Molecules incorporating a benzothiazole core scaffold inhibit the N-myristoyltransferase of Plasmodium falciparum.

Recombinant N-myristoyltransferase of Plasmodium falciparum (termed PfNMT) has been used in the development of a SPA (scintillation proximity assay) suitable for automation and high-throughput screening of inhibitors against this enzyme. The ability to use the SPA has been facilitated by development of an expression and purification system which yields considerably improved quantities of soluble active recombinant PfNMT compared with previous studies. Specifically, yields of pure protein have been increased from 12 microg x l(-1) to >400 microg x l(-1) by use of a synthetic gene with codon usage optimized for expression in an Escherichia coli host. Preliminary small-scale 'piggyback' inhibitor studies using the SPA have identified a family of related molecules containing a core benzothiazole scaffold with IC50 values 80% at a concentration of 10 microM

Insights into Cleavage Specificity from the Crystal Structure of Foot-and-Mouth Disease Virus 3C Protease Complexed with a Peptide Substrate.

Accepted versio

Design and Synthesis of High Affinity Inhibitors of Plasmodium falciparum and Plasmodium vivax N-Myristoyltransferases Directed by Ligand Efficiency Dependent Lipophilicity (LELP)

N-Myristoyltransferase (NMT) is an essential eukaryotic enzyme and an attractive drug target in parasitic infections such as malaria. We have previously reported that 2-(3-(piperidin-4-yloxy)benzo[b]thiophen-2-yl)-5-((1,3,5-trimethyl-1H-pyrazol-4-yl)methyl)-1,3,4-oxadiazole (34c) is a high affinity inhibitor of both Plasmodium falciparum and P. vivax NMT and displays activity in vivo against a rodent malaria model. Here we describe the discovery of 34c through optimization of a previously described series. Development, guided by targeting a ligand efficiency dependent lipophilicity (LELP) score of less than 10, yielded a 100-fold increase in enzyme affinity and a 100-fold drop in lipophilicity with the addition of only two heavy atoms. 34c was found to be equipotent on chloroquine-sensitive and -resistant cell lines and on both blood and liver stage forms of the parasite. These data further validate NMT as an exciting drug target in malaria and support 34c as an attractive tool for further optimization

Using a Non-Image-Based Medium-Throughput Assay for Screening Compounds Targeting N-myristoylation in Intracellular Leishmania Amastigotes

We have refined a medium-throughput assay to screen hit compounds for activity against N-myristoylation in intracellular amastigotes of Leishmania donovani. Using clinically-relevant stages of wild type parasites and an Alamar blue-based detection method, parasite survival following drug treatment of infected macrophages is monitored after macrophage lysis and transformation of freed amastigotes into replicative extracellular promastigotes. The latter transformation step is essential to amplify the signal for determination of parasite burden, a factor dependent on equivalent proliferation rate between samples. Validation of the assay has been achieved using the anti-leishmanial gold standard drugs, amphotericin B and miltefosine, with EC50 values correlating well with published values. This assay has been used, in parallel with enzyme activity data and direct assay on isolated extracellular amastigotes, to test lead-like and hit-like inhibitors of Leishmania Nmyristoyl transferase (NMT). These were derived both from validated in vivo inhibitors of Trypanosoma brucei NMT and a recent high-throughput screen against L. donovani NMT. Despite being a potent inhibitor of L. donovani NMT, the activity of the lead T. brucei NMT inhibitor (DDD85646) against L. donovani amastigotes is relatively poor. Encouragingly, analogues of DDD85646 show improved translation of enzyme to cellular activity. In testing the high-throughput L. donovani hits, we observed macrophage cytotoxicity with compounds from two of the four NMT-selective series identified, while all four series displayed low enzyme to cellular translation, also seen here with the T. brucei NMT inhibitors. Improvements in potency and physicochemical properties will be required to deliver attractive lead-like Leishmania NMT inhibitors

Selective Inhibitors of Protozoan Protein N-myristoyltransferases as Starting Points for Tropical Disease Medicinal Chemistry Programs

Inhibition of N-myristoyltransferase has been validated pre-clinically as a target for the treatment of fungal and trypanosome infections, using species-specific inhibitors. In order to identify inhibitors of protozoan NMTs, we chose to screen a diverse subset of the Pfizer corporate collection against Plasmodium falciparum and Leishmania donovani NMTs. Primary screening hits against either enzyme were tested for selectivity over both human NMT isoforms (Hs1 and Hs2) and for broad-spectrum anti-protozoan activity against the NMT from Trypanosoma brucei. Analysis of the screening results has shown that structure-activity relationships (SAR) for Leishmania NMT are divergent from all other NMTs tested, a finding not predicted by sequence similarity calculations, resulting in the identification of four novel series of Leishmania-selective NMT inhibitors. We found a strong overlap between the SARs for Plasmodium NMT and both human NMTs, suggesting that achieving an appropriate selectivity profile will be more challenging. However, we did discover two novel series with selectivity for Plasmodium NMT over the other NMT orthologues in this study, and an additional two structurally distinct series with selectivity over Leishmania NMT. We believe that release of results from this study into the public domain will accelerate the discovery of NMT inhibitors to treat malaria and leishmaniasis. Our screening initiative is another example of how a tripartite partnership involving pharmaceutical industries, academic institutions and governmental/non-governmental organisations such as Medical Research Council and Wellcome Trust can stimulate research for neglected diseases

Validation of N-myristoyltransferase as an antimalarial drug target using an integrated chemical biology approach

Malaria is an infectious disease caused by parasites of the genus Plasmodium, which leads to approximately one million deaths per annum worldwide. Chemical validation of new antimalarial targets is urgently required in view of rising resistance to current drugs. One such putative target is the enzyme N-myristoyltransferase, which catalyses the attachment of the fatty acid myristate to protein substrates (N-myristoylation). Here, we report an integrated chemical biology approach to explore protein myristoylation in the major human parasite P. falciparum, combining chemical proteomic tools for identification of the myristoylated and glycosylphosphatidylinositol-anchored proteome with selective small-molecule N-myristoyltransferase inhibitors. We demonstrate that N-myristoyltransferase is an essential and chemically tractable target in malaria parasites both in vitro and in vivo, and show that selective inhibition of N-myristoylation leads to catastrophic and irreversible failure to assemble the inner membrane complex, a critical subcellular organelle in the parasite life cycle. Our studies provide the basis for the development of new antimalarials targeting N-myristoyltransferase

Fragment-derived inhibitors of human N-myristoyltransferase block capsid assembly and replication of the common cold virus

Rhinoviruses (RVs) are the pathogens most often responsible for the common cold, and are a frequent cause of exacerbations in asthma, chronic obstructive pulmonary disease and cystic fibrosis. Here we report the discovery of IMP-1088, a picomolar dual inhibitor of the human N-myristoyltransferases NMT1 and NMT2, and use it to demonstrate that pharmacological inhibition of host-cell N-myristoylation rapidly and completely prevents rhinoviral replication without inducing cytotoxicity. The identification of cooperative binding between weak-binding fragments led to rapid inhibitor optimization through fragment reconstruction, structure-guided fragment linking and conformational control over linker geometry. We show that inhibition of the co-translational myristoylation of a specific virus-encoded protein (VP0) by IMP-1088 potently blocks a key step in viral capsid assembly, to deliver a low nanomolar antiviral activity against multiple RV strains, poliovirus and foot and-mouth disease virus, and protection of cells against virus-induced killing, highlighting the potential of host myristoylation as a drug target in picornaviral infections

Structural studies on DNP binding antibodies

This thesis is concerned with structural aspects of the recognition and effector functions of antibody molecules. The recognition process is investigated in the dinitrophenyl (DNP) binding mouse IgA produced by the myeloma MOPC 315. The studies on effector functions utilize a DNP binding mouse hybridoma IgG2a to examine the role of N-glycosylation in IgG. The combining site of protein 315. The involvement of tyrosyl residues in the combining site of protein 315 was examined by preparing specifically nitrated NO2-Tyr-33H and NO2-Tyr-34L derivatives of the Fv fragment of this protein. The ionizations of tnese derivatives were studied in the presence and absence of various DNP-ligands. Perturbations to the nitrotyrosine ionizations were found to be caused by the side chains of certain of these ligands, allowing an indication of the distance of these tyrosines from the bound hapten. On examination of the compatibility of these data with the model of the combining site of protein 315 proposed by Dower et al. (1977) (Biochem. J. 165, 207-225) it was found that while the location of Tyr-33H is consistent with this model, the position of Tyr-34L is not. A remodelled combining site using the modified ring-current treatment of Perkins and Dwek (1980) (Biochemistry 19, 245-258) is presented. This allows a better rationalization of the nitration data and of previous experimental observations on protein 315. The role of the conserved C 2 domain oligosaccharide of IgG. This was examined by a functional comparison of native IgG with an aglycosylated IgG preparation. Aglycosylation was acheived by cell culture of the hybridoma cells in the presence of the glycosylation inhibitor tunicamycin. The conditions for preparation and purification of this aglycosyl IgG are described. Aglycosylated IgG is found to be correctly assembled as an H2L2 unit. It retains the antigen binding and Staphylococcal protein A binding abilities of the native glycosylated molecule. Using an assay system designed specifically to overcome certain problems in comparing Clq binding to different preparations of IgG it was found that the aglycosylated preparation showed only slightly reduced affinity for Clq. In addition the aglycosylated IgG is able to activate bound Cl. The above results are consistent with the structure of the Fc region being only minimally altered in the absence of oligosaccharide. The structural integrity of the aglycosylated molecule may be compromised however, as its ability to bind to monocyte Fc receptor is significantly reduced. In addition the aglycosylated molecule becomes much more susceptible to proteolytic digestion. A computational model-building analysis of the quaternary structure of Fc allows an explanation of at least some of the effects of aglycosylation in terms of reduced conformational stability of the CH2 domains.</p

Structural studies on DNP binding antibodies

This thesis is concerned with structural aspects of the recognition and effector functions of antibody molecules. The recognition process is investigated in the dinitrophenyl (DNP) binding mouse IgA produced by the myeloma MOPC 315. The studies on effector functions utilize a DNP binding mouse hybridoma IgG2a to examine the role of N-glycosylation in IgG. The combining site of protein 315. The involvement of tyrosyl residues in the combining site of protein 315 was examined by preparing specifically nitrated NO2-Tyr-33H and NO2-Tyr-34L derivatives of the Fv fragment of this protein. The ionizations of tnese derivatives were studied in the presence and absence of various DNP-ligands. Perturbations to the nitrotyrosine ionizations were found to be caused by the side chains of certain of these ligands, allowing an indication of the distance of these tyrosines from the bound hapten. On examination of the compatibility of these data with the model of the combining site of protein 315 proposed by Dower et al. (1977) (Biochem. J. 165, 207-225) it was found that while the location of Tyr-33H is consistent with this model, the position of Tyr-34L is not. A remodelled combining site using the modified ring-current treatment of Perkins and Dwek (1980) (Biochemistry 19, 245-258) is presented. This allows a better rationalization of the nitration data and of previous experimental observations on protein 315. The role of the conserved C 2 domain oligosaccharide of IgG. This was examined by a functional comparison of native IgG with an aglycosylated IgG preparation. Aglycosylation was acheived by cell culture of the hybridoma cells in the presence of the glycosylation inhibitor tunicamycin. The conditions for preparation and purification of this aglycosyl IgG are described. Aglycosylated IgG is found to be correctly assembled as an H2L2 unit. It retains the antigen binding and Staphylococcal protein A binding abilities of the native glycosylated molecule. Using an assay system designed specifically to overcome certain problems in comparing Clq binding to different preparations of IgG it was found that the aglycosylated preparation showed only slightly reduced affinity for Clq. In addition the aglycosylated IgG is able to activate bound Cl. The above results are consistent with the structure of the Fc region being only minimally altered in the absence of oligosaccharide. The structural integrity of the aglycosylated molecule may be compromised however, as its ability to bind to monocyte Fc receptor is significantly reduced. In addition the aglycosylated molecule becomes much more susceptible to proteolytic digestion. A computational model-building analysis of the quaternary structure of Fc allows an explanation of at least some of the effects of aglycosylation in terms of reduced conformational stability of the CH2 domains.</p