Analiza bakterijskega encima Ddl in njegovih zaviralcev z uporabo prosto dostopnih bioinformacijskih orodij

Forštner, Patricia (author), Frlan, Rok (mentor)

Bakterijska odpornost velja za enega največjih svetovnih javnozdravstvenih problemov in je v naglem porastu. Neupravičena in neprimerna uporaba antibiotikov je prispevala k pojavu večkratno odpornih bakterij, katerih okužb ni več mogoče zdraviti s konvencionalnim protibakterijskim zdravljenjem. Zdravljenje takšnih okužb je omejeno, zato je nujen razvoj novih protibakterijskih učinkovin. Peptidoglikan, ki je glavna in esencialna komponenta bakterijske celične stene, pri tem predstavlja idealno tarčo za razvoj novih protibakterijskih učinkovin, saj z zaviranjem njegove biosinteze ali z njegovo specifično razgradnjo dosežemo selektivno toksično delovanje s prokariontsko celično lizo tako po Gramu negativnih kot po Gramu pozitivnih bakterij. V okviru naše magistrske naloge smo s pomočjo prosto dostopnih bioinformacijskih orodij in programov ovrednotili primernost bakterijskega encima D-alanin-D-alanin ligaze (Ddl), ki je pomemben encim v biosintezni poti peptidoglikana kot tarče za nove protibakterijske učinkovine. Encim smo analizirali z vidika lastnosti, ki so pomembne pri napovedovanju perspektivnosti tarč, kot so esencialnost, evolucijska ohranjenost in sposobnost tvorbe interakcij. Oblikovali smo seznam do sedaj znanih najbolj potencialnih zaviralcev encima in jih z namenom ocene njihove podobnosti z učinkovinami in spojino vodnico analizirali s sodobnimi pristopi farmacevtske kemije. Ugotovili smo, da je Ddl dokazano esencialen za 30 organizmov. S kombinacijo analize evolucijske ohranjenosti in tvorbe interakcij smo identificirali 11 ključnih aminokislin, za katere velja, da so evolucijsko glede na ostale položaje bolj ohranjene, so del aktivnega mesta, kjer tvorijo večino interakcij z ligandoma (ATP in dipeptid D-alanin-D-alanin) ter prispevajo k najvišjim deležem tvorbe interakcij vzdolž celotnega encima. 11 ključnih aminokislin so predstavljali aminokislinski ostanki: Glu-15, His-63, Glu-68, Lys-97, Lys-144, Ser-151, Glu-187, Glu-270, Asn-272, Gly-276 in Arg-255. Ugotovili smo, da so s stališča načrtovanja novih zaviralcev Ddl najbolj obetavne aminokisline, ki so v vezavnem mestu za substrat oz. produkt kemijske reakcije, ki jo katalizira Ddl in aminokisline, ki so v vezavnem mestu za fosfatni del molekule ATP. Naredili smo izbor petih najbolj perspektivnih zaviralcev, ki so celostno gledano na ovrednotene fizikalno-kemijske lastnosti, lipofilnost, vodotopnost, farmakokinetične lastnosti, podobnost z učinkovinami in druge lastnosti, vezane na farmacevtsko kemijo zaviralcev, kazali boljše lastnosti kot trenutno edina zdravilna učinkovina v klinični praksi z delovanjem na encim Ddl (cikloserin).

• Language: Slovenian
• Keywords: Ddl, esencialnost, evolucijska ohranjenost, zaviralci, bioinformacijska orodja
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2021
• PID: 20.500.12556/RUL-127422
• Publication date in RUL: 05.06.2021

Secondary language

• Language: English
• Title: Analysis of the Ddl bacterial enzyme and its inhibitors using freely available bioinformatics tools

Abstract

Bacterial resistance is one of the world’s biggest public health problems and it is getting worse. Improper and inappropriate use of antibiotics has led to the rise of multidrug-resistant bacteria, which cause infections that can no longer be treated with conventional antibacterial treatments. Treatment of such infections is limited, and therefore the development of novel antibacterial agents is necessary. Peptidoglycan being the essential and specific component of the bacterial wall, poses the ideal target for the development of new antimicrobial agents, as by inhibiting its biosynthesis or its specific degradation, selective toxicity is achieved by prokaryotic cell lysis of both, Gram-negative and Gram-positive bacteria. As part of our dissertation, we have evaluated the suitability of the bacterial enzyme D-alanine-D-alanine ligase (Ddl), which is an important enzyme in the biosynthetic pathway of peptidoglycan, as the target for new antibacterial agents, using freely available bioinformatics tools and programs. We have analyzed the enzyme in terms of properties that are important when predicting the prospects of suitable targets, such as essentiality, evolutionary conservation, and the ability to form interactions. We have established a list of the most potential enzyme inhibitors known to date, and analyze them, using modern approaches of the pharmaceutical chemistry in order to evaluate their drug-likeness and lead-likeness. We have found that the Ddl is proven to be essential for 30 organisms. With a combination of evolutionary analysis and formation of interactions, we have identified 11 key amino acids that are more evolutionary conserved than the other sites, are part of an active site where they form most interactions with ligands (ATP and D-alanine-D-alanine dipeptide) and contribute to the highest proportions of interaction formation along the whole enzyme. Amino acid residues that represented the 11 key amino acids: Glu-15, His-63, Glu-68, Lys-97, Lys-144, Ser-151, Glu-187, Glu-270, Asn-272, Gly-276 and Arg-255. We have selected the five most promising enzyme inhibitors that exhibited better physicochemical properties, lipophilicity, pharmacokinetic properties, drug-likeness and other properties related to the pharmaceutical chemistry of inhibitors, than the only current active substance in clinical practice that acts on the enzyme Ddl (cycloserine).

• Keywords: Ddl, essentiality, evolutionary conservation, inhibitors, bioinformatics tools