Chiara Cabrele

Peptides containing β-amino acid patterns: challenges and successes in medicinal chemistry.

The construction of bioactive peptides using β-amino acid-containing sequence patterns is a very promising strategy to obtain analogues that exhibit properties of high interest for medicinal chemistry applications. β-Amino acids have been shown to modulate the conformation, dynamics, and proteolytic susceptibility of native peptides. They can be either combined with α-amino acids by following specific patterns, which results in backbone architectures with well-defined orientations of the side chain functional groups, or assembled in de novo-designed bioactive β- or α,β-peptidic sequences. Such peptides display various biological functions, including antimicrobial activity, inhibition of protein-protein interactions, agonism/antagonism of GPCR ligands, and anti-angiogenic activity.

The Id-protein family in developmental and cancer-associated pathways

Inhibitors of DNA binding and cell differentiation (Id) proteins are members of the large family of the helix-loop-helix (HLH) transcription factors, but they lack any DNA-binding motif. During development, the Id proteins play a key role in the regulation of cell-cycle progression and cell differentiation by modulating different cell-cycle regulators both by direct and indirect mechanisms. Several Id-protein interacting partners have been identified thus far, which belong to structurally and functionally unrelated families, including, among others, the class I and II bHLH transcription factors, the retinoblastoma protein and related pocket proteins, the paired-box transcription factors, and the S5a subunit of the 26xa0S proteasome. Although the HLH domain of the Id proteins is involved in most of their protein-protein interaction events, additional motifs located in their N-terminal and C-terminal regions are required for the recognition of diverse protein partners. The ability of the Id proteins to interact with structurally different proteins is likely to arise from their conformational flexibility: indeed, these proteins contain intrinsically disordered regions that, in the case of the HLH region, undergo folding upon self- or heteroassociation. Besides their crucial role for cell-fate determination and cell-cycle progression during development, other important cellular events have been related to the Id-protein expression in a number of pathologies. Dysregulated Id-protein expression has been associated with tumor growth, vascularization, invasiveness, metastasis, chemoresistance and stemness, as well as with various developmental defects and diseases. Herein we provide an overview on the structural properties, mode of action, biological function and therapeutic potential of these regulatory proteins.

Inhibition of delta-secretase improves cognitive functions in mouse models of Alzheimer's disease.

δ-secretase, also known as asparagine endopeptidase (AEP) or legumain, is a lysosomal cysteine protease that cleaves both amyloid precursor protein (APP) and tau, mediating the amyloid-β and tau pathology in Alzheimers disease (AD). Here we report the therapeutic effect of an orally bioactive and brain permeable δ-secretase inhibitor in mouse models of AD. We performed a high-throughput screen and identified a non-toxic and selective δ-secretase inhibitor, termed compound 11, that specifically blocks δ-secretase but not other related cysteine proteases. Co-crystal structure analysis revealed a dual active site-directed and allosteric inhibition mode of this compound class. Chronic treatment of tau P301S and 5XFAD transgenic mice with this inhibitor reduces tau and APP cleavage, ameliorates synapse loss and augments long-term potentiation, resulting in protection of memory. Therefore, these findings demonstrate that this δ-secretase inhibitor may be an effective clinical therapeutic agent towards AD.

Stabilization of the Dimeric Birch Pollen Allergen Bet v 1 Impacts Its Immunological Properties

Background: Frequently reported dimerization of allergens may contribute to their allergenicity. Results: Polysulfide-bridged allergen dimers exhibit different allergenic properties compared with the monomer. Conclusion: The N-terminal region has a distinct susceptibility for modifications and impacts its protein-protein interaction characteristics. Significance: The crystal structures well mimic transient dimerization of the allergens in solution, providing a rational for effective IgE cross-linking on effector cells. Many allergens share several biophysical characteristics, including the capability to undergo oligomerization. The dimerization mechanism in Bet v 1 and its allergenic properties are so far poorly understood. Here, we report crystal structures of dimeric Bet v 1, revealing a noncanonical incorporation of cysteine at position 5 instead of genetically encoded tyrosine. Cysteine polysulfide bridging stabilized different dimeric assemblies, depending on the polysulfide linker length. These dimers represent quaternary arrangements that are frequently observed in related proteins, reflecting their prevalence in unmodified Bet v 1. These conclusions were corroborated by characteristic immunologic properties of monomeric and dimeric allergen variants. Hereby, residue 5 could be identified as an allergenic hot spot in Bet v 1. The presented results refine fundamental principles in protein chemistry and emphasize the importance of protein modifications in understanding the molecular basis of allergenicity.

Biomimetic soluble collagen purified from bones

Type I collagen has been extensively exploited as a biomaterial for biomedical applications and drug delivery; however, small molecular alterations occurring during the isolation procedure and its interaction with residual bone extracellular matrix molecules or proteins might affect the overall material biocompatibility and performance. The aim of the current work is to study the potential alterations in collagen properties and organization associated with the absence of proteoglycans, which mimic pathological conditions associated with age-related diseases. A new approach for evaluating the effect of proteoglycans on the properties of isolated type I collagen from the bone matrix is described. Additional treatment with guanidine hydrochloride was introduced to remove residual proteoglycans from the collagen matrix. The properties of the isolated collagen with/without guanidine hydrochloride treatment were investigated and compared with a commercial rabbit collagen as control. We demonstrate that the absence of proteoglycans in the isolated type I collagen affects its thermal properties, the extraction into its native structure, and its ability to hydrate and self-assemble into fibers. The fine control and tuning of all these features, linked to the absence of non-collagenous proteins as proteoglycans, offer the possibility of designing new strategies and biomaterials with advanced biomimetic properties aimed at regenerating bone tissue in the case of fragility and/or defects.

Impact of the amino acid sequence on the conformation of side chain lactam‐bridged octapeptides

Synthetic helical peptides are valuable scaffolds for the development of modulators of protein–protein interactions involving helical motifs. Backbone‐to‐side chain or side chain‐to‐side chain constraints have been and still are intensively exploited to stabilize short α‐helices. Very often, these constraints have been combined with backbone modifications induced by Cα‐tetrasubstituted, β‐, or γ‐amino acids, which facilitate the α‐peptide or α/β/γ‐peptide adopting an α‐helical conformation. In this work, we investigated the helical character of octapeptides that were cyclized by a Lys‐Asp‐(i,i + 4)‐lactam bridge. We started with two sequences extracted from the helix–loop–helix region of the Id proteins, which are inhibitors of cell differentiation during development and in cancer. Nineteen analogs containing the lactam bridge at different positions and displaying different amino acid core triads (i + 1,2,3) as well as outer residues were prepared by solid‐phase methodology. Their conformation in water and water/2,2,2‐trifluoroethanol mixtures was investigated by circular dichroism (CD) spectroscopy. The cyclopeptides could be grouped in helix‐prone and non‐helix‐prone structures. Both the amino acid core triad (i + 1,2,3) and the pendant residues positively or negatively affected the formation of a helical structure. Computational studies based on the NMR‐derived helical structure of a cyclopeptide containing Aib at position (i + 2) of the triad were generally in agreement with the secondary structure propensity of the cyclopeptides observed by CD spectroscopy. In conclusion, the Lys‐Asp‐(i,i + 4)‐lactam bridge may succeed or fail in the stabilization of short helices, depending on the primary structure. Moreover, computational methods may be valuable tools to discriminate helix‐prone from non‐helix‐prone peptide‐based macrolactams. Copyright

Visible‐light photoredox‐catalyzed desulfurization of thiol‐ and disulfide‐containing amino acids and small peptides

A scalable protocol for the desulfurization of cysteine by using visible light, the photocatalyst Ir(dF(CF3)ppy)2(dtb‐bpy)PF6 and triethylphosphite under biphasic reaction conditions has been developed. The loading of the catalyst can be as low as 0.01 mol%, which can be efficiently removed during the workup (≤0.3 ppm), giving rise to the corresponding desulfurized product in high yields. This method has been applied also to cystine, penicillamine, and reduced and oxidized glutathione. The desulfurization has been found to be pH sensitive, with an optimal pH value of 6.5 and 7.0 for the cysteine derivatives and glutathione, respectively. In addition, during the desulfurization of a decapeptide containing cysteine and methionine, concurrent oxidation of the two sulfur‐containing residues to disulfide and sulfoxide has been observed. Therefore, whereas the presented protocol allows a straightforward visible light‐mediated desulfurization of simple thiols by using very low catalyst loading and a cost‐effective trialkylphosphite as thiyl radical trapping agent, its application to complex substrates needs to be carefully validated. Copyright

Targeting of a helix-loop-helix transcriptional regulator by a short helical peptide

The Id proteins (Id1–4) are cell‐cycle regulators that play a key role during development, in cancer and vascular disorders. They contain a conserved helix‐loop‐helix (HLH) domain that folds into a parallel four‐helix bundle upon self‐ or hetero‐association with basic‐HLH transcription factors. By using such protein–protein interactions, the Id proteins inhibit cell differentiation and promote cell‐cycle progression. Accordingly, their supporting role in cancer has been convincingly demonstrated, which makes these proteins interesting therapeutic targets. Herein we present a short peptide containing an (i,i+4)‐lactam bridge and a hydrophobic (Φ) three‐residue motif Φ(i)−Φ(i+3)−Φ(i+6), which adopts a helical conformation in water, shows Id protein binding in the low‐micromolar range, penetrates into breast (MCF‐7 and T47D) and bladder (T24) cancer cells, accumulates in the nucleus, and decreases cell viability to ∼50u2009%. Thus, this cyclopeptide is a promising scaffold for the development of Id protein binders that impair cancer cell viability.

Complete NMR Assignment of Succinimide and Its Detection and Quantification in Peptides and Intact Proteins

Detecting and quantifying post-translational modifications (PTMs) in full-length proteins is a challenge, especially in the case of spontaneously occurring, nonenzymatic PTMs. Such a PTM is the formation of succinimide (Snn) in a protein that occurs spontaneously in prone primary sequences and leads typically to an equilibrium between Snn and its hydrolysis products isoaspartate (isoAsp) and aspartate. In order to detect these modifications in proteins by NMR spectroscopy, chemical shift assignments of reference compounds are required. We used peptide synthesis and 2D NMR spectroscopy to assign all 1H and 13C chemical shifts of Snn and isoAsp and found characteristic chemical shift correlations. To provide chemical shift reference data suitable for comparison with data of denatured proteins, we repeated the assignment in 7 M urea (pH 2.3) and in DMSO. Most characteristic of Snn are the two downfield shifted carbonyl chemical shifts, the chemical shift correlations of Cβ-Hβ of Snn and Cα-Hα of the succeeding residue which are clearly distinct from random coil chemical shift correlations. The characteristic 2D NMR fingerprints of Snn were used to detect and quantify this PTM in the model protein lysozyme, the biotherapeutic filgrastim, and the Fc part of immunoglobulin G1. Mass spectrometry (MS) was applied as an additional independent method. The orthogonality of the NMR and MS techniques allows cross-validation, which is especially important to search for subtle PTMs in proteins. Studying PTMs by NMR spectroscopy is a promising method to analyze proteins and peptides from natural sources, recombinant expression, or chemical synthesis.

Reduction of cancer cell viability by synergistic combination of photodynamic treatment with the inhibition of the Id protein family

The inhibitor of DNA binding and cell differentiation (Id) proteins are dominant negative regulators of the helix-loop-helix transcription factor family and play a key role during development as well as in vascular disorders and cancer. In fact, impairing the Id-protein activity in cancer cells reduces cell growth and even chemoresistance. Recently, we have shown that a synthetic Id-protein ligand (1Y) consisting of a cyclic nonapeptide can reduce the viability of the two breast cancer cell lines MCF-7 and T47D and of the bladder cancer cells T24 to about 50% at concentrations ≥100μM. Moreover, the cyclopeptide displays both proapoptotic and antiproliferative effects on MCF-7 cells. Herein, we show that the cyclopeptide does not induce cell death at the dose of 5μΜ, but it still inhibits MCF-7 and T24 cell proliferation, which correlates with an increased protein level of the cell-cycle regulator p27Kip1. Furthermore, 1Y-pretreated MCF-7, T47D, and T24 cells are more susceptible than untreated cells to the phototoxic effects of the three photosensitizers meta-tetra(hydroxyphenyl)chlorin, porfimer sodium, and hypericin, which are applied in photodynamic therapy (PDT). The combination of the Id-protein ligand with each of the light-activated photosensitizers shows synergistic effects on the reduction of cell viability. In conclusion, an Id-protein ligand with moderate cancer cell killing activity at concentrations ≥100μM can be applied at a 20-fold lower and barely toxic dose to raise the sensitivity of cancer cells towards phototoxicity associated with photodynamic treatment. This suggests the potential benefit of targeting the Id proteins in combined drug approaches for cancer therapy.