A Cyclized Helix‐Loop‐Helix Peptide as a Molecular Scaffold for the Design of Inhibitors of Intracellular Protein–Protein Interactions by Epitope and Arginine Grafting

The design of inhibitors of intracellular protein–protein interactions (PPIs) remains a challenge in chemical biology and drug discovery. We propose a cyclized helix‐loop‐helix (cHLH) peptide as a scaffold for generating cell‐permeable PPI inhibitors through bifunctional grafting: epitope grafting to provide binding activity, and arginine grafting to endow cell‐permeability. To inhibit p53–HDM2 interactions, the p53 epitope was grafted onto the C‐terminal helix and six Arg residues were grafted onto another helix. The designed peptide cHLHp53‐R showed high inhibitory activity for this interaction, and computational analysis suggested a binding mode for HDM2. Confocal microscopy of cells treated with fluorescently labeled cHLHp53‐R revealed cell membrane penetration and cytosolic localization. The peptide inhibited the growth of HCT116 and LnCap cancer cells. This strategy of bifunctional grafting onto a well‐structured peptide scaffold could facilitate the generation of inhibitors for intracellular PPIs.     

From Chemical Mutagenesis to Post‐Expression Mutagenesis: A 50 Year Odyssey

Site‐directed (gene) mutagenesis has been the most useful method available for the conversion of one amino acid residue of a given protein into another. Until relatively recently, this strategy was limited to the twenty standard amino acids. The ongoing maturation of stop codon suppression and related technologies for unnatural amino acid incorporation has greatly expanded access to nonstandard amino acids by expanding the scope of the translational apparatus. However, the necessity for translation of genetic changes restricts the diversity of residues that may be incorporated. Herein we highlight an alternative approach, termed post‐expression mutagenesis, which operates at the level of the very functional biomolecules themselves. Using the lens of retrosynthesis, we highlight prospects for new strategies in protein modification, alteration, and construction which will enable protein science to move beyond the constraints of the “translational filter” and lead to a true synthetic biology.     

环肽类组蛋白去乙酰化酶抑制剂

组蛋白乙酰化转移酶(HAT)和组蛋白去乙酰化酶(HDAC)调节组蛋白乙酰化程度，HDAC在基因表达和染色体形成等方面起着重要的调节作用。HDAC抑制剂能够引起肿瘤细胞生长停滞、诱导肿瘤细胞分化和调亡。通过对各种HDAC抑制剂结构及作用机制的研究有助于该类药物在临床上的应用和拓宽癌症治疗的适用范围。本文概述了近年来天然及合成的环肽类组蛋白去乙酰化酶抑制剂的研究进展。     

Biosynthetic Strategies for Macrocyclic Peptides

Macrocyclic peptides are predominantly peptide structures bearing one or more rings and spanning multiple amino acid residues. Macrocyclization has become a common approach for improving the pharmacological properties and bioactivity of peptides. A variety of ribosomal-derived and non-ribosomal synthesized cyclization approaches have been established. The biosynthesis of backbone macrocyclic peptides using seven new emerging methodologies will be discussed with regard to the features and strengths of each platform rather than medicinal chemistry tools. The mRNA display variant, known as the random nonstandard peptide integrated discovery (RaPID) platform, utilizes flexible in vitro translation (FIT) to access macrocyclic peptides containing nonproteinogenic amino acids (NAAs). As a new discovery approach, the ribosomally synthesized and post-translationally modified peptides (RiPPs) method involves the combination of ribosomal synthesis and the phage screening platform together with macrocyclization chemistries to generate libraries of macrocyclic peptides. Meanwhile, the split-intein circular ligation of peptides and proteins (SICLOPPS) approach relies on the in vivo production of macrocyclic peptides. In vitro and in vivo peptide library screening is discussed as an advanced strategy for cyclic peptide selection. Specifically, biosynthetic bicyclic peptides are highlighted as versatile and attractive modalities. Bicyclic peptides represent another type of promising therapeutics that allow for building blocks with a heterotrimeric conjugate to address intractable challenges and enable multimer complexes via linkers. Additionally, we discuss the cell-free chemoenzymatic synthesis of macrocyclic peptides with a non-ribosomal catalase known as the non-ribosomal synthetase (NRPS) and chemo-enzymatic approach, with recombinant thioesterase (TE) domains. Novel insights into the use of peptide library tools, activity-based two-hybrid screening, structure diversification, inclusion of NAAs, combinatorial libraries, expanding the toolbox for macrocyclic peptides, bicyclic peptides, chemoenzymatic strategies, and future perspectives are presented. This review highlights the broad spectrum of strategy classes, novel platforms, structure diversity, chemical space, and functionalities of macrocyclic peptides enabled by emerging biosynthetic platforms to achieve bioactivity and for therapeutic purposes.     

Structure‐Based Design of Inhibitors of Protein–Protein Interactions: Mimicking Peptide Binding Epitopes

Protein–protein interactions (PPIs) are involved at all levels of cellular organization, thus making the development of PPI inhibitors extremely valuable. The identification of selective inhibitors is challenging because of the shallow and extended nature of PPI interfaces. Inhibitors can be obtained by mimicking peptide binding epitopes in their bioactive conformation. For this purpose, several strategies have been evolved to enable a projection of side chain functionalities in analogy to peptide secondary structures, thereby yielding molecules that are generally referred to as peptidomimetics. Herein, we introduce a new classification of peptidomimetics (classes A–D) that enables a clear assignment of available approaches. Based on this classification, the Review summarizes strategies that have been applied for the structure‐based design of PPI inhibitors through stabilizing or mimicking turns, β‐sheets, and helices.     

Latest Advances Towards Ras Inhibition: A Medicinal Chemistry Perspective

Owing to their high occurrence rate across many human cancers and their lack of druggability so far, mutant forms of the signaling protein Ras are currently among the most attractive (and elusive) oncology targets. This strong appeal explains the sustained effort in the field, and the ensuing progress has rekindled optimism regarding the discovery of Ras inhibitors. In this Minireview, we discuss the most recent advances towards irreversible inhibitors, and highlight approaches to inhibitors of Ras–effector interactions that have been overshadowed by the current focus on direct Ras inhibition. At the same time, we provide a critical assessment from a medicinal chemistry perspective.     

Highly Stable,Amide‐Bridged Autoinducing Peptide Analogues that Strongly Inhibit the AgrC Quorum Sensing Receptor in Staphylococcus aureus

Blocking quorum sensing (QS) pathways has attracted considerable interest as an approach to suppress virulence in bacterial pathogens. Toward this goal, we recently developed analogues of a native autoinducing peptide (AIP‐III) signal that can inhibit AgrC‐type QS receptors and attenuate virulence phenotypes in Staphylococcus aureus. Application of these compounds is limited, however, as they contain hydrolytically unstable thioester linkages and have only low aqueous solubilities. Herein, we report amide‐linked AIP analogues with greatly enhanced hydrolytic stabilities and solubilities relative to our prior analogues, whilst maintaining strong potencies as AgrC receptor inhibitors in S. aureus. These compounds represent powerful tools for the study of QS.     

Application of the valence-universal coupled-cluster method based on various model spaces. II. Nonstandard solutions for Be

The impact of the choices of the complete model space (CMS) and of the orbital basis set on the existence, attainability, and properties of the nonstandard solutions of the valence-universal coupled-cluster (VU-CC) methods has been studied for the case of nonlinear equations corresponding to the atomically oriented form of these methods accounting for one- and two-electron excitations (VU-CCSD/R method) and applied to the Be atom. The results for five ¹S states are discussed. In addition to the previously applied CMS defined by the orbital set (2s, 2p) and (2s, 3s), we have employed the CMSs defined by the (2s, 2p, 3s) set. For each of the CMSs several nonstandard solutions are documented. It is found that the energies of the individual states corresponding to standard and nonstandard solutions differ very little. These energies are also almost independent on the choice of CMS. On the other hand, the energies of excited states disclose a strong dependence on the radial structure of the orbital basis set. It is also demonstrated that the magnitudes of the cluster amplitudes representing a set of states depend both on the choice of the CMS and whether they correspond to the standard or nonstandard solutions. © 1996 John Wiley & Sons, Inc.     

Amplification, mutation, and sequencing of a six-letter synthetic genetic system

The next goals in the development of a synthetic biology that uses artificial genetic systems will require chemistry-biology combinations that allow the amplification of DNA containing any number of sequential and nonsequential nonstandard nucleotides. This amplification must ensure that the nonstandard nucleotides are not unidirectionally lost during PCR amplification (unidirectional loss would cause the artificial system to revert to an all-natural genetic system). Further, technology is needed to sequence artificial genetic DNA molecules. The work reported here meets all three of these goals for a six-letter artificially expanded genetic information system (AEGIS) that comprises four standard nucleotides (G, A, C, and T) and two additional nonstandard nucleotides (Z and P). We report polymerases and PCR conditions that amplify a wide range of GACTZP DNA sequences having multiple consecutive unnatural synthetic genetic components with low (0.2% per theoretical cycle) levels of mutation. We demonstrate that residual mutation processes both introduce and remove unnatural nucleotides, allowing the artificial genetic system to evolve as such, rather than revert to a wholly natural system. We then show that mechanisms for these residual mutation processes can be exploited in a strategy to sequence "six-letter" GACTZP DNA. These are all not yet reported for any other synthetic genetic system.     

Inhibition of Ras Signaling by Blocking Ras–Effector Interactions with Cyclic Peptides

Ras genes are frequently activated in human cancers, but the mutant Ras proteins remain largely “undruggable” through the conventional small‐molecule approach owing to the absence of any obvious binding pockets on their surfaces. By screening a combinatorial peptide library, followed by structure–activity relationship (SAR) analysis, we discovered a family of cyclic peptides possessing both Ras‐binding and cell‐penetrating properties. These cell‐permeable cyclic peptides inhibit Ras signaling by binding to Ras‐GTP and blocking its interaction with downstream proteins and they induce apoptosis of cancer cells. Our results demonstrate the feasibility of developing cyclic peptides for the inhibition of intracellular protein–protein interactions and of direct Ras inhibitors as a novel class of anticancer agents.     

Exploring solvent versatility in immobilized cellulose-based chiral stationary phase for the enantioselective liquid chromatographic resolution of racemates

Chiralpak IB, a new chiral stationary phase (CSP) containing cellulose tris(3,5-dimethylphenylcarabamate) immobilized onto silica gel, is investigated for the direct enantioselective separation of a set of racemic N-alkylated barbiturates and analogs of thalidomide alkylated in position 3 of the piperidin-2,6-dione ring using different nonstandard solvents such as dichloromethane (DCM), ethyl acetate, THF, methyl tert-butyl ether as an eluent and diluent, respectively, in HPLC. The separation, resolution, and elution order of the investigated compounds were compared on both immobilized and coated cellulose tris(3,5-dimethylphenylcarbamate) CSPs (Chiralpak IB and Chiralcel OD, respectively) using a mixture of n-hexane/2-propanol (90:10 v/v) as mobile phase with different flow-rates and fixed UV detection at 254 nm. The effect of the immobilization of the cellulose tris-(3,5-dimethylphenylcarbamate) CSP on silica (Chiralpak IB) on the chiral recognition ability was noted as the coated phase (Chiralcel OD) possesses a higher resolving power in some cases than the immobilized one (Chiralpak IB). However, a few racemates, which were not or poorly resolved on the immobilized Chiralpak IB or the coated Chiralcel OD when using standard solvents were most efficiently resolved on the immobilized Chiralpak IB upon using nonstandard solvents. Furthermore, the immobilized phase withstands the nonstandard (prohibited) HPLC solvents mentioned previously when used as eluents or as a dissolving agent for the analyte itself. An example of inversion or apparent inversion of elution order on Chiralpak IB is reported. The direct analysis of a spiked plasma sample extracted using DCM on Chiralpak IB is also shown.     

Enzyme‐Degradable Self‐Assembled Hydrogels From Polyalanine‐Modified Poly(ethylene glycol) Star Polymers

The generation of a range of star‐shaped block copolymers composed of a biocompatible poly(ethylene glycol) (PEG) core tethered to a polyalanine (PAla) shell that possesses the capability to (reversibly) self‐assemble in water is described. The hydrogels formed offer a hydrophilic environment ideal for biological processes involving proteins and are able to withhold albumin for prolonged periods before its triggered release following the targeted material degradation by the proteolytic enzyme elastase. Consequently, the materials formed offer significant promise for the delivery of proteins, and possibly inhibitors, in response to a proteolytic enzyme overexpressed in chronic wounds.     

Stapled Peptides as HIF-1α/p300 Inhibitors: Helicity Enhancement in the Bound State Increases Inhibitory Potency

Protein–protein interactions (PPIs) control virtually all cellular processes and have thus emerged as potential targets for development of molecular therapeutics. Peptide-based inhibitors of PPIs are attractive given that they offer recognition potency and selectivity features that are ideal for function, yet, they do not predominantly populate the bioactive conformation, frequently suffer from poor cellular uptake and are easily degraded, for example, by proteases. The constraint of peptides in a bioactive conformation has emerged as a promising strategy to mitigate against these liabilities. In this work, using peptides derived from hypoxia-inducible factor 1 (HIF-1α) together with dibromomaleimide stapling, we identify constrained peptide inhibitors of the HIF-1α/p300 interaction that are more potent than their unconstrained sequences. Contrary to expectation, the increased potency does not correlate with an increased population of an α-helical conformation in the unbound state as demonstrated by experimental circular dichroism analysis. Rather, the ability of the peptide to adopt a bioactive α-helical conformation in the p300 bound state is better supported in the constrained variant as demonstrated by molecular dynamics simulations and circular dichroism difference spectra.     

Cyclic Peptides as Inhibitors of Amyloid Fibrillation

Many neurodegenerative diseases, like Parkinson’s, Alzheimer’s, or Huntington’s disease, occur as a result of amyloid protein fibril formation and cell death induced by this process. Cyclic peptides (CPs) and their derivatives form a new class of powerful inhibitors that prevent amyloid fibrillation and decrease the cytotoxicity of aggregates. The strategies for designing CPs are described, with respect to their amino acid sequence and/or conformational similarity to amyloid fibrils. The implications of CPs for the study and possible treatment of amyloid‐related diseases are discussed.     

Modulation of Src Kinase Activity by Selective Substrate Recognition with Pseudopeptidic Cages

The selective recognition of tyrosine residues in peptides is an appealing approach to inhibiting their tyrosine kinase (TK)-mediated phosphorylation. Herein, we describe pseudopeptidic cages that efficiently protect substrates from the action of the Src TK enzyme, precluding the corresponding Tyr phosphorylation. Fluorescence emission titrations show that the most efficient cage inhibitors strongly bind the peptide substrates with a very good correlation between the binding constant and the inhibitory potency. Structural insights and additional control experiments further support the proposed mechanism of selective supramolecular protection of the substrates. Moreover, the approach also works in a completely different kinase-substrate system. These results illustrate the potential of supramolecular complexes for the efficient and selective modulation of TK signaling.     

Synthesis of a Natural Product‐Like Compound Collection through Oxidative Cleavage and Cyclization of Linear Peptides

Massive efforts in molecular library synthesis have strived for the development of synthesis methodology which systematically delivers natural product‐like compounds of high spatial complexity. Herein, we present a conceptually simple approach that builds on the power of solid‐phase peptide synthesis to assemble precursor peptides (oligomers) designed to undergo oxidative cascade reactions. By harnessing the structural side‐chain diversity and inherent stereochemical features offered by readily available amino acids (monomers), a proof‐of‐concept collection of 54 skeletally and stereochemically diverse compounds was generated, and selected compounds were elaborated into isoform‐selective metalloprotease inhibitors.     

Structural Characterization of a Macrocyclic Peptide Modulator of the PD-1/PD-L1 Immune Checkpoint Axis

The clinical success of PD-1/PD-L1 immune checkpoint targeting antibodies in cancer is followed by efforts to develop small molecule inhibitors with better penetration into solid tumors and more favorable pharmacokinetics. Here we report the crystal structure of a macrocyclic peptide inhibitor (peptide 104) in complex with PD-L1. Our structure shows no indication of an unusual bifurcated binding mode demonstrated earlier for another peptide of the same family (peptide 101). The binding mode relies on extensive hydrophobic interactions at the center of the binding surface and an electrostatic patch at the side. An interesting sulfur/π interaction supports the macrocycle-receptor binding. Overall, our results allow a better understanding of forces guiding macrocycle affinity for PD-L1, providing a rationale for future structure-based inhibitor design and rational optimization.     

肽与寡核苷酸缀合物的合成及应用研究进展

寡核苷酸可作为基因表达的抑制剂和潜在的治疗药物,但许多类型的寡核苷酸为聚阴离子化合物,难以跨过细胞膜,而许多生物活性肽具有跨膜与核定位能力,通过合成的方法可以将这两种具有重要功能的生物高聚物以共价键连接在一起,从而实现药物的有效载运．本文综述了肽寡核苷酸缀合物的合成方法及其应用．     

Allosterically Regulated Phosphatase Activity from Peptide–PNA Conjugates Folded Through Hybridization

The importance of spatial organization in short peptide catalysts is well recognized. We synthesized and screened a library of peptides flanked by peptide nucleic acids (PNAs) such that the peptide would be constrained in a hairpin loop upon hybridization. A screen for phosphatase activity led to the discovery of a catalyst with >25‐fold rate acceleration over the linear peptide. We demonstrated that the hybridization‐enforced folding of the peptide is necessary for activity, and designed a catalyst that is allosterically controlled using a complementary PNA sequence.     

Direct Targeting of Rab‐GTPase–Effector Interactions

Small GTPases are molecular switches using GDP/GTP alternation to control numerous vital cellular processes. Although aberrant function and regulation of GTPases are implicated in various human diseases, direct targeting of this class of proteins has proven difficult, as GTPase signaling and regulation is mediated by extensive and shallow protein interfaces. Here we report the development of inhibitors of protein–protein interactions involving Rab proteins, a subfamily of GTPases, which are key regulators of vesicular transport. Hydrocarbon‐stapled peptides were designed based on crystal structures of Rab proteins bound to their interaction partners. These modified peptides exhibit significantly increased affinities and include a stapled peptide (StRIP3) that selectively binds to activated Rab8a and inhibits a Rab8a–effector interaction in vitro.