Multiple chemical scaffolds inhibit a promising Leishmania drug target

The need for an effective oral therapy for leishmaniasis is addressed through the study of the target N-myristoyltransferase from Leishmania major.

a series of low nanomolar inhibitors against TbNMT were identified that killed parasites very effectively with ED 50 values less than 5 nM (Frearson et al., 2010).
The major snag with such potent compounds in the HAT project was that they did not cross the blood brain barrier. In addition, there was a very poor (twofold) specificity for inhibition by the lead compound of TbNMT over the human enzyme (usually a target factor of approaching 100 would be desirable). In fact, the low specificity was found not to be a problem in cellular assays as the prodigious endocytotic activity makes T. brucei far more sensitive to NMT than human cells where no toxicity is observed. For HAT infection it is crucial that the drug crosses the brain barrier as trypanosomes invade the brain in the fatal stages of the disease; however, brain penetration is not an issue for leishmaniasis as the parasites take up residence inside human macrophagesproviding themselves with another layer of protection from both drugs and the mammalian immune system.
The four inhibitor X-ray structures presented in the York/ London paper have IC 50 values from 1.2 mM down to 30 nM. As might be expected, there is little difference between inhibition of the LmNMT and LdNMT values as the two enzymes only differ by 11 amino acids out of 421. Despite these minimal differences it was apparently impossible to obtain crystals of L. donovani NMT complexed with inhibitors and all crystallographic work was carried out using the L. major protein.
All crystal structures presented in this work are loaded with CoA-myristate in the active site and the inhibitors all bind in the adjacent substrate binding pocket (Fig. 1). There are remarkably few changes in protein structure upon ligand binding; the only notable feature being alternative conformations of the side chains of Tyr217 and His219. Despite the different chemical scaffolds, all compounds manage to squeeze into the substrate binding pocket burying over 80% their accessible surface area. Most also hydrogen bond with the catalytically important terminal carboxyl group of Leu421 (buried and out of site at the bottom of the substrate pocket in Fig. 1) as well as the side chain of a tyrosine at the top of the pocket. Encouragingly the four diverse ligands show reasonable specificity of between 10 and 200, over human NMT. This contrasts with the poor specificity but much enhanced binding of DDD646 which inhibits Tb, Ld and Lm NMT all with singledigit nanomolar affinities, but also inhibits the human enzyme almost equally well. There is no obvious structural explanation for the better specificities of the York/London inhibitors, as the binding site is highly conserved between the parasite and human structures -though there is hint that the orientation of the side chain of Tyr217 (highlighted in Fig. 1) may play a role. Compounds that show better specificity seem to have a preference for out-facing conformations of Tyr217 in the LmNMT structures while those inhibitors (like DDD646, Fig. 1) which allow Tyr217 to swing in are less specific (but can bind very tightly).
Specificity may well be one of the key issues in taking these compounds onto the next stage of selecting a preclinical antileishmania lead from these new scaffolds, and these structures provide an excellent starting point for the next wave of medicinal chemistry to try and enhance both potency and specificity.

Figure 1
Left: the X-ray structure of LmNMT (2wsa) in complex with DDD646 (magenta C atoms) occupying the substrate binding site. The adjacent active site is occupied by myristoyl-CoA (green C atoms). Tyr217 is highlighted in white. Right: the four ligands complexed with LmNMT described in the current paper and also found to occupy the substrate binding pocket.