Limits...
Targeting Ergosterol biosynthesis in Leishmania donovani: essentiality of sterol 14 alpha-demethylase.

McCall LI, El Aroussi A, Choi JY, Vieira DF, De Muylder G, Johnston JB, Chen S, Kellar D, Siqueira-Neto JL, Roush WR, Podust LM, McKerrow JH - PLoS Negl Trop Dis (2015)

Bottom Line: While these diseases are associated with significant morbidity and mortality, there are few adequate treatments available.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.

View Article: PubMed Central - PubMed

Affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, United States of America.

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.

Show MeSH

Related in: MedlinePlus

Susceptibility of half knockout strains to CYP51 inhibitors (EC50, μM): compounds with no significant differences between HKO + C and HKO + CYP strains.Values ± standard error are shown. a p-values are for comparison between HKO+C and HKO+CYP.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4359151&req=5

pntd.0003588.g003: Susceptibility of half knockout strains to CYP51 inhibitors (EC50, μM): compounds with no significant differences between HKO + C and HKO + CYP strains.Values ± standard error are shown. a p-values are for comparison between HKO+C and HKO+CYP.

Mentions: The persistence of CYP51-encoding pXNG4 plasmids even under GCV negative selection indicates that CYP51 is essential in L. donovani. Pharmacological inhibition of CYP51 should therefore lead to parasite growth arrest and death. The 4-aminopyridyl-based compound series of CYP51 inhibitors was derived from an initial hit in target-based high-throughput screening, followed by hit-to-lead optimization using structure-activity relationships (SAR), structure-property relationships (SPR), and biological and structural evaluation for T. cruzi CYP51 [34,35,36,41,42,43,44]. We tested 205 compounds from this series on wild-type intracellular L. donovani amastigotes by high content assay. Fifty-four compounds with over 60% activity at 10 μM were then used for dose-response experiments on wild-type L. donovani promastigotes and strains in which we modulated CYP51 expression (HKO, HKO+C and HKO+CYP). Representative compounds with the highest activity on promastigotes are shown in Fig. 3, Fig. 4. Activity on intracellular amastigotes is shown in S3 Table. No clear difference in EC50 values were observed between strains with ketoconazole and voriconazole controls, possibly due to their lower activity on L. donovani. In-house compounds were more potent in this assay than the commercial antifungal azoles. Overall, HKO+CYP strains were less sensitive to these 4-aminopyridyl-based inhibitors compared to HKO+C, indicating that these compounds inhibit Leishmania growth via a CYP51-mediated mechanism. This confirms that targeting CYP51 pharmacologically promotes inhibition of parasite growth, further supporting essentiality of CYP51 in L. donovani metabolism.


Targeting Ergosterol biosynthesis in Leishmania donovani: essentiality of sterol 14 alpha-demethylase.

McCall LI, El Aroussi A, Choi JY, Vieira DF, De Muylder G, Johnston JB, Chen S, Kellar D, Siqueira-Neto JL, Roush WR, Podust LM, McKerrow JH - PLoS Negl Trop Dis (2015)

Susceptibility of half knockout strains to CYP51 inhibitors (EC50, μM): compounds with no significant differences between HKO + C and HKO + CYP strains.Values ± standard error are shown. a p-values are for comparison between HKO+C and HKO+CYP.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359151&req=5

pntd.0003588.g003: Susceptibility of half knockout strains to CYP51 inhibitors (EC50, μM): compounds with no significant differences between HKO + C and HKO + CYP strains.Values ± standard error are shown. a p-values are for comparison between HKO+C and HKO+CYP.
Mentions: The persistence of CYP51-encoding pXNG4 plasmids even under GCV negative selection indicates that CYP51 is essential in L. donovani. Pharmacological inhibition of CYP51 should therefore lead to parasite growth arrest and death. The 4-aminopyridyl-based compound series of CYP51 inhibitors was derived from an initial hit in target-based high-throughput screening, followed by hit-to-lead optimization using structure-activity relationships (SAR), structure-property relationships (SPR), and biological and structural evaluation for T. cruzi CYP51 [34,35,36,41,42,43,44]. We tested 205 compounds from this series on wild-type intracellular L. donovani amastigotes by high content assay. Fifty-four compounds with over 60% activity at 10 μM were then used for dose-response experiments on wild-type L. donovani promastigotes and strains in which we modulated CYP51 expression (HKO, HKO+C and HKO+CYP). Representative compounds with the highest activity on promastigotes are shown in Fig. 3, Fig. 4. Activity on intracellular amastigotes is shown in S3 Table. No clear difference in EC50 values were observed between strains with ketoconazole and voriconazole controls, possibly due to their lower activity on L. donovani. In-house compounds were more potent in this assay than the commercial antifungal azoles. Overall, HKO+CYP strains were less sensitive to these 4-aminopyridyl-based inhibitors compared to HKO+C, indicating that these compounds inhibit Leishmania growth via a CYP51-mediated mechanism. This confirms that targeting CYP51 pharmacologically promotes inhibition of parasite growth, further supporting essentiality of CYP51 in L. donovani metabolism.

Bottom Line: While these diseases are associated with significant morbidity and mortality, there are few adequate treatments available.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.

View Article: PubMed Central - PubMed

Affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, United States of America.

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.

Show MeSH
Related in: MedlinePlus