Cutaneous Leishmaniasis (CL) affects the skin and organs of 12 million people in 98 countries. Drug misuse has led to resistance Leishmania parasites, for whom current treatment is inadequate; thus the need for a new drug is very urgent. Sterols are molecules on the membranes of the parasite that anchor molecules the parasite uses to evade and protects against the damaging host’s immune response. We have used chemical inhibitors to stop the production of the sterol, ergosterol in the parasite, but surprisingly, we instead found that the inhibitor increased ergosterol production. In depth analysis showed that the sterol, ergosta 7,22 dien- 3β-ol was significantly reduced, suggesting for the first time that it was produced from ergosterol and it was a novel target for an anti-leishmanial drug. This conversion in plants is carried out by the enzyme, 17β hydroxylsteroid dehydrogenase (17β HSD), and as the genome of L. mexicana contain the gene for this enzyme, we will now use a molecular approach to investigate the tractability of this enzyme for drug development. We will use a new molecular tool, the DiCre/LoxP system, to knockout the 17β HSD gene (KO) for this enzyme in the parasite to prove its role in parasite development, growth and virulence. We will carry out characterisation of the 17β HSD KO parasites compared to normal parasite using in vitro and in vivo model of CL. This will include comparative analysis of all classes of metabolites, and screening with novel 17β HSD inhibitors to determine if they are specific for the enzyme.