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Discovery of high affinity inhibitors of Leishmania donovani N-myristoyltransferase.

ABSTRACT: N-Myristoyltransferase (NMT) is a potential drug target in Leishmania parasites. Scaffold-hopping from published inhibitors yielded the serendipitous discovery of a chemotype selective for Leishmania donovani NMT; development led to high affinity inhibitors with excellent ligand efficiency. The binding mode was characterised by crystallography and provides a structural rationale for selectivity.

SUBMITTER: Rackham MD

PROVIDER: S-EPMC4757855 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Discovery of high affinity inhibitors of Leishmania donovani N-myristoyltransferase.

Rackham Mark D MD Yu Zhiyong Z Brannigan James A JA Heal William P WP Paape Daniel D Barker K Victoria KV Wilkinson Anthony J AJ Smith Deborah F DF Leatherbarrow Robin J RJ Tate Edward W EW

MedChemComm 20150819 10

N-Myristoyltransferase (NMT) is a potential drug target in Leishmania parasites. Scaffold-hopping from published inhibitors yielded the serendipitous discovery of a chemotype selective for Leishmania donovani NMT; development led to high affinity inhibitors with excellent ligand efficiency. The binding mode was characterised by crystallography and provides a structural rationale for selectivity. ...[more]

PMID: 26962429

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Pharmacological Validation of N-Myristoyltransferase as a Drug Target in Leishmania donovani.

Project description:Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and L. infantum, is responsible for ∼30 000 deaths annually. Available treatments are inadequate, and there is a pressing need for new therapeutics. N-Myristoyltransferase (NMT) remains one of the few genetically validated drug targets in these parasites. Here, we sought to pharmacologically validate this enzyme in Leishmania. A focused set of 1600 pyrazolyl sulfonamide compounds was screened against L. major NMT in a robust high-throughput biochemical assay. Several potent inhibitors were identified with marginal selectivity over the human enzyme. There was little correlation between the enzyme potency of these inhibitors and their cellular activity against L. donovani axenic amastigotes, and this discrepancy could be due to poor cellular uptake due to the basicity of these compounds. Thus, a series of analogues were synthesized with less basic centers. Although most of these compounds continued to suffer from relatively poor antileishmanial activity, our most potent inhibitor of LmNMT (DDD100097, K i of 0.34 nM) showed modest activity against L. donovani intracellular amastigotes (EC50 of 2.4 μM) and maintained a modest therapeutic window over the human enzyme. Two unbiased approaches, namely, screening against our cosmid-based overexpression library and thermal proteome profiling (TPP), confirm that DDD100097 (compound 2) acts on-target within parasites. Oral dosing with compound 2 resulted in a 52% reduction in parasite burden in our mouse model of VL. Thus, NMT is now a pharmacologically validated target in Leishmania. The challenge in finding drug candidates remains to identify alternative strategies to address the drop-off in activity between enzyme inhibition and in vitro activity while maintaining sufficient selectivity over the human enzyme, both issues that continue to plague studies in this area.

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Structure-based design of potent and selective Leishmania N-myristoyltransferase inhibitors.

Project description:Inhibitors of Leishmania N-myristoyltransferase (NMT), a potential target for the treatment of leishmaniasis, obtained from a high-throughput screen, were resynthesized to validate activity. Crystal structures bound to Leishmania major NMT were obtained, and the active diastereoisomer of one of the inhibitors was identified. On the basis of structural insights, enzyme inhibition was increased 40-fold through hybridization of two distinct binding modes, resulting in novel, highly potent Leishmania donovani NMT inhibitors with good selectivity over the human enzyme.

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N-myristoyltransferase from Leishmania donovani: structural and functional characterisation of a potential drug target for visceral leishmaniasis.

Project description:N-Myristoyltransferase (NMT) catalyses the attachment of the 14-carbon saturated fatty acid, myristate, to the amino-terminal glycine residue of a subset of eukaryotic proteins that function in multiple cellular processes, including vesicular protein trafficking and signal transduction. In these pathways, N-myristoylation facilitates association of substrate proteins with membranes or the hydrophobic domains of other partner peptides. NMT function is essential for viability in all cell types tested to date, demonstrating that this enzyme has potential as a target for drug development. Here, we provide genetic evidence that NMT is likely to be essential for viability in insect stages of the pathogenic protozoan parasite, Leishmania donovani, causative agent of the tropical infectious disease, visceral leishmaniasis. The open reading frame of L. donovani NMT has been amplified and used to overproduce active recombinant enzyme in Escherichia coli, as demonstrated by gel mobility shift assays of ligand binding and peptide-myristoylation activity in scintillation proximity assays. The purified protein has been crystallized in complex with the non-hydrolysable substrate analogue S-(2-oxo)pentadecyl-CoA, and its structure was solved by molecular replacement at 1.4 A resolution. The structure has as its defining feature a 14-stranded twisted beta-sheet on which helices are packed so as to form an extended and curved substrate-binding groove running across two protein lobes. The fatty acyl-CoA is largely buried in the N-terminal lobe, its binding leading to the loosening of a flap, which in unliganded NMT structures, occludes the protein substrate binding site in the carboxy-terminal lobe. These studies validate L. donovani NMT as a potential target for development of new therapeutic agents against visceral leishmaniasis.

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Discovery of pyridyl-based inhibitors of Plasmodium falciparum N-myristoyltransferase.

Project description:N-Myristoyltransferase (NMT) represents an attractive drug target in parasitic infections such as malaria due to its genetic essentiality and amenability to inhibition by drug-like small molecules. Scaffold simplification from previously reported inhibitors containing bicyclic cores identified phenyl derivative 3, providing a versatile platform to study the effects of substitution on the scaffold, which yielded pyridyl 19. This molecule exhibited improved enzyme and cellular potency, and reduced lipophilicity compared to inhibitor 3. Further structure-based inhibitor design led to the discovery of 30, the most potent inhibitor in this series, which showed single-digit nM enzyme affinity and sub-?M anti-plasmodial activity.

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Diverse modes of binding in structures of Leishmania major N-myristoyltransferase with selective inhibitors.

Project description:The leishmaniases are a spectrum of global diseases of poverty associated with immune dysfunction and are the cause of high morbidity. Despite the long history of these diseases, no effective vaccine is available and the currently used drugs are variously compromised by moderate efficacy, complex side effects and the emergence of resistance. It is therefore widely accepted that new therapies are needed. N-Myristoyltransferase (NMT) has been validated pre-clinically as a target for the treatment of fungal and parasitic infections. In a previously reported high-throughput screening program, a number of hit compounds with activity against NMT from Leishmania donovani have been identified. Here, high-resolution crystal structures of representative compounds from four hit series in ternary complexes with myristoyl-CoA and NMT from the closely related L. major are reported. The structures reveal that the inhibitors associate with the peptide-binding groove at a site adjacent to the bound myristoyl-CoA and the catalytic ?-carboxylate of Leu421. Each inhibitor makes extensive apolar contacts as well as a small number of polar contacts with the protein. Remarkably, the compounds exploit different features of the peptide-binding groove and collectively occupy a substantial volume of this pocket, suggesting that there is potential for the design of chimaeric inhibitors with significantly enhanced binding. Despite the high conservation of the active sites of the parasite and human NMTs, the inhibitors act selectively over the host enzyme. The role of conformational flexibility in the side chain of Tyr217 in conferring selectivity is discussed.

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