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Digital, Parasporin-2Aa1 (PS2Aa1) is a toxic protein of 37 KDa (30 kDa, activated form produced by proteolysis) that was shown to be cytotoxic against specific human cancer cells, although its mechanism of action has not been elucidated yet. In order to study the role of some native peptide fragments of proteins on anticancer activity, here we investigated the cytotoxic effect of peptide fragments from domain-1 of PS2Aa1 and one of the loops present in the binding region of the virus spike protein from Alphacoronavirus (HCoV-229E), the latter according to scientific reports, who showed interaction with the human APN (h-APN) receptor, evidence corroborated through computational simulations, and thus being possible active against colon cancer cells. Peptides namely P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa were synthesized using the Fmoc solid-phase synthesis and characterized by mass spectrometry (MS). Additionally, one region from loop 1 of HCoV-229E, Loop1-HCoV-229E, was also synthesized and characterized. The A4W-GGN5 anticancer peptide and 5-fluorouracil (5-FU) were taken as a control in all experiments. Circular dichroism revealed an α-helix structure for the peptides derived from PS2Aa1 (P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa) and β-laminar structure for the peptide derived from Alphacoronavirus spike protein Loop1-HCoV-229E. Peptides showed a hemolysis percentage of less than 20% at 100 μM concentration. Besides, peptides exhibited stronger anticancer activity against SW480 and SW620 cells after exposure for 48 h. Likewise, these compounds showed significantly lower toxicity against normal cells CHO-K1. The results suggest that native peptide fragments from Ps2Aa1 may be optimized as a novel potential cancer-therapeutic agents., Ciencias Médicas y de la Salud

En este trabajo de investigación se diseñaron y se sintetizaron ocho lipopéptidos con una longitud en la secuencia peptídica de 3 a 5 residuos de aminoácidos, conjugados con un ácido graso entre 12 y 14 átomos de carbono. La síntesis se realizó por la metodología en fase sólida Fmoc (9-Fluorenilmetoxicarbonil). El rendimiento de la reacción de síntesis se calculó de acuerdo con la cantidad de producto obtenido, el cual fue superior al 90% para todos los lipopéptidos. Las nuevas moléculas llamadas LIP 1, LIP 2, LIP 3, LIP 4, LIP 5, LIP 6, LIP 11 Y LIP 12, se caracterizaron verificando la pureza, la estructura secundaria y la masa molecular, mediante técnicas de cromatografía líquida de alta eficiencia (HPLC), dicroísmo circular (CD) y espectrometría de masas (MS), respectivamente, determinándose para todos los lipopéptidos una pureza mayor al 90%, una estructura secundaria random coil y se confirmó que la masa obtenida fue igual a la masa esperada en cada molécula. Los resultados obtenidos en la evaluación antimicrobiana de los ocho lipopéptidos, indicaron que dos de ellos, LIP 4 y LIP 12, presentan actividad antibacteriana contra una serie de 4 bacterias patógenas (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli y Pseudomonas aeruginosa) a concentraciones mínimas inhibitorias (CMI) entre 9,50 a 100 μM y 8,50 a 10,0 μM, respectivamente. Adicionalmente, no presentaron toxicidad en eritrocitos humanos a concentraciones entre 3,13 y 50,0 μM, ni en las larvas de mosquitos de la especie Aedes aegypti, a una concentración evaluada de 100 μM. Ambos lipopéptidos demostraron su potente actividad antibacteriana a 2 veces la concentración mínima inhibitoria, por medio de las micrografías obtenidas por microscopía electrónica de barrido (SEM). Se determinó la estabilidad a proteasas, presentes en el suero sanguíneo humano, al lipopéptido con mayor actividad, LIP 12, indicando que es estable durante 24 horas a 37 °C, ya que mediante un seguimiento por HPLC no se observó la aparición de nuevas especies. Se concluye que el lipopéptido LIP 12, es candidato a ser un medicamento antibacteriano, debido a los altos rendimientos de reacción de síntesis y por el efecto bactericida que presento frente a las 4 especies evaluadas. Abstract. In this research work, eight lipopeptides were designed and synthesized with a peptide sequence length between 3 to 5 amino acid residues, they were conjugated with a fatty acid between 12 and 14 carbon atoms. The synthesis was carried out by the Fmoc (9-Fluorenylmethoxycarbonyl) solid phase methodology. The yield of its reaction was calculated according to the amount of the product obtained, which was over 90% for all lipopeptides. The new molecules called LIP 1, LIP 2, LIP 3, LIP 4, LIP 5, LIP 6, LIP 11, and LIP 12 were characterized verifying the purity, the secondary structure, and the molecular mass through high performance liquid chromatography techniques (HPLC), circular dichroism (CD), and mass spectrometry (MS), respectively, determining for all lipopeptides a purity higher than 90% and a random coil secondary structure. It was confirmed that the collected mass is equal to the expected mass in each molecule. The results obtained in the antimicrobial evaluation of the eight lipopeptides indicated that two of them, LIP 4 and LIP 12, have antibacterial activity against a series of 4 pathogenic bacteria (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa) at minimum inhibitory concentrations (MIC) between 9,50 to 100 μM, and between 8,50 to 10,0 μM, respectively. Additionally, it does not present human erythrocyte toxicity between 3,13 to 50,0 μM, nor in the larvae of mosquitoes of the Aedes aegypti species, to an evaluated concentration of 100 μM. Both lipopeptides demonstrated their potent antibacterial activity at 2 times the minimum inhibitory concentration through micrographs by scanning electron microscopy (SEM). It was determined the stability to proteases presented in the human blood serum of the lipopeptide with the highest activity, LIP 12, indicating that it is stable at 37 °C during 24 hours, since a follow-up by HPLC does not present the appearance of new species. To conclude, the lipopeptide LIP 12 is a candidate to be an antibacterial medication due to the high reaction yields of the synthesis and the bactericidal effect that it presents against the four species evaluated. Magister en Ciencias - Química Maestría