Controlling oncogenic KRAS signaling pathways with a Palladium-responsive peptide

KRAS is the most frequently mutated RAS isoform and is implicated in highly lethal cancers (e.g., pancreatic, colorectal, lung adenocarcinoma). RAS functions as a GDP/GTP molecular switch regulated by GEFs (such as SOS1) and GAPs; oncogenic KRAS mutations (e.g., G12C, G12V) impair GTPase activity and drive persistent signaling that promotes proliferation and metastasis. Prior α-helix peptidomimetics derived from the SOS1 αH helix, stabilized by covalent constraints (hydrocarbon staples, helix-capping, mimics), can inhibit KRAS by blocking SOS1-mediated activation. However, constitutively active, permanently stapled helices lack stimulus-responsiveness. The study hypothesizes that metal coordination can endow a KRAS-binding peptide with switchable affinity, enabling spatiotemporal control. A bis-histidine mutant of the SOS1 αH helix (αH-His2) is designed to chelate Pd(II), which is expected to nucleate α-helix formation and enable folding-upon-binding to KRAS, providing a reversible, stimuli-responsive inhibitor.

Previous work established that conformationally stabilized α-helix peptidomimetics of the SOS1 αH helix can bind KRAS and inhibit SOS1-mediated activation (helix capping, hydrocarbon staples, proteomimetics). Metal-stabilized α-helices have precedent with various metal clips (e.g., Ru(III), Pd(II)) for helix nucleation, and the authors’ prior studies used metal chelation in peptides to control DNA binding and cell internalization. In contrast to permanent covalent constraints (hydrocarbon staples, hydrogen-bond surrogates, lactam bridges, cysteine crosslinks), a coordinative metal clip offers reversibility and external control. Concepts from intrinsically disordered proteins (IDPs) and trigger sequences suggest that partial preorganization may be sufficient to promote folding-upon-binding, motivating the design of a Pd(II)-responsive KRAS-binding helix.

Design: Based on structures of SOS1 αH helix inserted into KRAS (PDB 1NVW, 1BKD), residues Phe929–Asn944 were selected. Tyr933 and Ile937, solvent-exposed on the αH helix face, were mutated to histidines to create an i, i+4 bis-His chelation site for square-planar Pd(II). Two N-terminal Arg residues were added to enhance solubility and uptake, and the N-terminus was labeled with 6-carboxytetramethylrhodamine (TMR) for quantification and anisotropy readout (sequence: TMR-Ahx-RRFFGIHLTNHLKTEEGN). Peptide synthesis: Fmoc solid-phase synthesis on H-Rink amide ChemMatrix resin (0.1 mmol scale) using DIC/Oxime activation in DMF with microwave-assisted couplings (4 min at 90 °C). Fmoc deprotection with 20% piperidine in DMF (1 min, 75 °C). TMR was manually coupled (3 eq. TMR, 3 eq. HATU, 5 eq. DIEA, 60 min). Cleavage with TFA/CH2Cl2/H2O/TIS, ether precipitation, and preparative RP-HPLC purification (C18 column, 5–75% B over 40 min). UHPLC-MS used for quality control; ESI-MS in positive mode. Metalation: αH-His2 was mixed with 1 equiv cis-PdCl2(en) to form αH-His2[Pd], monitored by NMR (His ring proton shifts) and MS. Reversibility was tested using the Pd(II) chelator diethyldithiocarbamate (DEDTC) and re-addition of cis-PdCl2(en). Circular dichroism (CD): 20 µM peptide in 10 mM phosphate, pH 7.5, 100 mM NaCl, 10% TFE at 25 °C. Spectra from 300–195 nm; analysis of mean residue ellipticity at 222 nm. Conditions for switching: peptide alone, +1 eq Pd(II), +5 eq DEDTC, followed by +20 eq Pd(II). NMR spectroscopy: 1 mM peptide in H2O/D2O (90/10) at 298 K on 600 MHz spectrometer. 1D 1H, 2D TOCSY (8 kHz spin-lock, 50 ms) and NOESY (mixing 80–300 ms) for assignments. Sequential assignments and NOE-derived distance restraints used in CNS 1.2 for simulated annealing (implicit/explicit water refinements) to generate solution conformers. Pd coordination was not explicitly parameterized during structure calculation; His rotamers and Pd added for display. Computational modeling: Docking of Pd(II) to peptide with GOLD and GaudiMM to propose coordination modes. Metallated cores parametrized with AMBER MCPB.py using bonded model; QM at B3LYP/6-31G(d,p) (C,H,N) and SDD with f-polarization for Pd, with D3 dispersion and SMD(water) solvation. Charges by RESP; parameters by Seminario approach. Systems built with AMBER18 (ff14SB for peptide, GAFF for ligands), TIP3P water boxes (2700–3000 molecules), neutralized. After 10 ns classical MD, Gaussian accelerated MD (GaMD) for ≥2 µs per system (NVT, SHAKE; boost on dihedral and total potential). PCA used to assess conformational sampling. Simulations compared metal-free peptide and metallated αH-His2[Pd] with different His coordination modes. Fluorescence anisotropy: Jobin-Yvon Fluoromax-3; excitation 559 nm, emission 585 nm. Titration of 15 nM αH-His2 or αH-His2[Pd] with recombinant KRASWT (and mutants G12C, G12V) in 20 mM Tris-HCl pH 7.5, 100 mM NaCl at 25 °C. Anisotropy increases fitted to 1:1 binding model. Switching tested by adding 50 eq DEDTC to saturated complex, then 50 eq cis-PdCl2(en) to restore binding. Protein expression/purification: His-tagged KRAS (WT, G12V, G12C) expressed in BL21, Ni-NTA purification, buffer exchange to remove glycerol before assays. Nucleotide release assay: KRASWT loaded with mantGTP (10x excess, 1.5 h at 20 °C; MgCl2 added; free nucleotide removed by desalting). Intrinsic release monitored by decrease in fluorescence at 430 nm over 30 min (370 nm excitation) for 1 µM KRASWT/mantGTP with 10 µM αH-His2 or αH-His2[Pd]. Half-lives extracted; statistics by One-Way ANOVA. Cell studies: A549 cells cultured in DMEM + 5% FBS. Uptake: Cells incubated 30 min at 37 °C with 10 µM αH-His2 or preformed αH-His2[Pd] (1:1 premix for 10 min in water), washed, imaged by fluorescence microscopy (λex 550 nm; emission 590–650 nm). MAPK pathway inhibition: Serum-starved A549 cells treated 4 h with αH-His2, αH-His2[Pd], or cis-PdCl2(en) at indicated doses. Western blot for phospho-ERK1/2 (pERK) and total ERK1/2; densitometry to quantify pERK/ERK ratios.

• Metal-induced helicity: αH-His2 alone displays random coil CD with weak band at 222 nm (~−2200 deg cm² dmol⁻¹). Addition of 1 eq cis-PdCl2(en) increases the 222 nm signal to ~−5700 deg cm² dmol⁻¹, consistent with ~22% α-helical content. Addition of excess DEDTC diminishes helicity; subsequent Pd(II) re-addition restores helicity, demonstrating reversibility.
• NMR and MD: Free αH-His2 is largely unstructured with limited α-helix propensity in the central region. Upon Pd(II) coordination, long-range NOEs (Phe929, Ile932, Leu934) appear, indicating helix formation from Arg928–Phe930 and Gly931–Leu938 around His933/His937. GaMD (2 µs) shows stable α-helix in the Pd-bound core (His933–Glu941) with Nτ coordination on both histidines; metal-free peptide samples unfolded states. Alternative coordination modes yield less stable folds.
• KRAS binding: αH-His2 (apo) shows negligible binding to KRASWT by anisotropy. Metallopeptide αH-His2[Pd] binds tightly with Kd ≈ 240 nM (KRASWT). Similar affinities observed for mutants: Kd ≈ 345 nM (KRASG12C) and 294 nM (KRASG12V). DEDTC disrupts αH-His2[Pd]/KRASWT complex (anisotropy decrease), and re-addition of Pd(II) restores binding, confirming switchability (ANOVA, p < 0.001).
• Functional inhibition in vitro: In KRASWT/mantGTP complex, intrinsic half-life ~7 min. With 10 µM αH-His2, t1/2 ≈ 5 min. With 10 µM αH-His2[Pd], t1/2 ≈ 3 min, indicating accelerated nucleotide release/inhibition of exchange dynamics (ANOVA, p < 0.001).
• Cellular activity: αH-His2 exhibits poor uptake in A549 cells, whereas αH-His2[Pd] shows strong intracellular fluorescence after 30 min, indicating enhanced internalization. In serum-starved A549 cells, αH-His2[Pd] reduces phospho-ERK1/2 levels in a dose-responsive manner, while αH-His2 or cis-PdCl2(en) alone have no effect, demonstrating inhibition of the RAF-MEK-ERK pathway downstream of KRAS.

The study demonstrates that a bis-histidine mutant of the SOS1 αH helix can be converted into a switchable KRAS binder by coordinating Pd(II), which nucleates the α-helical conformation and enables folding-upon-binding. Unlike permanently stapled helices, Pd(II) coordination confers reversible control: chelation by DEDTC disengages the peptide from KRAS, and added Pd(II) restores binding. Despite only modest overall helicity in solution, the metallopeptide achieves sub-micromolar binding to KRASWT and oncogenic mutants, consistent with an IDP-like mechanism where partial preorganization by the metal lowers the barrier for coupled folding and binding on the protein surface. Functionally, the metallopeptide accelerates nucleotide release from KRAS in vitro and inhibits ERK phosphorylation in cells, supporting its capacity to modulate KRAS-driven signaling. This work provides a paradigm shift from maximizing helix preorganization toward exploiting dynamic, stimulus-responsive folding, potentially enabling spatiotemporal control in research and therapeutic contexts.

This proof-of-concept introduces a palladium-responsive, bis-histidine α-helix peptidomimetic (αH-His2[Pd]) that binds KRAS with high affinity only upon Pd(II) coordination, enabling reversible, switchable inhibition of KRAS signaling. Spectroscopic and computational data elucidate metal-induced helix nucleation and a folding-upon-binding mechanism. The metallopeptide shows cellular uptake and inhibits the RAF-MEK-ERK pathway in A549 cells, representing, to the authors’ knowledge, the first designed metallopeptide to modulate a signaling pathway in living cells. The approach opens avenues for creating dynamic α-helix peptidomimetics targeting diverse protein–protein interactions, with potential for externally controlled, spatiotemporally precise modulation. Future work could optimize sequences for potency/specificity, explore alternative metals/coordination chemistries, evaluate broader cellular contexts and in vivo efficacy, and integrate orthogonal triggers for multiplexed control.

The study is a proof-of-concept with most data from biophysical assays and a single cell line (A549); in vivo efficacy and specificity were not evaluated. The peptide exhibits only modest helicity in solution and requires Pd(II) for activity, making performance dependent on metal availability and vulnerable to chelators in complex biological environments. Potential off-target effects or toxicity of Pd(II) complexes were not assessed. Binding and pathway inhibition were demonstrated under defined conditions; generalizability across cell types and physiological contexts remains to be established.