Investigation of aminoacyl-tRNA syntetases as targets for antibiotics and how they develop resistance mechanism

VODITELJI PROJEKTA / PRINCIPAL INVESTIGATORS: prof. dr. sc. Ita Gruić Sovulj and Prof. dr. Nenad Ban

FINANCIRANJE / FUNDED BY:
Hrvatska zaklada za znanost / Croatian Science Foundation
Swiss National Science Foundation
(a bilateral Croatian-Swiss project)

TRAJANJE / DURATION: 01. 03. 2024. - 29. 02. 2028. / March 1st, 2024 to April 29th, 2028

ŠIFRA PROJEKTA / PROJECT IDENTIFIER: IP-2022-10-1400

AKRONIM / ACRONYM: ARSTARE

OPIS PROJEKTA

Sinteza proteina (translacija) središnji je biološki proces, učestalo inhibiran prirodnim i sintetskim antibioticima. Presudnu ulogu u translaciji imaju aminoacil-tRNA-sintetaze (AARS) koje povezuju aminokiseline s pripadnim tRNA za potrebe translacije na ribosomu. Pokazano je da su AARS dobre mete za razvoj antibiotika, a mupirocin kao inhibitor izoleucil-tRNA-sintetaze posjeduje odobrenje regulatornih agencija. U današnje vrijeme otkrivanje novih antibiotika je usporeno, dok pojava antibiotske rezistencije raste do alarmantnih razmjera. Stoga ovaj prijedlog projekta teži otkrivanju novih antibiotika iz skupine prirodnih spojeva usmjerenih na AARS. Za tu svrhu razvit ćemo novi stanični test za pretraživanje knjižnica prirodnih spojeva ili sirovih ekstrakata. Izolirat ćemo potencijalne antibiotike i ispitati mehanizam njihovog djelovanja na ciljnu sintetazu s kinetičkih i strukturnih aspekata. Istražit ćemo prirodne varijacije očuvanih motiva unutar aktivnog mjesta AARS i poveznicu s antibiotskom rezistencijom, te evolucijske prednosti takvih prirodnih varijacija. Proučavat ćemo osobitosti bakterijskih i eukariotskih AARS da bismo razjasnili osnove selektivnosti spram enzima patogena i humanih enzima. Prijedlog projekta temelji se na snažnoj suradnji glavnog prijavitelja (prof. Ita Gruić Sovulj) s velikim iskustvom u području reakcijskih mehanizama i biokemije AARS, prof. Nenada Bana (suprijavitelj s ETH Zürich) - vodećim ekspertom u strukturnoj biologiji translacije i prof. Jeffa Erringtona (suradnika s University of Sydney), svjetski priznatog bakterijskog genetičara i eksperta za prirodne spojeve kao terapeutike. Suradnja s eminentnim inozemnim znanstvenicima doprinijet će razvoju znanstvene izvrsnosti na Sveučilištu u Zagrebu i snažno će utjecati na znanstveno usavršavanje dva poslijedoktoranda i tri doktoranda koji će biti angažirani na projektu, te će pogodovati prijenosu vještina iz strukturne biologije i razvoja lijekova u Hrvatsku.

ABOUT THE PROJECT

Protein synthesis (translation) is a central biological process often targeted by natural and man-made antibiotics. Aminoacyl-tRNA synthetases (AARS) are key players that link a particular amino acid to its cognate tRNAs for delivery to the ribosome. AARSs are validated as good antibiotic targets, and mupirocin that inhibits isoleucyl-tRNA synthetase has been approved by FDA. The discovery of novel antibiotics occurs at a slow pace while antibiotic resistance increases rapidly. This contributes to the alarming situation in global health. Thus, this proposal aims to discover novel AARS-targeting antibiotics from the pool of natural products. In doing so, we will develop a new cell-based reporter assay for screening natural product libraries or crude extracts. The promising antibiotic(s) will be isolated and the mode of action against the target AARS kinetically and structurally examined. The evolutionary advantages of the natural variations in the most conserved motifs of AARS active sites and the link between these variations and antibiotic resistance will be explored. AARS bacterial- and eukaryote-specific features will be studied to understand the basis of selectivity for pathogen vs human enzymes. The proposal relies on strong collaborations of the main applicant (prof Ita Gruic Sovulj), highly experienced in the reaction mechanisms and biochemistry of AARSs, with prof. Nenad Ban (co-Applicant from ETH Zurich), a leading world expert in the structural biology of translation, and prof. Jeff Errington (a collaborator from the University of Sydney), a worldwide recognized bacterial geneticist also experienced in natural product therapeutics. Collaboration with such eminent scientists will promote scientific excellence at the University of Zagreb, strongly influence the scientific upbringing of two postdocs and three Ph.D students engaged with the project and bring novel expertise in structural biology and drug development to Croatia.

SURADNICI NA PROJEKTU / PROJECT TEAM:

Prirodoslovno-matematički fakultet, Zagreb

doc. dr. sc. Marko Močibob
doc. dr. sc. Morana Dulić
dr. sc. Igor Živković
Petra Kozulić, mag. chem.
Robert Pendelić, univ. mag. biol. mol.
Karla Peranić, mag. biol. exp.

University of Sydney, Australia

Prof. Jeff Errington
Dr. Yousef Dashti

ETH Zürich

Dr. Marc Leibundgut
Tanja Schönhut 

PUBLIKACIJE / PUBLICATIONS:

1. Živković, Igor; Dulic, Morana; Kozulic, Petra; Mocibob, Marko; Gruic‐Sovulj, Ita
   Kinetic characterization of amino acid activation by aminoacyl‐tRNA synthetases using radiolabelled γ‐[32P]ATP // FEBS Open Bio, (2024), 1-7. https://doi.org/10.1002/2211-5463.13903

2. Zivkovic, Igor; Gruic-Sovulj, Ita
   Exploring mechanisms of mupirocin resistance and hyper-resistance // Biochemical Society transactions, 52 (2024), 3; 1109-1120. https://doi.org/10.1042/BST20230581

POZVANA PREDAVANJA / INVITED LECTURES

1. Gruić Sovulj, Ita
   Aminoacyl-tRNA synthetases: mechanisms and antibiotic resistance
   Meeting of the EMBO communities, EMBO, October 29th - November 1st, 2024, Heidelberg, Germany

2. Gruić Sovulj, Ita
   Unexpected alteration in the signature motif of a class I aminoacyl-tRNA synthetase confers antibiotic hyper-resistance
   FEBS3+ Meeting: Exploring Molecular Frontiers, September 25th - 28th, 2024, Pula, Croatia

3. Živković, Igor; Maršavelski, Aleksandra; Gruić Sovulj, Ita
   How did negative catalysis tailor the specificity of isoleucyl-tRNA synthetases’s editing domain?
   FEBS3+ Meeting: Exploring Molecular Frontiers, September 25th - 28th, 2024, Pula, Croatia

4. Gruić Sovulj, Ita
   he Noncanonical Signature Motif of the Target Brings Antibiotic Hyper-Resistance
   Simpozij studenata bioloških usmjerenja / Symposium of Biology Students, May 25th -26th, 2024, Zagreb, Croatia

USMENA PRIOPĆENJA / ORAL PRESENTATIONS

1. Kozulić, Petra; Brkić, Alojzije; Leibundgut, Marc; Ban, Nenad; Gruić Sovulj, Ita
   Crucial residues for in vitro and in vivo function of Priestia megaterium isoleucyl-tRNA synthetase type 2
   Osmi mini simpozij medicinske i farmaceutske kemije HKD-a / 8th Mini Symposium of Section of Medicinal and Pharmaceutical Chemistry, November 19th, 2024, Zagreb, Croatia

POSTERSKA PRIOPĆENJA / POSTER PRESENTATIONS

1. Kozulić, Petra; Brkić, Alojzije; Leibundgut, Marc; Ban, Nenad; Gruić Sovulj, Ita
   Unique recognition of tRNA^Ile by isoleucyl-tRNA synthetase type 2 in Priestia megaterium
   FEBS3+ Meeting: Exploring Molecular Frontiers, September 25th - 28th, 2024, Pula, Croatia

2. Madunić, Ana; Živković, Igor; Močibob, Marko; Gruić Sovulj, Ita
   Atypical arginyl-tRNA synthetase of predatory bacteria Herpetosiphon aurantiacus and its potential protein interactors
   FEBS3+ Meeting: Exploring Molecular Frontiers, September 25th - 28th, 2024, Pula, Croatia

3. Kozulić, Petra; Brkić, Alojzije; Leibundgut, Marc; Ban, Nenad; Gruić Sovulj, Ita
   Molecular recognition of tRNA^Ile by the Priestia megaterium isoleucyl-tRNA synthetase type 2
   Ljetna konferencija Hrvatskog kemijskog društva Rijeka - Pula 2024 / Summer Conference of Croatian Chemical Society Rijeka - Pula 2024, September 5th and 6th, 2024, Rijeka, Croatia

OBRANJENI DIPLOMSKI RADOVI / DEFENDED MASTER THESES:

1. Knežević, Karla
   Optimizacija ekspresije i pročišćavanja atipičnih arginil-tRNA-sintetaza / Optimization of expression and purification of atypical arginyl-tRNA synthetases
   Gruić Sovulj, Ita (mentor). Zagreb, Prirodoslovno-matematički fakulte, Zagreb, 2024

2. Subota, Kristian
   Razvoj sustava in vivo za testiranje inhibicije triptofanil-tRNA sintetaze / Development of an in vivo tryptofanyl-tRNA synthetase inhibition assay
   Močibob, Marko (mentor). Zagreb, Prirodoslovno-matematički fakultet, Zagreb, 2025

NAGRADE I PRIZNANJA / AWARDS AND RECOGNITIONS

1. Madunić, Ana
   Potencijalni proteinski interaktori atipične arginil-tRNA-sintetaze predatorne bakterije Herpetosiphon aurantiacus / Potential protein interactors of atypical arginyl-tRNA synthetase from predatory bacterium Herpetosiphon aurantiacus
   Gruić Sovulj, Ita (mentor), Živković, Igor (komentor).
   Rektorova nagrada za individualni znanstveni i umjetnički rad, Sveučilište u Zagrebu, 2024. / Rector's Award for Individual Scientific and Artistic Work, University of Zagreb, 2024