Discovery and characterization of Z1: an imipridone-based ClpP agonist with potent anti-staphylococcal activity

To identify novel antibacterial agents targeting Staphylococcus aureus ClpP (SaClpP), a target-based screening model was established using the fluorogenic substrate Suc-LY-AMC, leading to the identification of a potent hit, Z1, from a proprietary compound library. SDS-PAGE-based protein degradation assays confirmed that Z1 significantly enhances the proteolytic activity of SaClpP. Quantitative analysis demonstrated that the activating potency EC₅₀ of Z1 toward SaClpP is markedly superior to that of the clinical candidate ONC212. Mechanistic investigations revealed that 10 μM Z1 significantly increased the melting temperature (ΔTm) of SaClpP by 18.5 °C, outperforming ONC212 (ΔTm = 15 °C). The intracellular target engagement was further corroborated by the cellular thermal shift assay (CETSA). Isothermal titration calorimetry (ITC) determined a dissociation constant (Kd) of 2.95 μM, indicating an exothermic binding process (ΔH = -14.1 kcal/mol). The binding affinity and interaction mode were further characterized using biolayer interferometry (BLI), three-dimensional fluorescence spectroscopy, and 50 ns molecular dynamics simulations, the latter of which showed a stable root-mean-square deviation (RMSD) within 0.3 nm. Biologically, Z1 exhibited potent antibacterial activity against Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 33591 (MIC = 0.5 μg/mL), outperforming ONC212 (MIC = 1 μg/mL). Notably, Z1 demonstrated a low frequency of resistance (FOR) at the 10^-8 level and exhibited significantly lower cytotoxicity against HepG2 cells compared to ONC212. Pharmacokinetic (PK) evaluation in mice revealed a favorable oral bioavailability (F) of 33.50% and an oral half-life (t_1/2) of 1.92 h. In a murine skin infection model, Z1 significantly reduced bacterial burden, diminished the infection area, and improved histopathological outcomes. These findings highlight Z1 as a promising ClpP agonist and support the strategy of targeting ClpP for the development of next-generation antibiotics.