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

Aguado, Mirtha Elisa; González-Matos, Maikel; Orbe, M. Elena de; Quintana, Juan F.; Field, Mark C.; González-Bacerio, Jorge

doi:10.1016/j.pep.2021.105877

Cited by 5 publications

(6 citation statements)

References 48 publications

(100 reference statements)

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“…24,25 Recombinant LmLAP (rLmLAP) was expressed in and purified from Escherichia coli. 23 The peptide LSTVIVR was used as a substrate and the enzyme reaction product STVIVR was directly measured by RapidFire-MS, using STVIVR* as internal standard. To ensure that our rLmLAP was enzymatically active, and we could detect the product of this reaction, we monitored the generation of product over time in assays using a fixed enzyme concentration (30 nM).…”

Section: ■ Resultsmentioning

confidence: 99%

“…To identify specific inhibitors of Lm LAP, we optimized a RapidFire-MS-based assay originally developed to identify inhibitors of T. cruzi LAP. , Recombinant Lm LAP (r Lm LAP) was expressed in and purified from Escherichia coli . The peptide LSTVIVR was used as a substrate and the enzyme reaction product STVIVR was directly measured by RapidFire-MS, using STVIVR* as internal standard.…”

Section: Resultsmentioning

confidence: 99%

“…Recombinant LmLAP (rLmLAP) was expressed in Escherichia coli and purified precisely as previously described. 23 Aminopeptidase Activity Assay by RapidFire-MS. Assays were carried out as previously described, 24 with some differences indicated below. The reaction mix consisted of 0 nM (control) and 30 nM rLmLAP in 1× PBS, pH 7.0, 0.005% (v/v) NP-40, plus 7.5 μL of LSTVIVR substrate peptide in the same buffer.…”

Section: ■ Discussionmentioning

confidence: 99%

“…33.2570; pI = 7.05) or basic (LmjF. 23.0950; pI = 9.5), with the active sites and C-terminal domains highly conserved. 18 The sequence identity percentages of the LmLAP enzyme vary from 92% to 96% when compared to other LAPs within the same family.…”

mentioning

confidence: 99%

“…21 In addition, leucine is a precursor for fatty acids and sterols biosynthesis. 22 Basic LmLAP can be inhibited by established LAP, aminopeptidase P and peptide-deformylase inhibitors, 19,23 suggesting that this enzyme (LmLAP from now) is potentially druggable and raising the possibility of selective inhibition in Leishmania. To test this hypothesis, we used RapidFire mass spectrometry (MS) to identify potent inhibitors of recombinant LmLAP.…”

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confidence: 99%

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Identification and Validation of Compounds Targeting Leishmania major Leucyl-Aminopeptidase M17

Aguado,

Carvalho,

Valdés-Tresanco

et al. 2024

ACS Infect. Dis.

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Leishmaniasis is a neglected tropical disease; there is currently no vaccine and treatment is reliant upon a handful of drugs suffering from multiple issues including toxicity and resistance. There is a critical need for development of new fitfor-purpose therapeutics, with reduced toxicity and targeting new mechanisms to overcome resistance. One enzyme meriting investigation as a potential drug target in Leishmania is M17 leucyl-aminopeptidase (LAP). Here, we aimed to chemically validate LAP as a drug target in L. major through identification of potent and selective inhibitors. Using RapidFire mass spectrometry, the compounds DDD00057570 and DDD00097924 were identified as selective inhibitors of recombinant Leishmania major LAP activity. Both compounds inhibited in vitro growth of L. major and L. donovani intracellular amastigotes, and overexpression of LmLAP in L. major led to reduced susceptibility to DDD00057570 and DDD00097924, suggesting that these compounds specifically target LmLAP. Thermal proteome profiling revealed that these inhibitors thermally stabilized two M17 LAPs, indicating that these compounds selectively bind to enzymes of this class. Additionally, the selectivity of the inhibitors to act on LmLAP and not against the human ortholog was demonstrated, despite the high sequence similarities LAPs of this family share. Collectively, these data confirm LmLAP as a promising therapeutic target for Leishmania spp. that can be selectively inhibited by drug-like small molecules.

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Section: ■ Resultsmentioning

confidence: 99%