Solid-phase synthesis and biological evaluation of des-hydroxy pseudouridimycin analogs

Area of Science:

• Medicinal Chemistry
• Structural Biology
• Microbiology

Background:

• Pseudouridimycin (PUM) is a C-nucleoside/peptide antibiotic effective against drug-resistant bacteria by inhibiting bacterial RNA polymerase (RNAP).
• PUM's clinical utility is limited by chemical instability, specifically self-immolative cleavage of its central hydroxamate bond.

Purpose of the Study:

• To determine the structural basis of PUM binding to bacterial RNAP.
• To design and synthesize chemically stabilized PUM analogs with enhanced inhibitory activity against RNAP.

Main Methods:

• Cryo-electron microscopy (cryo-EM) was used to visualize PUM and a stabilized analog bound to *Escherichia coli* RNAP.
• Solid-phase synthesis was employed to generate a library of 50 des-hydroxy PUM analogs with modifications at the Gln residue and Gdn-Gly tail.

Main Results:

• Cryo-EM structures revealed the binding conformation of PUM and its stabilized analog within the RNAP transcription complex.
• Several synthesized analogs demonstrated low-micromolar RNAP inhibitory activity.
• A *para*-substituted phenyl amidine analog (compound 54) exhibited potent inhibition with an IC50 of 0.95 μM.

Conclusions:

• The study provides a structural framework for understanding PUM-RNAP interactions.
• An efficient synthetic platform was established for developing stabilized PUM derivatives.
• These findings lay the groundwork for developing novel RNAP-targeted therapeutics against resistant bacterial infections.