Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.693870
Title: Novel fragment library approaches against metalloenzymes and their downstream biological targets
Author: Patel, Neki Vijay
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2012
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Abstract:
Rab27a is a small GTPase that is implicated in cancer and other diseases e.g. Griscelli syndrome. Rab27a is post-translationally modified (prenylated) by Rab geranylgeranyltransferase (RGGT), a prenyltransferase (PT). Prenylation is essential for Rab27a's function as a membrane-interacting regulator of vesicular trafficking. A link between Rab27a and matrix metalloproteinases (MMPs), enzymes that degrade extracellular matrix proteins, has been established. PTs and MMPs have also been implicated in disease, and selective inhibition of an enzyme in one of these classes may lead to novel treatments for a variety of conditions. This thesis describes the development of a novel library approach that exploits the metal-binding hydroxamic acid (HA) fragment (binds to the catalytic zinc ion in PTs or MMPs) linked to a variable peptide fragment (selectivity element to prevent toxicity due to broad-spectrum inhibition). The peptide fragment, a powerful method for achieving high conformational space, was generated as a 'one -bead one- peptide' library via solid phase peptide synthesis (SPPS), and azide- alkyne 'click chemistry' was exploited to incorporate the HA 'warhead'. This 'small molecule-peptide chimera library' enabled the simultaneous testing of a plethora of fragment combinations without the need for the laborious synthesis of each one. An efficient chimera library screening process was established using far -red wavelength dye-labelled enzyme and fluorescence activated cell sorting (FACS) to select the most intensely fluorescent beads (hits) for LC-MS/MS-based deconvolution. PEAKS software was used for the de novo peptide hit reconstruction. Screening was successfully conducted for several MMPs and PTs, and structure-activity relationships were identified. Novel activity assays were developed for two PTs (farnesyltransferase (FTase) and geranylgeranyltransferase-I (GGTase-I )), and the interaction of Rab27a (a prenylation target of RGGT) with downstream effectors, was explored. A fragment library was screened against Rab27a using differential scanning fluorimetry (DSF), several hits were identified, and NMR spectroscopy of Rab27a was also investigated.
Supervisor: Tate, Ed Sponsor: Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.693870  DOI: Not available
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