Claudia Marcela Parra Giraldo

Cervical cancer currently affects thousands of women worldwide. Consequently, the search for new peptide molecules, particularly chimeric peptides, has been proposed to treat this disease. In the present study, a library of chimeric peptides containing the RRWQWR sequence conjugated to functional peptides (such as anticancer peptides, cervical cancer cell‐targeting peptides, and cell‐penetrating peptides) was designed and synthesized. The most active chimera was optimized through chemical modifications, including the replacement of Arg with Lys, formation of dimers, changes in the linker, and deletion of amino acids at the N‐ and/or C‐terminus. All chimeric peptides were obtained via solid‐phase peptide synthesis and characterized by means of RP‐HPLC and mass spectrometry. The cytotoxicity of the chimeras against human cervical cancer cell lines HeLa and Ca Ski was evaluated in vitro. The results demonstrated that it is possible to obtain chimeric peptides from the RRWQWR core conjugated with functional peptides. Some peptides exhibited significant cytotoxic activity against the evaluated cell lines, demonstrating selectivity, rapid action, and concentration dependence. The chimeric peptide library allowed the identification of promising chimeric peptides such as KKWQWK‐Ahx‐RLLRRLLR. The identification of molecules with anticancer activity from sequences with lower activity represents a rapid, cost‐effective, and highly accurate strategy.

Palindromic antimicrobial peptides (PAMs) constitute versatile scaffolds for the design and optimization of anticancer agents with applications in therapy, diagnosis, and/or monitoring. In the present study, fluorolabeled peptides derived from the palindromic sequence RWQWRWQWR containing fluorescent probes, such as 2-Aminobenzoyl, 5(6)-Carboxyfluorescein, and Rhodamine B, were obtained. RP-HPLC analysis revealed that the palindromic peptide conjugated to Rhodamine B (RhB-RWQWRWQWR) exhibited the presence of isomers, likely corresponding to the open-ring and spiro-lactam forms of the fluorescent probe. This equilibrium is dependent on the peptide sequence, as the RP-HPLC analysis of dimeric peptide (RhB-RRWQWR-hF-KKLG)₂K-Ahx did not reveal the presence of isomers. The antibacterial activity of the fluorescent peptides depends on the probe attached to the sequence and the bacterial strain tested. Notably, some fluorescent peptides showed activity against reference strains as well as sensitive, resistant, and multidrug-resistant clinical isolates of E. coli, S. aureus, and E. faecalis. Fluorolabeled peptides 1-Abz (MIC = 62 µM), RhB-1 (MIC = 62 µM), and Abz-1 (MIC = 31 µM) exhibited significant activity against clinical isolates of E. coli, S. aureus, and E. faecalis, respectively. The RhB-1 (IC₅₀ = 61 µM), Abz-1 (IC₅₀ = 87 µM), and RhB-2 (IC₅₀ = 35 µM) peptides exhibited a rapid, significant, and concentration-dependent cytotoxic effect on HeLa cells, accompanied by morphological changes characteristic of apoptosis. RhB-1 (IC₅₀ = 18 µM) peptide also exhibited significant cytotoxic activity against breast cancer cells MCF-7. These conjugates remain valuable for elucidating the possible mechanisms of action of these novel anticancer peptides. Rhodamine-labeled peptides displayed cytotoxicity comparable to that of their unlabeled analogues, suggesting that cellular internalization constitutes a critical early step in their mechanism of action. These findings suggest that cell death induced by both unlabeled and fluorolabeled peptides proceeds predominantly via apoptosis and is likely contingent upon peptide internalization. Functionalization at the N-terminal end of the palindromic sequence can be evaluated to develop systems for transporting non-protein molecules into cancer cells.

Previous studies show that the palindromic peptide RWQWRWQWR, derived from bovine lactoferricin (LfcinB), exhibits selective in vitro cytotoxicity against cervical, breast, and prostate cancer cells.

Anacardium genus (Anacardiaceae) consists of approximately 20 species, and Anacardium microcarpum, Anacardium humile, and Anacardium occidentale are the most widely studied, because of their medicinal and nutraceutical properties. Their common name is cashew, marañon, or cajú. The leaves and flowers of A. occidentale have been used in traditional medicine to treat skin lesions and diarrhea and as anti-inflammatory agents. Previous results in our research group have shown that the leaf extract of A. occidentale possesses an interesting antifungal activity, inhibiting the proliferation and growth of Candida albicans. To identify a potential active marker for this extract, the purification of one of its major compounds by Centrifugal Partition Chromatography (CPC) was carried out in this work. First, the EtOAc fraction was separated using the CHCl3:MeOH:H2O (4:3.5:2) solvent system. We obtained 243 mg of the EtOAc fraction then it was solubilized in 5 mL of both phases. In a single CPC run, 12.5 mg of the major compound could be isolated from the extract, with a purity of over 90%, which was subsequently identified by LC-PDA-MS/MS as being the biflavone Agathisflavone showed a minimum inhibition concentration (MIC) of 250 μg/mL against C. albicans SC5314 and It did not show cytotoxicity activity in vitro. The CPC analysis described in this work allowed obtaining, in a single run, the major flavonoid of the A. occidentale leaf extract, which is now being evaluated for its antifungal potential against Candida albicans.

This study reports the synthesis, characterization, and antifungal evaluation of a series of pyridoxal and salicylaldehyde derivatives, using synthetic methodologies such as radical cyclizations and click chemistry. Compounds 6a and 6b, featuring a fused dihydrobenzoxepine-pyridine scaffold, demonstrated effective fungicidal activity with MIC values of 19 µg/mL against Cryptococcus neoformans 2807. Similarly, compound 6b exhibited notable activity with a MIC of 75 µg/mL against Candida auris PUJ-HUSI 537. Both compounds outperformed fluconazole (FLC) in these strains. In silico ADMET profiling revealed favorable pharmacokinetic properties, including blood–brain barrier penetration and drug-likeness parameters consistent with Lipinski’s rule of five. Cytotoxicity assays on human fibroblasts confirmed the low toxicity of compound 6a at the tested concentrations. These results highlight the potential of the fused dihydrobenzoxepine-pyridine scaffold as a promising antifungal candidate for further investigations.

Aim: To identify peptides derived from bovine lactoferricin (LfcinB) as potential therapeutics for colon cancer treatment. We systematically modified dimeric peptides to enhance their selectivity against colon cancer cells and reduce toxicity. We examined the effects of specific changes, such as substituting L-arginine (Arg) with L-ornithine (Orn) and/or D-Arg, on cytotoxic activity in colon cancer cells, as well as activity in prostate and cervical cancer cell lines. Additionally, we assessed the type of cell death induced and the in vivo toxicity of the dimeric peptides. Methods: The peptides were synthesized by manual solid-phase peptide synthesis, purified by reverse phase-solid phase extraction (RP-SPE), and characterized by RP-high performance liquid chromatography (RP-HPLC), and mass spectrometry (MS). Their cytotoxic effect on cancer and non-cancerous cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The most promising dimeric peptide underwent scale-up synthesis to yield approximately 1 g. The type of induced cell death was analyzed through cytometry assays, while preliminarily toxicity studies were conducted in Galleria mellonella, zebrafish, and CD1 mice. Results: Our findings demonstrated that dimeric peptides containing L-Orn or D-Arg residues exhibited potent and selective cytotoxic effects against colon cancer cells (Caco-2 and HT-29), prostate cancer cells (DU-145), and cervical adenocarcinoma (HeLa). Notably, these modified peptides showed minimal toxicity in human erythrocytes, HEK 293 cells or fibroblasts, and Galleria mellonella larvae. Peptide 3: (R-Orn-WQWRFKKLG)2-K-Ahx, emerged as particularly promising, preserving its integrity and anticancer activity during scaled-up synthesis. Furthermore, peptide 3 induced behavioral changes and sedation in CD1 mice and showed significantly lower toxicity in zebrafish. Conclusions: The results suggested that specific modifications of Arg/Orn residues in dimeric peptides enhance their cytotoxicity against colon cancer cells and reduce in vivo toxicity. These modified peptides hold promise as safe and effective therapeutic candidates, potentially expanding the treatment options available for cancer.

Extracellular vesicles (EVs) from Candida albicans can elicit immune responses, positioning them as promising acellular vaccine candidates. We characterized EVs from an avirulent C. albicans cell wall mutant (ecm33Δ) and evaluated their protective potential against invasive candidiasis. EVs from the yeast (YEVs) and hyphal (HEVs) forms of the SC5314 wild-type strain were also tested, yielding high survival rates with SC5314 YEV (91%) and ecm33 YEV immunization (64%). Surprisingly, HEV immunization showed a dual effect, resulting in 36% protection but also causing premature death in some mice. Proteomic analyses revealed distinct profiles among the top 100 proteins in the different EVs, which may explain these effects: a shared core of 50 immunogenic proteins such as Pgk1, Cdc19, and Fba1; unique, relevant immunogenic proteins in SC5314 YEVs; and proteins linked to pathogenesis, like Ece1 in SC5314 HEVs. Sera from SC5314 YEV-immunized mice showed the highest IgG2a titers and moderate IL-17, IFN-γ, and TNF-α levels, indicating the importance of both humoral and cellular responses for protection. These findings highlight the distinct immunogenic properties of C. albicans EVs, suggesting their potential in acellular vaccine development while emphasizing the need to carefully evaluate pathogenic risks associated with certain EVs.

Peptides containing the sequences ²⁰RRWQWR²⁵ and ²⁰RRWQWRMKKLG³⁰ derived from Bovine lactoferricin (LfcinB) were synthesized and their antibacterial effect against reference strains and sensitive and resistant clinical isolates of E. coli was evaluated. Tetra‐branched multiple antigen peptide (MAP) ((RRWQWR)₂‐K‐Ahx‐C)₂ exhibited significant antibacterial activity against sensitive, resistant, and multidrug‐resistant clinical isolates of E. coli. Peptide 3: RRWQWR‐Nal‐KKLG; MIC=16 μM, ²⁶[F]: (RRWQWRFKKLG)₂‐K‐Ahx; MIC=15 μM, 17: (RRWQWRFK)₂‐K‐Ahx; MIC=9 μM, and LfcinB (20–25)₂: (RRWQWR)₂‐K‐Ahx; MIC=11 μM exhibited the highest antibacterial activity against E. coli strains, with bactericidal effect and haemolytic effect at MIC less than 5 % and a therapeutic index >1. A synergistic effect of peptides ²⁶[F] and 17 with ciprofloxacin (CIP) or ceftriaxone (CEF) was observed. Prolonged treatment of E. coli ATCC 25922 with sublethal concentrations of CIP induced resistance in this strain, whereas some peptides did not induce resistance. These peptides can be considered to be promising candidates for treating infections caused by resistant strains of E. coli.

Extracellular vesicles (EVs) from Candida albicans can elicit immune responses, positioning them as promising acellular vaccine candidates. We characterized EVs from an avirulent C. albicans cell wall mutant ( ecm33Δ ) and evaluated their protective potential against invasive candidiasis. EVs from the yeast (YEVs) and hyphal (HEVs) forms of the SC5314 wild-type strain were also tested, yielding high survival rates with SC5314 YEVs (91%) and ecm33 YEVs immunization (64%). Surprisingly, HEV immunization showed a dual effect, resulting in 36% protection but also causing premature death in some mice. Proteomic analyses revealed distinct profiles among the top 100 proteins in the different EVs which may explain these effects: a shared core of 50 immunogenic proteins such as Pgk1, Cdc19, and Fba1; unique, relevant immunogenic proteins in SC5314 YEVs, and proteins linked to pathogenesis, like Ece1 in SC5314 HEVs. Sera from SC5314 YEVs-immunized mice showed the highest IgG2a titers and moderate IL-17, IFN-γ, and TNF-α levels, indicating the importance of both humoral and cellular responses for protection. These findings highlight the distinct immunogenic properties of C. albicans EVs, suggesting their potential in acellular vaccine development while emphasizing the need to carefully evaluate pathogenic risks associated with certain EVs.