Development of antagonists of growth hormone-mediated signal transduction

Abstract

Substantial evidence implicates the adverse effect of human growth hormone GH in cancer Currently pegvisomant is the only clinically available GH receptor GHR antagonist This thesis aimed to develop alternative approaches for inhibiting GH function in vitro and explored extraction approaches to identify small molecules that interact with growth hormone binding protein GHBP which is the extracellular domain of the receptor by liquid chromatography tandem mass spectrometry LC MS MS Small molecule GHR antagonists were screened based on an established discovery pipeline By testing the compound inhibition of GH dependent cell survival and using a counter screen one compound was identified from 24 potential compounds Further investigation included an AlphaScreen assay and western blot to examine inhibition of GH dependent STAT5 phosphorylation lactate dehydrogenase LDH assay to test compound cytotoxicity and an in vitro kinase screen SelectScreen to check compound inhibition against GH related kinases One compound was identified with reasonable inhibitory activity but significant off target effects A second round screen was performed and column chromatography purification was conducted to eliminate contaminants Compound 2 041 was identified with high potency against GH dependent molecular and cellular outcomes with few off target effects Three extraction strategies for small molecules that interact with GHBP were explored for LC MS MS analysis In each assay the compounds were first incubated with protein and then either collected from the supernatant or eluted from protein followed by signal detection by LC MS MS Using either a plate based or bead immobilised extraction approach there was no difference in the compound signal between control and protein incubated groups A final ultrafiltration extraction approach identified compounds that was enriched following incubation with GHBP However the signal abundance ratio for the compound in the GHBP incubation group versus the control group was inconsistent across repeat experiments Finally a neutralising anti GH antibody was generated by hybridoma approach and neutralising activity and specificity determined Initially a small panel of commercial anti GH antibodies was assessed for neutralising activity against GH by a cell viability assay then anti GH mouse antibodies were generated by using a hybridoma methodology 50 purified hybridoma fusion products and 10 hybridoma supernatants were screened in vitro for specificity and cross reactivity by ELISA and neutralisation against GH by cell viability assay Following a second subcloning phase one monoclonal antibody mAb 46 3 was selected Investigation of the antibody binding kinetics and cross reactivity demonstrated that mAb 46 3 had high affinity and specificity for GH without cross reacting with prolactin PRL or mouse GH mGH Investigation of GH signalling and function in cell based assays determined that mAb 46 3 had a strong neutralising effect on GH induced molecular and cellular effects In summary the work described in this thesis explored different approaches of inhibiting GH GHR signalling and developed novel inhibitors for use in preclinical studies with potential for further clinical development Substantial evidence implicates the adverse effect of GH in tumour development progression and treatment outcome The current studies provide new therapeutic approaches to inhibit GH that will serve as useful research tools and will broaden developing areas in cancer treatment.