Development of a High-Throughput Screening Assay to Identify Inhibitors of the Major M17-Leucyl Aminopeptidase from Trypanosoma cruzi Using RapidFire Mass Spectrometry

Expression in Escherichia coli, purification and kinetic characterization of LAPLm, a Leishmania major M17-aminopeptidase

“…LAPLm appears to be involved in nutritional supply, since the parasite lacks the biosynthetic pathways for branched side chain amino acids, including leucine [23]. Consistent with this crucial role, arphamenine A, a Trypanosoma cruzi acidic M17 LAP inhibitor [24], inhibits in vitro growth of related T. brucei brucei [12], suggesting functional conservation across kinetoplastids. Down-regulation of TbLAP1, a T. brucei M17 LAP involved in mitochondrial kinetoplast DNA segregation during cell division, generates a cytokinesis delay [16].…”

The Quest to Identify USP8 Inhibitors for Parkinson’s Disease, a PAINful Experience

“…The biochemical screening platform used for our diversity screen was based upon RapidFire mass spectrometry (RapidFire-MS) technology, which conducts solid phase extraction and allows for an accurate quantitation of enzyme reaction product. This screening platform has been successfully used by us and others for library screening in many targets including SARS-CoV-2 guanine-N7methyltransferase, 17 leucyl aminopeptidase, 18 acetyl-coenzyme A synthetase 19 and Sadenosylmethionine decarboxylase. 20 For our diversity screen, we developed a RapidFire-MS based assay to assess the activity of USP8 through the detection of Ac-KKTIPNDSRE, the de-ubiquitinated product of our model substrate (Ac-KK(Ub)TIPNDSRE).…”

Automated High-Throughput System Combining Small-Scale Synthesis with Bioassays and Reaction Screening

“…Solid-phase extraction (SPE)-based platforms (e.g., Agilent RapidFire), which provide online enrichment and sample cleanup, reducing ionization suppression and matrix effects but increasing analysis time (~8 s/sample). 13,41,42,53–55…”