Slide 1: Novel antibiotics inhibiting DNA polymerases from Gram-positive bacteria
Novel antibiotics inhibiting DNA polymerases from Gram-positive bacteria Mia Urem Leiden, The Netherlands MACHINES ON GENES 2025 Art by Ella Maru Studio Insert > Header & footer 1 5-Jun-25
Slide 2: The urgent need for novel antibiotics
The urgent need for novel antibiotics • Antibiotic resistance is a silent pandemic • Predicted to cause 10 million deaths annually by 2050 (WHO) • In the last 30 years only 1 novel antibiotic with new mode-of-action We need: • new chemotypes • new targets
Slide 3: DNA polymerase PolC: novel antibiotic target
DNA polymerase PolC: novel antibiotic target New chemotype • class of guanine analogues • specifically inhibit PolC New Target • PolC is the replicative DNA pol in Gram+ • Clinically unexploited • Distinct structure from human DNA polymerases Xu (2019) Bioorg Med Chem CDC: https://www.cdc.gov/antimi
Slide 4: First-in-class: Ibezapolstat (Ibz) entering Phase 3 trials
First-in-class: Ibezapolstat (Ibz) entering Phase 3 trials Phase 2 clinical trials • eradicates C. difficile infection (gut) Ibz • favourable safety profile • narrow spectrum → microbiome-sparing Garey (2022) Clin Infect Dis / Garey (2020) J Antimicrob Chemother PolC- guanine Ibz specifically inhibi
Slide 5: Modified core improves absorption & pharmacokinetics
Modified core improves absorption & pharmacokinetics Basis for next generation inhibitors • Screening: inhibition of bacterial growth → Gram+ specific • Assess candidates: inhibition of polymerase activity → PolC specific • ACX-801 was identified as a potent inhibitor Only PolC activity inhibited in
Slide 6: PolC-bound inhibitors adopt non-planar conformation
PolC-bound inhibitors adopt non-planar conformation • Cryo-EM structure to elucidate mode-of-action of Ibz and ACX-801 • Nucleobases: base-pair with dCMP in template strand • The N2-linked aromatic groups - perpendicular to the nucleobase - not observed in solution (NMR) PolC from E. faecium (VRE) -
Slide 7: Highly conserved aromatic residues form the binding pocket
Highly conserved aromatic residues form the binding pocket • π-π stacking interactions with N2 aromatic group • F1276 with N2 group and nucleobase • F1276 is a hotspot for lab-evolved mutations • Mutants retain active but decrease susceptibility
Slide 8: The inhibitors induce pocket formation by displacing the aromatic residues
The inhibitors induce pocket formation by displacing the aromatic residues • Pocket not observed in absence of inhibitor (apo) • H1185: not conserved in Gram- • F1276: hotspot for lab-evolved resistance mutations Movement required to create a ACX-801 vs Apo full inhibitor binding pocket
Slide 9: Key to inhibitor mode-of-action
Key to inhibitor mode-of-action: • Pocket formation through induced conformational change • Dependent on non-planar inhibitor conformation Guide rational design of new compounds with improved inhibitory activity
Slide 10: Acknowledgments
Acknowledgments POLSTOP2 team Annemieke Friggen, Nina Musch Wiep Klaas Smits, Meindert Lamers & students LUMC EM Facility Netherlands Centre for Electron Nanoscopy