Inhibitory effect of naphthoquine phosphate on Babesia gibsoni in vitro and Babesia rodhaini in vivo

Background Drug resistance and toxic side effects are major challenges in the treatment of babesiosis. As such, new drugs are needed to combat the emergence of drug resistance in Babesia parasites and to develop alternative treatment strategies. A combination of naphthoquine (NQ) and artemisinin is an antimalarial therapy in pharmaceutical markets. The present study repurposed NQ as a drug for the treatment of babesiosis by evaluating the anti-Babesia activity of naphthoquine phosphate (NQP) alone. Methods An in vitro growth inhibition assay of NQP was tested on Babesia gibsoni cultures using a SYBR Green I-based fluorescence assay. In addition, the in vivo growth inhibitory effect of NQP was evaluated using BALB/c mice infected with Babesia rodhaini. The parasitemia level and hematocrit values were monitored to determine the therapeutic efficacy of NQP and the clinical improvements in NQP-treated mice. Results The half maximal inhibitory concentration of NQP against B. gibsoni in vitro was 3.3 ± 0.5 μM. Oral administration of NQP for 5 consecutive days at a dose of 40 mg/kg of body weight resulted in significant inhibition of B. rodhaini growth in mice as compared with that of the control group. All NQP-treated mice survived, whereas the mice in the control group died between days 6 and 9 post-infection. Conclusion This is the first study to evaluate the anti-Babesia activity of NQP in vitro and in vivo. Our findings suggest that NQP is a promising drug for treating Babesia infections, and drug repurposing may provide new treatment strategies for babesiosis. Graphical Abstract

Improving the Yield of Xenocoumacin 1 by PBAD Promoter Replacement in Xenorhabdus nematophila CB6

Xenocoumacin 1 (Xcn1), which is produced by Xenorhabdus nematophila CB6, exhibits strong inhibition activity against plant pathogens, especially fungi and oomycetes. Therefore, it has attracted interest in developing it into a novel biofungicide applicable for plant protection. However, its low yield with concomitant high cost during the fermentation process limits its widespread application. In this study, we replaced the native promoter of xcnA with the arabinose-inducible araBAD promoter (PBAD), a well-known and widely used promoter for expressing heterologous genes, to evaluate its effects on Xcn1 yield and antimicrobial activity. Compared with wildtype strain, the fermentation yield of Xcn1 was improved from 68.5 mg/L to 249.7 mg/L (3.6-fold) and 234.9 mg/L (3.4-fold) at 0.5% and 1.0% L-arabinose concentration, respectively. We further explored the transcription level of the biosynthesis related genes of Xcn1 and found that their upregulation resulted in the yield improvement of Xcn1. Moreover, the antimicrobial activity of Xcn1 against Bacillus subtilis and Phytophthora capsici was determined by agar diffusion plate and growth inhibition assay, as expected, it was also found to be enhanced. The promoter-replacement strategy utilized here improves the yield of Xcn1 efficiently, which provides a basis for the industrial production of Xcn1.

Synthesis of small molecule inhibitors for the treatment of disease

<p>Three aspects of the protecting-group-free (PGF) synthesis of small molecules have been described in this thesis. In the first part, the PGF azasugar synthesis methodology was applied to 2-deoxy-D-glucose with the intention of selectively forming the six-membered azasugar 5-epi-fagomine. Surprisingly, four products were formed in the key I2-mediated carbamate annulation step, with a pyrrolidine being the major product after optimisation. This was formed in 15% yield. A mechanism that explains the formation of the four carbamates was proposed, which was supported by an investigation into related halocyclisation reactions. The next part of this thesis describes the development of a new PGF methodology for the synthesis of conduramines, another class of biologically interesting molecules. Conduramines are amino polyhydroxy cyclohexenes and some conduramines have glycosidase inhibitory activity. These molecules are also useful precursors to a variety of biologically useful molecules including aminocyclitols and azasugars. The key steps in the PGF synthesis of conduramines are a Vasella-Barbier amination, a reaction that forms new C-C and C-N bonds concomitantly, and a ring closing metathesis in the presence of free hydroxyl and amine groups. To this end, a 4-deoxy 3-conduramine was prepared in just four steps and in 27% yield. Finally, the preparation of an amine library and its biological testing for the identification of a new anti-tuberculosis drug is described. Two short syntheses were used to prepare alkenylamines and amines from the corresponding sugar, with various lipophilic groups attached to the amine. A 20-member amine library was prepared, and the compounds were tested for anti-mycobacterial activity in a mycobacterial growth inhibition assay. The most active compounds were subjected to further biological testing to determine their general cytotoxic properties. Two amines, arabinohexadecylamine and arabinohexadecylmethylamine, were identified as having the best potential for use as anti-tuberculosis drugs, and have been sent to Colorado State University for subsequent in vivo testing in a mouse model of tuberculosis.</p>

Synthesis of small molecule inhibitors for the treatment of disease

<p>Three aspects of the protecting-group-free (PGF) synthesis of small molecules have been described in this thesis. In the first part, the PGF azasugar synthesis methodology was applied to 2-deoxy-D-glucose with the intention of selectively forming the six-membered azasugar 5-epi-fagomine. Surprisingly, four products were formed in the key I2-mediated carbamate annulation step, with a pyrrolidine being the major product after optimisation. This was formed in 15% yield. A mechanism that explains the formation of the four carbamates was proposed, which was supported by an investigation into related halocyclisation reactions. The next part of this thesis describes the development of a new PGF methodology for the synthesis of conduramines, another class of biologically interesting molecules. Conduramines are amino polyhydroxy cyclohexenes and some conduramines have glycosidase inhibitory activity. These molecules are also useful precursors to a variety of biologically useful molecules including aminocyclitols and azasugars. The key steps in the PGF synthesis of conduramines are a Vasella-Barbier amination, a reaction that forms new C-C and C-N bonds concomitantly, and a ring closing metathesis in the presence of free hydroxyl and amine groups. To this end, a 4-deoxy 3-conduramine was prepared in just four steps and in 27% yield. Finally, the preparation of an amine library and its biological testing for the identification of a new anti-tuberculosis drug is described. Two short syntheses were used to prepare alkenylamines and amines from the corresponding sugar, with various lipophilic groups attached to the amine. A 20-member amine library was prepared, and the compounds were tested for anti-mycobacterial activity in a mycobacterial growth inhibition assay. The most active compounds were subjected to further biological testing to determine their general cytotoxic properties. Two amines, arabinohexadecylamine and arabinohexadecylmethylamine, were identified as having the best potential for use as anti-tuberculosis drugs, and have been sent to Colorado State University for subsequent in vivo testing in a mouse model of tuberculosis.</p>

Evaluation of Tuberculosis Vaccine Candidate, pcDNA3.1-rpfD using Mycobacterial Growth Inhibition Assay (MGIA)

Resuscitation-promoting factor D (RpfD) is a protein involved in the resuscitation of dormant bacteria. A new tuberculosis vaccine carrying the rpfD gene has been successfully constructed, pcDNA3.1-rpfD. It was demonstrated that this vaccine exhibits cellular and humoral immune responses. Therefore, within this study, the efficacy of this new vaccine candidate was evaluated using mycobacterial growth inhibition assay (MGIA). MGIA is a functional assay that measures the complex host immune response, peripheral blood mononuclear cell (PBMC) and splenocyte from BALB/c mice against mycobacteria. With BACTECTM MGITTM 960 automated system, the effect of vaccination on bacterial growth was reported as a time to positivity (TTP) in hours. The mean of TTP from the vaccinated group (both pcDNA3.1-rpfD and BCG) was higher than the negative control group. These results suggest that pcDNA3.1-rpfD may be effective in controlling tuberculosis growth and may provide a clue for the development of the tuberculosis vaccine. In addition, despite previous evidence that IFNγ was essential for tuberculosis immunity, IFNγ (interferon gamma) production was found not to be correlated with mycobacterial inhibition. Therefore, these findings offer an alternative method to evaluate vaccine candidates than the assessment using IFNγ only.

Synthesis and Fungicidal Activity of Hydrated Geranylated Phenols against Botrytis cinerea

Botrytis cinerea is a ubiquitous fungus that affects hundreds of plants, resulting in economic losses to the horticulture and fruit industry. The search for new antifungal agents is a matter of current interest. Thus, in this work a series of geranylated phenols in which the side alkyl chain has been hydrated have been synthesized, and their activity against B. cinerea has been evaluated. The coupling of phenol and geraniol has been accomplished under microwave irradiation obtaining the highest reaction yields in the shortest reaction times. Hydration of the side chain was carried out in dioxane with p-toluenesulfonic acid polymer-bound as the catalyst. All synthesized compounds were tested against B. cinerea using the growth inhibition assay and EC50 values were determined. The results show that activity depends on the number and nature of functional groups in the phenol ring and hydration degree of the geranyl chain. The most active compound is 1,4-dihydroquinone with one hydroxyl group attached at the end of the alkyl chain. Results from a molecular docking study suggest that hydroxyl groups in the phenol ring and alkyl chain are important in the binding of compounds to the active site, and that the experimental antifungal activity correlates with the number of H-bond that can be formed in the binding site.

The in vitro direct mycobacterial growth inhibition assay (MGIA) for the early evaluation of TB vaccine candidates and assessment of protective immunity: a protocol for non-human primate cells

The only currently available approach to early efficacy testing of tuberculosis (TB) vaccine candidates is in vivo preclinical challenge models. These typically include mice, guinea pigs and non-human primates (NHPs), which must be exposed to virulent M.tb in a ‘challenge’ experiment following vaccination in order to evaluate protective efficacy. This procedure results in disease development and is classified as ‘Moderate’ in severity under EU legislation and UK ASPA licensure. Furthermore, experiments are relatively long and animals must be maintained in high containment level facilities, making them relatively costly. We describe an in vitro protocol for the direct mycobacterial growth inhibition assay (MGIA) for use in the macaque model of TB vaccine development with the aim of overcoming some of these limitations. Importantly, using an in vitro assay in place of in vivo M.tb challenge represents a significant refinement to the existing procedure for early vaccine efficacy testing. Peripheral blood mononuclear cell and autologous serum samples collected from vaccinated and unvaccinated control animals are co-cultured with mycobacteria in a 48-well plate format for 96 hours. Adherent monocytes are then lysed to release intracellular mycobacteria which is quantified using the BACTEC MGIT system and colony-forming units determined relative to an inoculum control and stock standard curve. We discuss related optimisation and characterisation experiments, and review evidence that the direct NHP MGIA provides a biologically relevant model of vaccine-induced protection. The potential end-users of the NHP MGIA are academic and industry organisations that conduct the assessment of TB vaccine candidates and associated protective immunity using the NHP model. This approach aims to provide a method for high-throughput down-selection of vaccine candidates going forward to in vivo efficacy testing, thus expediting the development of a more efficacious TB vaccine and offering potential refinement and reduction to the use of NHPs for this purpose.

Antioxidant, Antifungal and Aphicidal Activity of Triterpenoids Spinasterol, 22,23-Dihydrospinasterol From Colocynthis (Citrullus Colocynthis L.) Leaves

Terpenoids from natural plants resources are valuable for diverse biological activities which exhibited important part in medical and agrochemicals industry. This study aimed to assess the antioxidant, antifungal and aphicidal activity of a mixture of Spinasterol, 22,23-dihydrospinasterol from Citrullus colocynthis leaves. 1, 1-diphenyl-2-picrylhydrazyl (DPPH) was used to assess the antioxidant activity whereas, antifungal activity was tested by mycelium growth inhibition assay on three pathogenic fungi Magnaporthe grisea, Rhizoctonia solani and Phytophthora infestans. Aphicidal activity against adults of Myzus persicae was also determined via In-vitro and In-vivo assays. The outcome of the study exposed that Spinasterol, 22, 23-dihydrospinasterol afforded moderate antioxidant activity even at lower concentrations i.e. 19.98, 31.52, 36.61 and 49.76% at 0.78, 3.0, 12.5 and 50µgmL− 1 respectively. However, reasonable fungicidal activity of Spinasterol, 22; 23-dihydrospinasterol was recorded as being EC50 values 129.5 and 206.1µgmL− 1 against R. solani and M. grisea respectively. On the other hand, Boscalid and Carbendazim being a positive control proved highly effective against all fungi except for M. grisea and P. infestans with EC50 values 868 and 272109µgmL− 1 respectively. The significant insecticidal activity was afforded via residual as well as greenhouse assay being LC50 values as 42.46, 54.86, 180.9 µgmL− 1 and 32.71, 42.46 and 173.8µgmL− 1 at 72, 48 and 24 h respectively. Moreover, antioxidant activity of Spinasterol, 22,23-dihydrospinasterol presented strong positive correlation versus antifungal and insecticidal activity. Spinasterol, 22,23-dihydrospinasterol possess good antioxidant and aphicidal activity with moderate fungicidal activity which could be a suitable candidate as an alternative to synthetic pesticidal agents.

Essential Oils of Sage, Rosemary, and Bay Laurel Inhibit the Life Stages of Oomycete Pathogens Important in Aquaculture

Saprolegnia parasitica, the causative agent of saprolegniosis in fish, and Aphanomyces astaci, the causative agent of crayfish plague, are oomycete pathogens that cause economic losses in aquaculture. Since toxic chemicals are currently used to control them, we aimed to investigate their inhibition by essential oils of sage, rosemary, and bay laurel as environmentally acceptable alternatives. Gas Chromatography–Mass Spectrometry (GC–MS) analysis showed that the essential oils tested were rich in bioactive volatiles, mainly monoterpenes. Mycelium and zoospores of A. astaci were more sensitive compared to those of S. parasitica, where only sage essential oil completely inhibited mycelial growth. EC50 values (i.e., concentrations of samples at which the growth was inhibited by 50%) for mycelial growth determined by the radial growth inhibition assay were 0.031–0.098 µL/mL for A. astaci and 0.040 µL/mL for S. parasitica. EC50 values determined by the zoospore germination inhibition assay were 0.007–0.049 µL/mL for A. astaci and 0.012–0.063 µL/mL for S. parasitica. The observed inhibition, most pronounced for sage essential oil, could be partly due to dominant constituents of the essential oils, such as camphor, but more likely resulted from a synergistic effect of multiple compounds. Our results may serve as a basis for in vivo experiments and the development of environmentally friendly methods to control oomycete pathogens in aquaculture.

Exploration of synergistic action of cell wall-degrading enzymes against Mycobacterium tuberculosis

Background The major global health threat tuberculosis is caused by Mycobacterium tuberculosis (Mtb). Mtb has a complex cell envelope – a partially covalently linked composite of polysaccharides, peptidoglycan and lipids, including a mycolic acid layer – which conveys pathogenicity but also protects against antibiotics. Given previous successes in treating gram-positive and -negative infections with cell wall degrading enzymes, we investigated such approach for Mtb. Objectives (i) Development of an Mtb microtiter growth inhibition assay that allows undisturbed cell envelope formation, to overcome the invalidation of results by typical clumped Mtb-growth in surfactant-free assays. (ii) Exploring anti-Mtb potency of cell wall layer-degrading enzymes. (iii) Investigation of the concerted action of several such enzymes. Methods We inserted a bacterial luciferase-operon in an auxotrophic Mtb strain to develop a microtiter assay that allows proper evaluation of cell wall degrading anti-Mtb enzymes. We assessed growth-inhibition by enzymes (recombinant mycobacteriophage mycolic acid esterase (LysB), fungal α-amylase and human and chicken egg white lysozymes) and combinations thereof, in presence or absence of biopharmaceutically acceptable surfactant. Results Our biosafety level-2 assay identified both LysB and lysozymes as potent Mtb-inhibitors, but only in presence of surfactant. Moreover, most potent disruption of the mycolic acid hydrophobic barrier was obtained by the highly synergistic combination of LysB, α-amylase and polysorbate 80. Conclusions Synergistically acting cell wall degrading enzymes are potently inhibiting Mtb – which sets the scene for the design of specifically tailored antimycobacterial (fusion) enzymes. Airway delivery of protein therapeutics has already been established and should be studied in animal models for active TB.