Promotion of quality standard of Chinese herbal medicine by the integrated and efficacy-oriented quality marker of Effect-constituent Index.

BACKGROUND: Multiple constituents have been applied currently as markers to control the quality of Chinese herbal medicine (CHM). However, those constituents are isolated from each other, failed to present their contribution differences to the bioeffect of CHM. Besides, a CHM for different clinic uses is often controlled by the same quality marker (Q-marker), which cannot correlate its efficacies differentially. PURPOSE: The study aims to promote the quality standard of CHM by the integrated and efficacy-oriented Q-marker of Effect-constituent Index (ECI). METHODS: With Coptidis Rhizoma (C. Rhizoma) as a case study, the Q-marker of ECI based on the integration of bioeffect and active constituents was developed. According to the efficacies of C. Rhizoma, we investigated its antibacterial and antineoplastic effects by microcalorimetry and MTT assay, respectively. High performance liquid chromatography was performed to determine the active constituents of C. Rhizoma extract simultaneously. ECIS of inhibition on Shigella dysenteriae (S. dysenteriae) and ECIH of inhibition on HepG2 cells were established by multi-indicator synthetic evaluation method. The organoleptic evaluation scores of C. Rhizoma samples were given by Delphi method. RESULTS: The correlation analysis showed that ECIS and ECIH were significantly correlated with the inhibiting effects of C. Rhizoma extract on the growth of S. dysenteriae (P < 0.01) and proliferation of HepG2 cells (P < 0.01), respectively. Moreover, ECI showed a good ability to distinguish and predict the bioeffect-based quality grade, whereas the organoleptic evaluation and chemical analysis failed to achieve it. Plus, some samples with lower ECIS showed higher ECIH and vice versa. CONCLUSIONS: The Q-marker of ECI is useful to associate different pharmacologic effects of C. Rhizoma containing multiple active constituents, which is beneficial for the improvement of quality standard of the CHM in an integrated, convenient, and differentiated way.

Tylophorine Analogs Allosterically Regulates Heat Shock Cognate Protein 70 And Inhibits Hepatitis C Virus Replication.

Tylophorine analogs have been shown to exhibit diverse activities against cancer, inflammation, arthritis, and lupus in vivo. In this study, we demonstrated that two tylophorine analogs, DCB-3503 and rac-cryptopleurine, exhibit potent inhibitory activity against hepatitis C virus (HCV) replication in genotype 1b Con 1 isolate. The inhibition of HCV replication is at least partially mediated through cellular heat shock cognate protein 70 (Hsc70). Hsc70 associates with the HCV replication complex by primarily binding to the poly U/UC motifs in HCV RNA. The interaction of DCB-3503 and rac-cryptopleurine with Hsc70 promotes the ATP hydrolysis activity of Hsc70 in the presence of the 3' poly U/UC motif of HCV RNA. Regulating the ATPase activity of Hsc70 may be one of the mechanisms by which tylophorine analogs inhibit HCV replication. This study demonstrates the novel anti-HCV activity of tylophorine analogs. Our results also highlight the importance of Hsc70 in HCV replication.

Study of Malformin C, a Fungal Source Cyclic Pentapeptide, as an Anti-Cancer Drug.

Malformin C, a fungal cyclic pentapeptide, has been claimed to have anti-cancer potential, but no in vivo study was available to substantiate this property. Therefore, we conducted in vitro and in vivo experiments to investigate its anti-cancer effects and toxicity. Our studies showed Malformin C inhibited Colon 38 and HCT 116 cell growth dose-dependently with an IC50 of 0.27±0.07µM and 0.18±0.023µM respectively. This inhibition was explicated by Malformin C's effect on G2/M arrest. Moreover, we observed up-regulated expression of phospho-histone H2A.X, p53, cleaved CASPASE 3 and LC3 after Malformin C treatment, while the apoptosis assay indicated an increased population of necrotic and late apoptotic cells. In vivo, the pathological study exhibited the acute toxicity of Malformin C at lethal dosage in BDF1 mice might be caused by an acute yet subtle inflammatory response, consistent with elevated IL-6 in the plasma cytokine assay. Further anti-tumor and toxicity experiments proved that 0.3mg/kg injected weekly was the best therapeutic dosage of Malformin C in Colon 38 xenografted BDF1 mice, whereas 0.1mg/kg every other day showed no effect with higher resistance, and 0.9mg/kg per week either led to fatal toxicity in seven-week old mice or displayed no advantage over 0.3mg/kg group in nine-week old mice. Overall, we conclude that Malformin C arrests Colon 38 cells in G2/M phase and induces multiple forms of cell death through necrosis, apoptosis and autophagy. Malformin C has potent cell growth inhibition activity, but the therapeutic index is too low to be an anti-cancer drug.

Function-oriented development of CXCR4 antagonists as selective human immunodeficiency virus (HIV)-1 entry inhibitors.

Motivated by the pivotal role of CXCR4 as an HIV entry co-receptor, we herein report a de novo hit-to-lead effort on the identification of subnanomolar purine-based CXCR4 antagonists against HIV-1 infection. Compound 24, with an EC50 of 0.5 nM against HIV-1 entry into host cells and an IC50 of 16.4 nM for inhibition of radioligand stromal-derived factor-1α (SDF-1α) binding to CXCR4, was also found to be highly selective against closely related chemokine receptors. We rationalized that compound 24 complementarily interacted with the critical CXCR4 residues that are essential for binding to HIV-1 gp120 V3 loop and subsequent viral entry. Compound 24 showed a 130-fold increase in anti-HIV activity compared to that of the marketed CXCR4 antagonist, AMD3100 (Plerixafor), whereas both compounds exhibited similar potency in mobilization of CXCR4(+)/CD34(+) stem cells at a high dose. Our study offers insight into the design of anti-HIV therapeutics devoid of major interference with SDF-1α function.

Correction: Virtual screening and optimization of Type II inhibitors of JAK2 from a natural product library.

Correction for 'Virtual screening and optimization of Type II inhibitors of JAK2 from a natural product library' by Dik-Lung Ma et al., Chem. Commun., 2014, 50, 13885-13888.

Virtual screening and optimization of Type II inhibitors of JAK2 from a natural product library.

Amentoflavone has been identified as a JAK2 inhibitor by structure-based virtual screening of a natural product library. In silico optimization using the DOLPHIN model yielded analogues with enhanced potency against JAK2 activity and HCV activity in cellulo. Molecular modeling and kinetic experiments suggested that the analogues may function as Type II inhibitors of JAK2.

Cryptopleurine analogs with modification of e ring exhibit different mechanism to rac-cryptopleurine and tylophorine.

Tylophorine analogs exhibit a broad range of pharmacological activities, including anti-cancer, anti-inflammatory, anti-autoimmune, and anti-virus effects. Structure-activity relationship study of different structure tylophorine analogs can provide further understanding of their biological activity. Modifications on the E ring of the quinolizidine moiety of cryptopleurine analogs changed the potency and the selective inhibitory effect on NF-κB, AP-1, and CRE signaling pathways. Functional cryptopleurine analogs showed potent inhibition of NF-κB signaling pathway in both HepG2 and HEK-293 cell lines. The E ring structure analogs also differed in suppression of protein translation, and expression of cyclin D1. Our results showed that DCB-3503 or Rac-cryptopleurine could be a scaffold for modification to yield compounds with different mechanisms of action.

Antitumor agents 294. Novel E-ring-modified camptothecin-4ß-anilino-4'-O-demethyl-epipodophyllotoxin conjugates as DNA topoisomerase I inhibitors and cytotoxic agents.

Two conjugates (1 and 2) of camptothecin (CPT) and 4ß-anilino-4'-O-demethylepipodophyllotoxin were previously shown to exert antitumor activity through inhibition of topoisomerase I (topo I). In this current study, two novel conjugates (1E and 2E) with an open E-ring in the CPT moiety were first synthesized and evaluated for biological activity in comparison with their intact E-ring congeners. This novel class of CPT-derivatives exhibits its antitumor effect against CPT-sensitive and -resistant cells, in part, by inhibiting topo I-linked DNA (TLD) religation. An intact E-ring was not essential for the inhibition of TLD religation, although conjugates with an open E-ring were less potent than the closed ring analogs. This lower religation potency resulted in decreased formation of protein-linked DNA breaks (PLDBs), and hence, less cell growth inhibition. In addition to their impact on topo I, conjugates 1E, 2, and 2E exhibited a minor inhibitory effect on topo II-induced DNA cleavage. The novel structures of 1E and 2E may present scaffolds for further development of dual function topo I and II inhibitors with improved pharmacological profiles and physicochemical properties.

Analysis of deoxyribonucleotide pools in human cancer cell lines using a liquid chromatography coupled with tandem mass spectrometry technique.

Endogenous ribonucleotides and deoxyribonucleotides play a critical role in cell function, and determination of their levels is of fundamental importance in understanding key cellular processes involved in energy metabolism and molecular and biochemical signaling pathways. In this study, we determined the respective ribonucleotide and deoxyribonucleotide pool sizes in different human cell lines using a simple sample preparation method and LC/MS/MS. This assay was used to determine alterations in deoxyribonucleotide pools in human pancreatic PANC1 cells in response to hypoxia and to treatment with either hydroxyurea or aphidicolin. The levels of all deoxyribonucleotide metabolites decreased with hypoxia treatment, except for dUMP, which increased by two-fold. This LC/MS/MS assay is simple, fast, and sensitive, and it represents a significant advance over previously published methodologies.

Synthesis of 4'-Ethynyl-2'-deoxy-4'-thioribonucleosides and Discovery of a Highly Potent and Less Toxic NRTI.

The synthesis of 4'-ethynyl-2'-deoxy-4'-thioribonucleosides was carried out utilizing an electrophilic glycosidation in which 4-ethynyl-4-thiofuranoid glycal 16 served as a glycosyl donor. Electrophilic glycosidation between 16 and the silylated nucleobases (N4-acetylcytosine, N6-benzoyladenine and N2-acetyl-O6-diphenylcarbamoylguanine) was carried out in the presence of N-iodosuccinimide (NIS) leading to the exclusive formation of the desired ß-anomers 29, 33 and 36. Anti-HIV studies demonstrated that these 4'-thio nucleosides were less cytotoxic to T-lymphocyte (i.e. MT-4 cells) than the corresponding 4'-ethynyl derivatives of 2'-deoxycytidine (44), 2'-deoxyadenosine (45) and 2'-deoxyguanosine (46). Comparison of the selectivity indices (SI) was made between 4'-thionucleosides (32, 41 and 43) and the corresponding 4'-oxygen analogues 44-46 by using the reported CC50 and EC50 values. In the case of cytosine and adenine nucleosides, comparable SI values were obtained: 32 (545) and 45 (458); 41 (>230) and 45 (1,630). In contrast, 4'-ethynyl-2'-deoxy-4'-thioguanosine 43 was found to possess a SI value of >18,200, which is twenty times better than that of 46 (933).

The four-herb Chinese medicine PHY906 reduces chemotherapy-induced gastrointestinal toxicity.

PHY906, a four-herb Chinese medicine formula first described 1800 years ago, decreases gastrointestinal toxicity induced by the chemotherapeutic drug CPT-11 (irinotecan), as shown in a phase I/II clinical study. Similarly, in a murine colon 38 allograft model, PHY906 increased the antitumor activity of CPT-11 while decreasing animal weight loss caused by CPT-11. Here, we have further examined the effect of PHY906 on the intestinal toxicity caused by CPT-11 in mice. PHY906 did not protect against the initial DNA damage and apoptosis triggered by CPT-11 in the intestine, but by 4 days after CPT-11 treatment, PHY906 had restored the intestinal epithelium by promoting the regeneration of intestinal progenitor or stem cells and several Wnt signaling components. PHY906 also potentiated Wnt3a activity in human embryonic kidney-293 cells. Furthermore, PHY906 exhibited anti-inflammatory effects in mice by decreasing the infiltration of neutrophils or macrophages, tumor necrosis factor-alpha expression in the intestine, and proinflammatory cytokine concentrations in plasma. Chemical constituents of PHY906 potently inhibited nuclear factor kappaB, cyclooxygenase-2, and inducible nitric oxide synthase. Our results show that the herbal medicine PHY906 can counteract the toxicity of CPT-11 via several mechanisms that act simultaneously.

Discovery of a synthetic dual inhibitor of HIV and HCV infection based on a tetrabutoxy-calix[4]arene scaffold.

A potential anti-HIV and HCV drug candidate is highly desirable as coinfection has become a worldwide public health challenge. A potent compound based on a tetrabutoxy-calix[4]arene scaffold that possesses dual inhibition for both HIV and HCV is described. Structural activity relationship studies demonstrate the effects of lower-rim alkylation in maintaining cone conformation and upper-rim interacting head groups on the calix[4]arene play key roles for its potent dual antiviral activities.

A novel class of meso-tetrakis-porphyrin derivatives exhibits potent activities against hepatitis C virus genotype 1b replicons in vitro.

Recent years have seen the rapid advancement of new therapeutic agents against hepatitis C virus (HCV) in response to the need for treatment that is unmet by interferon (IFN)-based therapies. Most antiviral drugs discovered to date are small molecules that modulate viral enzyme activities. In the search for highly selective protein-binding molecules capable of disrupting the viral life cycle, we have identified a class of anionic tetraphenylporphyrins as potent and specific inhibitors of the HCV replicons. Based on the structure-activity relationship studies reported herein, meso-tetrakis-(3,5-dicarboxy-4,4'-biphenyl) porphyrin was found to be the most potent inhibitor of HCV genotype 1b (Con1) replicon systems but was less effective against the genotype 2a (JFH-1) replicon. This compound induced a reduction of viral RNA and protein levels when acting in the low nanomolar range. Moreover, the compound could suppress replicon rebound in drug-treated cells and exhibited additive to synergistic effects when combined with protease inhibitor BILN 2061 or with IFN-alpha-2a. Our results demonstrate the potential use of tetracarboxyphenylporphyrins as potent anti-HCV agents.

Butyrate mediates nucleotide-binding and oligomerisation domain (NOD) 2-dependent mucosal immune responses against peptidoglycan.

The interaction between digestive tract microbiological flora and food has an important influence on human health. Butyrate is produced during the fermentation of dietary fibres by intestinal bacteria and plays an important role in the regulation of mucosal immunity. In this report, we studied the impact of butyrate on the defence mechanism against the bacterial membrane component peptidoglycan (PGN). Butyrate was found to enhance PGN-mediated IL-8 and GRO-alpha production. The expression of these chemokines required the activation of NF-kappaB and was dependent on the concentrations of butyrate and PGN. Butyrate was found to up-regulate nucleotide-binding and oligomerisation domain (NOD) 2, but not NOD1 or TLR2. NOD2 up-regulation was mediated by an increase in histone acetylation in the Nod2 promoter region, leading to enhanced PGN-induced IL-8 and GRO-alpha secretion. Knockdown of NOD2 and TLR2 by siRNA significantly reduced PGN-mediated chemokine production, suggesting that both NOD2 and TLR2 are required for maximal response. Our findings provide a better understanding of the mechanism by which butyrate regulates mucosal immunity for normal intestinal function. Based on the results of this study, we infer that dietary fibres can impact inflammatory bowel diseases.

Synthesis and anti-HIV activity of 4'-C-ethynyl-2'-deoxy-4'-thio-nucleosides.

Synthesis of 4'-C-ethynyl-2'-deoxy-4'-thionucleosides was carried out based on electrophilic glycosidation using 4-C-ethynyl-4-thiofuranoid glycal. The glycal 15 was prepared as follows: oxidative cleavage of 6 with Pb(OAc)(4) forming the aldehyde 7, aldol reaction of 7 and subsequent silylation to furnish 8, conversion of the formyl group of 8 into an ethynyl group, and finally beta-elimination of the resulting 14 with t-BuLi. The glycosyl donor 16 was prepared by silyl-protection of 15. Electrophilic glycosidation was performed between silylated N(4)-acetylcytosine and 16 in the presence of N-iodosuccinimide. Radical-mediated removal of the introduced iodine atom followed by deprotection gave 4'-C-ethynyl-2'-deoxy-4'-thiocytidine (18).

Impacts of baicalein analogs with modification of the 6th position of A ring on the activity toward NF-kappaB-, AP-1-, or CREB-mediated transcription.

The water extract of Scutellariae baicalensis Georgi (S. baicalensis) has potential anti-tumor and anti-inflammatory activities. A major flavonoid isolated from S. baicalensis, baicalein, was also found to have anti-tumor and anti-inflammatory activities. These biological activities could be due to their antioxidant action and/or effect on different signal transduction pathways. We investigated the effects of several baicalein analogs with a substitution of hydrogen of the hydroxyl group at the 6th position of A ring on three signal pathway mediated transcription (NF-kappaB, AP-1, and CREB) associated with inflammation and cancer growth. We found that the analogs with O-alkyl group of the different carbon chain length or O-benzyl activated NF-kappaB transcription without TNFalpha stimulation. Some of the analogs increased TNFalpha stimulated NF-kappaB transcription by two- to threefold. None of the analogs studied has major effect on AP-1 signal transduction with or without TPA stimulation. All of the analogs increased CREB transcription with forskolin stimulation up to twofold. However, they did not have a potent effect (less or about twofold activation) on intrinsic CREB signal transduction. The modification of baicalein at the 6th position of A ring was not correlated with change in these signal transduction pathways and cytotoxicity. Though, they are structural analogs, they are not functional analogs. Modification of baicalein at the 6th position could alter the specificity of action toward different cellular targets. Flavonoids could be chemophores in the development of drugs targeted at different signal transcriptional pathway.

Synthesis and anti-HIV activity of 4'-substituted 4'-thiothymidines: a new entry based on nucleophilic substitution of the 4'-acetoxy group.

Diacetoxylation of 1-(2,5-dideoxy-beta-L-glycero-pent-4-eno-4-thiofuranosyl)thymine (13) with Pb(OAc) 4 allowed introduction of an acetoxy leaving group to the 4'-position. Nucleophilic substitution of the resulting 4'-acetoxy derivative (14) with silicon reagents enabled us to prepare the 4'-phenylthio (17a), 4'-azido (18a), 4'-methoxy (20a), and 4'-allyl (21a) analogues of 4'-thiothymidine. 4'-Cyano ( 25a) and 4'-ethynyl (31) nucleosides were also synthesized from 3',5'-bis-O-TBDMS derivative (24). Among novel 4'-substituted 4'-thiothymidines, the 4'-azido (33), 4'-cyano (36), and 4'-ethynyl (37) derivatives were found to show potent inhibitory activity against HIV-1 and HIV-2. It is noteworthy that 36 and 37 were also inhibitory against replication of HIV variant resistant to 3TC (HIV-1 M184V), being as potent as against HIV-1 IIIB.

Structural analogs of tylophora alkaloids may not be functional analogs.

Phenanthroindolizidine-based tylophora alkaloids have been reported to have potential antitumor, anti-immuno and, anti-inflammatory activity. The structure-activity relationships of a series of tylophora alkaloids were studied to guide future drug design. Our results indicate that although these compounds are structural analogs, their potency of cytotoxicity, selectivity against NF-kappaB signaling pathway, and their inhibitory effects against protein and nucleic acid synthesis are different. Because they do not have an identical spectrum of targets, the studied compounds are structural, but may not be functional analogs.

Structure-activity studies of phenanthroindolizidine alkaloids as potential antitumor agents.

Five phenanthroindolizidine alkaloids (PA) were chemically synthesized and seven were isolated from Tylophora atrofolliculata. To facilitate future drug design of phenanthroindolizidine alkaloids as potential antitumor agents, we have explored the structure-activity relationships (SAR) of this class of compounds. We demonstrated that DCB-3503 and tylophorinidine (PA-7) were among the most active compounds against tumor growth both in vitro and in vivo. In the hepatocellular carcinoma cell line HepG2, the GI(50)s of DCB-3503 and PA-7 were 35+/-5 nM and 11+/-5 nM, respectively. DCB-3503 and PA-7 significantly inhibited HepG2 tumor growth in nude mice at a dose of 9 mg/kg given by intraperitoneal (ip) injections twice a day every third day for a total of four cycles (P<0.05 for DCB-3503 and P<0.01 for PA-7). Their potent antitumor activities correlated with their potent NF-kappaB-inhibitory effects and their cyclin D1 down-regulatory effects.

Synthesis and biological evaluation of 2',3'-didehydro-2',3'-dideoxy-9-deazaguanosine, a monophosphate prodrug and two analogues, 2',3'-dideoxy-9-deazaguanosine and 2',3'-didehydro-2',3'-dideoxy-9-deazainosine.

2',3'-Didehydro-2',3'-dideoxy-9-deazaguanosine (1), its monophosphate prodrug (2), and two analogues, 2',3'-dideoxy-9-deazaguanosine (3) and 2',3'-didehydro-2',3'-dideoxy-9-deazainosine (4), have been synthesized from benzoylated 9-deazaguanosine (5). Basic hydrolysis of 5, selective protection of the 2-amino and 5'-hydroxy functions with isobutyryl and silyl groups, respectively, followed by reaction with thiocarbonyldiimidazole gave the cyclic thiocarbonate, which, upon reaction with triethyl phosphite, followed by deprotection, afforded 1. Treatment of 1 with phenyl methoxyalaninylphosphochloridate and N-methylimidazole gave 2. Catalytic hydrogenation of 1 gave 3. Hydrodediazoniation of 1 with tert-butyl nitrite and tris(trimethylsilyl)silane gave 4. Compounds 1-4 were found to be inactive against the human immunodeficiency virus and exhibited minimal to no cytotoxic activity against the L1210 leukemia, CCRF-CEM lymphoblastic leukemia, and B16F10 melanoma in vitro.