Considerations for future screening approaches

5.2.1 Insights from the Rin1C/Rab5a Bodipy-TR-GTP nucleotide exchange assay Scientists planning on performing a HTS can certainly benefit from this study.

First of all it has to be kept in mind that the use of fluorophore labels influences the assay conditions in one way or the other. Therefore a fluorescence-independent control should be performed at an early stage after the screening, when a fluorescence-based method has been employed. By this, artefacts resulting from the use of fluorescently labelled material can be sorted out at an early time.

Next the use of a detergent in the screening buffer might have prevented hitting compounds that later turned out to be aggregators. This is, of course, only possible when the detergent does not interfere with the assay read-out, as it was seen in this study.

Lastly it has to be noted that in retrospect an assay monitoring the release of fluorescently labelled GDP (rather than the association of fluorescently labelled GTP) would have been preferable as in this setting CG3 05 A02 had no inhibitory effect.

5.2.2 Successful HTS approaches crucially depend on library size and quality

Many recent HTS approaches have been performed with libraries containing a way larger number of small molecules than the library used during this study250–253. Certainly a larger number of compounds raises the probability to find a suitable inhibitor.

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Moreover the appropriate storage of compound libraries was described to be as important as the HTS itself259. Reoccurring freeze-thaw cycles have been reported to induce compound precipitation and therefore change the compound concentration in the long term260. A mixture of water, DMSO and glycerol (10:45:45) was described to prevent compound precipitation and could be contemplable as a solvent for long-term storage261.

Compounds were also described to decay over longer storage periods, leading to the accumulation of impurities in the library262. Impurities can falsify screening results as they either might show effects in the screening assay, leading to false positive hits, or are inactive in contrast to the compound they originate from. The latter would result in false negative data, meaning missing out on a potentially promising compound.

Automated compound quality control should be undertaken at frequent intervals to assure the actual compound concentrations correspond to the theoretically desired library concentrations263. Solubility and purity should also be checked during quality controls. Library composition, age and storage conditions influence the outcome of HTS approaches and should always be considered during the planning of such.

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6 Conclusions

GTPases are molecular switches that are extensively involved in the regulation of gene expression, membrane trafficking and endocytosis18. They become activated by GEFs and inactivated via the hydrolysis of GTP with the help of catalytic GAPs12. Often a variety of different GEFs can activate the same GTPase and their distinct functions in the affected signalling cascades are not completely understood. The GTPase Rab5 can get activated by several GEFs, amongst them Rin1 and Rabex-5. Rab5 GEFs contain a catalytic Vps9 domain that mediates the nucleotide exchange100.

Small molecule inhibitors have proven to be valuable tools in the investigation of protein- protein interactions154. GTPases and GEFs are interesting and yet challenging targets due to their smooth surface that lacks obvious grooves and pockets suitable for small molecule interactions21,160,176. The homologous Vps9 domain shared by Rab5 GEFs makes it moreover difficult to target them specifically100. Small molecule inhibitors of Rab5 GEFs would help to shine light on the complex signalling networks around Rab5.

This study aimed on the identification of an inhibitor of the Rin1-mediated Rab5 activation in an in vitro high-throughput screening (HTS) approach. The screening yielded the small molecule CG3 05 A02 that specifically inhibited the Rin1C and Rabex-5GEF-mediated Bodipy-

TR-GTP nucleotide exchange on Rab5a without aggregating the proteins involved. The IC50

values of CG3 05 A02 in both cases were in the low micromolar range. The compound did not inhibit the Bodipy-TR-GTP nucleotide exchange between several other GEF/GTPase pairs, although the exchange mechanisms were described to be similar. However, it might still be able to inhibit other Vps9 domain containing Rab5 GEFs, as by now only Rin1C and Rabex- 5GEF have been tested. No unspecific inhibition could be seen in an unrelated insulin receptor

auto-phosphorylation assay and the compound did moreover not influence the interaction between Rin1 and ABL1. This interaction is mediated by the proline-rich region of Rin1 that is located adjacent to the Vps9 domain. Accordingly CG3 05 A02 did not show a domain- unspecific intramolecular effect on the proline-rich region of Rin1. A compound like this could, theoretically, be used to oppress tumorigenic effects mediated by Rab5 over- activation without influencing the tumour suppressive ABL1 signalling functions of Rin1123–

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During several control experiments it became, however, apparent that CG3 05 A02 does not inhibit the Rin1C and Rab3x-5GEF-mediated nucleotide exchange when radioactively labelled

GTP is being used. This unfortunately characterizes the compound as unsuitable for cellular or in vivo applications.

Structure-activity relationship analyses revealed the importance of a methoxy group that might perform a ring closure reaction with a neighbouring secondary amine. This group seems to be crucial for the compound to exhibit its inhibitory effect.

Binding studies failed to unravel whether Rin1C/Rabex-5GEF, Rab5a or the complex between

those proteins is the actual target of CG3 05 A02. However, loading of Rab5a with Bodipy- TR-GTP was not influenced by CG3 05 A02 and therefore GTP-competitive binding to Rab5a is unlikely. The compound could hypothetically bind close to the GTP binding site on the complex between Rin1C/Rabex-5GEF and Rab5a and prevent Bodipy-TR-GTP binding due to

steric hindrance. The Bodipy-TR moiety could prevent the nucleotide from entering the binding site when CG3 05 A02 is attached to the complex. This mode of action could also apply when the compound binds Rin1C and Rabex-5GEF at or adjacent to the Vps9 domain

and this protein-compound complex stays intact upon interaction with Rab5a. The much smaller GTP can, however, still enter the binding pocket, even when CG3 05 A02 is bound.

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To avoid investing in label-dependent compounds that might be found in future screenings a fluorescence-independent control should be performed promptly after the HTS. The right choice of the compound library to be screened is also an important issue that has to be thoroughly considered when a HTS should be performed259.

Identification of a possible binding site of CG3 05 A02 on Rin1C/Rabex-5GEF or the complex of

those with Rab5a in crystallization studies might pinpoint a protein cavity that could potentially be used for in silico HTS approaches. By this a second generation of inhibitors of the Rin1-mediated Rab5 activation could be identified. These second generation compounds could help to create a greater understanding of the signalling cascades involving Rab5 activation.

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