A Synthetic Bioactive Peptide Derived from the Asian Medicinal Plant Acacia catechu Binds to Dengue Virus and Inhibits Cell Entry

Dengue virus (DENV) infection has become a critically important globally prevalent infectious disease, especially in tropical and subtropical countries. Since neither currently exists, there is an urgent need for an effective vaccine to prevent, and a specific drug to treat DENV infection. Therapeutic peptides represent an attractive alternative for development into anti-DENV drugs due to their safety and their diverse biological and chemical properties. We recently reported novel bioactive peptides extracted from the Asian medicinal plant Acacia catechu that efficiently inhibited all four DENV serotypes. In this study, we investigated the anti-DENV activity of a synthetic bioactive peptide derived from this plant. The most effective peptide (designated Pep-RTYM) inhibited DENV infection with a half-maximal inhibition concentration value of 7.9 μM. Time-of-addition study demonstrated that Pep-RTYM interacted with DENV particles and inhibited cellular entry. Pep-RTYM at 50 μM significantly reduced DENV production in Vero-kidney epithelial cells about 1000-fold, but it could decrease the virus production in Huh7 hepatocyte cells approximately 40-fold. Binding of Pep-RTYM to DENV particles may prevent virus interaction with cellular receptor and subsequent virus entry. This finding suggests a potential role of Pep-RTYM in the development of a novel anti-DENV drug.     

Two novel ACE inhibitory peptides isolated from longan seeds: purification,inhibitory kinetics and mechanisms

Angiotensin converting enzyme (ACE) inhibition offers a useful means of managing hypertension, because ACE inhibitors (ACEIs) are known to serve as agents with antihypertensive properties in addition to generating positive metabolic and cardioprotective outcomes. However, current ACEIs are linked to adverse consequences, and so there is a requirement for effective but safer compounds, which might be achieved through chemical synthesis or the isolation of naturally obtained bioactive molecules. Protein hydrolysates with ACEI activity can be produced by the combined pepsin and pancreatin proteolysis (to mimic gastrointestinal digestion) of longan seed protein. This study examined longan seed protein hydrolysates, obtained from a sequential 3 h digestion with pepsin and then pancreatin. The resulting hydrolysate underwent sequential ultrafiltration membrane fractionation with a 10, 5, and 3 kDa molecular weight cut-off (MWCO). The permeate derived from the <3 kDa MWCO demonstrated the highest ACEI activity. This permeate subsequently underwent separation by reverse-phase high performance liquid chromatography to give the main fractions on the basis of differing elution times. The ACEI IC₅₀ values for these fractions were then identified. Quadrupole time-of-flight tandem mass spectrometry was employed to determine the peptide mass for the major peak (F₅), which was shown to be Glu–Thr–Ser–Gly–Met–Lys–Pro–Thr–Glu–Leu (ETSGMKPTEL) and Ile–Ser–Ser–Met–Gly–Ile–Leu–Val–Cys–Leu (ISSMGILVCL). These two peptides were stable over a temperature and pH range of −20 to 90 °C and 2–12, respectively, for 60 min. From the Lineweaver–Burk plot, both peptides inhibited ACE non-competitively. Molecular docking simulation of the peptides with ACE supported the formation of hydrogen bonds by the peptides with the ACE active pockets. This research indicates that it may be possible to use both of these peptides or longan seed protein hydrolysates in order to create ingredients for functional foods, or to produce pharmaceutical products, capable of lowering hypertension.

The isolation and subsequent identification of the two novel ACE-inhibitory peptides, ETSGMKPTEL, and ISSMGILVCL from the longan seeds were achieved. The inhibition mechanism was investigated by molecular docking.     

A Peptide Inhibitor Derived from the Conserved Ectodomain Region of DENV Membrane (M) Protein with Activity Against Dengue Virus Infection

Dengue virus (DENV) infection is a public health problem worldwide; thus, the development of a vaccine and anti‐DENV drugs is urgently needed. It has been observed that low levels of viremia in DENV‐infected individuals are associated with mild disease outcomes; therefore, reduction of DENV load should offer therapeutic benefits. Disruption of protein–protein interactions on the surface of DENV by a peptide that mimics part of its structural protein may affect stability of the virion structure and inhibit viral entry into host cells. To test this hypothesis, we generated a novel peptide inhibitor that mimics the conserved ectodomain region of DENV membrane (M) protein, MLH40 peptide, for DENV inhibition assays. MLH40 inhibited all four serotypes of the virus (DENV1–4) at half maximal inhibition concentration of 24–31 μm . MLH40 at 100 μm blocked DENV2 attachment to cells by 80%. The inhibitory activity of MLH40 against DENV was consistently observed with different cell types, including Vero, A549, and Huh7 cells. Prediction of MLH40 binding by a molecular docking program indicated that its N‐terminal loop may interact with DENV envelope (E) proteins and alter their dimer conformation. Thus, MLH40 may serve as a lead‐peptide inhibitor for the development of an anti‐DENV drug.     

Bioactivities of Jc‐SCRIP,a Type 1 Ribosome‐Inactivating Protein from Jatropha curcas Seed Coat

In this study, a type 1 RIP, designated as Jc‐SCRIP, was first isolated from the seed coat of Jatropha curcas Linn. It was purified by ammonium sulfate precipitation and chromatography on DEAE‐Sephacel? and CM‐cellulose columns. Purification fold of Jc‐SCRIP increased 113.8 times, and the yield was 1.13% of the total protein in the final step. It was shown to be a monomeric glycoprotein with a molecular mass of 38 938 Da, as determined by MALDI‐TOF/MS. It exhibited hemagglutination activity and possessed strong N‐glycosidase activity. The antimicrobial activity of Jc‐SCRIP was tested against nine human pathogenic bacteria and one fungus; the most potent inhibitory activity was against Staphylococcus epidermidis ATCC 12228, with minimum inhibitory concentration value of 0.20 μm . Jc‐SCRIP demonstrated in vitro cytotoxicity against human breast adenocarcinoma cell line (MCF‐7), a colon adenocarcinoma (SW620), and a liver carcinoma cell line (HepG2), with IC₅₀ values of 0.15, 0.25, and 0.40 mm , respectively. The results suggested that Jc‐SCRIP may be a potential natural antimicrobial and anticancer agent in medical applications.     

Human ScFv that block sodium ion channel activity of tetrodotoxin

Tetrodotoxin (TTX) is a heterocyclic guanidinium alkaloid (C₁₁H₁₇N₃O₈) with molecular mass of ∼320 Da. The TTX and toxic analogs block sodium ion activity of mammalian nerve cells resulting in failure to conduct nerve impulse which manifested clinically in host as variable degrees of organ paralysis. Human intoxication occurs after consuming food containing the toxins. Current treatment of the poisoning is supportive and symptomatic. There has been no specific drug or antidote for the TTX mediated malady. In this study, phage clones displaying human single chain antibody fragments (HuScFv) were selected from a human ScFv phage display library. HuScFv derived from phagemid transformed Escherichia coli clones (clones s16 and s35) bound to the TTX as tested by indirect ELISA and band shift assay. Homology modeling and molecular docking revealed that VL domain of the s16-HuScFv interacted with the hydroxyl groups of C6, C9, C10 and C11 of the TTX by means of Tyr 223, Ser226 and Tyr228, while the Asp53 and Asp55 of the VH domain of s35-HuScFv interacted with the positions 1 and 2 of the guanidinium group and the hydroxyl groups at C9 and C10 of the TTX. The s16- and s35-HuScFv neutralized the TTX bioactivity in nerve cell based- and mouse bio-assays. Moreover, the HuScFv could rescue the intoxicated mice from the TTX mediated lethality. Thus, the HuScFv derived from the transformed E. coli clones have high potential as a safe, effective and specific therapeutic remedy for TTX intoxication in humans and warrant further trials.     

Characterization of human semen alpha-L-fucosidases.

Human semen contains a large amount of alpha-L-fucosidase activity, the great majority of which is found in the seminal fluid. Immunocytochemical studies indicate that a small amount of semen fucosidase activity is present on the sperm plasma membrane, primarily in the posterior head region. Subcellular fractionation studies also indicate that sperm alpha-L-fucosidase is present in the plasma membrane-enriched fraction. Comparative characterization of human seminal fluid and sperm alpha-L-fucosidases indicates that seminal fluid alpha-L-fucosidase has a broad pH optimum curve with a number of near-equal maxima between pH 4.8 and 7.0 while sperm fucosidase has a major optimum between pH 3.4 and 4.0. Isoelectric focusing indicates that seminal fluid alpha-L-fucosidase contains three to six isoforms with isoelectric points (pI) of 5-7 while sperm fucosidase contains two distinct isoforms with pI values of 5. 2 +/- 0.2 and 7.0 +/- 0.2. Western blotting indicates that seminal fluid fucosidase contains a major protein band with a molecular mass ratio (M(r)) of approximately 56 kDa while sperm fucosidase contains a major protein band of approximately 51 kDa. The overall results indicate the presence of a low-abundance, plasma membrane-associated human sperm alpha-L-fucosidase, which is different in its properties from human seminal fluid alpha-L-fucosidase(s), and whose function is not yet known.     

Synthesis,HIV-1 RT inhibitory,antibacterial, antifungal and binding mode studies of some novel N-substituted 5-benzylidine-2,4-thiazolidinediones

Background

Structural modifications of thiazolidinediones at 3^rd and 5^th position have exhibited significant biological activities. In view of the facts, and based on in silico studies carried out on thiazolidine-2,4-diones as HIV-1- RT inhibitors, a novel series of 2,4-thiazolidinedione analogs have been designed and synthesized.

Methods

Title compounds were prepared by the reported method. Conformations of the structures were assigned on the basis of results of different spectral data. The assay of HIV-1 RT was done as reported by Silprasit et al. Antimicrobial activity was determined by two fold serial dilution method. Docking study was performed for the highest active compounds by using Glide 5.0.

Results

The newly synthesized compounds were evaluated for their HIV-1 RT inhibitory activity. Among the synthesized compounds, compound 24 showed significant HIV-1 RT inhibitory activity with 73% of inhibition with an IC₅₀ value of 1.31 μM. Compound 10 showed highest activity against all the bacterial strains.A molecular modeling study was carried out in order to investigate the possible interactions of the highest active compounds 24, 10 and 4 with the non nucleoside inhibitory binding pocket(NNIBP) of RT, active site of GlcN-6-P synthase and cytochrome P450 14-α-sterol demethylase from Candida albicans (Candida P450DM) as the target receptors respectively using the Extra Precision (XP) mode of Glide software.

Conclusion

A series of novel substituted 2-(5-benzylidene-2,4-dioxothiazolidin-3-yl)-N-(phenyl)propanamides (4–31) have been synthesized and evaluated for their HIV-1 RT inhibitory activity, antibacterial and antifungal activities. Some of the compounds have shown significant activity. Molecular docking studies showed very good interaction.