Optimized synthesis routes and biological application of N-peptide-6-amino-D-luciferin conjugates

In the recent years, numerous in vivo and in vitro analytical methods, based on fluorescence and bioluminescence, have been developed for various biological objectives, including immunoassays, gene expression assays, bioimaging, investigation of infectious diseases etc. Plate based, high-throughput viability assays addressing the detection of protease activity is in the focus of intensive research. The advantage of bioluminescent systems over fluorescent ones lies in their superior sensitivity and easy handling. In the bioluminescent methods, diverse sets of luciferases and their substrates, luciferins have been applied in different cellular and animal models, the most ubiquitous enzyme-substrate system is the American firefly (Photinus pyralis) luciferin-luciferase system. Substituting the 6-position hydroxyl group of native luciferin with an amino group, the resulting 6-amino-D-luciferin (2-(6-aminobenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid, hereinafter: aLuc) can form an amide bond with a peptide, while retaining the transport and bioluminescent properties of the original substrate, resulting in a good substrate for different important proteases.Due to this feature, these conjugates can be used for the determination of the enzymatic activity the following way: the protease enzyme to be measured recognizes the peptide part of the conjugate with the suitable peptide sequence, then cleaves the amide bond between the peptide and the aLuc, thus aLuc is released, which, in the presence of luciferase enzyme, emits light.The activity of the given protease enzyme can be determined from the amount of emitted light, as the emitted light is directly proportional to the activity of the enzyme. Unfortunately, the synthesis methods of these substrates published so far are complicated; and the very few commercially available conjugates are very expensive. The aim of this dissertation was to introduce novel routes for the scalable and economical synthesis of N-peptide-aLuc conjugates. The preparative work focused on preparing a precursor (6-amino-2-cyanobenzothiazole, 3), two conjugates (N-Z-Asp-Glu-Val-Asp-aLuc, 6, N-Fmoc-Gly-Pro-aLuc, 8) and an intermediate (NBoc- aLuc, 10)

Stability Test of PACAP in Eye Drops

PACAP is a neuropeptide with widespread distribution and diverse biological functions. It has strong cytoprotective effects mediated mainly through specific PAC1 receptors. Experimental data show protective effects of PACAP in the retina and cornea in several pathological conditions. Although intravitreal injections are a common practice in some ocular diseases, delivery of therapeutic agents in the form of eye drops would be more convenient and would lead to fewer side effects. We have previously shown that PACAP, in the form of eye drops, is able to pass through the ocular barriers and can exert retinoprotective effects. As eye drops represent a promising form of administration of PACAP in ocular diseases, it is important to investigate the stability of PACAP in solutions used in eye drops. In this study, the stability of PACAP1-27 and PACAP1-38 in eye drops was measured in four common media and a commercially available artificial tear solution at both room temperature and +4 °C. Mass spectrometry results show that the highest stability was gained with PACAP1-38 in water and 0.9% saline solution at +4 °C, representing 80–90% drug persistence after 2 weeks. PACAP1-38 in the artificial tear showed very fast degradation at room temperature, but was stable at +4 °C. In summary, PACAP1-38 has higher stability than PACAP1-27, with highest stability at +4 °C in water solution, but both peptides in each medium can be stored for relatively longer periods without significant degradation. These data can provide reference for future therapeutic use of PACAP in eye drops

Prolonged activity of the transposase helper may raise safety concerns during DNA transposon-based gene therapy

DNA transposon-based gene delivery vectors represent a promising new branch of randomly integrating vector development for gene therapy. For the side-by-side evaluation of the piggyBac and Sleeping Beauty systems—the only DNA transposons currently employed in clinical trials—during therapeutic intervention, we treated the mouse model of tyrosinemia type I with liver-targeted gene delivery using both transposon vectors. For genome-wide mapping of transposon insertion sites we developed a new next-generation sequencing procedure called streptavidin-based enrichment sequencing, which allowed us to identify approximately one million integration sites for both systems. We revealed that a high proportion of piggyBac integrations are clustered in hot regions and found that they are frequently recurring at the same genomic positions among treated animals, indicating that the genome-wide distribution of Sleeping Beauty-generated integrations is closer to random. We also revealed that the piggyBac transposase protein exhibits prolonged activity, which predicts the risk of oncogenesis by generating chromosomal double-strand breaks. Safety concerns associated with prolonged transpositional activity draw attention to the importance of squeezing the active state of the transposase enzymes into a narrower time window

Antiamnesic properties of analogs and mimetics of the tripeptide human urocortin 3

Amnesia is a deficit in memory caused by brain damage, disease, or trauma. Until now, there are no successful medications on the drug market available to treat amnesia. Short analogs and mimetics of human urocortin 3 (Ucn 3) tripeptide were synthesized and tested for their action against amnesia induced by eletroconvulsion in mice. Among the 16 investigated derivs. of Ucn 3 tripeptide, eight compds. displayed antiamnesic effect. Our results proved that the configuration of chiral center of glutamine does not affect the antiamnesic properties. Alkyl amide or isoleucyl amide at the C-terminus may lead to antiamnesic compds. As concerned the N-terminus, acetyl, Boc, and alkyl ureido moieties were found among the active analogs, but the free amino function at the N-terminus usually led to an inactive derivs. These observations may lead to the design and synthesis of small peptidomimetics and amino acid derivs. as antiamnesic drug candidates, although the elucidation of the mechanism of the action requires further investigations. [on SciFinder(R)

Synthesis of N-Peptide-6-Amino-d-Luciferin Conjugates with Optimized Fragment Condensation Strategy

Abstract: The synthesis of peptide-luciferin conjugates has a pivotal role in the development of bioluminescent detection systems that are based on the determination of protease enzyme activity. This work describes the optimized synthesis of an N-peptide-6-amino-d-luciferin conjugate (Fmoc-Gly-Pro-6-amino-d-luciferin) with a simple fragment condensation method in adequate yields. Fmoc-Gly-Pro-6-amino-d-luciferin was produced from a previously synthesized Fmoc-Gly-Pro-OH and also previously synthesized 6-amino-2-cyanobenzothiazole with an optimized method, to which conjugate cysteine was added in an also improved way. The resulting conjugate was successfully used in a bioluminescent system, in vitro, demonstrating the applicability of the method. Graphical Abstract: [Figure not available: see fulltext.]. © 2018, Springer Nature B.V