Natural Compound Targeting Research Articles

Applications of Monoclonal Antibodies Against Natural Compounds for Functional Analysis of Crude Drugs

This study summarizes the applications of monoclonal antibodies (MAbs) against bioactive natural compounds for the functional analysis of crude drugs. To date, we have established more than 40 kinds of MAbs against natural compounds and set up enzyme-linked immunosorbent assay (ELISA). The intracellular accumulation of harringtonine (HT) and its derivatives into cells was determined by ELISA using anti-HT MAb, and the uptake levels of these compounds conformed to their respective antiproliferative activities. Eastern blotting is an on-membrane quantitative analytical system that uses MAbs against natural compounds and it can also be used in a biological sample for pharmacokinetics study. Glycyrrhizin (GC) was detected in the serum of rats treated with GC by eastern blotting. Additionally, eastern blotting using MAb against 3-monoglucuronyl-glycyrrhetinic acid (3-MGA) could determine a potential causative agent of pseudoaldosteronism induced by excess intake of GC. The immunoaffinity column coupled with MAb made preparing an extract containing all components feasible except the target compound. Cell-based studies using GC-removed licorice extract, which was prepared by the immunoaffinity column conjugated with anti-GC MAb, revealed the potential functions of GC in licorice extract. Moreover, MAbs were used in determining cellular localization and target molecules of natural compounds. The combination of analysis using MAb and LC-MS/MS showed that aristolochic acid I (AA-I), a naturally occurring nephrotoxin and carcinogen, targets α-actinin-4 in the kidney cells. MAbs against natural compounds are useful tools to determine real pharmacological functions, cellular localization, and molecular targets of natural compounds in crude extracts.

Quantitative structure activity relationship and molecular simulations for the exploration of natural potent VEGFR-2 inhibitors: an in silico anti-angiogenic study

VEGFR-2 has recently become an eye-catching molecular target for the novel therapeutic designs against cancer for its well known role in persuading angiogenesis in tumor cells. The current study set sights on the exploration of novel potent natural compound targeting VEGFR-2 via computational ligand-based modeling and database screening followed by binding pattern analysis, reactivity site prediction and MD simulation studies. The known 53 VEGFR-2 inhibitors (with IC50 ranging from 0.7 nM to 9700 nM) were headed for development of Ligand based pharmacophore model using 3 D QSAR pharmacophore generation module of DS Client. Training set inhibitors (23 compounds) were exploited to create pharmacophore model based on their chemical features. The model was validated through 30 test set inhibitors and exploited further for screening of 62,082 natural compounds from InterBioscreen natural compound database. Screened compounds further went through Drug-Likeliness study, ADMET prediction, Binding pattern analysis, In silico prediction of reactivity sites, Biological activity spectra prediction, pan assay interference compound identification and MD simulation analysis. Out of 5 screened compounds, Compound A and Compound B exhibited highest binding energy judged against the standard drug “Sorafenib”. On further conducting reactivity site prediction, BAS prediction, and pan assay interference compound identification, Compound B exhibited better result which was carried forward for MD simulation study for 50 ns. MD simulation results suggested that Compound B exhibited more stable binding to the active site of VEGFR-2 without causing any conformational changes in protein-ligand complex. Thereby, the investigation proposes Compound B to hold potent antiangiogenic potential targeting VEGFR-2. Communicated by Ramaswamy H. Sarma

Sulforaphane inhibits gastric cancer stem cells via suppressing sonic hedgehog pathway

Sulforaphane (SFN) is the major component extracted from broccoli/broccoli sprouts. It has been shown to possess anti-cancer activity. Gastric cancer is common cancer worldwide. The objective of this work was to evaluate the inhibitory effect of SFN on gastric cancer by Sonic hedgehog (Hh) Pathway. The results found that tumorsphere formation and the expression levels of gastric cancer stem cells (CSCs) markers were significantly decreased after SFN treatment. SFN also exerted inhibitory effects by suppressing proliferation and inducing apoptosis in gastric CSCs. Intriguingly, SFN inhibited the activation of Sonic Hh, a key pathway in maintaining the stemness of gastric CSCs. Upregulation of Sonic Hh pathway diminished the inhibitory effects of SFN on gastric CSCs. Collectively, these data revealed that SFN could be a potent natural compound targeting gastric CSCs via suppression of Sonic Hh pathway, which might be an promising agent for gastric cancer intervention.

(-)-Epigallocatechin-3-Gallate Inhibits Colorectal Cancer Stem Cells by Suppressing Wnt/β-Catenin Pathway.

The beneficial effects of tea consumption on cancer prevention have been generally reported, while (−)-Epigallocatechin-3-gallate (EGCG) is the major active component from green tea. Cancer stem cells (CSCs) play a crucial role in the process of cancer development. Targeting CSCs may be an effective way for cancer intervention. However, the effects of EGCG on colorectal CSCs and the underlying mechanisms remain unclear. Spheroid formation assay was used to enrich colorectal CSCs from colorectal cancer cell lines. Immunoblotting analysis and quantitative real-time polymerase chain reaction were used to measure the alterations of critical molecules expression. Immunofluorescence staining analysis was also used to determine the expression of CD133. We revealed that EGCG inhibited the spheroid formation capability of colorectal cancer cells as well as the expression of colorectal CSC markers, along with suppression of cell proliferation and induction of apoptosis. Moreover, we illustrated that EGCG downregulated the activation of Wnt/β-catenin pathway, while upregulation of Wnt/β-catenin diminished the inhibitory effects of EGCG on colorectal CSCs. Taken together, this study suggested that EGCG could be an effective natural compound targeting colorectal CSCs through suppression of Wnt/β-catenin pathway, and thus may be a promising agent for colorectal cancer intervention.

Abstract 1340: Dextran-Catechin conjugate: An anticancer nano-modified natural compound targeting copper metabolism in neuroblastoma

Abstract Background: Neuroblastoma is an aggressive childhood cancer that is poorly responsive to therapy. Moreover, survivors experience long term side effects from their treatment highlighting the need for effective and less toxic therapies. Catechin is a natural polyphenol with anti-cancer properties and limited side effects. Low serum stability of Catechin limits its clinical use that we overcame by chemical functionalization with Dextran. However its mechanism of action is unknown. Here, we investigated the mechanism of action of Dextran-Catechin, and its efficacy against neuroblastoma in vitro and in vivo. Methods: Anticancer activity was tested in 4 independent neuroblastoma cell lines using the cell viability assay Alamar Blue and apoptosis was assessed using PARP cleavage. Gene expression was determined using qRT-PCR and protein expression of CTR1 by western blotting. Intracellular copper was measured by spectrophotometric analysis. Fluorescence-lifetime imaging microscopy was used to study NADH/NAD+ ratio to determine the induction of oxidative stress. Cellular levels of the antioxidant GSH was examined using a colorimetric assay. PET imaging studies with Cu64 were performed in a xenograft neuroblastoma model to monitor copper uptake in tumors. In vivo anticancer activity of Dextran-Catechin was assessed in human xenograft and syngeneic models of neuroblastoma. Results: The neuroblastoma cell lines SH-SY5Y, IMR-32, BE(2)C and doxorubicin-resistant BE(2)C-ADR were sensitive to Dextran-Catechin (IC50 9.7 μg/ml, 17.83 μg/ml, 16 μg/ml and 18.2 μg/ml, respectively) at concentrations that were not toxic to non-malignant MRC-5 cells. However, Cisplatin-resistant neuroblastoma cells, IMR-32-CisRes, were 2.5-fold resistant against Dextran-Catechin compared to the parental cells. Copper transporter 1 (CTR1) mediates cisplatin uptake and IMR-32-CisRes exhibited 50% lower expression of CTR1 and lower intracellular copper compared to the IMR-32 cells. In contrast, Dextran-Catechin sensitive neuroblastoma cells had elevated intracellular copper implicating copper in the activity of this conjugate. We demonstrated that Dextran-Catechin reacts with copper generating reactive oxygen species and inducing cancer cell death. Dextran-Catechin treatment caused a decrease of NADH/NAD+ ratio and GSH levels, confirming oxidative stress. PET imaging analysis of the IMR-32-neuroblastoma xenograft model revealed high accumulation of copper in the tumor mass. The high levels of copper were maintained in the tumor mass for up to 48h, while Cu64 was cleared by the other organs. Importantly, we showed that Dextran-Catechin significantly reduced tumor growth in human xenograft and syngeneic models of neuroblastoma with no evidence of side effects. Conclusion: Dextran-Catechin targets copper, inhibits tumour growth, and has therapeutic potential for cancers dependent on copper for their growth. Citation Format: Orazio Vittorio, Miriam Brandl, Giuseppe Cirillo, Kathleen Kimpton, Elizabeth Hinde, Hien T. T. Duong, Cyrille Boyer, Claudia Flemming, Eugene M.H. Yee, Naresh Kumar, Arvind Parmar, Giancarlo Pascali, Arnaud Charil, Michelle Haber, Murray Norris, Maria Kavallaris. Dextran-Catechin conjugate: An anticancer nano-modified natural compound targeting copper metabolism in neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1340.

Dextran-Catechin: An anticancer chemically-modified natural compound targeting copper that attenuates neuroblastoma growth.

Neuroblastoma is frequently diagnosed at advanced stage disease and treatment includes high dose chemotherapy and surgery. Despite the use of aggressive therapy survival rates are poor and children that survive their disease experience long term side effects from their treatment, highlighting the need for effective and less toxic therapies. Catechin is a natural polyphenol with anti-cancer properties and limited side effects, however its mechanism of action is unknown. Here we report that Dextran-Catechin, a conjugated form of catechin that increases serum stability, is preferentially and markedly active against neuroblastoma cells having high levels of intracellular copper, without affecting non-malignant cells. Copper transporter 1 (CTR1) is the main transporter of copper in mammalian cells and it is upregulated in neuroblastoma. Functional studies showed that depletion of CTR1 expression reduced intracellular copper levels and led to a decrease in neuroblastoma cell sensitivity to Dextran-Catechin, implicating copper in the activity of this compound. Mechanistically, Dextran-Catechin was found to react with copper, inducing oxidative stress and decreasing glutathione levels, an intracellular antioxidant and regulator of copper homeostasis. In vivo, Dextran-Catechin significantly attenuated tumour growth in human xenograft and syngeneic models of neuroblastoma. Thus, Dextran-Catechin targets copper, inhibits tumour growth, and may be valuable in the treatment of aggressive neuroblastoma and other cancers dependent on copper for their growth.

Broad-range inhibition of enterovirus replication by OSW-1, a natural compound targeting OSBP

Enteroviruses, e.g., polio-, coxsackie- and rhinoviruses, constitute a large genus within the Picornaviridae family of positive-strand RNA viruses and include many important pathogens linked to a variety of acute and chronic diseases. Despite their huge medical and economic impact, no approved antiviral therapy is yet available. Recently, the oxysterol-binding protein (OSBP) was implicated as a host factor for enterovirus replication. Here, we investigated the antiviral activity of the natural compound OSW-1, a ligand of OSBP that is under investigation as an anti-cancer drug. OSW-1 potently inhibited the replication of all enteroviruses tested, with IC50 values in the low nanomolar range, acted at the genome replication stage and was effective in all tested cell types of three different species. Importantly, OSBP overexpression rescued viral replication, demonstrating that the antiviral effect of OSW-1 is due to targeting OSBP. Together, we here report the anti-enterovirus activity of the natural anti-cancer compound OSW-1.

Abstract 3370: Natural compound targeting metabolism: A new insight for the treatment of triple negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is a highly diverse group associated with an aggressive phenotype, characterized by early relapse and chemotherapy as the only treatment option. Thus, there is an urgent need to identify alternative strategies for the treatment of TNBC. Use of aerobic glycolysis is important marker of cancer cells and thus monitoring and targeting metabolism in cancer cells is an expanding area of research. Lactate dehydrogenase (LDH) A and B are important enzymes in the metabolic pathway. Traditionally, LDH-A expression has been used to measure the glycolytic capacity of cancer cells however, more recently LDH-B has also been identified as an important biomarker of metabolism. LDH-A is over-expressed in breast cancer, while presence of LDH-B in tumors is contradictory. Higher expression of LDH-B has been reported in normal tissues, with lower expression also noted in breast and prostate tumors. Thus, clarification of the role of LDH-A and LDH-B in cancer metabolism is important in identifying potential targets for TNBC treatment. NF-κB plays an important role in modulating cancer metabolism through regulating the balance between glycolysis and mitochondrial respiration and is over-expressed in TNBCs. Thus, the focus of the present study is to determine the effect of an NF-KB inhibitor in altering cellular metabolism of aggressive breast cancer cells. Experimental Design: We measured the metabolic capacity of several breast cancer cell lines via measurement of mitochondrial membrane potential, ATP level and expression of LDH-A and LDH-B. We then evaluated the ability of the NF-kB inhibitor, panepoxydone (PP), to alter these markers of metabolism. Results: MCF-7 and MDA-MB-231 cells showed dose-dependent decreases in mitochondrial membrane potential, in the form of increased mitochondrial damage, along with decreased ATP levels, when treated with the NF-kB inhibitor, PP. Immunoblotting of LDH-A and LDH-B expression in MCF-7 and MDA-MB-231 cells showed high expression of LDH-A in both cell lines, while MDA-MB-231 cells had a higher level of LDH-A compared to LDH-B, MCF-7 cells had no LDH-B. When these cells were treated with PP, LDH-A levels was reduced in both cancer cell lines (3.5 -4 fold). Interestingly, elevated level of LDH-B was noticed in MDA-MB-231 cells (2 fold). We used Human mammary epithelial cells (HMEC) to check the basal level of LDH-A and LDH-B and almost similar level was observed. Furthermore, this increase in LDH-B was associated with decreased aggressiveness, as noted by decreased invasion and migration. Conclusion: All together this data highlights the role of targeting NF-kB in altering glycolysis through modulation of the membrane potential, ATP level and LDH-A and LDH-B levels. Additionally, it appears that simultaneous measurement, of both LDH-A and LDH-B, may be a more accurate predictor of the metabolic capacity of cancer cells and thus a reflection of their aggressiveness. Citation Format: Ritu Arora, David Schmitt, Steve McClellan, Ming Tan, Windy Dean-Colomb. Natural compound targeting metabolism: A new insight for the treatment of triple negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3370. doi:10.1158/1538-7445.AM2014-3370

Systems Biology Approaches in Identifying the Targets of Natural Compounds for Cancer Therapy

Natural compounds have been known to exert inhibitory effects on the development and progression of human cancers. However, the targets of these naturally occurring agents are largely elusive. Recently, systems biology approaches based on high-throughput technologies such as DNA microarrays have begun to be utilized for investigating the targets of drugs including natural compounds. Therefore, in this review article, we will briefly introduce the several systems biology approaches, and will discuss the application of these new technologies for identifying the therapeutic targets of natural compounds for supporting their roles in the prevention and/or treatment of human cancers. Furthermore, identification of the novel targets will be useful for designing more effective and targeted therapeutic strategies for achieving better treatment outcome in patients diagnosed with cancers.

In Silico Search for Drug Targets of Natural Compounds

Natural compounds represent a significant source for the development of novel medicines. Finding the target proteins for a natural compound is the most important step towards understanding its molecular mechanism for therapeutic usage. In fact, the search for target proteins could be considered the first step of the drug discovery and development pipeline. While experimental determination of compound-protein interactions remains very challenging, effective in silico approaches have been developed and have demonstrated appealing advantages, including their low-cost and capability to scale up easily. The goal of this article is to provide an introduction to in silico search for drug targets of natural compounds. I first review currently available natural compounds databases and human gene/protein databases, and the rapidly emerging databases for known drug-target interactions. These resources provide the 'materials' for in silico approaches and define the gold standard of 'positives' for evaluating them. I then introduce three classes of computational methods for target identification of natural compounds, namely molecular docking, quantitative structure-activity relationship (QSAR) modeling, and data mining and integrative analysis. Use of these methods is explained using real examples, and the advantages and disadvantages of each method are compared. As these state-of-the-art methods continue to mature amid significant challenges, this field appears poised for a period of significant growth, with untold benefits to drug discovery and natural product development.

Chemosensitization of Aflatoxigenic Fungi to Antimycin A and Strobilurin Using Salicylaldehyde, a Volatile Natural Compound Targeting Cellular Antioxidation System

Various species of fungi in the genus Aspergillus are the most common causative agents of invasive aspergillosis and/or producers of hepato-carcinogenic mycotoxins. Salicylaldehyde (SA), a volatile natural compound, exhibited potent antifungal and anti-mycotoxigenic activities to A. flavus and A. parasiticus. By exposure to the volatilized SA, the growth of A. parasiticus was inhibited up to 10-75% at 9.5mM≤SA≤16.0mM, while complete growth inhibition was achieved at 19.0mM≤SA. Similar trends were also observed with A. flavus. The aflatoxin production, i.e., aflatoxin B(1) and B(2) (AFB(1), AFB(2)) for A. flavus and AFB(1), AFB(2), AFG(1), and AFG(2) for A. parasiticus, in the SA-treated (9.5mM) fungi was reduced by ~13-45% compared with the untreated control. Using gene deletion mutants of the model yeast Saccharomyces cerevisiae, we identified the fungal antioxidation system as the molecular target of SA, where sod1Δ [cytosolic superoxide dismutase (SOD)], sod2Δ (mitochondrial SOD), and glr1Δ (glutathione reductase) mutants showed increased sensitivity to this compound. Also sensitive was the gene deletion mutant, vph2Δ, for the vacuolar ATPase assembly protein, suggesting vacuolar detoxification plays an important role for fungal tolerance to SA. In chemosensitization experiments, co-application of SA with either antimycin A or strobilurin (inhibitors of mitochondrial respiration) resulted in complete growth inhibition of Aspergillus at much lower dose treatment of either agent, alone. Therefore, SA can enhance antifungal activity of commercial antifungal agents required to achieve effective control. SA is a potent antifungal and anti-aflatoxigenic volatile that may have some practical application as a fumigant.

Molecular Targets of Natural Drug Substances: Idiosyncrasies and Preferences

All creatures - bacteria, plants, animals, humans - have many building blocks in common, down to biochemical structures and information processing languages. That is why natural substances seem to be better able to bind, and bind specifically, to drug targets in man, and to interact specifically with human biochemical networks. Property analyses of a large number of combinatorial and natural products and drugs have revealed the greater chemical similarity of the latter two. How about target preferences of natural products in comparison to synthetic drugs? On the basis of a comprehensive compilation and analysis of molecular targets of drug substances irrespective of their origin, the review categorises targets chemically and analyses the nature of drug targets. The dynamics of drug action are highlighted because an effective drug target comprises a biochemical system rather than a single molecule. The review is restricted to targets of natural compounds that are in use as therapeutic agents, comparing them with targets of marketed drugs in general. Differences are traced to historical, chemical, pharmacological, and social reasons. To give an example, the prevalence of natural products among antibacterial agents seems to be derived from, first, the necessity to have several hydrophilic binding sites for strong and lasting attachment to vital targets of bacteria, and synthetic drug candidates tend to be more hydrophobic than natural compounds. Second, other microorganisms are well equipped with - natural, of course - compounds with defensive or symbiotic functions that interfere with bacterial metabolism.

Molecular targets of natural drug substances

All creatures – bacteria, plants, animals, humans – have many building blocks in common, down to biochemical structures. That is why natural substances seem to be better able to bind, and bind specifically, to drug targets in man. This is supported, for instance, by a principal component analysis of a large number of combinatorial and natural products and drugs that revealed the greater chemical similarity of the latter two [1]. How about target preferences of natural products in comparison to synthetic drugs? Recently, a comprehensive compilation and analysis of molecular targets of drug substances irrespective of their origin was published [2]. The talk will categorise targets chemically and analyse the nature of drug targets. It will stress the dynamics of drug action because an effective drug target comprises a biochemical system rather than a single molecule. The focus will be on targets of natural compounds that are or were in use as therapeutic agents, and compare this with targets of drugs in general. Differences will be traced to historical, chemical, pharmacological, and social reasons. For example, the prevalence of natural products among antibacterial agents seems to be derived from first, the necessity to have several hydrophilic binding sites for strong and lasting attachment to vital targets of bacteria. Synthetic drug candidates tend to be more hydrophobic than natural compounds. Second, other microorganisms are well equipped with – natural, of course – compounds with defensive or symbiotic function that may be used as antibacterials.

Reverse Pharmacognosy: Identifying Biological Properties for Plants by Means of their Molecule Constituents: Application to Meranzin

Reverse pharmacognosy aims at finding biological targets for natural compounds by virtual or real screening and identifying natural resources that contain the active molecules. We report herein a study focused on the identification of biological properties of meranzin, a major component isolated from Limnocitrus littoralis (Miq.) Swingle. Selnergy, an IN SILICO biological profiling software, was used to identify putative binding targets of meranzin. Among the 400 screened proteins, 3 targets were selected: COX1, COX2 and PPARgamma. Binding tests were realised for these 3 protein candidates, as well as two negative controls. The predictions made by Selnergy were consistent with the experimental results, meaning that these 3 targets can be modulated by an extract containing this compound in a suitable concentration. These results demonstrate that reverse pharmacognosy and its inverse docking component is a powerful tool to identify biological properties for natural molecules and hence for plants containing these compounds.

Reverse Pharmacognosy: Application of Selnergy, a New Tool for Lead Discovery. The Example of ε-Viniferin

The aim of reverse pharmacognosy is to find new biological targets for natural compounds by virtual or real screening and identify natural resources that contain the active molecules. To demonstrate the applicability of this concept, we report here a study on epsilon-viniferin, an active ingredient for cosmetic development. Nevertheless, this natural substance is weakly defined in terms of biological properties. SELNERGY, an inverse docking computer software, was used to identify putative binding biological targets for epsilon-viniferin. Among the 400 screened proteins two targets were retained. For cosmetic application, cyclic nucleotide phosphodiesterase 4 (PDE4) was the most interesting candidate. Moreover, other PDE subtypes (1, 2, 3, 5 and 6) were not retained, indicating a selectivity for PDE4. The experimental binding tests on the 6 subtypes of PDE revealed a significant selectivity of epsilon-viniferin for the PDE4 subtype. This selectivity was confirmed by evaluation of epsilon-viniferin on the secretion of TNF-alpha and Interleukin-8. Our data demonstrated that epsilon-viniferin possesses anti-inflammatory properties by inhibiting PDE4 subtype. In conclusion, reverse pharmacognosy and its inverse docking component cannot only be integrated into a program for new lead discovery but is also a useful approach to find new applications for identified compounds.