Sortase A (SrtA) and some variants thereof have been used in a wide range of applications including fluorescent labeling, protein cyclization and immobilization due to their mild reaction conditions and high specificity. However, SrtA-catalyzed transpeptidation suffers the inherent limitation of being a reversible process which therefore requires an excessive amount of substrate to drive the reaction towards completion. Such an issue can prove prohibitive, especially in the case of high value-added substrate molecules. In this context, we disclose a novel substrate engineering strategy that enables to achieve high levels of SrtA-mediated protein modification with nearly stoichiometric amounts of substrate. Extension of the consensus sorting motif LPXTG with a positively charged peptidic module allows to achieve sequence-specific removal of by-products by applying the concept of electrostatic-assisted aminolysis reaction recently described by our group. The reaction equilibrium is driven to favor product formation, thereby greatly improving reaction yield.

Opioids are currently the most prescribed analgesic compounds. Although they are effective against moderate to severe acute pain, they come with several side effects. Recently, ACKR3 (Atypical Chemokine Receptor 3) was identified as having an affinity for endogenous opioid peptides. It is speculated that ACKR3 may act as a scavenger receptor for endogenous opioids, thereby reducing their analgesic effects. Modulating this receptor could therefore increase the availability of opioid peptides and, consequently, enhance their effects.Following this discovery, LIH383, a peptide agonist with pM-level potency, was developed.

In this project, LIH383 showed significant analgesic effects in the formalin test, a model of tonic pain. Additionally, LIH383 analogs were synthesized to better understand the relevant binding interactions and crucial molecular determinants for activity. So far, our initial structure-activity relationship (SAR) studies have shown that the N-terminal part of LIH383 is particularly sensitive to chemical modifications. The proteolytic stability of LIH383 was also evaluated, revealing a half-life of less than two minutes in rat plasma. Specifically, the dibasic Arg-Arg motif at the C-terminus is rapidly targeted by proteases, making it a preferred target. An SAR study will therefore be conducted to increase stability and thus the therapeutic potential of these compounds.

Nanocomposite hydrogels are gaining attention for their versatile applications. These materials can be fabricated from the co-assembly between peptides and inorganic nanoparticles resulting in hydrogels with enhanced mechanical and functional properties. A recent study has shown the co-assembly between laponite and linear peptide amphiphiles, resulting in hydrogels for potential applications in neovascularization and hierarchical mineralization.¹ The latter provides the basis for further exploration of nanosilicate-peptide interactions, paving the way for developing hydrogels tailored for various biomedical applications.

This study introduces a nanocomposite hydrogel formed by co-assembling cyclic octapeptide nanomaterials and nanosilicates (nSi). Cyclic peptide CP1 (cyclo-(D-Leu-Lys-D-Leu-Tyr)₂) self-assembled into elongated nanostructures under a pH-triggered mechanism and high peptide concentrations (1 - 2 %wt). Field emission SEM confirmed the formation of CP1 nanostructures, while FTIR validated the integration of CP1 and nSi within the hydrogel. CD spectroscopy indicated that the ß-sheet structure of CP1 was preserved upon nSi incorporation. Rheological analysis demonstrated enhanced elasticity compared to a nonpeptide control. Drug release studies showed a 40% release of vancomycin over 24 hours, sufficient to eradicate E. coli DH5-α. This novel organic-inorganic nanocomposite hydrogel has the potential to be used for diverse biomedical applications.

References:

1. Okesola et al. ACS Nano (2021). https://doi.org/10.1021/acsnano.0c09814

Background: As a lipid post-translational modification on proteins, prenylation is a critical process in regulating protein-membrane interactions. Ras proteins, a class of small GTPases are prenylated. Targeting Ras prenylation is a potential strategy to study Ras-related cancer. However, tumors which primarily contain K-Ras mutations, such as pancreatic adenocarcinoma, have a limited response to farnesyltransferase inhibitors. Therefore, new approaches to understand the mechanism of Ras prenylation in cancer cells is crucial.

Aims: The aim of this project is to construct a semi-synthetic K-Ras 4B protein using Expressed Protein Ligation (EPL) to investigate the biological behavior of Ras protein. In order to construct a semi-synthetic K-Ras 4B protein, K-Ras 4B ^1-174-MESNA ester has been purified. Solid phase peptide synthesis was used to prepare several forms of the C-terminal hypervariable region(HVR) for different applications. Ligation of those peptides with the aforementioned truncated K-Ras 4B protein thioester yielded several different forms of K-Ras 4B. This aim will be accomplished through the following two projects: A) Developing a method to study cell membrane localization of K-Ras 4B protein in cancer cells, and, B) Construction and structure elucidation of the Ras dimer and analysis of the effect of Ras clustering in cells.

The objective of this study was to reduce the formation of β-casomorphin-7 (BCM-7), an opioid peptide derived from β-casein A1, associated with inflammation and gastrointestinal disorders. To achieve this, strategies involving thermal treatments and bacterial fermentation with Lacticaseibacillus casei and Limosilactobacillus fermentum, combined with enzymatic hydrolysis, were evaluated. The thermal treatment at β-casomorphin-7 100°C for 30 minutes increased the concentration of soluble proteins to 7.58 ± 0.07 mg/mL, while autoclaving at 105°C for 15 minutes had a lesser impact, resulting in 3.24 ± 0.05 mg/mL, this method was chosen as the standard to minimize protein degradation and prepare the milk for subsequent processes. Bacterial fermentation preserved the proteins during digestion, while the control showed a significant drop in soluble protein concentrations, from 5.32 ± 0.05 mg/mL to 0.19 ± 0.01 mg/mL. SDS-PAGE evidenced the degradation of milk proteins, including β-casein. The ELISA showed that L. fermentum was more efficient in reducing BCM-7, decreasing its concentration from 0.22 ± 0.01 to 0.08 ± 0.01. The combination of L. casei and L. fermentum did not result in any additional significant reduction. Thus, fermentation combined with enzymatic hydrolysis proved effective in reducing the formation of BCM-7.

The development of theranostic agents has received increasing interest in recent literature. Radiolabelling of peptides with F-18 have shown its efficiency and reproducibility, and have been used extensively as PET imaging agents, for example [¹⁸F]-piflufolastat. On the therapeutic side, radioligands carrying Pb-212 have shown highly promising results in recent clinical trials.¹ This study highlights our effort towards the development of a PSMA-based tracer conjugated to an ammonium methylene trifluoroborate (AMBF₃) radioprosthetic and which also incorporates a novel chelator, for the chelation of the medical isotopes of lead. Such combination would allow orthogonal radiolabelling of the tracer with F-18 via isotopic exchange by virtue of the AMBF₃ radioprosthetic group.

We developed two PSMA-based tracers, [¹⁸F]-AT03 and [¹⁸F]-AT05, that incorporate multiple AMBF₃’s alongside chelator that should be capable of accommodating ^207/203/212Pb isotopes. Both candidate tracers are varied with linker arms of “medium” and “long”. Both tracers yielded high quality PET images and high tumour uptake values with minimal off target uptake. These results indicate a promising future in the translation to be used as theranostic agents.

1. Delpassand et al. Targeted α-emitter therapy with ²¹²Pb-DOTAMTATE for the treatment of metastatic SSTR-expressing neuroendocrine tumors: first-in-humans dose-escalation clinical trial. J Nucl Med. 2022;63:1326–1333.

The privileged role of turns in molecular recognition of peptides drives interest in mimicry of the backbone orientation and side chain pharmacophores of these important secondary structures. Previously, the stereoelectronic effects of semicarbazides have been used to induce turn geometry in azapeptides.¹ Moreover, the covalent constraint of α-amino-γ-lactam (Agl) residues, so-called Freidinger-Veber lactams, have been shown to favor beta-turns.^{2, 3} More recently, N-amino-imidazolinone (Nai) residues have been shown to combine stereoelectronic and covalent effects to augment potential to induce turn conformation.⁴ Focusing on the relevance of nitrogen chirality as a turn inducing factor in a model azapeptide, our presentation examines a minimal turn sequence to study turn formation without stereogenic carbon. Notably, diastereotopic glycine methylene proton signals have been observed to indicate pyramidal nitrogen for which the barrier for inversion was measured using variable temperature NMR spectroscopy and coalescence experiments. Efforts to crystalize and further characterize the model azapeptide conformation are under investigation.

Host defense peptides (HDPs) represent a group of essential compounds of innate immunity that can elicit strong anti-infective properties and immune regulation and are ubiquitously present in all organisms. These small positively charged amphipathic cationic peptides possess diverse roles regarding regulating and modulating the immune system, namely chemotaxis, cell differentiation, and pro- and anti-inflammatory cytokine production, by activating various intracellular cell signaling pathways. The activation of these pathways promotes wound healing, and acts as anticancer, and anti-infective therapeutic agents. This study aims to evaluate the immunomodulatory potential of HDP recently synthesized and characterized by the Ahmed group, originally derived from chicken Angiogenin 4. Our experimental procedures involve the treatment of mouse macrophages with mini chicken Angiogenin 4 (termed as mCA-4), evaluation of cytokine secretion, and modulation of two well-known immunoregulatory pathways, namely nuclear factor kappa B (NF-κB) and Mitogen-activated protein kinase (MAPK) by Western blot analysis. Our data indicate that mCA-4 is a pro-inflammatory peptide and activates these pathways, resulting in the secretion of pro-inflammatory mediators, including IL-1β, NO, and TNF-α, in treated macrophages. Future investigations will involve the evaluation of other intracellular pathways that may be involved in immunomodulation by this peptide, providing valuable insights into its therapeutic potential.

Bioactive peptides with anti-inflammatory activity have garnered growing interest due to their therapeutic potential in modulating the inflammatory response and as alternatives to traditional anti-inflammatory drugs. Frequently derived from food proteins, these peptides are released through hydrolysis and act on specific molecular pathways, such as COX-2. This study aimed to identify the peptidomic profile associated with the biochemical properties of these biopeptides using milk fermentation through bacterial co-culture. Following initial identification, in silico tools were employed to analyze the biochemical properties and predict the bioactivity of the peptides. A total of 730 peptides were identified, with 50.41% showing anti-inflammatory potential. Based on hydrophobicity, similar to that of aspirin (50.54%), 84 peptides were initially selected, narrowed down to 10 final biopeptides through rigorous screening for anti-inflammatory activity, low allergenicity, absence of toxicity, and good water solubility. The analysis revealed an average of 11.8 fragments, a molecular mass of 1400.26 Da, and an isoelectric point (pI) of 7.01, with an average hydrophobicity of 52.36%. The amino acid composition showed 6.20 hydrophobic residues and good solubility, suggesting effective interaction with cell membranes. These findings indicate the potential of biopeptides as safe and effective therapies, modulating inflammatory responses without adverse effects.

Vaccination remains the key strategy to control infectious diseases afflicting humans and animals. Owing to their biocompatibility and straightforward characterization, peptides have emerged as essential components of subunit vaccines. Over the last years, we reported a strategy to generate synthetic nanovaccines based on a chimeric peptide comprising a self-assembling sequence and an antigen, with the resulting nanoassemblies acting as an immunostimulator and a delivery system. In this study, we compared the 10-mer self-assembling peptide (I₁₀), originating from a naturally occurring amyloid peptide, to a de novo peptide sequence to conceive a nanoplatform for the delivery of a T-cell epitope model derived from ovalbumin, OVA_253-266. After synthesis, cleavage and purification, self-assembly was initiated by suspension of the lyophilized peptides in aqueous buffer. To characterize the supramolecular architecture of the resulting nanostructures, we used transmission electron microscopy and atomic force microscopy. Circular dichroism validated the secondary organization and the kinetic of self-assembly was followed using the fluorogenic probe ThT . Following the confirmation of the supramolecular assemblies, we immunized BALB/c mice intramuscularly with the nanoplatforms. Our results revealed the robustness of this novel supramolecular platform and confirmed that fully synthetic cross-beta-sheet nanoparticles can elicited strong antigen-specific immune response.