Fehérjék előállítása kémiai módszerekkel szerkezeti és funkcionális vizsgálatok céljára = Total synthesis of proteins for structural and functional studies

Szerkezeti és funkcionális vizsgálatokhoz szükséges specifikusan módosított fehérjék előállítására alkalmas lépésenkénti natív kémiai ligációt értem el két rekombináns fehérje és egy szintetikus, biotinnal jelzett peptid kapcsolásával. Az N-terminális reaktív csoport (Cys) átmenti védelmére Met-Met dipeptidet alkalmaztam, amelyet a következő ligációs reakció előtt enzimatikus úton, mutáns metionin aminopeptidáz enzimmel távolítottam el. A félszintetikus trimer terméket sztreptavidin affinitáskromatográfia segítségével izoláltam. A prion fehérje sejtmembránhoz horgonyzásához alkalmas koleszterin-trisz-etilén-glikol származékot állítottam elő. Konfokális mikroszkópiás vizsgálatok rámutattak, hogy a fluoreszceinnel jelzett koleszterin-trisz-etilén-glikol származék stabilan beágyazódik a liposzóma membránba, ami fuzionáltatható egér Zpl idegsejtekkel. Vagyis a horgonymimetikum alkalmas fehérjék sejtmembránhoz horgonyzására. | The structural and functional investigation of proteins requires specific protein derivatives. A consecutive native chemical ligation method was developed for the preparation of such unique molecules, and the ligation of two recombinant protein and one synthetic biotinylated peptide was achieved. For the temporary protection of the N-terminal reactive group (Cys), the dipeptide Met-Met was applied, that was enzymatically removed before the next ligation step by a mutant methionine aminopeptidase. The semisynthetic trimeric prduct was isolated by streptavidin affinity chromatography. A tris(ethylene glycol) derivative of cholesterol was prepared to investigate its possible application for the membrane anchoring of the prion protein. Confocal microscopic studies revealed that the fluorescent labeled cholesterol derivative associates effectively with liposome membranes, and this labeled liposomes can be fused to mouse neuronal cell lines. Thus, the amphipathic cholesterol molecule is presumably appropriate for the membrane anchoring of the prion protein

Discovery of orexant and anorexant agents with indazole scaffold endowed with peripheral antiedema activity

CB1 receptors and endocannabinoids are integrated components of neuronal networks controlling different organism’s functions, such as appetite and food intake in the hypothalamus. A series of Rimonabant/Fubinaca hybrids have been synthesized in solution as C-terminal amides, acids, methyl esters and N-methyl amides. These compounds have been studied in cannabinoid receptor binding assay and functional receptor assay in vitro, the most active among them as agonist (LONI 11) and antagonist (LONI 4) were tested in vivo to evaluate their ability to stimulate or suppress the feeding behavior after i.p. administration. For LONI 11 formalin test and tail flick tests after s.c. and i.c.v. routes respectively, were also performed in vivo with the aim to investigate the antinociceptive effect at the central or peripheral level. In the Zymosan-induced edema and hyperalgesia, LONI 11 reduced the % paw volume increase and % paw latency after s.c. administration, also suggesting a potential anti-inflammatory activity at the periphery. Keywords. Cannabinoid receptor, Rimonabant, food intake, anorexant agent, edema

Selective antiproliferative effect of C-2 halogenated 13α-estrones on cells expressing Organic anion-transporting polypeptide 2B1 (OATP2B1)

Organic anion-transporting polypeptide 2B1 (OATP2B1) is a multispecific transporter mediating the cellular uptake of steroids and numerous drugs. OATP2B1 is abundantly expressed in the intestine and is also present in various tumors. Increased steroid hormone uptake by OATP2B1 has been suggested to promote progression of hormone dependent tumors. 13 alpha-estrones are effective inhibitors of endogenous estrogen formation and are potential candidates to inhibit proliferation of hormone dependent cancers. Recently, we have identified a variety of 13 alpha/beta-estrone-based inhibitors of OATP2B1. However, the nature of this interaction, whether these inhibitors are potential transported substrates of OATP2B1 and hence may be enriched in OATP2B1overexpressing cells, has not yet been investigated. In the current study we explored the antiproliferative effect of the most effective OATP2B1 inhibitor 13 alpha/beta-estrones in control and OATP2B1-overexpressing A431 carcinoma cells. We found an increased antiproliferative effect of 3-O-benzyl 13 alpha/beta-estrones in both mock transfected and OATP2B1-overexpressing cells. However, C-2 halogenated 13 alpha-estrones had a selective OATP2B1-mediated cell growth inhibitory effect. In order to demonstrate that increased sensitization can be attributed to OATP2B1-mediated cellular uptake, tritium labeled 2-bromo-13 alpha-estrone was synthesized for direct transport measurements. These experiments revealed increased accumulation of [H-3]2-bromo-13 alpha-estrone due to OATP2B1 function. Our results indicate that C-2 halogenated 13 alpha-estrones are good candidates in the design of anti-cancer drugs targeting OATP2B1

Neuropeptidek radioaktív jelölése = Radioactive labelling of neuropeptides

Új jelölési módszereket fejlesztettünk ki neuropeptidek tríciummal való jelölésére. A módszerekhez új aminosavak és új neuropeptid prekurzorokra volt szükségünk. Aliciklikus béta-aminosavakat és ezek telítetlen analógjait szintetizáltunk vagy vásároltunk, racém vagy optikailag aktív formában. Az új aminosavakat felhasználtuk prekurzor peptidek szintéziséhez. A diasztereomer peptidek elválasztása radioaktív vagy nem radioaktív formában HPLC-vel történt. A radiojelölés általában trícium gázzal kettős kötés telítésével vagy dehalogénezéssel történt Pd katalizátorok jelenlétében. Az új módszerek között van a diszulfid kötést tartalmazó peptidek előállítása, valamint alaninban és valinban történő jelölés. Az aliciklikus béta-aminosavak radioaktív jelölése az első alkalommal valósult meg a jelöléstechnikában így ez a módszerünk úttörőnek tekinthető. Az így nyert radioaktív peptidek sokkal stabilabbak mint a természetes peptidek a biológiai rendszerekben. A 13 új radioaktív peptidünk (opioid peptidek, Substance P (1-7), Arg-vazopresszin) közül 3 peptidünk (Try-3H-ACPC-Phe-Phe-NH2, Dmt-3H-Pro-Phe-Phe-NH2, 3HDmt-DArg-Phe-Lys-NH2) kereskedelmi termék lett, ezen túlmenően radioaktív peptidjeink nemzetközi kutatási együttműködéseket és nemzetközi pályázatot is eredményezett. | Novel tritium labeling methods were developed for labeling of neuropeptides. New unnatural amino acids and neuropeptide precursors were needed for the labeling methods. Alicyclic-beta-amino acids and their analogues with unsaturated bond were synthesized or bought in raceme or optical pure forms. The new amino acids were used to the synthesis of the precursor peptides. The radio labeling occurred with tritium gas using catalytic saturation or dehalogenation methods using Pd catalyst. The diastereomer peptides in radioactive or no radioactive form were separated by HPLC. Among the new methods was the preparation of peptide containing disulphide bridge and radioactive labeling of peptides where the labels were in the alanine or valine residue. Pioneer method was developed when the label was introduced into the alicyclic beta-amino acids. These peptides were more stable than natural peptides in biological systems. Our 3 radioactive peptides (Dmt-DArg-Phe-Lys-NH2, Dmt-Pro-Phe-Phe-NH2, Tyr-Acpc-Phe-Phe-NH2) among 13 new peptides (opioid peptides, Substance P (1-7, 8Arg-vasopressin) became commercial product . Our radioactive peptides resulted in international research cooperation and international project

Ring size in cyclic endomorphin-2 analogs modulates receptor binding affinity and selectivity

The study reports the solid-phase synthesis and biological evaluation of a series of new side chain-to-side chain cyclized opioid peptide analogs of the general structure Tyr-[D-Xaa-Phe-Phe-Asp]NH2, where Xaa = Lys (1), Orn (2), Dab (3), or Dap (4) (Dab = 2,4-diaminobutyric acid, Dap = 2,3-diaminopropionic acid), containing 17- to 14-membered rings. The influence of the ring size on binding to the MOP, DOP and KOP opioid receptors was studied. In general, the reduction of the size of the macrocyclic ring increased the selectivity for the MOP receptor. The cyclopeptide incorporating Xaa = Lys displayed subnanomolar MOP affinity but modest selectivity over the KOP receptor, while the analog with the Orn residue showed increased affinity and selectivity for MOP. The analog with Dab was a weak MOP agonist and did not bind to the other two opioid receptors. Finally, the peptide with Xaa = Dap was completely MOP receptor-selective with subnanomolar affinity. Interestingly, the deletion of one Phe residue from 1 led to the 14-membered Tyr-c[D-Lys-Phe-Asp]NH2 (5), a potent and selective MOP receptor ligand. The in vitro potencies of the new analogs were determined in a calcium mobilization assay performed in Chinese Hamster Ovary (CHO) cells expressing human recombinant opioid receptors and chimeric G proteins. A good correlation between binding and the functional test results was observed. The influence of the ring size, solid support and the N-terminal protecting group on the formation of cyclodimers was studied