Hamann, Maren: The role of guanine nucleotide exchange factors (GEFs) in EGF-receptor signalling : Screening for a small molecule inhibitor of the Rin1-mediated Rab5 activation. - Bonn, 2018. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5n-50691
@phdthesis{handle:20.500.11811/7562,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5n-50691,
author = {{Maren Hamann}},
title = {The role of guanine nucleotide exchange factors (GEFs) in EGF-receptor signalling : Screening for a small molecule inhibitor of the Rin1-mediated Rab5 activation},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2018,
month = jun,

note = {The small GTPase Rab5 is a key regulator of early endosomal trafficking. It functions as a molecular switch that can be activated by guanine nucleotide exchange factors (GEFs) and inactivated via hydrolysis of GTP with the help of catalytic GTPase activating proteins (GAPs). GEFs mediate the exchange of GTPase-bound GDP for GTP. One of at least nine Rab5 GEFs, that have been identified until today, is Rin1. However, the differences in the exact roles of these GEFs are not completely understood. Rin1 is a multi-domain protein that has multiple signalling functions alongside the Rab5 activation. It is for example also involved in ABL kinase signalling, where it increases ABL activation. Rab5 over-activation has been reported to be involved in the genesis and progression of many different types of cancer and it could often be traced back to Rin1 over-expression. On the other hand Rin1 has been found to have a tumour repressive effect, mediated by its ABL kinase signalling function.
This study aimed on the identification of a small molecule inhibitor of the Rin1-mediated Rab5 activation. Such a small molecule inhibitor could be used as a tool to unravel parts of the complex signalling networks around Rab5. Ideally the inhibitor should be specific for Rin1 over other GEFs and moreover not influence its ABL kinase signalling function.
A high-throughput in vitro screening of more than 20 000 small molecules has been performed. The screening assay monitored the Rin1-catalyzed nucleotide exchange on Rab5a by the use of the fluorescently labelled GTP analogue Bodipy-TR-GTP. Amongst other primary hits the compound CG3 05 A02 was identified. During specificity and aggregation studies it turned out to be the most promising candidate.
Characterization of CG3 05 A02 revealed that its inhibitory effect in the Bodipy-TR-GTP nucleotide exchange assay is specific for Rin1 and Rabex-5 on Rab5a over several other GEF/GTPase pairs. The IC50 values were in both cases found to be in the low micromolar range. Rin1 and Rabex-5 share the homologous catalytic Vps9 domain and the compound might also be able to inhibit other Vps9 domain-containing GEFs. The compound did not induce aggregation of Rin1 or Rab5a and had no unspecific off-target effects in an unrelated insulin receptor auto-phosphorylation assay. It moreover did not influence the interaction between Rin1 and ABL1, indicating no intramolecular domain-unspecific inhibition. Unfortunately a control experiment with radioactively labelled GTP showed no inhibitory effect of the comound. The inhibition therefore depends on the use of Bodipy-TR-GTP but unlikely originates from CG3 05 A02 binding to the GTP analogue. In this case it would have inhibited the Bodipy-TR-GTP nucleotide exchange assay when GEFs/GTPases other than Rin1/Rabex-5 and Rab5 were used. This label-dependence classifies the compound unsuitable for cellular or in vivo application.
The actual target of CG3 05 A02 could not be finally addressed but Bodipy-TR-GTP binding to Rab5a in absence of a GEF was not influenced by the compound. This argues against GTP-competitive binding as the mechanism of inhibition. The compound potentially targets either directly the Vps9 domains of Rin1 and Rabex-5, areas adjacent to these, or the complex between the GEFs and Rab5. A mechanism based on steric hindrance involving the compound and the Bodipy-TR moiety of the GTP analogue was proposed as the mode of action.
Identification of a binding site for CG3 05 A02 in ongoing crystallization approaches could provide a starting point for in silico screenings that can result in a second generation of inhibitors of the Rin1-mediated Rab5 activation.},

url = {http://hdl.handle.net/20.500.11811/7562}
}

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