Thiol-to-amine cyclization reaction enables screening of large libraries of macrocyclic compounds and the generation of sub-kilodalton ligands.

Macrocyclic compounds are an attractive modality for drug development, but the limited availability of large, structurally diverse macrocyclic libraries hampers the discovery of leads. Here, we describe the discovery of efficient macrocyclization reactions based on thiol-to-amine ligations using bis-electrophiles, their application to synthesize and screen large libraries of macrocyclic compounds, and the identification of potent small macrocyclic ligands. The thiol-to-amine cyclization reactions showed unexpectedly high yields for a wide substrate range, which obviated product purification and enabled the generation and screening of an 8988 macrocycle library with a comparatively small effort. X-ray structure analysis of an identified thrombin inhibitor (K i = 42 ± 5 nM) revealed a snug fit with the target, validating the strategy of screening large libraries with a high skeletal diversity. The approach provides a route for screening large sub-kilodalton macrocyclic libraries and may be applied to many challenging drug targets.

Synthesis and characterization of fluorescent and photoactivatable MIP-1alpha ligands and interactions with chemokine receptors CCR1 and CCR5.

Photoaffinity and fluorescent analogues of the 70-amino acid chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) were designed, synthesized, characterized, and applied to probe MIP-1alpha interactions with the chemokine receptors CCR1 and CCR5. The photoactivatable MIP-1alpha ligand, BP-MIP-1alpha, and the fluorescent ligand, Flu-MIP-1alpha were prepared by selective chemical coupling of p-benzoylphenylthiocarbamyl or fluoresceinthiocarbamyl, respectively, at the N-terminus of MIP-1alpha. Both ligands BP-MIP-1alpha and Flu-MIP-1alpha retained high binding affinity and agonist potency at CCR1 and CCR5. Photoaffinity labeling of CCR1 and CCR5 receptors stably expressed in CHO cells resulted in specific covalent attachment of [(125)I]BP-MIP-1alpha and production of protein complexes of 54 and 48 kDa, respectively, on SDS-PAGE. This represents the first photo-cross-linking between a chemokine and its receptor. Flu-MIP-1alpha selectively labeled CCR1 or CCR5 receptors expressed in CHO cells and was used to characterize receptor binding domains. When bound to CCR1 or CCR5 receptors, the fluorescence signal of Flu-MIP-1alpha was quenched by collision with iodide indicating that the N-terminal end of MIP-1alpha is accessible to the solvent. These data strongly suggest that the N-terminal end of MIP-1alpha interacts with domains of CCR1 or CCR5 receptors located at the extracellular surface. The photoactivatable BP-MIP-1alpha described here should prove valuable for the identification of contact sites on receptors by photoaffinity labeling experiments.

Solid phase DNA amplification: characterisation of primer attachment and amplification mechanisms.

Different chemical methods used to attach oligonucleotides by their 5'-end on a glass surface were tested in the framework of solid phase PCR where surface-bound instead of freely-diffusing primers are used to amplify DNA. Each method was first evaluated for its capacity to provide a high surface coverage of oligonucleotides essentially attached via a 5'-specific linkage that satisfyingly withstands PCR conditions and leaves the 3'-ends available for DNA polymerase activity. The best results were obtained with 5'-thiol-modified oligonucleotides attached to amino-silanised glass slides using a heterobifunctional cross-linker reagent. It was then demonstrated that the primers bound to the glass surface using the optimal chemistry can be involved in attaching and amplifying DNA molecules present in the reaction mix in the absence of freely-diffusing primers. Two distinct amplification processes called interfacial and surface amplification have been observed and characterised. The newly synthesised DNA can be detected and quantified by radioactive and fluorescent hybridisation assays. These new surface amplification processes are seen as an interesting approach for attachment of DNA molecules by their 5'-end on a solid support and can be used as an alternative route for producing DNA chips for genomic studies.

Biophysical approaches to G protein-coupled receptors: structure, function and dynamics.

G protein-coupled receptors (GPCR) represent a large family of drug targets for which there is no high resolution structural information. In order to understand the mechanisms of ligand recognition and receptor activation, there is a strong need for novel biophysical methods. In this Perspective we provide an overview of recent experimental approaches used to explore the molecular architecture and dynamics of GPCR and their interactions with ligands and G proteins using biophysical, non-crystallographic, methods.

Fluorescent pseudo-peptide linear vasopressin antagonists: design, synthesis, and applications.

Fluoresceinyl and rhodamyl groups have been coupled by an amide link to side-chain amino groups at positions 1, 6, and 8 of pseudo-peptide linear vasopressin antagonists (Manning et al. Int. J. Pept. Protein Res. 1992, 40, 261-267) through different positions on the fluorophore, to give tetraethylrhodamyl-DTyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (2), 4-HOPh(CH2)2CO-DTyr(Me)-Phe-Gln-Asn-Lys(5-carboxyfl uoresceinyl)-Pro-A rg-NH2 (4), 4-HOPh(CH2)2CO-DTyr(Me)-Phe-Gln-Asn-Lys(5- or 6-carboxytetramethylrhodamyl)-Pro-Arg-NH2 (5, 6), 4-HOPh(CH2)2CO-DTyr(Me)-Phe-Gln-Asn-Arg-Pro-Lys(5- or 6- carboxyfluoresceinyl)-NH2 (8, 9), and 4-HOPh(CH2)2CO-DTyr(Me)-Phe-Gln-Asn-Arg-Pro-Lys(5- or 6- carboxytetramethylrhodamyl)-NH2 (10, 11). The closer to the C-terminus the fluorophore, the higher the affinities of the fluorescent derivatives for the human vasopressin V1a receptor transfected in CHO cells. The compound 10 has a Ki of 70 pM, as determined by competition experiments with [125I]-4-HOPhCH2CO-DTyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-NH2. It showed a good selectivity for human V1a receptor versus human OT (Ki = 1.2 nM), human vasopressin V1b (Ki approximately 27 nM), and human vasopressin V2 (Ki > 5000 nM) receptor subtypes. All fluorescent analogues were antagonists as shown by the inhibition of vasopressin induced inositol phosphate accumulation. These fluorescent ligands are efficient for labeling cells expressing the human V1a receptor subtype, as shown by flow cytofluorometric experiments or fluorescence microscopy. They are also appropriate tools for structural analysis of the vasopressin receptors by fluorescence.

Characterization of non-peptide antagonist and peptide agonist binding sites of the NK1 receptor with fluorescent ligands.

Ligand recognition of the NK1 receptor (substance P receptor) by peptide agonist and non-peptide antagonist has been investigated and compared by the use of fluorescent ligands and spectrofluorometric methods. Analogues of substance P (SP) labeled with the environment-sensitive fluorescent group 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) at either position 3, 8, or 11 or with fluorescein at the Nalpha position were synthesized and characterized. Peptides modified at the alpha-amino group or at positions 3 or 11 conserved a relatively good affinity for NK1 and agonistic properties. Modification at position 8 resulted in an 18, 000-fold decrease in affinity. A fluorescent dansyl analogue of the non-peptide antagonist CP96,345 was prepared and characterized. The quantum yield of fluorescence for dansyl-CP96,345 was much higher than for any of the dansyl-labeled peptides indicating that the micro-environment of the binding site is more hydrophobic for the non-peptide antagonist than for the peptide agonists. Comparison of collisional quenching of fluorescence by the water-soluble hydroxy-Tempo compound showed that dansyl-CP96,345 is buried and virtually inaccessible to aqueous quenchers, whereas dansyl- or fluoresceinyl-labeled peptides were exposed to the solvent. Anisotropy of all fluorescent ligands increased upon binding to NK1 indicating a restricted motional freedom. However, this increase in anisotropy was more pronounced for the dansyl attached to the non-peptide antagonist CP96,345 than for the fluorescent probes attached to different positions of SP. In conclusion, our data indicate that the environment surrounding non-peptide antagonist and peptide agonists are vastly different when bound to the NK1 receptor. These results support recent observations by mutagenesis and cross-linking work suggesting that peptide agonists have their major interaction points in the N-terminal extension and the loops forming the extracellular face of the NK1 receptor. Our data also suggest that neither the C terminus nor the N terminus of SP appears to penetrate deeply below the extracellular surface in the transmembrane domain of the receptor.

Fluorescent labeling of NK2 receptor at specific sites in vivo and fluorescence energy transfer analysis of NK2 ligand-receptor complexes.

A fluorescent unnatural amino acid was introduced biosynthetically at known sites into the G protein-coupled neurokinin (tachykinin) NK2 receptor by suppression of UAG nonsense codons with the aid of a chemically misacylated synthetic tRNA specifically designed for the incorporation of unnatural amino acids during heterologous expression in Xenopus oocytes. A systematic UAG-scanning mutagenesis in NK2 extra- or intracellular loops and proximal transmembrane domains established that readthrough at some UAG sites may represent a limitation to the range of applicability of the nonsense suppression methodology. Fluorescence-labeled NK2 mutants containing an unique fluorescent nitrobenzoxadiazoyl-diaminopropionic acid residue at known sites were shown to be functionnally active. Intermolecular distances were determined by measuring the fluorescence resonance energy transfer (FRET) between the fluorescent unnatural amino acid and a fluorescently labeled NK2 heptapeptide antagonist in a native membrane environment. These distances confirmed the seven transmembrane topology for G protein-coupled receptors and determined a structural model for NK2 ligand-receptor interactions. The peptide is inserted between the fifth and sixth transmembrane domains, thus suggesting that antagonism may be caused by preventing correct packing of the helices required for receptor function.

Probing the structure and function of the tachykinin neurokinin-2 receptor through biosynthetic incorporation of fluorescent amino acids at specific sites.

A general method for understanding the mechanisms of ligand recognition and activation of G protein-coupled receptors has been developed. A study of ligand-receptor interactions in the prototypic seven-transmembrane neurokinin-2 receptor (NK2) using this fluorescence-based approach is presented. A fluorescent unnatural amino acid was introduced at known sites into NK2 by suppression of UAG nonsense codons with the aid of a chemically misacylated synthetic tRNA specifically designed for the incorporation of unnatural amino acids during heterologous expression in Xenopus oocytes. Fluorescence-labeled NK2 mutants containing an unique 3-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-2,3-diaminopropionic acid (NBD-Dap) residue at either site 103, in the first extracellular loop, or 248, in the third cytoplasmic loop, were functionally active. The fluorescent NK2 mutants were investigated by microspectrofluorimetry in a native membrane environment. Intermolecular distances were determined by measuring the fluorescence resonance energy transfer (FRET) between the fluorescent unnatural amino acid and a fluorescently labeled NK2 heptapeptide antagonist. These distances, calculated by the theory of Förster, permit to fix the ligand in space and define the structure of the receptor in a molecular model for NK2 ligand-receptor interactions. Our data are the first report of the incorporation of a fluorescent unnatural amino acid into a membrane protein in intact cells by the method of nonsense codon suppression, as well as the first measurement of experimental distances between a G protein-coupled receptor and its ligand by FRET. The method presented here can be generally applied to the analysis of spatial relationships in integral membrane proteins such as receptors or channels.

Probing the binding domain of the NK2 receptor with fluorescent ligands: evidence that heptapeptide agonists and antagonists bind differently.

We have investigated the interaction of fluorescent peptide ligands with the G protein-coupled receptor NK2 using novel spectrofluorometric approaches. Several heptapeptide antagonists of structure PhCO-Xaa-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2 were labelled on position 1 (Xaa) with the environment-sensitive nitrobenzoxadiazole (NBD) probe, differing only in the length of the spacer between the NBD group and the peptide. Upon binding of the labelled antagonist to NK2 receptors stably expressed in Chinese hamster ovary (CHO) cells, an increase in NBD fluorescence was observed when the spacer length was less than 10 A. Collisional quenching experiments using iodide and Co2+ ions were performed to define the accessibility of the NBD group on bound ligands to the solvent. By comparing ligands with spacer arms of varying lengths, we found that the binding pocket is buried at a depth of 5-10 A. In contrast, N-terminally NBD-labelled agonists, decapeptide neurokinin A (NKA) or heptapeptide Nle10-NKA[4-10], bound to the NK2 receptor were accessible to the solvent. Binding of fluorescent ligands to the NK2 receptor was accompanied by an enhancement in the fluorescence anisotropy. The changes in fluorescence properties were used to determine the kinetic parameters of antagonist binding and dissociation. These results indicate that the binding site on the NK2 receptor for the amino-terminal end of the heptapeptide antagonists is buried in the hydrophobic pocket of the receptor protein and clearly distinct from the binding site for the amino-terminal end of agonists, which is accessible to the solvent.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and characterization of selective fluorescent ligands for the neurokinin NK2 receptor.

Several fluorescent probes for the NK2 receptor were designed, synthesized, and pharmacologically characterized. These fluorescent ligands are analogues of the selective NK2 heptapeptide antagonist N-alpha-benzoyl-Ala-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2 (1, GR94800). They were obtained by substitution of 2,n-diaminoalkyl amino acid (n = 3-6) for Ala1 and the subsequent coupling of the fluorophore NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) or fluoresceinthiocarbamyl to the N-omega amino group. The fluorescent derivatives retained high binding affinities for the NK2 receptor in transfected CHO cells. In contrast, fluorescent derivatives made by replacing the N-alpha-benzoyl group of 1 by NBD or fluorescein were considerably less active. The effect on ligand potency of varying the length of the spacer arm between the peptide moiety and the fluorescent group was also studied. The most potent fluorescent antagonists were N-alpha-benzoyl-Dab(gamma-NBD)-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2 (5B), pKi = 8.87 for NK2; N-alpha-benzoyl-Orn (delta-NBD)-Ala-D-Trp-Phe- D-Pro-Pro-Nle-NH2 (4B), pKi = 8.84; and N-alpha-benzoyl-Lys(epsilon-NBD)-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2 (3B), pKi = 8.83. These three compounds were highly selective for NK2 over NK3 and NK1 receptors. We show that these fluorescent ligands are useful tools for the detection of NK2 receptor expression by flow cytometry. Additionally, these fluorescent probes should prove valuable for fluorescence microscopy and study of ligand-receptor interaction by spectrofluorimetry.

Purification, cDNA cloning and heterologous expression of human phosphomannose isomerase.

Phosphomannose isomerase catalyses the interconversion of fructose-6-P and mannose-6-P and has a critical role in the supply of D-mannose derivatives required for many eukaryotic glycosylation reactions. Three classes of enzymes possessing phosphomannose-isomerase activity have been identified in bacteria and lower eukaryotes. We have purified human phosphomannose isomerase to homogeneity from placental tissue. Protein sequence information obtained from internal fragments of the protein was used to design degenerate oligonucleotides which were used to amplify a fragment of a human phosphomannose-isomerase cDNA. A full-length cDNA was isolated from a human testes lambda gt11 library using this fragment as a probe. The cDNA encoded a protein with significant sequence identity to fungal and some bacterial phosphomannose isomerases but was unrelated to those from other bacteria. Based on amino acid sequence identity we propose a classification system for enzymes with phosphomannose-isomerase activity. The cDNA, under the control of the GAL1 promoter, was expressed in a Saccharomyces cerevisiae strain from which the native gene encoding phosphomannose isomerase had been deleted. The human enzyme was found to be able to functionally substitute for the yeast enzyme. Phosphomannose-isomerase mRNA was found in all human tissues tested but was more highly expressed in heart, brain and skeletal muscle. The cDNA was expressed in Escherichia coli permitting the isolation of pure recombinant protein which will be used for kinetic and structural studies.

Role of cysteine62 in DNA recognition by the P50 subunit of NF-kappa B.

A powerful chemical modification procedure has been developed to define determinants of DNA recognition by the p50 subunit of NF-kappa B. Differential labelling with [14C] iodoacetate has identified a conserved cysteine residue, Cys62, that was protected from modification by the presence of an oligonucleotide containing the specific recognition site of the protein. To determine the importance of this cysteine residue, each of the conserved cysteines in p50 was changed to serine and the DNA binding properties of the mutant proteins determined. Scatchard analysis indicated that the C62S mutant bound to its DNA recognition site with a 10-fold larger dissociation constant than the wild type protein, while the other two mutants bound with an intermediate affinity. Dissociation rate constant measurements correlated well with the dissociation constants for the wild type, C119S, and C273S p50 proteins, whereas the p50 C62S-DNA complex dissociated anomalously quickly. Competition analyses with oligonucleotide variants of the DNA recognition site and nonspecific E. coli DNA revealed that the C62S p50 mutant had an altered DNA binding site specificity and was impaired in its ability to discriminate between specific and non-specific DNA. Thus the sulphydryl group of Cys62 is an important determinant of DNA recognition by the p50 subunit of NF-kappa B.

Biochemical characterization and solubilization of human NK2 receptor expressed in Chinese hamster ovary cells.

The human ileum neurokinin NK2 receptor has been stably expressed in Chinese hamster ovary (CHO) cells using the dihydrofolate reductase (DHFR) expression system. Amplified cell populations expressing approximately 7 x 10(5) NK2 receptors/cell were selected in the presence of the DHFR inhibitor methotrexate. Cross-linking of [125I]NKA to NK2 receptor transfected cells revealed a specifically labeled protein of apparent molecular weight 64 kDa by SDS-polyacrylamide gel electrophoresis. This protein was deglycosylated by the enzymes N-glycosidase F and endoglycosydase F to a protein of apparent molecular weight of 39 kDa. The NK2 receptor was solubilized in an active form from CHO cell membranes using the zwitterionic detergent CHAPS. This method represents a valuable approach for the production of significant amounts of NK2 receptor protein from mammalian cells.

Partial characterization of natural and recombinant human soluble CD23.

The purification to homogeneity of an active soluble 25 kDa fragment of CD23, produced in insect cells using the baculovirus expression system, is described. Peptide mapping and analysis by Edman degradation and mass spectrometry permitted partial characterization of the protein. A total of 165 out of 172 residues, including N-terminal and C-terminal regions, were mapped. The positions of the two disulphide bonds in the IgE-binding region were also determined: residue 110 is joined to residue 124, and residue 42 to residue 133. Natural CD23 25 kDa fragment was also analysed and found to possess the same disulphide bond arrangement. These results extend the previously noted sequence similarity with lectins to elements of secondary structure.

Purification and characterization of biologically active human recombinant 37 kDa soluble CD23 (sFc epsilon RII) expressed in insect cells.

Human recombinant soluble 37 kDa CD23 has been expressed in insect cells and secreted into the culture medium using the IL-2 leader sequence. The 37 kDa CD23 was purified 600-fold to homogeneity by monoclonal antibody affinity chromatography and gel filtration. The pure protein is monomeric, glycosylated, depleted of one N terminal amino acid and contains four disulphide bonds. It degrades into smaller fragments of 33, 29 and 25 kDa if purified in the absence of protease inhibitors. The same pattern of proteolytic fragments is observed when the pure preparation is incubated at room temperature for 3 weeks. Physical characterization of the 37 kDa CD23 by circular dichroism indicates that the protein contains mainly beta sheet and 20% of alpha helical structures. Specific binding of IgE to natural CD23 (low affinity IgE receptor) was inhibited by purified recombinant 37 kDa CD23. Moreover, purified recombinant 37kDa CD23 and interleukin-1 promoted the survival of germinal centre B cells.

Purification and partial characterization of rat factor D.

Rat factor D has been purified to homogeneity (10,559-fold) from serum by chromatography on CM-Sepharose Fast Flow, phenyl-Sepharose CL-4B and Mono S and has been shown to resemble its human and mouse counterparts both in substrate specificity and in its susceptibility to inhibition by the organophosphorous inhibitor di-isopropylfluorophosphate. The rat enzyme, however, is heavily glycosylated and binds to wheat-germ lectin-Sepharose 6MB and 5-hydroxytryptamine-agarose, but not to concanavalin A-Sepharose 4B. All of the carbohydrate chains are N-linked. Enzymic removal of this carbohydrate decreased the Mr by approx. 15,000. The deglycosylated rat enzyme had the same mobility as native human factor D on SDS/PAGE, corresponding to an Mr of 24,500. N-Terminal sequence analysis of the first 30 amino acids of rat factor D highlighted the sequence similarity with human factor D (greater than 76%) and, in particular, with mouse adipsin (greater than 93%).

Human interleukin-5 expressed in Escherichia coli: assignment of the disulfide bridges of the purified unglycosylated protein.

Human interleukin-5 is a homodimer; each subunit contains two cysteine residues that form two inter-subunit disulfide bonds. The topology of the disulfides in recombinant human interleukin-5 produced in Escherichia coli was studied by proteolytic digestion and peptide mapping. Disulfide linked peptides containing cysteine 42 linked to cysteine 84 were isolated. This indicated that cysteines 42 and 84 of one subunit were linked in an antiparallel manner to cysteines 84 and 42 of the other subunit.

Structure and partial amino acid sequence of calf thymus DNA topoisomerase II: comparison with other type II enzymes.

The partial amino acid sequence of p140 calf thymus DNA topoisomerase II was determined by analysis of cyanogen bromide peptides. Five peptides were aligned and shared extensive homology with sequences derived from cDNA clones for the human topoisomerase II isoenzyme forms. Less homology was seen with the Drosophila, yeast and bacterial type II enzymes. Calf and human enzymes shared epitopes allowing isolation of a cDNA clone to human topoisomerase II isoenzyme alpha. Our results indicate that calf thymus p140 topoisomerase II is an active N-terminal proteolytic fragment of the native p180 enzyme and demonstrate that mammalian type II enzymes exhibit close sequence similarity.

Tumor necrosis factor inhibitor: purification, NH2-terminal amino acid sequence and evidence for anti-inflammatory and immunomodulatory activities.

The urine of some febrile patients has been shown to contain a tumor necrosis factor-alpha-inhibiting activity (TNF-alpha INH) when tested in a cytotoxicity assay using the TNF-susceptible cell line L-929. The inhibitor was purified to homogeneity using a simple three-step procedure which included a TNF-alpha affinity column, cation exchange and reverse-phase chromatography. The NH2-terminal amino acid sequence of the inhibitor showed no sequence similarity with proteins in the data bases used. Using gel filtration, it was shown that TNF-alpha and the inhibitor form a stable complex which eluted with a molecular weight of about 75,000. This value corresponds to the sum of the inhibitor (approximately 30,000) and TNF-alpha (approximately 45,000-50,000) molecular weight. The TNF-alpha INH blocked prostaglandin E2 production by dermal fibroblasts in a dose-dependent manner, providing evidence for antiinflammatory activity. TNF-alpha INH also blocked class I antigen expression in a dose-dependent manner as measured using the human Colo 205 tumor cell line. Furthermore, TNF-alpha INH affected TNF-alpha synergism with IFN-gamma-induced HLA-DR antigen expression but had no effect on IFN-gamma activity. The data presented demonstrate that TNF-alpha bioactivity can be regulated at the protein level.

Purification and characterization of a methionine-specific aminopeptidase from Salmonella typhimurium.

An aminopeptidase specific for methionine (peptidase M) has been purified from wild-type and mutant Salmonella typhimurium strains. Recombinant peptidase M was also purified from Escherichia coli. These preparations were characterized with respect to their physicochemical properties using analytical ultracentrifugation, SDS/PAGE, isoelectric focusing, titration curve analysis, amino acid analysis, N-and C-terminal sequencing and various spectroscopic methods. Peptidase M activity is stimulated by Co2+, in agreement with previous studies using crude extracts of Salmonella. The purified preparations did not contain significant amounts of any metal. Enzymically important metal is loosely associated and lost during enzyme purification. Peptidase M was shown to contain seven free sulphydryl residues none of which are involved in either intra-or inter-molecular disulphide bonds. Most appear solvent-accessible as evidenced by their reactivity under native conditions. Limited modification of the sulphydryl residues with either iodoacetamide or 5,5'-dithiobis(2-nitrobenzoic acid) led to inactivation. Several cysteines were shown to be labelled to various degrees by peptide mapping of inactivated S-[14C]carboxymethylated protein. Whether cysteine modification affects enzymic activity directly (blocking an active site) or indirectly (by causing conformational change) remains to be established.