Synthetic 10FN3-based mono- and bivalent inhibitors of MDM2/X function

Abstract

Engineered non-antibody scaffold proteins constitute a rapidly growing technology for diagnostics and modulation/perturbation of protein function. Here, we describe the rapid and systematic development of high-affinity 10FN3 domain inhibitors of the MDM2 and MDMX proteins. These are often overexpressed in cancer and represent attractive drug targets. Using facile in vitro expression and pull-down assay methodology, numerous design iterations addressing insertion site(s) and spacer length were screened for optimal presentation of an MDM2/X dual peptide inhibitor in the 10FN3 scaffold. Lead inhibitors demonstrated robust, on-target cellular inhibition of MDM2/X leading to activation of the p53 tumor suppressor. Significant improvement to target engagement was observed by increasing valency within a single 10FN3 domain, which has not been demonstrated previously. We further established stable reporter cell lines with tunable expression of EGFP-fused 10FN3 domain inhibitors, and showed their intracellular location to be contingent on target engagement. Importantly, competitive inhibition of MDM2/X by small molecules and cell-penetrating peptides led to a readily observable phenotype, indicating significant potential of the developed platform as a robust tool for cell-based drug screening.

Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain

“…It is now understood that FN3 domains incorporate multiple 'association zones' across the entire beta-sheet structure [13] ( Figure 1C), which has led to development of the 'side' surface including the FG loop, F and G strands to produce a concave binding surface [3,14,15] and the 'south' AB, CD and EF loops [5,16,17]. In line with its natural function, most of the surface of the FN3 molecule is amenable to evolution for association with target proteins and bivalent monobodies have been developed which make concurrent use of separate binding surfaces [18].…”

“…The current field of monobody development also benefits from mature technologies that can deliver binding reagents on demand [37] through yeast surface display [38], phage display [39] and mRNA display [40]. The scaffold is also viable for grafting and evolution from single peptide 'anchors' [18,[41][42][43] or CDR-like loop grafting between FN3 scaffolds [44][45][46].…”

Development and Differentiation in Monobodies Based on the Fibronectin Type 3 Domain

“…It is now understood that FN3 domains incorporate multiple 'association zones' across the entire beta-sheet structure [13], which has led to development of the 'side' surface including the FG loop, F and G strands to produce a concave binding surface [3,14,15] and the 'south' AB, CD and EF loops [5,16,17]. In line with its natural function, most of the surface of the FN3 molecule is amenable to evolution for association with target proteins [ Figure 1C] and bivalent monobodies have been developed which make concurrent use of separate binding surfaces [18].…”

“…The current field of monobody development benefits from mature technologies that can deliver binding reagents on demand [36] through yeast surface display [37], phage display [38] and mRNA display [39]. The scaffold is also viable for grafting and evolution from single peptide 'anchors' [18,[40][41][42] or CDR-like loop grafting between FN3 scaffolds [43][44][45].…”

“…Here the small monobody size overcomes some limitations of antibodies, resulting in greater tumour penetration and limited circulation [64]. These advantages were validated as part of human safety studies with an 18 F loaded Adnectin, where imaging could be completed on the same day as injection and antibody-based reagents may take several days of radiation burden to clear non-specific binding [66,67]. This was further confirmed after loading the Adnectin with 64 Cu where the monobody was able to provide same-day PET visualisation of tumour cells, resulting in an increase in the monobody uptake in tumours over 24 hours postinjection [61,68].…”