, QFL and YP wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgements

The authors would like to thank Prof. Jun Xu (Sun Yat-sen University, China) and Prof. Yanze Liu (IMPLAD, China) for assistance in preparing the manuscript and Dr. Rong Zhao (National Yang-Ming University, Taiwan) for assistance in analyzing the pathway. This work was supported by National Natural Science Foundation of China (Grant No. 81072995), and Peking Union Medical College Youth Fund (Grant No. 3332013079).

Competing interests

The authors declare that they have no competing interests.

Abbreviations

CHARMM

chemistry at Harvard Macromolecular Mechanics

MOE

molecular operating environment

RMSD

root mean square deviation

MMFF

Merck molecular force field

PDB

Protein Data Bank

KEGG

Kyoto Encyclopedia of genes and genomes

TTD

Therapeutic Target Database

HTML

hypertext markup language

Contributor Information

Qifang Lei, Email: leiqifang66@163.com.

Haibo Liu, Email: hbliu@implad.ac.cn.

Yong Peng, Email: ypeng@implad.ac.cn.

Peigen Xiao, Email: xiaopg@public.bta.net.cn.

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