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

Vanadium: a re-emerging environmental hazard

Vanadium (V) is a contaminant which has been long confined to the annals of regulatory history. This follows the reduction of its historical primary source (fossil fuel emissions) since the 1970s (e.g., by 80% in the UK). However, V is quickly becoming an important strategic resource which promises its return to environmental prominence because of changing industrial practices and emerging waste streams. We discuss below (i) what makes V a re-emerging environmental and human health hazard of global interest, (ii) the knowledge gaps that currently restrict prediction of environmental effect and mitigation, and (iii) opportunities for the community to address these gaps toward reducing the risk of an impending environmental hazard

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

Twenty-three unsolved problems in hydrology (UPH) – a community perspective

This paper is the outcome of a community initiative to identify major unsolved scientific problems in hydrology motivated by a need for stronger harmonisation of research efforts. The procedure involved a public consultation through on-line media, followed by two workshops through which a large number of potential science questions were collated, prioritised, and synthesised. In spite of the diversity of the participants (230 scientists in total), the process revealed much about community priorities and the state of our science: a preference for continuity in research questions rather than radical departures or redirections from past and current work. Questions remain focussed on process-based understanding of hydrological variability and causality at all space and time scales. Increased attention to environmental change drives a new emphasis on understanding how change propagates across interfaces within the hydrological system and across disciplinary boundaries. In particular, the expansion of the human footprint raises a new set of questions related to human interactions with nature and water cycle feedbacks in the context of complex water management problems. We hope that this reflection and synthesis of the 23 unsolved problems in hydrology will help guide research efforts for some years to come

Clozapine's functional mesolimbic selectivity is not duplicated by the addition of anticholinergic action to haloperidol: a brain stimulation study in the rat

This study examined whether the anticholinergic potency of the clinically superior antipsychotic drug clozapine contributes to clozapine's anatomically-selective functional inhibition of the mesolimbic dopamine (DA) system, using an electrical brain-stimulation reward (BSR) paradigm in rats that has been previously shown to be highly sensitive to clozapine's mesolimbic functional selectivity. Rats were chronically administered saline, clozapine, haloperidol, or haloperidol plus the anticholinergic compound trihexyphenidyl, and threshold sensitivity of the mesolimbic and nigrostriatal DA systems was assessed using the BSR paradigm, to infer degree of functional DA blockade produced by the chronic drug regimens. Chronic saline produced no change in either DA system. Congruent with previous findings, chronic clozapine powerfully inhibited the mesolimbic DA system but spared the nigrostriatal DA system. Also congruent with previous findings, chronic haloperidol powerfully inhibited both DA systems. Compared to chronic haloperidol alone, chronic haloperidol plus chronic trihexyphenidyl exerted diminished anti-DA action in both the mesolimbic and nigrostriatal DA systems. These results suggest that clozapine's anticholinergic potency is not an adequate explanation for its functional mesolimbic selectivity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46341/1/213_2005_Article_BF02246960.pd