Streptococcus Mutans sortāzes A ekspresija, attīrīšana un raksturošana zāļu vielu skrīningam - Ģederte, Lote (Latvijas Universitāte, 2019)
Slimības, ko izraisa patogēni, īpaši multirezistenti, piemēram, Streptococcus mutans, ir nepieciešams apkarot. Multirezistentu grampozitīvu patogēnu baktēriju transpeptidāzes - sortāzes A - mērķproteīna inhibēšana mazina patogēnu saistīšanās spēju - virulenci - un līdz ar to arī rezistenci. Bakalaura darbā Streptococcus mutans sortāze A tiek izmantota kā instruments, lai pārbaudītu novatoriskus ķīmiskus savienojumus, kam piemīt spēja inhibēt sortāzi A. Mērķis bija, izmantojot zāļu vielu skrīninga metodi, atlasīt ķīmiskas vielas, kam piemīt sortāzes A inhibēšana un kuras varētu izmantot, lai apkarotu multirezistentu grampozitīvu patogēnu izraistītas slimības. Streptococcus mutans sortāzes A 40N-gala delēcijas rekombinants konstrukts tika ekspresēts, iegūstot proteīnu, kas tika attīrīts un kam tika pārbaudīta enzimātiskā aktivitāte. Zāļu vielu skrīningam izmantoja konstruktu ar papildus sešiem histidīniem, jo tas labāk ekspresējās. No eventuāliem, iepriekš zāļu skrīningā atlasītiem 14 inhibitoriem pēc detalizētas pārbaudes tika izraudzītas divi, kam piemīt vislabākā Streptococcus mutans sortāzes A delta N40 inhibēšanas spēja. Tās ir 4-(dimethylamino)-2-fluoro-N-[4-(pyrrolidin-1-yl) pyrimidin-5-yl]benzamide (C17H20FN5O) [7.viela] un 3-(1,3-dihydro-2-benzofuran-5-yl)-1-(4 hydroxyphenyl)urea (C15H14N2O3) [10.viela]. No šīm divām vielām specifiskākā Streptococcus mutans virulences ierobežošanā bija 7., jo tai bija specifiski augsta inhibēšana, tā praktiski neietekmē baktēriju augšanu un ir maz toksiska eikariotiskām šūnām. Bakalaura darbu izstrādāju Latvijas Biomedicīnas pētījumu un studiju centrā, Strukturālās bioloģijas pētījumu A. Leončika grupā.

It is necessary to combat diseases, caused by pathogens, especially multiresistants like Streptococcus mutans. Determination of enzymatic activity and inhibition of transpeptidase - sortase A - targetprotein reduces patogene potency of binding – virulence - and therefore also prevents the development of resistance. Streptococcus mutans sortaze A in the bachelor’s work is used as a tool to examine innovative, selected chemical compounds, which have the ability to inhibit sortase A. Main objective was to use drug substance screening method to sort chemicals with sortase A inhibition sortase A inhibitors with inhibitory properties and which could be used to combat multi-resistant gram-positive pathogens. The sortase A protein was expressed with the Streptococcus mutans sortase A 40 N-terminal domain deletion recombinant construct obtaining protein which was purified and whom enzymatic activity was tested. The resulting protein was purified and concentrated. The construct with additional six histidines was used for the screening of drug substances, because of its expressional properties. Two substances of the eventual previously selected in drug substance screening method 14 inhibitors with the best Streptococcus mutans sortase A delta 40 inhibitory properties were selected by detailed testing. These substances are: 4-(dimethylamino)-2-fluoro-N-[4-(pyrrolidin-1-yl)pyrimidin-5-yl]benzamide (C17H20FN5O) [7. substance] and 3-(1,3-dihydro-2-benzofuran-5-yl)-1-(4-hydroxyphenyl)urea (C15H14N2O3) [10. substance]. From the two selected substances the most specific one for the Streptococcus mutans virulence limiting was the number 7. because it has a high inhibition, which practically does not inhibit bacterial growth and is little toxic to eukaryotic cells. Bachelor’s thesis was developed in Latvian Biomedical Research and Study Center, A. Leončika Structural biology research group.
Streptococcus Mutans Sortase A expression, purification and representation of drug substances
Keywords: Bioloģija; Streptococcus mutans; sortāze A; multirezistence; virulence; zāļu vielu skrīnigs