In Silico-based Screen Synergistic Drug Combinations From Herb Medicines: A Case Using Cistanche Tubulosa-Ⅰ

Neurogenic neuroinflammation is defined as orchestrated actions of innate and adaptive immune cells, vascular cells, and neurons triggered by pathological states and enhanced neuronal activity in the central nervous system (CNS). It is likely to play a role in the priming of CNS inflammatory reactions by conditions such as pain, psychological stress, and epilepsy or become a pathogenic factor in neurodegenerative diseases. The current agents for the treatment of neuroinflammation mostly belong to monotherapy, including dopamine, somatostatin, neuropeptide, adenosine, and so on. However, for example, long-term use of the common but old drugs COX inhibitors, Nonsteroidal Anti-inflammatory Drugs (NSAIDs), might cause adverse side effects, gastrointestinal lesions, or cardiovascular risks, and clinical trial results remain unsatisfactory particularly, so there is an unmet need for new treatments of neuroinflammation. 

NATURAL CISTANCHE TUBULOSA FOR TREATING ALZHEIMER'S DISEASE PHGS75% ECH 30% ACT 12%

The novel therapy of drug combinatorial may be a surging strategy to meet the needs of the development of novel drugs as well as overcome hurdles in the treatments of complex diseases. Combination or multicomponent therapy could fulfill the above requirements, in which two or more drugs are used together, with the listed advantages: higher efficacy, minimal cross-resistance, low dose while fewer side effects, and less toxicity when compared to single-drug agents6. It has been used in the treatment of complex diseases for nearly 30 years. Intriguing, drug combination therapy is also applied in the research on neurological diseases, for instance, a combination of glimepiride and ibuprofen could effectively reduce inflammation in Alzheimer's Disease (AD), or ketamine/atropine might lower pro-inflammatory protein expression in epileptic mice. Synergistic drug combinations may therefore bring new inspiration for tracing effective treatments for neuroinflammation. Hence, how can we crack the hard nut to achieve the optimal combinatorial drugs?

Nowadays, the existing approaches to screen out drug combinations are as follows, systematic surveys of drug pairs in vitro such as the high throughput screening method11 and the 'Multiplex Screening for Interacting Compounds' (MuSIC) or evaluations of the pairwise drug combinations with large-scale experiments. Nevertheless, the excessive consumption of manpower, natural resources, and time of arduous empirical testing may be an inevitable issue in the evaluation of effective drug combinations. In response to conquering the shortcomings, approaches based on network analysis, especially genetic interaction networks chemical systems biology data, and molecular and pharmacological data also emerge for combinatorial drug discovery. Furthermore, an increasing body of investigators proposed novel network approaches to predict optimal combinations and offered the corresponding experimental validation in the meantime, so integrating network prediction with experimental validation may be a new trend in the field of combinational prediction.

Herbal medicines involve considerable numbers of formulas (Fang-Ji in Mandarin) and chemical ingredients, which form a natural products database so that it could afford innovative clues, and fundamental biology data for the development of drug combinations, as Professor Li and Professor Liu introduced holistic analysis methods based on integrated biology to decipher the molecular mechanisms of herbal medicines: Liu-Wei-Di-Huang pill or Reducing Injection. In our previous work, we found not only two representative herbs Lonicera japonica and Fructus Forsythiae show synergistic effects on infuenza or inflammation, but also compounds rutin and amentoflavone present synergistic effects in preventing depression.

In our current work, we develop a system pharmacology approach to discover the synergistic drug combinations among compounds from the herb Cistanche tubulosa (SCHENK) R. WIGHT, the following steps are proposed: firstly, we pick out bioactive compounds through drug-likeness prediction, which are used as baits to fsh the related targets. And then, inspired by a "network target"-based paradigm to prioritize synergistic agent combinations in a high throughput way, we acquire effective drug combinations among the potential compounds based on an in-house algorithm that is termed Probability Ensemble Approach (PEA)26 with a high training efficiency, extensive applicability, and two quantitative indexes to describe the property of a drug combination. Finally, we use the obtained targets and the compounds of candidate pairs to build a network/pathway and then provide analysis to encode the mechanism of Cistanche tubulosa on neuroinflammation holistically. As an example, it is the first time to screen out effective drug combinations from natural products based on system pharmacology through integrating computational methods and experimental validation to approve the reliability of the prediction. We believe that this may help to personalize neuroinflammation treatment, enhance our understanding of effective neuroprotective development, and aid future preclinical research.

CISTANCHE TUBULOSA FOR REGULATING NEUROINFLAMMATION IN PARKINSON'S DISEASE PHGS75% ECH 30% ACT 12%

Results 

Targets of Cistanche tubulosa

To get the targets related to neuroinflammation we firstly achieve the ingredients in Cistanche tubulosa (SCHENK) R. WIGHT by searching the TCMSP database (http://lsp.nwu.edu. cn/), which results in 103 compounds (Supplementary Table S1, See Materials and Methods). Then, we analyze their drug-likeness by applying the DL prediction model constructed in our previous work (See Materials and Methods). In this way, we achieve 63 potential bioactive compounds (Supplementary Table S2) with DL index ≥0.18. Subsequently, using the SysDT and WES algorithms, we identify 117 targets of these potential bioactive compounds. Finally, 43 potential targets (Supplementary Table S3) closely related to neuroinflammation are retrieved after deleting noise and errors, by mapping the 117 targets of the compounds to the CTD database.

GOBP enrichment analysis for targets

To check whether the proteins targeted by the potential bioactive compounds are closely related to neuroinflammation, we perform GOBP enrichment analysis by mapping targets to DAVID. Fig. 1 shows a GO tree representing the results of significantly enriched GOBP terms (P value≤0.05), where the targets are categorized into 20 different groups, such as positive regulation of vascular smooth muscle cell proliferation and positive regulation of leukocyte migration. Among these groups, chemical synaptic transmission, inflammatory response, cell-cell signaling, and so on are all closely associated with neuroinflammation. For instance, the synaptic alterations occurring during neuroinflammatory diseases are largely mediated by inflammatory cytokines released from infiltrating T cells and from activated microglia, and are responsible, at least in part, for irreversible dendritic pathology. Collectively, these observations suggest that the predicted targets probably can contribute to the treatment of neuroinflammation.

Compound-target network construction and analysis

Generally, feasible and effective combination therapies are combinations of bioactive compounds with ideal pharmacokinetic properties, which can balance the disease network by regulating specific targets. Therefore, we further construct a static compound-target network to check their topological relations. As shown in Fig. 2, the bipartite compound-target (C-T) network exhibits 482 interactions between 63 compounds and 43 targets in a visually appealing manner. We analyze the nodes' degree in the compound-target network which results in an average degree per compound of 11.209 and 7.651 per target, respectively. We observe that among the 63 compounds, 38 of them adjust more than 7 targets (larger than the average degree), manifesting the potential synergistic effects among them.

For example, syringin (mol30) interacts with the largest number of targets, which may play a hub role in the network. Interestingly, a study proves that the phenolic constituent syringin isolated from Euonymus alatus (Tunb.) Sieb. (Celastraceae) has an anti-neuroinflammatory effect by inhibiting NO production27. 2′-acetylacteoside (mol42) is in control of 10 different targets (degree=10). Among these targets, HSPB1 (also known as HSP27), as an example, is a molecular chaperone that displays neuroprotective properties in many disease and injury models. For verbascoside (mol33), the neuroprotective properties of this bioactive compound involve modulation of transcription factors and 

consequently altered gene expression, resulting in the downregulation of inflammation. It is worth noting that although the topology property of the network does not bias toward echinacoside (mol41, degree=7), it is a potential novel orally active compound for regulating neuroinflammation and related signals in Parkinson's disease and may provide a new prospect for clinical treatment. Taken together, these results indicate that the screened potential active compounds are all related to neuroinflammation and can be regarded as the data set for predicting drug combinations.

Compound-Target-Pathway network construction and analysis

By employing the PEA algorithm, we get 10 different drug combinations (Table 1.), which involve 12 compounds. These drug combinations are all with high synergy probability, which represents the possibility of inducing synergy between two compounds. 

Therefore, up to a certain extent, these compounds are considered to be the pharmacological fundamental substances of Cistanche tubulosa. Based on the target information of these compounds and the pathways derived from DAVID, we construct the compound-target-pathway (C-T-P) network to preliminarily light on the molecular mechanisms of these compounds. The resulting compound-target-pathway network consists of 47 nodes and 99 edges. As illustrated in Fig. 3, these compounds can act on not only the proteins of the upstream but also the downstream pathways associated with neuroinflammation, especially the direct indicators of inflammation markers.

In particular, the vast majority of compounds target the upstream proteins such as HSPB1, HTR2A, NTSR1, and others, indicating the macro regulation of these compounds for the treatment of neuroinflammation. For instance, the compound echinacoside (mol41) targets the VEGF pathway through protein HSPB1(Supplementary Figure 1). Fortunately, studies show that HSPB1 has significant cytoprotective properties in several models of neurological disease in vivo or in vitro and it may play a role in anti-infammatory effect by regulating the nuclear factor-κB (NF-κB) signaling pathway. In addition, a study also demonstrates that R-Ras could regulate angiogenic activities of endothelial cells in part via inhibition of the p38 mitogen-activated protein kinase (p38 MAPK)-HSPB1 axis of the VEGF signaling pathway.

We can get that compound tubuloside A (mol56) to target the Calcium signaling pathway through protein NTSR1 in the network. It is all known that calcium ions (Ca2+) are a universal second messenger in the immune system cells. The reduction in synaptic activity or increased extrasynaptic N-methyl-D-aspartic acid (NMDA) receptor signaling may lead to nuclear calcium dyshomeostasis, thereby increasing the occurrence of neurodegeneration and cognitive dysfunction. Furthermore, the target protein NTSR1 is a member of the large superfamily of G-protein coupled receptors, and signaling is generated through binding G proteins that may activate a phosphatidylinositol-calcium second messenger system and downstream MAP kinases.

Another example is that compound 2′-acetylacteoside (mol42) targets the GnRH signaling pathway through protein PRKCD. PRKCD, one of the PKC isoforms, is a major mediator of the activation of extracellular regulated protein kinases1/2 MAPK (ERK1/2 MAPK), c-Jun N-terminal protein kinases MAPK (JNK MAPK) and p38 MAPK by gonadotropin-releasing hormone (GnRH)40. And, as key processes in the GnRH-stimulated signaling network, the downstream MAPK cascades and arachidonic acid(AA) metabolites could produce an inflammatory protein, COX-2. As for kankanoside O (mol18) and syringin (mol30), they interact directly with the downstream proteins PTGS2, NOS2, and other inflammatory indicators, which is an intuitive refect of the compounds' efficacy. A study shows syringin could suppress the production of tumor necrosis factor-α (TNF-α) in Lipopolysaccharides (LPS)-stimulated RAW264.7 cells42. The other studies demonstrate that syringin could also lower NO concentration and NOS activity43 or the production of prostaglandin E244. Isoacteoside (mol43) targeted MMP9, is an important player in the central nervous system, and would be a putative mediating enzyme for neuropsychiatric disorders such as schizophrenia and bipolar illness. The degradation of NF-κB and phosphorylation of p38, ERK1/2, JNK MAPK, or Akt (Protein Kinase B) of the upstream signaling pathways could modulate the MMP9 gene expression and inhibition of MMP9 that may reduce the expression of inducible nitric oxide synthase (iNOS) in activated cells.

NATURAL CISTANCHE TUBULOSA FOR REDUCING NEUROINFLAMMATION PHGS75% ECH 30% ACT 12%

Pathway analysis

An incorporated "Neuroinflammation pathway" is constructed by integrating the key pathways obtained through compound-target-pathway network analysis, including the Alzheimer's disease pathway, Calcium signaling pathway, GnRH signaling pathway, VEGF signaling pathway, and the Serotonergic synapse. Of the 43 targets, 19 can be mapped onto the "Neuroinflammation pathway". As shown in Fig. 4, the Cistanche tubulosa represents the targets of the active compounds that distribute in the "Neuroinflammation pathway". The "Neuroinflammation pathway" is classified into 13 different therapeutic modules, such as cell death, apoptosis, inflammation, and neuroprotection. In this study, we take cell death, inflammation, and neuroprotection modules as examples to clarify the mechanism of Cistanche tubulosa for neuroinflammation.

Cell death module

Targets located in the Alzheimer's disease pathway are mainly involved in the cell death process, suggesting that cell death is closely related to neuroinflammation. Studies demonstrate that neuroinflammation mediated by microglia contributes to neuronal cell death, which is not restricted to a specific disease but implicated in various diseases such as ischemia, Parkinson's disease, and Alzheimer's disease. As shown in Fig. 4, 

targets APP, GRIN2B, and MAPT are regulated by the active compounds from Cistanche tubulosa indicating that they can inhibit cell death and thereby offer a treatment for neuroinflammation. Interestingly, the APP intracellular domain impairs adult neurogenesis in transgenic mice by inducing neuroinflammation. The inability of MAPT to properly regulate neuronal microtubule dynamics and thus mediate neuronal cell death. All these indicate that Cistanche tubulosa may treat neuroinflammation by inhibiting cell death.

Inflammation module

The term neuroinflammation is the inflammatory reactions in the CNS in response to neuronal activity1. In this study, we detect that the GnRH signaling pathway and the VEGF signaling pathway are involved in the inflammatory module (Fig. 4). For instance, MMP2 in the GnRH signaling pathway belongs to the MMPs family, which is expressed in physiological situations and pathological conditions involving inflammation. And MMPs regulate several functions related to inflammation including bioavailability and activity of inflammatory cytokines and chemokines. In addition, the VEGF signaling pathway can lead to the generation of NOS2, moreover, it participates in the acute inflammatory response to LPS by multiple mechanisms: involvement in proinflammatory cytokine signaling and alteration of the expression of various genes that affect inflammatory-immune responses to LPS. Collectively, all these indicate that Cistanche tubulosa may cure neuroinflammation by regulating the inflammatory system.

NATURAL CISTANCHE TUBULOSA FOR  ANTI-ALZHEIMER'S DISEASE PHGS75% ECH 30% ACT 12%