Discovery of a Novel HIV-1 Integrase/p75 Interacting Inhibitor by Docking Screening, Biochemical Assay, and in Vitro Studies.

Protein-protein interaction between lens epithelium-derived growth factor (LEDGF/p75) and HIV-1 integrase becomes an attractive target for anti-HIV drug development. The blockade of this interaction by small molecules could potentially inhibit HIV-1 replication. These small molecules are termed as LEDGINs; and several newly identified LEDGINs have been reported to significantly reduce HIV-1 replication. Through this project, we have finished the docking screening of the Maybridge database against the p75 binding site of HIV-1 integrase using both DOCK and Autodock Vina software. Finally, we have successfully identified a novel scaffold LEDGINs inhibitor DW-D-5. Its antiviral activities and anticatalytic activity of HIV-1 integrase are similar to other LEDGINs under development. We demonstrated that the combination of DW-D-5 and FDA approved anti-HIV drugs resulted in additive inhibitory effects on HIV-1 replication, indicating that DW-D-5 could be an important component of combination pills for clinic use in HIV treatment.

Herbalog: A tool for target-based identification of herbal drug efficacy through molecular docking.

BACKGROUND: Traditionally, molecular docking is primarily employed to screen pure compounds; the top-ranking chemicals are subsequently selected for experimental validation. Unlike synthetic chemicals, most natural products are commercially unavailable. The isolation and purification of each natural product is extremely time-consuming, which has restricted the screening of lead compounds from natural products. PURPOSE: We developed a protocol, Herbalog, to facilitate the identification of bioactive phytochemicals through molecular docking. METHODS: We wrote a script using Python and Autodock Vina for docking; ligand displacement and adipolysis assays were used to determine the anti-fatty acid binding protein (FABP) 4 activity of bioactive extracts. An ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometry system was applied for identifying major peaks of bioactive extracts. RESULTS: Herbalog, a natural product database, contains 5,112 phytochemicals from 197 common herbs and a script that counts the number of hits from docking in each herb and calculates the hit rate of herbs. Herbalog prioritizes herbs according to their hit rates, and top-ranking herb candidates contain a large repertoire of hits. We used Herbalog as a screening tool and identified labdane diterpenoids from Andrographis paniculata as leading candidates of FABP4 inhibitors. CONCLUSION: Herbalog facilitates the discovery of herbs that possess the highest number of inhibitors or activators against target proteins, which reduces the sample preparation time for preliminary validation.

Jujube-containing herbal decoctions induce neuronal differentiation and the expression of anti-oxidant enzymes in cultured PC12 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: The fruit of Ziziphus jujuba (Mill.), known as Jujuba Fructus (JF) or jujube, is a well-known Traditional Chinese Medicine (TCM) for blood nourishment and sedation effect. Apart from prescribing as single herb alone, JF is very often being included in multi-herbal decoctions to prolong, enhance and harmonize pharmaceutical effects of decoctions while at the same time reducing toxicity. Here, we aimed to compare the protective and differentiating activities of three chemically standardized jujube-containing decoctions, including Guizhi Tang (GZT), Neibu Dangguijianzhong Tang (NDT) and ZaoTang (ZOT) in cultured PC12 cells. MATERIALS AND METHODS: The protein expressions of neurofilaments, including NF68, NF160 and NF200, under the herbal treatment were revealed by western blot. The determination of neurite outgrowth in cultured PC12 cells upon the treatment of herbal extracts was performed by light microscope equipped with a phase-contrast condenser and SPOT imaging software. The protective effect against tBHP-induced cytotoxicity under the herbal treatment was measured by MTT assay. A luciferase reporter construct carrying four repeats of anti-oxidant response element (ARE) and a downstream luciferase reporter gene luc2P was transfected into PC12 cells to study the transcriptional activation of ARE. The mRNA expression of antioxidant enzymes under the herbal treatment was analyzed by quantitative real-time PCR. RESULTS: These jujube-containing decoctions processed similar neuro-protective and brain beneficial properties. The herbal treatment induced the protein expression of neurofilaments. Neurite outgrowth was observed under the herbal treatment. In parallel, the pre-treatment of herbal extracts protected PC 12 cells against oxidative stress-induced apoptosis in a dose-dependent manner. Moreover, the herbal treatments triggered the mRNA expressions of relevant anti-oxidation genes, i.e. glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase modulatory subunit (GCLM), glutathione S-transferase (GST) and NAD(P)H quinone oxidoreductase (NQO1) via the activation of anti-oxidant response element (ARE). CONCLUSION: The results therefore demonstrated neuro-protective and differentiating properties of the three closely related decoctions, and which subsequently illustrated the enhancement function of jujube within a multi-herbal decoction.

Authentication of Cordyceps sinensis by DNA Analyses: Comparison of ITS Sequence Analysis and RAPD-Derived Molecular Markers.

Cordyceps sinensis is an endoparasitic fungus widely used as a tonic and medicinal food in the practice of traditional Chinese medicine (TCM). In historical usage, Cordyceps specifically is referring to the species of C. sinensis. However, a number of closely related species are named themselves as Cordyceps, and they are sold commonly as C. sinensis. The substitutes and adulterants of C. sinensis are often introduced either intentionally or accidentally in the herbal market, which seriously affects the therapeutic effects or even leads to life-threatening poisoning. Here, we aim to identify Cordyceps by DNA sequencing technology. Two different DNA-based approaches were compared. The internal transcribed spacer (ITS) sequences and the random amplified polymorphic DNA (RAPD)-sequence characterized amplified region (SCAR) were developed here to authenticate different species of Cordyceps. Both approaches generally enabled discrimination of C. sinensis from others. The application of the two methods, supporting each other, increases the security of identification. For better reproducibility and faster analysis, the SCAR markers derived from the RAPD results provide a new method for quick authentication of Cordyceps.

Calycosin orchestrates the functions of Danggui Buxue Tang, a Chinese herbal decoction composing of Astragali Radix and Angelica Sinensis Radix: An evaluation by using calycosin-knock out herbal extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Danggui Buxue Tang (DBT) is a classical Chinese herbal decoction containing two herbs, Astragali Radix (AR) and Angelicae Sinensis Radix (ASR), which serves as dietary supplement for treating women menopausal syndromes. Pharmacological studies indicate that DBT has estrogenic, erythropoietic and osteogenic properties; however, the action mechanism for this complex herbal decoction is not known. Calycosin, a major flavonoid in AR, shares similar structure with ß-estradiol, and thus which is hypothesized to be the critical compound of DBT. Here, we aim to investigate the role of calycosin in DBT in terms of its biological functions by using a calycosin-depleted DBT decoction (DBT(Δcal)). The biological functions of DBT(Δcal) and parental DBT were systematically compared. MATERIALS AND METHODS: In order to standardize DBT decoction, four chemical markers were determined and quantified by HPLC. A semi-preparative HPLC method was utilized to prepare DBT(Δcal). The authenticity of DBT(Δcal) was evaluated by LC-QQQ-MS/MS. To reveal the effect of calycosin on DBT functions, several cell assays related to the known properties of DBT were revealed, including estrogenic, erythropoietic and osteogenic functions. RESULTS: As compared to parental DBT, the estrogenic, erythropoietic and osteogenic abilities were markedly reduced in DBT(Δcal). However, calycosin alone did not show significant responses. CONCLUSIONS: Our results suggest that calycosin is a bioactive chemical in DBT decoction, and which could play a key linker in orchestrating multi-components of DBT as to achieve maximal functions. These discoveries should be invaluable in drug development and in investigating the modernization of traditional Chinese medicine from a new perspective.

Herb-drug interaction between an anti-HIV Chinese herbal SH formula and atazanavir in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: With the prevalent use of highly active antiretroviral therapy (HAART) for AIDS patients since 1996, the mortality of HIV/AIDS patients has been remarkably decreased. With long-term use of HAART, drug resistance and side effects of antiretrovirals have been frequently reported, which not only reduce the efficacy, but also decreases the tolerance of patients. Traditional herbal medicine has become more popular among HIV/AIDS patients as adjuvant therapy to reduce these adverse effects of HAART. SH formula is a Chinese herbal formula consisting of five traditional Chinese herbs including Morus alba L., Glycyrrhiza glabra L., Artemisia capillaris Thumb., Astragalus membranaceus Bge., and Carthamus tinctorius L. SH formula is clinically used for HIV treatment in Thailand. However, the possible pharmacokinetic interactions between these Chinese herbs and antiretroviral drugs have not been well documented. The aim of this study was to investigate the potential herb-drug interaction between SH herbal Chinese formula and the antiretroviral drug atazanavir (ATV). MATERIALS AND METHODS: The combination effect of SH formula and ATV on HIV protease was studied in HIV-1 protease inhibition assay in vitro. The inhibition of SH formula on rat CYP3A2 was assessed by detecting the formation of 1'-OH midazolam from midazolam in rat liver microsomes in vitro. The in vivo pharmacokinetic interaction between SH formula and ATV was investigated by measuring time-dependent plasma concentrations of ATV in male Sprague-Dawley rats with liquid chromatography-mass spectrometry. RESULTS: Through the in vitro HIV-1 protease inhibition assay, combination of SH formula (41.7-166.7 µg/ml) and ATV (16.7-33.3 ng/ml) showed additive inhibition on HIV-1 protease activity than SH formula or ATV used alone. In vitro incubation assay indicated that SH formula showed a weak inhibition (IC50=231.2 µg/ml; Ki=98.2 µg/ml) on CYP3A2 activity in rat liver microsomes. In vivo pharmacokinetic study demonstrated that SH formula did not affect the metabolism of ATV in rats. CONCLUSIONS: Our study demonstrated for the first time that there is no metabolism-based herb-drug interaction between SH formula and ATV in rats, but this combination enhances the inhibition potentials against HIV protease activity. This observation may support the combinational use of anti-HIV treatment in human.

Structural characterization and immunomodulatory effect of a polysaccharide HCP-2 from Houttuynia cordata.

Immunomodulation of natural polysaccharides has been the hot topic of research in recent years. In order to explore the immunomodulatory effect of Houttuynia cordata Thunb., the water extract was studied and a polysaccharide HCP-2 with molecular weight of 60,000 Da was isolated by chromatography using DEAE Sepharose CL-6B and Sephacryl S-500 [corrected] HR columns. The structure characterization of HCP-2 was performed by Fourier transform infrared spectroscopy (FTIR), acidic hydrolysis, PMP derivation, HPLC analysis and nuclear magnetic resonance spectra (NMR). HCP-2 was elucidated as a pectic polysaccharide with a linear chain of 1,4-linked α-D-galacturonic acid residues in which part of the 6-carboxyl groups were methyl esterified and part of 2-hydroxyl groups were acetylated. The bioactivity assays showed that HCP-2 could increase the secretions of interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), macrophage inhibitory protein-1α (MIP-1α), macrophage inhibitory protein-1ß (MIP-1ß), and RANTES (regulated on activation, normal T cell expressed and secreted) in human peripheral blood mononuclear cells (PBMCs), which play critical roles in the innate immune system and shape the adaptive immunity. Our results implied that HCP-2 could be an immune enhancer.

Interaction study of two diterpenes, cryptotanshinone and dihydrotanshinone, to human acetylcholinesterase and butyrylcholinesterase by molecular docking and kinetic analysis.

Alzhemier's disease (AD) is a common form of dementia in the ageing population which is characterized by depositions of amyloids and a cholinergic neurotransmission deficit in the brain. Current therapeutic intervention for AD is primarily based on the inhibition of brain acetylcholinesterase (AChE) to restore the brain acetylcholine level. Cryptotanshinone (CT) and dihydrotanshinone (DT) were diterpenoids extracted from Salvia miltiorrhiza Bge. having anti-cholinesterase activity. Here we characterized the inhibition property of these two diterpenoids towards human AChE and butyrylcholinesterase (BChE). Both CT and DT were found to be mixed non-competitive inhibitors for human AChE and an uncompetitive inhibitor for human BChE. The docking analyses of CT and DT into the active sites of both cholinesterases indicate that they interact with the allosteric site inside the active-site gorge mainly by hydrophobic interactions.

Cryptotanshinone, an acetylcholinesterase inhibitor from Salvia miltiorrhiza, ameliorates scopolamine-induced amnesia in Morris water maze task.

Alzheimer's disease (AD) is a common form of dementia which is characterized by the deposition of amyloids in affected neurons and a cholinergic neurotransmission deficit in the brain. The current therapeutic intervention for AD is primarily based on the inhibition of brain acetylcholinesterase (AChE) to restore the brain acetylcholine level. Cryptotanshinone (CT) is a diterpene extracted from the root of Salvia miltiorrhiza, a herb that is commonly prescribed in Chinese medicine to treat cardiovascular disease. In the present study, we demonstrated that CT is an inhibitor of both human acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) with IC(50) values of 4.09 and 6.38 microM, respectively. The IC(50) ratio of CT for BuChE:AChE was 1.56. CT inhibited human AChE in a reversible manner, and the inhibition showed the characteristics of mixed-type as both the KM and V(max) were affected by CT. The effect of CT on learning impairment in scopolamine-treated rats was also evaluated by the acquisition protocol of the Morris water maze. The task learning ability of scopolamine-treated rats was significantly reversed by CT (5 mg/kg), and the CT-fed rats were able to develop a spatial searching strategy comparable to that of the control animals. In addition, chronic CT treatment did not cause hepatotoxicity as measured by blood alanine transferase (ALT) level. Our findings demonstrate the ability of CT to improve task learning in rats with scopolamine-induced cognitive impairment. These results suggest that CT has the potential as a therapeutic drug for treating AD.