Immunoproteomically identified GBAA_0345, alkyl hydroperoxide reductase subunit C is a potential target for multivalent anthrax vaccine

Anthrax is caused by the spore‐forming bacterium Bacillus anthracis, which has been used as a weapon for bioterrorism. Although current vaccines are effective, they involve prolonged dose regimens and often cause adverse reactions. High rates of mortality associated with anthrax have made the development of an improved vaccine a top priority. To identify novel vaccine candidates, we applied an immunoproteomics approach. Using sera from convalescent guinea pigs or from human patients with anthrax, we identified 34 immunogenic proteins from the virulent B. anthracis H9401. To evaluate vaccine candidates, six were expressed as recombinant proteins and tested in vivo. Two proteins, rGBAA_0345 (alkyl hydroperoxide reductase subunit C) and rGBAA_3990 (malonyl CoA‐acyl carrier protein transacylase), have afforded guinea pigs partial protection from a subsequent virulent‐spore challenge. Moreover, combined vaccination with rGBAA_0345 and rPA (protective antigen) exhibited an enhanced ability to protect against anthrax mortality. Finally, we demonstrated that GBAA_0345 localizes to anthrax spores and bacilli. Our results indicate that rGBAA_0345 may be a potential component of a multivalent anthrax vaccine, as it enhances the efficacy of rPA vaccination. This is the first time that sera from patients with anthrax have been used to interrogate the proteome of virulent B. anthracis vegetative cells.     

Evaluation of a new antigen for diagnosis of Helicobacter pylori infection in stool of adult and children

Background: Nowadays, there is an increasing interest in noninvasive methods to diagnose Helicobacter pylori infection. Indeed, they can profitably replace endoscopy in predicting the diagnosis. The stool antigen test for H. pylori is a noninvasive immunoassay to diagnose active infection with this bacterium in human fecal samples. The aim of this study was detection of alkyl hydroperoxide reductase protein (AhpC) antigen by immunoblotting in stool samples for diagnosis of H. pylori. Materials and Methods: Chromosomal DNA from H. pylori was isolated. AhpC gene was amplified by PCR, These amplicons were cloned into pTZ57R/T cloning vector then subcloned into pQE30 expression vector and overexpressed using isopropyl‐beta‐D‐thiogalactopyranoside in E. coli M15. AhpC protein was purified by affinity chromatography. Rabbits were immunized with the purified AhpC protein for the production of antibodies. To determine the accuracy of the test for diagnosing H. pylori infection from stool, we evaluated 84 patients (6–81 years old) using Western blot analysis by rabbit anti‐AhpC antibody. Positive rapid urease test on biopsy samples was considered as the gold standard. Results: AhpC gene was overexpressed, and AhpC protein was purified. Rabbit anti‐AhpC antibody produced after immunization with the purified AhpC protein. By immunoblotting, we detected AhpC protein in the positive stool samples. The test showed a 83.3% sensitivity (95% CI: 69.8–92.5%) and a 91.7% specificity (95% CI: 77.5–98.2). Among the children, the sensitivity was 88.2% (95% CI: 63.6–98.5) and the specificity was 100% (95% CI: 69.2–100); in adults, the sensitivity and specificity were 80.6% (95% CI: 62.5–92.5) and 88.5% (95% CI: 69.8–97.6), respectively. Conclusions: Using of AhpC antigen for diagnosis of H. pylori infection is a useful noninvasive method, accurate in adolescents and children, and can be used for the development of a stool antigen detection kit for H. pylori.     

Synthesis of Isosteviol analogues as potential protective agents against Doxorubicin-induced cardiomyopathy in zebrafish embryos

Doxorubicin (DOX) is a powerful anthracycline antibiotic agent which is widely used to treat various types of cancers. Despite efficacy, it displays severe cardiotoxic side effects. Discovery of novel and effective protective agents against DOX-induced cardiotoxicity has been a subject of great interest. Herein, we report the synthesis of two series of analogues of Isosteviol (ISV) 1 with modifications at C-16, C-19 positions as the first series and at C-15, C-16 positions as the other series. Interestingly second series analogues have shown a potential protective effect against DOX-induced cardiotoxicity in zebrafish embryos in vivo. Further, we have demonstrated that the synthesized new analogues of ISV, prevented the morphological distortions caused due to DOX cardiotoxicity in zebrafish heart and the associated cardiac impairments.     

Immune potential of a novel multiple-epitope vaccine to FMDV type Asia 1 in guinea pigs and sheep

To develop a safe and efficient recombinant subunit vaccine to foot-and-mouth disease virus(FMDV)type Asia 1 in sheep,a tandem repeated multiple-epitope gene consisting of residues 137-160 and 197-211 of the VP1 gene of FMDV was designed and artificially synthesized.The biologically functional molecule,the ovine IgG heavy constant region(oIgG)as a protein carrier was introduced for design of the multiple-epitope recombinant vaccine and recombinant expression plasmids pET-30a-RE and pET-30a-RE-oIgG were successfully constructed.The recombinant proteins,RE and RE-oIgG,were expressed as a formation of inclusion bodies in E.coli.The immune potential of this vaccine regime in guinea pigs and sheep was evaluated.The results showed that IgG could significantly enhance the immune potential of antigenic epitopes.The recombinant protein RE-oIgG could not only elicit the high levels of neutralizing antibodies and lymphocytes proliferation responses in the vaccinated guinea pigs,but confer complete protection in guinea pigs against virus challenge.Although the recombinant protein RE could not confer protection in the vaccinated animals,it could delay the appearance of the clinical signs and reduce the severity of disease.Inspiringly,the titers of anti-FMDV neutralizing antibodies elicited in sheep vaccinated with RE-oIgG was significantly higher than that for the RE vaccination.Therefore,we speculated that this vaccine formulation may be a promising strategy for designing a novel vaccine against FMDV in the future.     

SAR optimization studies on a novel series of 2-anilinopyrimidines as selective inhibitors against triple-negative breast cancer cell line MDA-MB-468

Triple-negative breast cancers (TNBCs) account for approximately 15% of breast cancer cases and exhibit an aggressive clinical behavior. In this study, we designed and synthesized two series of 2-anilinopyrimidines based on the structure of our previously reported compound 1 that act as a selective inhibitor of the basal-like TNBC cell line MDA-MB-468. Through the fine-tuning of 1, cyclic and acyclic amines at 4-position of the pyrimidine core were turned out to be crucial for the selectivity. An extensive analysis of structure-activity relationships of the analogs revealed that aminoalkyl groups at the end of the propyl chain are amenable to modification. Among the newly synthesized analogs, compound 38, bearing 4-chloropiperidinyl and cyclohexyl groups, was found to be the most potent and selective, and was about three times more potent and selective than 1 was against the TNBC cells.     

Novel amino acid-substituted diphenylpyrimidine derivatives as potent BTK inhibitors against B cell lymphoma cell lines

A new family of diphenylpyrimidine derivatives bearing an amino acid substituent were identified as potent BTK inhibitors. Among them, compound 7b, which features an l-proline substituent, was identified as the strongest BTK inhibitor, with an IC₅₀ of 8.7?nM. Compound 7b also displayed similar activity against B-cell lymphoma cell lines as ibrutinib. Moreover, 7b exhibited low cytotoxic activity against normal PBMC cells. In addition, the acridine orange/ethidium bromide (AO/EB) staining assay, Western blot analysis and flow cytometry analysis also showed its effectiveness in interfering with B-cell lymphoma cell growth. The molecular simulation performance showed that 7b forms additional strong hydrogen bonds with the BTK protein. All these findings provided new clues about the pyrimidine scaffold as an effective BTK inhibitor for the treatment of B-cell lymphoma.     

Inhibition of mouse SP2/0 myeloma cell growth by the B7-H4 protein vaccine

B7-H4 is a member of B7 family of co-inhibitory molecules and B7-H4 protein is found to be overexpressed in many human cancers and which is usually associated with poor survival. In this study, we developed a therapeutic vaccine made from a fusion protein composed of a tetanus toxoid (TT) T-helper cell epitope and human B7-H4IgV domain (TT-rhB7-H4IgV). We investigated the anti-tumor effect of the TT-rhB7-H4IgV vaccine in BALB/c mice and SP2/0 myeloma growth was significantly suppressed in mice. The TT-rhB7-H4IgV vaccine induced high-titer specific antibodies in mice. Further, the antibodies induced by TT-rhB7-H4IgV vaccine were capable of depleting SP2/0 cells through complement-dependent cytotoxicity (CDC) in vitro. On the other hand, the poor cellular immune response was irrelevant to the therapeutic efficacy. These results indicate that the recombinant TT-rhB7-H4IgV vaccine might be a useful candidate of immunotherapy for the treatment of some tumors associated with abnormal expression of B7-H4. [BMB Reports 2014; 47(7): 399-404]     

Highly tunable thiosulfonates as a novel class of cysteine protease inhibitors with anti-parasitic activity against Schistosoma mansoni

The development of a new class of cysteine protease inhibitors utilising the thiosulfonate moiety as an SH specific electrophile is described. This moiety has been introduced into suitable amino acid derived building blocks, which were incorporated into peptidic sequences leading to very potent i.e. sub micromolar inhibitors of the cysteine protease papain in the same range as the vinyl sulfone based inhibitor K11777. Therefore, their inhibitory effect on Schistosoma mansoni, a human blood parasite, that expresses several cysteine proteases, was evaluated. The homophenylalanine side chain containing compounds 27–30 and especially 36 showed promising activities compared with K11777 and warrant further investigations of these peptidic thiosulfonate inhibitors as new potential anti-parasitic compounds.     

Synthesis and biological evaluation of 3-carbamate smilagenin derivatives as potential neuroprotective agents

Studies indicated that smilagenin, isolated from Anemarrhena asphodeloides Bunge, could improve cognitive impairment and exhibit neuroprotective activity. On the basis of the structure of smilagenin, a series of derivatives were synthesized and evaluated for their neuroprotective effects of H₂O₂-induced, oxygen glucose deprivation-induced neurotoxicity in SH-SY5Y cells and LPS-induced NO production in RAW264.7 cells. Structure activity relationship of derivatives revealed that benzyl-substituted piperazine formate derivatives showed the potent neuroprotective activity such as A12. These findings may provide new insights for the development of neuroprotective agents against Alzheimer’s disease.     

Total synthesis of dryocrassin ABBA and its analogues with potential inhibitory activity against drug-resistant neuraminidases

The stems of Dryopteris crassirhizoma, one of the main components of Lianhua-Qingwen Formula (LQF) was traditionally used for heat-clearing and detoxifying. Dryocrassin ABBA is a key antiviral component in the herbal medicine while the compound is hard to get in large amounts with the features of homologous compounds, polyphenol groups, and low contents. Therefore, the present work aims to seek influenza H7N9 virus inhibitors from natural source by synthesis of dryocrassin ABBA and its analogues. As a result, total synthesis of the compound was achieved in nine steps with an over-all yield of 4.6%. Neuraminidases (NAs) inhibitory activities of the synthesized product and its analogues were evaluated afterward. Comparing with the positive control, OSV (9.6 μM), it was very exciting that dryocrassin ABBA and its analogues (b5 and e2) showed better NAs inhibitory activity against Anhui H7N9 with IC₅₀ values of 3.6 μM, 2.5 μM and 1.6 μM. For the highly resistant Shanghai N9, these compounds can also show medium inhibitory activities. Docking results indicated the direct interaction of synthesized 3 hits with the key K294 by hydrogen bonds, but no direct interaction of OSV with the key K294 was observed in Shanghai N9. This study suggested that dryocrassin ABBA and its analogues especially AB, which consisted of polyphenol groups may have beneficial effects on treating avian influenza H7N9 virus.     

Promises and pitfalls of Pseudomonas aeruginosa lipopolysaccharide as a vaccine antigen

Antibodies directed to the Pseudomonas aeruginosa lipopolysaccharide (LPS) O-antigens have clearly shown to mediate the most effective immunity to infection caused by LPS-smooth strains. Such strains are major causes of disease in immunocompromised hosts such as burn or cancer patients, individuals in intensive care units, and those who utilize extended-wear contact lenses. Yet producing an effective vaccine composed of non-toxic, immunogenic polysaccharides has been challenging. The chemical diversity among the different O-antigens representative of the 20 major serotypes, plus additional diversity among some O-antigens representing variant subtype antigens, translates into a large degree of serologic variability that increases the complexity of O-antigen specific vaccines. Further complications come from the poor immunogenicity of the major protective epitope expressed by some O-antigens, and a large degree of diversity in animal responses that preclude predicting the optimal vaccine formulation from such studies. Nonetheless human trials over the years of vaccines eliciting O-antigen immunity have been encouraging, though no vaccine has yet been fully evaluated and found to be clinically efficacious. Newer vaccine approaches such as using polysaccharide-protein conjugates and passive therapy with monoclonal or polyclonal immune sera offer some additional means to try and produce an effective immunotherapeutic reagent for this problematic pathogen.     

Synthesis and immunological activity of an oligosaccharide-conjugate as a vaccine candidate against Group A Streptococcus

The synthesis and immunogenicity of a tetanus toxoid (TT)-conjugate of the hexasaccharide portion of the cell-wall polysaccharide (CWPS) of the Group A Streptococcus (GAS) is described. The synthesis relies on the reaction of an allyl glycoside of the hexasaccharide with cysteamine, followed by the reaction of the resultant amine with diethyl squarate to give the monoethyl squarate adduct. Subsequent reaction with the lysine ε-amino groups on TT gives the glycoconjugate containing 30 hexasaccharide haptens per TT molecule. The immunogenicity in mice is similar to that obtained with a native CWPS–TT conjugate, validating the glycoconjugate as a vaccine candidate against GAS infections.     

DNA vaccine with discontinuous T‐cell epitope insertions into HSP65 scaffold as a potential means to improve immunogenicity of multi‐epitope Mycobacterium tuberculosis vaccine

DNA‐based vaccine is a promising candidate for immunization and induction of a T‐cell‐focused protective immune response against infectious pathogens such as Mycobacterium tuberculosis (M. tb). To induce multi‐functional T response against multi‐TB antigens, a multi‐epitope DNA vaccine and a ‘protein backbone grafting’ design method is adopted to graft five discontinuous T‐cell epitopes into HSP65 scaffold protein of M. tb for enhancement of epitope processing and immune presentation. A DNA plasmid with five T‐cell epitopes derived from ESAT‐6, Ag85B, MTB10.4, PPE25 and PE19 proteins of H37Rv strain of M. tb genetically inserted into HSP65 backbone was constructed and designated as pPES. After confirmation of its in vitro expression efficiency, pPES DNA was i.m. injected into C57BL/6 mice with four doses of 50 µg DNA followed by mycobacterial challenge 4 weeks after the final immunization. It was found that pPES DNA injection maintained the ability of HSP65 backbone to induce specific serum IgG. ELISPOT assay demonstrated that pPES epitope‐scaffold construct was significantly more potent to induce IFN‐γ⁺ T response to five T‐cell epitope proteins than other DNA constructs (with epitopes alone or with epitope series connected to HSP65), especially in multi‐functional‐CD4⁺ T response. It also enhanced granzyme B⁺ CTL and IL‐2⁺ CD8⁺ T response. Furthermore, significantly improved protection against Mycobacterium bovis BCG challenge was achieved by pPES injection compared to other DNA constructs. Taken together, HSP65 scaffold grafting strategy for multi‐epitope DNA vaccine represents a successful example of rational protein backbone engineering design and could prove useful in TB vaccine design.     

Design,synthesis and biological research of novel N-phenylbenzamide-4-methylamine acridine derivatives as potential topoisomerase I/II and apoptosis-inducing agents

A series of novel N-phenylbenzamide-4-methylamine acridine derivatives were designed and synthesized based initially on the structure of amsacrine (m-AMSA). Molecular docking suggested that the representative compound 9a had affinity for binding DNA topoisomerase (Topo) II, which was comparable with that of m-AMSA, and furthermore that 9a could have preferential interactions with Topo I. After synthesis of 9a and analogues 9b-9f, these were all tested in vitro and the synthesized compounds displayed potent antiproliferative activity against three different cancer cell lines (K562, CCRF-CEM and U937). Among them, compounds 9b, 9c and 9d exhibiting the highest activity with IC₅₀ value ranging from 0.82 to 0.91 μM against CCRF-CEM cells. In addition, 9b and 9d also showed high antiproliferative activity against U937 cells, with IC₅₀ values of 0.33 and 0.23 μM, respectively. The pharmacological mechanistic studies of these compounds were evaluated by Topo I/II inhibition, western blot assay and cell apoptosis detection. In summary, 9b effectively inhibited the activity of Topo I/II and induced DNA damage in CCRF-CEM cells and, moreover, significantly induced cell apoptosis in a concentration-dependent manner. These observations provide new information and guidance for the structural optimization of more novel acridine derivatives.     

The identification of nitrate reductase fromEscherichia coli as the antigen for a monoclonal antibody of unknown specificity

The immunoaffinity chromatography of total membrane proteins fromEscherichia coli helped determine the specificity of the monoclonal antibody 3A6 that was obtained upon immunization of mice with nicotinamide nucleotide transhydrogenase preparations and reacted with an unknownE. coli antigen. Proteins with apparent molecular masses of 150, 45, and 20 kDa were isolated and identified byN-terminal sequencing as the subunits of nitrate reductase. This conclusion was confirmed by immunoblotting with the 3A6 antibody of the proteins from theE. coli cells grown upon induction of nitrate reductase. It was shown that the 3A6 antibody specifically recognizes the α subunit of nitrate reductase, and the formation of the enzyme-antibody complex does not result in a loss of the enzyme catalytic activity.     

Design and synthesis of functionalized coumarins as potential anti-austerity agents that eliminates cancer cells' tolerance to nutrition starvation

Human pancreatic tumor cells have inherent ability to tolerate nutrition starvation which enables them to survive in the hypovascular tumor microenvironment. Discovery of agents that selectively inhibit the cancer cells’ tolerance to nutrition starvation leading to cancer cell death is a new anti-austerity approach in anti-cancer drug discovery. A series of coumarins derivatives were synthesized and evaluated for their anti-austerity activity against PANC-1 human pancreatic cancer cell line. The compound 7-Hydroxy-2-oxo-2H-chromene-3-carboxylic acid (3-phenylpropyl)amide (2c) showed highly potent selective cytotoxicity against PANC-1 cells under nutrient-deprived conditions, with a PC₅₀ value of 0.44 μM, without exhibiting toxicity in normal, nutrient-rich medium. Compound 2c caused dramatic alterations in PANC-1 cell morphology, leading to cell death. The compound 2c was found to inhibit PANC-1 cell migration and colony formation in a concentration-dependent manner. The compound 2c is a lead structure for the anti-austerity drug development against pancreatic cancer.     

Antitumor effects of a xenogeneic survivin bone marrow derived dendritic cell vaccine against murine GL261 gliomas

Survivin is a member of the inhibitor of apoptosis protein family. Gliomas and many other tumors express survivin at high levels; whereas, normal fully differentiated cells generally do not. Therefore, survivin represents a tumor-specific target for cancer vaccine therapy. It has been shown that it is possible to produce a MHC-I-restricted cellular immunologic response to survivin vaccines. To study differences in immunogenicity between murine and human survivin proteins, we vaccinated C57BL/6 mice with bone marrow dendritic cells (BMDC) transfected with expression vectors containing the murine and human survivin genes. Mice vaccinated with BMDCs expressing a truncated human survivin protein developed cytotoxic T lymphocyte to subcutaneous GL261 glioma cells and exhibited prolonged tumor-free survival compared to mice vaccinated with BMDCs transfected with vector alone (P<0.01). While mice challenged with intracerebral GL261 cells had increased survival, no cures were observed. In contrast, vaccinated mice that fully resisted subcutaneous tumor challenge were rendered resistant to intracerebral GL261 re-challenge. BMDCs transfected with the full-length human survivin molecule were significantly more effective at prolonging survival than BMDCs expressing the full-length murine survivin gene (P=0.0175). Therefore, xenogeneic differences between human and murine sequences might be exploited to develop more immunogenic tumor vaccines.     

Reductive detoxification of acrolein as a potential role for aldehyde reductase (AKR1A) in mammals

Aldehyde reductase (AKR1A), a member of the aldo–keto reductase superfamily, suppresses diabetic complications via a reduction in metabolic intermediates; it also plays a role in ascorbic acid biosynthesis in mice. Because primates cannot synthesize ascorbic acid, a principle role of AKR1A appears to be the reductive detoxification of aldehydes. In this study, we isolated and immortalized mouse embryonic fibroblasts (MEFs) from wild-type (WT) and human Akr1a-transgenic (Tg) mice and used them to investigate the potential roles of AKR1A under culture conditions. Tg MEFs showed higher methylglyoxal- and acrolein-reducing activities than WT MEFs and also were more resistant to cytotoxicity. Enzymatic analyses of purified rat AKR1A showed that the efficiency of the acrolein reduction was about 20% that of glyceraldehyde. Ascorbic acid levels were quite low in the MEFs, and while the administration of ascorbic acid to the cells increased the intracellular levels of ascorbic acid, it had no affect on the resistance to acrolein. Endoplasmic reticulum stress and protein carbonylation induced by acrolein treatment were less evident in Tg MEFs than in WT MEFs. These data collectively indicate that one of the principle roles of AKR1A in primates is the reductive detoxification of aldehydes, notably acrolein, and protection from its detrimental effects.