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

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
• Názvy látek
• antiparazitární látky MeSH
• leucylaminopeptidasa MeSH
• trypanocidální látky * MeSH

Therapeutic agents with novel mechanisms of action are urgently needed to counter the emergence of drug-resistant infections. Several decades of research into proteases of disease agents have revealed enzymes well suited for target-based drug development. Among them are the three recently validated proteolytic targets: proteasomes of the malarial parasite Plasmodium falciparum, aspartyl proteases of P. falciparum (plasmepsins) and the Sars-CoV-2 viral proteases. Despite some unfulfilled expectations over previous decades, the three reviewed targets clearly demonstrate that selective protease inhibitors provide effective therapeutic solutions for the two most impacting infectious diseases nowadays-malaria and COVID-19.

• Klíčová slova
• infectious diseases, inhibition, parasites, protease, therapy,
• MeSH
• aspartátové endopeptidasy metabolismus   MeSH
• COVID-19 enzymologie   metabolismus   MeSH
• farmakoterapie COVID-19 * MeSH
• inhibitory proteas farmakologie   MeSH
• lidé MeSH
• malárie farmakoterapie   enzymologie   metabolismus   MeSH
• Plasmodium falciparum účinky léků   patogenita   MeSH
• proteasomový endopeptidasový komplex účinky léků   MeSH
• SARS-CoV-2 účinky léků   patogenita   MeSH
• vyvíjení léků metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• aspartátové endopeptidasy MeSH
• inhibitory proteas MeSH
• plasmepsin MeSH Prohlížeč
• proteasomový endopeptidasový komplex MeSH

Schistosomiasis, a parasitic disease caused by blood flukes of the genus Schistosoma, is a global health problem with over 200 million people infected. Treatment relies on just one drug, and new chemotherapies are needed. Schistosoma mansoni cathepsin B1 (SmCB1) is a critical peptidase for the digestion of host blood proteins and a validated drug target. We screened a library of peptidomimetic vinyl sulfones against SmCB1 and identified the most potent SmCB1 inhibitors reported to date that are active in the subnanomolar range with second order rate constants (k2nd) of ∼2 × 105 M-1 s-1. High resolution crystal structures of the two best inhibitors in complex with SmCB1 were determined. Quantum chemical calculations of their respective binding modes identified critical hot spot interactions in the S1' and S2 subsites. The most potent inhibitor targets the S1' subsite with an N-hydroxysulfonic amide moiety and displays favorable functional properties, including bioactivity against the pathogen, selectivity for SmCB1 over human cathepsin B, and reasonable metabolic stability. Our results provide structural insights for the rational design of next-generation SmCB1 inhibitors as potential drugs to treat schistosomiasis.

• Klíčová slova
• Schistosoma mansoni, cathepsin B, cysteine peptidase, drug target, parasite, vinyl sulfone inhibitor,
• MeSH
• kathepsin B * MeSH
• lidé MeSH
• Schistosoma mansoni MeSH
• schistosomóza * farmakoterapie   MeSH
• sulfony farmakologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• divinyl sulfone MeSH Prohlížeč
• kathepsin B * MeSH
• sulfony MeSH

Schistosomiasis is one of a number of chronic helminth diseases of poverty that severely impact personal and societal well-being and productivity. Peptidases (proteases) are vital to successful parasitism, and can modulate host physiology and immunology. Interference of peptidase action by specific drugs or vaccines can be therapeutically beneficial. To date, research on peptidases in the schistosome parasite has focused on either the functional characterization of individual peptidases or their annotation as part of global genome or transcriptome studies. We were interested in functionally characterizing the complexity of peptidase activity operating at the host-parasite interface, therefore the excretory-secretory products of key developmental stages of Schistosoma mansoni that parasitize the human were examined. Using class specific peptidase inhibitors in combination with a multiplex substrate profiling assay, a number of unique activities derived from endo- and exo-peptidases were revealed in the excretory-secretory products of schistosomula (larval migratory worms), adults and eggs. The data highlight the complexity of the functional degradome for each developmental stage of this parasite and facilitate further enquiry to establish peptidase identity, physiological and immunological function, and utility as drug or vaccine candidates.

• Klíčová slova
• Excretion, Fluke, Inhibitor, Parasite, Protease, Secretion,
• MeSH
• interakce hostitele a patogenu MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• ovum metabolismus   MeSH
• proteasy metabolismus   MeSH
• proteiny červů metabolismus   MeSH
• proteolýza MeSH
• Schistosoma mansoni růst a vývoj   metabolismus   fyziologie   MeSH
• schistosomiasis mansoni parazitologie   MeSH
• sekvence aminokyselin MeSH
• stadia vývoje MeSH
• substrátová specifita MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• proteasy MeSH
• proteiny červů MeSH