ARTICLE https://doi.org/10.1038/s42004-022-00691-7 OPEN Controlling oncogenic KRAS signaling pathways with a Palladium-responsive peptide Soraya Learte-Aymamí1, Pau Martin-Malpartida2, Lorena Roldán-Martín3, Giuseppe Sciortino3,4, José R. Couceiro1, Jean-Didier Maréchal3, Maria J. Macias2,5, José L. Mascareñas 1 ✉ & M. Eugenio Vázquez 1 ✉ RAS oncoproteins are molecular switches associated with critical signaling pathways that regulate cell proliferation and differentiation. Mutations in the RAS family, mainly in the KRAS 1234567890():,; isoform, are responsible for some of the deadliest cancers, which has made this protein a major target in biomedical research. Here we demonstrate that a designed bis-histidine peptide derived from the αH helix of the cofactor SOS1 binds to KRAS with high affinity upon coordination to Pd(II). NMR spectroscopy and MD studies demonstrate that Pd(II) has a nucleating effect that facilitates the access to the bioactive α-helical conformation. The binding can be suppressed by an external metal chelator and recovered again by the addition of more Pd(II), making this system the first switchable KRAS binder, and demonstrates that folding-upon-binding mechanisms can operate in metal-nucleated peptides. In vitro experi- ments show that the metallopeptide can efficiently internalize into living cells and inhibit the MAPK kinase cascade. 1 Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CiQUS), Departamento de Química Orgánica, Universidade de Santiago de Compostela, Santiago de Compostela 15705, Spain. 2 Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona 08028, Spain. 3 Insilichem, Departament de Química, Universitat Autònoma de Barcelona, Cerdanyola 08193, Spain. 4 Institute of Chemical Research of Catalonia (ICIQ), The Barcelona Institute of Science and Technology, Tarragona 43007, Spain. 5 Institució Catalana de Recerca i Estudis Avançats (ICREA), Passeig Lluís Companys 23, Barcelona 08010, Spain. ✉email: joseluis.mascarenas@usc.es; eugenio.vazquez@usc.es COMMUNICATIONS CHEMISTRY | (2022)5:75 | https://doi.org/10.1038/s42004-022-00691-7 | www.nature.com/commschem 1 ARTICLE COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00691-7 R AS proteins are oncogenic supervillains mutated in almost half of all cancers1. Many of these mutations drive some of the most lethal cancer types, including about 95% of pan- creatic, 45% of colorectal cancers, and 33% of lung adenocarcinomas2. More specifically, KRAS, the most frequently mutated isoform, is responsible for most RAS cancers and gen- erates highly aggressive and life-threatening phenotypes3. These features have made KRAS a key target in the search for new antitumor agents for over three decades4,5. Physiologically, RAS proteins are binary molecular switches that oscillate between an inactive, GDP-bound, state and an active RAS-GTP complex, aided by specific guanine nucleotide exchange factor proteins (GEFs), such as SOS1, which stimulate the GDP/GTP exchange and activation, and GTPase-activating proteins (GAPs) that cat- alyze the hydrolysis of GTP and inactivation in the RAS-GDP state. Certain mutations, such as KRASG12C or KRASG12V, compromise the GTPase activity of RAS proteins, locking them in their active state, thereby inducing the aberrant activation of Fig. 1 Design of the metal responsive KRAS peptide inhibitor. a Detail of several signaling pathways that ultimately lead to uncontrolled the αH helix of SOS1 complexed with RAS (PDB code 1BKD) indicating the cell growth and proliferation, invasiveness, angiogenesis, and residues Tyr933 and Ile937 that are replaced by the metal chelating His metastasis6,7. Activation of RAS-GDP by SOS1 is induced by the residues; the structure of SOS1 is shown in white. b Sequence of the natural insertion of the αH helix of the exchange factor, which opens up αH helix of SOS1 and the mutant peptide αH-His2 used in this study, the GTP binding pocket and reduces the affinity for the highlighting the two engineered His residues. c Conformational control by trinucleotide8. It has been shown, that conformationally restricted Pd(II) chelation. d Wenxiang diagram of the αH-His279,80, indicating the peptidomimetics of the SOS1 αH helix featuring helix-capping residues aligned in the outer face of the αH helix, including the two histidine modifications9,10, hydrocarbon staples11, and SOS1 protein residues in consecutive turns of the α-helix. mimics12, bind KRAS with high affinity, and competitively inhibit RAS activation by SOS1. were synthesized following standard Fmoc solid-phase peptide In contrast to these constitutively active inhibitors, locked into synthesis methods on an automated microwave synthesizer24,25. stable α-helical conformations by permanent linkages, stimuli- responsive peptides that change their affinity in response to an Addition of Pd(II) increases the α-helical content of the pep- external input could offer new opportunities in basic research, as tide αH-His2. Mixing the peptide αH-His2 with 1 equiv. of cis- well as for spatiotemporal controlled therapeutic applications. PdCl2(en) gave the expected metallopeptide, αH-His2[Pd] from Thus, going beyond the classic metal-stabilized α-helices13–16, now on, as confirmed by NMR by following the chemical shift and considering our previous studies in the field of DNA changes observed at the His ring protons, and by MS (see below binding17–20, we hypothesized that metal chelation could be and Fig. S2). Circular dichroism studies showed that metal applied for the development of stimuli-responsive KRAS peptide coordination moderately increases the α-helical content of the inhibitors21,22. Indeed, here we show that the peptide αH-His2, peptide. Thus, a solution of αH-His2 displayed the typical CD engineered to include two i,i + 4 His chelating residues in a short signature of a random coil peptide and a low-intensity band at fragment of the SOS1 αH helix, binds KRAS only after addition of 222 nm (≈−2200 deg cm2 dmol−1); addition of one equivalent of a Pd(II) source. We also show that such coordination is rever- cis-PdCl2(en) induced a modest increase in the negative intensity sible, and that the metallopeptide αH-His2[Pd] is capable of of this band (≈−5 700 deg cm2 dmol−1) that is consistent with a inhibiting KRAS-activated pathways in live cells. Moreover, we relatively small α-helical content of about 22% (Fig. 2, left; also found that complete preorganization of the peptide α-helix is Fig. S3)26. Earlier experiments in our laboratory show that square not required for efficient binding and inhibition, and that αH- planar cis-Pd(II) complexes display the ideal coordination prop- His2[Pd] operates through a coupled folding-upon-binding erties to promote the effective folding of the helical conformation mechanism, typical of Intrinsically Disordered Proteins. while other geometry (trans-[Pd(PPh3)2Cl2]), or other metal ions, such as Cu(II), Co(II), Ni(II), Fe(II), do not stabilize the active conformation22. Interestingly, the addition of a small excess of the Results Pd(II) chelator diethyldithiocarbamate (DEDTC), gave rise to a Design and synthesis of the metal-responsive KRAS peptide marked reduction in the helicity, supporting the key role of the inhibitor. Based on the reported examples of SOS1 αH metal in stabilizing the α-helical conformation. Subsequent peptidomimetics11, as well as on the structural details of the SOS1 αH addition of cis-PdCl2(en) led to a recovery of the helicity, thus inserted between the switch regions of KRAS (PDB codes 1NVW confirming that the system can be easily switched between the and 1BKD)8,23, we chose the fragment comprising SOS1 residues metallo-stapled and the metal-free forms (Fig. 2, right). Phe929 to Asn944, which spans the full αH helix for our experi- ments. Inspection of the hypothetical αH/RAS complex revealed that positions Tyr933 and Ile937 would be located in the solvent-exposed NMR spectroscopy demonstrates that the αH-His2 core face of such αH helix fragment, and distanced so that their side sequence partially folds into an α-helical conformation upon chains, when replaced by His residues, would be in register and metal chelation. We characterized in detail the conformational appropriately oriented to chelate a Pd(II) center (Fig. 1). In addition effects of the Pd(II) coordination by NMR spectroscopy. All to the Tyr933His and Ile937His mutations, the αH sequence in our peptide residues were unambiguously assigned combining spin final design included two additional Arg residues in its N-terminus, system identification in the TOCSY experiment and sequential for increased solubility and cellular uptake, and the sequence was assignment via NOEs (SI, pages S7-S11). In agreement with the capped with 6-carboxytetramethylrhodamine (TMR) for quantifica- CD studies, we found that the free peptide αH-His2 is mostly tion and fluorescence monitoring (Fig. 1, αH-His2). The peptides unstructured in solution, with a certain propensity to populate an 2 COMMUNICATIONS CHEMISTRY | (2022)5:75 | https://doi.org/10.1038/s42004-022-00691-7 | www.nature.com/commschem COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00691-7 ARTICLE Fig. 2 Circular Dichroism studies show that the α-helical content of the peptide αH-His2 can be switched. a Circular dichroism of a 20 µM solution of αH-His2 (dashed line) and the spectrum 30 min after addition of 1 eq. of cis-PdCl2(en); b CD signal at 222 nm for: A 20 µM solution of αH-His2; B same solution after addition of Pd(II) and formation of the αH-His2[Pd] metallopeptide; C solution in B after addition of 5 eq. of DEDTC; D solution in C after addition of 20 eq. of cis-PdCl2(en). The experiments were carried out in 10 mM phosphate buffer pH 7.5, 100 mM of NaCl and 10% TFE, 25 °C. Mean residue molar ellipticity ([Θ]MRE) was calculated considering an 18-mer. α-helical conformation in the central part of the sequence, as deduced from the pattern of NH-NH NOEs observed for residues 929-940. The addition of cis-PdCl2(en) (up to 3 equiv.) led to a significant folding of the peptide chain, as deduced from several long-range NOEs between the side chains of Phe929, Ile932, and Leu934. These NOEs were absent in the free peptide and suggest that in addition to locking the α-helical conformation in the Fig. 3 NMR spectroscopy and computational studies confirm the vicinity of the coordinating residues His933 and His937—from nucleation of the α-helix in αH-His2[Pd]. a Experimental NMR residues Gly931 to Leu938—metal chelation also promotes the conformation in solution (lowest energy) in yellow; NOEs are indicated as formation of a N-terminal helical conformation from residues dotted lines superimposed with a representative conformation of the MD Arg928 to Phe930 (Fig. 3, Methods). simulation (in blue). b, c Aromatic-side chain regions of the NOESY spectra. b native peptide. c in the presence of cis-PdCl2(en). Residues Molecular modeling provides further insight into the con- participating in NOEs are labeled. In the left panel only intra-residue NOEs formational dynamics and metal coordination of the peptide are detected whereas in the right panel, both intra and inter residue NOEs αH-His2. To understand the conformational effect of the metal are observed, indicating the presence of a helical conformation upon Pd chelation in the α-helical conformation, we ran a series of 2 µs binding. PDB structures available as Supplementary Data 1. Gaussian accelerated molecular dynamics (GaMD) simulations of the peptide αH-His2 in its free form and coordinated with Pd(II). transition to a random coil is observed (Figs. S4b and S5b). A For the latter, the starting model was generated by performing comparison between the MD most sampled conformation of the protein-ligand docking of the Pd(II) metal center to the peptide αH-His2[Pd] and the lowest energy NMR conformer in solution using approaches updated for metal ligands27–30. The simulations shows an excellent agreement (Fig. 3). An additional control MD show in all cases a largely disordered N-terminal region (from run with an alternative coordination mode (Nτ-His933/Nπ- residues Arg927 to Ile932). In the case of the metal-free peptide, His937) showed a marginally stable helical fold with frequent followed by an unstable C-terminal α-helix that undergoes mul- deviations of the ideal α-helix geometry and even completely tiple folding/unfolding cycles until, at the end of the simulation, unfolded structures. Such poor structuring effect is clearly shown the conformational sampling leads to a long-lasting ensemble of by the representative structure of the most populated sub- unfolded structures (Supplementary Information, Figs. S4a and ensemble, which shows a kink in the α-helix at the coordination S5a). This indicates that the peptide has some intrinsic helical site (Figs. S4c and S5c). Altogether, the computational study propensity, albeit insufficient to reach a stable fold. This result is provides an excellent match with the spectroscopic data, con- supported by the experimental evidence that points to a largely firming the enhancement of α-helical conformation through dual disordered state in this metal-free form. In contrast, the simula- coordination of the His residues through their Nτ atoms. tion of the metallopeptide αH-His2[Pd] with Nτ coordination in both His led to a more stable fold with substantial α-helical The metallopeptide αH-His2[Pd] binds KRASwt. The above propensity throughout the 2 µs simulation. The core region from studies demonstrate that coordination to the Pd(II) modulates the residues His933 to Glu941, where the coordination occurs, rarely conformation of the peptide αH-His2 in solution, nucleating the deviates from a canonical α-helix and when it does, it adopts formation of an incipient α-helix. We anticipated that this metal- other organized secondary structures (e.g., 310 helix). No induced folding might facilitate the interaction of the peptide COMMUNICATIONS CHEMISTRY | (2022)5:75 | https://doi.org/10.1038/s42004-022-00691-7 | www.nature.com/commschem 3 ARTICLE COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00691-7 Fig. 4 Binding of αH-His2[Pd] to KRAS, and RAS-binding switching Fig. 5 Binding of the metallopeptide αH-His2[Pd] to KRAS inhibits the studied by fluorescence anisotropy. a Normalized emission anisotropy at nucleotide release process. Preformed αH-His2[Pd] increases the 559 nm of a 15 nM solution of αH-His2[Pd] with increasing concentrations displacement of mantGTP bound to KRASwt. a normalized emission at of KRASwt. The best fit to a 1:1 binding model is also shown43,44; 430 nm. Black triangles: intrinsic nucleotide displacement curve of a 1 μM b anisotropy values for: A, a saturated αH-His2[Pd]/KRAS complex; B, solution of the preformed KRASwt/mantGTP complex; white circles, same same solution after addition of 50 eq. of DEDTC; C, solution in B after solution in the presence of 10 μM αH-His2; black circles: same KRASwt/ addition of 50 eq. of cis-Cl2Pd(en). Data are mean ± SEM for experiments mantGTP solution in the presence of 10 μM αH-His2[Pd]. b half-life of the performed in technical triplicate and are representative of at least three kinetic traces shown on the left. A intrinsic KRASwt/mantGTP complex; B biological replicates performed with independent preparations of with αH-His2; C with αH-His2[Pd]. Data were subjected to a One-Way recombinant KRASwt. All experiments carried out at 25 °C. Data were ANOVA statistical analysis, ***p < 0.001. subjected to a One-Way ANOVA statistical analysis, ***p < 0.001. These results are also consistent with the region of the metal clip with KRAS. Indeed, mixing a 15 nM solution of the unstructured being highly structured, as shown by NMR and MD studies, αH-His2 in absence of Pd(II) with increasing concentrations of despite the low total helical content of the metallopeptide as wild-type KRASwt did not produce any increase in the fluores- measured by CD. cence anisotropy of the TMR fluorophore at 559 nm, indicating that the apopeptide, αH-His2, has low binding affinity for The metallopeptide αH-His2[Pd] inhibits KRASwt GTP KRASwt. In contrast, when the titration was repeated with a exchange. Having confirmed the high affinity of the complex αH- preformed palladium/peptide complex, αH-His2[Pd], we His2[Pd] for KRASwt, we studied its effect in the nucleotide observed a marked increase in the anisotropy, which is consistent release process associated with biological inactivation of this with binding to KRASwt and formation of a larger complex with protein38. Therefore, we first measured the intrinsic nucleotide slower rotational diffusion. The increase in anisotropy could be release rate by monitoring the fluorescence emission of the fitted to a simple 1:1 binding model with an approximate dis- complex of KRASwt with the GTP analog mantGTP, 2′-/3′-O-(N- sociation constant of 240 nM (Fig. 4a). Similar titration profiles methylanthraniloyl) guanosine-5′-triphosphate, which displays were obtained when αH-His2[Pd] was incubated with the onco- low fluorescence in solution, but becomes strongly emissive when genic mutants KRASG12C and KRASG12V, which displayed also associated with KRASwt21,22. As expected, the KRASwt/mantGPT tight binding with dissociation constants of 345 nM and 294 nM, complex was very stable, and fluorescent mantGTP was released respectively (Fig. S6). Importantly, the addition of DEDTC to the very slowly, with a half-life of about 7 min (Fig. 5). The half-life of solution of the complex αH-His2[Pd]/KRASwt18,31,32, resulted in this complex was somewhat decreased by the presence of the free a marked decrease of the TMR anisotropy, indicating the dis- peptide αH-His2 (Fig. 5 right, t1/2 ≈ 5 min), but the addition of assembly of the metallopeptide αH-His2[Pd], and disengagement the preformed metallopeptide αH-His2[Pd] drastically increased of αH-His2 from KRASwt. Furthermore, the addition of fresh cis- the rate of nucleotide release, so that the measured half-time of PdCl2(en) to that solution restored the original anisotropy, the KRASwt/mantGPT complex was reduced to about 3 min indicating that the released peptide αH-His2 was still able to under the same conditions (Fig. 5, right). chelate Pd(II) metal center and to bind once again as αH- His2[Pd] to KRASwt (Fig. 4b), thereby confirming the reversibility The metallopeptide αH-His2[Pd] inhibits the ERK1/2 signaling of the system. pathway in cells. The coordinative basis of our stapled peptide Taken together, these data confirm that the palladium clip is raised the question of whether it could be used in the complex working as a facilitator of the helix folding rather than as a cellular environment. Thus, to assess its capacity to antagonize traditional staple in which the covalent link fixes the bioactive α- the KRAS activation in living mammalian cells, we first mon- helical conformation33. In other words, the coordination to the itored its cellular uptake by fluorescence microscopy. Incubation Pd(II) appears to provide just enough thermodynamic stabiliza- of lung carcinoma A549 cells with αH-His2 revealed a rather poor tion for the peptide to display folding-upon-binding properties internalization (Fig. 6a), but when this peptide was previously such as those typically observed for Intrinsically Disordered incubated with cis-PdCl2(en), the resulting metallopeptide, αH- Proteins34,35. A similar effect has been described for short trigger His2[Pd], showed an intense intracellular fluorescence signal sequences that mediate the folding of coiled-coil peptides and (Fig. 6b). Therefore, the palladium clip facilitates the allow the peptides to surpass a critical threshold of stability36,37. internalization39. We then analyzed the activation of the MAPK 4 COMMUNICATIONS CHEMISTRY | (2022)5:75 | https://doi.org/10.1038/s42004-022-00691-7 | www.nature.com/commschem COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00691-7 ARTICLE basic research and biomedicine. In this context, we envisioned the application of metal coordination to provide increased con- formational stability and stimuli-responsive properties to a short α-helix peptidomimetic. As model system, we chose the SOS1 αH helix/KRAS complex because of the intrinsic relevance of the KRAS GTPase in cancer as well as because there are clear precedents of short peptides derived from the αH helix that selectively bind to KRAS21,22. Furthermore, the available structural data of the SOS1/KRAS complex allowed us to rationally engineer the parent sequence to introduce the required residues for Pd(II) coordination in the exposed face of the αH helix in its complex with KRAS54–56. Thus, inspection of the reported structures of the SOS1/KRAS complexes suggested the replacement of Tyr933 and Ile937 with two His residues to create the metal-binding site33; these muta- tions, together with two additional Arg residues at the N-terminus ensured the solubility of the resulting peptide Fig. 6 αH-His2[Pd] is internalized and promotes the inhibition of the (RRFFGIHLTNHLKTEEGN). No further optimization of the MAPK kinase cascade. a, b Effect of Pd(II) coordination in cellular sequence to increase its intrinsic α helical propensity was internalization (A549 cells). a Incubation with αH-His2 (10 µM, 30 min attempted. Indeed, the software AGADIR, accessible via http:// at 37 °C); b incubation with the preformed αH-His2[Pd] complex, prepared agadir.crg.es/, predicts that the sequence of the αH-His2 peptide by mixing an equimolar mixture of αH-His2 and cis-PdCl2(en) in water at 25 °C and pH 7 is only 12% helical56–58. for 10 min (10 μM, 30 min at 37 °C). The cells were washed with PBS Having at hand the desired peptide, αH-His2, we used a before being observed under the microscope. Incubations were made in combination of circular dichroism, NMR spectroscopy, and Dulbecco´s modified Eagle medium (DMEM) completed with 5% of fetal computational methods to study its coordination properties and bovine serum (FBS-DMEM). λexc = 550 nm, λem = 590-650 nm; c Scheme the effect of Pd(II) binding in its folding. Both CD and NMR of the MAPK pathway and the proposed inhibition of the SOS1-mediated indicated that the free peptide αH-His2 was largely unstructured activation of KRAS by αH-His2[Pd] that should lead to reduced in solution and also showed a very modest stabilization of the α phosphorylation of ERK; d Addition of cis-PdCl2(en) to αH-His2 led to a helical conformation in the presence of the metal ion. Thus, palladium peptide complex that inhibits the RAF-MEK-ERK mitogen- addition of Pd(II) results in a small increase in the negative molar activated protein kinase (MAPK) pathway downstream of KRAS in living ellipticity signal at 222 nm from ≈−2 200 to −5 700 deg cm2 cells, as deduced from the decrease in the amount of phosphorylated pERK. dmol−1, and in the appearance of several long-range NOEs Serum-starved A549 cells were incubated either with αH-His2, the between the side chains of Phe929, Ile932, and Leu934. Molecular preformed metallopeptide αH-His2[Pd], or with cis-PdCl2(en) at the Dynamics studies confirm the Nτ coordination of both His indicated doses for 4 h. Cellular lysates were subjected to Western blot residues, and the partial folding of the peptide from residues analysis by using antibodies to phospho- and total ERK1/2. Densitometry Arg928 to Phe930. These effects were reversible, and addition of a measurement of the Western blot bands. The ratio between phospho- Pd(II) chelator reduced the helical content of the peptide. ERK1/2 and total ERK1/2 are indicated as fold change with respect to Despite the modest effect of Pd(II) chelation in the overall activation conditions (without FBS, lane 2). Activation percentage is shown helical content of the αH-His2 peptide, Pd(II) coordination led to above each column. Original WB and micrographies available as a dramatic change in the affinity for KRAS, so that while no Supplementary Data 2. significant binding was observed by fluorescence anisotropy when the free peptide αH-His2 was incubated with KRAS, titrations with the metallopeptide αH-His2[Pd] resulted in clear binding RAF-MEK-ERK cascade, a well-documented KRAS effector isotherms with tight dissociation constants of 345 nM and pathway implicated in cell proliferation, survival, and differ- 294 nM for KRASG12C and KRASG12V, respectively. This effect is entiation (Fig. 6c)40. As expected, incubation of serum-starved consistent with an induced folding-upon-binding mechanism A549 cells41 with either the apopeptide αH-His2 or with cis- facilitated by the nucleation of the bioactive α helical conforma- PdCl2(en) failed to induce changes in the levels of ERK phos- tion when the Pd(II) coordinates the two His residues. Therefore, phorylation, as measured by blotting the cell lysates with pERK although the coordination of the metal ion does not provide specific antibodies. In contrast, the addition of the preformed enough energy to fold the metallopeptide αH-His2[Pd] into an α- complex αH-His2[Pd] to the cell culture led to a significant helix by itself, it is enough to do so in the presence of KRAS and decrease in the levels of phosphorylated ERK (Fig. 6d), thus the additional interactions with the protein that stabilize the demonstrating that the palladapeptide is capable of inhibiting bound helix. This behavior reproduces that of Intrinsically Dis- ERK phosphorylation pathways in a dose-responsive manner. ordered Proteins or regions of proteins, which are typically involved in protein-protein interactions regulating signaling pathways, that rely on their transition from a disordered to a Discussion folded state upon recognition of their physiological partners58. Peptidomimetics, particularly α-helix mimics, are promising This mechanism allows high specificity, ultra-sensitivity, and platforms for the modulation of “undruggable” protein targets42. switching behavior59, which might also be desirable properties in To date, the α-helical peptide inhibitors reported in the literature designed peptide inhibitors. As expected from the initial coordi- typically rely on the covalent linkages43–45, including carbon nation tests, addition of a Pd(II) chelator disassembles the staples46–48, hydrogen bond surrogates49–51, lactam bridges52,53, metallopeptide αH-His2[Pd] and takes apart the αH-His2[Pd]/ or cysteine crosslinks52,53, among others54,55. These covalent KRAS complex. Importantly, the formation of the αH-His2[Pd]/ modifications afford peptides that are constitutively active, but KRAS complex has functional consequences, so that the inhibi- having at hand stimuli-responsive peptidomimetics, which can be tion of the GTPase activity of KRAS can be modulated in vitro by (de)activated at will, would open interesting opportunities both in the presence of Pd(II). COMMUNICATIONS CHEMISTRY | (2022)5:75 | https://doi.org/10.1038/s42004-022-00691-7 | www.nature.com/commschem 5 ARTICLE COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00691-7 Finally, the preformed metallopeptide αH-His2[Pd] was Fluorescence Anisotropy and switch experiments. Measurements were made internalized by A549 cells, as demonstrated by fluorescence with a Jobin-Yvon Fluoromax-3, (DataMax 2.20), coupled to a Wavelength Elec- tronics LFI − 3751 temperature controller, and using a 1 mL Hellma micro cuvette. microscopy, and the peptide inhibited the MAPK RAF-MEK- Settings: integration time, 2.0 s; excitation slit width, 5.0 nm; emission slit width, ERK cascade in a dose-responsive manner, as demonstrated by 20.0 nm; excitation wavelength, 559 nm; emission wavelength, 585 nm. Aliquots of the measured decrease in the levels of phosphorylated ERK. To a stock solution of protein were added onto 15 nM solutions of αH-His2 or αH- our knowledge, this is the first demonstration of a designed His2[Pd] in Tris-HCl buffer 20 mM, pH 7.5, 100 mM NaCl, and the anisotropy was metallopeptide that can modulate a signaling pathway in living recorded after each addition. Experimental data correspond to the mean of three independent titration experiments and are representative of at least three biological cells. replicates performed with independent preparations of recombinant proteins. All In this proof-of-concept work we challenge the current para- experiments carried out at 25 °C. Before use the corresponding purified protein, the digm for the design of α-helix peptidomimetics, which seeks to glycerol was removed using a Zeba Spin Desalting Columns (Thermo fisher) that maximize the α-helical content to avoid the entropic penalty had been equilibrated with anisotropy buffer (Tris-HCl buffer 20 mM, pH 7.5, 100 mM NaCl). For the switch experiment, after completing the titration at associated with the folding of the peptide into its bioactive helical saturating concentrations of the protein, 50 eq. of DEDTC (relative to the metal- conformation. We demonstrate that such drastic preorganization lopeptide) was added to the solution and the anisotropy was recorded. After that, is not required and that marginally stable helices can act as 50 eq. of cis-PdCl2(en) was added (relative to the metallopeptide) and the aniso- effective inhibitors through a folding-upon-binding mechanism, tropy was recorded again. just like the natural Intrinsically Disordered Proteins that exploit this process to modulate protein activity. Moreover, we also High-resolution NMR. NMR experiments were recorded on a Bruker Avance III 600- report, for the first time, a strategy to obtain a switchable α-helical MHz spectrometer (IRB Barcelona) equipped with a quadruple (1H, 13C, 15N, 31P) resonance cryogenic probe head and a z-pulse field gradient unit at 298 K using a peptidomimetics that modify their folding and binding affinity to 1 mM solution of αH-His2 peptide—in the presence or absence of cis-PdCl2(en)—1D their targets in response to an external stimulus, namely, the proton spectra were recorded with a sweep width of 12000 Hz and 32 k data points. A presence of a Pd(II) metal ion. We applied this strategy for the total of 16 scans were accumulated with an acquisition time of 2.05 s. A Watergate w5 design of a short peptide derived from the αH helix of the composite pulse was used to suppress the water signal. 1H 2D-TOCSY and NOESY experiments were acquired in 90% H2O/10% D2O and used to assign the spin systems cofactor SOS1 that binds the oncogenic protein KRAS with high corresponding to the peptide resonances61–63. For the 2D-NOESY experiments, mixing affinity upon coordination to Pd(II). NMR and MD studies times of 300, 150, and 80 ms were acquired to minimize the impact of spin-diffusion in provide a detailed atomistic picture of the nucleating effect of the the assignments. Spin-locking fields of 8 kHz and 50 ms mixing time was used for the metal center and support a folding-upon binding mechanism 2D-TOCSY experiments. All 2D spectral widths were 8000 Hz. The data size was 512 points in F1, indirect dimension, and 2048 points in F2, direct dimension. For each F1 typical of Intrinsically Disordered Proteins60. In consonance with value, 48 transients were accumulated in the NOESY and 32 in the TOCSY experi- the metal-induced activation, the metal-free peptide, αH-His2, ments respectively. Data were processed with a combination of exponential and shifted is biologically inactive in cellular assays, but the metallopeptide sine-bell window functions for each dimension followed by automated baseline- and αH-His2[Pd] induced a significant inhibitory effect in KRAS- phase correction using TopSpin 3.5 (© Bruker 2020). The 512 × 2k data matrices were promoted pathways. Our approach uncovers new possibilities for zero-filled to 2k × 2k (NOESY and TOCSY). Structure Calculation: To prove that the primary structure (composition and connectivity) corresponds to the theoretical the design of dynamic α-helix peptidomimetics that exploit the sequence, we have followed the sequence assignment strategy63. We identified the folding-upon-binding mechanism and can be extended to other characteristic spin system of every residue in the sequence, using the 2D-TOCSY biological targets. experiment, and every spin system was connected to the following one via NOEs observed from the side chain of a given residue (i) to the amide proton of the following residue (i + 1) as well as from the amide proton of (i) to the amide of (i + 1). The full spin analysis as well as the assignment of the NOEs were carried out manually using Methods CARA software64,65. Distance restraints derived from the NOESY experiments were Peptide synthesis. The peptide αH-His2 was synthesized on a Liberty Blue Lite used for the NMR-based model building of the peptide in solution using unambigu- automatic microwave assisted peptide synthesizer from CEM Corporation, fol- ously assigned peaks exclusively and the program CNS 1.2 (Crystallography and NMR lowing the manufacturer’s recommended procedures. The synthesis was on a system)66. The protocol consisted of an implicit water simulated-annealing of 0.1 mmol scale using a 0.5 mmol/g load H-Rink amide ChemMatrix resin. Amino 120 structures using 8,000 cooling steps followed by an explicit water refinement of the acids were coupled in 5-fold excess using DIC (N,N'-Diisopropylcarbodiimide) as calculated structures using all experimental restraints during 1200 steps. The Pd activator, Oxime as base, and DMF as solvent. Couplings were conducted for 4 min coordination was not explicitly included in the calculation. To display the metal at 90 °C. Deprotection of the temporal Fmoc protecting group was performed with coordination, we manually optimized the His rotamers to facilitate the coordination 20% piperidine in DMF for 1 min at 75 °C. TMR was manually coupled to the and the Pd was added to the final model displayed in Fig. 2. N-terminus of the sequence by incubation of the resin-bound peptide with 3 eq. of 5-carboxytetramethylrhodamine (0.15 mmol, 64.5 mg), 3 eq. of HATU, and 5 eq. of DIEA 0.2 M in DMF for 60 min. Cleavage/deprotection was done by treating the Computational methods. Ligand-protein docking exploration with GOLD30, and resin-bound peptide for 2 h with 900 μL TFA, 50 μL CH2Cl2, 25 μL H2O and 25 μL GaudiMM28, was first performed to discern the possible coordination modes of the TIS (1 mL of cocktail/40 mg resin). The resin was filtered, and the filtrate was Pd(II) complex with the αH-His2 peptide. Then, the αH-His2[Pd] cores were added onto ice-cold diethyl ether. After ~20 min, the precipitate was centrifuged parametrized with the Amber tool MCPB.py67, based on the bonded model and washed again with of ice-cold ether. The solid residue was dried under argon approach. QM calculations were performed with the DFT formalism as imple- and redissolved in water/CH3CN (1:1) for HPLC purification on a semipreparative mented in Gaussian 09, combined with the hybrid functional B3LYP68. The basis Agilent 1100 series LC using a Phenomenex Luna–C18 (250 × 10 mm) reverse-phase set employed was 6-31 g(d,p) for C, H and N atoms, while the SDD basis set with f- column and a linear gradient (5 to 75% B over 40 min, 4 mL/min.; A: H2O 0.1% polarization function was used for the metal ion, with a pseudo-potential for the TFA, B: CH3CN 0.1% TFA). Collected fractions with pure products were freeze- core electrons69. All these calculations were combined with the Grimme’s D3 dried to afford the desired peptide. The peptide was analyzed by analytical correction for dispersion70. The complexes were embedded in a solvent-polarizable UHPLC-MS with an Agilent 1200 series LC/MS using an SB C18 (1.8 μm, dielectric continuum model (SMD, water)71. Once these QM calculations were 2.1 × 50 mm) analytical column from Phenomenex with a linear gradient from 5 to obtained, the parameters were created for those atoms coordinating the Pd(II) ion 95% of solvent B for 20 min at a flow rate of 0.35 mL/min (A: water with 0.1% TFA, through Seminario’s approach72. The point charges were computed based on the B: acetonitrile with 0.1% TFA). Compounds were detected by UV absorption at RESP protocol73. Once the parameters were obtained, the systems were set up with 222, 270, and 330 nm. Electrospray Ionization Mass Spectrometry (ESI/MS) was AMBER18 tleap to build the topology and the coordinates file. The force field used performed with an Agilent 6120 Quadrupole LC/MS model in positive scan mode for the protein atoms was the ff14SB74, improved force field recommended for the using direct injection of the purified peptide solution into the MS detector. simulation of peptides. For the remaining atoms, the GAFF force field was applied; while the parameters obtained from the MCPB.py calculation were used for the Pd(II) ion. Explicit water TIP3P solvent was also considered in the complexes75, introducing neutrality by the addition of three and one chloride ions for the Circular Dichroism. Circular Dichroism was measured on a Jasco-715 coupled metallic and free peptide, respectively. The systems were embedded into a cubic with a thermostat Nestlab RTE-111 using a 2 mm Hellma cuvette at 25 °C and the box containing between 2700 and 3000 water molecules, 10 Å from the protein to following settings: Acquisition range, 300-195 nm; band width, 2.0 nm; resolution, the edge of the box. The topology and the coordinates files achieved for each 0.2 nm; accumulation, 5 scans; sensitivity, 10 mdeg; response time, 0.25 s; speed, complex were then used for a first 10 ns classical Molecular Dynamics (MD) 100 nm/min. The spectra are the average of 5 scans and were processed using the simulation. The coordinates of the last structure were used for the Gaussian “smooth” macro in the program KaleidaGraph (v 3.5 by Synergy Software). accelerated Molecular Dynamics (GaMD) simulations76, which allows an extensive 6 COMMUNICATIONS CHEMISTRY | (2022)5:75 | https://doi.org/10.1038/s42004-022-00691-7 | www.nature.com/commschem COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00691-7 ARTICLE exploration of the conformational space. The AMBER ff14SB force field in an NVT Reporting summary. Further information on research design is available in the Nature ensemble was also used with SHAKE algorithm. A boost on both dihedral a total Research Reporting Summary linked to this article. potential energy was applied, producing GaMDs of at least 2 μs for each system. To prove the correct exploration of the conformational space, PCA analysis were applied to the trajectories. Despite GaMD allows extensive conformational exploration, simulations were started from different putative folded geometries of Data availability the peptide to assure unbiased predictions to evaluate the progression of the system The PDB structures of the NMR and Molecular Dynamics studies are available as from the two extreme situations (data not shown). Supplementary Data 1; the original WB and microscope images are available as Supplementary Data 2. All other datasets generated and analyzed during the current study are available from the corresponding authors on reasonable request. Protein Expression and Purification. The constructs pDNA2.0 6H-TEV-KRAS, pDNA2.0 6H-TEV-KRAS G12V and pDNA2.0 6H-FLAG-TEV-KRAS G12C were Received: 3 March 2022; Accepted: 10 June 2022; purchased from Addgene. BL21 cells (Invitrogen) were transform with the con- structs and grown in Luria broth (LB) to OD 600 0.7 and induced with 250 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 16 h at 16 °C. Cells were pelleted and resuspended in lysis buffer (20 mM sodium phosphate, pH 8.0, 500 mM NaCl, 10 mM imidazole, 1 mM 2-mercaptoethanol (BME), 5% (vol/vol) glycerol). After centrifugation, the pellet was frozen until use. Then, protease inhibitor (EDTA free) and lysozyme (1 mg/mL) were added, and the pellet was sonicated for 6 min. References (35 sec. on /10 sec. off). Protein was purified over an IMAC (immobilized metal 1. Sanchez-Vega, F. et al. Oncogenic signaling pathways in the cancer genome affinity chromatography) following HisPur Ni-NTA protocols (Thermo Fisher). Atlas. Cell 173, 321–337.e10 (2018). Protein was concentrated in a 10-kDa Amicon Ultra-15 (Millipore) aliquoted with 2. Forbes, S. A. et al. COSMIC: exploring the world’s knowledge of somatic 15% glycerol, and then flash-frozen and stored at −20 °C. mutations in human cancer. Nucleic Acids Res. 43, D805–D811 (2015). 3. Hobbs, G. A., Der, C. J. & Rossman, K. L. RAS isoforms and mutations in cancer at a glance. J. Cell Sci. 129, 1287–1292 (2016). 4. Ledford, H. Cancer: the Ras renaissance. Nature 520, 278–280 (2015). Nucleotide Release assays. Loading of fluorescent-labeled GDP (mantGTP) to 5. Spiegel, J., Cromm, P. M., Zimmermann, G., Grossmann, T. N. & Waldmann, KRASwt was conducted following previous reports8,77. Purified KRASwt was buffer- H. Small-molecule modulation of Ras signaling. Nat. Chem. Biol. 10, 613–622 exchanged in Zeba Spin Desalting Columns (Thermo Fisher) in loading buffer (2014). (20 mM Tris-HCl [pH 7.5], 50 mM NaCl, 4 mM EDTA and 1 mM DTT). The 6. Wittinghofer, A. & Waldmann, H. Ras—a molecular switch involved in tumor resulted eluted KRASwt, after measuring their concentration, was incubated with formation. Angew. Chem. Int. Ed. 39, 4192–4214 (2000). 10-fold molar excess of mantGTP (Abcam) for 1.5 h at 20 °C in the dark. Reactions 7. Rajalingam, K., Schreck, R., Rapp, U. R. & Albert, S. Ras oncogenes and their were supplemented with 10 mM MgCl2 and incubated for 1 h on ice. Free downstream targets. Biochim. Biophys. Acta 1773, 1177–1195 (2007). nucleotide was removed using a Zeba Spin Desalting Columns (Thermo Fisher) 8. Margarit, S. M. et al. Structural evidence for feedback activation by Ras.GTP of that had been equilibrated with the reaction buffer (20 mM Tris-HCl, pH 7.5, the Ras-specific nucleotide exchange factor SOS. Cell 112, 685–695 (2003). 50 mM NaCl, 1 mM MgCl2 and 1 mM DTT). The effect of peptides on the intrinsic 9. Patgiri, A., Yadav, K. K., Arora, P. S. & Bar-Sagi, D. An orthosteric inhibitor of rate of nucleotide release was monitored using the decrease in fluorescence with time as mantGTP dissociates from KRAS in a 100 μL reaction mixture (96-well the Ras-Sos interaction. Nat. Chem. Biol. 7, 585–587 (2011). plate) of 1 μM KRASwt/mantGPT complex and 10 μM of αH-His2 or αH-His2[Pd]. 10. Yoo, D. Y., Hauser, A. D., Joy, S. T., Bar-Sagi, D. & Arora, P. S. Covalent Fluorescence was excited at 370 nm and emission was monitored at 430 nm during targeting of Ras G12C by rationally designed peptidomimetics. ACS Chem. 30 min, using a Tecan Infinite M Plex plate reader. The data were processed using Biol. 15, 1604–1612 (2020). the program GraphPad Prism. 11. Leshchiner, E. S. et al. Direct inhibition of oncogenic KRAS by hydrocarbon- stapled SOS1 helices. Proc. Natl Acad. Sci. USA 112, 1761–1766 (2015). 12. Hong, S. H. et al. A Sos proteomimetic as a pan-Ras inhibitor. Proc. Natl. Acad. Sci. USA 118, e2101027118 (2021). Cell internalization and inhibition of ERK1/2. All steps were performed on a 13. Ghadiri, M. R. & Fernholz, A. K. Peptide architecture. Design of stable α- sterile clean bench Telstar AV-100 at rt. Solutions stored in a fridge were warmed helical metallopeptides via a novel exchange-inert ruthenium(III) complex. J. beforehand in a water bath (37 °C). Unless otherwise specified, all incubations were Am. Chem. Soc. 112, 9633–9635 (1990). performed in DMEM containing 5% of fetal bovine serum (FBS-DMEM). Cell 14. Ghadiri, M. R. & Choi, C. Secondary structure nucleation in peptides. Culture: A549 cell line was cultured in DMEM (Dulbecco´s modified Eagle´s Transition metal ion stabilized α-helices. J. Am. Chem. Soc. 112, 1630–1632 medium), 5 mM glutamine, penicillin (100 units/mL) and streptomycin (100 units/ (1990). mL), all from Invitrogen. Proliferating cultures were maintained in a 5% CO2 15. Kelso, M. J., Hoang, H. N., Appleton, T. G. & Fairlie, D. P. The first solution humidified incubator at 37 °C. For all the experiments, cells were seeded in the stucture of a single α-helical turn. A pentapeptide α-helix stabilized by a metal corresponding well at the indicated concentration 2 days before treatment. A549 clip. J. Am. Chem. Soc. 122, 10488–10489 (2000). cells were seeded on glass-bottom plates 48 h (150.000 cell/ml) before treatment. 16. Ma, M. T., Hoang, H. N., Scully, C. C. G., Appleton, T. G. & Fairlie, D. P. Culture medium was removed and DMEM containing 5% fetal bovine serum (FBS- Metal clips that induce unstructured pentapeptides to be α-helical in water. J. DMEM) and αH-His2 or αH-His2[Pd] (10 µM) were added. The metallopeptide was made just before the addition to cells by pre-incubating with metal complexes Am. Chem. Soc. 131, 4505–4512 (2009). (1:1) in water for 10 min. After 30 min of incubation with cells, these were washed 17. Rodríguez, J., Mosquera, J., Vázquez, M. E. & Mascareñas, J. L. Nickel- twice with PBS and replaced with fresh FBS-DMEM to observe under the promoted recognition of long DNA sites by designed peptide derivatives. microscope with adequate filters. Digital pictures of the different samples were Chemistry 22, 13474–13477 (2016). taken under identical conditions of gain and exposure. A549 cells were seeded on 18. Rodríguez, J., Mosquera, J., García-Fandiño, R., Vázquez, M. E. & Mascareñas, glass-bottom plates (150.000 cell/ml) 48 h before treatment. Culture medium was J. L. A designed DNA binding motif that recognizes extended sites and spans removed, the cells was serum-starved and αH-His2, αH-His2[Pd] or cis-PdCl2(en) two adjacent major grooves. Chem. Sci. 7, 3298–3303 (2016). were added at different concentrations and incubated for 4 h. After two washes 19. Learte-Aymamí, S., Rodríguez, J., Vázquez, M. E. & Mascareñas, J. L. with PBS, the cells were lysed in Laemmli buffer containing Tris pH 6,8 1 M, Assembly of a ternary metallopeptide complex at specific DNA sites mediated Glycerol, SDS, Bromophenol Blue, and β-mercaptoethanol, and heated at 95 °C for by an AT-hook adaptor. Chemistry 26, 8875–8878 (2020). 10 min. The samples were separated by SDS-12.5% polyacrylamide gel and 20. Gómez-González, J. et al. Dynamic stereoselection of peptide helicates and transferred to nitrocellulose membrane, to probed by Western blotting. Levels of their selective labeling of DNA replication foci in cells*. Angew. Chem. Int. Ed. total ERK2 and phosphorylated ERK were detected with anti-ERK1/2 and phos- Engl. 60, 8859–8866 (2021). pho-ERK1/2) antibodies, respectively and revealed with LuminataTM Classico 21. Learte-Aymamí, S., Vidal, C., Gutiérrez-González, A. & Mascareñas, J. L. Western HRP substrate following manufacter´s protocol. The visualization was Intracellular reactions promoted by Bis(histidine) miniproteins stapled using performed with ChemiDoc MP imaging system by Biorad, and the processing of palladium(II) complexes. Angew. Chem. Int. Ed. Engl. 59, 9149–9154 (2020). the images and densitometry quantification was carried out with ImageLab 22. Learte-Aymamí, S., Curado, N., Rodríguez, J., Vázquez, M. E. & Mascareñas, J. program. L. Metal-dependent DNA recognition and cell internalization of designed, basic peptides. J. Am. Chem. Soc. 139, 16188–16193 (2017). 23. Boriack-Sjodin, P. A., Margarit, S. M., Bar-Sagi, D. & Kuriyan, J. The Fluorescence microscopy. All images were obtained with an Andor Zyla mounted structural basis of the activation of Ras by Sos. Nature 394, 337–343 (1998). on a Nikon TiE. LED excitation wavelength 550 nm. Filter cube TRITC-B-000 24. Coin, I., Beyermann, M. & Bienert, M. Solid-phase peptide synthesis: from (Semrock): BP 543/22 nm, LP 593/40 nm, and DM 562 nm. Images were further standard procedures to the synthesis of difficult sequences. Nat. Protoc. 2, processed with Image J. 3247–3256 (2007). COMMUNICATIONS CHEMISTRY | (2022)5:75 | https://doi.org/10.1038/s42004-022-00691-7 | www.nature.com/commschem 7 ARTICLE COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00691-7 25. Vanier, G. S. Microwave-assisted solid-phase peptide synthesis based on the 54. Horne, W. S. & Grossmann, T. N. Proteomimetics as protein-inspired scaffolds Fmoc protecting group strategy (CEM). Methods Mol. Biol. 1047, 235–249 with defined tertiary folding patterns. Nat. Chem. 12, 331–337 (2020). (2013). 55. Rezaei Araghi, R. & Keating, A. E. Designing helical peptide inhibitors of 26. Shepherd, N. E., Hoang, H. N., Abbenante, G. & Fairlie, D. P. Single turn protein-protein interactions. Curr. Opin. Struct. Biol. 39, 27–38 (2016). peptide α helices with exceptional stability in water. J. Am. Chem. Soc. 127, 56. Muñoz, V. & Serrano, L. Elucidating the folding problem of helical peptides 2974–2983 (2005). using empirical parameters. II. Helix macrodipole effects and rational 27. Jones, G., Willett, P., Glen, R. C., Leach, A. R. & Taylor, R. Development and modification of the helical content of natural peptides. J. Mol. Biol. 245, validation of a genetic algorithm for flexible docking. J. Mol. Biol. 267, 275–296 (1995). 727–748 (1997). 57. Muñoz, V. & Serrano, L. Elucidating the folding problem of helical peptides 28. Rodríguez-Guerra Pedregal, J., Sciortino, G., Guasp, J., Municoy, M. & using empirical parameters. III. Temperature and pH dependence. J. Mol. Biol. Maréchal, J.-D. GaudiMM: A modular multi-objective platform for molecular 245, 297–308 (1995). modeling. J. Comput. Chem. 38, 2118–2126 (2017). 58. Muñoz, V. & Serrano, L. Elucidating the folding problem of helical peptides 29. Di Meo, T. et al. Functionalized artificial bidomain proteins based on an α- using empirical parameters. Nat. Struct. Biol. 1, 399–409 (1994). solenoid protein repeat scaffold: a new class of artificial diels–alderases. ACS 59. Teilum, K., Olsen, J. G. & Kragelund, B. B. On the specificity of Omega 4, 4437–4447 (2019). protein–protein interactions in the context of disorder. Biochem. J. 478, 30. Sciortino, G., Garribba, E. & Maréchal, J.-D. Validation and applications of 2035–2050 (2021). protein-ligand docking approaches improved for metalloligands with multiple 60. Tsomaia, N. Peptide therapeutics: targeting the undruggable space. Eur. J. vacant sites. Inorg. Chem. 58, 294–306 (2019). Med. Chem. 94, 459–470 (2015). 31. Kuzmic, P. Program DYNAFIT for the analysis of enzyme kinetic data: 61. Bax, A. & Davis, D. G. Practical aspects of two-dimensional transverse NOE application to HIV proteinase. Anal. Biochem. 237, 260–273 (1996). spectroscopy. J. Magn. Reson. 63, 207–213 (1985). 32. Kuzmič, P. In Methods in Enzymology Vol. 467 (ed. Abelson, J.) 247–280 62. Macura, S. & Ernst, R. R. Elucidation of cross relaxation in liquids by two- (Academic Press, 2009). dimensional N.M.R. spectroscopy. Mol. Phys. 41, 95–117 (1980). 33. Toto, A. et al. Templated folding of intrinsically disordered proteins. J. Biol. 63. Wuthrich, K. NMR of Proteins and Nucleic Acids. (John Wiley & Sons, 1986). Chem. 295, 6586–6593 (2020). 64. Ramirez-Espain, X., Ruiz, L., Martin-Malpartida, P., Oschkinat, H. & Macias, 34. Kammerer, R. A. et al. An autonomous folding unit mediates the assembly of M. J. Structural characterization of a new binding motif and a novel binding two-stranded coiled coils. Proc. Natl Acad. Sci. USA 95, 13419–13424 (1998). mode in group 2 WW domains. J. Mol. Biol. 373, 1255–1268 (2007). 35. Lee, D. L., Lavigne, P. & Hodges, R. S. Are trigger sequences essential in the 65. Martín-Gago, P. et al. A tetradecapeptide somatostatin dicarba-analog: folding of two-stranded alpha-helical coiled-coils? J. Mol. Biol. 306, 539–553 synthesis, structural impact and biological activity. Bioorg. Med. Chem. Lett. (2001). 24, 103–107 (2014). 36. Rojas, R. J., Kimple, R. J., Rossman, K. L., Siderovski, D. P. & Sondek, J. 66. Brunger, A. T. Version 1.2 of the crystallography and NMR system. Nat. Established and emerging fluorescence-based assays for G-protein function: Ras- Protoc. 2, 2728–2733 (2007). superfamily GTPases. Comb. Chem. High. Throughput Screen. 6, 409–418 (2003). 67. Li, P. & Merz, K. M. Jr MCPB.py: A python based metal center parameter 37. Jeganathan, S., Müller, M. P., Ali, I. & Goody, R. S. Assays for nucleotide builder. J. Chem. Inf. Model. 56, 599–604 (2016). competitive reversible and irreversible inhibitors of Ras GTPases. Biochemistry 68. Becke, A. D. Density‐functional thermochemistry. III. The role of exact 57, 4690–4699 (2018). exchange. J. Chem. Phys. 98, 5648–5652 (1993). 38. Hiratsuka, T. New ribose-modified fluorescent analogs of adenine and 69. Bergner, A., Dolg, M., Küchle, W., Stoll, H. & Preuß, H. Ab initio energy- guanine nucleotides available as substrates for various enzymes. Biochim. adjusted pseudopotentials for elements of groups 13–17. Mol. Phys. 80, Biophys. Acta 742, 496–508 (1983). 1431–1441 (1993). 39. Roberts, P. J. & Der, C. J. Targeting the Raf-MEK-ERK mitogen-activated 70. Grimme, S., Antony, J., Ehrlich, S. & Krieg, H. A consistent and accurate ab protein kinase cascade for the treatment of cancer. Oncogene 26, 3291–3310 initio parametrization of density functional dispersion correction (DFT-D) for (2007). the 94 elements H-Pu. J. Chem. Phys. 132, 154104 (2010). 40. Jung, Y. D., Nakano, K., Liu, W., Gallick, G. E. & Ellis, L. M. Extracellular 71. Marenich, A. V., Cramer, C. J. & Truhlar, D. G. Universal solvation model signal-regulated kinase activation is required for up-regulation of vascular based on solute electron density and on a continuum model of the solvent endothelial growth factor by serum starvation in human colon carcinoma defined by the bulk dielectric constant and atomic surface tensions. J. Phys. cells. Cancer Res. 59, 4804–4807 (1999). Chem. B 113, 6378–6396 (2009). 41. Miller, M. The importance of being flexible: the case of basic region leucine 72. Seminario, J. M. Calculation of intramolecular force fields from second- zipper transcriptional regulators. Curr. Protein Pept. Sci. 10, 244–269 (2009). derivative tensors. Int. J. Quantum Chem. 60, 1271–1277 (1996). 42. de Araujo, A. D. et al. Comparative α-helicity of cyclic pentapeptides in water. 73. Bayly, C. I., Cieplak, P., Cornell, W. & Kollman, P. A. A well-behaved Angew. Chem. Int. Ed. Engl. 53, 6965–6969 (2014). electrostatic potential based method using charge restraints for deriving 43. Bernal, F., Tyler, A. F., Korsmeyer, S. J., Walensky, L. D. & Verdine, G. L. atomic charges: the RESP model. J. Phys. Chem. 97, 10269–10280 (1993). Reactivation of the p53 tumor suppressor pathway by a stapled p53 peptide. J. 74. Maier, J. A. et al. ff14SB: improving the accuracy of protein side chain and Am. Chem. Soc. 129, 2456–2457 (2007). backbone parameters from ff99SB. J. Chem. Theory Comput. 11, 3696–3713 44. Cromm, P. M., Spiegel, J. & Grossmann, T. N. Hydrocarbon stapled peptides (2015). as modulators of biological function. ACS Chem. Biol. 10, 1362–1375 (2015). 75. Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W. & Klein, M. 45. Walensky, L. D. & Bird, G. H. Hydrocarbon-stapled peptides: principles, L. Comparison of simple potential functions for simulating liquid water. J. practice, and progress. J. Med. Chem. 57, 6275–6288 (2014). Chem. Phys. 79, 926–935 (1983). 46. Chapman, R. N., Dimartino, G. & Arora, P. S. A highly stable short alpha- 76. Miao, Y. et al. Improved reweighting of accelerated molecular dynamics helix constrained by a main-chain hydrogen-bond surrogate. J. Am. Chem. simulations for free energy calculation. J. Chem. Theory Comput. 10, Soc. 126, 12252–12253 (2004). 2677–2689 (2014). 47. Patgiri, A., Jochim, A. L. & Arora, P. S. A hydrogen bond surrogate approach 77. McCarthy, M. J. et al. Discovery of high-affinity noncovalent allosteric KRAS for stabilization of short peptide sequences in α-helical conformation. Acc. inhibitors that disrupt effector binding. ACS Omega 4, 2921–2930 (2019). Chem. Res. 41, 1289–1300 (2008). 78. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory 48. Patgiri, A., Menzenski, M. Z., Mahon, A. B. & Arora, P. S. Solid-phase research and analysis. J. Comput. Chem. 25, 1605–1612 (2004). synthesis of short α-helices stabilized by the hydrogen bond surrogate 79. Chou, K. C., Zhang, C. T. & Maggiora, G. M. Disposition of amphiphilic approach. Nat. Protoc. 5, 1857–1865 (2010). helices in heteropolar environments. Proteins 28, 99–108 (1997). 49. Harrison, R. S. et al. Downsizing human, bacterial, and viral proteins to short 80. Chou, K.-C., Lin, W.-Z. & Xiao, X. Wenxiang: a web-server for drawing water-stable alpha helices that maintain biological potency. Proc. Natl Acad. wenxiang diagrams. Nat. Sci. (Irvine) 03, 862–865 (2011). Sci. USA 107, 11686–11691 (2010). 50. Hoang, H. N. et al. A novel long-range n to π* interaction secures the smallest known α-helix in water. Angew. Chem. Int. Ed. Engl. 58, 18873–18877 (2019). Acknowledgements 51. Shepherd, N. E., Abbenante, G. & Fairlie, D. P. Consecutive cyclic Financial support from the Spanish grants RTI2018-099877-B-I00, CTQ2015-70698-R, pentapeptide modules form short α-helices that are very stable to water and Orfeo-cinqa network CTQ2016-81797-REDC, Red the Péptidos en Biomedicina y denaturants. Angew. Chem. Int. Ed. 43, 2687–2690 (2004). Nanociencia, RED2018-102417-T; Centro Singular de Investigación de Galicia accred- 52. Fairlie, D. P. & Dantas de Araujo, A. Review stapling peptides using cysteine itation 2019-2022, ED431G 2019/03 and the European Union (European Regional crosslinking. Biopolymers 106, 843–852 (2016). Development Fund - ERDF) and AGAUR (SGR-50); the European Research Council 53. Jackson, D. Y., King, D. S., Chmielewski, J., Singh, S. & Schultz, P. G. General (Advanced Grant No. 340055), are gratefully acknowledged. S. Learte-Aymamí thanks approach to the synthesis of short α-helical peptides. J. Am. Chem. Soc. 113, the MINECO for her Ph.D. fellowship. G. Sciortino thanks the Spanish MICINN Juan de 9391–9392 (1991). la Cierva program, FJC2019-039135-I. We also acknowledge institutional funding from 8 COMMUNICATIONS CHEMISTRY | (2022)5:75 | https://doi.org/10.1038/s42004-022-00691-7 | www.nature.com/commschem COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00691-7 ARTICLE MINECO through the Centers of Excellence Severo Ochoa Award given to IRB Barcelona, Correspondence and requests for materials should be addressed to José L. Mascareñas or as well as from the CERCA Program of the Generalitat de Catalunya. M.J.M. is an ICREA M. Eugenio Vázquez. Programme Investigator. Molecular graphics performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the UCSF, with Peer review information Communications Chemistry thanks the anonymous reviewers support from NIH P41-GM10331178. We also want to thank Dr. Huy Hoang and Prof. for their contribution to the peer review of this work. Peer reviewer reports are available. David P. Fairlie, from The University of Queensland, Brisbane, for sharing the NMR structure of the peptide Ac-[KAAAD]-NH2 used in our preliminary modeling. Finally, Reprints and permission information is available at http://www.nature.com/reprints we also thank R. Menaya-Vargas for her technical assistance with cell experiments. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author contributions Conceptualization, M.E.V. and J.L.M; methodology, J.R.C., J.-D.M., M.J.M., J.L.M., and M.E.V.; Validation, S.L.A., P.M.M., L.R.M., G.S., J.R.C., J.-D.M., M.J.M., J.L.M., and M.E.V.; Open Access This article is licensed under a Creative Commons formal analysis, S.L.A., P.M.M., L.R.M., G.S., J.R.C., J.-D.M, and M.J.M.; investigation, S.L.A., P.M.M., M.J.M., L.R.M. and G.S.; resources, M.E.V., J.L.M., M.J.M. and J.-D.M.; data Attribution 4.0 International License, which permits use, sharing, curation, S.L.A., P.M.M., L.R.M., G.S. and J.R.C.; writing—original draft, M.E.V., J.L.M., adaptation, distribution and reproduction in any medium or format, as long as you give M.J.M. and J.-D.M.; writing—review and editing, S.L.A., P.M.M., L.R.M., G.S., J.R.C., appropriate credit to the original author(s) and the source, provide a link to the Creative J.-D.M., M.J.M., J.L.M., and M.E.V.; visualization, J.R.C., J.-D.M, M.J.M., J.L.M., and M.E.V.; Commons license, and indicate if changes were made. The images or other third party supervision, J.R.C., J.-D.M., M.J.M., J.L.M., and M.E.V.; project administration, M.E.V.; material in this article are included in the article’s Creative Commons license, unless funding acquisition, M.E.V., J.L.M., M.J.M. and J.-D.M. Author Contributions are detailed indicated otherwise in a credit line to the material. If material is not included in the as Supplementary Note 1 in the Supplementary Information file. article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ Competing interests licenses/by/4.0/. The authors declare no competing interests. © The Author(s) 2022 Additional information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s42004-022-00691-7. COMMUNICATIONS CHEMISTRY | (2022)5:75 | https://doi.org/10.1038/s42004-022-00691-7 | www.nature.com/commschem 9