Asa-peptiidsideme moodustumine aktivaatoriga trifosgeen

Peptiidid on head ravimikandidaadid, kuid nende kasutamine ravimitena on piiratud kiire lagunemise tõttu elusorganismides. Seega on arendatud peptidomimeetikuid, nt asa-peptiide, mis oleksid ensümaatilisele degradatsioonile vastupidavamad. Asa-peptiidide bioaktiivseid omadusi on aga vähe uuritud, kuna nende sünteesimiseks puuduvad sobilikud meetodid. Bakalaureusetöö eesmärgiks oli hinnata kineetilise meetodiga aktivaatori trifosgeen efektiivsust asa-peptiidsideme sünteesil mudelpeptiidis H-Ala-asaAla-Phe-NH2. Selgus, et asa-peptiidsideme moodustumise reaktsioon ei lähe lõpuni ja on aeglane ning võrreldes teiste asa-peptiidsünteesis kasutatud aktivaatoritega, on reaktsiooni saagis madal. Seega tuleks efektiivsemaks asa-peptiidide sünteesimeetodi välja töötamiseks testida veel tahkefaasilisel peptiidisünteesil kasutatud sidumisreagente või arendada sootuks uusi aktivaatoreid

Asa-aminohappe struktuuri mõju asa-peptiidi sünteesile kasutades aktivaatorit trifosgeen

Asa-peptiidid on peptidomimeetikud, mis on arendatud vältimaks takistusi, mis esinevad peptiidide kasutamisel ravimikandidaatidena. Olemasolevad meetodid ei võimalda aga asapeptiidide sünteesi sobiva saagisega. Käesolevas magistritöös uuriti asa-aminohappe (asaGly, asaAla, asaLeu, asaVal) struktuuri mõju sidumisreaktsiooni efektiivsusele mudel asa-peptiidis H-asaAH-Ala-Phe-NH2 aktivaatoriga BTC. Lisaks uuriti ühe asa-aminohappe (asaAla) dipeptiidile H-Val-Phe-NH-vaik sidumise näitel, kas lisatavale asa-aminohappele eelneva aminohappe struktuur mõjutab sidumise efektiivsust. Selgus, et seotava asa-aminohappe ega sellele järjestuses eelneva aminohappe kõrvalahela struktuur praktiliselt ei mõjuta sidumisreaktsiooni kiirust ning mõjutab reaktsiooni saagist väga vähesel määral. Seega võib asa-aminohapete lisamiseks peptiidjärjestusse aktivaatoriga BTC rakendada ühesugust eeskirja sõltumata asa-aminohappe või sellele eelneva aminohappe alküül-kõrvalahela struktuurist. AsaGly sidumiseks peptiidjärjestusse on aga sobivam kasutada aktivaatorit DSC

Enhancing Cellular Uptake of Native Proteins through Bio-Orthogonal Conjugation with Chemically Synthesized Cell-Penetrating Peptides

The potential for native proteins to serve as a platform for biocompatible, targeted, and personalized therapeutics in the context of genetic and metabolic disorders is vast. Nevertheless, their clinical application encounters challenges, particularly in overcoming biological barriers and addressing the complexities involved in engineering transmembrane permeability. This study is dedicated to the development of a multifunctional nanoentity in which a model therapeutic protein is covalently linked to a cell-penetrating peptide, NickFect 55, with the objective of enhancing its intracellular delivery. Successful binding of the nanoentity fragments was achieved through the utilization of an intein-mediated protein-trans splicing reaction. Our research demonstrates that the fully assembled nanoentity-containing protein was effectively internalized by the cells, underscoring the potential of this approach in overcoming barriers associated with protein-based therapeutics for the treatment of genetic disorders

Modification of the linker amino acid in the cell-penetrating peptide NickFect55 leads to enhanced pDNA transfection for in vivo applications

Despite numerous efforts over the last three decades, nucleic acid-based therapeutics still lack delivery platforms in the clinical stage. Cell-penetrating peptides (CPPs) may offer solutions as potential delivery vectors. We have previously shown that designing a “kinked” structure in the peptide backbone resulted in a CPP with efficient in vitro transfection properties. Further optimization of the charge distribution in the C-terminal part of the peptide led to potent in vivo activity with the resultant CPP NickFect55 (NF55). Currently, the impact of the linker amino acid was further investigated in the CPP NF55, with the aim to discover potential transfection reagents for in vivo application. Taking into account the expression of the delivered reporter in the lung tissue of mice, and the cell transfection in the human lung adenocarcinoma cell line, the new peptides NF55-Dap and NF55-Dab* have a high potential for delivering nucleic acid-based therapeutics to treat lung associated diseases, such as adenocarcinoma.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)19/20907-72022/3056-

Predicting Transiently Expressed Protein Yields: Comparison of Transfection Methods in CHO and HEK293

Therapeutic proteins are currently at the apex of innovation in pharmaceutical medicine. However, their industrial production is technically challenging and improved methods for transient transfection of mammalian cell cultures are necessary. We aimed to find a fast, microliter-scale transfection assay that allows the prediction of protein expression in the transient production settings. We used an array of lipid, polymeric and cell-penetrating peptide transfection reagents, and compared their performance in various high throughput transfection assays to their performance in protein (antibody) expression in professional protein-producer cell lines. First, we show that some of the most frequently used microliter-scale transfection efficacy assays fail to predict performance in the protein production in milliliter and liter scale settings. We found that CHO suspension culture post-transfection EGFP(+) population and SEAP quantitation correlate with large-scale protein production, whereas the adhesion culture assays and transfection of pLuc are non-predictive. Second, we demonstrated that cell-penetrating peptide-based transfection achieves significantly higher protein yields compared to PEI and lipoplex methods in both CHO and HEK293 producer cell lines. In this work we demonstrate a CPP-based transient protein expression approach that significantly outperformed the current industry standard workhorse method of PEI

Aggregation limiting cell-penetrating peptides derived from protein signal sequences

Alzheimer’s disease (AD) is the most common neurodegenerative disease (ND) and the leading cause of dementia. It is characterized by non-linear, genetic-driven pathophysiological dynamics with high heterogeneity in the biological alterations and the causes of the disease. One of the hallmarks of the AD is the progression of plaques of aggregated amyloid-β (Aβ) or neurofibrillary tangles of Tau. Currently there is no efficient treatment for the AD. Nevertheless, several breakthroughs in revealing the mechanisms behind progression of the AD have led to the discovery of possible therapeutic targets. Some of these include the reduction in inflammation in the brain, and, although highly debated, limiting of the aggregation of the Aβ. In this work we show that similarly to the Neural cell adhesion molecule 1 (NCAM1) signal sequence, other Aβ interacting protein sequences, especially derived from Transthyretin, can be used successfully to reduce or target the amyloid aggregation/aggregates in vitro. The modified signal peptides with cell-penetrating properties reduce the Aβ aggregation and are predicted to have anti-inflammatory properties. Furthermore, we show that by expressing the Aβ-EGFP fusion protein, we can efficiently assess the potential for reduction in aggregation, and the CPP properties of peptides in mammalian cells.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)19/20907-72022/3056-