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 fit-for-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.
- Klíčová slova
- Leishmania, M17 leucyl-aminopeptidase, RapidFire-MS, drug discovery, target validation,
- MeSH
- antiprotozoální látky * farmakologie chemie MeSH
- Leishmania donovani enzymologie účinky léků genetika MeSH
- Leishmania major * enzymologie účinky léků genetika MeSH
- lidé MeSH
- protozoální proteiny * antagonisté a inhibitory chemie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antiprotozoální látky * MeSH
- protozoální proteiny * MeSH
The Leishmania major leucyl-aminopeptidase (LAPLm), a member of the M17 family of proteases, is a potential drug target for treatment of leishmaniasis. To better characterize enzyme properties, recombinant LAPLm (rLAPLm) was expressed in Escherichia coli. A LAPLm gene was designed, codon-optimized for expression in E. coli, synthesized and cloned into the pET-15b vector. Production of rLAPLm in E. coli Lemo21(DE3), induced for 4 h at 37 °C with 400 μM IPTG and 250 μM l-rhamnose, yielded insoluble enzyme with a low proportion of soluble and active protein, only detected by an anti-His antibody-based western-blot. rLAPLm was purified in a single step by immobilized metal ion affinity chromatography. rLAPLm was obtained with a purity of ~10% and a volumetric yield of 2.5 mg per liter, sufficient for further characterization. The aminopeptidase exhibits optimal activity at pH 7.0 and a substrate preference for Leu-p-nitroanilide (appKM = 30 μM, appkcat = 14.7 s-1). Optimal temperature is 50 °C, and the enzyme is insensitive to 4 mM Co2+, Mg2+, Ca2+ and Ba2+. However, rLAPLm was activated by Zn2+, Mn2+ and Cd2+ but is insensitive towards the protease inhibitors PMSF, TLCK, E-64 and pepstatin A, being inhibited by EDTA and bestatin. Bestatin is a potent, non-competitive inhibitor of the enzyme with a Ki value of 994 nM. We suggest that rLAPLm is a suitable target for inhibitor identification.
- Klíčová slova
- Expression in E. coli, IMAC, Kinetic characterization, Leucyl aminopeptidases, pET-15b vector,
- MeSH
- aminopeptidasy * biosyntéza chemie genetika izolace a purifikace MeSH
- Escherichia coli * genetika metabolismus MeSH
- kinetika MeSH
- Leishmania major * enzymologie genetika MeSH
- protozoální proteiny * biosyntéza chemie genetika izolace a purifikace MeSH
- rekombinantní proteiny biosyntéza chemie genetika izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- aminopeptidasy * MeSH
- protozoální proteiny * MeSH
- rekombinantní proteiny MeSH
Leishmania major is the main causative agent of cutaneous leishmaniasis in the Old World. In Leishmania parasites, the lack of transcriptional control is mostly compensated by post-transcriptional mechanisms. Methylation of arginine is a conserved post-translational modification executed by Protein Arginine Methyltransferase (PRMTs). The genome from L. major encodes five PRMT homologs, including the cytosolic protein associated with several RNA-binding proteins, LmjPRMT7. It has been previously reported that LmjPRMT7 could impact parasite infectivity. In addition, a more recent work has clearly shown the importance of LmjPRMT7 in RNA-binding capacity and protein stability of methylation targets, demonstrating the role of this enzyme as an important epigenetic regulator of mRNA metabolism. In this study, we unveil the impact of PRMT7-mediated methylation on parasite development and virulence. Our data reveals that higher levels of LmjPRMT7 can impair parasite pathogenicity, and that deletion of this enzyme rescues the pathogenic phenotype of an attenuated strain of L. major. Interestingly, lesion formation caused by LmjPRMT7 knockout parasites is associated with an exacerbated inflammatory reaction in the tissue correlated with an excessive neutrophil recruitment. Moreover, the absence of LmjPRMT7 also impairs parasite development within the sand fly vector Phlebotomus duboscqi. Finally, a transcriptome analysis shed light onto possible genes affected by depletion of this enzyme. Taken together, this study highlights how post-transcriptional regulation can affect different aspects of the parasite biology.
- MeSH
- delece genu MeSH
- Leishmania major enzymologie genetika metabolismus MeSH
- leishmanióza kožní parazitologie patologie MeSH
- myši MeSH
- neutrofily fyziologie MeSH
- proteinmethyltransferasy genetika metabolismus MeSH
- protozoální proteiny metabolismus MeSH
- regulace genové exprese enzymů MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Intramural MeSH
- Názvy látek
- proteinmethyltransferasy MeSH
- protozoální proteiny MeSH
Trypanosoma brucei is an important human pathogen. In this study, we have focused on the characterization of FtsH protease, ATP-dependent membrane-bound mitochondrial enzyme important for regulation of protein abundance. We have determined localization and orientation of all six putative T.brucei FtsH homologs in the inner mitochondrial membrane by in silico analyses, by immunofluorescence, and with protease assay. The evolutionary origin of these homologs has been tested by comparative phylogenetic analysis. Surprisingly, some kinetoplastid FtsH proteins display inverted orientation in the mitochondrial membrane compared to related proteins of other examined eukaryotes. Moreover, our data strongly suggest that during evolution the orientation of FtsH protease in T. brucei varied due to both loss and acquisition of the transmembrane domain.
- Klíčová slova
- AAA protease, Evolution, FtsH, Mitochondrion, Phylogeny, Trypanosoma,
- MeSH
- Arabidopsis klasifikace enzymologie genetika MeSH
- Euglena gracilis klasifikace enzymologie genetika MeSH
- Euglena longa klasifikace enzymologie genetika MeSH
- exprese genu MeSH
- fylogeneze MeSH
- izoenzymy chemie genetika metabolismus MeSH
- konzervovaná sekvence MeSH
- Leishmania major klasifikace enzymologie genetika MeSH
- lidé MeSH
- mitochondriální membrány chemie enzymologie MeSH
- mitochondriální proteiny chemie genetika metabolismus MeSH
- mitochondrie enzymologie genetika MeSH
- molekulární evoluce * MeSH
- myši MeSH
- proteasy chemie genetika metabolismus MeSH
- proteinové domény MeSH
- protozoální proteiny chemie genetika metabolismus MeSH
- Saccharomyces cerevisiae klasifikace enzymologie genetika MeSH
- Trypanosoma brucei brucei klasifikace enzymologie genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- izoenzymy MeSH
- mitochondriální proteiny MeSH
- proteasy MeSH
- protozoální proteiny MeSH
Leishmaniasis is an infectious disease caused by protozoan parasites of the genus Leishmania. There is no vaccine against human leishmaniasis and the treatment of the disease would benefit from a broader spectrum and a higher efficacy of leishmanicidal compounds. We analyzed the leishmanicidal activity and the mechanism of action of the calcium ionophore, calcimycin. L. major promastigotes were coincubated with calcimycin and the viability of the cells was assessed using resazurin assay. Calcimycin displayed dose-dependent effect with IC50 = 0.16 μM. Analysis of propidium iodide/LDS-751 stained promastigotes revealed that lower concentrations of calcimycin had cytostatic effect and higher concentrations had cytotoxic effect. To establish the mechanism of action of calcimycin, which is known to stimulate activity of mammalian constitutive nitric oxide synthase (NOS), we coincubated L. major promastigotes with calcimycin and selective NOS inhibitors ARL-17477 or L-NNA. Addition of these inhibitors substantially decreased the toxicity of calcimycin to Leishmania promastigotes. In doing so, we demonstrated for the first time that calcimycin has a direct leishmanicidal effect on L. major promastigotes. Also, we showed that Leishmania constitutive Ca2+/calmodulin-dependent nitric oxide synthase is involved in the parasite cell death. These data suggest activation of Leishmania nitric oxide synthase as a new therapeutic approach.
- MeSH
- aktivace enzymů MeSH
- calcimycin farmakologie MeSH
- ionofory kalciové farmakologie MeSH
- Leishmania major enzymologie MeSH
- leishmanióza kožní farmakoterapie enzymologie MeSH
- protozoální proteiny metabolismus MeSH
- synthasa oxidu dusnatého metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- calcimycin MeSH
- ionofory kalciové MeSH
- protozoální proteiny MeSH
- synthasa oxidu dusnatého MeSH
The Leishmania metalloproteinase GP63 has been reported to play important roles mainly in resistance of promastigotes to complement-mediated lysis and in interaction with macrophage receptors. On the other hand, its function in insect vectors is still unclear. We compared the structure and dosage of gp63 genes and the activity of GP63 in Leishmania major Yakimoff et Schokhor strains and lines differing in virulence for mice and ability to develop in sand flies. The results demonstrate considerable variability in amount and proteolytical activity of GP63 among L. major strains although genomic changes in the gp63 locus were not found. Attenuated LV561/AV line showed low amount and low enzymatic activity of GP63. Serial passages of attenuated parasites through either Phlebotomus duboscqi Neveu-Lemaire or through mice led to a recovery of GP63 proteolytical activity to the level present in virulent LV561/V line. Overexpression of GP63 was found in two L major strains (L119, Neal) with defective lipophosphoglycan (LPG); both these strains were capable to cause mice infection but unable to survive and multiply in sand flies. Differences were found also in karyotypes and in amount of minichromosomes amplified in some lines of the LV561 strain. The results suggest that parasite virulence is not simply correlated with the activity of GP63; however, this enzyme plays a significant role in association with other surface molecules, especially LPG. Overexpression of GP63 can compensate LPG defect in the vertebrate host but in sand flies both molecules fulfil quite different functions and the defect in LPG is lethal for the parasite. On the other hand, linear minichromosomes of about 200 kb found in some lines of the LV561 strain are associated with development in vitro and in the vector but they are not essential for the infection of the vertebrate host.
- MeSH
- glykosfingolipidy metabolismus MeSH
- karyotypizace MeSH
- křečci praví MeSH
- Leishmania major enzymologie genetika patogenita MeSH
- lidé MeSH
- mapování chromozomů metody MeSH
- metaloendopeptidasy biosyntéza genetika metabolismus MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- Phlebotomus parazitologie MeSH
- protozoální DNA genetika MeSH
- Psychodidae parazitologie MeSH
- zvířata MeSH
- Check Tag
- křečci praví MeSH
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- glycoprotein gp63, Leishmania MeSH Prohlížeč
- glykosfingolipidy MeSH
- lipophosphonoglycan MeSH Prohlížeč
- metaloendopeptidasy MeSH
- protozoální DNA MeSH
