García Pérez, Angela (2018) Investigation of Siglec8 as a therapeutic target to treat asthma. PhD thesis, University of Warwick.

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Abstract

Siglecs (Sialic Acid Immunoglobulin-like Lectins) are immune system protein receptors that play important roles in the down-regulation of cellular signals upon Sialic Acid ligand binding. At present, the human Siglec family is composed of 16 members expressed by specific immune system cellular types. Due to their highly restrictive expression patterns and key regulatory function, Siglecs have been pointed out as interesting pharmaceutical targets to treat certain immune diseases where those specific cellular types are involved.

This work is focused on Siglec8 which is expressed on the surface of mast cells and eosinophils and has demonstrated to induce their apoptosis upon ligand binding. This effect could be exploited to develop therapeutics that target and reduce the levels of eosinophils in the inflammatory diseases such as asthma where they are the main effectors. The aim of this thesis is to design some Siglec8-ligand mimetic small molecules, test their binding to the receptor and obtain affinity data about the interaction.

We managed to express the Siglec8 Ig-like V-type carbohydrate binding domains in Escherichia coli, refold and purify them in solution and perform a biophysical characterization by means of different techniques such as Fluorescence, Absorbance, Mass Spectrometry (MS), Circular Dichroism (CD) and Single-Angle Neutron Scattering (SAXS). We also designed nine small Siglec8-ligand mimetic molecules and tested their binding to the receptor domains using the Octet technology and equilibrium dialysis-1 D H⁺ NMR measurements. We found the ligand Phospho-Tyrosine to be our best Siglec8 binder and estimated a dissociation constant around 100 μM for the interaction. Thus, the binding information obtained in this work could lead to the development of higher affinity and specific molecules that target Siglec8.

This thesis also encompassed an industrial collaboration with the company Mologic Ltd where anti-Siglec8 antibodies where developed with chronic lung disease diagnostic purposes.

Item Type:	Thesis or Dissertation (PhD)
Subjects:	Q Science > QD Chemistry
Library of Congress Subject Headings (LCSH):	Asthma -- Treatment, Lectins -- Therapeutic use, Pharmaceutical chemistry, Glycoproteins, Sialic acids, Drug targeting, Binding sites (Biochemistry), Cell receptors
Official Date:	March 2018
Dates:	Date Event March 2018 Submitted
Institution:	University of Warwick
Theses Department:	Department of Chemistry
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Dixon, Ann M.
Sponsors:	Engineering and Physical Sciences Research Council ; Mologic Ltd.
Format of File:	pdf
Extent:	163 pages : illustrations, charts
Language:	eng

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