Single-Chain Fragment Variables Targeting Leukocidin ED Can Alleviate the Inflammation of Staphylococcus aureus-Induced Mastitis in Mice

Staphylococcus aureus is a vital bovine mastitis pathogen causing huge economic losses to the dairy industry worldwide. In our previous studies, leukotoxin ED (LukED) was detected in most S. aureus strains isolated from bovine mastitis. Here, four single-chain fragment variables (scFvs) (ZL8 and ZL42 targeting LukE, ZL22 and ZL23 targeting LukD) were obtained using purified LukE and LukD proteins as the antigens after five rounds of bio-panning. The complementarity-determining region 3 (CDR3) of the VH domain of these scFvs exhibited significant diversities. In vitro, the scFvs significantly decreased LukED-induced cell killing by inhibiting the binding of LukED to chemokine receptors (CCR5 and CXCR2) and reduced the death rates of bovine neutrophils and MAC-T cells caused by LukED and S. aureus (p < 0.05). In an S. aureus-induced mouse mastitis model, histopathology and MPO results revealed that scFvs ameliorated the histopathological damages and reduced the infiltration of inflammatory cells (p < 0.05). The ELISA and qPCR assays showed that scFvs reduced the transcription and expression levels of Tumor Necrosis Factor-alpha (TNF-α), interleukin-1β (IL-1β), IL-6, IL-8 and IL-18 (p < 0.05). The overall results demonstrated the protective anti-inflammatory effect of scFvs in vitro and in vivo, enlightening the potential role of scFvs in the prevention and treatment of S. aureus-induced mastitis.

Towards Synthesis of Simplified Analogues of Pateamine A

<p>Pateamine A (22) is a natural product that was isolated from a marine sponge inhabiting the coast of New Zealand. It exhibits potent inhibition of protein synthesis and nonsense-mediated mRNA decay through binding with eIF4A isoforms. Due to the scarcity of pateamine A (22) in the natural source and the low yield of total synthesis of pateamine A, it is necessary to prepare structurally simplified analogues which would allow further research on structure-activity relationships (SAR) of pateamine A (22). Based on the structure-activity relationship studies reported by Romo and co-workers, a simplified triazole analogue 182 lacking methyl groups was synthesized by Hemi Cumming, a previous Ph.D. student who studied at Victoria University of Wellington. The antiproliferative activity of this analogue was found to be significantly lower than that of pateamine A, suggesting that the thiazole embedded within the molecule or the excised methyl groups are crucial for its potency. Therefore, to further explore the necessary features for its selective activity for eIF4A isoforms, new thiazole analogues 183 – 186 and triazole analogues (10S)-and (10R)-analogue 187 were targeted in this project. The preparation of the thiazole-containing macrocyclic core of analogues 183 and 184 was achieved. It features: (1) gold-catalysed thiazole formation through coupling between an alkyne fragment and a thioamide fragment; (2) preparation of the Z,E-dienoate moiety by base-induced ring-opening of a δ-substituted-α, β-unsaturated lactone; and (3) a modified Mukaiyama macrolactonisation. The synthesis of the triazole-containing macrocyclic core of (10S)-analogue 187 was completed. It features: (1) a copper-catalysed triazole formation through 1,3-dipolar cycloaddition between an alkyne fragment and an azide fragment; (2) preparation of the Z,E-dienoate moiety by base-induced ring-opening of δ-substituted-α, β-unsaturated lactone; and (3) a modified Mukaiyama macrolactonisation. Studies on the preparation of a side-chain fragment with suitable functionalities to allow coupling with the various macrocycles through olefination reactions were also conducted. The attachment of the side-chain fragment onto the macrocyclic cores for the synthesis of the targeted analogues 183 and 184 and (10S)-analogue 187 will be investigated in future work. These experimental results will inform the synthesis of new generation analogues to further study the key structures required for effective binding to the protein target eIF4A and selectivity between isoforms.</p>

Towards Synthesis of Simplified Analogues of Pateamine A

<p>Pateamine A (22) is a natural product that was isolated from a marine sponge inhabiting the coast of New Zealand. It exhibits potent inhibition of protein synthesis and nonsense-mediated mRNA decay through binding with eIF4A isoforms. Due to the scarcity of pateamine A (22) in the natural source and the low yield of total synthesis of pateamine A, it is necessary to prepare structurally simplified analogues which would allow further research on structure-activity relationships (SAR) of pateamine A (22). Based on the structure-activity relationship studies reported by Romo and co-workers, a simplified triazole analogue 182 lacking methyl groups was synthesized by Hemi Cumming, a previous Ph.D. student who studied at Victoria University of Wellington. The antiproliferative activity of this analogue was found to be significantly lower than that of pateamine A, suggesting that the thiazole embedded within the molecule or the excised methyl groups are crucial for its potency. Therefore, to further explore the necessary features for its selective activity for eIF4A isoforms, new thiazole analogues 183 – 186 and triazole analogues (10S)-and (10R)-analogue 187 were targeted in this project. The preparation of the thiazole-containing macrocyclic core of analogues 183 and 184 was achieved. It features: (1) gold-catalysed thiazole formation through coupling between an alkyne fragment and a thioamide fragment; (2) preparation of the Z,E-dienoate moiety by base-induced ring-opening of a δ-substituted-α, β-unsaturated lactone; and (3) a modified Mukaiyama macrolactonisation. The synthesis of the triazole-containing macrocyclic core of (10S)-analogue 187 was completed. It features: (1) a copper-catalysed triazole formation through 1,3-dipolar cycloaddition between an alkyne fragment and an azide fragment; (2) preparation of the Z,E-dienoate moiety by base-induced ring-opening of δ-substituted-α, β-unsaturated lactone; and (3) a modified Mukaiyama macrolactonisation. Studies on the preparation of a side-chain fragment with suitable functionalities to allow coupling with the various macrocycles through olefination reactions were also conducted. The attachment of the side-chain fragment onto the macrocyclic cores for the synthesis of the targeted analogues 183 and 184 and (10S)-analogue 187 will be investigated in future work. These experimental results will inform the synthesis of new generation analogues to further study the key structures required for effective binding to the protein target eIF4A and selectivity between isoforms.</p>

Anti-Staphylococcus aureus Single-Chain Fragment Variables Play a Protective Anti-Inflammatory Role In Vitro and In Vivo

Staphylococcus aureus is a causative agent of bovine mastitis, capable of causing significant economic losses to the dairy industry worldwide. This study focuses on obtaining single-chain fragment variables (scFvs) against the virulence factors of S. aureus and evaluates the protective effect of scFvs on bovine mammary epithelial (MAC-T) cells and mice mammary gland tissues infected by S. aureus. After five rounds of bio-panning, four scFvs targeting four virulence factors of S. aureus were obtained. The complementarity-determining regions (CDRs) of these scFvs exhibited significant diversities, especially CDR3 of the VH domain. In vitro, each of scFvs was capable of inhibiting S. aureus growth and reducing the damage of MAC-T cells infected by S. aureus. Preincubation of MAC-T cells with scFvs could significantly attenuate the effect of apoptosis and necrosis compared with the negative control group. In vivo, the qPCR and ELISA results demonstrated that scFvs reduced the transcription and expression of Tumor Necrosis Factor alpha (TNF-α), interleukin-1β (IL-1β), IL-6, IL-8, and IL-18. Histopathology and myeloperoxidase (MPO) results showed that scFvs ameliorated the histopathological damages and reduced the inflammatory cells infiltration. The overall results demonstrated the positive anti-inflammatory effect of scFvs, revealing the potential role of scFvs in the prevention and treatment of S. aureus infections.

In Vivo Human Single‐Chain Fragment Variable Phage Display‐Assisted Identification of Galectin‐3 as a New Biomarker of Atherosclerosis

Background Atherosclerosis is a complex pathology in which dysfunctional endothelium, activated leucocytes, macrophages, and lipid‐laden foam cells are implicated, and in which plaque disruption is driven by many putative actors. This study aimed to identify accurate targetable biomarkers using new in vivo approaches to propose tools for improved diagnosis and treatment. Methods and Results Human scFv (single‐chain fragment variable) selected by in vivo phage display in a rabbit model of atherosclerosis was reformatted as scFv fused to the scFv‐Fc (single‐chain fragment variable fused to the crystallizable fragment of immunoglobulin G format) antibodies. Their reactivity was tested using flow cytometry and immunoassays, and aorta sections from animal models and human carotid and coronary artery specimens. A pool of atherosclerotic proteins from human endarterectomies was co‐immunoprecipitated with the selected scFv‐Fc followed by mass spectrometry for target identification. Near‐infrared fluorescence imaging was performed in Apoe −/− mice after injection of an Alexa Fluor 647–labeled scFv‐Fc‐2c antibody produced in a baculovirus system with 2 additional cysteine residues (ie, 2c) for future coupling to nano‐objects for theranostic applications. One scFv‐Fc clone (P3) displayed the highest cross‐reactivity against atherosclerotic lesion sections (rabbit, mouse, and human) and was chosen for translational development. Mass spectrometry identified galectin‐3, a β‐galactoside‐binding lectin, as the leader target. ELISA and immunofluorescence assays with a commercial anti‐galectin‐3 antibody confirmed this specificity. P3 scFv‐Fc‐2c specifically targeted atherosclerotic plaques in the Apoe −/− mouse model. Conclusions These results provide evidence that the P3 antibody holds great promise for molecular imaging of atherosclerosis and other inflammatory pathologies involving macrophages. Recently, galectin‐3 was proposed as a high‐value biomarker for the assessment of coronary and carotid atherosclerosis.

scFv-Anti-LDL(-)-Metal-Complex Multi-Wall Functionalized-Nanocapsules as a Promising Tool for the Prevention of Atherosclerosis Progression

Atherosclerosis can be originated from the accumulation of modified cholesterol-rich lipoproteins in the arterial wall. The electronegative LDL, LDL(-), plays an important role in the pathogenesis of atherosclerosis once this cholesterol-rich lipoprotein can be internalized by macrophages, contributing to the formation of foam cells, and provoking an immune-inflammatory response. Herein, we engineered a nanoformulation containing highly pure surface-functionalized nanocapsules using a single-chain fragment variable (scFv) reactive to LDL(-) as a ligand and assessed whether it can affect the LDL(-) uptake by primary macrophages and the progression of atherosclerotic lesions in Ldlr−/− mice. The engineered and optimized scFv-anti-LDL(-)-MCMN-Zn nanoformulation is internalized by human and murine macrophages in vitro by different endocytosis mechanisms. Moreover, macrophages exhibited lower LDL(-) uptake and reduced mRNA and protein levels of IL1B and MCP1 induced by LDL(-) when treated with this new nanoformulation. In a mouse model of atherosclerosis employing Ldlr−/− mice, intravenous administration of scFv-anti-LDL(-)-MCMN-Zn nanoformulation inhibited atherosclerosis progression without affecting vascular permeability or inducing leukocytes-endothelium interactions. Together, these findings suggest that a scFv-anti-LDL(-)-MCMN-Zn nanoformulation holds promise to be used in future preventive and therapeutic strategies for atherosclerosis.