Utilization of achiral alkenyl amines for the preparation of high affinity Grb2 SH2 domain-binding macrocycles by ring-closing metathesis

A family of previously reported ring-closing metathesis (RCM)-derived macrocycles that exhibit potent Grb2 SH2 domain-binding affinity is characterized by stereoselectively-introduced upper ring junctions that bear bicyclic aryl substituents. However, the synthetic complexity of these macrocycles presents a potential limit to their therapeutic application. Therefore, the current study was undertaken to simplify these macrocycles through the use of achiral 4-pentenylamides as ring-forming components. A series of macrocycles (5a-f) was prepared bearing both open and cyclic constructs at the upper ring junction. The Grb2 SH2 domain-binding affinities of these macrocycles varied, with higher affinities being obtained with cyclo-substituents. The most potent analogue (5d) contained a cyclohexyl group and exhibited Grb2 SH2 domain-binding affinity (K(D) = 1.3 nM) that was nearly equal to the parent macrocycle (2), which bore a stereoselectively-introduced naphthylmethyl substituent at the upper ring junction (K(D) = 0.9 nM). The results of this study advance design considerations that should facilitate the development of Grb2 SH2 domain-binding antagonists.     

Synthesis of N-Fmoc 3-(4-(di-(tert-butyl)phosphonomethyl)phenyl)pipecolic acid as a conformationally constrained phosphotyrosyl mimetic suitably protected for peptide synthesis

Phosphonomethylphenylalanine (Pmp, 2) has shown wide utility as a hydrolytically stable phosphtyrosyl (pTyr, 1) mimetic, particularly in Src homology 2 (SH2) domain-binding peptides. (2S,3R)-3-(4-(phosphonomethyl)phenyl)pipecolic acid (3) represents a variant of Pmp having φ and χ₁ torsion angles constrained through incorporation into the piperidinyl ring structure contained within pipecolic acid. Reported herein is the enantioselective preparation of 3, in an orthogonally protected form (4) suitable for use in peptide synthesis. Stereochemistries at both the 2- and 3-positions are derived inductively from a single chiral center provided by the commercially available Evans chiral auxiliary, (4S)-4-benzyl-1,3-oxazolidin-2-one. Incorporation of 4 into a Grb2 SH2 domain-directed tripeptide (18) showed that Grb2 SH2 domain-binding affinity was reduced relative to the parent Pmp-containing tripeptide (19). Although conformational constraint did not enhance affinity in this case, novel amino acid analogue 4 may serve as a useful tool for the induction of defined phosphotyrosyl geometry in peptides directed at other signal transduction targets.     

Synthesis of a phosphotyrosyl analogue having χ1, χ2 and φ angles constrained to values observed for an SH2 domain-bound phosphotyrosyl residue

Src homology 2 (SH2) domains provide connectivity in protein-tyrosine kinase (PTK)-dependent signaling through their high affinity association with phosphotyrosyl (pTyr)-containing peptide sequences. Because recognition of pTyr residues is central to SH2 domain-binding affinity, design of pTyr-mimicking residues has been one component of SH2 domain signaling antagonist development. Reported herein is the synthesis of (±)-(rel-1R,2R,5S)-3-acetyl-1,2,3,4,5,6-hexahydro-8-O-phosphoryl-1,5-methano-3-benzazocine-2-carboxylic acid methyl ester (3c) as a monomeric pTyr-mimicking analogue that constrains three torsion angles (χ₁=168°; χ₂=−85°; φ₁=−113°) to values approximating those observed for a pTyr residue bound to the Grb2 SH2 domain (χ₁=182°; χ₂=−89°; φ₁=−132°). Compound 3c differs from our previously reported analogue, (±)-(rel-1R,2R,5S)-3-acetyl-1,2,3,4,5,6-hexahydro-1-methyl-1,5-methano-3-benzazocin-8-ol, in lacking a methyl substituent at the bridgehead 1-position. Molecular modeling studies had indicated that this methyl group could potentially hinder SH2 domain binding. Synthesis of the desmethyl derivative was achieved by formation of the methanobenzazocine ring system using an intramolecular electrophilic cyclization that proceeds through an activated acyliminium intermediate. Importantly, the correct relative (2R) stereochemistry at the ‘α-carboxyl’-bearing carbon is obtained through base-catalyzed equilibration of a (2S/2R) diastereomeric mixture that results from intramolecular ring closure. Comparison of Grb2 SH2 domain-binding affinity of 3c (IC₅₀=1167 μM) with conformationally flexible phosphorylated (±)-N-acetyl-tyrosine methyl ester (15; IC₅₀=1469 μM) revealed no apparent enhancement in affinity. This apparent ineffectiveness of ‘local conformational constraint’ on SH2 domain-binding affinity of the monomeric pTyr mimetic is consistent with previous reports obtained by conformationally constraining pTyr-mimicking residues that were contained within peptide platforms. Although not providing high binding affinity in its current form, the novel 1,5-methano-3-benzazocine ring system may afford a novel platform for further elaboration and development of small molecule SH2 domain signaling antagonists.     

Utilization of 3'-carboxy-containing tyrosine derivatives as a new class of phosphotyrosyl mimetics in the preparation of novel non-phosphorylated cyclic peptide inhibitors of the Grb2-SH2 domain

A new class of phosphotyrosyl (pTyr) mimetics, distinct from the conventional pTyr mimetic design of adding non-hydrolyzable acidic functionalities to the 4'-position of phenylalanine, was created by introducing carboxy-containing groups to the 3'-position of tyrosine. The effect of the chain length of the carboxy substituent was examined. Reported herein is the chiral pool synthesis of the new pTyr mimetics, and their first use in a novel non-phosphorylated Grb2-SH2 domain binding motif with the 5-amino-acid sequence Xx1-Leu-(3'-substituted-Tyr)-Ac6c-Asn. The highest affinity was exhibited by the 3-L-(2-carboxyethyl)tyrosine-containing sulfoxide-cyclized peptide , with an IC50 = 1.1 microM, providing a promising new template for further development of potent Grb2-SH2 domain inhibitors with reduced charge and peptidic nature, but improved selectivity and bioavailability.     

CH/pi hydrogen bonds determine the selectivity of the Src homology 2 domain to tyrosine phosphotyrosyl peptides: an ab initio fragment molecular orbital study

The CH/pi hydrogen bond is a weak molecular force occurring between CH groups (soft acids) and pi-systems (soft bases), and has been recognized to be important in the interaction of proteins with their specific ligands. For instance, it is well known that Src homology-2 protein (SH2) recognizes its specific pTyr peptide in two key regions, pTyr-binding region and specificity-determining region, by the use of attractive molecular forces, including the CH/pi hydrogen bond. We hypothesized that the CH/pi hydrogen bond plays a key role in determining the selectivity of SH2 proteins, and studied this issue by the ab initio fragment molecular orbital (FMO) method. The FMO calculations were carried out, at the HF/6-31G* and MP2/6-31G* level, for SH2 domains of Src, Grb2, P85alpha(N), Syk, and SAP, in complex with corresponding pTyr peptides. CH/pi hydrogen bonds have in fact been found to be important in stabilizing the structure of the complexes. We conclude that the CH/pi hydrogen bond plays an indispensable role in the recognition of SH2 domains with their specific pTyr peptides, thus playing a vital role in the signal transduction system.     

Olefin metathesis in the design and synthesis of a globally constrained Grb2 SH2 domain inhibitor

One drawback frequently associated with olefin metathesis-mediated peptide macrocyclization, the loss of side chain functionality at sites of ring closure, may be circumvented by incorporation of side chain functionality within the ring-closing olefin segments. This approach is demonstrated in the preparation of a macrocyclic Grb2 SH2 domain antagonist designed as a conformationally constrained beta-bend mimic.     

Cyclic peptidyl inhibitors of Grb2 and tensin SH2 domains identified from combinatorial libraries

Cyclic peptides provide attractive lead compounds for drug discovery and excellent molecular probes in biomedical research. In this work, a novel method has been developed for the high-throughput synthesis, screening, and identification of cyclic peptidyl ligands against macromolecular targets. Support-bound cyclic phosphotyrosyl peptide libraries containing randomized amino acid sequences and different ring sizes (theoretical diversity of 3.2 x 10(6)) were synthesized and screened against the SH2 domains of Grb2 and tensin. Potent, selective inhibitors were identified from the libraries and were generally more effective than the corresponding linear peptides. One of the inhibitors selected against the Grb2 SH2 domain inhibited human breast cancer cell growth and disrupted actin filaments. This method should be applicable to the development of cyclic peptidyl inhibitors against other protein domains, enzymes, and receptors.     

Cyclic phosphopeptides for interference with Grb2 SH2 domain signal transduction prepared by ring-closing metathesis and phosphorylation

Cyclic phosphopeptides were prepared using ring-closing metathesis followed by phosphorylation. These cyclic phosphopeptides were designed to interact with the SH2 domain of Grb2, which is a signal transduction protein of importance as a target for antiproliferative drug development. Binding of these peptides to the Grb2 SH2 domain was evaluated by a surface plasmon resonance assay. High affinity binding to the Grb2 SH2 domain was maintained upon macrocyclization, thus indicating that this method can be used to assemble high affinity cyclic phosphopeptides that interfere with signal transduction cascades.     

Concise and enantioselective synthesis of Fmoc-Pmp(But)2-OH and design of potent Pmp-containing Grb2-SH2 domain antagonists

[reaction: see text] L-Phosphonomethylphenylalanine (L-Pmp) is an important phosphatase-resistant pTyr analogue. A most concise and stereoselective approach to the synthesis of the suitably protected Fmoc-Pmp(Bu(t))(2)-OH was developed in order to incorporate the functionally significant L-Pmp residue into peptides and peptidomimetics efficiently using standard Fmoc protocol. With this key building block, we are able to efficiently synthesize a series of potent Pmp-containing Grb2-SH2 domain antagonists, which can be used as chemotherapeutic leads for the treatment of erbB2-overexpressed breast cancer.     

Unexpected relative aqueous solubilities of a phosphotyrosine analogue and two phosphonate derivatives

Phosphotyrosine (pTyr) is an essential component of biological signaling, often being a determinant of protein-protein interactions. Accordingly, a number of drug discovery efforts targeting signal transduction pathways have included phosphotyrosine and analogues as essential components of the lead compounds. Toward the goal of improved biological efficacy, the phosphonate and difluoro phosphonate analogues of pTyr have been employed in inhibitor design because of their stability to hydrolysis and enhanced binding affinity in certain cases. To quantitate the contribution of aqueous solubility of pTyr, phosphonomethyl phenylalanine (Pmp), and difluorophosphonomethyl phenylalanine (F(2)Pmp) to their relative binding affinities, free energy perturbation calculations were undertaken on the mimetics phenol phosphate (PP), benzyl phosphonate (BP), and difluorobenzyl phosphonate (F(2)BP), including development of empirical force field parameters compatible with the CHARMM all-atom force fields. Notably, it is shown that the most favorably solvated compound of the series is BP, followed by PP, with F(2)BP the least favorably solvated for both the mono- and dianionic forms of the compounds. The molecular origin of this ordering is shown to be due to changes in charge distribution, in the comparatively larger size of the fluorine atoms, as well as in differences of local solvation between PP and BP. The implications of the differences in aqueous solubility toward the relative binding potencies of pTyr-, Pmp-, and F(2)Pmp-containing peptide ligands are discussed. Our results indicate that one general principle explaining the efficacy of selective fluorination to enhance binding affinities may lie in the ability of fluorine atoms to increase the hydrophobicity of a ligand while maintaining its capability to form hydrogen bonds.     

Synthesis of a new conformation-constrained L-tyrosine analogue as a potential scaffold for SH2 domain ligands

The enantioselective synthesis of a new tricyclic tyrosine analogue is reported. This conformation-constrained SH2 domain ligand scaffold 2 was designed on the basis of the natural ligand, whose structure contains the elements of a tyrosine moiety having chi(1) and chi(2) angles constrained to values observed for a phosphotyrosyl (pTyr) residue bound to the p56(lck) SH2 domain. It represents a unique, highly constrained amino acid, which may be of value in signal transduction studies. Three key steps, an asymmetric tandem Michael addition, an intramolecular Friedel-Crafts reaction, and an intramolecular Mannich reaction, were successfully applied in the presented synthetic route.     

Calorimetric and structural studies of 1,2,3-trisubstituted cyclopropanes as conformationally constrained peptide inhibitors of Src SH2 domain binding.

Isothermal titration calorimetry and X-ray crystallography have been used to determine the structural and thermodynamic consequences associated with constraining the pTyr residue of the pYEEI ligand for the Src Homology 2 domain of the Src kinase (Src SH2 domain). The conformationally constrained peptide mimics that were used are cyclopropane-derived isosteres whereby a cyclopropane ring substitutes to the N-Calpha-Cbeta atoms of the phosphotyrosine. Comparison of the thermodynamic data for the binding of the conformationally constrained peptide mimics relative to their equivalent flexible analogues as well as a native tetrapeptide revealed an entropic advantage of 5-9 cal mol(-1) K(-1) for the binding of the conformationally constrained ligands. However, an unexpected drop in enthalpy for the binding of the conformationally constrained ligands relative to their flexible analogues was also observed. To evaluate whether these differences reflected conformational variations in peptide binding modes, we have determined the crystal structure of a complex of the Src SH2 domain bound to one of the conformationally constrained peptide mimics. Comparison of this new structure with that of the Src SH2 domain bound to a natural 11-mer peptide (Waksman et al. Cell 1993, 72, 779-790) revealed only very small differences. Hence, cyclopropane-derived peptides are excellent mimics of the bound state of their flexible analogues. However, a rigorous analysis of the structures and of the surface areas at the binding interface, and subsequent computational derivation of the energetic binding parameters, failed to predict the observed differences between the binding thermodynamics of the rigidified and flexible ligands, suggesting that the drop in enthalpy observed with the conformationally constrained peptide mimic arises from sources other than changes in buried surface areas, though the exact origin of the differences remains unclear.     

Phosphopeptide selective coordination complexes as promising SRC homology 2 domain mimetics

Src Homology 2 (SH2) domains are the paradigm of phosphotyrosine (pY) protein recognition modules and mediate numerous cancer-promoting protein-protein complexes. Effective SH2 domain mimicry with pY-binding coordination complexes offers a promising route to new and selective disruptors of pY-mediated protein-protein interactions. We herein report the synthesis and in vitro characterization of a library of coordination complex SH2 domain proteomimetics. Compounds were designed to interact with phosphopeptides via a two-point interaction, principally with pY, and to make secondary interactions with pY+2/3, thereby achieving sequence-selective discrimination. Here, we report that lead mimetics demonstrated high target phosphopeptide affinity (K(a) ～ 10(7) M(-1)) and selectivity. In addition, biological screening in various tumor cells for anticancer effects showed a high degree of variability in cytotoxicity among receptors, which supported the proposed two-point binding mode. Several receptors potently disrupted cancer cell viability in breast cancer, prostate cancer, and acute myeloid leukemia cell lines.     

Inhibitors of tyrosine kinase proteins induced Ras signaling pathway as potential anti-tumor agents

Cellular signaling pathways induced by growth-factor receptors with tyrosine kinase activity are frequently deregulated in cancer. Anti-tumor agents that inhibit their enzymatic tyrosine kinase activity have been designed and are now used in human chemotherapy. We review here our data constituting an alternative way to interrupt over-expressed signaling pathway by inhibiting protein-protein interactions. In our approach, the adaptor protein Grb2 over-expressed in connection with HER2/ErbB2/neu in Ras signaling pathway was chosen as a target. Peptides and peptidomimetics with very high affinities for either SH3 or SH2 domains of Grb2 were rationally designed from structural data. We describe their synthesis, their capacity to interrupt the signaling pathway and their anti-proliferative activity. To cite this article: M. Vidal et al., C. R. Chimie 8 (2005).     

Protein-ligand interactions: thermodynamic effects associated with increasing nonpolar surface area

Thermodynamic parameters were determined for complex formation between the Grb2 SH2 domain and Ac-pTyr-Xaa-Asn derived tripeptides in which the Xaa residue is an α,α-cycloaliphatic amino acid that varies in ring size from three- to seven-membered. Although the six- and seven-membered ring analogs are approximately equipotent, binding affinities of those having three- to six-membered rings increase incrementally with ring size because increasingly more favorable binding enthalpies dominate increasingly less favorable binding entropies, a finding consistent with an enthalpy-driven hydrophobic effect. Crystallographic analysis reveals that the only significant differences in structures of the complexes are in the number of van der Waals contacts between the domain and the methylene groups in the Xaa residues. There is a positive correlation between buried nonpolar surface area and binding free energy and enthalpy, but not with ΔC(p). Displacing a water molecule from a protein-ligand interface is not necessarily reflected in a favorable change in binding entropy. These findings highlight some of the fallibilities associated with commonly held views of relationships of structure and energetics in protein-ligand interactions and have significant implications for ligand design.     

Enantioselective synthesis of protected L-4-[sulfonamido(difluoromethyl)]phenylalanine and L-4-[sulfonamido(methyl)]phenylalanine and an examination of hexa- and tripeptide platforms for evaluating pTyr mimics for PTP1B inhibition

The first enantioselective syntheses of L-4-(sulfonamidomethyl)phenylalanine and L-[sulfonamido(difluoromethyl)]phenylalanine suitably protected for peptide syntheses are described. A key step in the synthesis of L-(sulfonamidomethyl)phenylalanine was an oxidative chlorination on Ac-L-Phe(4-CH2SCOCH3)-OEt to give crude Ac-L-Phe(4-CH2SO2Cl)-OEt, which could be reacted with amines to give the corresponding sulfonamides. Key to the preparation of L-[sulfonamido(difluoromethyl)]phenylalanine was a highly enantioselective reaction involving William's auxiliary and a benzylic bromide intermediate. These amino acids were incorporated into two peptide sequences, DADE-X-LNH2 and FmocGlu(OBn)-X-LNH2, which have previously been employed as platforms for assessing pTyr mimics for inhibition of protein tyrosine phosphatase 1B (PTP1B). Inhibition studies with these and other peptides and PTP1B revealed that good inhibition could be obtained using the tripeptide platform, although the presence of a pTyr mimic was not required for good inhibition. These results suggest that the FmocGlu(OBn)-X-LNH2 tripeptide platform is not suitable for assessing pTyr mimics for PTP1B inhibition.     

Synthetic inhibitors of proline-rich ligand-mediated protein-protein interaction: potent analogs of UCS15A

The proline-rich motif in proteins is known to function as a ligand sequence that binds to protein modules such as SH3, WW, and several other protein interaction domains. These proline-rich ligand-mediated protein-protein interactions (abbreviated PLPI) are important in many signaling pathways that are involved in various diseases. Our previous studies showed that UCS15A, produced by Streptomyces species, inhibited PLPI. Here we report on synthetic analogs of UCS15A that show more potent activity than UCS15A in inhibiting PLPI. A synthetic analog, compound 2c, blocked in vitro PLPI of Sam68-Fyn-SH3 as well as in vivo PLPI of Grb2-Sam68 and Grb2-Sos1. Activation of MEK was also inhibited by compound 2c. Unlike UCS15A, compound 2c was an order of magnitude less cytotoxic and did not cause morphological changes in treated cells.     

Gas-phase electrophilic addition promoted by CH(3)S(+)=CH(2) ions on aromatic systems

The gas-phase methylenation reaction between CH(3)S(+)=CH(2) and alkylbenzenes, aniline, phenol and alkyl phenyl ethers, which yields [M + CH](+) and CH(3)SH, has been studied by Fourier transform ion cyclotron resonance (FT-ICR) techniques and computational chemistry at the DFT level. The methylthiomethyl cation is less reactive than methoxymethyl and, unlike the latter, is unreactive toward benzene. The calculations suggest that reaction with toluene should proceed primarily by addition at the para and ortho positions resulting in a benzyl-type ion. Reaction with aniline-2,3,4,5,6-d(5) reveals that elimination of CH(3)SD is kinetically favored by a factor of 5 over elimination of CH(3)SH. Experiments with C(6)H(6)ND(2) and theoretical calculations suggest that methylenation at the nitrogen atom is energetically favorable and likely, but the observed results may reflect some H/D scrambling, which occurs after attack at a ring position. By comparison, reaction with phenol-2,3,4,5,6-d(5) reveals that methylenation followed by elimination of CH(3)SD is kinetically favored by a factor of 3.8 over elimination of CH(3)SH. For phenol, the theoretical calculations suggest that attack by CH(3)S(+)=CH(2) at the para or ortho position is the only low-energy pathway for methylenation. However, a low-energy pathway for hydrogen scrambling is predicted by the calculations originating from the exit complex, [CH(3)SH(...) CH(2)=C(6)H(4)=OH](+), of reaction at a ring position.     

Binuclear Terbium(III) Complex as a Probe for Tyrosine Phosphorylation

By using the luminescence from binuclear complexes of Tb^III ( Tb₂‐L¹ and Tb₂‐L² ), phosphorylated Tyr residue in peptides was selectively detected in neutral aqueous solutions. Neither the non‐phosphorylated Tyr, pSer, pThr, nor the other phosphate‐containing biomolecules tested affected the luminescence intensity to any notable extent. Upon the binding of the pTyr to these Tb^III complexes, the luminescence from the metal ion was notably promoted, as the light energy absorbed by the benzene ring of pTyr is efficiently transferred to the Tb^III center. The binding activity of the binuclear Tb^III complexes towards pTyr is two orders of magnitude larger than that of the corresponding mononuclear complex. These binuclear complexes were successfully used for real‐time monitoring of enzymatic phosphorylation of a peptide by a tyrosine kinase.     

Novel superalkali superhalogen compounds (Li3)+(SH)- (SH=LiF2, BeF3, and BF4) with aromaticity: new electrides and alkalides.

Optimized structures, with all real frequencies, of superalkali superhalides (Li(3))(+)(SH)(-) (SH=LiF(2), BeF(3), and BF(4)), are obtained, for the first time, at the B3LYP/aug-cc-pVDZ and MP2/aug-cc-pVDZ computational levels. These superalkali superhalides possess three characteristics that are significantly different from normal alkali halides. 1) They have a variety of structures, which come from five bonding mode types: edge-face, edge-edge, face-face, face-edge, and staggered face-edge. We find that the bonding mode type closely correlates with the Li(3)-SH bond energy. 2) The valence electrons on the Li(3) ring are pushed out by the (SH)(-) anion, and become excess electrons, conferring alkalide or electride characteristics on these Li(3)-SH species, depending on the bonding mode type. 3) The highest occupied molecular orbital of each Li(3)-SH species is a doubly occupied delocalized sigma bonding orbital on the Li(3) ring, which indicates its aromaticity. It is noticeable that the maximum negative nucleus-independent chemical shift value (about -10 ppm) moves out from the center of the Li(3) ring, owing to repulsion by the SH(-) anion. We find that these superalkali superhalides are not only complicated "supermolecules", but are also a new type of alkalide or electride, with aromaticity.