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derivative), was also present in the NCC003 library. Of interest, Irinotecan had

no effect on the ERBB4/PIK3R1 co-culture assay. Irinotecan is a pro-drug that has to be activated in the liver (Xie et al., 2003). As the co-culture assay cannot provide a metabolic functionality that is present in the liver, I tested the activated Irinotecan-metabolite SN38 (Chabot 1998). SN38 did not show any inhibitory or activatory activity in the ERBB4/PIK3R1 co-culture assay (Figure 66). The analysis of Irinotecan and SN 38 suggests that the measured effect is Topotecan-specific and not a general effect of Camptothecin analogues.

5.8.4.4 Summary validation Topotecan

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NTEV-tevS-GV CTEV Drug IC50

ERBB1 ERBB1 EGF 0.5

ERBB4 PIK3R1 co-culture assay 0.4

ERBB4 PIK3R1 SN38 90.6

Table 16: Summary of IC50 values

From these data, Topotecan was found to be an inhibitor of ERBB dimerisation and adapter recruitment. Binding of Topotecan to ERBB receptors has to be further validated. However, the severe side effects of Topotecan, resulting from its anti-topoisomerase I effects, makes the usage of Topotecan for the treatment of psychiatric patients highly unlikely (Alimonti et al., 2004). To use the potential of Topotecan in this setting, an in-depth analysis of the underlying mechanisms related to the toxicity has to be performed. Only then, it might be worth to look at new lead structures for further developments.

Figure 59: ERBB4/PIK3R1 co-culture assay

A) ERBB4 was fused to NTEV-tevS-GV-2HA; PIK3R1 was fused to CTEV-2HA. Cells were stimulated with 10ng/ml EGFld.

Figure 60: Technical controls for Topotecan

A) Data set from the NCC003 screen. Topotecan significantly inhibits Nrg1-typeI-stimulated ERBB4/PIK3R1signalling. B) Renilla luciferase assay. Cells were transfected with 20ng/well TK-Rluc. C) GV control assay in PC12 cells. Cells were transfected with 20ng/well CMV-GV, UAS-Fluc, and TK-Rluc. D) TM-TEV/TM-GV control assay in PC12 cells. Cells were transfected with 20ng/well TM-TEV and TM-GV.E) Datasets shown from (B) to (D) were merged. The ERBB4/PIK3R1 assay is shown in red.

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Figure 61: Vertical validation Topotecan ERBB4/adapter recruitment

A) ERBB4/adapter recruitment. ERBB4 was fused to NTEV-tevS-GV-2HA; adapters were fused to CTEV-2HA. Cells were stimulated with 10ng/ml EGFld. B-F) ERBB4/adapter recruitment. ERBB4 was fused to NTEV-tevS-GV-2HA; adapters were fused to CTEV-2HA. PC12 cells were stimulated with 10ng/ml EGFld. G) Comparison of ERBB4/adapter recruitment assays to ERBB4-PIK3R1 recruitment assay (red). ERBB/adapter recruitment for SHC1, GRB2, and STAT5A are less efficiently inhibited compared to the ERBB4/PIK3R1 recruitment.

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Figure 62: Homo and heterodimerisation of ERBB receptors

A) ERBB homo- and heterodimerisation. ERBBx/ERBBy split TEV assay in PC12 cells.

ERBBx was fused to NTEV-tevS-GV-2HA; ERBBy was fused to CTEV-2HA. Cells were stimulated with 10 ng/ml EGFld. B-K) ERBB homo- and heterodimerisations. ERBBx was fused to NTEV-tevS-GV-2HA; ERBBy was fused to CTEV-2HA. PC12 cells were stimulated with 10 ng/ml EGFld. L) Comparison of ERBB homo- and heterodimerisation assays to ERBB4-PIK3R1 recruitment. ERBB homo/heterodimerisations are less efficiently inhibited compared to ERBB4-PIK3R1 recruitment.

Figure 63: Summary on Topotecan validation

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Figure 64: Horizontal validation ERBB2/ERBB3/adapters

A) Schematic representation ERBB2/ERBB3 heterodimerisation. B) Assay performance.

ERBB2/ERBB3 heterodimerise after stimulation with 10 ng/ml EGFld and recruit adapters as indicated. C) Comparison of the z’-factors obtained from the assays. D-H) ERBB2/ERBB3/various adapters. ERBB2 was fused to a V5 tag, ERBB3 was fused to NTEV-tevS-GV-2HA; adapters were fused to CTEV-2HA. PC12 cells were stimulated with 10 ng/ml EGFld.

Figure 65: Horizontal validation for Topotecan’s effect on ERBB2/ERBB4/adapters A) Schematic representation ERBB2/ERBB4 heterodimerisation. B) Assay performance.

ERBB2/ERBB4 heterodimerise after stimulation with 10 ng/ml EGFld and recruit adapters as indicated. C) Comparison of the z’-factors obtained from the assays. D-H) ERBB2/ERBB4/various adapters. ERBB2 was fused to a V5 tag, ERBB4 was fused to NTEV-tevS-GV-2HA; adapters were fused to CTEV-2HA. PC12 cells were stimulated with 10ng/ml EGFld.

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Figure 66: Effect of Topotecan derivatives Irinotecan and SN38 on the ERBB4/PIK3R1 split TEV assay

A) Comparison of the chemical structures of Camptothecin derivates. Chemical structures of Camptothecin, Topotecan, Irinotecan, SN38. B) Data set from the NCC003 screen.

The difference between Topotecan and Irinotecan on the ERBB4/PIK3R1co-culture screen is shown. C-D) Individual effects of Topotecan (C) and SN38 (D).

ERBB4 was fused to NTEV-tevS-GV-2HA; PIK3R1was fused to CTEV-2HA. Cells were stimulated with 10ng/ml EGFld.

Figure 67: Effect of Topotecan on ERBB1/1 dimerisation induced by EGF

A) ERBB1 was fused to NTEV-tevS-GV-2HA; ERBB1 was fused to CTEV-2HA. PC12 cells were stimulated with 1 ng/ml EGF. B) Activation window of the EGF-induced ERBB1/1 assay. Setup as in (A).

Figure 68: Effect of Topotecan on the FRB/FKBP interaction induced by Rapamycin A) TM-FRB was fused to NTEV-tevS-GV, FKBP was fused to CTEV. PC12 cells were stimulated with 10nM Rapamycin. B) Activation window of the Rapamycin-induced FRB/FKBP assay. Setup as in (A).

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