Multivalent binding kinetics resolved by fluorescence proximity sensing

Abstract

Multivalent protein interactors are an attractive modality for probing protein function and exploring novel pharmaceutical strategies. The throughput and precision of state-of-the-art methodologies and workflows for the effective development of multivalent binders is currently limited by surface immobilization, fluorescent labelling and sample consumption. Using the gephyrin protein, the master regulator of the inhibitory synapse, as benchmark, we exemplify the application of Fluorescence proximity sensing (FPS) for the systematic kinetic and thermodynamic optimization of multivalent peptide architectures. High throughput synthesis of +100 peptides with varying combinatorial dimeric, tetrameric, and octameric architectures combined with direct FPS measurements resolved on-rates, off-rates, and dissociation constants with high accuracy and low sample consumption compared to three complementary technologies. The dataset and its machine learning-based analysis deciphered the relationship of specific architectural features and binding kinetics and thereby identified binders with unprecedented protein inhibition capacity; thus, highlighting the value of FPS for the rational engineering of multivalent inhibitors.

Stabilisation of β-Catenin-WNT signalling by USP10 in APC-truncatedcolorectal cancer drives cancer stemness and enables super-competitor signalling

“…Recent studies have reported that tumour progression, loss-of-heterozygosity, and deregulation of WNT signalling are indeed affected by the length of the remaining tumour suppressor (Albuquerque et al, 2002). Furthermore, aberrant WNT signalling, caused by APC truncation, is a prerequisite for the establishing of a super competitor cell, manifesting CRC onset (Flanagan et al, 2021b; Schulte et al, 2022; van Neerven et al, 2021). Despite our growing understanding of the underlying genetic causalities of colorectal cancer, the identification of mechanisms at the basis of this phenomenon and identification of suitable therapeutic targets presents a challenge that yet needs to be overcome (Guren, 2019).…”

“…To confirm the interaction and map the USP10 binding site required for β-Catenin interaction, we conducted a µSPOT protein binding assay (Schulte et al, 2022). Here, the intrinsically disordered regions (IDR) of USP10, along with the IDR sequences of a known β-Catenin binder, USP7 (Novellasdemunt et al, 2017), and an additional DUB comprised of large unstructured regions, USP36 were displayed as overlapping peptide libraries and probed with recombinant β-Catenin (Figure 3E).…”

Molecular characterization of AIFM2/FSP1 inhibition by iFSP1-like molecules

“…This was achieved using the minimal iFSP1 pharmacophore with a piperazine group (iFSP1-Pip) as an exit vector protruding from the FSP1 binding pocket (Fig. 4A) for conjugation to a SulfoCyanine5 [22]. The resulting fluorescent tracer "FSP1-Tracer" enabled the detection of FSP1 binding and further highlights the precursor as starting point for the design of advanced iFSP1-based effectors.…”

Stabilisation of β-Catenin-WNT signalling by USP10 in APC-truncated colorectal cancer drives cancer stemness and enables super-competitor signalling

“…To con rm the interaction and map the USP10 binding site required for β-Catenin interaction, we conducted a µSPOT protein binding assay 29 . Here, the intrinsically disordered regions (IDR) of USP10, along with the IDR sequences of a known β-Catenin binder, USP7 12 , and an additional DUB comprised of large unstructured regions, USP36 were displayed as overlapping peptide libraries and probed with recombinant β-Catenin (Fig.…”