Tick-transmitted Babesia are a major global veterinary threat and an emerging risk to humans. Unlike their Plasmodium relatives, these erythrocyte-infecting Apicomplexa have been largely overlooked and lack specific treatment. Selective targeting of the Babesia proteasome holds promise for drug development. In this study, we screened a library of peptide epoxyketone inhibitors derived from the marine natural product carmaphycin B for their activity against Babesia. Several of these compounds showed activity against both the asexual and sexual blood stages of Plasmodium falciparum. These compounds inactivate β5 proteasome subunit activity in the lysates of Babesia divergens and Babesia microti in the low nanomolar range. Several compounds were tested with the purified B. divergens proteasome and showed IC50 values comparable to carfilzomib, an approved anticancer proteasome inhibitor. They also inhibited B. divergens growth in bovine erythrocyte cultures with solid EC50 values, but importantly, they appeared less toxic to human cells than carfilzomib. These compounds therefore offer a wider therapeutic window and provide new insights into the development of small proteasome inhibitors as selective drugs for babesiosis.

• Publikační typ
• časopisecké články MeSH

The proteasome is a proteolytic enzyme complex essential for protein homeostasis in mammalian cells and protozoan parasites like Trichomonas vaginalis (Tv), the cause of the most common, non-viral sexually transmitted disease. Tv and other protozoan 20S proteasomes have been validated as druggable targets for antimicrobials. However, low yields and purity of the native proteasome have hindered studies of the Tv 20S proteasome (Tv20S). We address this challenge by creating a recombinant protozoan proteasome by expressing all seven α and seven β subunits of Tv20S alongside the Ump-1 chaperone in insect cells. The recombinant Tv20S displays biochemical equivalence to its native counterpart, confirmed by various assays. Notably, the marizomib (MZB) inhibits all catalytic subunits of Tv20S, while the peptide inhibitor carmaphycin-17 (CP-17) specifically targets β2 and β5. Cryo-electron microscopy (cryo-EM) unveils the structures of Tv20S bound to MZB and CP-17 at 2.8 Å. These findings explain MZB's low specificity for Tv20S compared to the human proteasome and demonstrate CP-17's higher specificity. Overall, these data provide a structure-based strategy for the development of specific Tv20S inhibitors to treat trichomoniasis.

• MeSH
• elektronová kryomikroskopie * MeSH
• inhibitory proteasomu * farmakologie   chemie   MeSH
• lidé MeSH
• molekulární modely MeSH
• proteasomový endopeptidasový komplex * metabolismus   MeSH
• protozoální proteiny metabolismus   antagonisté a inhibitory   genetika   chemie   MeSH
• rekombinantní proteiny * metabolismus   genetika   MeSH
• Trichomonas vaginalis * účinky léků   genetika   enzymologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• inhibitory proteasomu * MeSH
• proteasomový endopeptidasový komplex * MeSH
• protozoální proteiny MeSH
• rekombinantní proteiny * MeSH

Proteasomes are essential for protein homeostasis in mammalian cells1-4 and in protozoan parasites such as Trichomonas vaginalis (Tv).5 Tv and other protozoan 20S proteasomes have been validated as druggable targets.6-8 However, in the case of Tv 20S proteasome (Tv20S), biochemical and structural studies were impeded by low yields and purity of the native proteasome. We successfully made recombinant Tv20S by expressing all seven α and seven β subunits together with the Ump-1 chaperone in insect cells. We isolated recombinant proteasome and showed that it was biochemically indistinguishable from the native enzyme. We confirmed that the recombinant Tv20S is inhibited by the natural product marizomib (MZB)9 and the recently developed peptide inhibitor carmaphycin-17 (CP-17)8,10. Specifically, MZB binds to the β1, β2 and β5 subunits, while CP-17 binds the β2 and β5 subunits. Next, we obtained cryo-EM structures of Tv20S in complex with these covalent inhibitors at 2.8Å resolution. The structures revealed the overall fold of the Tv20S and the binding mode of MZB and CP-17. Our work explains the low specificity of MZB and higher specificity of CP-17 towards Tv20S as compared to human proteasome and provides the platform for the development of Tv20S inhibitors for treatment of trichomoniasis.

• Publikační typ
• časopisecké články MeSH
• preprinty 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.

• Klíčová slova
• Babesia, Carfilzomib, Cytotoxicity, Epoxyketone, Proteasome,
• 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
• lékové transportní systémy * 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
• 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
• Názvy látek
• carfilzomib MeSH Prohlížeč
• epoxomicin MeSH Prohlížeč
• inhibitory proteasomu MeSH
• kyseliny boronové MeSH
• oligopeptidy MeSH
• proteasomový endopeptidasový komplex MeSH
• proteom MeSH