Targeting Ergosterol Biosynthesis in Leishmania donovani: Essentiality of Sterol 14alpha-demethylase

Abstract

Leishmania protozoan parasites (Trypanosomatidae family) are the causative agents of cutaneous, mucocutaneous and visceral leishmaniasis worldwide. While these diseases are associated with significant morbidity and mortality, there are few adequate treatments available. Sterol 14alpha-demethylase (CYP51) in the parasite sterol biosynthesis pathway has been the focus of considerable interest as a novel drug target in Leishmania. However, its essentiality in Leishmania donovani has yet to be determined. Here, we use a dual biological and pharmacological approach to demonstrate that CYP51 is indispensable in L. donovani. We show via a facilitated knockout approach that chromosomal CYP51 genes can only be knocked out in the presence of episomal complementation and that this episome cannot be lost from the parasite even under negative selection. In addition, we treated wild-type L. donovani and CYP51-deficient strains with 4-aminopyridyl-based inhibitors designed specifically for Trypanosoma cruzi CYP51. While potency was lower than in T. cruzi, these inhibitors had increased efficacy in parasites lacking a CYP51 allele compared to complemented parasites, indicating inhibition of parasite growth via a CYP51-specific mechanism and confirming essentiality of CYP51 in L. donovani. Overall, these results provide support for further development of CYP51 inhibitors for the treatment of visceral leishmaniasis.

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

“…in 2015 who found that L . donovani parasites were able to tolerate > 50% reduction in CYP51 protein levels with no apparent effects on parasite phenotype [11]. These authors finally confirmed essentiality of CYP51 in Leishmania by obtaining complete loss of chromosomal CYP51 genes only in the presence of an episomal source of CYP51.…”

“…brucei BSF. As Tb CYP51 and Ld CYP51 proteins share 84% of identity, we used the anti- Ld CYP51 antibody described by McCall and colleagues [11] to probe the kinetoplastids used in this study by western-blot. A band at the expected size was observed demonstrating that Tb CYP51 is expressed in T .…”

“…Among the Trypanosomatidae , the essentiality of CYP51 has been demonstrated for Leishmania donovani [11] and Leishmania major [58]. In the latter study, CYP51-null mutants were viable but show defects in growth rate, cytokinesis defects and hypersensitivity to heat stress.…”

“…Membranes were blocked in blocking solution (tris-buffered saline (TBS), 0.2% Tween-20, 5% skimmed milk powder) for 1 hour, and incubated overnight at 4°C with the primary antibody anti-CYP51 (A kind gift from Dr. James. H. McKerrow, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, U.S.A) [11] diluted in blocking solution at 1:5,000. After 3 washes (10 min) in TBS, 0.2% Tween-20, the membranes were incubated for 1 hour at room temperature with anti-rabbit HRP (horseradish peroxidase)-conjugated secondary antibody (Sigma A-9169) diluted at 1:10,000 in blocking solution.…”

“…Sterol 14alpha-demethylase (cytochrome P450 family 51 (CYP51)) catalyzes removal of a 14alpha-methyl group from lanosterol [9,10]. Recently, CYP51 has been shown as essential in Leishmania donovani [11]. Nevertheless, data are limited regarding T .…”