The making of the minibody: An engineered β-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 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 β-protein aimed at retaining some desirable features of immunogloblin variable domains, such as tolerance to sequence variability in selected regions of the protein and predictability of 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 syntyhesis and expression in E. coli and charactgerized 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 soubility. We were able to overcome this problem both by further; mutagenesis of the framework and by addition of a solublizing 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.     

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

Summary 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 -helical portion of a 26-residue consensus peptide (CP1) belonging to the Cys²-Hys² zinc-finger family. The result was shown to be a highly homogeneous -helical library (Bianchi et al., 1995). The structures of the parent compound (CP1) and of a representative member (CP1m) 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.Abbreviations t-Bu tert-butyloxycarbonyl - Fmoc 9-fluorenylmethoxycarbonyl     

Structural characterization of the interactions of optimized product inhibitors with the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein by NMR and modelling studies

The interactions of peptide inhibitors, obtained by the optimization of N-terminal cleavage products of natural substrates, with the protease of human hepatitis C virus (HCV) are characterized by NMR and modelling studies. The S-binding region of the enzyme and the bound conformation of the ligands are experimentally determined. The NMR data are then used as the experimental basis for modelling studies of the structure of the complex. The S-binding region involves the loop connecting strands E2 and F2, and appears shallow and solvent-exposed. The ligand binds in an extended conformation, forming an antiparallel beta-sheet with strand E2 of the protein, with the P1 carboxylate group in the oxyanion hole.     

A loop-mimetic inhibitor of the HCV-NS3 protease derived from a minibody

We have been interested for some time in establishing a strategy for deriving lead compounds from macromolecule ligands such as minibody variants. A minibody is a minimized antibody variable domain whose two loops are amenable to combinatorial mutagenesis. This approach can be especially useful when dealing with 'difficult' targets. One such target is the NS3 protease of hepatitis C virus (HCV), a human pathogen that is believed to infect about 100 million individuals worldwide and for which an effective therapy is not yet available. Based on known inhibitor specificity (residues P6-P1) of NS3 protease, we screened a number of minibodies from our collection and we were able to identify a competitive inhibitor of this enzyme. We thus validated an aspect of recognition by HCV NS3 protease, namely that an acid anchor is necessary for inhibitor activity. In addition, the characterization of the minibody inhibitor led to the synthesis of a constrained hexapeptide mimicking the bioactive loop of the parent macromolecule. The cyclic peptide is a lead compound prone to rapid optimization through solid phase combinatorial chemistry. We therefore confirmed that the potential of turning a protein ligand into a low molecular weight active compound for lead discovery is achievable and can complement more traditional drug discovery approaches.     

Inhibiting viral proteases: challenges and opportunities

Inhibitor design against viral targets must take into account the peculiar characteristics of viral biology-in particular, the plasticity of their replicative machinery. This includes maturational cleavage of the polyprotein, which is mediated by virally encoded proteases. Designing against a movable target is particularly challenging, but at the same time it offers new opportunities. Here we describe our experience with the NS3/4A (NS: nonstructural) serine protease of human hepatitis C virus (HCV). By extensive use of combinatorial peptide libraries, various inhibitor types were generated, including product inhibitors, serine traps, P-P' inhibitors, and prime side inhibitors. The latter represent a first case for a serine protease. A key finding, derived from structural studies utilizing these inhibitors, was that NS3 is an induced-fit protease, requiring both the NS4A cofactor protein and the substrate to fully activate its catalytic machinery. In the absence of cofactor and/or substrate, NS3 exists in solution as a large conformational ensemble, which can be matched by a correspondingly large set of peptide inhibitors, each one stabilizing a given conformer. In the perspective of inhibiting viral proteases in general, we suggest that combinatorial ligand ensembles may be a powerful tool, to contrast the adaptive potential of the viral quasispecies.     

Interleukin 1B gene polymorphism is associated with baseline C-reactive protein levels in healthy individuals

C-reactive protein (CRP) is a sensitive marker of inflammation induced by both IL-6 and IL-1. Thus, genetic variation in these genes could be associated with the variety in C-reactive protein levels, and therefore with the severity of the entire inflammatory response. Even a subtle elevation in baseline CRP levels in healthy individuals has been found to significantly increase the risk for cardiovascular diseases. Therefore, to find out the possible role of pro-inflammatory cytokines in CRP baseline regulation we conducted a study of 338 healthy blood donors whose CRP levels were determined and whose single nucleotide polymorphisms of IL1A(C/T)-889, IL1B(C/T)-511, IL1B(C/T) + 3954, IL6(G/C)-174 and ILRN (a VNTR) both genotyped and haplotyped. The data revealed an association between CRP levels and the IL1B + 3954 genotype. Also, the bilocus haplotype IL1B-511*1/IL1B + 3954*2 was more frequent in subjects with below median CRP levels (< 0.72 mg/l), and composite genotype analysis of IL1B-511/IL1B + 3954 supported this finding. Our findings suggest that in healthy people, basal CRP levels are regulated by IL1B but not by IL6 genetics.     

GacS sensor domains pertinent to the regulation of exoproduct formation and to the biocontrol potential of Pseudomonas fluorescens CHA0

In the root-colonizing biocontrol strain CHA0 of Pseudomonas fluorescens, cell density-dependent synthesis of extracellular, plant-beneficial secondary metabolites and enzymes is positively regulated by the GacS/GacA two-component system. Mutational analysis of the GacS sensor kinase using improved single-copy vectors showed that inactivation of each of the three conserved phosphate acceptor sites caused an exoproduct null phenotype (GacS-), whereas deletion of the periplasmic loop domain had no significant effect on the expression of exoproduct genes. Strain CHA0 is known to synthesize a solvent-extractable extracellular signal that advances and enhances the expression of exoproduct genes during the transition from exponential to stationary growth phase when maximal exoproduct formation occurs. Mutational inactivation of either GacS or its cognate response regulator GacA abolished the strain's response to added signal. Deletion of the linker domain of the GacS sensor kinase caused signal-independent, strongly elevated expression of exoproduct genes at low cell densities. In contrast to the wild-type strain CHA0, the gacS linker mutant and a gacS null mutant were unable to protect tomato plants from crown and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici in a soil-less microcosm, indicating that, at least in this plant-pathogen system, there is no advantage in using a signal-independent biocontrol strain.     

Optimization of the P'-region of peptide inhibitors of hepatitis C virus NS3/4A protease

Infection by Hepatitis C Virus (HCV) leads to a slowly progressing disease that over two decades can lead to liver cirrhosis or liver cancer. Currently, one of the most promising approaches to anti-HCV therapy is the development of inhibitors of the NS3/4A protease, which is essential for maturation of the viral polyprotein. Several substrate-derived inhibitors of NS3/4A have been described, all taking advantage of binding to the S subsite of the enzyme. Inspection of the S' subsite of NS3/4A shows binding pockets which might be exploited for inhibitor binding, but due to the fact that ground-state binding to the S' subsite is not used by the substrate, this does not represent a suitable starting point. We have now optimized S'-binding in the context of noncleavable decapeptides spanning P6-P4'. Binding was sequentially increased by introduction of the previously optimized P-region [Ingallinella et al. (1998) Biochemistry 37, 8906-8914], change of the P4' residue, and combinatorial optimization of positions P2'-P3'. The overall process led to an increase in binding of more than 3 orders of magnitude, with the best decapeptide showing IC(50) < 200 pM. The binding mode of the decapeptides described in the present work shares features with the binding mode of the natural substrates, together with novel interactions within the S' subsite. Therefore, these peptides may represent an entry point for a novel class of NS3 inhibitors.     

Novel potent apoA-I peptide mimetics that stimulate cholesterol efflux and pre-β particle formation in vitro

Reverse cholesterol transport (RCT) is believed to be the primary mechanism by which HDL and its major protein apoA-I protect against atherosclerosis. Starting from the inactive 22-amino acid peptide representing the consensus sequence of the class A amphipathic helical repeats of apoA-I, we designed novel peptides able to mobilize cholesterol from macrophages in vitro, and to stimulate the formation of ‘nascent HDL’ particles, with potency comparable to the entire apoA-I protein.     

Positive control of swarming, rhamnolipid synthesis, and lipase production by the posttranscriptional RsmA/RsmZ system in Pseudomonas aeruginosa PAO1

In Pseudomonas aeruginosa, the small RNA-binding, regulatory protein RsmA is a negative control element in the formation of several extracellular products (e.g., pyocyanin, hydrogen cyanide, PA-IL lectin) as well as in the production of N-acylhomoserine lactone quorum-sensing signal molecules. RsmA was found to control positively the ability to swarm and to produce extracellular rhamnolipids and lipase, i.e., functions contributing to niche colonization by P. aeruginosa. An rsmA null mutant was entirely devoid of swarming but produced detectable amounts of rhamnolipids, suggesting that factors in addition to rhamnolipids influence the swarming ability of P. aeruginosa. A small regulatory RNA, rsmZ, which antagonized the effects of RsmA, was identified in P. aeruginosa. Expression of the rsmZ gene was dependent on both the global regulator GacA and RsmA, increased with cell density, and was subject to negative autoregulation. Overexpression of rsmZ and a null mutation in rsmA resulted in quantitatively similar, negative or positive effects on target genes, in agreement with a model that postulates titration of RsmA protein by RsmZ RNA.