Study on the chemical constituents and mechanism of Kai-Xin-San based on UPLC-Q-Exactive MS and network pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Vascular disease (VD) is a kind of common disease harmful to the health of the middle-aged and elderly, which has the characteristics of long treatment cycle and high recurrence rate, and without effective method to treat so far. Traditional Chinese medicine (TCM) has the characteristics of multi-components and multi-targets to treat diseases. Kai-Xin-San is a TCM formula applied for treating psychiatric diseases such as depression in China for thousands of years, and it has been used in clinical treatment of VD. But up to now, its active composition and mechanism are not clear. AIM OF THE STUDY: To explore the effective components of Kai-Xin-San, investigate the effect of Kai-Xin-San on angiogenesis, screen and verify the related targets and possible mechanisms of Kai-Xin-San against VD. MATERIALS AND METHODS: UPLC-Q-Exactive Orbitrap MS was performed to identify the chemical components of Kai-Xin-San. The mechanism of multi-components, multi-targets, and multi-pathways of Kai-Xin-San in the treatment of VD were explored by network pharmacology. And then, quail chick chorioallantoic membrane (qCAM) assays were used to evaluate the vascular protective activity of Kai-Xin-San. Evaluation of angiogenesis by calculating the relative vessels area. The levels of VEGFA and Akt1 in qCAM were measured by RT-PCR. Twenty-five male SD rats were randomly divided into the sham group, model group, Donepezil (0.45 mg/kg) group, Kai-Xin-San low dose group (0.1575 g/kg), Kai-Xin-San high dose group (0.63 g/kg). Two-vessel occlusion (2-VO) rat model is established to evaluate the therapeutic effect of Kai-Xin-San pretreatment. Hematoxylin-eosin (HE) staining is conducted to detect the morphological changes of neurons in the hippocampus. RESULTS: Data showed that 62 compounds were identified in Kai-Xin-San. The network pharmacology results showed 73 compounds in Kai-Xin-San play a role in the treatment of VD, such as Ginsenoside Rh4, kaempferol, and Poricoic acid C. A total of 7 main targets are predicted, including Akt1, TNF and so on. Kai-Xin-San could increase VEGFA and Akt1 expression, promote angiogenesis and regulate the PI3K-Akt signaling pathway. The results depict that Kai-Xin-San has dose-dependently improved the cognitive function in 2-VO model rats. It has also been showed that Kai-Xin-San can rescue neuron damage in the hippocampus. CONCLUSION: The complex chemical components of Kai-Xin-San play a synergistic role in the treatment of VD, and involve multiple pathways and targets. To protect blood vessels by promoting angiogenesis is one of the potential mechanisms of Kai-Xin-San in the treatment of VD. This study reveals that Kai-Xin-San protects the 2-VO model rats from ischemic injury by alleviating neuron damage in the hippocampus.

Multi-Omics Analysis Reveals the Gut Microbiota Characteristics of Diarrheal Piglets Treated with Gentamicin.

The involvement of alterations in gut microbiota composition due to the use of antibiotics has been widely observed. However, a clear picture of the influences of gentamicin, which is employed for the treatment of bacterial diarrhea in animal production, are largely unknown. Here, we addressed this problem using piglet models susceptible to enterotoxigenic Escherichia coli (ETEC) F4, which were treated with gentamicin. Gentamicin significantly alleviated diarrhea and intestinal injury. Through 16s RNS sequencing, it was found that gentamicin increased species richness but decreased community evenness. Additionally, clear clustering was observed between the gentamicin-treated group and the other groups. More importantly, with the establishment of a completely different microbial structure, a novel metabolite composition profile was formed. KEGG database annotation revealed that arachidonic acid metabolism and vancomycin resistance were the most significantly downregulated and upregulated pathways after gentamicin treatment, respectively. Meanwhile, we identified seven possible targets of gentamicin closely related to these two functional pathways through a comprehensive analysis. Taken together, these findings demonstrate that gentamicin therapy for diarrhea is associated with the downregulation of arachidonic acid metabolism. During this process, intestinal microbiota dysbiosis is induced, leading to increased levels of the vancomycin resistance pathway. An improved understanding of the roles of these processes will advance the conception and realization of new therapeutic and preventive strategies.

Novel Natural Carrier-Free Self-Assembled Nanoparticles for Treatment of Ulcerative Colitis by Balancing Immune Microenvironment and Intestinal Barrier.

Ulcerative colitis (UC) is a chronic inflammatory illness affecting the colon and rectum, with current treatment methods being unable to meet the clinical needs of ulcerative colitis patients. Although nanomedicines are recognized as promising anti-inflammatory medicines, their clinical application is limited by their high cost and unpredictable safety risks. This study reveals that two natural phytochemicals, berberine (BBR) and hesperetin (HST), self-assemble directly to form binary carrier-free multi-functional spherical nanoparticles (BBR-HST NPs) through noncovalent bonds involving electrostatic interactions, π-π stacking, and hydrogen bonding. Because of their synergistic anti-inflammatory activity, berberine-hesperetin nanoparticles (BBR-HST NPs) exhibit significantly better therapeutic effects on UC and inhibitory effects on inflammation than BBR and HST at the same dose by regulating the immune microenvironment and repairing the damaged intestinal barrier. Furthermore, BBR-HST NPs exhibit good biocompatibility and biosafety. Thus, this study proves the potential of novel natural anti-inflammatory nanoparticles as therapeutic agents for UC, which could promote the progress of drug development for UC and eventually benefit patients who suffering from it.

Immunomodulatory Effects of Microcin C7 in Cyclophosphamide-Induced Immunosuppressed Mice.

Microcin C7 (McC) as a viable immunomodulator peptide can be a potential solution for pathogenic microbial infection in the post-antibiotic era and has gained substantial attention. This study was designed to evaluate the immunomodulatory activity of Microcin C7 in a cyclophosphamide (CTX)-induced immunodeficient mouse model. We show that Microcin C7 treatment significantly alleviated the CTX-caused body weight loss, improved the feed and water consumption to improve the state of the mice, and elevated the absolute number and proportion of peripheral blood lymphocytes as well as the level of hemoglobulin. We further aim to characterize the phenotypes of the immune function and intestinal health profiles. The results demonstrate that Microcin C7 treatment increased serum levels of immunoglobulin A (IgA), IgG, interleukin 6, and hemolysin, promoted splenic lymphocyte proliferation induced by concanavalin A and LPS, and enhanced the phagocytosis of peritoneal macrophages immunized by sheep red blood cells. Additionally, Microcin C7 treatment decreased levels of diamine oxidase and d-lactate, ameliorated CTX-induced intestinal morphological damage, and increased the levels of zonula occluden 1, occludin, claudin-1, mucin 2, and secretary IgA in the jejunum and colon. Moreover, Microcin C7 administration is sufficient to reverse CTX-induced intestinal microbiota dysbiosis by increasing the number of Lactobacillus and Bifidobacterium, decreasing the number of Escherichia coli in colonic contents. Collectively, our results demonstrate that Microcin C7 may have protective and immunomodulatory functions and could be a potential candidate used in animal feed, functional foods, and immunological regimens..

[Therapeutic effect of ursodeoxycholic acid-berberine supramolecular nanoparticles on ulcerative colitis based on supramolecular system induced by weak bond].

Ulcerative colitis(UC) is a recurrent, intractable inflammatory bowel disease. Coptidis Rhizoma and Bovis Calculus, serving as heat-clearing and toxin-removing drugs, have long been used in the treatment of UC. Berberine(BBR) and ursodeoxycholic acid(UDCA), the main active components of Coptidis Rhizoma and Bovis Calculus, respectively, were employed to obtain UDCA-BBR supramolecular nanoparticles by stimulated co-decocting process for enhancing the therapeutic effect on UC. As revealed by the characterization of supramolecular nanoparticles by field emission scanning electron microscopy(FE-SEM) and dynamic light scattering(DLS), the supramolecular nanoparticles were tetrahedral nanoparticles with an average particle size of 180 nm. The molecular structure was described by ultraviolet spectroscopy, fluorescence spectroscopy, infrared spectroscopy, high-resolution mass spectrometry, and hydrogen-nuclear magnetic resonance(H-NMR) spectroscopy. The results showed that the formation of the supramolecular nano-particle was attributed to the mutual electrostatic attraction and hydrophobic interaction between BBR and UDCA. Additionally, supramolecular nanoparticles were also characterized by sustained release and pH sensitivity. The acute UC model was induced by dextran sulfate sodium(DSS) in mice. It was found that supramolecular nanoparticles could effectively improve body mass reduction and colon shortening in mice with UC(P<0.001) and decrease disease activity index(DAI)(P<0.01). There were statistically significant differences between the supramolecular nanoparticles group and the mechanical mixture group(P<0.001, P<0.05). Enzyme-linked immunosorbent assay(ELISA) was used to detect the serum levels of tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6), and the results showed that supramolecular nanoparticles could reduce serum TNF-α and IL-6 levels(P<0.001) and exhibited an obvious difference with the mechanical mixture group(P<0.01, P<0.05). Flow cytometry indicated that supramolecular nanoparticles could reduce the recruitment of neutrophils in the lamina propria of the colon(P<0.05), which was significantly different from the mechanical mixture group(P<0.05). These findings suggested that as compared with the mechanical mixture, the supramolecular nanoparticles could effectively improve the symptoms of acute UC in mice. The study provides a new research idea for the poor absorption of small molecules and the unsatisfactory therapeutic effect of traditional Chinese medicine and lays a foundation for the research on the nano-drug delivery system of traditional Chinese medicine.

Anti-tumor effects of novel alkannin derivatives with potent selectivity on comprehensive analysis.

BACKGROUND: Therapeutic approaches based on glycolysis and energy metabolism of tumor cells are new promising strategies for the treatment of cancer. Currently, researches on the inhibition of pyruvate kinase M2, a key rate limiting enzyme in glycolysis, have been corroborated as an effective cancer therapy. Alkannin is a potent pyruvate kinase M2 inhibitor. However, its non-selective cytotoxicity has affected its subsequent clinical application. Thus, it needs to be structurally modified to develop novel derivatives with high selectivity. PURPOSE: Our study aimed to ameliorate the toxicity of alkannin through structural modification and elucidate the mechanism of the superior derivative 23 in lung cancer therapy. METHODS: On the basis of the principle of collocation, different amino acids and oxygen-containing heterocycles were introduced into the hydroxyl group of the alkannin side chain. We examined the cell viability of all derivatives on three tumor cells (HepG2, A549 and HCT116) and two normal cells (L02 and MDCK) by MTT assay. Besides, the effect of derivative 23 on the morphology of A549 cells as observed by Giemsa and DAPI staining, respectively. Flow cytometry was performed to assess the effects of derivative 23 on apoptosis and cell cycle arrest. To further assess the effect of derivative 23 on the Pyruvate kinase M2 in glycolysis, an enzyme activity assay and western blot assay were performed. Finally, in vivo the antitumor activity and safety of the derivative 23 were evaluated by using Lewis mouse lung cancer xenograft model. RESULTS: Twenty-three novel alkannin derivatives were designed and synthesized to improve the cytotoxicity selectivity. Among these derivatives, derivative 23 showed the highest cytotoxicity selectivity between cancer and normal cells. The anti-proliferative activity of derivative 23 on A549 cells (IC50 = 1.67 ± 0.34 µM) was 10-fold higher than L02 cells (IC50 = 16.77 ± 1.44 µM) and 5-fold higher than MDCK cells (IC50 = 9.23 ± 0.29 µM) respectively. Subsequently, fluorescent staining and flow cytometric analysis showed that derivative 23 was able to induce apoptosis of A549 cells and arrest the cell cycle in the G0/G1 phase. In addition, the mechanistic studies suggested derivative 23 was an inhibitor of pyruvate kinase; it could regulate glycolysis by inhibiting the activation of the phosphorylation of PKM2/STAT3 signaling pathway. Furthermore, studies in vivo demonstrated derivative 23 significantly inhibited the growth of xenograft tumor. CONCLUSION: In this study, alkannin selectivity is reported to be significantly improved following structural modification, and derivative 23 is first shown to be able to inhibit lung cancer growth via the PKM2/STAT3 phosphorylation signaling pathway in vitro, indicating the potential value of derivative 23 in treating lung cancer.

Explore bioactive ingredients and potential mechanism of Houpo Mahuang decoction for chronic bronchitis based on UHPLC-Q exactive orbitrap HRMS, network pharmacology, and experiment verification.

ETHNOPHARMACOLOGICAL RELEVANCE: Chronic bronchitis (CB) affects a growing number of people and may be linked to lung function impairment. The traditional Chinese medicine formula Houpo Mahuang Decoction (HPMHD) has been used for clinical treatment of respiratory diseases for thousands of years. Until now, its bioactive ingredients, potential targets and molecular mechanism remain unclear. AIM OF THE STUDY: To investigate the effect of HPMHD on the treatment of CB and explore the bioactive ingredients and possible mechanisms of HPMHD against CB. MATERIALS AND METHODS: UHPLC-Q Exactive Orbitrap HRMS was performed to analyze the chemical components of HPMHD. The mechanism of multiple components, targets and pathways of HPMHD in the treatment of chronic bronchitis were explored by network pharmacology. Additionally, CB mice model induced by lipopolysaccharide (LPS) and smoking was used to evaluate the anti-chronic bronchitis activity of HPMHD in vivo. Pulmonary pathology was determined by hematoxylin and eosin (H&E) measurement. The levels of TNF-α and IL-6 in lung were measured by ELISA. The immunofluorescence experiments were carried out for the expression of IL-1ß, TNF-α, IL-6 and NF-κB p-P65/P65 in lung. Western blot assays were performed to quantify and visualize the protein expression of NF-κB p-P65/P65 in mice lung. RESULTS: Data showed that 79 compounds were identified in HPMHD. The network pharmacology results showed 53 compounds were hinted their effectivity for the treatment of chronic bronchitis with HPMHD, such as ephedrine, schisantherin A, and honokiol. The main targets were predicted as 37 genes, including TNF, TP53, IL6 and so on. HPMHD ameliorated lung damages in mice and inhibited the NF-κB signaling pathway, one of the pathways plotted by KEGG pathway enrichment analysis, by reducing IL-1ß, TNF-α and IL-6 expression and significantly downregulating the NF-κB p-P65/P65. CONCLUSION: In summary, the complex chemical components of HPHMD was successfully elucidate by UHPLC-Q Exactive Orbitrap HRMS. The study based on network pharmacology and experiment verification indicated that HPMHD can decreased inflammatory response in lung to treat CB. The underlying mechanism may be related to the reduction of inflammation by down-regulated the NF-κB pathways.

Synthesis and biological evaluation of novel sinomenine derivatives as anti-inflammatory and analgesic agent.

Sinomenine (SIN) has long been known as an anti-inflammatory drug, while poor efficiency and large-dose treatment had limited its further application. A series of novel SIN derivatives 1-26 were designed and synthesized to improve its anti-inflammatory activity. The anti-inflammatory activity evaluation showed most of the derivatives exhibited enhanced anti-inflammatory activity in vitro compared to SIN. Compound 17 significantly inhibited LPS-induced secretion of pro-inflammatory factors NO (IC50 = 30.28 ± 1.70 µM), and suppressed the expression of iNOS, IL-6 and TNF-α in RAW264.7 cells. Moreover, compound 17 showed excellent anti-inflammatory in mouse paw edema. Immunohistochemistry results revealed that compound 17 exerted anti-inflammatory activity by inhibiting the pro-inflammatory cytokine TNF-α. Furthermore, compound 17 exhibited an analgesic effect in vivo. The results attained in this study indicated that compound 17 had the potential to be developed into an anti-inflammation and analgesic agent.

Effects of robot-assisted therapy on upper limb and cognitive function in patients with stroke: study protocol of a randomized controlled study.

BACKGROUND: Impairments in upper limb motor function and cognitive ability are major health problems experienced by stroke patients, necessitating the development of novel and effective treatment options in stroke care. The aim of this study is to examine the effects of robot-assisted therapy on improving upper limb and cognitive functions in stroke patients. METHODS: This will be a single-blinded, 2-arm, parallel design, randomized controlled trial which will include a sample size of 86 acute and subacute stroke patients to be recruited from a single clinical hospital in Shanghai, China. Upon qualifying the study eligibility, participants will be randomly assigned to receive either robot-assisted therapy or conventional therapy with both interventions being conducted over a 6-week period in a clinical rehabilitation setting. In addition to comprehensive rehabilitation, the robot-assisted therapy group will receive a 30-min Armguider robot-assisted therapy intervention 5 days a week. Primary efficacy outcomes will include Fugl-Meyer Assessment for Upper Extremity (FMA-UE) and Mini-Mental Status Examination (MMSE). Other secondary outcomes will include Trail Making Test (TMT), Auditory Verbal Learning Test (AVLT), Digit Symbol Substitution Test (DSST), and Rey-Osterrieth Complex Figure Test (ROCFT). All trial outcomes will be assessed at baseline and at 6-week follow-up. Intention-to-treat analyses will be performed to examine changes from baseline in the outcomes. Adverse events will be monitored throughout the trial period. DISCUSSION: This will be the first randomized controlled trial aimed at examining the effects of robot-assisted therapy on upper limb and cognitive functions in acute and subacute stroke patients. Findings from the study will contribute to our understanding of using a novel robotic rehabilitation approach to stroke care and rehabilitation. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR2100050856 . Registered on 5 September 2021.

Microbiota Transplantation in an Antibiotic-Induced Bacterial Depletion Mouse Model: Reproducible Establishment, Analysis, and Application.

The fecal bacteria transplantation (FMT) technique is indispensable when exploring the pathogenesis and potential treatments for microbiota-related diseases. For FMT clinical treatments, there are already systematic guidelines for donor selection, fecal bacterial separation, FMT frequency, and infusion methods. However, only a few studies have demonstrated the use of standardized FMT procedures for animal models used in theoretical research, creating difficulties for many new researchers in this field. In the present paper, we provide a brief overview of FMT and discuss its contribution to the current understanding of disease mechanisms that relate to microbiota. This protocol can be used to generate a commonly used FMT mouse model and provides a literature reference of customizable steps.

Biosynthetic Microcin J25 Exerts Strong Antibacterial, Anti-Inflammatory Activities, Low Cytotoxicity Without Increasing Drug-Resistance to Bacteria Target.

Multidrug resistant (MDR) bacterial infection has emerged, raising concerns about untreatable infections, and posing the highest health risks. Antimicrobial peptides (AMPs) are thought to be the best remedy for this problem. Here, we showed biosynthetic microcin J25 (MccJ25) exhibited excellent bactericidal activity against standard and clinically relevant veterinary MDR strains with high stability, no cytotoxicity, and no increase in drug resistance. Analysis of antimicrobial mechanism possessed by sensitive enterotoxigenic Escherichia coli (ETEC) based on electron microscopy and Sytox Green methods was carried out. Results showed excellent activity against ETEC was due to permeabilizing bacterial membranes and strong affinity. MccJ25 exhibited high endotoxin-neutralizing activity in both in vivo and in vitro environments, and mice exposed to lipopolysaccharide (LPS) showed decreased plasma LPS levels and improved survival after administration of MccJ25. In an LPS-treated mouse septicemia model, MccJ25 treatment significantly alleviated inflammatory responses by inhibiting proinflammatory factor secretion and expression. In a mouse E. coli infection model, administration of MccJ25 effectively improved host defense against clinically source cocktail of multidrug-resistant E. coli strains induced intestinal inflammation and bacteria dissemination. Results of studies on anti-inflammatory mechanisms showed that MccJ25 downregulated nuclear factor kappa B kinase and mitogen-activated protein kinase, thereby reducing the production of toll-like receptor 4, myeloid differentiation factor 88 and decreasing the key proinflammatory cytokines. These findings clarify MccJ25 may be an ideal antibacterial/antiendotoxic drug candidate that has the potential to further guide the development of anti-inflammatory and/or antimicrobial agents in the war against MDR bacterial infection.

Effects of Antimicrobial Peptide Microcin C7 on Growth Performance, Immune and Intestinal Barrier Functions, and Cecal Microbiota of Broilers.

Microcin C7 is an antimicrobial peptide produced by Escherichia coli, composed of a heptapeptide with a modified adenosine monophosphate. This study was performed to evaluate the effects of Microcin C7 as a potential substrate to traditional antibiotics on growth performance, immune functions, intestinal barrier, and cecal microbiota of broilers. In the current study, 300 healthy Arbor Acres broiler chicks were randomly assigned to one of five treatments including a corn-soybean basal diet and basal diet supplemented with antibiotic or 2, 4, and 6 mg/kg Microcin C7. Results showed that Microcin C7 significantly decreased the F/G ratio of broilers; significantly increased the levels of serum cytokine IL-10, immunoglobulins IgG and IgM, and ileal sIgA secretion; significantly decreased the level of serum cytokine TNF-α. Microcin C7 significantly increased villus height and V/C ratio and significantly decreased crypt depth in small intestine of broilers. Microcin C7 significantly increased gene expression of tight junction protein Occludin and ZO-1 and significantly decreased gene expression of pro-inflammatory and chemokine TNF-α, IL-8, IFN-γ, Toll-like receptors TLR2 and TLR4, and downstream molecular MyD88 in the jejunum of broilers. Microcin C7 significantly increased the number of Lactobacillus and decreased the number of total bacteria and Escherichia coli in the cecum of broilers. Microcin C7 also significantly increased short-chain fatty acid (SCFA) and lactic acid levels in the ileum and cecum of broilers. In conclusion, diet supplemented with Microcin C7 significantly improved growth performance, strengthened immune functions, enhanced intestinal barrier, and regulated cecal microbiota of broilers. Therefore, the antimicrobial peptide Microcin C7 may have the potential to be an ideal alternative to antibiotic.

Self-Assembled Nanoparticles of Natural Phytochemicals (Berberine and 3,4,5-Methoxycinnamic Acid) Originated from Traditional Chinese Medicine for Inhibiting Multidrug-Resistant Staphylococcus aureus.

BACKGROUND: In the field of antibacterial, nanomaterials are favored by researchers because of their unique advantages. Medicinal plants, especially traditional Chinese medicine, are considered to be an important source of new chemicals with potential therapeutic effects, as well as an important source for the discovery of new antibiotics. MRSA is endangering people's lives as a kind of multidrug-resistant Staphylococcus aureus, which is resistant to tetracycline, amoxicillin, norfloxacin and other first-line antibiotics. It is a hotspot to find good anti-drug-resistant bacteriae, nature-originated nanomaterials with good biocompatibility. OBJECTIVE: We reported the formation of phytochemical nanoparticles (NPs) by the self-assembly of berberine (BBR) and 3,4,5-methoxycinnamic acid (3,4,5-TCA) from Chinese herb medicine, which had good antibacterial activity against MRSA. METHODS AND RESULTS: We found that NPs had good antibacterial activity against MRSA; especially, its antibacterial activity was better than first-line amoxicillin, norfloxacin and its self-assembling precursors on MRSA. When the concentration reached 0.1 µmol/mL, the inhibition rate of NPs reached 94.62%, which was higher than that of BBR and the other two antibiotics (p < 0.001). It was observed by Field-Emission Scanning Electron Microscopy (FESEM) that NPs could directly adhere to the bacterial surface, which might be an important aspect of the antibacterial activity of NPs. Meanwhile, we further analyzed that the self-assembly was formed by hydrogen bonds and π-π stacking through Ultraviolet-Visible (UV-vis), Fourier Transform Infrared Spectroscopy (FTIR), hydrogen Nuclear Magnetic Spectrum (1H NMR), and powder X-ray Diffraction (pXRD). NPs' morphology was observed by FESEM and TEM. The particle size and surface charge were characterized by Dynamic Light Scattering (DLS); and the surface charge was -31.6 mv, which proved that the synthesized NPs were stable. CONCLUSION: We successfully constructed a naturally self-assembled nanoparticle, originating from traditional Chinese medicine, which had a good antibacterial activity for MRSA. It is a promising way to obtain natural nanoparticles from medicinal plants and apply them to antibacterial therapy.

Betulinic Acid-Nitrogen Heterocyclic Derivatives: Design, Synthesis, and Antitumor Evaluation in Vitro.

Betulinic acid (BA) is a star member of the pentacyclic triterpenoid family, which exhibits great prospects for antitumor drug development. In an attempt to develop novel antitumor candidates, 21 BA-nitrogen heterocyclic derivatives were synthetized, in addition to four intermediates, 23 of which were first reported. Moreover, they were screened for in-vitro cytotoxicity against four tumor cell lines (Hela, HepG-2, BGC-823 and SK-SY5Y) by a standard methylthiazol tetrazolium (MTT) assay. The majority of these derivatives showed much stronger cytotoxic activity than BA. Remarkably, the most potent compound 7e (the half maximal inhibitory concentration (IC50) of which was 2.05 ± 0.66 µM) was 12-fold more toxic in vitro than BA-treated Hela. Furthermore, multiple fluorescent staining techniques and flow cytometry collectively revealed that compound 7e could induce the early apoptosis of Hela cells. Structure-activity relationships were also briefly discussed. The present study highlighted the importance of introducing nitrogen heterocyclic rings into betulinic acid in the discovery and development of novel antitumor agents.

The Complex Energy Landscape of the Protein IscU.

IscU, the scaffold protein for iron-sulfur (Fe-S) cluster biosynthesis in Escherichia coli, traverses a complex energy landscape during Fe-S cluster synthesis and transfer. Our previous studies showed that IscU populates two interconverting conformational states: one structured (S) and one largely disordered (D). Both states appear to be functionally important because proteins involved in the assembly or transfer of Fe-S clusters have been shown to interact preferentially with either the S or D state of IscU. To characterize the complex structure-energy landscape of IscU, we employed NMR spectroscopy, small-angle x-ray scattering (SAXS), and differential scanning calorimetry. Results obtained for IscU at pH 8.0 show that its S state is maximally populated at 25°C and that heating or cooling converts the protein toward the D state. Results from NMR and DSC indicate that both the heat- and cold-induced S→D transitions are cooperative and two-state. Low-resolution structural information from NMR and SAXS suggests that the structures of the cold-induced and heat-induced D states are similar. Both states exhibit similar (1)H-(15)N HSQC spectra and the same pattern of peptidyl-prolyl peptide bond configurations by NMR, and both appear to be similarly expanded compared with the S state based on analysis of SAXS data. Whereas in other proteins the cold-denatured states have been found to be slightly more compact than the heat-denatured states, these two states occupy similar volumes in IscU.

pH-induced conformational change of IscU at low pH correlates with protonation/deprotonation of two conserved histidine residues.

IscU, the scaffold protein for the major iron-sulfur cluster biosynthesis pathway in microorganisms and mitochondria (ISC pathway), plays important roles in the formation of [2Fe-2S] and [4Fe-4S] clusters and their delivery to acceptor apo-proteins. Our laboratory has shown that IscU populates two distinct, functionally relevant conformational states, a more structured state (S) and a more dynamic state (D), that differ by cis/trans isomerizations about two peptidyl-prolyl peptide bonds [Kim, J. H., Tonelli, M., and Markley, J. L. (2012) Proc. Natl. Acad. Sci. U.S.A., 109, 454-459. Dai Z., Tonelli, M., and Markley, J. L. (2012) Biochemistry, 51, 9595-9602. Cai, K., Frederick, R. O., Kim, J. H., Reinen, N. M., Tonelli, M., and Markley, J. L. (2013) J. Biol. Chem., 288, 28755-28770]. Here, we report our findings on the pH dependence of the D ⇄ S equilibrium for Escherichia coli IscU in which the D-state is stabilized at low and high pH values. We show that the lower limb of the pH dependence curve results from differences in the pKa values of two conserved histidine residues (His10 and His105) in the two states. The net proton affinity of His10 is about 50 times higher and that of His105 is 13 times higher in the D-state than in the S-state. The origin of the high limb of the D ⇄ S pH dependence remains to be determined. These results show that changes in proton inventory need to be taken into account in the steps in iron-sulfur cluster assembly and transfer that involve transitions of IscU between its S- and D-states.

Metamorphic protein IscU alternates conformations in the course of its role as the scaffold protein for iron-sulfur cluster biosynthesis and delivery.

IscU from Escherichia coli, the scaffold protein for iron-sulfur cluster biosynthesis and delivery, populates a complex energy landscape. IscU exists as two slowly interconverting species: one (S) is largely structured with all four peptidyl-prolyl bonds trans; the other (D) is partly disordered but contains an ordered domain that stabilizes two cis peptidyl-prolyl peptide bonds. At pH 8.0, the S-state is maximally populated at 25 °C, but its population decreases at higher or lower temperatures or at lower pH. The D-state binds preferentially to the cysteine desulfurase (IscS), which generates and transfers sulfur to IscU cysteine residues to form persulfides. The S-state is stabilized by Fe-S cluster binding and interacts preferentially with the DnaJ-type co-chaperone (HscB), which targets the holo-IscU:HscB complex to the DnaK-type chaperone (HscA) in its ATP-bound from. HscA is involved in delivery of Fe-S clusters to acceptor proteins by a mechanism dependent on ATP hydrolysis. Upon conversion of ATP to ADP, HscA binds the D-state of IscU ensuring release of the cluster and HscB. These findings have led to a more complete model for cluster biosynthesis and delivery.

Metamorphic protein IscU changes conformation by cis-trans isomerizations of two peptidyl-prolyl peptide bonds.

IscU from Escherichia coli, the scaffold protein for iron-sulfur cluster biosynthesis and transfer, populates two conformational states with similar free energies and with lifetimes on the order of 1 s that interconvert in an apparent two-state reaction. One state (S) is structured, and the other (D) is largely disordered; however, both play essential functional roles. We report here nuclear magnetic resonance studies demonstrating that all four prolyl residues of apo-IscU (P14, P35, P100, and P101) are trans in the S state but that two absolutely conserved residues (P14 and P101) become cis in the D state. The peptidyl-prolyl peptide bond configurations were determined by analyzing assigned chemical shifts and were confirmed by measurements of nuclear Overhauser effects. We conclude that the S ⇄ D interconversion involves concerted trans-cis isomerization of the N13-P14 and P100-P101 peptide bonds. Although the D state is largely disordered, we show that it contains an ordered domain that accounts for the stabilization of two high-energy cis peptide bonds. Thus, IscU may be classified as a metamorphic protein.