Screening for small molecule inhibitors

A functional nucleotide exchange assay was to be established with regard to screening for small molecule inhibitors of the Rin1-mediated Rab5 activation. The compounds were, as in most HTS approaches, only tested in single values. Therefore the assay had to be exact and sensitive in the identification of hits and also robust enough to avoid false positives²³². This required a wide span between the signals of the positive control (GEF-mediated nucleotide exchange) and the negative control (no GEF-mediated nucleotide exchange)²³³. The assay properties could be quantified by use of the Z’ factor. It describes the assay quality and enables to judge, whether an assay is in general suitable for HTS approaches²³⁴.

3.3.1 Establishing the Bodipy-TR-GTP nucleotide exchange screening assay

The fluorescently labelled GTP analogue Bodipy-TR-GTP has previously been used in similar screening approaches and led to promising results^214,235. The Rin1C/Rab5a Bodipy-TR-GTP exchange assay was chosen to be optimized for HTS. Rin1C catalysed the nucleotide exchange on Rab5a in a concentration-dependent manner (cp. figure 16a), similar as previously described for Rabex-5¹³¹. First, different concentrations of Rin1C were tested again over a longer period to find conditions with a linear phase long enough to allow appropriate calculation of the slope. Figure 17 shows the Rin1C-mediated nucleotide exchange on Rab5a. As expected, higher concentrations of Rin1C again led to a faster nucleotide exchange. The reaction reached saturation at approx. 2100 fluorescence units.

The use of 200 nM Rin1C resulted in a linear increase of the fluorescence intensity over the first 20 minutes and was therefore chosen as appropriate to be used during the screening.

Figure 17: The Rin1C-mediated Bodipy-TR-GTP nucleotide exchange on Rab5a. Rin1C dose-dependently catalysed the nucleotide exchange on Rab5a. The nucleotide exchange was measured by monitoring the Bodipy-TR-GTP fluorescence at 595 / 620 nm (Ex. / Em.). Without Rin1C almost no nucleotide binding occurred (black curve). 1 µM Rab5a and 2 µM Bodipy-TR-GTP have been used. 200 nM Rin1C resulted in a linear increase of the fluorescence intensity over the first 1200 seconds.

53 3.3.2 Assay optimization for HTS

To increase the sample throughput, the assay had to be converted to an endpoint measurement instead of measuring the kinetics. This allowed the measurement of a whole 384 well plate at once in a time-delayed manner as described in section 7.2.3.1.1. The fluorescence intensity was measured once directly after Bodipy-TR-GTP addition (t = 0 min.) and then again after 20 minutes. From those values, the difference in fluorescence intensity (ΔFI) was calculated for each well. A small ΔFI could be measured for the negative control without Rin1C while the ΔFI in presence of 200 nM Rin1C was much higher. Figure 18a and b illustrate the conversion from the kinetic to the endpoint measurement. Since the library compounds were dissolved in dimethyl sulfoxide (DMSO), the DMSO tolerance had to be tested. The results are shown in Figure 18c. There was no significant difference between 0 – 6 % DMSO detectable. 4 % DMSO was necessary to use 40 µM as the final compound concentration and was found to be tolerated in the Bodipy-TR-GTP exchange assay.

Figure 18: Adjustment of the Bodipy-TR-GTP assay for a HTS approach. a → b: Conversion of the kinetic measurement to an endpoint measurement. ΔFI could be calculated from the fluorescence intensities at t = 0 min. and t = 20 min. c: DMSO tolerance of the Bodipy-TR-GTP exchange assay. There was no significant difference in the ΔFI between 0 – 6 % DMSO. The significance was calculated by one-way ANOVA, p < 0.05 using GraphPad Prism software.

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With this conversion, the assay was expected to be suitable for HTS. During the screening the hits should exhibit lower ΔFI values compared to the DMSO controls (32 per plate).

Other important considerations were the type of plate to be used, the buffer conditions and the order of sample preparation. Bodipy-TR-GTP turned out to be sticky on most plastic surfaces, therefore only plates with non-binding surface (NBS) yielded reasonable results.

Moreover the use of detergents (tested: Tween-20, Triton X-100, Nonidet P-40, sodium cholate, CHAPS and IGEPAL CA-630, data not shown) dramatically increased the fluorescence signal of Bodipy-TR-GTP and simultaneously the error between replicates, leading to bad assay quality. In terms of assay preparation order, robot-assisted addition of compounds/DMSO into the plates followed by mixing Rab5a and Rin1C in exchange buffer and manual addition of the proteins gave the best results (lowest variance between replicates). Bodipy-TR-GTP was finally injected at the Tecan infinite M1000 pro plate reader directly before starting the time-delayed measurement.

3.3.3 Calculation of the Z’ factor

To minimize the probability of false positives and false negatives during a HTS, the screening assay has to meet certain quality criteria. False positives would be time- and material consuming to be re-tested after the screening while false negatives would mean missing out on promising compounds. The Z’ factor, a statistical parameter to judge assay quality, has been calculated as follows:

𝑍^′𝑓𝑎𝑐𝑡𝑜𝑟 = 1 −3(𝜎_𝑝+ 𝜎_𝑛)

|𝜇_𝑝− 𝜇_𝑛|

It is defined in terms of the means (µ) and the standard deviations (σ) of the positive (p) and the negative (n) controls. The closer the Z’ factor to its maximum 1, the higher the assay quality. A Z’ factor between 0.5 and 1 defines an excellent assay that is suitable for HTS approaches²³⁴.

A 384 well plate with 192 positive controls and 192 negative controls (without Rin1C) has been measured in the time-delayed setup described in section 7.2.3.1.1. The corresponding results are shown in figure 19. The Z’ factor was calculated to be 0.57 for the Bodipy-TR-GTP screening assay, indicating suitability for HTS.

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Figure 19: Determination of the Z’ factor for the Bodipy-TR-GTP screening assay. 192 positive controls (green dots, 4% DMSO, 1 µM Rab5a, 200 nM, Rin1C and 2 µM Bodipy-TR-GTP) and 192 negative controls (red squares, without Rin1C) were measured in the time-delayed screening setup followed by calculation of the means ± standard deviations. The Z’ factor was calculated to be 0.57.

3.3.4 The compound library

An in-house compound library has been used for the screening. It contained 20 328 compounds in total of which the majority has been purchased at ComGenex. The library also contained many additional molecules that have been synthesized by members of the groups of Prof. Dr. M. Famulok, Prof. Dr. M. Gütschow and other groups from the University of Bonn.

3.3.5 High-throughput screening results

The screening of 20 328 compounds was performed under the conditions described in section 3.3.2 with 32 DMSO controls per plate (in columns 1 and 24). During the evaluation the ΔFI values for each compound sample and each control sample were calculated.

Compounds that resulted in ≥ 80 % inhibition (equalling ≤ 20 % residual nucleotide exchange activity) compared to the DMSO controls from the same plate were defined as hits. This procedure identified 239 primary hits, which correlates to 1.2 % of the total compounds.

3.3.6 Identification of secondary hits

The primary hits were re-screened manually as duplicates in two different concentrations:

40 µM and 10 µM. By this, 26 secondary hits showing ≥ 50 % inhibition (at 40 µM) could be identified. This equals 10.9 % of the primary hits. Correspondingly 213 (89.1 %) of the primary hits were found to be false positives of which some compounds had to be excluded due to strong quenching of the Bodipy-TR-GTP fluorescence signal. Quenchers usually interfere with the read-out rather than with the nucleotide exchange reaction. The results of the re-screen are depicted in figure 20. The 26 secondary hits were then tested from frozen 10 mM compound stock solutions to assure the inhibition does not result from degradation

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products in the library plates. Degradation could have occurred during several thaw-freezing cycles in earlier screening approaches. Only 10 of the 26 secondary hits were found to inhibit the nucleotide exchange when the stock solution was tested. Due to insufficient information on possible degradation byproducts, the 16 compounds that did not show an inhibitory effect were omitted from further investigation. The 10 remaining compounds were chosen to be characterized. A complete list of the secondary hits is provided in the appendix (section 8.3).

Figure 20: Manual re-screening of the 239 primary hit compounds. The positive controls (4 % DMSO, 1 µM Rab5a200 nM, Rin1C and 2 µM Bodipy-TR-GTP) are shown as green dots and the negative controls (without Rin1C) as red squares. The re-screened compounds (40 µM, mean of duplicates) are depicted as blue triangles.

Compounds that exhibited ≥ 50 % inhibition at 40 µM were defined as secondary hits. Those are represented by the 26 blue triangles beneath the dotted line. The grey diamonds represent the compounds tested at a concentration of 10 µM (mean of duplicates). Only four molecules showed ≥ 50 % inhibition at 10 µM.