Biomedical subjects

A Pessi

Publications and source records attributed to A Pessi.

At least 19 recordsLinked to original sources

PubMed1996

Selection of potent inhibitors of farnesyl-protein transferase from a synthetic tetrapeptide combinatorial library.

Inhibitors of farnesyl-protein transferase (FPTase) show promise as anticancer agents. Based on the sequence of the protein substrates of FPTase (the CAAX sequence), potent and selective peptidomimetic inhibitors have been developed; these compounds share with the peptide substrate a free thiol and a C-terminal carboxylate. We have used a synthetic tetrapeptide combinatorial library to screen for new leads devoid of these features: the peptides were C-terminally amidated, and no free thiol was included in the combinatorial building blocks. To compensate for this negative bias, an expanded set of 68 amino acids was used, including both L and D as well as many non-coded residues. Sixteen individual tetrapeptides derived from the consensus were synthesized and tested; all were active, showing IC50 values ranging from low micromolar to low nanomolar. The most active peptide, D-tryptophan-D-methionine-D-4-chlorophenylalanine-L-gamma- carboxyglutamic acid (Ki = 2 nM), is also very selective showing little inhibitory activity against the related enzyme geranylgeranyl-protein transferase type I (IC50 > 50 microM). In contrast to CAAX-based peptidomimetics, D-tryptophan-D-methionine-D-4-chlorophenylalanine-L-gamma-carboxyglut amic acid appeared to mimic the isoprenoid substrate farnesyl diphosphate as determined by kinetic and physical measurements. D-Tryptophan-Dmethionine-D-4-chlorophenylalanine-L-gamma- carboxyglutamic acid was a competitive inhibitor of FPTase with respect to farnesyl diphosphate substrate and uncompetitive with respect to CAAX substrate. Furthermore, we demonstrated that FPTase undergoes ligand dependent conformational changes in its circular dichroism spectrum and that D-tryptophan-D-methionine-D-4-chlorophenylalanine-L-gamma- carboxyglutamic acid induced a conformational change identical to that observed with farnesyl diphosphate ligand.

Alkyl and Aryl Transferases↗

PubMed1996

Use of a conformationally restricted secondary structural element to display peptide libraries: a two-stranded alpha-helical coiled-coil stabilized by lactam bridges.

A model for an alpha-helical peptide library based on a lactam bridged stabilized two-stranded alpha-helical coiled-coil is described. Sites for library display were incorporated in the middle of the peptide sequence of the most solvent accessible sites of a coiled-coil. A comparison was made between this coiled coil and a native coiled-coil based on the same sequence but lacking the lactam bridges. A lactam bridged peptide where the hydrophobic repeat consisted of all alanine residues, such that the tendency to dimerize would be diminished, was also prepared. This enabled us to determine the role tertiary interactions play in maintaining library positions in a helical conformation. The consensus sequence derived from a Zn finger library screened against an IgA reactive against the lipopolysaccharide of Shigella flexneri was transplanted into the peptides. CD spectroscopy revealed that although both coiled-coils are highly helical at 100 microM, the lactam bridge enhanced dimerization and allow the peptide to maintain its coiled-coil conformation at lower peptide concentrations. The helical content of the alanine based peptide was 69% and was independent of concentration over a range of 1.4 to 1410 microM. Urea denaturation studies indicated that the coiled-coils were considerably more stable than the alanine based peptide and that the lactam bridge coiled-coil was more stable than the native coiled coil by 1.6 kcal/mol. The lactam bridged coiled-coil was found to inhibit binding of the Zn finger peptide to the IgA in a concentration dependent manner with an IC50 of 5.0 microM whereas the peptide lacking the lactam bridges was much less effective in inhibiting binding. The alanine peptide was less active than the lactam bridged coiled-coil but more effective than the native coiled-coil with an IC50 of 16 microM. The versatility of the lactam stabilized coiled-coil template was demonstrated by incorporating five Gly residues into the library display sites. While the native coiled-coil adopted a random conformation the lactam bridged coiled-coil was 59% helical. Incorporation of a Cys-Gly-Gly linker to the N terminus and formation of a disulfide bond stabilized the peptide to the extent that it adopted a highly helical coiled-coil (94%) with a [urea]1/2 value of 5.9 M. Since the five glycine residues represent one of the most destabilizing combinations of amino acids that would be encountered at the five library display sites (with the exception of Pro), this stabilized coiled-coil should maintain its folded conformation regardless of the amino acids occupying the library positions.

Amino Acid Sequence↗

PubMed1996

A continuous assay of hepatitis C virus protease based on resonance energy transfer depsipeptide substrates.

Hepatitis C virus (HCV) is the major causative agent of non-A non-B hepatitis, an important health problem with an estimated 50 million people infected worldwide. Among the possible targets for therapeutic intervention, the serine protease contained within the N-terminal region of nonstructural protein 3 (NS3 protease) is so far the best characterized. In vitro characterization of synthetic substrates based on all the natural cleavage sites (as well as a series of analogs) has consistently revealed poor kinetic parameters, making them unsuitable for sensitive high-throughput screening. To overcome these difficulties, we have recently developed depsipeptide substrates incorporating an ester bond between residues P1 and P&prime1. Due to ready transesterification of the scissile bond to the acyl-enzyme intermediate, these substrates showed very high kcat/Km values, enabling detection of activity with subnanomolar NS3 concentrations. We have used the same principle to synthesize internally quenched depsipeptide fluorogenic substrates based on resonance energy transfer between the donor/acceptor couple 5-[(2'-aminoethyl)amino]naphthalene sulfonic acid/4-[[4'-(dimethylamino)phenyl]azo]benzoic acid, and developed a continuous assay for NS3 activity. Substrate cleavage is linear with enzyme concentration: depending on the conditions chosen, we estimated a detection limit for NS3 between 1 nM and 250 pM. The suitability of the assay for evaluation of inhibitors was established using as competitor a tridecapeptide corresponding to the natural NS4A/4B cleavage site; this gave an IC50 of 30 microM, well in agreement with the previously found Km value (40 microM).

Chromatography, High Pressure Liquid↗

PubMed1996

Synthetic depsipeptide substrates for the assay of human hepatitis C virus protease.

Hepatitis C virus (HCV) is the major etiological agent of both parenterally transmitted and sporadic non-A, non-B hepatitis. The disease is a major health problem with an estimated 50 million people infected worldwide, a high percentage of whom become chronically infected and are at high risk for liver cirrhosis. The serine protease contained within the N-terminal region of the nonstructural protein 3 (NS3 protease) of HCV is considered a promising target for the development of an antiviral therapy. A prime requisite to study in detail the biochemistry of the protease as well as develop inhibitors is the availability of a fast and sensitive in vitro assay of enzyme activity. However, due to their low kcat/Km values, synthetic peptide substrates based on the natural cleavage sites appear unsuitable for this purpose. We show here that appropriate substrates can be obtained by substituting the scissile amide bond with an ester linkage. The resulting depsipeptides show >100-fold improvement in kcat/Km values, up to 13,000 M-1 s-1, enabling detection of activity with subnanomolar NS3 concentrations. The ester substrates are obtained in high yield entirely by solid-phase synthesis using commercially available materials, without the need for any preassembled building blocks.(c) 1996 Academic Press, Inc.

Amino Acid Sequence↗

PubMed1996

Probing the tertiary structure of proteins by limited proteolysis and mass spectrometry: the case of Minibody.

A strategy that combines limited proteolysis experiments and mass spectrometric analysis of the fragments generated has been developed to probe protease-accessible sites on the protein surface. This integrated approach has been employed to investigate the tertiary structure of the Minibody, a de novo designed 64-residue protein consisting of a beta-sheet scaffold based on the heavy-chain variable-domain structure of a mouse immunoglobulin and containing two segments corresponding to the hypervariable H1 and H2 regions. The low solubility of the protein prevented a detailed characterization by NMR and/or X-ray. Different proteases were used under strictly controlled conditions and the cleavage sites were mapped onto the anticipated Minibody model, leading to the identification of the most exposed regions. A single-residue mutant was constructed and characterized, following the same procedure, showing a slightly higher correspondence with the predicted model. This strategy can be used to effectively supplement NMR and X-ray investigations of protein tertiary structure, where these procedures cannot provide definitive data, or to verify and refine protein models.

Amino Acid Sequence↗

PubMed1996

High-resolution solution structure of two members of a conformationally homogeneous combinatorial peptide library based on the classical zinc-finger motif.

We describe the high-resolution structure by NMR of two peptides that belong to a combinatorial library based on the zinc-finger motif. The library represents, to the best of our knowledge, the first example of a conformationally homogeneous peptide library and was obtained by introducing random residues in five positions of the alpha-helical portion of a 26-residue 'consensus' peptide (CP1) belonging to the Cys(2)-Hys2 zinc-finger family. The result was shown to be a highly homogeneous alpha-helical library (Bianchi et al., 1995). The structures of the parent compound (CPI) and of a representative member (CPlm) that was selected by screening the library with a monoclonal antibody are compared in detail as an example of the very high stability of the zinc-finger scaffold upon sequence variability. The two peptides exhibit an extremely high degree of structural similarity. The use of this type of conformationally constrained combinatorial library might represent a step forward in the design of peptidomimetics, as it considerably accelerates the process of the identification of the spatial relationship among the pharmacophoric groups.

Magnetic Resonance Spectroscopy↗

PubMed1996

Modulated 5-fluorouracil (5-FU) regimens in advanced colorectal cancer: a critical review of comparative studies.

Several modifications to the administration schedule of 5-fluorouracil (5-FU) alone or in combination with other agents have been investigated in advanced colorectal cancer. Biochemical modulation of 5-FU with leucovorin (LV) increases response rate compared with 5-FU alone, but without improvement of overall survival. The best treatment schedule and optimal dose of LV remain unclear, although low doses seem equally as effective as high doses, with the advantage of reduced cost. Methotrexate can increase the activity of 5-FU to a similar degree as LV and a recent meta-analysis showed a slight improvement in survival. The combination of 5-FU + interferon has been disappointing, with phase III trials showing similar activity to 5-FU + LV, but with high toxicity. Other modulators (e.g. hydroxyurea, N-phosphonacetyl-L-aspartate, dipyridamole) show promising but sometimes conflicting results. Standardisation of assessment criteria should be considered when comparing these data to the activity of new drugs such as 'Tomudex' (raltitrexed, previously known as ZD1694), CPT-11 and oxaliplatin.

Antimetabolites, Antineoplastic↗

PubMed1996

Activity of purified hepatitis C virus protease NS3 on peptide substrates.

The protease domain of the hepatitis C virus (HCV) protein NS3 was expressed in Escherichia coli, purified to homogeneity, and shown to be active on peptides derived from the sequence of the NS4A-NS4B junction. Experiments were carried out to optimize protease activity. Buffer requirements included the presence of detergent, glycerol, and dithiothreitol, pH between 7.5 and 8.5, and low ionic strength. C- and N-terminal deletion experiments defined a peptide spanning from the P6 to the P4' residue as a suitable substrate. Cleavage kinetics were subsequently measured by using decamer P6-P4' peptides corresponding to all intermolecular cleavage sites of the HCV polyprotein. The following order of cleavage efficiency, in terms of kcat/Km, was determined: NS5A-NS5B > NS4A-NS4B >> NS4B-NS5A. A 14-mer peptide containing residues 21 to 34 of the protease cofactor NS4A (Pep4A 21-34), when added in stoichiometric amounts, was shown to increase cleavage rates of all peptides, the largest effect (100-fold) being observed on the hydrolysis of the NS4B-NS5A decamer. From the kinetic analysis of cleavage data, we conclude that (i) primary structure is an important determinant of the efficiency with which each site is cleaved during polyprotein processing, (ii) slow cleavage of the NS4B-NS5A site in the absence of NS4A is due to low binding affinity of the enzyme for this site, and (iii) formation of a 1:1 complex between the protease and Pep4A 21-34 is sufficient and required for maximum activation.

Amino Acid Sequence↗

PubMed1995

Identification of biologically active peptides using random libraries displayed on phage.

The construction of new and increasingly diverse libraries, as well as the implementation of more powerful selection schemes, has led to the identification of linear peptides that mimic complex epitopes. Phage display techniques are allowing the selection of disease-related peptides, which reproduce the antigenic and immunogenic properties of natural antigens, using whole sera from patients. The range of applications of phage technology has been extended to include the search for peptides binding to molecules other than antibodies, such as cell receptors and enzymes.

Amino Acid Sequence↗

PubMed1994

High level expression and rational mutagenesis of a designed protein, the minibody. From an insoluble to a soluble molecule.

We recently described the design and chemical synthesis of the minibody, a 61-residue metal binding beta-protein with a novel fold. Characterization of the polypeptide by circular dichroism spectroscopy, size exclusion chromatography, and metal binding studies showed the molecule to be folded, monomeric, globular and able to bind metals. The main obstacle which prevented a more detailed characterization was the very low solubility of the protein in water (about 10 microM). To address this problem, we used two independent approaches: (1) mutagenesis of the beta-sheet framework residues and (2) addition of a solubilizing motif, made of three lysine residues, at the N or C termini. Engineering and production of mutants was facilitated by the achievement of high level expression of the protein in Escherichia coli. Both approaches led to minibody variants with a solubility ranging from tenfold higher up to millimolar levels. For the best-characterized variant obtained so far, the thermodynamic stability calculated from denaturant-induced transition is identical to that of the parent, poorly soluble, molecule.

Amino Acid Sequence↗

PubMed1994

The making of the minibody: an engineered beta-protein for the display of conformationally constrained peptides.

Conformationally constraining selectable peptides onto a suitable scaffold that enables their conformation to be predicted or readily determined by experimental techniques would considerably boost the drug discovery process by reducing the gap between the discovery of a peptide lead and the design of a peptidomimetic with a more desirable pharmacological profile. With this in mind, we designed the minibody, a 61-residue beta-protein aimed at retaining some desirable features of immunoglobulin variable domains, such as tolerance to sequence variability in selected regions of the protein and predictability of the main chain conformation of the same regions, based on the 'canonical structures' model. To test the ability of the minibody scaffold to support functional sites we also designed a metal binding version of the protein by suitably choosing the sequences of its loops. The minibody was produced both by chemical synthesis and expression in E. coli and characterized by size exclusion chromatography, UV CD (circular dichroism) spectroscopy and metal binding activity. All our data supported the model, but a more detailed structural characterization of the molecule was impaired by its low solubility. We were able to overcome this problem both by further mutagenesis of the framework and by addition of a solubilizing motif. The minibody is being used to select constrained human IL-6 peptidic ligands from a library displayed on the surface of the f1 bacteriophage.

Amino Acid Sequence↗

PubMed1993

Conformational study of a short Pertussis toxin T cell epitope incorporated in a multiple antigen peptide template by CD and two-dimensional NMR. Analysis of the structural effects on the activity of synthetic immunogens.

The immunogenic efficacy of multiple antigen peptides, MAPs, i.e. branched molecules in which multiple copies of a given immunogenic peptide are attached on a scaffold of lysine residues via both alpha and epsilon linkages, has been repeatedly demonstrated, but little is known about the structural arrangement of these peptide constructs. A conformational characterization was therefore performed for a known T cell epitope of the S1 subunit of Pertussis toxin, whose sequence is predicted to form alpha-helix. The peptide DNVLDHLTGR, its N-acetylated and C-amidated analogue and a tetrabranched MAP based on the N-acetylated peptide were prepared and studied by CD and two-dimensional 1H-NMR. No evidence of helical structure was obtained in water for the isolated peptides. In contrast, in triflouroethanol, the isolated epitopes fold into a helical structure spanning the segment Val3-Thr8 in the uncapped molecule and encompassing also the N-terminal region in the capped analogue. The mobile C-terminal region tends to adopt a distorted turn arrangement in both peptides due to the folding of Arg10 guanidinium over the backbone. No distortion of the helix structure was observed for the single-copy epitope in the four-branched MAP molecule in trifluoroethanol: each peptide chain is equivalent within the MAP and shows an even more regular helical pattern than the isolated end-blocked sequence. A slight difference was located at the junction with the lysine scaffold: the peptide bond to epsilon NH was found in a much more extended conformation than the corresponding link to alpha NH. These structural results correlate with in vitro T cell stimulatory activity of the three compounds examined and provide arguments supporting the previous suggestion that MAP tetramers are unlikely to elicit an immune response specific for the synthetic template assembly, a feature necessary to retain the advantage of the polymeric epitope presentation.

Amino Acid Sequence↗

PubMed1993

Mimicking of discontinuous epitopes by phage-displayed peptides, I. Epitope mapping of human H ferritin using a phage library of constrained peptides.

We have constructed a random nonapeptide library in the N-terminal region of the major coat protein VIII of bacteriophage f1, with two cysteines flanking the insert, and preliminary data suggest that many of the clones display at least some of their peptides in cyclized form. This library was used to select oligopeptides binding to the monoclonal antibody (mAb) H107, recognising the assembled native conformation of recombinant human H-subunit ferritin (H Fer), whose three-dimensional structure is known. Comparison of the selected oligopeptides with one another allowed us to derive two consensus sequences characterized by conserved amino acid (aa) residues. Analysis of the distribution of the aa side chains exposed on the surface of H Fer reveals that most of the aa defining both consensus sequences are present either at the end of the big loop or at the end of the A helix. These two regions of the H Fer, though separated in the linear sequence, are very close in the folded molecule. Interestingly, each consensus sequence derived from the selected phage-displayed peptides is characterized by aa present both at the end of the big loop and at the end of the A helix. These two H Fer regions are good candidates for mimicry by the selected peptides and therefore for constituting part of the H107 epitope. To provide support to this hypothesis, we constructed several H Fer mutants carrying point mutations in different positions of these two regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

A designed metal-binding protein with a novel fold.

A major challenge in protein design is to create stable scaffolds into which tailored functions can be introduced. Here we present the design, synthesis and characterization of a 61-residue all-beta protein: the minibody. We used a portion of the heavy chain variable domain of an immunoglobulin as a template, obtaining a molecule with a novel beta-sheet scaffold and two regions corresponding to the hypervariable loops H1 and H2. To exploit the potential for creating functional centres in the minibody, we engineered a metal-binding site into it. This site is formed by one histidine in H1 and two in H2. The protein is folded, compact and able to bind metal, thus representing the first designed beta-protein with a novel fold and a tailored function. By randomizing the sequence of the hypervariable loops, we are using the minibody scaffold to construct a conformationally constrained peptide library displayed on phage.

Amino Acid Sequence↗

PubMed1993

Chemical synthesis of a designed beta-protein through the flow-polyamide method.

A designed, 61-residue long, metal-binding protein was synthesized through the flow-polyamide method. This protein, named Minibody (Mini-antibody), contains a beta-sheet scaffold and two regions corresponding to immunoglobulin hypervariable loops (H1 and H2), onto which a metal binding site was engineered. The protein is extremely hydrophobic, with a 70% beta structure. Accordingly, it was anticipated, and actually found, to be a "difficult sequence" for all its length. Comparison of the standard, "minimum redundancy" protocol (single coupling, low excess of activated species) with a different protocol (double and triple coupling plus capping) showed that the two produced approximately the same amount of full-length product (3.7% after extensive purification). Capping was effective in blocking the unreacted amino groups, converting most failure sequences into truncated ones, a possible aid to implement affinity-type chromatographic protocols. Purification was complicated by the very low solubility of the molecule, coupled to a high tendency to aggregate even in concentrated chaotropic media (8 M urea). Nevertheless, a multi-dimensional purification scheme produced a highly homogeneous product, with the expected Ion Spray mass spectrum. The Minibody produced by recDNA methods showed identical chromatographic, spectroscopic and biochemical properties to those of the synthetic product.

Amino Acid Sequence↗

PubMed1993

Affinity purification of a difficult-sequence protein. Implications for the inclusion of capping in synthetic protocols.

A biotinylated derivative of a designed, difficult-sequence protein (the Minibody, Bianchi, E., Tramontano, A., Sollazzo, M. & Pessi, A. (1993) Int. J. Peptide Protein Res. 41, 385-393) which represented only 3.7% of the crude, cleaved material was quantitatively recovered with about 70% purity, in a single step, by affinity chromatography on immobilised avidin. Purification to homogeneity was then easily achieved by preparative HPLC. This highly effective purification scheme must be contrasted with the previously shown multidimensional, low-yield chromatographic protocol. Since no facilitation of the purification had been obtained by capping alone, this result suggests that capping is useful only in conjunction with affinity chromatography.

Amino Acid Sequence↗

PubMed1992

Use of human universally antigenic tetanus toxin T cell epitopes as carriers for human vaccination.

Synthetic constructs were assembled as multiple Ag peptide systems containing repetitive sequences of Plasmodium falciparum and Plasmodium berghei, the causative agents of human and murine malaria respectively, and two universal human tetanus toxin T cell epitopes 830-843 and 947-967. These constructs were tested for antibody production in mice and for their capacity to stimulate human PBL and tetanus toxin-specific T cell clones. A high antibody titer can be obtained in mice when multiple Ag peptide systems are injected in various adjuvants or in PBS alone. Furthermore, all constructs can activate PBL from every donor tested. However, a variable response was obtained when different clones specific for the two tetanus toxin universal epitopes were used. These constructs may represent possible candidates for a malaria vaccine.

Amino Acid Sequence↗

PubMed1992

Anti-circumsporozoite protein antibodies measure age related exposure to malaria in Kataragama, Sri Lanka.

Antibodies to two peptides DDAAD and (NANP)40 representing the repetitive sequence of circumsporozoite antigens (CS protein) of P. vivax and P. falciparum respectively were measured in a cohort of 149 and 107 individuals respectively at four, 6 monthly blood surveys performed on residents of Kataragama, a P. vivax malaria endemic region in southern Sri Lanka. The prevalence of antibodies to the CS protein of both species was relatively low being less than 20% to either peptide in the population as a whole, this being consistent with the low entomological inoculation rates in the area. A marked age related prevalence pattern was evident, with the prevalence of antibodies increasing with age to reach between 25 to 30% in the 25-50 year age group in both P. vivax and P. falciparum. The population had had a life long exposure to P. vivax malaria but not to P. falciparum, an epidemic of which occurred in this region a few months prior to the beginning of this study. Nevertheless, the age-related prevalence of these antibodies was identical with respect to the two species. This suggests that the age-related prevalence pattern reflected differences in inoculation rates between the age groups due to differences in exposure to inoculation rather than an age acquired response resulting from a cumulative experience over several years. An analysis of antibody prevalence in individuals showed first, that sporozoite inoculations must have been clustered rather than homogeneously distributed in the population and secondly, that sero-conversion did not correlate with malaria infections in these individuals.

Adolescent↗