Unveiling the “invisible” druggable conformations of GDP-bound inactive Ras

The prevalent view on whether Ras is druggable has gradually changed in the recent decade with the discovery of effective inhibitors binding to cryptic sites unseen in the native structures. Despite the promising advances, therapeutics development toward higher potency and specificity is challenged by the elusive nature of these binding pockets. Here we derive a conformational ensemble of guanosine diphosphate (GDP)-bound inactive Ras by integrating spin relaxation-validated atomistic simulation with NMR chemical shifts and residual dipolar couplings, which provides a quantitative delineation of the intrinsic dynamics up to the microsecond timescale. The experimentally informed ensemble unequivocally demonstrates the preformation of both surface-exposed and buried cryptic sites in Ras•GDP, advocating design of inhibition by targeting the transient druggable conformers that are invisible to conventional experimental methods. The viability of the ensemble-based rational design has been established by retrospective testing of the ability of the Ras•GDP ensemble to identify known ligands from decoys in virtual screening.

Situated in a central position of the complex intracellular signaling network, Ras proteins play critical roles in regulating cell growth, differentiation, migration and apoptosis through cycling between the guanosine diphosphate (GDP)-bound inactive and guanosine triphosphate (GTP)-bound active forms (1, 2). Aberrant signaling caused by oncogenic mutations in Ras that break this physiological balance can result in uncontrolled cell proliferation and ultimately the development of human malignancies (3, 4). Despite its well-established role in tumorigenesis and the extensive efforts to target this oncoprotein in past decades, clinically approved therapies remain unavailable. One obstacle to the development of anti-Ras drugs lies in the native structures of active and inactive Ras that lack apparently druggable pockets for high-affinity interactions with inhibitory compounds (5–7).Both the active and inactive forms of Ras, however, are inherently flexible, populating rare conformers distinct from the native structures and presenting alternative opportunities for drug discovery (8–11). For example, in GTP-bound active Ras, a major and minor state (termed states 2 and 1, respectively) coexist in solution and exchange on a millisecond timescale, with state 1 showing surface roughness unobserved in the major state (12–18). The direct visibility of state 1 in the one-dimensional ³¹P NMR spectra of active Ras largely facilitated its early discovery and characterization (12, 19). And the available mutants of H-Ras (e.g., T35A), or the homolog M-Ras, which predominantly assume the state 1 conformation, further promoted the atomic-resolution studies of its structure and internal dynamics, as well as the concomitant drug discovery efforts targeting this low-populated conformer (17, 18, 20).In comparison to the intensive studies on active Ras, research on the dynamics of GDP-bound inactive Ras has lagged far behind, presumably due to its high degree of spectral homogeneity with little sign of resonance splitting or exchange broadening at room temperature (21). The previously reported cryptic pockets for covalent and noncovalent inhibitors of Ras•GDP (22–24), which are unseen in the compound-free structure, nevertheless indicate that the inactive form is also structurally plastic. The recent relaxation-based NMR experiments carried out at low temperature successfully captured the intrinsic microsecond timescale motions in Ras•GDP, which map to regions that overlap with those rearranged on the binding of inhibitors (11). However, the structural information of the transiently formed excited state, in the form of chemical shifts, is not available from the relaxation measurements, owing to the fast exchange rate on the chemical shift timescale. Moreover, unlike the case of active Ras, there are no known mutations that can stabilize the excited state of Ras•GDP for investigations using conventional biophysical techniques. Thus far, the sparsely populated conformations of inactive Ras derived from its microsecond dynamics remain poorly understood, precluding structure-based rational drug discovery.To address these challenges, in this work we constructed a solution ensemble of Ras•GDP by integrating atomistic computer simulation with diverse NMR experimental parameters containing complementary information about the intrinsic protein motions on timescales from picoseconds to microseconds. This NMR-based ensemble well covers the slow dynamics as probed by spin relaxation and provides an atomic-resolution delineation of thermally accessible conformations, including those bearing surface or buried pockets similar to the cryptic pockets previously observed in the inhibitor-bound forms. The utility of the Ras•GDP ensemble in the development of inhibitors is demonstrated by ensemble-based virtual screening, which achieves an impressive level of enrichment of known binders.