Fragment-based exploration of the 14-3-3/Amot-p130 interface

Abstract

The modulation of protein-protein interactions (PPIs) has developed into a well-established field of drug discovery. Despite the advances achieved in the field, many PPIs are still deemed as ‘ undruggable’ targets and the design of PPIs stabilizers remains a significant challenge. The application of fragment-based methods for the identification of drug leads and to evaluate the ‘ tractability’ of the desired protein target has seen a remarkable development in recent years. In this study, we explore the molecular characteristics of the 14-3-3/Amot-p130 PPI and the conceptual possibility of targeting this interface using X-ray crystallography fragment-based screening. We report the first structural elucidation of the 14-3-3 binding motif of Amot-p130 and the characterization of the binding mode and affinities involved. We made use of fragments to probe the ‘ ligandability’ of the 14-3-3/Amot-p130 composite binding pocket. Here we disclose initial hits with promising stabilizing activity and an early-stage selectivity toward the Amot-p130 motifs over other representatives 14-3-3 partners. Our findings highlight the potential of using fragments to characterize and explore proteins' surfaces and might provide a starting point toward the development of small molecules capable of acting as molecular glues .

Contemporary biophysical approaches for studying 14-3-3 protein-protein interactions

“…In isolation, each of the techniques FP, FRET, ITC, and SPR act as a unique lens through which to study 14-3-3 PPIs, but these methods alone do not provide a full picture. Other approaches not discussed here (e.g., microscale thermophoresis (Centorrino et al, 2022;Kozeleková et al, 2022), differential scanning fluorimetry (Cossar et al, 2021;Waløen et al, 2021;Joshi et al, 2022), analytical ultracentrifugation (Horvath et al, 2021;Leysen et al, 2021;Neves et al, 2021;Pohl et al, 2021;Srdanović et al, 2022), single angle x-ray scattering (Kast and Dominguez, 2019;Leysen et al, 2021) also contribute new understanding and support hypotheses. NMR (Kuusk et al, 2019;Neves et al, 2021) and native mass spectrometry (Bellamy-Carter et al, 2021) are also important techniques that shows great promise for studying 14-3-3 PPIs.…”

“…Synthetic peptides that mimic the binding motifs of the phosphorylated partner protein to 14-3-3 have a sufficiently small size relative to 14-3-3 and can be modified to incorporate a fluorescent label. Their use in place of the natural protein partner makes FP a particularly versatile technique for studying In this review of recent 14-3-3 literature, we have identified 15 studies that used FP as part of the analytical workflow (Soini et al, 2021a;Cossar et al, 2021;Falcicchio et al, 2021;Gogl et al, 2021;Guillory et al, 2021;Horvath et al, 2021;Ruks et al, 2021;Sijbesma et al, 2021;Centorrino et al, 2022;Hazegh Nikroo et al, 2022;Joshi et al, 2022;Srdanovic et al, 2022;Srdanović et al, 2022;Stevers et al, 2022). Because it is such a well-established technique, here we focus on select examples that demonstrate how FP can be used to address three key challenges in current 14-3-3 research: (i) Quantifying the cooperative nature of 14-3-3 molecular glues; (ii) Understanding 14-3-3 isoform specificity and partner protein binding mechanisms; (iii) Studying the kinetic characteristics of 14-3-3 PPIs.…”

“…FP has been widely used to screen and profile molecular glues from established examples such as fusicoccin A through to novel fragments, macrocycles and peptide-conjugates. Typically, the effect of a glue is quantified either: 1) by performing 14-3-3 protein titrations in the presence of a fixed concentration of the glue or inhibitor and partner peptide, and comparing the effective K d observed to that of a control experiment (see in Figure 2A ); or 2) by performing ligand titrations to fixed concentrations of 14-3-3 protein and partner peptide to calculate “EC 50 ” values (see in Figure 2B ) ( Soini et al, 2021a ; Falcicchio et al, 2021 ; Guillory et al, 2021 ; Sijbesma et al, 2021 ; Wolter et al, 2021 ; Centorrino et al, 2022 ; Srdanovic et al, 2022 ; Stevers et al, 2022 ). However, both of these approaches are concentration dependent and the values obtained differ depending on experimental design (i.e., the concentration of the fluorophore in protein titrations, or the concentration of the 14-3-3 protein in ligand titrations).…”

Fragment Screening Yields a Small‐Molecule Stabilizer of 14‐3‐3 Dimers That Modulates Client Protein Interactions

“…[17,18] Fragment approaches targeting the 14-3-3 interface with various protein interaction partners using both covalent and non-covalent fragments have been reported. [19][20][21][22][23][24][25][26] Whereas the 14-3-3/ERα interface has previously been targeted by stabilizing covalent fragments, [22] here we target the 14-3-3/ERα interaction with stabilizing non-covalent fragments. Our in-house fragment library of 1600 non-covalent fragments was screened using a homogenous time-resolved FRET assay (HTRF).…”

“…Fragment screening is an approach that is complementary to high‐throughput screening and is able to efficiently sample chemical space of protein binders by using small libraries of small molecules [17,18] . Fragment approaches targeting the 14‐3‐3 interface with various protein interaction partners using both covalent and non‐covalent fragments have been reported [19–26] . Whereas the 14‐3‐3/ERα interface has previously been targeted by stabilizing covalent fragments, [22] here we target the 14‐3‐3/ERα interaction with stabilizing non‐covalent fragments.…”