Reverse interactomics: decoding protein-protein interactions with combinatorial peptide libraries

Identification of binding partners is the crucial first step towards understanding the biological function of a protein. Many protein-protein interactions occur via modular domains that recognize short peptide motifs in their target proteins. Here we describe a chemical/bioinformatics approach for predicting the binding partners of modular domains. The optimal binding motif(s) of a protein domain is identified by screening a combinatorial peptide library. The resulting consensus sequence is used to search protein and genomic databases for potential binding proteins, which are subsequently confirmed (or disproved) by conventional protein binding assays (e.g. pull-down and co-immunoprecipitation).     

Solution-phase combinatorial synthesis of nonpeptide bradykinin antagonists

We describe the solution-phase combinatorial synthesis and pharmacological effect of fifty N,N(')-substituted-N"-1-(4-chlorobenzhydryl)piperazine iminodiacetic acid triamide derivatives as nonpeptide B2 antagonists. The synthesized compounds were tested for their antibradykinin activity by utilizing guinea-pig ileum smooth muscle. Most of the compounds showed antagonistic effects on bradykinin induced contraction. N-acetyl-N(')-(4-methylbenzyl)-N"-1-(4-chlorobenzhydryl)piperazine iminodiacetic acid triamide (A3B1C1) showed the 46% inhibition at 100nM.     

De novo proteins from designed combinatorial libraries

Combinatorial libraries of de novo amino acid sequences can provide a rich source of diversity for the discovery of novel proteins with interesting and important activities. Randomly generated sequences, however, rarely fold into well-ordered proteinlike structures. To enhance the quality of a library, features of rational design must be used to focus sequence diversity into those regions of sequence space that are most likely to yield folded structures. This review describes how focused libraries can be constructed by designing the binary pattern of polar and nonpolar amino acids to favor proteins that contain abundant secondary structure, while simultaneously burying hydrophobic side chains and exposing hydrophilic side chains to solvent. The "binary code" for protein design was used to construct several libraries of de novo proteins, including both alpha-helical and beta-sheet structures. The recently determined solution structure of a binary patterned four-helix bundle is well ordered, thereby demonstrating that sequences that have neither been selected by evolution (in vivo or in vitro) nor designed by computer can form nativelike proteins. Examples are presented demonstrating how binary patterned libraries have successfully produced well-ordered structures, cofactor binding, catalytic activity, self-assembled monolayers, amyloid-like nanofibrils, and protein-based biomaterials.     

Inhibiting protein-protein interactions using designed molecules

Continuing investigations into protein-protein interactions have revealed their key role in regulating a wide range of cellular processes. Although efforts to modulate these interactions are more challenging and much less mature than work on conventional drug discovery pathways, significant progress has been made on several fronts. Highlights of recent advances involve peptide-based inhibitors, including sidechain and backbone cross-linked agents, and peptide scaffolds, as well as small-molecule inhibitors of protein-protein interactions, such as those containing terephthalate or bis-imidazole scaffolds.     

Mapping protein-protein interactions with combinatorial biology methods

Extremely diverse, DNA-encoded libraries of peptides and proteins have been constructed that include a linkage between each polypeptide and the encoding DNA. Library members can be selected by virtue of a particular binding specificity, and their protein sequence can be deduced from the sequence of the cognate DNA. Such combinatorial biology methods have proven invaluable in both identifying natural protein-protein interactions and also in mapping the specificities and energetics of these interactions in fine detail.     

Solution-phase combinatorial synthesis and evaluation of piperazine-2,5-dione derivatives

An efficient one-pot synthesis of a 61-membered combinatorial chemistry library of piperazine-2,5-diones was accomplished. Results of combinatorial synthesis, purification, analysis, and biological evaluation are described.     

De novo heme proteins from designed combinatorial libraries.

We previously reported the design of a library of de novo amino acid sequences targeted to fold into four-helix bundles. The design of these sequences was based on a "binary code" strategy, in which the patterning of polar and nonpolar amino acids is specified explicitly, but the exact identities of the side chains is varied extensively (Kamtekar S, Schiffer JM, Xiong H, Babik JM, Hecht MH, 1993, Science 262:1680-1685). Because of this variability, the resulting collection of amino acid sequences may include de novo proteins capable of binding biologically important cofactors. To probe for such binding, the de novo sequences were screened for their ability to bind the heme cofactor. Among an initial collection of 30 binary code sequences, 15 are shown to bind heme and form bright red complexes. Characterization of several of these de novo heme proteins demonstrated that their absorption spectra and resonance Raman spectra resemble those of natural cytochromes. Because the design of these sequences is based on global features of polar/ nonpolar patterning, the finding that half of them bind heme highlights the power of the binary code strategy, and demonstrates that isolating de novo heme proteins does not require explicit design of the cofactor binding site. Because bound heme plays a key role in the functions of many natural proteins, these results suggest that binary code sequences may serve as initial prototypes for the development of large collections of functionally active de novo proteins.     

The Bead blot: a method for identifying ligand-protein and protein-protein interactions using combinatorial libraries of peptide ligands

Small molecules that bind proteins can be used as ligands for protein purification and for investigating protein-protein and protein-drug interactions. Unfortunately, many methods used to identify new ligands to desired proteins suffer from common shortcomings, including the requirement that the target protein be purified and/or the requirement that the ligands be selected under conditions different from those under which it will be used. We have developed a new method called the Bead blot that can (i) select ligands to unpurified proteins, including trace proteins, present in complex materials (e.g., unfractionated plasma); (ii) select ligands to multiple proteins under a variety of conditions in a single experiment; and (iii) be used with libraries of different types of ligands. In the Bead blot, a library of ligands, synthesized on chromatography resin beads, is incubated with a starting material containing a target protein for which a ligand is sought. The proteins in the material bind to their complementary ligands according to specific affinity interactions. Then the protein-loaded beads are immobilized in a porous matrix, and the proteins are directionally eluted from the beads and captured on a membrane superimposed on the beads. The location of the target protein on the membrane is determined, and because the position of the protein(s) on the membrane reflects the position of the bead(s) in the matrix, the bead that originally bound the protein is identified, with subsequent elucidation of the ligand sequence. Ligands to several targets can be identified in one experiment. Here we demonstrate the broad utility of this method by the selection of ligands that purify plasma protein complexes or that remove pathogens from whole blood with very high affinity constants. We also select ligands to a protein based on competitive elution.     

High-throughput and multiplexed protein array technology: protein-DNA and protein-protein interactions

Miniaturized protein arrays address protein interactions with various types of molecules in a high-throughput and multiplexed fashion. This review focuses on achievements in the analysis of protein-DNA and protein-protein interactions. The technological feasibility of protein arrays depends on the different factors that enable the arrayed proteins to recognize molecular partners and on the specificity of the interactions involved. Proteome-scale studies of molecular interactions require high-throughput approaches for both the production and purification of functionally active proteins. Various solutions have been proposed to avoid non-specific protein interactions on array supports and to monitor low-abundance molecules. The data accumulated indicate that this emerging technology is perfectly suited to resolve networks of protein interactions involved in complex physiological and pathological phenomena in different organisms and to develop sensitive tools for biomedical applications.     

Computationally designed peptide inhibitors of protein-protein interactions in membranes

We recently reported a computational method (CHAMP) for designing sequence-specific peptides that bind to the membrane-embedded portions of transmembrane proteins. We successfully applied this method to design membrane-spanning peptides targeting the transmembrane domains of the alpha IIb subunit of integrin alpha IIbbeta 3. Previously, we demonstrated that these CHAMP peptides bind specifically with reasonable affinity to isolated transmembrane helices of the targeted transmembrane region. These peptides also induced integrin alpha IIbbeta 3 activation due to disruption of the helix-helix interactions between the transmembrane domains of the alpha IIb and beta 3 subunits. In this paper, we show the direct interaction of the designed anti-alpha IIb CHAMP peptide with isolated full-length integrin alpha IIbbeta 3 in detergent micelles. Further, the behavior of the designed peptides in phospholipid bilayers is essentially identical to their behavior in detergent micelles. In particular, the peptides assume a membrane-spanning alpha-helical conformation that does not disrupt bilayer integrity. The activity and selectivity of the CHAMP peptides were further explored in platelets, comfirming that anti-alpha IIb activates wild-type alpha IIbbeta 3 in whole cells as a result of its disruption of the protein-protein interactions between the alpha and beta subunits in the transmembrane regions. These results demonstrate that CHAMP is a successful chemical biology approach that can provide specific tools for probing the transmembrane domains of proteins.     

A phage display system designed to detect and study protein-protein interactions.

Analysing protein-protein interactions is critical in proteomics and drug discovery. The usage of 2-Hybrid (2lambda) systems is limited to an in vivo environment. We describe a bacteriophage 2-Hybrid system for studying protein interactions in vitro. Bait and prey are displayed as fusions to the surface of phage lambda that are marked with different selectable drug-resistant markers. An interaction of phages in vitro through displayed proteins allows bacterial infection by two phages resulting in double drug-resistant bacterial colonies at very low multiplicity of infections. We demonstrate interaction of the protein sorting signal Ubiquitin with the Vps9-CUE, a Ubiquitin binding domain, and by the interaction of (Gly-Glu)(4) and (Gly-Arg)(4) peptides. Interruptions of the phage interactions by non-fused (free) bait or prey molecules show how robust and unique our approach is. We also demonstrate the use of Ubiquitin and CUE display phages to find binding partners in a lambda-display library. The unique usefulness to 2lambda is also described.     

Intracellular kinase inhibitors selected from combinatorial libraries of designed ankyrin repeat proteins

The specific intracellular inhibition of protein activity at the protein level allows the determination of protein function in the cellular context. We demonstrate here the use of designed ankyrin repeat proteins as tailor-made intracellular kinase inhibitors. The target was aminoglycoside phosphotransferase (3')-IIIa (APH), which mediates resistance to aminoglycoside antibiotics in pathogenic bacteria and shares structural homology with eukaryotic protein kinases. Combining a selection and screening approach, we isolated 198 potential APH inhibitors from highly diverse combinatorial libraries of designed ankyrin repeat proteins. A detailed analysis of several inhibitors revealed that they bind APH with high specificity and with affinities down to the subnanomolar range. In vitro, the most potent inhibitors showed complete enzyme inhibition, and in vivo, a phenotype comparable with the gene knockout was observed, fully restoring antibiotic sensitivity in resistant bacteria. These results underline the great potential of designed ankyrin repeat proteins for modulation of intracellular protein function.     

Solution-phase synthesis of a combinatorial monocyclic β-lactam library: Potential protease inhibitors

A 126-member library of monocyclic β-lactams was generated in parallel fashion by solution-phase Ugi four-component condensation reaction between β-amino acids, aldehydes, and isocyanides. The library was designed to identify potential human leukocyte elastase inhibitors. The approach is also capable of optimizing the lead compounds generated in the original library.     

Solution-phase parallel synthesis and screening of anti-tumor activities from fenbufen and ethacrynic acid libraries

The derivatives with fenbufen and ethacrynic acid core compounds was synthesized through a facial preparation of 1-amino-4-azidobutane. The subsequent coupling with 102 members of carboxylic acids afforded amide products. The in situ screening using colorimetric assay with 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide showed that fenbufen but not ethacrynic acid butyl amide members displayed the cytotoxicities to tumor cells substantially, including two human cell lines (MCF7 and A549) and two murine cell lines (C26 and TRAMP-C1). Three fenbufen analogs were found to have a good anti-tumor activity comparable to cisplatin.     

Methods of screening combinatorial libraries using immobilized or restrained receptors

The screening of combinatorial libraries for compounds with high affinity toward drug receptors is currently a major center of attention. We describe methods recently developed for library screening that involve “constrained” receptors (either immobilized onto a surface or restrained to a compartment by some physical means). These include affinity selection chromatography, ultrafiltration assays, the scintillation proximity assay, a variety of interfacial optical techniques (surface plasmon resonance and its relatives, among others), the quartz crystal microbalance, the jet ring cell, and new interferometric assays using porous silicon to immobilize the receptor. We note some trends in assay development involving assays of membrane-bound complexes, and the coupling of two analytical methods to expand the assay resolution.     

Label-free sensing and atomic force spectroscopy for the characterization of protein-DNA and protein-protein interactions: application to estrogen receptors

In this paper we describe a new surface plasmon resonance (SPR) biosensor dedicated to potential estrogenic compounds prescreening, by developing an estrogen receptor (ER) specific DNA chip. Through the covalent binding of a DNA strain wearing the estrogen response element (ERE) to an activated 6-mercapto-1-hexadecanoic acid and 11-mercapto-1-undecanol self-assembled monolayer on gold surface, the SPR biosensor allows to detect specifically, quickly, and without any labeling the binding of ER in the presence of estrogen. In parallel, we investigated the ER interaction with itself, in order to study the formation of ER dimer apparently needed to activate the gene expression through ERE interaction. For that, we engaged force spectroscopy experiments that allowed us to prove that ER needs estrogen for its dimerization. Moreover, these ER/ER intermolecular measurements enabled to propose an innovative screening tool for anti-estrogenic compounds, molecules of interest for hormono-dependent cancer therapy.