doktorska disertacija
Rok Frlan (Avtor), Slavko Pečar (Mentor), Aleš Obreza (Komentor)

Povzetek

Zaradi naraščajoče rezistence na protibakterijske učinkovine postajata v današnjem času načrtovanje in sinteza novih protibakterijskih učinkovin vedno bolj aktualna. Med najbolj privlačnimi tarčami so encimi, udeleženi v biosintezi peptidoglikana, ki je ključna sestavina bakterijske celične stene, zato lahko vsaka motnja v tej makromolekuli vodi v propad bakterijske celice. Nekatere od začetnih stopenj v biosintezi peptidoglikana katalizirajo encimi iz skupine Mur ligaz (MurC, MurD, MurE in MurF), ki smo jih izbrali kot tarče pri našem raziskovalnem delu. V okviru doktorske naloge smo ovrednotili hipotezo, da so derivati sulfonohidrazidov lahko potencialni inhibitorji Mur ligaz. Pri tem smo uporabili sodobne pristope farmacevtske kemije, s katerimi smo načrtovali in sintetizirali več strukturno različnih novih sulfonohidrazidnih inhibitorjev Mur ligaz ter jih biokemijsko ovrednotili. V začetnem delu smo se osredotočili zlasti na encim MurC, za katerega ni bilo znanih veliko inhibitorjev, kasneje pa smo načrtovali tudi inhibitorje za ostale Mur ligaze. Eden od pristopov pri načrtovanju novih spojin je bilo posnemanje strukture znanih inhibitorjev MurC, benziliden rodaninov, ki smo jih uporabili kot spojine vodnice. Tiazolidin-2,4-dionski obroč v spojini vodnici smo najprej nadomestili s strukturno sorodnim necikličnim sulfonohidrazonskim skeletom, nato smo preko modifikacij vseh delov molekule poskušali povečati inhibitorno aktivnost na MurC. p-klorobenziltio substituent v spojini vodnici smo tako nadomestili z različnimi arilsulfonati, arilamidi in arilestri. Sintetizirali smo serijo spojin, za katere nam je Urad RS za zaščito intelektualne lastnine za omenjene spojine podelil patent št. SI22254, Evropska patentna organizacija pa je v obravnavo sprejela patent št. EP1845083 (A2). Kljub temu, da je bilo za te spojine sprva ugotovljena inhibitorna aktivnost v mikromolarnem območju, se je kasneje izkazalo, da gre po vsej verjetnosti za t.i.promiskuitetne inhibitorje.Vzporedno smo se lotili racionalnega načrtovanja novih inhibitorjev MurC na osnovi strukture aktivnega mesta encima. Z uporabo programske opreme za virtualno rešetanje in na podlagi objavljene kristalne strukture encima MurC s produktom encimsko katalizirane reakcije smo na osnovi sidranja in povezovanja strukturnih fragmentov v aktivnem mestu načrtovali in sintetizirali spojine s sulfonohidrazidnim skeletom, ki naj bi se vezale v vezavno mesto za difosfatni del substrata (oz. produkta). S kritičnim vrednotenjem rezultatov biokemijskih testiranj smo določili osnovne strukturne zahteve za doseganje dobre inhibitorne aktivnosti na MurC. Prva serija spojin, ki je vsebovala levcinski fragment, je izkazovala inhibitorno aktivnost v srednjem μM koncentracijskem območju in nam je služila kot osnova za nadaljnje delo. Enega prvih sulfonohidrazidnih inhibitorjev MurC (IC50 = 240 μM) smo testirali tudi na bakterijskih kulturah, kjer smo ugotovili šibko protibakterijsko aktivnost (MIC = 128 μg/mL, S. aureus).Sledeče modifikacije so bile usmerjene tako v povečanje topnosti molekul kot tudi v izboljšanje inhibitorne aktivnosti spojin. V ta namen smo levcin zamenjali s treoninom,sulfonohidrazidni fragment pa smo pri nekaterih spojinah nadomestili s hidrazidnim. Uporabili smo različno substituirane aromatske obročne sisteme z namenom povečanja možnosti tvorbe H-vezi med aktivnim mestom encima in polarnimi skupinami na aromatskih obročih. Eden od poskusov doseganja večje inhibitorne aktivnosti je bila tudi rigidizacija spojine, kjer smo namesto levcina uporabili prolin. Kljub dobremu prileganju v aktivno mesto pa nobena od sintetiziranih spojin v tej seriji ni izkazovala inhibitorne aktivnosti. V nadaljevanju smo nato sintetizirali spojine, ki so poleg mimetikov difosfatnega dela substrata (oz. produkta) vsebovale tudi krajše peptidne fragmente, ki so posnemali peptidne dele naravnih substratov encimov MurB, MurC, MurD in Mur E. Z namenom tvorbe močnejših interakcij z aktivnim mestom encimov smo pri nekaterih inhibitorjih namesto karboksilnega dela peptidnih fragmentov uporabili hidroksamske kisline. Kot mimetik difosfatnega dela substrata (oz. produkta) smo v tem primeru uporabili različne mimetike difosfatnega dela substrata (oz. produkta): sulfonamidokarbamatni, sulfonamidotreonin in sulfonamidotreoninohidrazidni mimetik. Spojine smo rigidizirali s fenilnim obročem, katerega smo substituirali s prej omenjenimi mimetiki difosfatnega dela substratov (oz. produktov) in s peptidnimi fragmenti. Na ta način nam je uspelo sintetizirati več serij spojin, od katerih je ena spojina izkazovala inhibitorno aktivnost na MurC v srednjem μM območju. Z opisanimi pristopi nam je uspelo sintetizirati kar nekaj spojin, ki smo jih testirali in vitro na izoliranih bakterijskih encimih. Ob tem smo ugotovili, da so nekatere spojine izkazovale inhibitorno aktivnost v srednjem mikromolarnem koncentracijskem območju.Tekom te doktorske disertacije smo ugotovili, da je sulfonohidrazidni fragment primeren gradnik za načrtovanje inhibitorjev ligaz Mur. Z uporabo pristopov strukturno podprtega načrtovanja smo namreč dosegli, da so se spojine s sulfonohidrazidnim delom v našem modelnem sistemu prilegale v bližino vezavnega mesta za difosfat. eHITS se je pri tem izkazal kot primerno orodje za načrtovanje novih učinkovin, saj nam je omogočil napoved aktivnosti z več kot 50% zanesljivostjo. Z uporabo eHITS in iz rezultatov biokemijskih testov smo spoznali, da se lahko sulfonohidrazidni fragment zamenja s strukturno sorodnim hidrazidnim fragmentom, vendar pri tem tvegamo zmanjšanje topnosti spojin. Prav tako smo ugotovili, da so se kot najbolj optimalne za doseganje inhibitorne aktivnosti izkazale spojine z levcinom v svoji osnovni strukturi. S pomočjo modifikacij v aromatskem delu smo spoznali, da ta del molekule dopušča različne variacije in posledično omogoča izboljševanje inhibitorne aktivnosti tako na MurC kot tudi na MurD. Omenjena spoznanja, dajejo pomemben doprinos k razumevanju odnosa med strukturo in delovanjem pri načrtovanju novih inhibitorjev encimov, udeleženih v biosintezi bakterijske celične stene.

Ključne besede

bakterije;učinkovine, protibakterijske;rezistenca;sulfonohidradini derivati, sinteza;sintezne spojine;inhibitorji Mur ligaz;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.08 - Doktorska disertacija
Organizacija: UL FFA - Fakulteta za farmacijo
Založnik: [R. Frlan]
UDK: 547.057
COBISS: 2542449 Povezava se bo odprla v novem oknu
Št. ogledov: 292
Št. prenosov: 43
Ocena: 0 (0 glasov)
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Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: Design, synthesis and evaluation of sulfonohydrazide inhibitors of Mur ligases
Sekundarni povzetek: Due to increasing resistance to antibacterial agents the design and synthesis of new antibacterial agents is becoming a new focal point of the field. Among the most validated targets for new antibacterial compounds are enzymes which are a part of peptidoglycan biosynthetic pathway constituting an essential part of the bacterial cell wall and thus any anomaly in this macromolecule can lead to cell lysis. Some of the initial stages of the peptidoglycan biosynthesis are catalyzed by enzymes from the Mur ligase family (MurC, MurD, MurE and MurF), which were chosen as targets in our research. We evaluated a hypothesis stating that sulfonohydrazides could be designed to inhibit Mur ligases. Various structurally diverse new sulphonohydraside inhibitors of Mur ligases were designed, synthesized and biochemically evaluated using modern approaches in medicinal chemistry. Initially, we focused mainly on MurC enzyme for which only few inhibitors had been previously known. However, during our continuous research inhibitors of other Mur ligazes were also designed. One of the approaches was to use benzylidenerodanines as lead compounds which are well-documented MurC inhibitors. Leads were then further modified on the thioxothiazolidin-4-one ring which was first replaced by structurally similar non-cyclic sulfonohydrazone moiety. All other parts of molecule were then modified in an attempt to increase the inhibitory activity on MurC. p-chlorobenzil substituent in the lead compound was thus replaced by various arylsulfonates, aryamides, and arylesters. We have also acquired the right to the Slovenian patent by the The Slovenian Intellectual Property Office. However, despite the manifested inhibitory activity in the micromolar region it has been afterwards discovered that this inhibitory activity was most probably the result of a nonspecific promiscuous binding activity. On a parallel line a more rational design based on the structure of the enzyme’s active site to new MurC inhibitors was attempted. Using published X-ray crystal structures of MurC complexed with the reaction product, computer based virtual screening and de novo design we prepared new sulfonohydrazide moiety based inhibitors, which, according to our model, were designed to bind into diphosphate binding pocket with sulfonohydrazide moiety. Basic structural requirements for good inhibitory activity against MurC using critical evaluation of of biochemical testing results were depicted. The initial hits with leucine as a building block were having the inhibitory activity in the middle micromolar range and were used in further studies. One of the first inhibitors with IC50 of 240 μM was also found to posses weak antibacterial activity with MIC of 128 μg/mL against S. aureus. Further modifications were directed towards increased solubility of compounds as well as towards their increased inhibitory activity. Leucine was therefore replaced by threonine and in some compounds sulfonohydrazide was also replaced by hydrazide. With the aim of increasing the number of H-bonds between the compounds and the enzyme, differently substituted arenes were used. Rigidisation of compounds where proline was used instead of leucine was also one of the attempts in increasing the potency. However, despite promising docking results no compound was found active. In a search for new compounds with the potential to inhibit MurB, MurC, MurD and MurE, we also designed a series of compounds which consisted of peptide mimetic, diphosphate mimetic and phenyl substitute for sugar residue, which orients both mimetics towards the proper conformation and, to some level restricts the molecule’s degrees of freedom. Sulfonylcarbamates, sulfonamidothreonine and sulfonamidothreoninohydrazine were used as pyrophosphate mimetics. The hydroxamic acid functionality, a well-known metal complex forming group, was also introduced into some of the mimetics, in order to form strong interactions with the Mg2+ in the enzymes’ active sites. This series of compounds was tested and one was found to be active against MurC in middle μM range. With the procedures described above we managed to synthesize several inhibitors with their inhibitory activities in the middle μM range. In the course of our scientific research we found sulfonohydrazide to be a suitable fragment in the design of novel inhibitors of Mur ligases. Using structure-based design the compunds were efficiently docked into the vicinity of diphosphate binding pocket. Furthermore, using eHITS we were able to predict inhibitory activity with over 50% accuracy and therefore this docking software proved to be a suitable tool for the design of new inhibitors. According to the results of the eHITS and of biochemical testing it was concluded that sulfonohydrazide fragment could be replaced with structurally similar hydrazide fragment, however, a risk of lower solubility of designed compounds was present. In addition, leucine was found to be the most suitable building block for the inhibitory activity. It has also been established that both phenyl fragments enable several modifications which can be used to tune up the inhibitory activity towards MurC as well as MurD. These important discoveries, which have been made in the disertation, contribute significantly to the understanding of the structure activity relationship in the design of novel inhibitors of Mur ligases, which participate in bacterial cell wall biosynthesis.
Vrsta dela (COBISS): Doktorska disertacija
Komentar na gradivo: Univ. Ljubljana, Fak. za farmacijo
Strani: 152 str.
ID: 12895811