Here, we present remarkable epoxyketone-based proteasome inhibitors with low nanomolar in vitro potency for blood-stage Plasmodium falciparum and low cytotoxicity for human cells. Our best compound has more than 2,000-fold greater selectivity for erythrocytic-stage P. falciparum over HepG2 and H460 cells, which is largely driven by the accommodation of the parasite proteasome for a D-amino acid in the P3 position and the preference for a difluorobenzyl group in the P1 position. We isolated the proteasome from P. falciparum cell extracts and determined that the best compound is 171-fold more potent at inhibiting the β5 subunit of P. falciparum proteasome when compared to the same subunit of the human constitutive proteasome. These compounds also significantly reduce parasitemia in a P. berghei mouse infection model and prolong survival of animals by an average of 6 days. The current epoxyketone inhibitors are ideal starting compounds for orally bioavailable anti-malarial drugs.

• MeSH
• antimalarika * farmakologie   MeSH
• inhibitory proteasomu chemie   MeSH
• lidé MeSH
• myši MeSH
• Plasmodium falciparum MeSH
• Plasmodium * MeSH
• proteasomový endopeptidasový komplex chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Cathepsin L is a key host cysteine protease utilized by coronaviruses for cell entry and is a promising drug target for novel antivirals against SARS-CoV-2. The marine natural product gallinamide A and several synthetic analogues were identified as potent inhibitors of cathepsin L with IC50 values in the picomolar range. Lead molecules possessed selectivity over other cathepsins and alternative host proteases involved in viral entry. Gallinamide A directly interacted with cathepsin L in cells and, together with two lead analogues, potently inhibited SARS-CoV-2 infection in vitro, with EC50 values in the nanomolar range. Reduced antiviral activity was observed in cells overexpressing transmembrane protease, serine 2 (TMPRSS2); however, a synergistic improvement in antiviral activity was achieved when combined with a TMPRSS2 inhibitor. These data highlight the potential of cathepsin L as a COVID-19 drug target as well as the likely need to inhibit multiple routes of viral entry to achieve efficacy.

• MeSH
• antivirové látky chemická syntéza   chemie   farmakologie   MeSH
• biologické přípravky chemická syntéza   chemie   farmakologie   MeSH
• buňky A549 MeSH
• Cercopithecus aethiops MeSH
• COVID-19 farmakoterapie   metabolismus   MeSH
• inhibitory cysteinových proteinas chemická syntéza   chemie   farmakologie   MeSH
• kathepsin L antagonisté a inhibitory   metabolismus   MeSH
• kationické antimikrobiální peptidy chemická syntéza   chemie   farmakologie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• molekulární konformace MeSH
• proteomika MeSH
• SARS-CoV-2 účinky léků   MeSH
• Vero buňky MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou 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

The worldwide COVID-19 pandemic caused by the coronavirus SARS-CoV-2 urgently demands novel direct antiviral treatments. The main protease (Mpro) and papain-like protease (PLpro) are attractive drug targets among coronaviruses due to their essential role in processing the polyproteins translated from the viral RNA. In this study, we virtually screened 688 naphthoquinoidal compounds and derivatives against Mpro of SARS-CoV-2. Twenty-four derivatives were selected and evaluated in biochemical assays against Mpro using a novel fluorogenic substrate. In parallel, these compounds were also assayed with SARS-CoV-2 PLpro. Four compounds inhibited Mpro with half-maximal inhibitory concentration (IC50) values between 0.41 μM and 9.0 μM. In addition, three compounds inhibited PLpro with IC50 ranging from 1.9 μM to 3.3 μM. To verify the specificity of Mpro and PLpro inhibitors, our experiments included an assessment of common causes of false positives such as aggregation, high compound fluorescence, and inhibition by enzyme oxidation. Altogether, we confirmed novel classes of specific Mpro and PLpro inhibitors. Molecular dynamics simulations suggest stable binding modes for Mpro inhibitors with frequent interactions with residues in the S1 and S2 pockets of the active site. For two PLpro inhibitors, interactions occur in the S3 and S4 pockets. In summary, our structure-based computational and biochemical approach identified novel naphthoquinonal scaffolds that can be further explored as SARS-CoV-2 antivirals.

• MeSH
• antivirové látky * farmakologie   chemie   MeSH
• COVID-19 MeSH
• inhibitory proteas * farmakologie   chemie   MeSH
• koronavirové proteasy 3C * antagonisté a inhibitory   MeSH
• koronavirové proteasy podobné papainu * antagonisté a inhibitory   MeSH
• lidé MeSH
• naftochinony * chemie   farmakologie   MeSH
• papain MeSH
• SARS-CoV-2 * účinky léků   enzymologie   MeSH
• simulace molekulového dockingu MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Host-cell cysteine proteases play an essential role in the processing of the viral spike protein of SARS coronaviruses. K777, an irreversible, covalent inactivator of cysteine proteases that has recently completed phase 1 clinical trials, reduced SARS-CoV-2 viral infectivity in several host cells: Vero E6 (EC50< 74 nM), HeLa/ACE2 (4 nM), Caco-2 (EC90 = 4.3 μM), and A549/ACE2 (<80 nM). Infectivity of Calu-3 cells depended on the cell line assayed. If Calu-3/2B4 was used, EC50 was 7 nM, but in the ATCC Calu-3 cell line without ACE2 enrichment, EC50 was >10 μM. There was no toxicity to any of the host cell lines at 10-100 μM K777 concentration. Kinetic analysis confirmed that K777 was a potent inhibitor of human cathepsin L, whereas no inhibition of the SARS-CoV-2 cysteine proteases (papain-like and 3CL-like protease) was observed. Treatment of Vero E6 cells with a propargyl derivative of K777 as an activity-based probe identified human cathepsin B and cathepsin L as the intracellular targets of this molecule in both infected and uninfected Vero E6 cells. However, cleavage of the SARS-CoV-2 spike protein was only carried out by cathepsin L. This cleavage was blocked by K777 and occurred in the S1 domain of the SARS-CoV-2 spike protein, a different site from that previously observed for the SARS-CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of cathepsin L-mediated viral spike protein processing.

• MeSH
• antivirové látky farmakologie   MeSH
• Cercopithecus aethiops MeSH
• fenylalanin farmakologie   MeSH
• glykoprotein S, koronavirus chemie   metabolismus   MeSH
• inhibitory cysteinových proteinas farmakologie   MeSH
• internalizace viru účinky léků   MeSH
• kathepsin L antagonisté a inhibitory   metabolismus   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• nádorové buněčné linie MeSH
• piperaziny farmakologie   MeSH
• proteinové domény MeSH
• proteolýza MeSH
• SARS-CoV-2 účinky léků   MeSH
• tosylové sloučeniny farmakologie   MeSH
• Vero buňky MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Research Support, N.I.H., Extramural MeSH

Babesiosis is a tick-transmitted zoonosis caused by apicomplexan parasites of the genus Babesia. Treatment of this emerging malaria-related disease has relied on antimalarial drugs and antibiotics. The proteasome of Plasmodium, the causative agent of malaria, has recently been validated as a target for anti-malarial drug development and therefore, in this study, we investigated the effect of epoxyketone (carfilzomib, ONX-0914 and epoxomicin) and boronic acid (bortezomib and ixazomib) proteasome inhibitors on the growth and survival of Babesia. Testing the compounds against Babesia divergens ex vivo revealed suppressive effects on parasite growth with activity that was higher than the cytotoxic effects on a non-transformed mouse macrophage cell line. Furthermore, we showed that the most-effective compound, carfilzomib, significantly reduces parasite multiplication in a Babesia microti infected mouse model without noticeable adverse effects. In addition, treatment with carfilzomib lead to an ex vivo and in vivo decrease in proteasome activity and accumulation of polyubiquitinated proteins compared to untreated control. Overall, our results demonstrate that the Babesia proteasome is a valid target for drug development and warrants the design of potent and selective B. divergens proteasome inhibitors for the treatment of babesiosis.

• MeSH
• Babesia microti účinky léků   genetika   růst a vývoj   MeSH
• Babesia účinky léků   genetika   růst a vývoj   MeSH
• babezióza farmakoterapie   MeSH
• buněčné linie MeSH
• inhibitory proteasomu aplikace a dávkování   škodlivé účinky   farmakologie   terapeutické užití   MeSH
• kyseliny boronové farmakologie   MeSH
• makrofágy účinky léků   parazitologie   MeSH
• modely nemocí na zvířatech MeSH
• myši MeSH
• oligopeptidy farmakologie   MeSH
• proteasomový endopeptidasový komplex účinky léků   MeSH
• proteom účinky léků   genetika   MeSH
• systémy cílené aplikace léků * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• validační studie MeSH

BACKGROUND: Serine proteases are important virulence factors for many pathogens. Recently, we discovered a group of trypsin-like serine proteases with domain organization unique to flatworm parasites and containing a thrombospondin type 1 repeat (TSR-1). These proteases are recognized as antigens during host infection and may prove useful as anthelminthic vaccines, however their molecular characteristics are under-studied. Here, we characterize the structural and proteolytic attributes of serine protease 2 (SmSP2) from Schistosoma mansoni, one of the major species responsible for the tropical infectious disease, schistosomiasis. METHODOLOGY/PRINCIPAL FINDINGS: SmSP2 comprises three domains: a histidine stretch, TSR-1 and a serine protease domain. The cleavage specificity of recombinant SmSP2 was determined using positional scanning and multiplex combinatorial libraries and the determinants of specificity were identified with 3D homology models, demonstrating a trypsin-like endopeptidase mode of action. SmSP2 displayed restricted proteolysis on protein substrates. It activated tissue plasminogen activator and plasminogen as key components of the fibrinolytic system, and released the vasoregulatory peptide, kinin, from kininogen. SmSP2 was detected in the surface tegument, esophageal glands and reproductive organs of the adult parasite by immunofluorescence microscopy, and in the excretory/secretory products by immunoblotting. CONCLUSIONS/SIGNIFICANCE: The data suggest that SmSP2 is secreted, functions at the host-parasite interface and contributes to the survival of the parasite by manipulating host vasodilatation and fibrinolysis. SmSP2 may be, therefore, a potential target for anti-schistosomal therapy.

• MeSH
• fibrinolýza účinky léků   MeSH
• hemokoagulace účinky léků   MeSH
• hemostatika antagonisté a inhibitory   MeSH
• krevní tlak účinky léků   MeSH
• molekulární modely MeSH
• plazminogen účinky léků   MeSH
• proteinové domény MeSH
• proteiny červů chemie   genetika   farmakologie   MeSH
• proteolýza účinky léků   MeSH
• rekombinantní proteiny MeSH
• Schistosoma mansoni enzymologie   MeSH
• schistosomiasis mansoni parazitologie   MeSH
• sekvence aminokyselin MeSH
• serinové endopeptidasy chemie   genetika   farmakologie   MeSH
• tkáňový aktivátor plazminogenu účinky léků   MeSH
• vazodilatace účinky léků   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The opportunistic fungal pathogen Cryptococcus neoformans is a major cause of mortality in immunocompromised individuals, resulting in more than 600,000 deaths per year. Many human fungal pathogens secrete peptidases that influence virulence, but in most cases the substrate specificity and regulation of these enzymes remains poorly understood. The paucity of such information is a roadblock to our understanding of the biological functions of peptidases and whether or not these enzymes are viable therapeutic targets. We report here an unbiased analysis of secreted peptidase activity and specificity in C. neoformans using a mass spectrometry-based substrate profiling strategy and subsequent functional investigations. Our initial studies revealed that global peptidase activity and specificity are dramatically altered by environmental conditions. To uncover the substrate preferences of individual enzymes and interrogate their biological functions, we constructed and profiled a ten-member gene deletion collection of candidate secreted peptidases. Through this deletion approach, we characterized the substrate specificity of three peptidases within the context of the C. neoformans secretome, including an enzyme known to be important for fungal entry into the brain. We selected a previously uncharacterized peptidase, which we term Major aspartyl peptidase 1 (May1), for detailed study due to its substantial contribution to extracellular proteolytic activity. Based on the preference of May1 for proteolysis between hydrophobic amino acids, we screened a focused library of aspartyl peptidase inhibitors and identified four high-affinity antagonists. Finally, we tested may1Δ strains in a mouse model of C. neoformans infection and found that strains lacking this enzyme are significantly attenuated for virulence. Our study reveals the secreted peptidase activity and specificity of an important human fungal pathogen, identifies responsible enzymes through genetic tests of their function, and demonstrates how this information can guide the development of high affinity small molecule inhibitors.

• MeSH
• aspartátové proteasy metabolismus   MeSH
• Cryptococcus neoformans enzymologie   patogenita   MeSH
• faktory virulence metabolismus   MeSH
• fungální proteiny metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• imunoblotting MeSH
• koncentrace vodíkových iontů MeSH
• kryptokokóza enzymologie   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• modely nemocí na zvířatech MeSH
• myši MeSH
• proteasy metabolismus   MeSH
• proteomika MeSH
• stanovení celkové genové exprese MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články 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.

• MeSH
• interakce hostitele a patogenu MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• ovum metabolismus   MeSH
• proteasy metabolismus   sekrece   MeSH
• proteiny červů metabolismus   sekrece   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

To identify the gut-associated tick aspartic hemoglobinase, this work focuses on the functional diversity of multiple Ixodes ricinus cathepsin D forms (IrCDs). Out of three encoding genes representing Ixodes scapularis genome paralogs, IrCD1 is the most distinct enzyme with a shortened propeptide region and a unique pattern of predicted post-translational modifications. IrCD1 gene transcription is induced by tick feeding and is restricted to the gut tissue. The hemoglobinolytic role of IrCD1 was further supported by immunolocalization of IrCD1 in the vesicles of tick gut cells. Properties of recombinantly expressed rIrCD1 are consistent with the endo-lysosomal environment because the zymogen is autoactivated and remains optimally active in acidic conditions. Hemoglobin cleavage pattern of rIrCD1 is identical to that produced by the native enzyme. The preference for hydrophobic residues at the P1 and P1' position was confirmed by screening a novel synthetic tetradecapeptidyl substrate library. Outside the S1-S1' regions, rIrCD1 tolerates most amino acids but displays a preference for tyrosine at P3 and alanine at P2'. Further analysis of the cleavage site location within the peptide substrate indicated that IrCD1 is a true endopeptidase. The role in hemoglobinolysis was verified with RNAi knockdown of IrCD1 that decreased gut extract cathepsin D activity by >90%. IrCD1 was newly characterized as a unique hemoglobinolytic cathepsin D contributing to the complex intestinal proteolytic network of mainly cysteine peptidases in ticks.

• MeSH
• genetická transkripce fyziologie   MeSH
• genom fyziologie   MeSH
• hemoglobiny genetika   metabolismus   MeSH
• kathepsin D genetika   metabolismus   MeSH
• klíště enzymologie   genetika   MeSH
• posttranslační úpravy proteinů fyziologie   MeSH
• proteiny členovců genetika   metabolismus   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• střeva enzymologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH