Authors

JD Kumar¹; M Wheldon¹; SA Hawley²; C Kerr¹; L Spinelli²; C McGinnis¹; S Boomkamp¹; EA Fordyce¹; F Cunningham¹; C Mackenzie¹; DA Cantrell²; DA Hardie²; DW Gray¹; 
¹ Drug Discovery Unit, School of Life Sciences, University of Dundee, Dundee, UK, UK;  ² Cell Signalling and Immunology, School of Life Science, University of Dundee, UK, UK

Discussion

The α1-subunit of the AMPK (adenosine 5′ monophosphate-activated protein kinase) complex is frequently activated in tumour cells. To date, identifying a specific AMPK α1 inhibitor has been challenging due to inhibitors targeting the generic ATP binding pocket resulting in promiscuity and off-target effects. Here we propose to use an activator specifically targeting the allosteric drug and metabolite (AdAM) site of AMPK α1 as a POI binder and to generate Proteolysis Targeting Chimeras (PROTAC) to drive targeted degradation of the AMPK α1. A novel plate-based chemistry approach was used to generate a ‘crude’ library of 123 PROTACs using the AMPK activator MK8722 as a targeted protein warhead, coupled to various linkers and E3 ligase binders. PROTACs were screened in HEK293 cells and protein degradation quantified by automated Western Blot analysis. 18 PROTACs were identified showing >30% degradation of AMPK α1 and re-confirmed in dose-response tests. Based on these data, 4 potential PROTAC hits were selected for re-synthesis and purification. These PROTACs, and their corresponding POI binders, were further profiled in a cell-free phosphorylation assay. Most of the individual POI binders showed considerable activation (35 – 155-fold) of the naïve AMPK α1β1ϒ1 complex whereas significantly reduced activation effect was observed in corresponding PROTACs. PROTAC DDD02388041 and its POI binder showed minimum activation of AMPK. Further, DDD02388007, but not DDD02388041, caused a dose-dependent increase in phosphorylated acetyl-CoA carboxylase, a downstream signalling target of AMPK. Further studies are currently underway in T cells and preliminary data from these will also be presented. In summary, we report the successful identification of promising AMPK α1-targeting PROTAC molecules using a plate-based chemistry approach for the high throughput synthesis of a PROTAC library coupled with a cellular protein degradation assay.