ABS 016: Inhibition of RNase P by Analogs of Z25711365

Eli Groothuis ¹, Tingyi Zhu ¹, Michael Harris ¹

¹ Division of Chemical Biology, University of Florida

The Van Wickle Journal (2026) Volume 2, ABS016

Introduction: Antimicrobial resistance (AMR) is an escalating global health crisis driven by the rapid emergence of multidrug-resistant organisms, including the ESKAPE pathogens, which account for a large proportion of hospital-acquired infections worldwide. These bacteria possess an exceptional ability to evade existing antibiotics, highlighting the urgent need for therapeutics with novel mechanisms of action. One promising approach is targeting noncoding RNA (ncRNA), which offers structurally diverse binding sites for small molecules and may reduce the likelihood of resistance developing through traditional protein-target mutations.

This study explores the inhibition of Ribonuclease P (RNase P), an essential ribozyme composed of catalytic RNA and protein components that processes precursor transfer RNA (tRNA) by removing the 5′ leader sequence. Because RNase P activity is indispensable for bacterial survival, it represents a compelling antimicrobial target. The small molecule Z25711365 (Compound 5) was previously identified as an inhibitor of RNase P assembly in Staphylococcus aureus, with an IC₅₀ of K = 47.6895 ± 12.45459 μM, indicating moderate potency. Building on this result, structural analogues of Compound 5 were examined to identify the molecular features required for effective inhibition.

Methods: This experiment was conducted by building the enzyme-inhibitor complex using assembly mechanisms, then adding the t-RNA substrate and quenching the reaction at different time points ranging from 15 seconds to 30 minutes. Enzymatic activity was evaluated using gel electrophoresis assays that separated processed from unprocessed tRNA, enabling visualization of 5′ leader cleavage. Lastly, the gels were scanned to determine concentration of the fluorescent tag on the 5' leader sequence to determine the rate of the reaction over the time points collected.

Results: The results show that Z104557902 (Compound 5.01), which represents the minimal scaffold of Compound 5, showed no detectable inhibitory activity, likely due to the absence of functional groups necessary for productive target binding. Z26272197 (Compound 5.3), which represents a scaffold where a thiophene replaces the benzene on Compound 5, showed inhibitory activity with an IC₅₀ of K = 19.46 ± 1.95 μM.

Discussion: Overall, these findings establish a foundation for ncRNA-targeted antibiotic development. Future work will examine additional analogues, including Z26272197 (Compound 5.3), to clarify structure–activity relationships and optimize conditions for selective, specific RNase P inhibition. Future work will also investigate the mechanism of binding between the inhibitor and RNase P subunits.