Previous studies showed that the QDF peptide derived from cowpea β-vignin have the potential to lower cholesterol synthesis through a statin-like regulation mechanism, in vitro. In this study, we showed the effects of daily oral administration of the QDF peptide in rats fed high saturated-fat diet (HC) by 21 days. Rats were divided into the following groups: i. Dyslipidemic (DG), received the HC diet (glucose, 30 g/100 g; hydrogenated lard, 30 g/100 g; and cholesterol, 0.5 g/100 g).ii. HC+QDF, received HC diet, plus QDF peptide (10 mg/bw/day), and iii. HC+SVT, received HC diet, plus simvastatin (10 mg/bw/day). Food intake and feeding efficiency ratio were similar among DG group and treatment groups, indicating that these parameters were not affected. The growth animals’ simvastatin treatment had minor (16%) gain weight at 21^st day. HC+QDF group had lower (-23%) plasma triglycerides concentration than DG group, but there were no differences between total cholesterol concentration. HC+SVT group had a decrease in the total cholesterol (-21%) and triacylglycerols concentrations (-23%), but glucose concentration showed significant increase (+27%) compared to DG. Results showed that administration of QDF peptide promoted reduction of triglycerides in plasma but didn’t affected glucose concentration in rats fed rich-fat and sugar diet.

Mesobuthus cyprius, one of the two endemic scorpions in Cyprus, belongs in the family of Buthidae which is geographically distributed worldwide and is the largest of the scorpion families. Moreover, from a clinical perspective, Buthidae is the most important scorpion family as several members of this family are toxic to mammals and can be dangerous to humans. Even though Mesobuthus Cyprius was discovered in 2000 using molecular phylogenetics there are no other published data regarding the peptide and protein composition, the toxicity, or any other activity of the venom. It is impressive, that a broad variety of bioactive substances in scorpion venoms may be considered as a source for drug discovery and development.

Direct NMR spectroscopic analysis of unpurified biological extracts is a powerful tool for the discovery of natural products especially in complex mixtures like venoms. It permits partial or complete structural characterization of its major components as well as of many minor components. Herein, we report the application of 1D and 2D NMR spectroscopy for the first time in the analysis of the Mesobuthus Cyprius venom and the identification of a wide range of biomolecules and peptides. Samples were dissolved in D₂O and based on COSY, TOCSY, NOESY, HSQC and HMBC spectra, structures were proposed for the venom’s major components as well as for many minor components. Final identification of individual compounds can be accomplished through synthesis of proposed structures and additional Liquid Chromatography - Mass Spectrometry Analysis.

Cocoa is recognized worldwide for its health-promoting properties, mainly attributed to its high phenolic compound content. Cocoa proteins, albeit a minor fraction, could present interesting food and nutrition applications. However, to date, few studies report information about it. This work aimed to investigate the cocoa protein fraction as a source of bioactive peptides. The bioactive potential of cocoa proteins and their sensory profile, allergenicity, and bioactive peptides released during gastrointestinal protein hydrolysis were analyzed using in silico techniques. Proteins already-known sequences (30 in total) were retrieved from public databases. Cocoa proteins primarily showed potential bitter (29–51%) and sweet (15–30%) flavor sequences. None of the studied proteins exhibited predicted allergenicity. Cocoa proteins' degree of hydrolysis increased from the gastric (4–21%) to the intestinal (27–51%) phase. Consequently, most bioactive peptides were released after gastrointestinal digestion. The peptides released after in silico digestion showed antidiabetic (DPP-IV inhibition), antihypertensive (ECA inhibition), and antioxidant activities. These biological activities were associated with the presence of Leu, Pro, and Val (antidiabetic), Ala, Gly, and Val (antihypertensive), and Ala, Trp, and Tyr (antioxidant). Most bioactive peptides (56–63%) exhibited high gastrointestinal absorption. The results proved that cocoa could be used as a protein source and potentially bioactive peptides to prevent cardiometabolic diseases.

Leishmaniasis is a protozoan disease, which occurs in 12 to 15 million people worldwide and infects greater than 1 million new cases annually.¹ The limitations in availability of effective drugs and the rise of drug resistant strains has created an urgent need for new anti-leishmanial therapy.² In the light of the limitations of contemporary leishmaniasis therapy, the N-methylated almiramide lipo-peptides represent promising leads due to relatively high anti-leishmanial activity and low host toxicity due to theirs likely mechanism of action involving disruption of the vital energy machinery proteins of the glycosome.^3,4 With a focus on the influences of N-methylation and conformation on the antiprotozoan activity, our presentation provides a structure-activity relationship study of the almiramide peptides employing resistant parasite strains and murine macrophages.

References

1. Burza, S.; Croft, S. L.; Boelaert, M., Leishmaniasis. The Lancet 2019, 392 (10151), 951-970.
2. Uliana, S. R.; Trinconi, C. T.; Coelho, A. C., Chemotherapy of leishmaniasis: present challenges. Parasitology 2018, 145 (4), 464-480.
3. Sanchez, L. M.; Lopez, D.; Vesely, B. A.; Della Togna, G.; Gerwick, W. H.; Kyle, D. E.; Linington, R. G., Almiramides A− C: discovery and development of a new class of leishmaniasis lead compounds. J. Med. Chem. 2010, 53 (10), 4187-4197.
4. Sanchez, L. M.; Knudsen, G. M.; Helbig, C.; De Muylder, G.; Mascuch, S. M.; Mackey, Z. B.; Gerwick, L.; Clayton, C.; McKerrow, J. H.; Linington, R. G. Examination of the mode of action of the almiramide family of natural products against the kinetoplastid parasite Trypanosoma brucei. J. Nat. Prod. 2013, 76 (4), 630-641.

Insect consumption has nowadays become a new research field in food science and nutrition. Insects have been proposed as a promising alternative to cover the growing demand for protein by the population. This study aimed to evaluate the protein content and profile of six insects and peptide release during gastrointestinal digestion, assessing both proteins and peptides' physicochemical and biological properties. Protein concentrates from mealworm (Tenebrio molitor), beetle (Phyllophaga rugosa), caterpillar (Nudaurelia melanops), ant (Oecophylla smaradigna), locust (Locusta migratoria), and cricket (Acheta domesticus) flours were characterized by SDS-PAGE. identified proteins were hydrolyzed (pepsin, trypsin, and chymotrypsin) in silico to obtain the potential peptides released. Physicochemical properties and biological activity were evaluated using . Protein content varied from 36 to 42%, and the main proteins identified among insects were cytochrome c oxidase subunit I, acyl-CoA Δ-9 desaturase, and α-amylase. Insect proteins were rich in Leu, Ala, Gly, and Ile and deficient in Cys and Trp. Locust tropomyosin exhibited shared allergenic 22 epitopes with lobster tropomyosin. Gastrointestinal hydrolysis of each insect protein mainly released free amino acids (22–55%), di- and tri-peptides (22–55% and 17–31%, respectively) being peptides longer than four amino acids less (12–42%). Peptides presented antioxidant and DPP-IV and ECA inhibitory potential. Results revealed the feasibility of insect proteins as sources of bioactive peptides. Forthcoming studies are required to expand the knowledge on insects' proteins and peptides and validate their use by the food industry and gastronomy.

The Wittig reaction can be used for late stage functionalization of proteins and peptides to ligate glycans, pharmacophores, and other natural or unnatural functionalities do not present in polypeptides made of 20 natural amino acids. It is not obvious how peptide sequence influences the rate and outcome of Wittig ligation. Our group has previously employed the Wittig reaction to modify N-terminal aldehydes in Genetically-Encoded (GE) peptide libraries. It was found that the reaction rate strongly depends on the nature of the two N-terminal amino acids: specifically peptides with penultimate Pro-Pro exhibit a significantly decreased reaction rate. A plausible mechanistic hypothesis is that the hydrogen bonds inside the peptide backbone stabilize the transition state (TS) of the Wittig reaction and accelerate this reaction. I synthesized two model isoteric peptide substrates—CHO-Ala-Ala and CHO-Sarcosine-Sarcosine—with and without primary amides. The DFT calculation of the geometry of transition states in model peptide CHO-Ala-Ala indeed detected a contribution of hydrogen bond between backbone N-H and the betaine in the late TS while CHO-Sarcosine-Sarcosine does not have this characteristic. I measured reaction rates with model ylide and compare the observed results to the energy and geometry of the Wittig reaction TSs for CHO-Ala-Ala and CHO-Sarcosine-Sarcosine obtained in DFT calculation.

Antimicrobial resistance is a public health problem, requiring the development of new therapeutic agents. Antimicrobial peptides derived from Bovine Lactoferricin (LfcinB) have exhibited antibacterial and antifungal effects. Lipopeptides and peptides containing non-natural amino acids, derived from LfcinB (21-25) Pal: RWQWRWQWR, were synthesized by SPPS-Fmoc/tBu and characterized by RP-HPLC and MALDI-TOF mass spectrometry. The antimicrobial activity was tested against reference strains E. coli ATCC 11775 and 25922, S. aureus ATCC 25923, C. albicans SC 5314, C. auris 435 and 537, as well as against the clinical isolate of C. albicans 256 which is resistant to fluconazole. The peptides containing non-natural amino acids exhibited lower minimum inhibitory concentrations (MIC), and minimum fungicidal concentration (MFC) / minimum bactericidal concentration (MBC) values ​​for most of the strains except C. auris. The lipopeptides did not show activity at the concentrations evaluated, however, molecules containing amino-isobutyric or lauric acid combined with fluconazole showed an additivity effect against the clinical isolate C. albicans (256), finding decreases of two to eight times in the MIC values compared with the MIC of the fluconazole only. The results suggest that these molecules could be candidates to develop combined therapies against strains resistant to fluconazole. All the molecules are synthetically viable and the functionalization with non-natural amino acids is a positive modification to enhance the antimicrobial activity.

SARS-CoV-2 is the viral cause of the most significant global health crisis of our generation, causing over 144 million confirmed cases and 3 million deaths in the year following the declaration of a public health emergency of international concern by the WHO. While the first mRNA vaccines were deployed, the use of stapled peptides to investigate more synthetically accessible inhibitors has sparked rampant interest since as such peptides can have well-defined secondary structures and small molecule-like pharmacokinetic properties. Peptide stapling allows shorter amino acid sequences to mimic the interactions found at the surfaces of protein-protein interactions. In the environment of a rapidly growing field our lab has developed a unique stapling technology we coined Fluorescent Isoindole Crosslinking (FlICk) which employs ortho-phthalaldehyde (OPA) to staple an amine bearing side chain and a thiol bearing side chain in a dehydrative aromatization reaction to yield an isoindole, a chemical moiety which adds value beyond just rigidification through the emergent fluorescence of the staple itself, directly yielding lead molecules which do not have to be further derivatized with fluorophores for use as probes. The reaction conditions are highly benign, operating at room temperature and at ambient conditions close to physiological pH, thus lending themselves naturally to cyclization of unprotected peptides. In the process of developing this chemistry we were able to produce both monocycles and bicycles with biological activity (on melanocortin stimulating hormone and α-amanitin platforms, respectively.) With the emergence of the COVID-19 pandemic in 2020 we were compelled to apply our newly developed technology to what may be the most significant global health crisis of our time. The SARS-CoV-2 virus enters human cells via the interaction of the spike-RBD proteins on its surface with the Angiotensin Converting Enzyme 2 (ACE2) on the host cell surface. The viral protein interacts with the α-helical N-terminal domain of ACE2, a portion of the protein we sought to interrogate with stapled peptide surrogates as such molecules could act as decoys for virus, blocking attachment and thus entry to host cells. We developed an ELISA for testing new lead molecules and set off by examining linear sequences that were direct cut-outs from the ACE2 protein and found that none inhibited the ACE2-Spike interaction at the concentrations tested. We found that while we could smoothly apply our FlICk chemistry to produce stapled helical mimics, such monocyclic compounds were also inactive at the concentrations tested. Thus, we sought to expand the capabilities of FlICk and explore the possibility of introducing a double staple on an unprotected linear sequence and were pleased to find that the resulting double FlICked peptides were our first hits for successfully inhibiting the Spike-ACE2 interaction (at μM concentrations.) We are rapidly developing a pseudovirus neutralization assay (PNA) to validate these initial hits in-vivo while simultaneously pressing lead development in search of better inhibitors. Additionally, we are beginning work on examining the interactions of our ACE2 mimicking stapled peptides with COVID-19 variant spike proteins, which have an even greater affinity for ACE2 than the original viral protein. While this fact has grave implications for public health and has led to their increased transmissivity, it also offers the potential that peptide mimics of ACE2, such as ours, could be more effective treatments against these mutant strains.

The main aim of this study was to assess the ability of producing extracellular laccases of fungi isolated from Algerian’s saline soil and use them for an eventual low coast laccase’s production using agro-industrial wastes as substrate. Three out of twenty-three isolates displayed laccase-producing ability as measured by selective agar plate assays and were all halotolerant. Among them, one strain grows and decolorizes the Olive oil wastewater agar medium. The efficient strain was identified as Penicillium chrysogenum. This latter was highly halotolerant fungus due to its ability to grow from 0 to 21% (w/v) of NaCl with optimal growth showed at 15 % NaCl of liquid medium. The enzyme production was carried out, in the dark, at 25° C on diluted olive oil wastewater under Submerged fermentation (SmF) static condition for ten days and on olive pomace under Solid Stat Fermentation (SSF) for seven days. Both of the olive-oil wastes were adequate for laccase activities with 183 and 203 U/L on respectively 10% and 20% unsupplemented olive mill wastewaters and 12,46 U/g and 11,56 U/g on the untreated grinded and ungrinded olive pomace correspondingly. These results might suggest the possible use of the strain Penicillium chrysogenum for the economic production of laccases using phenolic agro-industrial wastes.

Recently, some studies have indicated that legume-derived protein hydrolysates can generate bioactive peptides with antitumoral effect. Hence, the present study evaluates the impact of cowpea β-vignin protein hydrolysate (BVPH) and its fractions on breast, liver and prostate cancer cell proliferation, in vitro. β-vignin was isolated, purified by size exclusion chromatographic process and analyzed by SDS-PAGE. The BVPH was produced by in vitro digestion of the protein using commercial pepsin and pancreatic enzymes under previously established conditions. BVPH was further separated by ultrafiltration into three peptide fractions (30-10, 10-3 and < 3 kDa) and tested on MDA-MB-231, Hep-G2 and DU-145 cells, in concentrations that ranged between 12.5–200 μg/ml. BVPH inhibited cancer cell lines up to 72.7%, although there was no statistical difference in the inhibition of MDA-MB-231 and DU-145 cells among different concentrations. The 10-3 kDa peptide fraction presented better antiproliferative effect against breast (IC50=0.33 μg/ml) as well as prostate cancer cells (IC50=4.37 μg/ml). However, in liver cells, the 30-10 kDa peptide fraction showed the greatest antiproliferative activity (IC50=231.79 μg/ml). Also, a dose-dependent effect was observed. The results observed in the present study suggest that peptides derived from β-vignin protein from cowpea bean have a cytotoxic effect on breast, liver and prostate cancer cells. In this sense, complementary studies are being carried out in order to identify the peptides are responsible for this effect.