Določanje protibakterijske aktivnosti izbranih naravnih spojin in zaviralcev proteina FtsZ

Oblak, Petra (avtor), Pajk, Stane (mentor), Hrast, Martina (komentor)

Protibakterijske učinkovine za zdravljenje bakterijskih okužb delujejo na principu selektivne toksičnosti: uničujejo patogene celice, na gostiteljeve pa nimajo vpliva. Antibiotiki so protibakterijske učinkovine naravnega ali polsinteznega, kemoterapevtiki pa popolnoma sinteznega izvora. Občutljivost bakterijskega seva na učinkovino opišemo z minimalno inhibitorno koncentracijo (MIK) in minimalno baktericidno koncentracijo. Široka, pretirana in napačna uporaba antibiotikov ter neustrezen nadzor bakterijskih okužb so privedli do pojava genetske in biokemijske odpornosti bakterij, ki predstavljajo veliko nevarnost za naše zdravje. Zato sta iskanje in razvoj novih protibakterijskih učinkovin nujna. V okviru magistrske naloge smo ovrednotili protibakterijsko aktivnost različnih spojin. Z mikrodilucijsko metodo na mikrotitrskih ploščah smo jim določili minimalne inhibitorne koncentracije. Flavonoidi niso pokazali bistvenega delovanja na Escherichia coli (MIK > 0,25 mM), na Staphylococcus aureus pa je najmočneje deloval FLAV-9 (MIK = 0,031 mM). Aktivnost rastlinskih ekstraktov smo testirali na bakterije in kvasovko Candida albicans, na katero ekstrakti niso pokazali učinka. Na S. aureus je z MIK ⡤ 3,91 μg/mL najmočneje deloval ekstrakt šentjanževke (EK-17), ki je imel najmočnejši učinek tudi na rast multirezistentnih MRSA. Med testiranimi β-laktamskimi antibiotiki je pokazal učinek na E. coli (MIK = 1 μg/mL ) in S. aureus (MIK = 0,5 μg/mL) le imipenem. Ciljanje in zaviranje bakterijskih iztočnih črpalk sta možnosti za zmanjšanje pojava in širjenja odpornosti. Izmed 9-ih testiranih zaviralcev iztočnih črpalk je na testirani bakteriji najmočneje deloval karbonil cianid m-klorofenil hidrazon (EPI-7). Filamentni temperaturno občutljivi protein Z (FtsZ) je zelo ohranjena bakterijska beljakovina s pomembno vlogo pri delitvi celice. Testirani zaviralci proteina so bili različno substituirani heterocikli, ki so večinoma vsebovali 1,4-benzodioksan, povezan preko metilenoksi mosta z 2,6-difluorobenzamidom. Močno delovanje sta na S. aureus in MRSA-e pokazala zaviralca FZ100 in FZ95. S testom šahovnice na mikrotitrskih ploščah smo ocenili potencialno kombinacijo zaviralcev iztočnih črpalk in zaviralcev proteina FtsZ kot terapevtsko možnost za zdravljenje bakterijskih okužb. Izračunane vrednosti indeksa delne inhibitorne koncentracije so ustrezale območju 0,5 – 4 (indiferentnost), kar pomeni, da zaviralca ne delujeta sinergistično, nimata vpliva drug na drugega in njun skupni učinek ni večji kot učinek posameznega zaviralca.

• Jezik: Slovenski jezik
• Ključne besede: protibakterijske učinkovine, bakterijska odpornost, minimalna inhibitorna koncentracija, protein FtsZ, zaviralec iztočne črpalke
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2021
• PID: 20.500.12556/RUL-127513
• Datum objave v RUL: 11.06.2021

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Determination of antibacterial activity of selected natural compounds and FtsZ protein inhibitors

Izvleček

Antibacterial agents for the treatment of bacterial infections work on the principle of selective toxicity: they destroy pathogenic cells and have no effect on the host. Antibiotics are antibacterial agents of natural or semi-synthetic, while chemotherapeutics are of synthetic origin. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration describe the susceptibility of a bacterial strain to the drug. Extensive and excessive misuse of antibiotics and inadequate control of bacterial infections have led to the emergence of genetically and biochemically resistant bacteria, which pose a great danger to our health. Therefore, the search for and development of new antibacterial agents has become necessary. In this master's thesis, we evaluated the antibacterial activity of various compounds, for which we determined the minimum inhibitory concentrations on the microtiter plates by the microdilution method. Flavonoids did not show significant activity on Escherichia coli (MIC > 0.25 mM), while FLAV-9 (MIC = 0.031 mM) had strong effect on Staphylococcus aureus. The activity of plant extracts was tested for bacteria and the yeast Candida albicans, on which the extracts showed no effect. St. John's wort extract (EK-17) had the strongest effect on S. aureus with MIC ⡤ 3.91 μg/mL, and also had the strongest effect on the growth of multidrug-resistant MRSA. Among the β-lactam antibiotics tested, only imipenem showed an effect on E. coli (MIC = 1 μg/mL) and S. aureus (MIC = 0.5 μg/mL). Targeting and inhibiting bacterial efflux pumps are options to reduce the occurrence and spread of resistance. Among 9 tested efflux pump inhibitors, carbonyl cyanide m-chlorophenyl hydrazone (EPI-7) had the strongest effect on the tested bacterium. Filamentous temperature-sensitive protein Z (FtsZ) is a highly conserved bacterial protein with an important role in cell division. The protein inhibitors were variously substituted heterocycles, which mostly contained 1,4-benzodioxane linked via a methyleneoxy bridge to 2,6-difluorobenzamide. FZ100 and FZ95 inhibitors showed strong activity on S. aureus and MRSAs. A potential combination of efflux pump inhibitors and FtsZ protein inhibitors as a therapeutic option for the treatment of bacterial infections was evaluated by a checkerboard test on microtiter plates. The calculated values of the fractional inhibitory concentration index corresponded to the range 0.5 – 4 (indifference). This means that the inhibitors do not act synergistically, have no effect on each other and their combined effect is as great as the effect of an individual inhibitor.

• Ključne besede: antibacterial agents, bacterial resistance, minimum inhibitory concentration, FtsZ protein, efflux pump inhibitor