Trypanosoma brucei CYP51: Essentiality and Targeting Therapy in an Experimental Model

Abstract

Trypanosoma brucei gambiense is the main causative agent of Human African Trypanosomiasis (HAT), also known as sleeping sickness. Because of limited alternatives and treatment toxicities, new therapeutic options are urgently needed for patients with HAT. Sterol 14alpha-demethylase (CYP51) is a potential drug target but its essentiality has not been determined in T. brucei. We used a tetracycline-inducible RNAi system to assess the essentiality of CYP51 in T. brucei bloodstream form (BSF) cells and we evaluated the effect of posaconazole, a well-tolerated triazole drug, within a panel of virulent strains in vitro and in a murine model. Expression of CYP51 in several T. brucei cell lines was demonstrated by western blot and its essentiality was demonstrated by RNA interference (CYP51RNAi) in vitro. Following reduction of TbCYP51 expression by RNAi, cell growth was reduced and eventually stopped compared to WT or non-induced cells, showing the requirement of CYP51 in T. brucei. These phenotypes were rescued by addition of ergosterol. Additionally, CYP51RNAi induction caused morphological defects with multiflagellated cells (p<0.05), suggesting cytokinesis dysfunction. The survival of CYP51RNAi Doxycycline-treated mice (p = 0.053) and of CYP51RNAi 5-day pre-induced Doxycycline-treated mice (p = 0.008) were improved compared to WT showing a CYP51 RNAi effect on trypanosomal virulence in mice. The posaconazole concentrations that inhibited parasite growth by 50% (IC50) were 8.5, 2.7, 1.6 and 0.12 μM for T. b. brucei 427 90–13, T. b. brucei Antat 1.1, T. b. gambiense Feo (Feo/ITMAP/1893) and T. b. gambiense Biyamina (MHOM/SD/82), respectively. During infection with these last three virulent strains, posaconazole-eflornithine and nifurtimox-eflornithine combinations showed similar improvement in mice survival (p≤0.001). Our results provide support for a CYP51 targeting based treatment in HAT. Thus posaconazole used in combination may represent a therapeutic alternative for trypanosomiasis.

Mutations in CFAP43 and CFAP44 cause male infertility and flagellum defects in Trypanosoma and human

“…Cells were cultured at 37 °C and 5% CO 2 and transfected and cloned as described in ref. 62 in medium supplemented with puromycin (0.1 μg/ml) or blasticidin (10 μg/ml) for constitutive expression of myc-tagged proteins and 10TY1-tagged proteins respectively, and with phleomycin (2.5 μg/ml) for RNAi-inducible cells. RNAi interference was induced with tetracycline (10 μg/ml).…”

“…For WT level of expression of 3 x myc C-terminal tagged proteins (TbCFAP44 myc , TbCFAP43 myc ), parental cells were transfected as in ref. 62 with a tagging cassette that was obtained by PCR using a set of long primers containing 80 nucleotides from the 5′-UTR and 3′-UTR flanking regions of the Tb CFAP44 and Tb CFAP43 open reading frame (ORF) and pMOTag23M vector as template 20 (Supplementary Table 6 ). For RNAi, fragments of TbCFAP44 ORF (bp 1,880–2,309) and TbCFAP43 ORF (bp 131–596) were cloned into p2T7tiB 63 and transfected into TbCFAP44 myc , TbCFAP43 myc cell lines generating the cell lines TbCFAP44 myc RNAi and TbCFAP43 myc RNAi (Supplementary Table 6 ).…”

Unpacking the Pathogen Box—An Open Source Tool for Fighting Neglected Tropical Disease

“…The 1,2,4‐triazoles 1 and 2 are both known antifungal pesticides, which, like the more popular 1,2,4‐triazole antifungals fluconazole ( 4 ) and posaconazole ( 5 , MMV688774), inhibit CYP51 in the sterol biosynthesis pathway. CYP51 has been validated as a target in T. brucei , with 5 displaying the capacity to inhibit the Tb variant of this enzyme, while X‐ray co‐crystal studies of CYP51 from T. cruzi and T. brucei with compounds 4 (PDB IDs: https://www.rcsb.org/structure/2WX2 and https://www.rcsb.org/structure/2WUZ) and 5 (PDB ID: https://www.rcsb.org/structure/3K1O) indicate that the respective active sites are nearly identical . However, while the CYP51 enzymes from T. cruzi and T. brucei share 83 % identity, both species have demonstrated distinct differences in substrate specificity .…”

Heme-deficient metabolism and impaired cellular differentiation as an evolutionary trade-off for human infectivity in Trypanosoma brucei gambiense

“…Being involved in the sterol metabolism (16,17), the hemoprotein CYP51 is considered to be essential and, therefore, a promising drug target against different trypanosomatids (18,19,20). Ketoconazole is one of the broadly used compounds which binds to the active site of CYP51 and inhibits its activity (21).…”