Razvoj protibakterijskih učinkovin z delovanjem na validirane tarče v biosintezi peptidoglikana / Development of antibacterial compounds targeting validated enzymes in peptidoglycan biosynthesis

Okužbe, ki jih povzročajo bakterije, odporne na antibiotike, še vedno predstavljajo izziv zdravstvenim sistemom  po vsem svetu. Soočamo se z naraščajočo odpornostjo Gram-pozitivnih in Gram-negativnih patogenov, ki povzroča okužbe v bolnišnicah in skupnostih. Tako imenovane “superbakterije” so odporne na več antibiotikov hkrati in predstavljajo velik svetovni zdravstveni problem. Glede na to da imamo na obzorju le nekaj novih protimikrobnih zdravil in se soočamo z vse pogostejšo odpornostjo na več zdravil, moramo podvojiti naša prizadevanja pri iskanju novih protimikrobnih zdravil. Zato je nujno, da farmacevtski kemiki razvijamo nove protibakterijske spojine vodnice in klinične kandidate, ki delujejo na validirane protibakterijske tarče, saj le to omogoča razvoj novih protimikrobnih zdravil. Peptidoglikan je makromolekula, ki je bistvena za preživetje bakterij in edinstvena za bakterijsko celično steno. Encimi, vključeni v njegovo biosintezno pot, predstavljajo možne tarče za odkrivanje novih protimikrobnih zdravil. Med znotrajceličnimi encimi biosinteze peptidoglikana sta le dva encima validirana kot protibakterijski tarči z zdravili, ki se uporabljata v kliniki. MurA je validiran s fosfomicinom, ki se uporablja za zdravljenje urinalnih infekcij. DdlB je validiran s cikloserinom, ki je zdravilo drugega izbora za zdravljenje tuberkuloze. Ta dva encima bosta v tem projektu predstavljala tarči za načrtovanje novih zaviralcev na osnovi fragmentov (FBDD). S pomočjo FBDD indentificiramo nizko molekularne ligande (~150 Da), ki se vežejo na biološko pomembne makromolekule. Ko določimo eksperimentalni tridimenzionalni način vezave teh fragmentov, ga lahko uporabimo za optimizacijo fragmentov v bolj aktivne spojine vodnice. Poleg klasičnega pristopa FBDD, kjer iščemo fragmente z nekovalentnimi interakcijami s tarčnim encimom, bomo uporabili tudi inovativen pristop, pri katerem se FBDD uporablja za razvoj kovalentnih inhibitorjev. Kovalentni inhibitorji so spojine, ki so zasnovane tako, da se kovalentno vežejo na specifično molekulsko tarčo in tako zavirajo njeno biološko funkcijo. S skrbnim načrtovanjem so nedavno v kovalentne inhibitorje vpeljali visoko stopnjo selektivnosti, kar je privedlo do številnih kliničnih kandidatov in odobrenih zdravil. V tem projektu želimo doseči dva glavna cilja: cilj 1 je izvesti rešetanje knjižnice kovalentnih in nekovalentnih fragmentov na tarčnih encimih MurA in DdlB, cilj 2 pa je izbrati najboljše zadetke-fragmente in jih razviti v vodnice. Najprej bomo knjižnice fragmentov potestirali na ciljne encime in nato določili načine vezave fragmentov z rentgensko kristalografijo ali NMR. S pomočjo strukturno podprtega načrtovanja bomo generirali ideje za izboljšavo in rast fragmentov ter izboljšane spojine sintetizirali. Vse načrtovane spojine bomo farmakološko ovrednotili. Delo bo potekalo ponavljajoče se in tako omogočalo iterativni proces odkrivanja novih učinkovin, ki je sestavljen iz načrtovanja, sinteze, biokemijskega in strukturnega vrednotenja, pri čemer bo slednje uporabljeno za začetek nove razvojne iteracije.

ANG

Infections caused by antibiotic-resistant bacteria continue to challenge health-care systems worldwide. We face a growing resistance of Gram-positive and Gramnegative pathogens that cause infections in hospitals and in the community, with the so-called antibiotic-resistant ‘superbugs’ that now represent a major global health problem. The obstacles of having only a few new antimicrobials on the horizon and facing increasing frequency of multidrug resistance mean that we must redouble our efforts in the search for new antimicrobials. There is therefore an urgent need that medicinal chemists develop new antibacterial lead compounds and clinical candidates that bind to validated antibacterial drug targets, as this will enable development of novel antimicrobial drugs. Peptidoglycan is a macromolecule that is essential for bacterial survival and unique to the bacterial cell wall. The enzymes involved in its biosynthetic pathway constitute potential targets for the discovery of new antimicrobials. Among the intracellular enzymes of peptidoglycan biosynthesis, only two enzymes are validated antibacterial targets by inhibitors that are in clinical use: MurA is validated by fosfomycin, which is used for the treatment of urinary infections, and DdlB is validated by cycloserine, which is a second line drug for the treatment of tuberculosis. Therefore, these two enzymes will be targeted in the present Project by fragment-based drug discovery (FBDD). FBDD identifies low-molecular-weight ligands (~150 Da) that bind to biologically important macromolecules. The three-dimensional experimental binding mode of these fragments is determined and is used to facilitate their optimization into potent lead compounds. In addition to classical FBDD approach, where fragments with noncovalent interactions with the target protein are sought, we will also use an innovative approach, where FBDD is used to develop covalent inhibitors. Covalent inhibitors are compounds designed to bind covalently to a specific molecular target and thereby supress its biological function. By careful design, a high degree of selectivity has recently been built into covalent inhibitors, resulting in many clinical candidates and approved drugs. During this Project, we aim to achieve two major objectives: objective 1 is to perform screening of a library of covalent and non-covalent fragments on target enzymes MurA and DdlB, and objective 2 is to perform fragment-to-lead development of selected hit-fragments. First, libraries of fragments will be screened on target enzymes, and then the binding modes of fragments will be determined with X-ray crystallography or NMR. In the next step, structure-based design will be used to generate the ideas how to improve and grow the fragments, and these improved compounds will be synthesized. All target compounds will be pharmacologically evaluated. The work will be iterative and will proceed in a typical drug discovery iterative loop consisting of design, synthesis, biochemical evaluation and structural evaluation, where the latter will be used to start new development iteration.