Acurx Pharmaceuticals, Inc. · NASDAQ:ACXP
Acurx Pharmaceuticals — 8th International Clostridioides difficile Symposium (ICDS) 2024
15 slides from Acurx Pharmaceuticals's conference presentation, Sep 17, 2024
Slide 1: Structure of the replicative polymerase PolC in complex with ibezapolstat and a related inhibitor
Structure of the replicative polymerase PolC in complex with the anti-CDI agent ibezapolstat and a related inhibitor Mode of action and mechanism of resistance Wiep Klaas Smits Leiden University Center of Infectious Diseases LEIDEN, THE NETHERLANDS Created with Biorender
Slide 2: Conflict of interest statement
Conflict of interest statement The POLSTOP2 project is co-funded by a PPP Acurx Pharmaceuticals is a allowance made available by Health~Holland, Consortium partner in this project. Top Sector Life Sciences & Health, to stimulate public-private partnerships. Please do not share unpublished experiment
Slide 3: There is an urgent need for novel antimicrobials against C. difficile
•. There is an urgent need for novel antimicrobials against C. difficile • C. difficile epidemiology is influenced by antimicrobial resistance • Fluoroquinolone resistance in epidemic lineages • Resistance to therapeutics is increasingly reported • Resistance to fidaxomicin, vancomycin (VAN) and met
Slide 4: Ibezapolstat is a promising anti-CDI agent
Ibezapolstat is a promising anti-CDI agent • Ibezapolstat (IBZ, ACX-362E, GLS-362E) • Novel target: replicative DNA polymerase (PolC; a.k.a. DNA pol IIIC) • Improved activity towards C. difficile compared to “ancestral” HPUra • No pre-existing (cross)resistance (new-to-nature) • Phase 2: no recurren
Slide 5: Ibezapolstat also has activity vs other G+ priority pathogens
Ibezapolstat also has activity vs other G+ priority pathogens, suggesting broader utility for the class • PolC is conserved in Bacillota (Firmicutes) • Similar minimal inhibitory concentrations MRSA • No cross-resistance with existing classes of skin infections, blood infections, antimicrobials obse
Slide 6: A medium-throughput screen identified novel PolC-inhibitors
A medium-throughput screen identified novel PolC-inhibitors with activity against PolC of VRE and MRSA • Based on quenching of fluorescence in a primer extension reaction • Screen of >50 ACX inhibitors (guanine analogs) selected for inhibitory activity against MRSA/VRE/PRSP • IC50 – IBZ: 9 µM; ACX-8
Slide 7: 3D structure of PolC:inhibitor complex resolved by CryoEM at 2.8Å
3D structure of PolC:inhibitor complex was resolved by CryoEM at 2.8Å resolution • Exonuclease-inactivated PolC, N-terminal 6xHistag • Fork-mimicking oligonucleotide as DNA • First PolC structure with an intact exonuclease domain • Similar to Xray structure of PolC catalytic domain from Geobacillus
Slide 8: PolC:inhibitor complex provides direct evidence for mechanism as guanine analog
PolC:inhibitor complex provides direct evidence for a mechanism as guanine analog • ACX-801 density ~ guanine of dGTP, H-bond with template cytosine • Distant from catalytic residues for triphosphate hydrolysis Evans (2008) Proc Natl Acad Sci U S A 8 8th ICDS, Bled, Slovenia 17-Sep-24
Slide 9: PolC:inhibitor complexes show an unusual conformation of the inhibitor
PolC:inhibitor complexes show an unusual conformation of the inhibitor at the binding site • Pocket for moiety crucial for activity • Strong bend in molecule • Conservation in proximal residues • Similar for ACX-801 (2.8Å) and IBZ (3.1Å) template C ACX-801 IBZ ACX-801 ACX-801 Butler (1990) Nucleic A
Slide 10: polC mutations identified in strains with reduced susceptibility to PolC inhibitors
polC mutations are identified in strains with reduced susceptibility to PolC inhibitors • Multiple novel ACX compounds (CRO) or IBZ (LUMC) on multiple organisms • Whole genome sequencing (PE150) + SNP calling • The equivalent positions in E. faecium PolC: D1103Y, A1281T, F1276L/I/S • Phenylalanine m
Slide 11: Resistance positions are adjacent to the PolC-inhibitor binding site
Structure shows that resistance positions are located adjacent to the PolC-inhibitor binding site • Conserved A1281 and F1276 are within 4Å of ACX-801 binding A1281 • Non-conserved D1103 is located further away (~9-10Å) F1276 • May affect DNA binding by PolC, necessary for drug interactions D1103 •
Slide 12: Mutations in PolC affect in vitro polymerase activity and inhibition
Mutations in PolC affect in vitro polymerase activity and inhibition by PolC-inhibitors • A1281T: no activity in our real time assay; F1276L/S: clear polymerase activity (not shown) • F1276L: poorly inhibited by IBZ and ACX-801 • F1276S: poorly inhibited by ACX-801, but modestly by IBZ • Some mutati
Slide 13: polC p.F1258 mutations in C. difficile reduce susceptibility to ibezapolstat
Introduction of polC p.F1258 mutations in C. difficile leads to reduced susceptibility towards ibezapolstat • Plasmid carrying wild type or mutant polC alleles under native promoter • Correlates with observed effect on E. faecium PolC inhibition by IBZ • F1258L also identified in passage experiments
Slide 14: Key take-away points
Key take-away points • The active site in PolC is highly conserved between PolC’s of Bacillota • IBZ also inhibits E. faecium PolC (incl. VRE) -> IBZ does not foster VRE emergence • CryoEM structures of PolC from E. faecium in complex with ACX-801 and IBZ show an unexpected conformation of the inhib
Slide 15: Acknowledgements
Acknowledgements • The POLSTOP2 team: Mia Urem, Annemieke Friggen, Nina Musch and Meindert Lamers • Acurx Pharmaceuticals • LUMC’s EM Facility • Netherlands Centre for Electron Nanoscopy
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