Identification of active main metabolites of anti‐infective inhibitors of the macrophage infectivity potentiator protein by liquid chromatography using mass detection

• Published in: Archiv der Pharmazie (Weinheim) 2024-08, Vol.357 (8), p.e2400032-n/a
• Main Authors: Lohr, Theresa, Scheuplein, Nicolas Julian, Jenkins, Christopher, Norville, Isobel, Erk, Christine, Stapf, Maximilian, Kirchner, Lukas, Sarkar‐Tyson, Mitali, Holzgrabe, Ulrike
• Format: Article
• Language: English
• Subjects: active metabolites; Chromatography; Drug development; gram‐negative bacteria; LC/MS‐MS; liver microsomes; macrophage infectivity potentiator protein (Mip); Metabolites; Virulence
• ISSN: 0365-6233; 1521-4184; 1521-4184
• DOI: 10.1002/ardp.202400032

Due to increasing antibiotic resistance, the development of anti‐infectives with new mechanisms of action is crucial. Virulence factors such as the “macrophage infectivity potentiator” (Mip) protein, which catalyzes the folding of proline‐containing proteins by means of their cis–trans isomerase (PPIase) activity, have come into focus as a potential new target. Since the inhibition of Mip by small molecules has been shown to lead to reduced virulence and survival in vitro, especially of Gram‐negative bacteria such as Burkholderia pseudomallei (Bp), Neisseria meningitidis (Nm), and Neisseria gonorrhoeae (Ng), or Coxiella burnetii (Cb), among many others, a library of Mip inhibitors was developed. As drug metabolism has a significant impact on the overall therapeutic outcome, this report describes the biotransformation of the most potent Mip inhibitors. Therefore, the anti‐infectives were treated using human liver microsomes in vitro. Liquid chromatography with tandem mass spectrometry (LC/MS‐MS) methods were applied to identify the metabolites and quantify the metabolic degradation of the hit compounds. Active metabolites, N‐oxides, were found, leading to new opportunities for further drug development. The metabolism of inhibitors of the virulence factor, macrophage infectivity potentiator protein, was investigated and active metabolites, N‐oxides, were found. Experiments with human liver microsomes reveal a sixfold longer half‐life of the main and active metabolite 4b (t1/2 = 54 min) compared with its parent compound 4a (t1/2 = 9 min).