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Effects of a Serine Protease Inhibitor N-p-Tosyl-L-phenylalanine Chloromethyl Ketone (TPCK) on Leishmania amazonensis and Leishmania infantum.

ABSTRACT: Studies have previously demonstrated the importance of serine proteases in Leishmania. A well-known serine protease inhibitor, TPCK, was used in the present study to evaluate its in vitro and in vivo antileishmanial effects and determine its mechanism of action. Despite slight toxicity against mammalian cells (CC₅₀ = 138.8 µM), TPCK was selective for the parasite due to significant activity against L. amazonensis and L. infantum promastigote forms (IC₅₀ = 14.6 and 31.7 µM for L. amazonensis PH8 and Josefa strains, respectively, and 11.3 µM for L. infantum) and intracellular amastigotes (IC₅₀ values = 14.2 and 16.6 µM for PH8 and Josefa strains, respectively, and 21.7 µM for L. infantum). Leishmania parasites treated with TPCK presented mitochondrial alterations, oxidative stress, modifications in lipid content, flagellar alterations, and cytoplasmic vacuoles, all of which are factors that could be considered as contributing to the death of the parasites. Furthermore, BALB/c mice infected with L. amazonensis and treated with TPCK had a reduction in lesion size and parasite loads in the footpad and spleen. In BALB/c mice infected with L. infantum, TPCK also caused a reduction in the parasite loads in the liver and spleen. Therefore, we highlight the antileishmanial effect of the assessed serine protease inhibitor, proposing a potential therapeutic target in Leishmania as well as a possible new alternative treatment for leishmaniasis.

SUBMITTER: Machado PA

PROVIDER: S-EPMC9320531 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Effects of a Serine Protease Inhibitor N-p-Tosyl-L-phenylalanine Chloromethyl Ketone (TPCK) on Leishmania amazonensis and Leishmania infantum.

Machado Patrícia de A PA Gomes Pollyanna S PS Carneiro Monique P D MPD Midlej Victor V Coimbra Elaine S ES de Matos Guedes Herbert L HL

Pharmaceutics 20220629 7

Studies have previously demonstrated the importance of serine proteases in Leishmania. A well-known serine protease inhibitor, TPCK, was used in the present study to evaluate its in vitro and in vivo antileishmanial effects and determine its mechanism of action. Despite slight toxicity against mammalian cells (CC50 = 138.8 µM), TPCK was selective for the parasite due to significant activity against L. amazonensis and L. infantum promastigote forms (IC50 ...[more]

PMID: 35890269

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Project description:BackgroundLeishmaniases are neglected tropical diseases with great clinical and epidemiological importance. The current chemotherapy available for the treatment of leishmaniasis presents several problems, such as adverse effects, toxicity, long treatment time, and parasite resistance. The discovery of new therapeutic alternatives is extremely essential, and the discovery of cellular targets is a tool that helps in the development of new drugs. Serine proteases emerge as important virulence factors in the Leishmania genus, as they participate in important processes involved in their infectivity, virulence, and survival. In this work, we evaluated the leishmanicidal effect of different serine protease inhibitors (Benzamidine, PF-429242, PMSF, TLCK, and TPCK). Additionally, we determined the implication of pretreatment with these inhibitors on the entry and survival of parasites within macrophages, as well as the conversion of promastigotes into amastigotes, to discover the importance of serine proteases in the establishment of infection and, consequently, as targets for new drugs for Leishmania.ResultsIn general, the inhibitors had low toxicity in host macrophages, and three showed some effect in promastigote and amastigote forms of L. amazonensis (PF-429242, TLCK, and TPCK). Using a short incubation interval, we pretreated L. amazonensis promastigotes with these five compounds before in vitro infection. Pretreatment with PF-429242, TLCK, and TPCK considerably compromised the survival of these parasites inside host macrophages, without altering the entry of promastigotes into these cells and differentiation into amastigotes. In addition, treatment with PF-429242 and TPCK was able to reduce the serine proteases' enzymatic activity using subtilisin substrate on L. amazonensis promastigote lysate.ConclusionsThis work highlights the importance of serine proteases in L. amazonensis as a possible target for new therapeutic alternatives in Leishmania spp.

Project description:Chemotherapy against visceral leishmaniasis is associated with high toxicity and drug resistance. Leishmania parasites are purine auxotrophs that obtain their purines from exogenous sources. Nucleoside hydrolases release purines from nucleosides and are drug targets for anti-leishmanial drugs, absent in mammal cells. We investigated the substrate specificity of the Leishmania (L.) donovani recombinant nucleoside hydrolase NH36 and the inhibitory effect of the immucillins IA (ImmA), DIA (DADMe-ImmA), DIH (DADMe-ImmH), SMIH (SerMe-ImmH), IH (ImmH), DIG (DADMe-ImmG), SMIG (SerMe-ImmG) and SMIA (SerME-ImmA) on its enzymatic activity. The inhibitory effects of immucillins on the in vitro multiplication of L. (L.) infantum chagasi and L. (L.) amazonensis promastigotes were determined using 0.05-500 μM and, when needed, 0.01-50 nM of each drug. The inhibition on multiplication of L. (L.) infantum chagasi intracellular amastigotes in vitro was assayed using 0.5, 1, 5 and 10 μM of IA, IH and SMIH. The NH36 shows specificity for inosine, guanosine, adenosine, uridine and cytidine with preference for adenosine and inosine. IA, IH, DIH, DIG, SMIH and SMIG immucillins inhibited L. (L.) infantum chagasi and L. (L.) amazonensis promastigote growth in vitro at nanomolar to micromolar concentrations. Promastigote replication was also inhibited in a chemically defined medium without a nucleoside source. Addition of adenosine decreases the immucillin toxicity. IA and IH inhibited the NH36 enzymatic activity (Ki = 0.080 μM for IA and 0.019 μM for IH). IA, IH and SMIH at 10 μM concentration, reduced the in vitro amastigote replication inside mice macrophages by 95% with no apparent effect on macrophage viability. Transmission electron microscopy revealed global alterations and swelling of L. (L.) infantum chagasi promastigotes after treatment with IA and IH while SMIH treatment determined intense cytoplasm vacuolization, enlarged vesicles and altered kinetoplasts. Our results suggest that IA, IH and SMIH may provide new chemotherapy agents for leishmaniasis.

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Dataset Information

Effects of a Serine Protease Inhibitor N-p-Tosyl-L-phenylalanine Chloromethyl Ketone (TPCK) on Leishmania amazonensis and Leishmania infantum.

Publications

Effects of a Serine Protease Inhibitor N-<i>p</i>-Tosyl-L-phenylalanine Chloromethyl Ketone (TPCK) on <i>Leishmania amazonensis</i> and <i>Leishmania infantum</i>.

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