9P5I

E. coli Dihydropteroate Synthase in complex with pterin-based inhibitor

This is a non-PDB format compatible entry.

Summary for 9P5I

• Entry DOI: 10.2210/pdb9p5i/pdb
• Related: 9P4A
• Descriptor: Dihydropteroate synthase, 7-amino-1-methyl-3-{[(1H-tetrazol-5-yl)amino]methyl}pyrimido[4,5-c]pyridazine-4,5(1H,6H)-dione (3 entities in total)
• Functional Keywords: inhibitor, transferase
• Biological source: Escherichia coli
• Total number of polymer chains: 2
• Total formula weight: 60270.83

Authors

Snoke, H.E.,Reeve, S.M.,Lee, R.E. (deposition date: 2025-06-18, release date: 2025-11-05, Last modification date: 2025-11-26)

Primary citation

Snoke, H.E.,Reeve, S.M.,Dharuman, S.,Wallace, M.J.,Loudon, V.C.,Zhao, Y.,Bowling, J.J.,Murphy, P.A.,Waddell, B.,Lee, R.B.,Bulitta, J.B.,Lee, R.E.
Development of Pyrimido Pyridazine Analogs through Increased Whole Cell Target Engagement of the Dihydropteroate Synthase Pterin Binding Site in Gram-Negative Bacteria.
Acs Infect Dis., 11:3237-3250, 2025

PubMed Abstract: Dihydropteroate synthase (DHPS) is a critical enzyme in the folate biosynthetic pathway of bacteria, fungi, and protozoans. Sulfonamides successfully target the aminobenzoic acid (ABA) binding site of DHPS, forming a false product that obstructs the formation of 7,8-dihydropteroate and disrupts subsequent reactions in the pathway. Pyrimido[4,5-]pyridazine-based inhibitors target the pterin binding site of DHPS, demonstrating high target affinity but minimal antimicrobial activity, which has previously been attributed to poor permeability without detailed analysis. In this study, we investigate the permeability limitations of our pyrimido pyridazine series in Gram-negative bacteria within the context of whole cell target engagement and cellular accumulation. To evaluate their whole cell target engagement against DHPS (DHPS), we developed a robust luminescence-based HiBiT cellular thermal shift assay and combined it with surface plasmon resonance and an LC-MS/MS-based accumulation assay. This orthogonal assay platform was used to reevaluate the SAR of our Legacy pyrimido pyridazine compound series against DHPS and to facilitate the design of an exploratory series of compounds with improved permeability. From this series, we found that the removal or replacement of the negatively charged carboxylic acid pyrimido pyridazine side chain with a thiotetrazole or a nitrile group resulted in increased accumulation, improved whole cell target engagement, and moderate antimicrobial activity against .

PubMed: 41116192
DOI: 10.1021/acsinfecdis.5c00635

Experimental method

X-RAY DIFFRACTION (2.141 Å)