Small molecules can stabilise the native, intrinsically disordered state of the Alzheimer's amyloid-β42 (Aβ42) peptide through a dynamic 'disordered binding' mechanism in which both partners remain unstructured: enthalpically favourable transient π-stacking contacts are coupled to a global increase in the peptide's conformational entropy. This mechanism was established computationally (Koopman-model kinetics from unbiased molecular dynamics) only for the single pair Aβ42–10074-G5, and its predicted microsecond-scale state transitions and nanosecond-scale contact lifetimes have not been tested experimentally. The open problem is to determine whether this native-state stabilisation mechanism generalises to other intrinsically disordered protein (IDP)–small-molecule systems — as distinct from folding-on-binding or entropic collapse — and to validate the predicted binding kinetics by direct experimental observables such as NMR relaxation dispersion or single-molecule FRET. Later literature provides computational support for entropic expansion in a second system (α-synuclein–fasudil) but also counterexamples where small molecules induce partially folded collapsed states (androgen receptor transactivation domain), and a 2026 review by the source paper's own authors confirms that no clinically approved drug targets any disordered protein, so the generalisation and validation questions remain open.

Background

Intrinsically disordered proteins (IDPs) do not adopt a single stable folded structure under physiological conditions but populate a dynamic ensemble of interconverting conformations. The 42-residue amyloid-β peptide (Aβ42), central to Alzheimer's disease, is such an IDP: its monomeric form is a heterogeneous conformational ensemble, and drug-discovery efforts have struggled to target it because classical drug design assumes well-defined binding pockets. One successful strategy for folded proteins is pharmacological stabilisation of the native state — for example tafamidis stabilises the folded transthyretin tetramer in amyloidosis (Burton et al., 2021). For IDPs it has been proposed that, since the disordered state is the free-energy minimum of an 'inverted' landscape, small molecules that stabilise this disordered native state could be easier to find than molecules that force the peptide into a single structure (Heller, Sormanni & Vendruscolo, 2015; Heller, Bonomi & Vendruscolo, 2018). In this 'entropic expansion' picture, the ligand gains affinity from transient, weakly specific contacts while the protein's conformational entropy increases, in contrast to folding-on-binding or entropic collapse mechanisms where binding induces local structure or compaction.

The small molecule 10074-G5 (originally identified as an inhibitor of the c-Myc–Max protein–protein interaction) binds monomeric Aβ42 and sequesters it from aggregation; its binding thermodynamics were characterised experimentally and with restrained molecular dynamics simulations (Heller et al., 2020, Science Advances). Because those simulations used time-dependent restraints and biases, they could address thermodynamics but not kinetics. A kinetic ensemble of apo Aβ42 was then constructed from tens of microseconds of unbiased molecular dynamics using a Koopman model — a data-driven kinetic model, built with variational deep-learning methods, that approximates the system's slow dynamical modes and transition times between metastable states (an approach closely related to Markov state models) (Löhr et al., 2021, Nature Computational Science).

The source paper (Löhr, Kohlhoff, Heller, Camilloni & Vendruscolo, 2022, ACS Chemical Neuroscience) extended this to the Aβ42–10074-G5 complex using unbiased molecular dynamics simulations and Koopman-based kinetic modelling. Its central finding is a 'disordered binding' mechanism: neither the peptide nor the ligand adopts a defined bound structure; instead, enthalpically favourable local π-stacking interactions (aromatic contact lifetimes of at most tens of nanoseconds) are coupled to entropically advantageous global effects, with the slowest transitions between the peptide's metastable disordered states occurring on microsecond timescales. The authors contrast this with the 'shuttling' diffusion of the small molecule fasudil on α-synuclein described by Robustelli et al. (2022), and present the complex as 'a possible example of the previously proposed entropic expansion mechanism'; the abstract observes that 'It is still unclear, however, whether this approach can be applied to intrinsically disordered proteins', and the outlook anticipates 'a drug discovery strategy for disordered proteins based on the stabilisation of their native states through the disordered binding mechanism'. The paper's Discussion enumerates two methodological limitations: (i) the lack of a framework that incorporates experimental data (e.g., NMR chemical shifts) into Koopman-based kinetic models without perturbing the dynamics and that permits enhanced sampling techniques such as metadynamics (an accelerated sampling method that biases selected collective variables) without prior knowledge of the states — a framework that 'would make the study of these systems easier and more accurate'; and (ii) the limited accuracy of fixed-charge molecular-mechanics force fields, in which π–π interactions are only indirectly approximated by electrostatic and hydrophobic terms, with polarizable force fields (models including induced dipoles) proposed instead, which 'may offer a computationally affordable alternative that could more accurately model this type of binding'. The Discussion also acknowledges that 'the concept of disordered binding is difficult to probe, as the tools suitable to detecting small changes in the conformational ensemble of disordered proteins are still in their infancy'; the paper's own experimental validation was limited to back-calculating NMR chemical shifts from the simulated ensembles, and the Introduction notes that microscopic kinetics in the form of contact lifetimes 'have, to the best of our knowledge, never been calculated for this kind of interaction' and that a quantitative kinetic study 'may allow a more targeted approach to the design of both drugs and better experiments to probe their binding modalities'. Whether the disordered binding mechanism generalises to other IDP–small-molecule systems, and whether its purely computational kinetic predictions survive direct experimental test, therefore remain open. Resolving these questions is the prerequisite for the drug-discovery strategy the authors envision: stabilising disordered proteins in their native functional states.

Problem Statement

Determine whether the disordered binding mechanism of native-state stabilisation, established computationally for the single pair Aβ42–10074-G5, is a general mode of small-molecule recognition of intrinsically disordered proteins, and validate its predicted microscopic kinetics experimentally. Concretely: (1) for IDP–small-molecule systems beyond Aβ42–10074-G5, establish whether small molecules can stabilise the native disordered ensemble through the disordered binding mode — both partners remaining disordered, with enthalpically favourable transient local contacts (notably π-stacking) coupled to a global increase in conformational entropy — as distinct from folding-on-binding or entropic collapse; this mechanistic criterion — both partners remaining disordered, transient π-stacking contacts, and a global increase in conformational entropy — is what counts as instantiating the disordered binding mode, and the mode's generality is to be assessed over a small representative set of additional IDP–small-molecule systems rather than by exhaustive enumeration, the case for generality (or for its limits) resting on whether each examined system meets this criterion or instead displays folding-on-binding or entropic collapse; and (2) test the kinetic predictions of the Aβ42–10074-G5 Koopman model — microsecond-scale interconversion between metastable disordered states and small-molecule contact lifetimes of at most tens of nanoseconds — by direct experimental observables such as NMR relaxation dispersion or single-molecule FRET, thereby placing the disordered binding mechanism, or its refutation, on an experimental footing; this component is satisfied by designing and justifying a discriminating experiment — the choice of observable, the timescale it accesses, and the predicted read-out that would confirm or refute each kinetic prediction — rather than by newly performed measurements. Studies towards this end face the source's acknowledged enabling limitations — the absence of a framework that incorporates experimental data into Koopman-based kinetic models without perturbing the dynamics while allowing enhanced sampling without prior knowledge of states, and the inaccuracy of fixed-charge force fields for π-mediated interactions — which constrain how reliably either component can be answered but are not themselves deliverables of this problem.

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