The cloning of human genes using cDNA phage display and small-molecule chemical probes

The cloning of genes based on protein function has become a powerful tool for protein discovery and should play an important role in proteomics in general. We have recently reported a technique for the functional identification of protein targets by combining traditional affinity chromatography with cDNA phage display. This procedure, referred to as display cloning, directly couples biologically active natural products to the gene of their protein cellular target. We now report the cloning of a human gene, the domain of F1 ATP synthase, using a synthetic scaffold molecule which serves as a prototype for a diverse chemical library. The ability to select genes from cDNA libraries using probes from combinatorial libraries would greatly increase the number of small molecule/protein interactions that can be identified. This method might prove valuable in furthering our understanding of biology and its application toward drug development.     

The powerful combination of phage surface display of cDNA libraries and high throughput screening

Phage surface display of cDNA libraries facilitates cloning, expression and rapid selection of functional gene products physically linked to their genetic information through gene product-ligand interactions. Efficient screening technologies based on selective enrichment of clones expressing desired gene products allows, within a short time, the isolation of all ligand-specific clones that are present in a library. Manual identification of clones by restriction analysis and random sequencing is unlike to be successful for the isolation of gene products derived from rare mRNA species resulting from selection of the libraries using polyvalent ligands like serum from patients. Here we describe rapid handling of large numbers of individual clones selected from molecular libraries displayed on phage surface using the power of robotics-based high throughput screening. The potential of the combination of cDNA-phage surface display, with selection for specific interactions by functional screening and robotic technology is illustrated by the isolation of more sequences potentially encoding IgE-binding proteins than postulated from Western blot analyses using extracts derived from raw material of complex allergenic sources. The subsequent application of functional enrichment and robotics-based screening will facilitate the rapid generation of information about the repertoire of protein structures involved in allergic diseases.     

Evidence for the Preservation of Native Inter- and Intra-Molecular Hydrogen Bonds in the Desolvated FK-Binding Protein·FK506 Complex Produced by Electrospray Ionization

It is now well established that electrospray ionization (ESI) is capable of introducing noncovalent protein assemblies into a desolvated environment, thereby allowing their analysis by mass spectrometry. The degree to which native interactions from the solution phase are preserved in this environment is less clear. Site-directed mutagenesis of FK506-binding protein (FKBP) has been employed to probe specific intra- and inter-molecular interactions within the complex between FKBP and its ligand FK506. Collisional activation of wild-type and mutant-FKBP?FK506 ions, generated by ESI, demonstrated that removal of native protein-ligand interactions formed between residues Asp37, Tyr82, and FK506 significantly destabilized the complex. Mutation of Arg42 to Ala42, or Tyr26 to Phe26 also resulted in lower energy dissociation of the FKBP·FK506 complex. Although these residues do not form direct H-bonds to FK506, they interact with Asp37, ensuring its correct orientation to associate with the ligand. Comparison with solution-based affinity measurements of these mutants has been discussed, including the stabilization afforded by ordered water molecules. Ion mobility spectrometry (IMS) has been employed to provide gas-phase structural information on the unfolding of the complexes. The [M + 6H](6+) complexes of the wild-type and mutants have been shown to resist unfolding and retain compact conformations. However, removal of the basic Arg42 residue was found to induce significant structural weakening of the [M + 7H](7+) complex when raised to dissociation-level energies. Overall, destabilization of the FKBP·FK506 complex, resulting from targeted removal of specific H-bonds, provides evidence for the preservation of these interactions in the desolvated wild-type complex.     

Frameshifting in the p6 cDNA phage display system

Phage display is a powerful technique that enables easy identification of targets for any type of ligand. Targets are displayed at the phage surface as a fusion protein to one of the phage coat proteins. By means of a repeated process of affinity selection on a ligand, specific enrichment of displayed targets will occur. In our studies using C-terminal display of cDNA fragments to phage coat protein p6, we noticed the occasional enrichment of targets that do not contain an open reading frame. This event has previously been described in other phage display studies using N-terminal display of targets to phage coat proteins and was due to uncommon translational events like frameshifting. The aim of this study was to examine if C-terminal display of targets to p6 is also subjected to frameshifting. To this end, an enriched target not containing an open reading frame was selected and an E-tag was coupled at the C-terminus in order to measure target display at the surface of the phage. The tagged construct was subsequently expressed in 3 different reading frames and display of both target and E-tag measured to detect the occurrence of frameshifting. As a result, we were able to demonstrate display of the target both in the 0 and in the +1 reading frame indicating that frameshifting can also take place when C-terminal fusion to minor coat protein p6 is applied.     

Simultaneous arming and structure/activity studies of natural products employing O-H insertions: an expedient and versatile strategy for natural products-based chemical genetics

The identification of "druggable" targets is an immediate opportunity and challenge in the post-genomic era. Natural products are enduring tools for basic cellular studies and leads for identifying medically relevant protein targets. However, their use for these studies is often hampered by limited quantities and a lack of selective and mild monofunctionalization reactions. The development of selective methods that could simultaneously equip the natural product with a reactive group for subsequent conjugation to reporter tags and provide important structure-activity relationship (SAR) information, requiring only a knowledge of functional groups present in the natural product, could significantly decrease the time between bioactive natural product isolation and target identification. Herein, we report such a strategy that enables simultaneous arming and SAR studies of alcohol-containing natural products involving both chemo- and site-selective ("chemosite" selective) and site-nonselective O-H insertion reactions with rhodium carbenoids derived from alkynyl diazo acetates. This strategy was applied to a diverse set of natural products, and general guidelines for predicting chemosite selectivity were formulated. A subsequent Sharpless-Hüisgen [3 + 2] cycloaddition reaction with the appended alkyne allows for attachment of a variety of reporter tags. Using this strategy, we synthesized a novel FK506-biotin conjugate that enabled pull-down of the entire "immunosuppressive complex" including FKBP12, calcineurins A and B, and calmodulin. In addition, the potential for a chemoselective but site-nonselective process was shown with both gibberellic acid methyl ester and brefeldin A using only achiral rhodium catalysts.     

Identification of calpain substrates by ORF phage display

Substrate identification is the key to defining molecular pathways or cellular processes regulated by proteases. Although phage display with random peptide libraries has been used to analyze substrate specificity of proteases, it is difficult to deduce endogenous substrates from mapped peptide motifs. Phage display with conventional cDNA libraries identifies high percentage of non-open reading frame (non-ORF) clones, which encode short unnatural peptides, owing to uncontrollable reading frames of cellular proteins. We recently developed ORF phage display to identify endogenous proteins with specific binding or functional activity with minimal reading frame problem. Here we used calpain 2 as a protease to demonstrate that ORF phage display is capable of identifying endogenous substrates and showed its advantage to re-verify and characterize the identified substrates without requiring pure substrate proteins. An ORF phage display cDNA library with C-terminal biotin was bound to immobilized streptavidin and released by cleavage with calpain 2. After three rounds of phage selection, eleven substrates were identified, including calpastatin of endogenous calpain inhibitor. These results suggest that ORF phage display is a valuable technology to identify endogenous substrates for proteases.     

Involvement of some large immunophilins and their ligands in the protection and regeneration of neurons: a hypothetical mode of action

The powerful immunosuppressive drugs such as FK506 and its derivatives induce some regeneration and protection of neurons from ischaemic brain injury and some other neurological disorders. The drugs form complexes with diverse FKBPs but apparently the FKBP52/FK506 complex was shown to be involved in the protection and regeneration of neurons. We used several different sequence attributes in searching diverse genomic databases for similar motifs as those present in the FKBPs. A Fortran library of algorithms (Par_Seq) has been designed and used in searching for the similarity of sequence motifs extracted from the multiple sequence alignments of diverse groups of proteins (query motifs) and the target motifs which are encoded in various genomes. The following sequence attributes were used in the establishment of the degree of convergence between: (A) amino acid (AA) sequence similarity (ID) of the query/target motifs and (B) their: (1) AA composition (AAC); (2) hydrophobicity (HI); (3) Jensen-Shannon entropy; and (4) AA propensity to form a particular secondary structure. The sequence hallmark of two different groups of peptidylprolyl cis/trans isomerases (PPIases), namely tetratricopetide repeat (TPR) motifs, which are present in the heat-shock cyclophilins and in the large FK506-binding proteins (FKBPs) were used to search various genomic databases. The Par_Seq algorithm has revealed that the TPR motifs have similar sequence attributes as a number of hydrophobic sequence segments of functionally unrelated membrane proteins, including some of the TMs from diverse G protein-coupled receptors (GPCRs). It is proposed that binding of the FKBP52/FK506 complex to the membranes via the TPR motifs and its interaction with some membrane proteins could be in part responsible for some neuro-regeneration and neuro-protection of the brain during some ischaemia-induced stresses.     

High-throughput screening of surface displayed gene products

With the human genome project approaching completion, there is a growing interest in functional analysis of gene products. The characterization of large numbers of proteins, their expression patterns and in vivo localisations, demands the use of automated technology that maintains a logistic link to the encoding genes. As a complementary approach, phage display is used for recombinant protein expression and the selection of interacting (binding) molecules. Cloning of libraries in filamentous bacteriophage or phage mid vectors provides a physical link between the expressed protein and its encoding DNA sequence. High-throughput technology for automated library handling and phage display selection has been developed using picking-spotting robots and a module for pin-based magnetic particle handling. This system enables simultaneous interaction screening of libraries and the selection of binders to different target molecules at high throughput. Target molecules are either displayed on high-density filter membranes (protein filters) or tag-bound to magnetic particles and can be handled as native ligands. Binding activity is confirmed by magnetic particle ELISA in the microtitre format. The whole procedure from immobilisation of target molecules to confirmed clones of binders is automatable. Using this technology, we have selected human scFv antibody fragments against expression products of human cDNA libraries.     

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.     

Synthesis of calcineurin-resistant derivatives of FK506 and selection of compensatory receptors

We used olefin metathesis to synthesize C40 derivatives of FK506 and measured their ability, when complexed to FKBP12, to inhibit calcineurin's phosphatase activity. We identified modular dimerization domains (CABs) containing segments of the calcineurin A and B polypeptides. These CABs respond to FK506 both when overexpressed in mammalian cells and in yeast or mammalian three-hybrid assays. Using chemical genetic selection, we identified compensatory mutant CABs that respond to a calcineurin-resistant FK506 derivative at concentrations well below the response threshold for CABs containing only wild-type calcineurin sequence. These reagents provide a small molecule-protein combination orthogonal to existing dimerizer systems and may be used with existing systems to increase the complexity of induced-proximity experiments. This new use of the "bump-hole" strategy protects target cells from complications arising from the inhibition of endogenous calcineurin.     

A Bioorthogonal Small‐Molecule‐Switch System for Controlling Protein Function in Live Cells

Chemically induced dimerization (CID) has proven to be a powerful tool for modulating protein interactions. However, the traditional dimerizer rapamycin has limitations in certain in vivo applications because of its slow reversibility and its affinity for endogenous proteins. Described herein is a bioorthogonal system for rapidly reversible CID. A novel dimerizer with synthetic ligand of FKBP′ (SLF′) linked to trimethoprim (TMP). The SLF′ moiety binds to the F36V mutant of FK506‐binding protein (FKBP) and the TMP moiety binds to E. coli dihydrofolate reductase (eDHFR). SLF′‐TMP‐induced heterodimerization of FKBP(F36V) and eDHFR with a dissociation constant of 0.12 μM . Addition of TMP alone was sufficient to rapidly disrupt this heterodimerization. Two examples are presented to demonstrate that this system is an invaluable tool, which can be widely used to rapidly and reversibly control protein function in vivo.     

Detection of noncovalent FKBP-FK506 and FKBP-Rapamycin complexes by capillary electrophoresis-mass spectrometry and capillary electrophoresis-tandem mass spectrometry

The well known biospecific noncovalent receptor-ligand association complexes between the immunophilin FKBP and the immunosuppressive drugs FK506 and Rapamycin (RM) were investigated by on-line capillary electrophoresis-mass spectrometry (CE-MS) under selected ion monitoring (SIM) conditions and by CE-MS with tandem mass spectrometry (CE-MS/MS) under selected reaction monitoring (SRM) conditions. Solutions of hFKBP (33.3 µM) were dissolved in 50 mM ammonium acetate at pH 7.5. Samples that contained 100 µM of FK506 or RM also were prepared under the same solution conditions. By using these aqueous pH neutral conditions, samples were analyzed by SIM CE-MS and SRM CE-MS and the target complexes were separated by CE with mass spectrometer detection of the individual complexes between FKBP and FK506 [hFKBP + FK506 + 7HJ⁷⁺ as well as FKBP and RM [hFKBP + RM + 7HJ⁷⁺. In an experiment where a mixture of FK506 and RM was analyzed in the presence of FKBP, a nine-to-one ratio of ion current abundances between the RM and FK506 complexes was observed as reported in the literature from other studies. These results suggest that CE-MS and CE-MS/MS may be yet another analytical method for studying noncovalent interactions of biologically important macromolecules under physiological conditions.     

Design and screening of M13 phage display cDNA libraries

The last decade has seen a steady increase in screening of cDNA expression product libraries displayed on the surface of filamentous bacteriophage. At the same time, the range of applications extended from the identification of novel allergens over disease markers to protein-protein interaction studies. However, the generation and selection of cDNA phage display libraries is subjected to intrinsic biological limitations due to their complex nature and heterogeneity, as well as technical difficulties regarding protein presentation on the phage surface. Here, we review the latest developments in this field, discuss a number of strategies and improvements anticipated to overcome these challenges making cDNA and open reading frame (ORF) libraries more readily accessible for phage display. Furthermore, future trends combining phage display with next generation sequencing (NGS) will be presented.     

Functional drift of sequence attributes in the FK506-binding proteins (FKBPs)

Diverse members of the FK506-binding proteins (FKBPs) group and their complexes with different macrocyclic ligands of fungal origins such as FK506, rapamycin, ascomycin, and their immunosuppressive and nonimmunosuppressive derivatives display a variety of cellular and biological activities. The functional relatedness of the FKBPs was estimated from the following attributes of their aligned sequences: 1 degrees conservation of the consensus sequence; 2 degrees sequence similarity; 3 degrees pI; 4 degrees hydrophobicity; 5 degrees amino acid hydrophobicity and bulkiness profiles. Analyses of the multiple sequence alignments and intramolecular interaction networks calculated from a series of structures of the FKBPs revealed some variations in the interaction clusters formed by the AA residues that are crucial for sustaining peptidylprolyl cis/trans isomerases (PPIases) activity and binding capacity of the FKBPs. Fine diversification of the sequences of the multiple paralogues and orthologues of the FKBPs encoded in different genomes alter the intramolecular interaction patterns of their structures and allowed them to gain some selectivity in binding to diverse targets (functional drift).     

Nocardiopsins: New FKBP12‐Binding Macrolide Polyketides from an Australian Marine‐Derived Actinomycete,Nocardiopsis sp.

A marine‐derived actinomycete, Nocardiopsis sp. (CMB‐M0232), obtained from a sediment sample collected at a depth of 55 m off the coast of Brisbane, Australia, yielded two new macrolide polyketides. Structures for nocardiopsins A and B were assigned by detailed spectroscopic analysis, degradation and chemical derivatization. A Marfey’s analysis revealed an unexpected acid‐mediated partial racemization of the L ‐pipecolic acid incorporated within the nocardiopsins. The scope of this racemization was assessed against a selection of natural and synthetic N‐acyl pipecolic acids. While the nocardiopsins are not antibacterial, antifungal or cytotoxic, they do exhibit low‐micromolar binding to the immunophilin FKBP12, consistent with their structural and biosynthetic relationship to the immunosuppressive agents FK506 and rapamycin. The nocardiopsins represent a new point of entry into what has been a valuable, exclusive and reclusive region of bioactive chemical space—that surrounding the FK506/rapamycin pharmacophore.     

Genome-wide high-throughput mining of natural-product biosynthetic gene clusters by phage display

We have developed a phage-display method for high-throughput mining of bacterial gene clusters encoding the natural-product biosynthetic enzymes, polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). This method uses the phosphopantetheinyl transferase activity of Sfp to specifically biotinylate NRPS and PKS carrier-protein domains expressed from a library of random genome fragments fused to a gene encoding a phage coat protein. Subsequently, the biotinylated phages are enriched through selection on streptavidin-coated plates. Using this method, we isolated phage clones from the multiple NRPS and PKS gene clusters encoded in the genomes of Bacillus subtilis and Myxococcus xanthus. Due to the rapid and unambiguous identification of carrier domains, this method will provide an efficient tool for high-throughput cloning of NRPS and PKS gene clusters from many individual bacterial genomes and multigenome environmental DNA.     

Shotgun phage display cloning

Shotgun phage display cloning is a useful tool for studying interactions between bacterial and host proteins. Libraries are constructed by cloning randomly fragmented prokaryotic DNA into phage mid-vectors. Theoretically, these libraries will consist of phages that together display all proteins encoded by the bacterial genome. Selecting a gene III-based library, made from Staphylococcus aureus DNA, against IgG and fibronectin resulted in 20-40% positive clones after two pannings. Increasing the number of fusion proteins per phage particle by using gene VIII-based display, increased the frequency of correct clones to 75-100%.     

Functional genomics in cancer research: identification of target genes of the Epstein-Barr virus nuclear antigen 2 by subtractive cDNA cloning and high-throughput differential screening using high-density agarose gels

In the past, the identification and isolation of phenotype-associated genes was a difficult and time-consuming task. However, recent improvements of methods that are designed to isolate differentially expressed genes have remarkably speeded up the process of target gene isolation. The ultimate goal of functional genomics is to apply these technologies to clone phenotype-associated genes irrespective of the availability of probes (e.g., antibodies) and an intimate knowledge of biological background. We demonstrate the use of a novel subtractive cDNA cloning approach for the isolation and characterization of target genes of the Epstein-Barr virus nuclear antigen 2 (EBNA2). Two different subtractive cDNA libraries specific for two different time periods following activation of a conditional estrogen receptor/EBNA2 (ER/EBNA2) fusion protein were generated. Comparison of the two libraries by cross-hybridization experiments allowed the differentiation between direct and indirect target genes of EBNA2 and led to the identification of a novel direct target gene of EBNA2.     

Picomolar FKBP inhibitors enabled by a single water-displacing methyl group in bicyclic [4.3.1] aza-amides

Methyl groups can have profound effects in drug discovery but the underlying mechanisms are diverse and incompletely understood. Here we report the stereospecific effect of a single, solvent-exposed methyl group in bicyclic [4.3.1] aza-amides, robustly leading to a 2 to 10-fold increase in binding affinity for FK506-binding proteins (FKBPs). This resulted in the most potent and efficient FKBP ligands known to date. By a combination of co-crystal structures, isothermal titration calorimetry (ITC), density-functional theory (DFT), and 3D reference interaction site model (3D-RISM) calculations we elucidated the origin of the observed affinity boost, which was purely entropically driven and relied on the displacement of a water molecule at the protein–ligand–bulk solvent interface. The best compounds potently occupied FKBPs in cells and enhanced bone morphogenic protein (BMP) signaling. Our results show how subtle manipulation of the solvent network can be used to design atom-efficient ligands for difficult, solvent-exposed binding pockets.

Enhancement by displacement. A single methyl group displaces a water molecule from the binding site of FKBPs, resulting in the most potent binders known, outperforming the natural products FK506 and rapamycin in biochemical and cellular assays.