An object-oriented library for computational protein design

Recent advances in computational protein design have established it as a viable technique for the rational generation of stable protein sequences, novel protein folds, and even enzymatic activity. We present a new and object-oriented library of code, written specifically for protein design applications in C(++), called EGAD Library. The modular fashion in which this library is written allows developers to tailor various energy functions and minimizers for a specific purpose. It also allows for the generation of novel protein design applications with a minimal amount of code investment. It is our hope that this will permit labs that have not considered protein design to apply it to their own systems, thereby increasing its potential as a tool in biology. We also present various uses of EGAD Library: in the development of Interaction Viewer, a PyMOL plug-in for viewing interactions between protein residues; in the repacking of protein cores; and in the prediction of protein-protein complex stabilities.     

Synthesis of a novel macrocyclic library: discovery of an IGF-1R inhibitor

We present a new approach for the conversion of active sequences of proteins and peptides into small molecules. A library of macrocyclic disulfide molecules was made, in which the active pharmacophores of the parent peptide are preserved while the size of the macromolecular scaffold on which the pharmacophores are arranged is varied. This enables a systematic search for macromolecules in which the pharmacophores are in an appropriate conformation for biological activity. We developed two procedures for the synthesis of such libraries from building blocks that include commercial amino acids and functionalized aldehydes. Chemical synthesis using the "tea-bag" method gave a library with higher diversity, but low yields, compared to the manual synthesis of the library, in which the compounds were synthesized in individual vessels and the yield and purity improved dramatically. As a proof of concept, we synthesized a 34-member library derived from the sequence of the activation loop of insulin-like growth factor-1 receptor. Selected compounds were screened, and one was found to be biologically active in the low micromolar range. The concept presented here may prove particularly useful in cases where the pharmacophores are known but need to be systematically screened for a spatial arrangement that will enable biological activity.     

Gain-of-function analogues of the pore-forming peptide melittin selected by orthogonal high-throughput screening

We recently developed an orthogonal, high-throughput assay to identify peptides that self-assemble into potent, equilibrium pores in synthetic lipid bilayers. Here, we use this assay as a high-throughput screen to select highly potent pore-forming peptides from a 7776-member rational combinatorial peptide library based on the sequence of the natural pore-forming peptide toxin melittin. In the library we varied ten critical residues in the melittin sequence, chosen to test specific structural hypotheses about the mechanism of pore formation. Using the new high-throughput assay, we screened the library for gain-of-function sequences at a peptide to lipid ratio of 1:1000 where native melittin is not active. More than 99% of the library sequences were also inactive under these conditions. A small number of library members (0.1%) were highly active. From these we identified 14 potent, gain-of-function, pore-forming sequences. These sequences differed from melittin in only 2-6 amino acids out of 26. Some native residues were highly conserved and others were consistently changed. The two factors that were essential for gain-of-function were the preservation of melittin's proline-dependent break in the middle of the helix and the improvement and extension the amphipathic nature of the α-helix. In particular the highly cationic carboxyl-terminal sequence of melittin, is consistently changed in the gain-of-function variants to a sequence that it is capable of participating in an extended amphipathic α-helix. The most potent variants reside in a membrane-spanning orientation, in contrast to the parent melittin, which is predominantly surface bound. This structural information, taken together with the high-throughput tools developed for this work, enable the identification, refinement and optimization of pore-forming peptides for many potential applications.     

Screening for protease substrate by polyvalent phage display

Proteases are key regulators of many physiological and pathological processes [1,2], and are recognized as important and tractable drug candidates. Consequently, knowledge of protease substrate recognition and specificity promotes identification of biologically relevant substrates, helps elucidating a protease's biological function, and the design of specific inhibitors. Traditional methods for establishing substrate recognition profiles involve the identification of the scissile bond within a given protein substrate by proteomic methods such as Edman degradation. Then, synthetic peptide variants of this sequence can be screened in an iterative fashion to arrive at more optimized substrates. Even though it can be fruitful, this iterative strategy is biased toward the original substrate sequence and it is also tremendously cumbersome. Furthermore, it is not amenable to high throughput analysis. In 1993, Matthew & Wells presented a method for the use of monovalent "substrate phage" libraries for discovering peptide substrates for proteases, in which more than 10(7) potential substrates can be tested concurrently [3]. A library of fusion proteins was constructed containing randomized substrate sequences placed between a binding domain and the gene III coat protein of the filamentous phage, M13, which displays the fusion protein and packages the gene coding for it inside. Each fusion protein was displayed as a single copy on filamentous phagemid particles (substrate phage). This method allows one to rapidly survey the substrate recognition and specificity of individual or closely related members of proteases. Over the past decade, substrate phage screening has shown terrific utility in rapidly determining protease specificity and characterization of substrate recognition profile of proteases. In some cases, the structural insights of the catalytic domain were obtained from comparison of substrate specificity among closely related family of proteases [4-6]. The number of proteases (from various classes) characterized by this approach testifies to its power. Since the initial development of substrate phage library, different versions of the substrate phage cloning vectors have been constructed to further improve the utility of substrate phage display. This review will provide an overview of the construction of substrate phage display libraries, screening of substrate phage libraries, examples of application, summary and future directions.     

Discovery of a new phosphotyrosine mimetic for PTP1B using breakaway tethering

Protein tyrosine phosphatases play important roles in many signaling cascades involved in human disease. The identification of druglike inhibitors for these targets is a major challenge, and the discovery of suitable phosphotyrosine (pY) mimetics remains one of the key difficulties. Here we describe an extension of tethering technology, "breakaway tethering", which is ideally suited for discovering such new chemical entities. The approach involves first irreversibly modifying a protein with an extender that contains both a masked thiol and a known pY mimetic. The extender is then cleaved to release the pY mimetic, unmasking the thiol. The resulting protein is screened against a library of disulfide-containing small molecule fragments; any molecules with inherent affinity for the pY binding site will preferentially form disulfides with the extender, allowing for their identification by mass spectrometry. The ability to start from a known substrate mimimizes perturbation of protein structure and increases the opportunity to probe the active site using tethering. We applied this approach to the anti-diabetic protein PTP1B to discover a pY mimetic which belongs to a new molecular class and which binds in a novel fashion.     

SSC: a tool for constructing libraries for systematic screening of conformers

Even a relatively small molecule with 10-20 atoms might have a few local minima, which correspond to different conformers. The number of local minima quickly increases with molecular size and the most common algorithms, driven by calculated forces, frequently identify a minimum, which is closest to the initial structure, rather than the most stable conformer. Here we discuss how to perform a systematic search of the conformational space for a chain-like molecule. Our approach is fully automated and a user has control which chemical bonds will be probed and with which increments. Moreover, whole fragments of the molecule, which are adjacent to each selected rotational bond, are rotated in a properly selected cylindrical coordinate system and unchemical hybridizations and some "clashes" between neighboring groups, which are common when standard Z-matrices are used, are avoided. A library of potentially relevant conformers is created with a tool, which we call SSC, denoting Systematic Screening of Conformers. Each member of the library is prescreened at a predefined level of theory and the most promising conformers are identified. Finally, they are further evaluated at a higher level of theory to identify the most stable structures and their physicochemical properties. As an example, we demonstrate the results of this approach for 2'-deoxycytidine.     

Hydrazone- and hydrazide-containing N-substituted glycines as peptoid surrogates for expedited library synthesis: application to the preparation of Tsg101-directed HIV-1 budding antagonists

Replacing the Pro6 in the p6(Gag)-derived 9-mer "P-E-P-T-A-P-P-E-E" with N-substituted glycine (NSG) residues is problematic. However, incorporation of hydrazone amides ("peptoid hydrazones") can be readily achieved in library fashion. Furthermore, reduction of these hydrazones to N-substituted "peptoid hydrazides" affords a facile route to library diversification. This approach is demonstrated by application to Tsg101-binding compounds designed as potential HIV budding antagonists. [reaction: see text]     

Selection of enantioselective acyl transfer catalysts from a pooled peptide library through a fluorescence-based activity assay: an approach to kinetic resolution of secondary alcohols of broad structural scope.

An assay employing a fluorescently labeled split and pool peptide library has been applied to the discovery of a new class of octapeptide catalysts for the kinetic resolution of secondary alcohols. A highly diverse library of peptide-based catalysts was synthesized on solid-phase synthesis beads such that each individual bead was co-functionalized with (i) a uniform loading of a pH-sensitive fluorophore and (ii) a unique peptide-based catalyst. The library was then screened for activity in acylation reactions employing (+/-)-sec-phenylethanol as the substrate and acetic anhydride as the acylation agent. From the most active catalysts, a lead peptide (4) was identified that provides a selectivity-factor (k(rel)) of 8.2 upon resynthesis and evaluation under homogeneous conditions. A "directed" second-generation split and pool peptide library was synthesized such that the new peptide sequences in the library were biased toward the lead structure. Random samples of the second generation library were screened in single bead assays that revealed several new peptide-based catalysts that afford improved selectivities in kinetic resolutions. Peptide catalyst 13 proves effective for the kinetic resolution of sec-phenylethanol (k(rel) = 20), as well as eight other secondary alcohols of a broad substrate scope (k(rel) = 4 to >50).     

Scanning conformational space with a library of stereo- and regiochemically diverse aminoglycoside derivatives: the discovery of new ligands for RNA hairpin sequences

A library of stereo- and regiochemically diverse aminoglycoside derivatives was screened at 1 microM using surface plasmon resonance (SPR) against RNA hairpin models of the bacterial A-site, and the HIV viral TAR and RRE sequences. In order to double the stereochemical diversity of the library, the compounds were screened against both enantiomers of each of these sequences. Remarkably, this initial screen suggested that the same four aminoglycoside derivatives bound most tightly to all three of the RNAs, suggesting that these compounds were good RNA binders which, nonetheless, discriminated poorly between the RNA sequences. The interactions between selected isomeric aminoglycoside derivatives and the RNA hairpins were then studied in more detail using an SPR assay. Three isomeric tight-binding aminoglycoside derivatives, which had been identified from the initial screen, were found to bind more tightly to the RNA hairpins (with K(D) values in the range 0.23 to 4.7 microM) than a fourth isomeric derivative (which had K(D) values in the range 6.0 to 30 microM). The magnitude of the tightest RNA-aminoglycoside interactions stemmed, in large part, from remarkably slow dissociation of the aminoglycosides from the RNA targets. The three tight-binding aminoglycoside derivatives were found, however, to discriminate rather poorly between alternative RNA sequences with, at best, around a twenty-fold difference in affinity for alternative RNA hairpin sequences. Within the aminoglycoside derivative library studied, high affinity for an RNA target was not accompanied by good discrimination between alternative RNA sequences.     

Combinatorial library of peptide isosters based on Diels-Alder reactions: identification of novel inhibitors against a recombinant cysteine protease from Leishmania mexicana

A combinatorial split-and-mix library of peptide isosters based on a Diels-Alder reaction was synthesized as a "one-bead-two-compounds" library and encoded by ladder synthesis for facile analysis by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. In the "one-bead-two-compounds" library approach, each bead contains a library member as a putative protease inhibitor along with a fluorescence-quenched substrate for the protease. When the library was screened with CPB2.8 DeltaCTE, a recombinant cysteine protease from L. mexicana, several beads containing compounds with inhibitory activity could be selected from the library and analyzed by MALDI-TOF MS for structure elucidation. Two types of inhibitors were revealed. One novel class of inhibitors had the bicyclic Diels-Alder product isosteric element incorporated internally in a peptide, while the other type was an N-terminal alpha,beta-unsaturated ketone Michael acceptor used as starting material for the Diels-Alder reaction. Selected hit sequences and constructed consensus sequences based on the observed frequencies of amino acids in different subsites were resynthesized and assayed in solution for inhibitor activity and were shown to have IC(50) values in the high nanomolar to low micromolar range.     

Identification of alpha-galactosyl epitope mimetics through rapid generation and screening of C-linked glycopeptide library

A general methodology has been established for rapid generation and screening of combinatorial glycopeptide library and subsequent mass spectrometric sequencing to identify the mimetics of Galalpha(1,3)Gal epitopes. Using this approach, several active glycopeptide sequences were recognized and found to inhibit the binding of human natural anti-Gal antibodies with comparable IC(50)s to synthetic Galalpha(1,3)Gal oligosaccharides. The most active glycopeptides detected from the library included Gal-Tyr-Trp-Arg-Tyr, Gal-Thr-Trp-Arg-Tyr, and Gal-Arg-Trp-Arg-Tyr. These glycopeptides showed higher affinities to anti-Gal antibodies than known Galalpha(1,3)Gal peptide mimetics, such as DAHWESWL and SSLRGF. Our results suggest that, by combining a peptide sequence (the "functional" mimic part) with a terminal alpha-linked galactose moiety (the "structural" mimic part), the resulting glycopeptide could be a very good Galalpha(1,3)Gal mimetic. Analysis of these active glycopeptides provided first-hand information regarding the binding site of anti-Gal antibodies to facilitate the structurally based design of more potent and stable inhibitors.     

Secondary Structure Libraries for Artificial Evolution Experiments

Methods of artificial evolution such as SELEX and in vitro selection have made it possible to isolate RNA and DNA motifs with a wide range of functions from large random sequence libraries. Once the primary sequence of a functional motif is known, the sequence space around it can be comprehensively explored using a combination of random mutagenesis and selection. However, methods to explore the sequence space of a secondary structure are not as well characterized. Here we address this question by describing a method to construct libraries in a single synthesis which are enriched for sequences with the potential to form a specific secondary structure, such as that of an aptamer, ribozyme, or deoxyribozyme. Although interactions such as base pairs cannot be encoded in a library using conventional DNA synthesizers, it is possible to modulate the probability that two positions will have the potential to pair by biasing the nucleotide composition at these positions. Here we show how to maximize this probability for each of the possible ways to encode a pair (in this study defined as A-U or U-A or C-G or G-C or G.U or U.G). We then use these optimized coding schemes to calculate the number of different variants of model stems and secondary structures expected to occur in a library for a series of structures in which the number of pairs and the extent of conservation of unpaired positions is systematically varied. Our calculations reveal a tradeoff between maximizing the probability of forming a pair and maximizing the number of possible variants of a desired secondary structure that can occur in the library. They also indicate that the optimal coding strategy for a library depends on the complexity of the motif being characterized. Because this approach provides a simple way to generate libraries enriched for sequences with the potential to form a specific secondary structure, we anticipate that it should be useful for the optimization and structural characterization of functional nucleic acid motifs.     

Recent advances on catalytic asymmetric difunctionalization of 1,3-dienes

1,3-Dienes and derivatives are either feedstock chemicals or easily available materials. Catalytic difunctionalization of 1,3-diene is one of the most powerful methods for carboncarbon bond formation with rapid increase of the molecular complexity and synthetic value in an atom economic way. By choosing proper metals and chiral ligands, a variety of catalytic asymmetric difunctionalization of conjugated dienes in a highly regioselective fashion have been reported. In this digest review, we will summarize recent advances on this topic based on different metals. We will also introduce unique phenomena that include reversal of regio- and diastereoselectivity.     

Solid phase combinatorial library of phosphinic peptides for discovery of matrix metalloproteinase inhibitors

A solid phase combinatorial library of 165,000 phosphinic peptide inhibitors was prepared and screened for activity against MMP-12. The inhibitors of the library had the structure XXX-Gpsi(PO2H-CH2)L-XXX, in which X is an arbitrary amino acid and Gpsi(PO2H-CH2)L is a Gly-Leu phosphinic dipeptide analogue. The library was constructed as a one-bead-two-compounds library so that every bead contained a common quenched fluorogenic substrate and a different putative inhibitor. In addition, the inhibitor part was prepared by ladder synthesis. After incubation with MMP-12, beads containing active inhibitors were selected, and the inhibitor sequences were recorded using MALDI-TOF MS. Statistical analysis of the sequences obtained from 86 beads gave rise to a consensus sequence which was resynthesized along with 20 related sequences. Three truncated sequences and 16 sequences originally present on beads were also resynthesized. The inhibitors were investigated in an enzyme kinetic assay with MMP-12 showing that the compounds derived from the consensus sequence were strong inhibitors with Ki values down to 6 nM, whereas the sequences originally present on beads varied in potency with Ki values from micromolar to nanomolar. Truncated sequences derived from the consensus sequence were poor inhibitors of MMP-12.     

Combinatorial synthesis of bimetallic complexes with three halogeno bridges

Methods for the synthesis of bimetallic complexes in which two different metal fragments are connected by three chloro or bromo bridges are reported. The reactions are general, fast, and give rise to structurally defined products in quantitative yields. Therefore, they are ideally suited for generating a library of homo- and heterobimetallic complexes in a combinatorial fashion. This is of special interest for applications in homogeneous catalysis. Selected members of this library were synthesized and comprehensively characterized; single-crystal X-ray analyses were performed for 15 new bimetallic compounds.     

A combinatorial approach to minimal peptide models of a metalloprotein active site

Screening of a "one-bead-one-compound" peptide library containing biomimetic His/Cys ligands has led to the discovery of sequences that hydrolyze ester substrates in combination with Zn2+.     

毛细管电泳法高效筛选8-氧代鸟嘌呤DNA糖基化酶的核酸适配体

8-氧代鸟嘌呤DNA糖基化酶(OGG1)是人体中重要的功能蛋白,在修复DNA氧化性损伤过程中起关键作用。氧化应激等引起的氧化损伤易导致炎症反应的发生,对OGG1的抑制可以一定程度上起到缓解作用;对癌细胞OGG1的抑制有望作为癌症治疗的新方法。目前的研究多集中于小分子对OGG1功能的影响和调控,而OGG1的适配体筛选尚未见报道。作为功能配体,适配体具有合成简单、高亲和力及高特异性等优点。该文筛选了OGG1的核酸适配体,结合毛细管电泳高效快速的优点建立了两种基于毛细管电泳-指数富集进化(CE-SELEX)技术的筛选方法:同步竞争法和多轮筛选法。同步竞争法利用单链结合蛋白(SSB)与核酸库中单链核酸的强结合能力,与目标蛋白OGG1组成竞争体系,并通过增加SSB浓度来增加竞争筛选压力,以去除与OGG1弱结合的核酸序列,一步筛选即可获得与OGG1强结合的核酸序列。多轮筛选法在相同孵育条件和电泳条件下,经3轮筛选获得OGG1的核酸适配体。比较两种筛选方法的筛选结果,筛选结果中频次最高的3条候选核酸适配体序列一致,其解离常数(K_D)值在1.71~2.64 μmol/L之间。分子对接分析结果表明候选适配体1(Apt 1)可能与OGG1中具有修复氧化性损伤功能的活性口袋结合。通过对两种筛选方法的对比,证明同步竞争法更加快速高效,对其他蛋白核酸适配体筛选方法的选择具有一定的指导意义。得到的适配体有望用于OGG1功能调控,以抑制其修复功能。     

Combinatorial solid-phase synthesis and screening of a diverse tripodal triazacyclophane (TAC)-based synthetic receptor library showing a remarkable selectivity towards a d-Ala-d-Ala containing ligand

A large and diverse library of a TAC-based tripodal synthetic receptor library (6) has been prepared by split-mix synthesis on the solid phase. Each receptor of the 46,656-member TAC-based library (6) is attached to a solid support bead and contains three different dipeptide arms. On-bead screening for binding of a d-Ala-d-Ala containing ligand (7) by the TAC-based library (6) was performed in phosphate buffer (0.2 N, pH=7.0). Remarkable selectivity for particular library members was observed. The best binding members from the screening were manually selected using fluorescence microscopy and subjected to structural analysis by sequencing. The thus determined binding sequences showed a high consensus.     

A comparison of different functions for predicted protein model quality assessment

In protein structure prediction, a considerable number of models are usually produced by either the Template-Based Method (TBM) or the ab initio prediction. The purpose of this study is to find the critical parameter in assessing the quality of the predicted models. A non-redundant template library was developed and 138 target sequences were modeled. The target sequences were all distant from the proteins in the template library and were aligned with template library proteins on the basis of the transformation matrix. The quality of each model was first assessed with QMEAN and its six parameters, which are C_β interaction energy (C_beta), all-atom pairwise energy (PE), solvation energy (SE), torsion angle energy (TAE), secondary structure agreement (SSA), and solvent accessibility agreement (SAE). Finally, the alignment score (score) was also used to assess the quality of model. Hence, a total of eight parameters (i.e., QMEAN, C_beta, PE, SE, TAE, SSA, SAE, score) were independently used to assess the quality of each model. The results indicate that SSA is the best parameter to estimate the quality of the model.     

Construction of a protein-detection system using a loop peptide library with a fluorescence label

Construction of a novel protein-detection system was carried out using a designed peptide library with fluorescent labels based on loop structures. As a basic model study, detection of alpha-amylase using fluorescent-labeled peptides derived from an active loop of tendamistat was examined. The detection methods for proteins with immobilized peptides as well as peptides in solution have been successfully established. Based on these results, a loop peptide library that has various turn sequences grafted on a stable loop structure has been constructed. Various proteins with recognition patterns corresponding, for instance, to "protein fingerprints" could be detected using an immobilized peptide library. The present results suggest that the system can be applied to the development of a peptide microarray that behaves as a protein chip.