LEUCINE AMINOPEPTIDASE
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BACKGROUND: Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and leads to ~10,000 deaths each year. Nifurtimox and benznidazole are the only two drugs available but have significant adverse effects and limited efficacy. New chemotherapeutic agents are urgently required. Here we identified inhibitors of the acidic M17 leucyl-aminopeptidase from T. cruzi (LAPTc) that show promise as novel starting points for Chagas disease drug discovery. METHODOLOGY/PRINCIPAL FINDINGS: A RapidFire-MS screen with a protease-focused compound library identified novel LAPTc inhibitors. Twenty-eight hits were progressed to the dose-response studies, from which 12 molecules inhibited LAPTc with IC50 < 34 μM. Of these, compound 4 was the most potent hit and mode of inhibition studies indicate that compound 4 is a competitive LAPTc inhibitor, with Ki 0.27 μM. Compound 4 is selective with respect to human LAP3, showing a selectivity index of >500. Compound 4 exhibited sub-micromolar activity against intracellular T. cruzi amastigotes, and while the selectivity-window against the host cells was narrow, no toxicity was observed for un-infected HepG2 cells. In silico modelling of the LAPTc-compound 4 interaction is consistent with the competitive mode of inhibition. Molecular dynamics simulations reproduce the experimental binding strength (-8.95 kcal/mol), and indicate a binding mode based mainly on hydrophobic interactions with active site residues without metal cation coordination. CONCLUSIONS/SIGNIFICANCE: Our data indicates that these new LAPTc inhibitors should be considered for further development as antiparasitic agents for the treatment of Chagas disease.
- MeSH
- antiparazitární látky terapeutické užití MeSH
- Chagasova nemoc * farmakoterapie MeSH
- leucylaminopeptidasa chemie farmakologie terapeutické užití MeSH
- lidé MeSH
- objevování léků MeSH
- trypanocidální látky * terapeutické užití MeSH
- Trypanosoma cruzi * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Leucyl aminopeptidases (LAPs) are involved in multiple cellular functions, which, in the case of infectious diseases, includes participation in the pathogen-host cell interface and pathogenesis. Thus, LAPs are considered good candidate drug targets, and the major M17-LAP from Trypanosoma cruzi (LAPTc) in particular is a promising target for Chagas disease. To exploit LAPTc as a potential target, it is essential to develop potent and selective inhibitors. To achieve this, we report a high-throughput screening method for LAPTc. Two methods were developed and optimized: a Leu-7-amido-4-methylcoumarin-based fluorogenic assay and a RapidFire mass spectrometry (RapidFire MS)-based assay using the LSTVIVR peptide as substrate. Compared with a fluorescence assay, the major advantages of the RapidFire MS assay are a greater signal-to-noise ratio as well as decreased consumption of enzyme. RapidFire MS was validated with the broad-spectrum LAP inhibitors bestatin (IC50 = 0.35 μM) and arphamenine A (IC50 = 15.75 μM). We suggest that RapidFire MS is highly suitable for screening for specific LAPTc inhibitors.
- MeSH
- Chagasova nemoc diagnóza enzymologie parazitologie MeSH
- hmotnostní spektrometrie MeSH
- kinetika MeSH
- leucylaminopeptidasa genetika izolace a purifikace MeSH
- lidé MeSH
- rychlé screeningové testy * MeSH
- sekvence aminokyselin genetika MeSH
- substrátová specifita MeSH
- Trypanosoma cruzi enzymologie izolace a purifikace patogenita MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We evaluated whether changes in fine root non-structural carbohydrate reserves of Fagus sylvatica and Pinus sylvestris trees influence potential enzymatic activities of their ectomycorrhizal symbionts from winter towards spring reactivation, and whether these changes influence potential soil enzymatic activities. We analyzed sugar and starch concentrations in the fine roots of Fagus sylvatica and Pinus sylvestris and potential activities of ß-glucosidase, ß-xylosidase, and cellobiohydrolase (as proxies for carbon-degrading enzymes) as well as leucine aminopeptidase and chitinase (as proxies for nitrogen-degrading enzymes) of their dominant ectomycorrhizal symbionts as well as in the soil. Sugar concentrations in the fine roots were significantly positively correlated with enzymatic activities of the ectomycorrhizal symbionts. In Pinus sylvestris, both carbon- and nitrogen-degrading enzyme activities showed significant positive correlations with fine root sugar concentrations. In Fagus sylvatica, fine root sugar concentrations were explicitly positively correlated with the activity of nitrogen-degrading enzymes. The chitinase activity in the soil was found to be strongly positively correlated with the enzymatic activity of the ectomycorrhizal symbionts as well as with fine root sugar concentrations. Fine root carbohydrate concentrations of Fagus sylvatica and Pinus sylvestris trees and enzymatic activities of their associated ectomycorrhizal fungi are connected. The specific nutrient demand of the tree species during spring reactivation may affect ectomycorrhizal enzymatic activity via carbon mobilization in the fine roots of Fagus sylvatica and Pinus sylvestris. Moreover, our results suggest that trees indirectly contribute to the degradation of fungal necromass by stimulating ectomycorrhizal chitinase activity in the soil.
- MeSH
- borovice lesní * MeSH
- buk (rod) * MeSH
- kořeny rostlin MeSH
- mykorhiza * MeSH
- sacharidy MeSH
- Publikační typ
- časopisecké články MeSH
The kinetoplast (k), the uniquely packaged mitochondrial DNA of trypanosomatid protists is formed by a catenated network of minicircles and maxicircles that divide and segregate once each cell cycle. Although many proteins involved in kDNA replication and segregation are now known, several key steps in the replication mechanism remain uncharacterized at the molecular level, one of which is the nabelschnur or umbilicus, a prominent structure which in the mammalian parasite Trypanosoma brucei connects the daughter kDNA networks prior to their segregation. Here we characterize an M17 family leucyl aminopeptidase metalloprotease, termed TbLAP1, which specifically localizes to the kDNA disk and the nabelschur and represents the first described protein found in this structure. We show that TbLAP1 is required for correct segregation of kDNA, with knockdown resulting in delayed cytokinesis and ectopic expression leading to kDNA loss and decreased cell proliferation. We propose that TbLAP1 is required for efficient kDNA division and specifically participates in the separation of daughter kDNA networks.
- MeSH
- buněčný cyklus fyziologie MeSH
- kinetoplastová DNA genetika MeSH
- leucylaminopeptidasa genetika metabolismus MeSH
- mitochondriální DNA genetika MeSH
- mitochondrie metabolismus ultrastruktura MeSH
- protozoální DNA genetika MeSH
- protozoální proteiny metabolismus MeSH
- replikace DNA fyziologie MeSH
- Trypanosoma brucei brucei genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Ligands containing bulky aliphatic P1 residues exhibit a high affinity towards cytosolic leucine aminopeptidase, a bizinc protease of biomedical significance. According to this specificity, a series of phosphonic and phosphinic compounds have been put forward as novel putative inhibitors of the enzyme. These phosphonic and phosphinic compounds were derivatives of methionine and norleucine as both single amino acids and dipeptides. The designed inhibitors were synthesised and tested towards the peptidase isolated from porcine kidneys using an improved separation procedure affording superior homogeneity. Unexpectedly, organophosphorus derivatives of methionine and norleucine exhibited moderate activity with K(i) values in the micromolar range.
- MeSH
- aktivace enzymů účinky léků MeSH
- fosfor chemie MeSH
- inhibitory enzymů farmakologie MeSH
- ledviny enzymologie MeSH
- leucylaminopeptidasa antagonisté a inhibitory MeSH
- methionin chemie farmakologie MeSH
- molekulární struktura MeSH
- norleucin chemie farmakologie MeSH
- prasata MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In the present study, we describe in detail the synthesis of a relatively rare class of phosphorus compounds, α-carboxyphosphinopeptides. We prepared several norleucine-derived α-carboxyphosphinic pseudopeptides of the general formula Nle-Ψ[PO(OH)]-Gly. These compounds could have important applications as transition state-mimicking inhibitors for methionine or leucine aminopeptidases or other enzymes. For the preparation of the key α-carboxyphosphinate protected precursors, we investigated, compared and improved two different synthetic methods described in literature: the Arbuzov reaction of a silylated N-protected phosphinic acid with a bromoacetate ester and the nucleophilic addition of a mixed O-methyl S-phenyl N-protected phosphonic acid or a methyl N-protected phosphonochloridate with tert-butyl lithioacetate. We also prepared two N-Fmoc protected synthons, Fmoc-Nle-Ψ[PO(OH)]-Gly-COOH and Fmoc-Nle-Ψ[PO(OAd)]-Gly-COOH, and demonstrated that these precursors are suitable building blocks for the solid-phase synthesis of α-carboxyphosphinopeptides.
