Sequence-Selective Molecular Recognition of the C-Terminal CaaX

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Sequence-Selective Molecular Recognition of the C‑Terminal CaaXBoxes of Rheb and Related Ras-Proteins by Synthetic Receptors Peter M. Düppe,† Thao Tran Thi Phuong,† Jasmin Autzen,† Miriam Schöpel,‡ King Tuo Yip,‡ Raphael Stoll,*,‡ and Jürgen Scherkenbeck*,† †

University of Wuppertal, Gaußstraße 20, D-42119 Wuppertal, Germany Ruhr-University of Bochum, Universitätsstraße 150, D-44780 Bochum, Germany

‡

S Supporting Information *

ABSTRACT: Constitutive activation of Ras-proteins plays an important role in the development of aggressive colorectal carcinomas and several other types of cancer. Despite some progress in recent years in the case of K-Ras4B, until now not a single small molecule inhibitor has been identified that binds efficiently to Rheb and interrupts the protein−protein interactions with mTOR. We describe here a complementary approach that aims at inhibiting membrane insertion of Rheb and related Ras proteins by masking the crucial C-terminal CaaX-box with peptidomimetic receptors identified in combinatorial solid-phase libraries.

of benign hamartomatous tumors in the brain, kidneys, heart, lungs, skin, or eyes. Furthermore, Rheb has been reported to be involved in the rapid development of aggressive and drugresistant lymphomas.9−11 Despite numerous attempts to identify small molecules that interfere with the binding sites of Ras with either GEF, GAP or GTP, no drug has entered the development phase.12−18 The reasons for this failure are manifold.19,20 On the one side, the search for small molecules that inhibit the protein−protein interactions between Ras and its effectors with a small molecule is a daunting task due to the large interaction area and weak contributions of each residue to the overall binding energy.21,22 On the other side, tightly or even irreversibly binding GTP analogues bear the risk of high toxicity due to interactions with many other GTPases. Another strategy aims at inhibiting the membrane insertion and localization of Ras-proteins, a prerequisite for normal biological function. Ras-proteins are post-translationally lipidated by prenyl transferases, which recognize the C-terminal CaaX-box of Ras-proteins and transfer farnesyl and/or palmitoyl residues to Cys181 located at the carboxyterminus.23,24 During the past decade, several inhibitors of farnesyl transferases (FTIs) have been identified, and at least a few reached clinical development. However, all FTIs bear the general risk to inactivate unselectively also those farnesyl transferases not involved in Ras-protein prenylation. In

Ras-GTPases (rat adeno sarcoma) belong to the GTP-binding proteins that are involved in numerous cellular processes such as signal transduction, apoptosis, cell growth, and cell regulation.1 Ras proteins switch between an inactive GDPbound “off” state and a GTP-bound “on” state. The exchange of GDP by GTP in the activation step is catalyzed by guanine nucleotide exchange factors (GEFs), while the inactivation of Ras-proteins through hydrolysis of GTP to GDP is catalyzed by GTPase activating proteins (GAPs). Oncogenic Ras-proteins are constitutively active due to a resistance to GAP-mediated GTP hydrolysis. Mutated Ras-proteins are found in about 30% of all human cancers. In particular, hyperactive K-Ras is implicated in 60% of pancreas and colorectal carcinoma.2,3 Thus, proteins of the Ras superfamily have been considered drug targets ever since they were discovered more than 30 years ago. Rheb (Ras homologue enriched in brain) belongs to a unique family within the Ras superfamily of GTPases.4,5 Rheb has been identified as a molecular switch in many cellular processes such as cell volume growth, cell cycle progression, neuronal axon regeneration, autophagy, nutritional deprivation, oxygen stress, and cellular energy status. Rheb activity is regulated by insulin and other growth factors that stimulate the GTP loading of Rheb via inhibition of the tuberous sclerosis complex, a tumor suppressor protein complex (TSC1 and TSC2) that acts as a Rheb GTPase-activating protein.6 The effects of Rheb are mediated via the mammalian target of rapamycin (mTOR), which exists in two different multiprotein complexes, mTORC2 and mTORC1, the latter of which is activated by elevated levels of Rheb.7,8 Hyperactivation of Rheb proteins plays an important role in tuberous sclerosis, which causes the formation © 2014 American Chemical Society

Received: March 19, 2014 Accepted: May 23, 2014 Published: May 23, 2014 1755

dx.doi.org/10.1021/cb5002075 | ACS Chem. Biol. 2014, 9, 1755−1763

ACS Chemical Biology

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molecules is almost impossible. In fact, still the most promising strategy to generate host molecules with the desired recognition properties is to screen combinatorial compound libraries. Based upon the pioneering work of Still and co-workers, a limited number of amino acid and peptide recognizing receptors has been published.28−30 However, almost all published receptor structures are based solely on proteinogenic amino acids, and thus their value as lead structures for the development of a pharmaceutical drug is questionable. We report here for the first time more drug-like receptors, containing D-, β-, and other nonproteinogenic amino acids able to bind sequence-selectively to the C-terminal CSVM sequence of Rheb in an aqueous environment. Sequence-selectivity studies with analogous receptors for H-Ras and K-Ras4B allow a deeper insight into the general requirements for CaaX-box recognition. The structures of hits identified in our one-beadone-compound (OBOC) library were elucidated directly by mass spectrometry, which does not require tedious tagging strategies.

addition, K-Ras4B, one most important oncogenic Rasproteins, can be prenylated alternatively by geranyl−geranyl transferases. Very recently, it was demonstrated that inhibition of the prenyl-binding protein PDEδ with small molecules efficiently interrupts the spatial organization and signaling of farnesylated K-Ras4B.25 A largely neglected, complementary strategy to interfere with the Ras-prenylation process comprises the sequence-selective recognition of the CaaX-box of a specific Ras-protein by a synthetic small receptor molecule and subsequent formation of a supramolecular complex that prevents the farnesyl transferase from transferring the prenyl residue onto the Cys residue. A major advantage of this concept is that CaaX-boxes of specific Ras-proteins differ in sequence and can be selectively blocked. Potential inhibitors are therefore expected to be less toxic compared to farnesyl transferase inhibitors. Remarkably, only one study that deals with the inhibition of Ras-proteins by masking the CaaX-box has been published until now.26 For several reasons Rheb appears to be an ideal target for a molecular recognition approach. First, only one small-molecule inhibitor of Rheb with a rather low affinity has been reported until today.18 Second, it has been demonstrated that a correct membrane attachment and localization is also essential for the normal function of Rheb comparably to Ras.27 Third, native Rheb is only monofarnesylated, and thus membrane attachment cannot be accomplished by a second prenyl or palmitoyl residue. Last but not least, the C-terminus of Rheb (Ser175− Met184) is highly flexible and completely solvent-exposed, an important prerequisite for molecular recognition by a suitable receptor molecule (Table 1).7

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RESULTS AND DISCUSSION Synthesis of the Receptor Library and Fluorescent CaaX-Boxes. A strong supramolecular anion complexation in water represents a considerable challenge, because the major driving forces for this process are electrostatic interactions that are weakened in polar solvents due to the competing formation of H-bonds with the solvent molecules. Hydrophobic and aromatic interactions on the other hand become particularly strong in aqueous solution but are less specific and therefore difficult to design and control. In the recent past several carboxylate recognizing motives have been published, mainly based on amino acid, urea, pyridine, or pyrrole scaffolds.26,31 Currently, the most efficient carboxylate recognizing scaffold is a guanidinio pyrrole developed by Schmuck.32,33 Receptors carrying this headgroup (hg) have been reported to bind sequence-selectively the dipeptide D-Ala-D-Ala, the recognition unit of the antibiotic vancomycin, with a KA value of 3.3 × 105 M−1 in a buffered aqueous solution. Inspired by these impressive results, we also used the Schmuck headgroup 2 for our library consisting of 8000 compounds (Scheme 1, Table 2). We regard this intermediate library size as adequate because very large libraries are extremely difficult to analyze, while on the other hand, in

Table 1. CaaX-Box Sequences of Selected Ras-Proteins CaaX

protein

CVVM CVIM CVLS CKVL CSVM

N-Ras K-Ras4B H-Ras RhoB Rheb

Due to the complex influence of polar, nonpolar, and ionic forces involved between interacting peptides or proteins, the rational design of sequence-selective synthetic receptor Scheme 1. Preparation of the Screening Librarya

a Reagents and conditions: (i) 3 equiv guanidinio pyrrole 2, 3 equiv HBTU (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), 3 equiv HOBT (1-hydroxybenzotriazole), 4.5 equiv DIPEA (N,N′-diisopropylethylamine), DMF (dimethylformamide), 12 h RT, (each coupling step was repeated once); (ii) TFA (trifluoroacetic acid)/TIPS (triisopropylsilane)/H2O, 95:2.5:2.5.

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Table 2. Building Blocks for Positions 1−3a aa2

aa1

L/D-Lys

aa3

L/D/β-Ala

L/D/β-Ala

L/D-Arg

L/D-Val

L/D-Val

L/D-Leu

L-His

L-His

L/D-Phe

L-Ile

L-Ile

L/D-Val

L/D-Pro

L/D-Pro

L/D-Thr

L-Tyr

L-Tyr

L/D-Ala

L/D-Phe

L/D-Asn

Gly L-Lys L-Ser Ac5c GABA L-Tic Inp L-ThzMe2

L/D-Phe Gly L-Lys L-Ser Ac5c GABA L-Tic Inp L-ThzMe2

L-Thz L-ThzMe2 L-His L-Ser

Scheme 2. Solution Synthesis of the NBD-Labeled CaaX-Box of Rheba

a Ac5c: 1-aminocyclopentane-1-carboxylic acid; GABA: 4-aminobutyric acid; L-Tic: L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; Inp: isonipecotic acid; L-Thz: L-thiazolidine-4-carboxylic acid; LThzMe2: L-2,2-dimethylthiazolidine-4-carboxylic acid.

small libraries that encompass only a few hundred compounds the structural diversity appears to be too narrow to generate exactly the functionality required for an optimal sequenceselective recognition process. The sequence-selectivity in our library is accomplished by a variable tripeptide unit that should allow interactions at least with the ultimate three C-terminal amino acids of a given CaaX-box. In addition to the proteinogenic L-amino acids, also the corresponding D-enantiomers, β-amino acids, and other unusual amino acids were utilized as building blocks. The screening library was synthesized on Tentagel macrobeads using the basic cleavable HMBA (4-(hydroxymethyl)benzoic acid) linker. This is perfectly orthogonal to the acidic conditions needed for the cleavage of the Boc group (tertbutoxycarbonyl group) used to protect the guanidinio residue in the headgroup. DIC (N,N′-diisopropylcabodiimide)/HOBT (1-hydroxybenzotriazole) activation was used for the Cterminal amino acid and HBTU (2-(1H-benzotriazole-1-yl)1,1,3,3-tetramethyluronium hexafluorophosphate)/HOBT for the coupling of the remaining residues. Following the “split and combine” principle, the overall library was prepared on a Bhodan Miniblock system as an assembly of 20 sublibraries. Each sublibrary comprised a mixture of 400 compounds with a definite residue in the C-terminal position 3 and randomized positions 2 and 1 (Table 2). The Schmuck-receptor was prepared according to literature procedures and coupled to the N-termini of the split-and-combine libraries after removal of the Fmoc group.34 The CaaX-boxes of Rheb (CSVM), H-Ras (CVLS), and KRas4B (CVIM) were synthesized either in solution according to Scheme 2 or on solid phase. Due to its high fluorescence quantum yield and stability under aqueous conditions, we used NBD (4-chloro-7-nitrobenzofurazan) as fluorescence label, which was introduced to the CaaX-boxes via a spacer, prepared from NBD-chloride 5 and 6-aminohexanoic acid (6).35 The methyl ester of the final tetrapeptide 10 was selectively cleaved with Me3SnOH.36 More basic conditions such as LiOH in THF/H2O or LiI in DMF (100 °C) always resulted in extensive cleavage of the base-labile Fmoc group (fluorenylme-

a

Reagents and conditions: (i) 3 equiv NaHCO3, MeOH, H2O, 1 h 50 °C, 94%; (ii) 1.1 equiv HATU (2-(7-aza-1H-benzotriazole-1-yl)1,1,3,3-tetramethyluronium hexafluorophosphate), 3 equiv DIPEA, DMF, Ar, 1 h 0 °C → RT, 16 h RT, 51%; (iii) 2.1 equiv Me3SnOH, 1,2-dichloroethane, 32%; (iv) TFA/TIPS/H2O (95:2.5:2.5), 2 h, 84%.

thoxycarbonyl group), whereas in enzymatic cleavages with Candida antarctica lipase no product was formed at all. On-Bead Screening of the Receptor Library. In order to exclude beads that showed non-desired interactions with the fluorophore, an aliquot (70 mg, around 8000 beads) of each sublibrary was incubated (16 h) with a 0.1 μM solution of the NBD methyl ester in 10% DMSO (dimethyl sulfoxide)/90% bis-tris buffer (5 μM). Fluorescent beads were removed manually from all sublibraries. In a second series of pretests the optimal screening concentration was determined. Thus, fluorescence-labeled Rheb-, K-Ras, and H-Ras CaaX-boxes were incubated at 0.01, 0.1, and 1 μM concentrations with the L-Ala sublibrary. At the lowest possible screening concentration of 0.01 μM only a low percentage of the beads produced a significant fluorescence (Figure 1). The screening was then conducted with 8.5 mg portions of resin (around 950 beads) for all 20 sublibraries, which were incubated (16 h) in separate vials with 500 μL of a 0.01 μM solution of the labeled CaaX-boxes of Rheb, H-Ras, and K-Ras in 10% DMSO/90% bis-tris buffer (5 μM) exactly as in the pretests. A small amount of DMSO (10%) was needed in the screening solution due to the limited solubility of the NBDlinked CaaX-boxes in pure water. Since the actual screening concentrations were even 1 order of magnitude lower as in the pretest, potentially remaining unspecific interactions with the fluorescence dye can be excluded. Overall, 60 incubations were performed corresponding to around 57,000 beads. At a CaaX1757



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problem analytical construct resins have been developed that contain additional functionality with MS-enhancing properties.38−40 For instance, to achieve the necessary sensitivity for detection in ESI (electrospray ionization) positive-ion mode, a charged group, typically an amine moiety, is introduced into the linker system. The fact that all our compounds already carry a guanidine moiety, which is positively charged under physiologic conditions, allowed us to determine the molecular masses and sequences of the majority of our library members directly without the need for a complex MS-enhancing linker or cumbersome tagging strategies. Thus, fluorescent single beads were transferred in 1 mL vials with a conical bottom and washed repeatedly. Then, the receptor was cleaved off from the resin with propylamine (3 μL, 12 h). After evaporation of the amine the sample was dissolved in NH4OAc buffer and analyzed by MS spectrometry (ESI source, ion-trap). In the example shown in Figure 2, the molecular mass of 567 allowed two sequences, 11a and 11b, which could be distinguished explicitly by their MS1−MS5 spectra, proving sequence 11b to be the correct one. MS analyses from 100 strong fluorescent beads afforded 72 molecular masses, which agreed with the theoretically possible MS values, and 56 sequence proposals. Since MS analysis does not provide any information on the stereochemistry of the residues, an additional solid-phase single-compound library (altogether 74 members) including all combinations of stereoisomers was prepared. The largest number of highly fluorescent beads in this library was found for the CaaX-box of Rheb followed by H-Ras and KRas4B, the latter of which gave only 9 sequences with low to intermediate fluorescence (Table 3). Again, almost all strong fluorescent Rheb CaaX-box receptors carry a D-Lys residue in the C-terminal position 3 and frequently a second one in positions 1 or 2. The position for the second Lys appears uncritical as can be seen from a comparison of the sequences 13/14 and 15/16. In the presence of only one Lys in the receptor, position 3 is clearly preferred over position 2 or 1. On

Figure 1. Determination of the optimal screening concentration.

box concentration of 0.01 μM, only the very small number of 270 beads (5 × 107 (0.38)d ∼ 1.0 × 107 (2.44)d 1.2 × 106 (0.36)d 3.0 × 105 (6.18)d 2.5 × 105 (1.62)d

≤10 1,150 (0.25)d 4,100 (0.52)d

a

Determined by NMR titrations in d6-DMSO. bDetermined by NMR titrations in d6-DMSO/H2O 6:4. cUV titrations in DMSO. dRoot-mean-square error (RMSE) in %. 1759



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plished with receptors completely devoid of proteinogenic amino acids.41,42 NMR and UV Measurements. For each CaaX-box, selected strongly fluorescent receptors were resynthesized in milligram amounts and the KA values were determined by NMR titrations in pure d6-DMSO and a d6-DMSO (60%)/H2O (40%) mixture under strict control of pH. The DMSO/H2O system used is a standard solvent system in the field of molecular recognition and guarantees a complete solubility of substrates and receptors even at those concentrations needed for NMR studies.43 Addition of increasing amounts of a solution of the tetramethylammonium (TMA) salts of the respective CaaX-box to the receptor picrate salts (1.55 mM) resulted in concentration-dependent significant shifts of specific proton resonances in the receptor structure. The KA values were calculated using the software package WINEQNMR2 from the shifts of the pyrrole-hydrogens 3 and 4 and the NH of the guanidinium amide and were found to depend most on the CaaX-box concentration.44 In pure DMSO, however, the receptor CaaX-box associations were too strong to determine the KA values exactly. In a DMSO (60%)/H2O (40%) solution the association is reduced considerably because of the competing water molecules, which has led us to extract KA values from the NMR titration data. Receptor 12 with the highest fluorescence overall binds to the Rheb CaaX-box even in an aqueous environment with a KA value of 3 × 104 M−1 (Table 4). A comparable KA was found for sequence 21 in which AC5C was replaced by L-Lys. It should be noted that even these reduced KA values are in the range of the strongest small-molecule protein−protein inhibitors of Ras-proteins.15,16,22 On the contrary, the exchange of L-Pro for D-Pro (15) or particularly D-Ala (13) drastically reduced binding affinity to the CSVM sequence of Rheb in aqueous DMSO. Albeit on a lower level, the best H-Ras receptor NH2CO-D-Leu-L-Ser-L-Serhg (24) shows a significant sequence-selectivity over the Rheb CaaX-sequence in aqueous solution. UV titrations, in general better suited for strong associations, confirm receptor 12 in agreement with the UV and NMR data as the best one with a KA value >5 × 107 M−1 in pure DMSO, followed by receptor 13 (KA 1.0 × 107 M−1). Somewhat surprising is the 2 orders of magnitude lower KA value of receptor 21 compared to receptor 12 in pure DMSO, albeit both receptors show a strong association in aqueous DMSO. This is a clear hint that the parameters that determine the association strength in water considerably differ from those in DMSO. STD (saturation transfer difference) NMR spectra clearly corroborate that receptor 12 also binds to full lengths Rheb protein in complex with GDP in an aqueous buffer solution with not more than 5% of DMSO (Figure 3). In particular, the lipophilic CH2 groups of Pro, Lys, and AC5C show strong STD signals, indicating large nonpolar association areas comparable to the structurally related receptor 21 (Figure 5b). Modeling Studies. Monte Carlo conformational searches in CHCl3 and H2O followed by minimizations were used to locate low-energy binding and therefore preferred conformations of the receptor CaaX-box complexes to get a better understanding of the strongly varying KA values obtained by the NMR titration experiments in aqueous DMSO solution. In general, the simulations reveal a dense network of hydrogen bonds and electrostatic interactions for all complexes in CHCl3. In H2O, usually a complete reorientation of the receptor and

Figure 3. Interactions of receptor 12 with full length Rheb protein. (a) 1D proton reference spectrum of receptor 12. (b) STD spectrum of receptor 12 in the presence of GDP-bound Rheb.

CaaX-box is observed with a reduced number of electrostatic interactions and H-bonds. Instead, the nonpolar contact surfaces in the receptor CaaX-box complexes increase significantly. However, the extent of reorientation changes individually for each complex studied. In full agreement with the high KA value (>105 M−1) of receptor 24 in pure DMSO, significant shift changes of the αhydrogen of Ser2, OH of Ser1, the pyrrole hydrogens, and the amide hydrogen of the guanidine function in the 1H NMR spectrum provide strong evidence for a tight association throughout the whole receptor sequence and the Rheb CaaXbox. This finding is corroborated by Monte Carlo simulations, which show a network of several H-bonds between the receptor and the Rheb CaaX-box in CHCl3 and additional ionic interactions between the Met 4-carboxy group and the guanidinio moiety. In the bound state, the CaaX-box adopts a rigid β-turn-like structure that is stabilized by two intramolecular H-bonds between Cys1-NH/Met4-carboxylate and Cys1-NHCO/Met4NH (Figure 4a). Further intermolecular Hbonds are formed between Val3-CO (Rheb)/Ser2-NH (receptor) and both Val3-NH (Rheb) and Ser2-OH (Rheb) with the pyrrole-CO (receptor) group. These results obtained by 1H NMR studies and Monte Carlo conformational searches are confirmed by MD simulations (10 ns) in CHCl3, which show an almost identical H-bond pattern with an additional contact between Ser2-OH (receptor) and Val2CO (Rheb). On the contrary, in an aqueous DMSO (60%) solution the backbone and side-chain shifts disappear, indicating only a weak association as has been found before by NMR titration experiments. Our modeling studies demonstrate a completely different lowest-energy conformation in water with only one backbone H-bond between the guanidinio-CO (receptor) and the Val3-NH (Rheb) left (Figure 4b). In particular, the intramolecular H-bond between Cys1-NH and Met4-carboxylate is lost, which causes a flexible CaaX-box conformation. Analogous to receptor 24, receptor 21 also shows a tight network of intra- and intermolecular hydrogen bonds in a nonpolar environment. Each residue of the CSVM-tetrapeptide is bound by at least one H-bond to receptor 21. In particular, the carboxylate function of Met4 is fixed by five H-bridges, two of which are formed intramolecularly to Ser2-NH and Ser2-βOH (Figure 5). The side-chain of Lys1 (receptor) folds back to 1760



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Figure 4. Calculated low-energy conformations of the receptor 24/Rheb CaaX-box complex in CHCl3 (a) and H2O (b).

Figure 5. Calculated low-energy conformations of the receptor 21/Rheb CaaX-box complex in CHCl3 (a,c) and H2O (b,d). (c,d) Partial charges mapped on molecular surface. Red, negatively charged; green, neutral; blue, positively charged.

extend into the solvent. Thus, in the bound state, the CSVMsequence adopts a rigid conformation in a simulated CHCl3 solution. Again, the arrangement of receptor 21 and the Rheb CaaXbox in water changes completely. Now, the side chain of Lys1 (receptor) is directed into the solvent, while Lys3 folds back to

form a H-bond to the acetyl oxygen of Cys1 (CaaX-box). Additionally, the NH function of Cys1 is linked to the carbonyloxygen of Lys1 by another H-bridge. Both the acetyl-NH and the neighboring NH of Lys3 (receptor) form H-bonds to the Val3 (CaaX-box) and Ser2 (CaaX-box) carbonyl oxygen atoms. This arrangement causes the side-chain of Lys3 (receptor) to 1761



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electrostatically stabilize the carboxylate anion of Met4 together with the guanidinio scaffold. Similar to receptor 24, only one backbone H-bond betweenVal3-NH (CaaX-box) and the pyrrole-CO remains in aqueous solution (Figure 5). Therefore, at first glance the relatively high KA value of receptor 21 (KA = 19800 M−1) in contrast to receptor 24 (KA = 320 M−1) appears enigmatic. However, in water nonpolar interactions become increasingly important for complex formation. From Figure 5d it is evident that receptor 21 forms a large lipophilic interface including Pro2 as well as the side chains of D-Lys and L-Lys of the receptor together with Met, Val, and the Cys of the CaaXbox. On the contrary, in a nonpolar solvent the dominant driving forces are electrostatic and polar forces. As shown in Figure 4c, receptor 21 adopts a torus-like shape in CHCl3 with most of the polar interactions embedded in the interior of the receptor−substrate complex and the lipophilic residues oriented toward the solvent. As a consequence, the nonpolar interaction area is reduced. The same factors explain the low KA value of receptor 24 and others, which form only a small lipophilic contact surface in water with the Rheb CaaX-box. Obviously, a delicate combination of polar and lipophilic interactions determines the association of each receptor CaaXbox complex. Summary and Conclusions. The inhibition of protein− protein interactions by small molecules is still regarded a formidable challenge in medicinal research. Despite some progress in the case of K-Ras4B in recent years, not a single small molecule inhibitor has entered the clinical phase. A complementary approach that aims at masking the C-terminal CaaX-box, which is crucial for membrane insertion and normal function of Rheb, has the potential to overcome the principal problem of finding small molecules that bind efficiently onto the surface of Ras-proteins and inhibit the interactions with effector proteins of the GTPase signaling cascade. By a combination of the Schmuck headgroup with nonproteinogenic amino acids and turn-inducing Pro residues, we succeeded in identifying a receptor (12) for the C-terminal Rheb CaaX-box with a KA value of 3 × 104 M−1 in aqueous DMSO solution. STD NMR spectra confirm that receptor 12 also binds to the full length Rheb protein in an aqueous buffer with only 5% DMSO as cosolvent. Our molecular modeling studies provide a plausible model for the sequence-selective binding to the CaaX-boxes of K-Ras4B, H-Ras, and Rheb in water and a nonpolar environment. The pincer-like receptors 12 and 21 provide excellent starting points for complete peptidomimetic, sequence-selective receptors. A stronger association in aqueous solution should be achieved by rigid β-turn mimetics and extending the recognition unit to the last 5 to 6 C-terminal residues of Rheb and other related Ras proteins. We believe that our work not only represents an important contribution to the principles of molecular recognition of peptides but may also provide a promising new approach for the selective inactivation of Ras proteins.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to the Deutsche Krebshilfe (109776 and 109777) for generous financial support. REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Experimental details of library and single compound syntheses including complete characterizations as well as descriptions of the UV and NMR titration procedures. This material is available free of charge via the Internet at http://pubs.acs.org. 1762



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Sequence-Selective Molecular Recognition of the C-Terminal CaaX

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