High-throughput drug screening to identify novel small molecules to rescue Noonan syndrome phenotypes in Drosophila

Perundurai S Dhandapany1, Susumu Hirabayashi2, Elizabeth Horvath1, Kimihiko Oishi1, Ross L Cagan2 and Bruce D. Gelb1
1Departments of Pediatrics and Genetics & Genomic Sciences and the Child Health and Development Institute, 2Department of Developmental and Regenerative Biology, Mount Sinai Medical School, One Gustave L. Levy Place, Box 1020, NY 10029, USA

BACKGROUND: Noonan syndrome (NS) is a pleiomorphic developmental disorder, for which increased RAS pathway signaling is the primary cause. Gain-of-function mutations in PTPN11, encoding SHP-2, are the major cause. While powerful Ras signaling inhibitors are in development for cancer, such drugs may not be useful for long-term use in children with NS. To find drugs that restore normal Ras signaling in NS, we are using a transgenic Drosophila model with the D61G mutation in corkscrew mutation (cswD61G)the fly orthologue of PTPN11.

AIMS: We aimed to establish conditions for high-throughput drug screening with the cswD61G fly and to screen a limited panel of biologically relevant drugs to demonstrate proof of principle.

DESIGN/METHODS: To establish conditions of pupal lethality, which is optimal for high-throughput chemical screening, we crossed the UAS-cswD61G allele with several different Gal4 drivers (daughterless, actin, tubulin and patched) and cultured at temperatures ranging from 18-29oC to alter transgene expression levels. After establishing optimal driver and culture conditions, we performed a screen employing a panel of biologically relevant drugs at two concentrations (50 and 100 µM).

RESULTS/CONCLUSIONS: Most drivers were embryonic lethal, precluding their use, but patched-Gal4 exhibited 100% lethality at 23 °C, semi-viability at 21 °C, and full viability at 18 °C. Using 23 °C as the “barely lethal” condition, we screened 14 drugs/chemicals acting on RAS, NOTCH, mTOR and PI3 kinase signaling. The mTOR and PI3 kinase inhibitors, rapamycin and wortmannin, respectively, failed to rescue lethality. The remaining 12 compounds achieved rescue at varying extents. Sunitinib, which is a multiple kinase inhibitor including RTK, had maximal rescue of 60% at 50 µM. Sorafenib, a multiple RTK and RAF inhibitor, rescued more consistently nearly 40% of pupae, so will be used for future chemical library screening. Of interest, we also observed rescue by inhibitors of JNK (SP5600125), AKT (10_DEBC), Rac (NSC23766), and Notch/Presinilin (DAPT). We are now initiating a screen of  640 FDA-approved compounds using a 96-well plate format with robotic dispensing of the embryos.