Schistosomiasis, caused by a parasitic blood fluke of the genus Schistosoma, is a global health problem for which new chemotherapeutic options are needed. We explored the scaffold of gallinamide A, a natural peptidic metabolite of marine cyanobacteria that has previously been shown to inhibit cathepsin L-type proteases. We screened a library of 19 synthetic gallinamide A analogs and identified nanomolar inhibitors of the cathepsin B-type protease SmCB1, which is a drug target for the treatment of schistosomiasis mansoni. Against cultured S. mansoni schistosomula and adult worms, many of the gallinamides generated a range of deleterious phenotypic responses. Imaging with a fluorescent-activity-based probe derived from gallinamide A demonstrated that SmCB1 is the primary target for gallinamides in the parasite. Furthermore, we solved the high-resolution crystal structures of SmCB1 in complex with gallinamide A and its two analogs and describe the acrylamide covalent warhead and binding mode in the active site. Quantum chemical calculations evaluated the contribution of individual positions in the peptidomimetic scaffold to the inhibition of the target and demonstrated the importance of the P1' and P2 positions. Our study introduces gallinamides as a powerful chemotype that can be exploited for the development of novel antischistosomal chemotherapeutics.
- Klíčová slova
- Schistosoma mansoni, acrylamide inhibitor, cathepsin B, cysteine protease, drug target, parasite,
- MeSH
- kathepsin B * antagonisté a inhibitory metabolismus MeSH
- krystalografie rentgenová MeSH
- molekulární modely MeSH
- Schistosoma mansoni * enzymologie účinky léků MeSH
- schistosomicidy farmakologie chemie MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- kathepsin B * MeSH
- schistosomicidy MeSH
Rhodesain is the major cysteine protease of the protozoan parasite Trypanosoma brucei and a therapeutic target for sleeping sickness, a fatal neglected tropical disease. We designed, synthesized and characterized a bimodal activity-based probe that binds to and inactivates rhodesain. This probe exhibited an irreversible mode of action and extraordinary potency for the target protease with a kinac /Ki value of 37,000 M-1 s-1 . Two reporter tags, a fluorescent coumarin moiety and a biotin affinity label, were incorporated into the probe and enabled highly sensitive detection of rhodesain in a complex proteome by in-gel fluorescence and on-blot chemiluminescence. Furthermore, the probe was employed for microseparation and quantification of rhodesain and for inhibitor screening using a competition assay. The developed bimodal rhodesain probe represents a new proteomic tool for studying Trypanosoma pathobiochemistry and antitrypanosomal drug discovery.
- Klíčová slova
- activity-based probes, affinity chromatography, cysteine proteases, imaging agents, peptidomimetics, rhodesain, size exclusion chromatography,
- MeSH
- biotin MeSH
- cysteinové proteasy * MeSH
- fluorescence MeSH
- proteomika MeSH
- Trypanosoma brucei brucei * MeSH
- Trypanosoma * MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- biotin MeSH
- cysteinové proteasy * MeSH
- rhodesain MeSH Prohlížeč
The cysteine protease cathepsin K is a target for the treatment of diseases associated with high bone turnover. Cathepsin K is mainly expressed in osteoclasts and responsible for the destruction of the proteinaceous components of the bone matrix. We designed various fluorescent activity-based probes (ABPs) and their precursors that bind to and inactivate cathepsin K. ABP 25 exhibited extraordinary potency (kinac/Ki = 35,300 M-1s-1) and selectivity for human cathepsin K. Crystal structures of cathepsin K in complex with ABP 25 and its nonfluorescent precursor 21 were determined to characterize the binding mode of this new type of acrylamide-based Michael acceptor with the particular orientation of the dibenzylamine moiety to the primed subsite region. The cyanine-5 containing probe 25 allowed for sensitive detection of cathepsin K, selective visualization in complex proteomes, and live cell imaging of a human osteosarcoma cell line, underlining its applicability in a pathophysiological environment.
- MeSH
- akrylamidy chemická syntéza chemie metabolismus MeSH
- fluorescenční barviva chemická syntéza chemie metabolismus MeSH
- fluorescenční mikroskopie MeSH
- inhibitory cysteinových proteinas chemická syntéza chemie metabolismus MeSH
- katalytická doména MeSH
- kathepsin K antagonisté a inhibitory chemie metabolismus MeSH
- konfokální mikroskopie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- racionální návrh léčiv MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- akrylamidy MeSH
- CTSK protein, human MeSH Prohlížeč
- fluorescenční barviva MeSH
- inhibitory cysteinových proteinas MeSH
- kathepsin K 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
Azapeptide nitriles are postulated to reversibly covalently react with the active-site cysteine residue of cysteine proteases and form isothiosemicarbazide adducts. We investigated the interaction of azadipeptide nitriles with the cathepsin B1 drug target (SmCB1) from Schistosoma mansoni, a pathogen that causes the global neglected disease schistosomiasis. Azadipeptide nitriles were superior inhibitors of SmCB1 over their parent carba analogs. We determined the crystal structure of SmCB1 in complex with an azadipeptide nitrile and analyzed the reaction mechanism using quantum chemical calculations. The data demonstrate that azadipeptide nitriles, in contrast to their carba counterparts, undergo a change from E- to Z-configuration upon binding, which gives rise to a highly favorable energy profile of noncovalent and covalent complex formation. Finally, azadipeptide nitriles were considerably more lethal than their carba analogs against the schistosome pathogen in culture, supporting the further development of this chemotype as a treatment for schistosomiasis.
- Klíčová slova
- azapeptide inhibitors, cysteine proteases, protein structures, schistosomiasis, structure−activity relationships,
- MeSH
- kathepsin B MeSH
- proteasy * MeSH
- Schistosoma mansoni * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Názvy látek
- kathepsin B MeSH
- proteasy * MeSH
Schistosomiasis caused by parasitic blood flukes of the genus Schistosoma is a global health problem with over 200 million people infected. Schistosoma mansoni cathepsin B1 (SmCB1) is a gut-associated protease critical for digestion of host blood proteins as a source of nutrients. SmCB1 is a validated drug target, and inhibitors of SmCB1 represent promising anti-schistosomals. A comprehensive structural and functional characterization of SmCB1 provides a starting point for the rational design of selective and potent SmCB1 inhibitors. Here, we report optimized protocols for (1) the production of recombinant SmCB1 in the Pichia pastoris expression system and its purification, (2) the measurement of SmCB1 activity and inhibition in a kinetic fluorescence assay, and (3) the preparation and crystallization of SmCB1 in complex with a model vinyl sulfone inhibitor, and the determination of its crystal structure.
- Klíčová slova
- Activity assay, Cathepsin B, Crystal structure, Expression, Inhibition, Parasite, Protease, Schistosoma mansoni,
- MeSH
- aktivace enzymů MeSH
- elektroporace MeSH
- exprese genu MeSH
- genetické vektory metabolismus MeSH
- glykosylace MeSH
- kathepsin B antagonisté a inhibitory chemie izolace a purifikace metabolismus MeSH
- kinetika MeSH
- krystalizace MeSH
- mutace genetika MeSH
- rekombinantní proteiny izolace a purifikace metabolismus MeSH
- Saccharomycetales genetika MeSH
- Schistosoma mansoni enzymologie MeSH
- transformace genetická MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- kathepsin B MeSH
- rekombinantní proteiny MeSH
BACKGROUND: Blood flukes of the genus Schistosoma cause schistosomiasis, a parasitic disease that infects over 240 million people worldwide, and for which there is a need to identify new targets for chemotherapeutic interventions. Our research is focused on Schistosoma mansoni prolyl oligopeptidase (SmPOP) from the serine peptidase family S9, which has not been investigated in detail in trematodes. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that SmPOP is expressed in adult worms and schistosomula in an enzymatically active form. By immunofluorescence microscopy, SmPOP is localized in the tegument and parenchyma of both developmental stages. Recombinant SmPOP was produced in Escherichia coli and its active site specificity investigated using synthetic substrate and inhibitor libraries, and by homology modeling. SmPOP is a true oligopeptidase that hydrolyzes peptide (but not protein) substrates with a strict specificity for Pro at P1. The inhibition profile is analogous to those for mammalian POPs. Both the recombinant enzyme and live worms cleave host vasoregulatory, proline-containing hormones such as angiotensin I and bradykinin. Finally, we designed nanomolar inhibitors of SmPOP that induce deleterious phenotypes in cultured schistosomes. CONCLUSIONS/SIGNIFICANCE: We provide the first localization and functional analysis of SmPOP together with chemical tools for measuring its activity. We briefly discuss the notion that SmPOP, operating at the host-parasite interface to cleave host bioactive peptides, may contribute to the survival of the parasite. If substantiated, SmPOP could be a new target for the development of anti-schistosomal drugs.
- MeSH
- DNA primery genetika MeSH
- Escherichia coli MeSH
- fluorescenční mikroskopie MeSH
- hydrolýza MeSH
- imunoblotting MeSH
- katalytická doména genetika MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- molekulární modely * MeSH
- prolyloligopeptidasy MeSH
- regulace genové exprese enzymů fyziologie MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- Schistosoma mansoni enzymologie MeSH
- serinové endopeptidasy genetika metabolismus MeSH
- stanovení celkové genové exprese MeSH
- substrátová specifita MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA primery MeSH
- prolyloligopeptidasy MeSH
- rekombinantní proteiny MeSH
- serinové endopeptidasy MeSH
Cathepsin B1 (SmCB1) is a digestive protease of the parasitic blood fluke Schistosoma mansoni and a drug target for the treatment of schistosomiasis, a disease that afflicts over 200 million people. SmCB1 is synthesized as an inactive zymogen in which the N-terminal propeptide blocks the active site. We investigated the activation of the zymogen by which the propeptide is proteolytically removed and its regulation by sulfated polysaccharides (SPs). We determined crystal structures of three molecular forms of SmCB1 along the activation pathway: the zymogen, an activation intermediate with a partially cleaved propeptide, and the mature enzyme. We demonstrate that SPs are essential for the autocatalytic activation of SmCB1, as they interact with a specific heparin-binding domain in the propeptide. An alternative activation route is mediated by an S. mansoni asparaginyl endopeptidase (legumain) which is downregulated by SPs, indicating that SPs act as a molecular switch between both activation mechanisms.
- MeSH
- glykosaminoglykany metabolismus MeSH
- kathepsin B metabolismus MeSH
- molekulární modely MeSH
- proteiny červů metabolismus MeSH
- Schistosoma mansoni metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- glykosaminoglykany MeSH
- kathepsin B MeSH
- proteiny červů MeSH
The quantum mechanics (QM)-based scoring function that we previously developed for the description of noncovalent binding in protein-ligand complexes has been modified and extended to treat covalent binding of inhibitory ligands. The enhancements are (i) the description of the covalent bond breakage and formation using hybrid QM/semiempirical QM (QM/SQM) restrained optimizations and (ii) the addition of the new ΔG(cov)' term to the noncovalent score, describing the "free" energy difference between the covalent and noncovalent complexes. This enhanced QM-based scoring function is applied to a series of 20 vinyl sulfone-based inhibitory compounds inactivating the cysteine peptidase cathepsin B1 of the Schistosoma mansoni parasite (SmCB1). The available X-ray structure of the SmCB1 in complex with a potent vinyl sulfone inhibitor K11017 is used as a template to build the other covalently bound complexes and to model the derived noncovalent complexes. We present the correlation of the covalent score and its constituents with the experimental binding data. Four outliers are identified. They contain bulky R1' substituents structurally divergent from the template, which might induce larger protein rearrangements than could be accurately modeled. In summary, we propose a new computational approach and an optimal protocol for the rapid evaluation and prospective design of covalent inhibitors with a conserved binding mode.
- MeSH
- cysteinové proteasy chemie metabolismus MeSH
- inhibitory cysteinových proteinas chemie farmakologie MeSH
- krystalografie rentgenová MeSH
- kvantová teorie * MeSH
- molekulární modely MeSH
- molekulární struktura MeSH
- Schistosoma mansoni enzymologie MeSH
- sulfony chemie farmakologie MeSH
- vinylové sloučeniny chemie farmakologie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- cysteinové proteasy MeSH
- inhibitory cysteinových proteinas MeSH
- sulfony MeSH
- vinylové sloučeniny MeSH
Schistosomiasis caused by a parasitic blood fluke of the genus Schistosoma afflicts over 200 million people worldwide. Schistosoma mansoni cathepsin B1 (SmCB1) is a gut-associated peptidase that digests host blood proteins as a source of nutrients. It is under investigation as a drug target. To further this goal, we report three crystal structures of SmCB1 complexed with peptidomimetic inhibitors as follows: the epoxide CA074 at 1.3 Å resolution and the vinyl sulfones K11017 and K11777 at 1.8 and 2.5 Å resolutions, respectively. Interactions of the inhibitors with the subsites of the active-site cleft were evaluated by quantum chemical calculations. These data and inhibition profiling with a panel of vinyl sulfone derivatives identify key binding interactions and provide insight into the specificity of SmCB1 inhibition. Furthermore, hydrolysis profiling of SmCB1 using synthetic peptides and the natural substrate hemoglobin revealed that carboxydipeptidase activity predominates over endopeptidolysis, thereby demonstrating the contribution of the occluding loop that restricts access to the active-site cleft. Critically, the severity of phenotypes induced in the parasite by vinyl sulfone inhibitors correlated with enzyme inhibition, providing support that SmCB1 is a valuable drug target. The present structure and inhibitor interaction data provide a footing for the rational design of anti-schistosomal inhibitors.
- MeSH
- hemoglobiny chemie MeSH
- hydrolýza účinky léků MeSH
- inhibitory proteas chemie MeSH
- kathepsin D antagonisté a inhibitory chemie genetika MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- peptidomimetika chemie MeSH
- peptidy chemie MeSH
- proteiny červů antagonisté a inhibitory chemie genetika MeSH
- Schistosoma mansoni enzymologie genetika MeSH
- schistosomiasis mansoni farmakoterapie enzymologie genetika MeSH
- systémy cílené aplikace léků * 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, U.S. Gov't, Non-P.H.S. MeSH
- Názvy látek
- hemoglobiny MeSH
- inhibitory proteas MeSH
- kathepsin D MeSH
- peptidomimetika MeSH
- peptidy MeSH
- proteiny červů MeSH
