Investigating the anti-inƒammatory activity of a fragment of Chaperonin 60.1 derived from Mycobacterium tuberculosis

  • Francis Alexandre Wei Ming Man

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

Chaperonin 60.1 (Cpn60.1) from Mycobacterium tuberculosis is a protein that has evolved from the traditional role of chaperone in protein folding to a role in extracellular signalling. While non-essential to the growth and survival of the bacteria, Cpn60.1 is essential to the formation of granuloma in tuberculosis [150]. Cpn60.1 has been shown to induce cytokine production in human monocytes [201], but also to prevent leukocyte migration in murine models of allergic lung inflƒammation [300].
Peptide 4, a fragment of Cpn60.1, has been shown to be responsible for the cytokineinducing activity of Cpn60.1 in monocytes [149] and has been shown in our laboratory to prevent leukocyte migration in models of allergic and non-allergic inƒflammation. We believe that Peptide 4 would be an interesting lead compound or drug candidate for the treatment of diseases in which leukocyte in€ltration plays a major role, such as chronic obstructive pulmonary disease (COPD) or acute respiratory distress syndrome (ARDS). However, most of the knowledge regarding Peptide 4 originates from in vivo experiments and little is known about its mechanism of action. In addition, due to the size and nature of Peptide 4, there is potential for improvement in its drug-likeness while retaining its anti-inƒflammatory activity.
The Œprincipal objective of this thesis was to develop in vitro screening assays for shorter fragments of the biologically active Peptide 4, as a preliminary step in developing a novel anti-inflƒammatory drug candidate. A number of functions of human neutrophils and mouse bone marrow cells were explored, such as adhesion molecule expression, cytoskeleton reorganisation, chemotaxis, adhesion to extracellular matrix proteins, platelet-leukocyte aggregate formation, and granule release. ThŒese assays were performed on isolated cells and whole blood.
Because in vivo experiments revealed an e‚ffect of Peptide 4 on the expression of endothelial adhesion molecules and circulating pro-inƒammatory cytokines, the peptide was also assayed in cultured human endothelial cells. Adhesion molecule expression was measured by ƒow cytometry and immunoƒfluorescence microscopy. Cytokine release was measured by ELISA. Labelled peptides were used in attŠempts to observe the binding to cells.
In each case, the focus was on fi€nding an assay that would be convenient for screening purposes and relevant to the intended use of the compound as a drug for the treatment of human diseases.
In addition, solid-phase synthesis of peptides derived from Peptide 4, including scrambled and ƒfluorescent derivatives, was performed. Most synthesised peptides were tested in vivo for early structure-activity relationship studies. Some of the peptides contained flƒuorescent labels or a�ffinity tags for imaging purposes. Some peptides were analysed by circular dichroism spectroscopy to obtain information about their secondary structure and behaviour in diff‚erent environments.
Several assays were developed in murine and human leukocytes. Œe results showed that Peptide 4 did not aff‚ect selectin or integrin expression, migration toward chemoattractants, adhesion to €bronectin or endothelial cells, cell polarisation or the formation of platelet-leukocyte aggregates. Similarly, Peptide 4 did not a‚ffect the expression of adhesion molecules or the release of IL-6 and IL-8 by HUVECs. Altogether, the results suggested the eff‚ect of Peptide 4 was not directly mediated by neutrophils or endothelial cells.
Experiments with ƒfluorescent peptides were inconclusive, as consistent binding to the cells could not be achieved. In vivo testing of synthetic derivatives of Peptide 4 showed that a free N-terminus was important for biological activity, and that the lysine sidechain could be used as an anchor point for functional groups such as biotin or flƒuorescent dyes.
In vivo studies also pointed towards an increased release of monocyte-attŠracting cytokines, and it was demonstrated that lung-resident phagocytes are essential for to the biological activity of Peptide 4.
Date of Award2016
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorClive Page (Supervisor) & Sukhi Bansal (Supervisor)

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