Linear Aliphatic Dialkynes as Alternative Linkers for Double‐Click Stapling of p53‐Derived Peptides

We investigated linear aliphatic dialkynes as a new structural class of i,i+7 linkers for the double‐click stapling of p53‐based peptides. The optimal combination of azido amino acids and dialkynyl linker length for MDM2 binding was determined. In a direct comparison between aliphatic and aromatic staple scaffolds, the aliphatic staples resulted in superior binding to MDM2 in vitro and superior p53‐activating capability in cells when using a diazidopeptide derived from phage display. This work demonstrates that the nature of the staple scaffold is an important factor that can affect peptide bioactivity in cells.     

Cyclization of Peptides through a Urea Bond: Application to the Arg‐Gly‐Asp Tripeptide

Various synthetic cyclopeptides bind different cellular proteins with high affinity and specificity. In this study, we designed a new series of cyclic tetrapeptides containing the RGD sequence, a ligand for the α_vβ₃ integrin receptor, in which the ring closure was performed through a urea bond between the α‐amino group of the peptide and either the α‐ or the ε‐amino group of an additional lysine. Interestingly, we showed that the urea‐closed peptide had a higher affinity for α_vβ₃ receptors than a reference pentacyclopeptide. Moreover, the synthetic strategy allows coupling of the resulting cyclic tetrapeptide through the carboxylic acid moiety of its lysine residue to fluorescent molecules or drugs. In addition, this strategy could be easily adapted for the cyclization of any other peptides.     

Improving Binding Affinity and Stability of Peptide Ligands by Substituting Glycines with D‐Amino Acids

Improving the binding affinity and/or stability of peptide ligands often requires testing of large numbers of variants to identify beneficial mutations. Herein we propose a type of mutation that promises a high success rate. In a bicyclic peptide inhibitor of the cancer‐related protease urokinase‐type plasminogen activator (uPA), we observed a glycine residue that has a positive ? dihedral angle when bound to the target. We hypothesized that replacing it with a D ‐amino acid, which favors positive ? angles, could enhance the binding affinity and/or proteolytic resistance. Mutation of this specific glycine to D ‐serine in the bicyclic peptide indeed improved inhibitory activity (1.75‐fold) and stability (fourfold). X‐ray‐structure analysis of the inhibitors in complex with uPA showed that the peptide backbone conformation was conserved. Analysis of known cyclic peptide ligands showed that glycine is one of the most frequent amino acids, and that glycines with positive ? angles are found in many protein‐bound peptides. These results suggest that the glycine‐to‐D ‐amino acid mutagenesis strategy could be broadly applied.     

Crystal Structures of BapA Complexes with β‐Lactam‐Derived Inhibitors Illustrate Substrate Specificity and Enantioselectivity of β‐Aminopeptidases

β‐Aminopeptidases have exclusive biocatalytic potential because they react with peptides composed of β‐amino acids, which serve as building blocks for the design of non‐natural peptidomimetics. We have identified the β‐lactam antibiotic ampicillin and the ampicillin‐derived penicilloic acid as novel inhibitors of the β‐aminopeptidase BapA from Sphingosinicella xenopeptidilytica (K_i values of 0.69 and 0.74 mM , respectively). We report high‐resolution crystal structures of BapA in noncovalent complexes with these inhibitors and with the serine protease inhibitor 4‐(2‐aminoethyl)benzenesulfonyl fluoride. All three inhibitors showed similar binding characteristics; the aromatic moiety extended into a hydrophobic binding pocket of the active site, and the free amino group formed a salt bridge with Glu133 of BapA. The exact position of the inhibitors and structural details of the ligand binding pocket illustrate the specificity and the enantioselectivity of BapA‐catalyzed reactions with β‐peptide substrates.     

Directed Evolution of Scanning Unnatural‐Protease‐Resistant (SUPR) Peptides for in Vivo Applications

Peptides typically have poor biostabilities, and natural sequences cannot easily be converted into drug‐like molecules without extensive medicinal chemistry. We have adapted mRNA display to drive the evolution of highly stable cyclic peptides while preserving target affinity. To do this, we incorporated an unnatural amino acid in an mRNA display library that was subjected to proteolysis prior to selection for function. The resulting “SUPR (scanning unnatural protease resistant) peptide” showed ≈500‐fold improvement in serum stability (t =160 h) and up to 3700‐fold improvement in protease resistance versus the parent sequence. We extended this approach by carrying out SUPR peptide selections against Her2‐positive cells in culture. The resulting SUPR4 peptide showed low‐nanomolar affinity toward Her2, excellent specificity, and selective tumor uptake in vivo. These results argue that this is a general method to design potent and stable peptides for in vivo imaging and therapy.     

Design of Cyclic Peptides Featuring Proline Predominantly in the cis Conformation under Physiological Conditions

Turns are secondary‐structure elements that are omnipresent in natively folded polypeptide chains. A large variety of four‐residue β‐turns exist, which differ mainly in the backbone dihedral angle values of the two central residues i+1 and i+2. The βVI‐type turns are of particular biological interest because the i+2 residue is always a proline in the cis conformation and might thus serve as target of peptidyl prolyl cis/trans isomerases (PPIases). We have designed cyclic hexapeptides containing two proline residues that predominantly adopt the cis conformation in aqueous solution. NMR data and MD calculations indicated that the cyclic peptide sequences c‐(‐D Xaa‐Ser‐Pro‐D Xaa‐Lys‐Pro‐) result in highly symmetric backbone structures when both prolines are in the cis conformation and the D ‐amino acids are either alanine or phenylalanine residues. Replacement of the serine residue either by phosphoserine or by tyrosine compromises this symmetry, but further increases the cis conformation content of both prolines. As a result, we obtained a cyclic hexapeptide that exists almost exclusively as the cis‐Pro/cis‐Pro conformer but shows no cis/trans interconversion even in the presence of the PPIase Pin1, apparently due to an energetically quite favorable but highly restricted conformational space.     

Probing the Peptidylglycine α‐Hydroxylating Monooxygenase Active Site with Novel 4‐Phenyl‐3‐butenoic Acid Based Inhibitors

Specific inhibition of the copper‐containing peptidylglycine α‐hydroxylating monooxygenase (PHM), which catalyzes the post‐translational modification of peptides involved in carcinogenesis and tumor progression, constitutes a new approach for combating cancer. We carried out a structure–activity study of new compounds derived from a well‐known PHM substrate analogue, the olefinic compound 4‐phenyl‐3‐butenoic acid (PBA). We designed, synthesized, and tested various PBA derivatives both in vitro and in silico. We show that it is possible to increase PBA affinity for PHM by appropriate functionalization of its aromatic nucleus. Compound 2 d , for example, bears a meta‐benzyloxy substituent, and exhibits better inhibition features (K_i=3.9 μM , k_inact/K_i=427 M ^?1 s^?1) than the parent PBA (K_i=19 μM , k_inact/K_i=82 M ^?1 s^?1). Docking calculations also suggest two different binding modes for PBA derivatives; these results will aid in the development of further PHM inhibitors with improved features.     

A One‐Pot “Triple‐C” Multicyclization Methodology for the Synthesis of Highly Constrained Isomerically Pure Tetracyclic Peptides

A broadly applicable one‐pot methodology for the facile transformation of linear peptides into tetracyclic peptides through a chemoenzymatic peptide synthesis/chemical ligation of peptides onto scaffolds/copper(I)‐catalyzed reaction (CEPS/CLIPS/CuAAC; “triple‐C”) locking methodology is reported. Linear peptides with varying lengths (≥14 amino acids), comprising two cysteines and two azidohomoalanines (Aha), were efficiently cyclized head‐to‐tail by using the peptiligase variant omniligase‐1 (CEPS). Subsequent ligation–cyclization with tetravalent (T4_1/2) scaffolds containing two bromomethyl groups (CLIPS) and two alkyne functionalities (CuAAC) yielded isomerically pure tetracyclic peptides. Sixteen different functional tetracycles, derived from bicyclic inhibitors against urokinase plasminogen activator (uPA) and coagulation factor XIIa (FXIIa), were successfully synthesized and their bioactivities evaluated. Two of these (FF‐T4_1/2) exhibited increased inhibitory activity against FXIIa, compared with a bicyclic control peptide. The corresponding hetero‐bifunctional variants (UF/FU‐T4_1/2), with a single copy of each inhibitory sequence, exhibited micromolar activities against both uPA and FXIIa; thus illustrating the potential of the “bifunctional tetracyclic peptide” inhibitor concept.     

Design,Synthesis, and Biological Evaluation of 3‐Benzazepin‐1‐ols as NR2B‐Selective NMDA Receptor Antagonists

Cleavage and reconstitution of a bond in the piperidine ring of ifenprodil ( 1 ) leads to 7‐methoxy‐2,3,4,5‐tetrahydro‐1H‐3‐benzazepin‐1‐ols, a novel class of NR2B‐selective NMDA receptor antagonists. The secondary amine 7‐methoxy‐2,3,4,5‐tetrahydro‐1H‐3‐benzazepin‐1‐ol ( 12 ), which was synthesized in six steps starting from 2‐phenylethylamine 3 , represents the central building block for the introduction of several N‐linked residues. A distance of four methylene units between the basic nitrogen atom and the phenyl residue in the side chain results in high NR2B affinity. The 4‐phenylbutyl derivative 13 (WMS‐1405, K_i=5.4 nM ) and the conformationally restricted 4‐phenylcyclohexyl derivative 31 (K_i=10 nM ) represent the most potent NR2B ligands of this series. Whereas 13 shows excellent selectivity, the 4‐phenylcyclohexyl derivative 31 also interacts with σ₁ (K_i=33 nM ) and σ₂ receptors (K_i=82 nM ). In the excitotoxicity assay the phenylbutyl derivative 13 inhibits the glutamate‐induced cytotoxicity with an IC₅₀ value of 360 nM , indicating that 13 is an NMDA antagonist.     

Exploring Non‐obvious Hydrophobic Binding Pockets on Protein Surfaces: Increasing Affinities in Peptide–Protein Interactions

A 42‐residue polypeptide conjugated to a small‐molecule organic ligand capable of targeting the phosphorylated side chain of Ser15 was shown to bind glycogen phosphorylase a (GPa) with a K_D value of 280 nm . The replacement of hydrophobic amino acids by Ala reduced affinities, whereas the incorporation of l ‐2‐aminooctanoic acid (Aoc) increased them. Replacing Nle5, Ile9 and Leu12 by Aoc reduced the K_D value from 280 to 27 nm . “Downsizing” the 42‐mer to an undecamer gave rise to an affinity for GPa an order of magnitude lower, but the undecamer in which Nle5, Ile9 and Leu12 were replaced by Aoc showed a K_D value of 550 nm , comparable with that of the parent 42‐mer. The use of Aoc residues offers a convenient route to increased affinity in protein recognition as well as a strategy for the “downsizing” of peptides essentially without loss of affinity. The results show that hydrophobic binding sites can be found on protein surfaces by comparing the affinities of polypeptide conjugates in which Aoc residues replace Nle, Ile, Leu or Phe with those of their unmodified counterparts. Polypeptide conjugates thus provide valuable opportunities for the optimization of peptides and small organic compounds in biotechnology and biomedicine.     

β‐Aminopeptidase‐Catalyzed Biotransformations of β2‐Dipeptides: Kinetic Resolution and Enzymatic Coupling

We have previously shown that the β‐aminopeptidases BapA from Sphingosinicella xenopeptidilytica and DmpA from Ochrobactrum anthropi can catalyze reactions with non‐natural β³‐peptides and β³‐amino acid amides. Here we report that these exceptional enzymes are also able to utilize synthetic dipeptides with N‐terminal β²‐amino acid residues as substrates under aqueous conditions. The suitability of a β²‐peptide as a substrate for BapA or DmpA was strongly dependent on the size of the C_α substituent of the N‐terminal β²‐amino acid. BapA was shown to convert a diastereomeric mixture of the β²‐peptide H‐β²hPhe‐β²hAla‐OH, but did not act on diastereomerically pure β²,β³‐dipeptides containing an N‐terminal β²‐homoalanine. In contrast, DmpA was only active with the latter dipeptides as substrates. BapA‐catalyzed transformation of the diastereomeric mixture of H‐β²hPhe‐β²hAla‐OH proceeded along two highly S‐enantioselective reaction routes, one leading to substrate hydrolysis and the other to the synthesis of coupling products. The synthetic route predominated even at neutral pH. A rise in pH of three log units shifted the synthesis‐to‐hydrolysis ratio (v_S/v_H) further towards peptide formation. Because the equilibrium of the reaction lies on the side of hydrolysis, prolonged incubation resulted in the cleavage of all peptides that carried an N‐terminal β‐amino acid of S configuration. After completion of the enzymatic reaction, only the S enantiomer of β²‐homophenylalanine was detected (ee>99 % for H‐(S)‐β²‐hPhe‐OH, E>500); this confirmed the high enantioselectivity of the reaction. Our findings suggest interesting new applications of the enzymes BapA and DmpA for the production of enantiopure β²‐amino acids and the enantioselective coupling of N‐terminal β²‐amino acids to peptides.     

Incorporation of 2,3‐Diaminopropionic Acid into Linear Cationic Amphipathic Peptides Produces pH‐Sensitive Vectors

Nonviral vectors that harness the change in pH in endosomes, are increasingly being used to deliver cargoes, including nucleic acids, into mammalian cells. Here we present evidence that the pK_a of the β‐NH₂ in 2,3‐diaminopropionic acid (Dap) is sufficiently lowered, when Dap is incorporated into peptides, that its protonation state is sensitive to the pH changes that occur during endosomal acidification. The lowered pK_a of around 6.3 is stabilized by the increased electron‐withdrawing effect of the peptide bonds, by intermolecular hydrogen bonding and from contributions arising from the peptide conformation. These include mixed polar/apolar environments, Coulombic interactions and intermolecular hydrogen bonding. Changes in the charged state are therefore expected between pH 5 and 7, and large‐scale conformational changes are observed in Dap‐rich peptides, in contrast to analogues containing lysine or ornithine, when the pH is altered through this range. These physical properties confer a robust gene‐delivery capability on designed cationic amphipathic peptides that incorporate Dap.     

Conversion of Low‐Affinity Peptides to High‐Affinity Peptide Binders by Using a β‐Hairpin Scaffold‐Assisted Approach

Affinity maturation of protein‐targeting peptides is generally accomplished by homo‐ or heterodimerization of known peptides. However, applying a heterodimerization approach is difficult because it is not clear a priori what length or type of linker is required for cooperative binding to a target. Thus, an efficient and simple affinity maturation method for converting low‐affinity peptides into high‐affinity peptides would clearly be advantageous for advancing peptide‐based therapeutics. Here, we describe the development of a novel affinity maturation method based on a robust β‐hairpin scaffold and combinatorial phage‐display technology. With this strategy, we were able to increase the affinity of existing peptides by more than four orders of magnitude. Taken together, our data demonstrate that this scaffold‐assisted approach is highly efficient and effective in generating high‐affinity peptides from their low‐affinity counterparts.     

The Cyclic Cystine Ladder of Theta‐Defensins as a Stable,Bifunctional Scaffold: A Proof‐of‐Concept Study Using the Integrin‐Binding RGD Motif.

Peptides have the specificity and size required to target the protein–protein interactions involved in many diseases. Some cyclic peptides have been utilised as scaffolds for peptide drugs because of their stability; however, other cyclic peptide scaffolds remain to be explored. θ‐Defensins are cyclic peptides from mammals; they are characterised by a cyclic cystine ladder motif and have low haemolytic and cytotoxic activity. Here we demonstrate the potential of the cyclic cystine ladder as a scaffold for peptide drug design by introducing the integrin‐binding Arg‐Gly‐Asp (RGD) motif into the θ‐defensin RTD‐1. The most active analogue had an IC₅₀ of 18 nM for the α_vβ₃ integrin as well as high serum stability, thus demonstrating that a desired bioactivity can be imparted to the cyclic cystine ladder. This study highlights how θ‐defensins can provide a stable and conformationally restrained scaffold for bioactive epitopes in a β‐strand or turn conformation. Furthermore, the symmetry of the cyclic cystine ladder presents the opportunity to design peptides with dual bioactive epitopes to increase activity and specificity.     

Evaluation of (4‐Arylpiperidin‐1‐yl)cyclopentanecarboxamides As High‐Affinity and Long‐Residence‐Time Antagonists for the CCR2 Receptor

Animal models suggest that the chemokine ligand 2/CC‐chemokine receptor 2 (CCL2/CCR2) axis plays an important role in the development of inflammatory diseases. However, CCR2 antagonists have failed in clinical trials because of a lack of efficacy. We previously described a new approach for the design of CCR2 antagonists by the use of structure–kinetics relationships (SKRs). Herein we report new findings on the structure–affinity relationships (SARs) and SKRs of the reference compound MK‐0483, its diastereomers, and its structural analogues as CCR2 antagonists. The SARs of the 4‐arylpiperidine group suggest that lipophilic hydrogen‐bond‐accepting substituents at the 3‐position are favorable. However, the SKRs suggest that a lipophilic group with a certain size is desired [e.g., 3‐Br: K_i=2.8 nM , residence time (t_res)=243 min; 3‐iPr: K_i=3.6 nM , t_res=266 min]. Alternatively, additional substituents and further optimization of the molecule, while keeping a carboxylic acid at the 3‐position, can also prolong t_res; this was most prominently observed in MK‐0483 (K_i=1.2 nM , t_res=724 min) and a close analogue (K_i=7.8 nM ) with a short residence time.     

Rigid Peptide Macrocycles from On‐Resin Glaser Stapling

Peptide macrocycles are widely utilized in the development of high affinity ligands, including stapled α‐helices. The linear rigidity of a 1,3‐diynyl linkage provides an optimal distance (7 Å) between β‐carbons of the i,i+4 amino acid side chains, thus suggesting its utility in stabilizing α‐helical structures. Here, we report the development of an on‐resin strategy for an intramolecular Glaser reaction between two alkyne‐terminated side chains by using copper chloride, an essential bpy‐diol ligand, and diisopropylethylamine at room temperature. The efficiency of this ligation was illustrated by the synthesis of (i,i+4)‐, (i,i+5)‐, (i,i+6)‐, and (i,i+7)‐stapled BCL‐9 α‐helical peptides using the unnatural amino acid propargyl serine. Overall, this procedurally simple method relies on inexpensive and widely available reagents to generate low molecular weight 23‐, 26‐, 29‐, and 32‐membered peptide macrocycles.     

Arginine Arrangement of Bacteriophage λ N‐Peptide Plays a Role as a Core Motif in GNRA Tetraloop RNA Binding

A simple α‐helical N‐model‐peptide was designed to investigate the role of the arginine‐rich motif of bacteriophage λ N‐peptide in selective binding with boxB RNA. The five‐arginine arrangement of native N‐peptide was retained; all other residues were replaced with alanine. In vitro selection of RNA (30 random‐nucleotide region) was carried out with N‐model‐peptide immobilized on a 27 MHz quartz‐crystal microbalance (QCM). Selected RNAs were evaluated on the same QCM plate to obtain binding constants (K_a=10⁷–10⁸ M ^?1). Many selected RNAs contained GNR(N)A‐type loops (similar to the boxB RNA motif recognized by the native N‐peptide). Fragments and minimal RNAs containing the GNRA‐type loop also bound to N‐model‐peptide (K_a=10⁶–10⁷ M ^?1). The RNA recognition specificity of the peptide was studied by changing the “closing” U–A base pair and one base in the tetraloop of the RNA aptamers, and by peptide mutations (18th residue of N‐model‐peptide). It was concluded that the five‐arginine arrangement of the peptide performs selective recognition of the GNRA tetraloop and GNR(N)A pentaloop RNA structures, and that substitution of another functional amino acid residue at the 18th position in N‐peptide adds the recognition ability for a loop‐RNA sequence.     

Ribosomal Synthesis of Natural‐Product‐Like Bicyclic Peptides in Escherichia coli

Methods to access natural‐product‐like macrocyclic peptides can disclose new opportunities for the exploration of this important structural class for chemical biology and drug discovery applications. Here, the scope and mechanism of a novel strategy for directing the biosynthesis of thioether‐bridged bicyclic peptides in bacterial cells was investigated. This method entails split intein‐catalyzed head‐to‐tail cyclization of a ribosomally produced precursor peptide, combined with inter‐side‐chain crosslinking through a genetically encoded cysteine‐reactive amino acid. This strategy could be successfully applied to achieve formation of structurally diverse bicyclic peptides with high efficiency and selectivity in Escherichia coli. Insights into the sequence of reactions underlying the peptide bicyclization process were gained from time‐course experiments. Finally, the potential utility of this methodology toward the discovery of macrocyclic peptides with enhanced functional properties was demonstrated through the isolation of a bicyclic peptide with sub‐micromolar affinity for streptavidin.     

Targeting tumour proliferation with a small-molecule inhibitor of AICAR transformylase homodimerization

Aminoimidazole carboxamide ribonucleotide transformylase/ inosine monophosphate cyclohydrolase (ATIC) is a bifunctional homodimeric enzyme that catalyzes the last two steps of de novo purine biosynthesis. Homodimerization of ATIC, a protein–protein interaction with an interface of over 5000 Ų, is required for its aminoimidazole carboxamide ribonucleotide (AICAR) transformylase activity, with the active sites forming at the interface of the interacting proteins. Here, we report the development of a small‐molecule inhibitor of AICAR transformylase that functions by preventing the homodimerization of ATIC. The compound is derived from a previously reported cyclic hexapeptide inhibitor of AICAR transformylase (with a K_i of 17 μM ), identified by high‐throughput screening. The active motif of the cyclic peptide is identified as an arginine‐tyrosine dipeptide, a capped analogue of which inhibits AICAR transformylase with a K_i value of 84 μM . A library of nonnatural analogues of this dipeptide was designed, synthesized, and assayed. The most potent compound inhibits AICAR transformylase with a K_i value of 685 nM , a 25‐fold improvement in activity from the parent cyclic peptide. The potential for this AICAR transformylase inhibitor in cancer therapy was assessed by studying its effect on the proliferation of a model breast cancer cell line. Using a nonradioactive proliferation assay and live cell imaging, a dose‐dependent reduction in cell numbers and cell division rates was observed in cells treated with our ATIC dimerization inhibitor.     

Cyclic Peptides Made by Linking Synthetic and Genetically Encoded Fragments

Cyclic peptides can be highly valuable as bioactive molecules, both for biomedical applications and in basic research. We introduce a new fragment‐based approach to access cyclic peptide structures in which one fragment is of synthetic origin and the other is genetically encoded. The synthetic peptide, which can contain one or more non‐proteinogenic building blocks, is coupled to the recombinantly expressed peptide through two bonds, one formed by protein trans‐splicing with a split intein and the other by oxime ligation. Semisynthetic macrocycles were obtained with high efficiency for various sequences and ring sizes; they can be prepared in quantities sufficient for initial bioactivity tests. We also prepared lipidated and d ‐amino‐acid‐containing peptides that were inspired by the peptide antibiotic daptomycin. Such structures are not accessible by other methods that harness the power of simple genetic diversification in the DNA‐encoded part of the peptide.