Competitive, connective and allosteric Nanobodies that modulate the SOS1•RAS protein-protein interactions and tune the nucleotide exchange rate as a starting point for drug design

Abstract

Protein-protein interactions (PPIs) are central in cell metabolism but research tools for the structural and functional characterization of these PPIs are often missing. Here we introduce novel and broadly applicable immunization (Cross-link PPIs and immunize llamas, ChILL) and selection strategies (Display and co-selection, DisCO) for the discovery of diverse Nanobodies that either stabilize or disrupt PPIs in a single experiment. We applied ChILL and DisCO to identify competitive, connective or fully allosteric Nanobodies that inhibit or facilitate the formation of the SOS1-RAS complex and modulate the nucleotide exchange rate on this pivotal GTPase in vitro and RAS signalling in cellulo. One of these connective Nanobodies fills a cavity that was previously identified as the binding pocket for a series of therapeutic lead compounds. The long complementarity-determining region (CDR3) that penetrates this binding pocket serves as an innovative pharmacophore for extending the repertoire of potential leads.

Nanobody CDR3 mimetics enhance SOS1-catalyzed nucleotide exchange on RAS

“…In the used set-up, Nb14 showed some inhibitory effect on the formation of the RAS:GDP:SOS1 complex ( k 1 decreased 12-fold, Figure 6B and C), whereas a significant acceleration of the rate limiting transition towards the nucleotide free RAS:SOS1 complex was observed ( k 2 enhanced 474-fold). 9 Interestingly, both peptides 8 and 10 displayed a similar effect on the catalytic cycle, although less pronounced (Figures 6B and C). These peptides have a limited effect on the formation of RAS:GDP:SOS1 ( k 1 decreased 2- to 4-fold), but a major impact on the rate limiting nucleotide release ( k 2 enhanced 37- to 50-fold).…”

“…3 As one of their many interesting applications, nanobodies emerged as useful tools to stabilize conformationally flexible proteins 7 and protein-protein complexes, 8 such as the recent discovery of nanobodies modulating the RAS:SOS1 protein complex. 9…”

“…17 Similarly to these small molecules, Nanobody14 (Nb14) enhances nucleotide exchange on RAS by interaction with the RAS:SOS1 complex. 9 Hence, we explored the feasibility to develop downsized peptide mimetics of Nb14 by structurally mimicking one of its CDRs.…”

“…(A) Crystal structure of the RAS:SOS1:Nb14 complex showing the binding interfaces involving Nb14. 9 (B) Zoom on the CDR loops. (C) Interaction of the truncated CDR3 (Ile99-Ala105) with SOS1.…”

“…In addition, their effect on the RAS:SOS1 nucleotide exchange catalytic cycle was investigated by measuring all microscopic rate constants in a similar set-up as previously used for Nb14 (Figure S7). 9 The catalytic cycle is a four step mechanism starting with the formation of the RAS:GDP:SOS1 complex ( K D1 , Figure 6A), followed by the dissociation of GDP towards the intermediate RAS:SOS1 complex ( K D2 ). The subsequent binding of GTP followed by the accumulation of RAS:GTP, can be considered symmetrical to the first two steps of the catalytic cycle.…”