Andrew Woodland

A novel multiple-stage antimalarial agent that inhibits protein synthesis

There is an urgent need for new drugs to treat malaria, with broad therapeutic potential and novel modes of action, to widen the scope of treatment and to overcome emerging drug resistance. Here we describe the discovery of DDD107498, a compound with a potent and novel spectrum of antimalarial activity against multiple life-cycle stages of the Plasmodium parasite, with good pharmacokinetic properties and an acceptable safety profile. DDD107498 demonstrates potential to address a variety of clinical needs, including single-dose treatment, transmission blocking and chemoprotection. DDD107498 was developed from a screening programme against blood-stage malaria parasites; its molecular target has been identified as translation elongation factor 2 (eEF2), which is responsible for the GTP-dependent translocation of the ribosome along messenger RNA, and is essential for protein synthesis. This discovery of eEF2 as a viable antimalarial drug target opens up new possibilities for drug discovery.

Identification of Inhibitors of the Leishmania cdc2-Related Protein Kinase CRK3

New drugs are urgently needed for the treatment of tropical parasitic diseases such as leishmaniasis and human African trypanosomiasis (HAT). This work involved a high‐throughput screen of a focussed kinase set of ∼3400 compounds to identify potent and parasite‐selective inhibitors of an enzymatic Leishmania CRK3–cyclin 6 complex. The aim of this study is to provide chemical validation that Leishmania CRK3–CYC6 is a drug target. Eight hit series were identified, of which four were followed up. The optimisation of these series using classical SAR studies afforded low‐nanomolar CRK3 inhibitors with significant selectivity over the closely related human cyclin dependent kinase CDK2.

Identification of small molecule inhibitors of pre-mRNA splicing.

Background: There is a need for new small molecule pre-mRNA splicing inhibitors as biotools. Results: High throughput screening resulted in the identification of small molecule splicing inhibitors that are active in vitro and in cells. Conclusion: New small molecules for studying pre-mRNA splicing in vitro and in cells are identified. Significance: Small drug-like molecules are identified that modulate splicing in vitro and in cells. Eukaryotic pre-mRNA splicing is an essential step in gene expression for all genes that contain introns. In contrast to transcription and translation, few well characterized chemical inhibitors are available with which to dissect the splicing process, particularly in cells. Therefore, the identification of specific small molecules that either inhibit or modify pre-mRNA splicing would be valuable for research and potentially also for therapeutic applications. We have screened a highly curated library of 71,504 drug-like small molecules using a high throughput in vitro splicing assay. This identified 10 new compounds that both inhibit pre-mRNA splicing in vitro and modify splicing of endogenous pre-mRNA in cells. One of these splicing modulators, DDD00107587 (termed “madrasin,” i.e. 2-((7methoxy-4-methylquinazolin-2-yl)amino)-5,6-dimethylpyrimidin-4(3H)-one RNAsplicing inhibitor), was studied in more detail. Madrasin interferes with the early stages of spliceosome assembly and stalls spliceosome assembly at the A complex. Madrasin is cytotoxic at higher concentrations, although at lower concentrations it induces cell cycle arrest, promotes a specific reorganization of subnuclear protein localization, and modulates splicing of multiple pre-mRNAs in both HeLa and HEK293 cells.

From On-Target to Off-Target Activity: Identification and Optimisation of Trypanosoma brucei GSK3 Inhibitors and Their Characterisation as Anti-Trypanosoma brucei Drug Discovery Lead Molecules

Human African trypanosomiasis (HAT) is a life‐threatening disease with approximately 30 000–40 000 new cases each year. Trypanosoma brucei protein kinase GSK3 short (TbGSK3) is required for parasite growth and survival. Herein we report a screen of a focused kinase library against T. brucei GSK3. From this we identified a series of several highly ligand‐efficient TbGSK3 inhibitors. Following the hit validation process, we optimised a series of diaminothiazoles, identifying low‐nanomolar inhibitors of TbGSK3 that are potent in vitro inhibitors of T. brucei proliferation. We show that the TbGSK3 pharmacophore overlaps with that of one or more additional molecular targets.

Discovery of Inhibitors of Trypanosoma brucei by Phenotypic Screening of a Focused Protein Kinase Library.

A screen of a focused kinase inhibitor library against Trypanosoma brucei rhodesiense led to the identification of seven series, totaling 121 compounds, which showed >50 % inhibition at 5 μm. Screening of these hits in a T. b. brucei proliferation assay highlighted three compounds with a 1H‐imidazo[4,5‐b]pyrazin‐2(3H)‐one scaffold that showed sub‐micromolar activity and excellent selectivity against the MRC5 cell line. Subsequent rounds of optimisation led to the identification of compounds that exhibited good in vitro drug metabolism and pharmacokinetics (DMPK) properties, although in general this series suffered from poor solubility. A scaffold‐hopping exercise led to the identification of a 1H‐pyrazolo[3,4‐b]pyridine scaffold, which retained potency. A number of examples were assessed in a T. b. brucei growth assay, which could differentiate static and cidal action. Compounds from the 1H‐imidazo[4,5‐b]pyrazin‐2(3H)‐one series were found to be either static or growth‐slowing and not cidal. Compounds with the 1H‐pyrazolo[3,4‐b]pyridine scaffold were found to be cidal and showed an unusual biphasic nature in this assay, suggesting they act by at least two mechanisms.

Optimisation of the Anti-Trypanosoma brucei Activity of the Opioid Agonist U50488

Screening of the Sigma–Aldrich Library of Pharmacologically Active Compounds (LOPAC) against cultured Trypanosoma brucei, the causative agent of African sleeping sickness, resulted in the identification of a number of compounds with selective antiproliferative activity over mammalian cells. These included (+)‐(1R,2R)‐U50488, a weak opioid agonist with an EC50 value of 59 nM as determined in our T. brucei in vitro assay reported previously. This paper describes the modification of key structural elements of U50488 to investigate structure–activity relationships (SAR) and to optimise the antiproliferative activity and pharmacokinetic properties of this compound.

Differential control of Toll-like receptor 4–induced interleukin-10 induction in macrophages and B cells reveals a role for p90 ribosomal S6 kinases

Increasing evidence has linked dysregulated interleukin (IL)-10 production by IL-10+ve B cells to autoimmunity, highlighting the importance of improving the understanding of the regulation of IL-10 production in these cells. In both B cells and myeloid cells, IL-10 can be produced in response to Toll-like receptor (TLR) agonists. In macrophages, previous studies have established that mitogen- and stress-activated protein kinases (MSKs) regulate IL-10 production via the phosphorylation of cAMP response element–binding (CREB) protein on the IL-10 promoter. We found here that although MSKs are activated in peritoneal B cells in response to TLR4 agonists, neither MSKs nor CREB are required for IL-10 production in these cells. Using a combination of chemical inhibitors and knockout mice, we found that IL-10 induction in B cells was regulated by an ERK1/2- and p90 ribosomal S6 kinase-dependent mechanism, unlike in macrophages in which p90 ribosomal S6 kinase was not required. This observation highlights fundamental differences in the signaling controlling IL-10 production in B cells and macrophages, even though these two cell types respond to a common TLR stimulus.

Corrigendum: A novel multiple-stage antimalarial agent that inhibits protein synthesis

This corrects the article DOI: 10.1038/nature14451

Discovery of super soft-drug modulators of sphingosine-1-phosphate receptor 1

Graphical abstract

Screening a protein kinase inhibitor library against Plasmodium falciparum

BackgroundProtein kinases have been shown to be key drug targets, especially in the area of oncology. It is of interest to explore the possibilities of protein kinases as a potential target class in Plasmodium spp., the causative agents of malaria. However, protein kinase biology in malaria is still being investigated. Therefore, rather than assaying against individual protein kinases, a library of 4731 compounds with protein kinase inhibitor-like scaffolds was screened against the causative parasite, Plasmodium falciparum. This approach is more holistic and considers the whole kinome, making it possible to identify compounds that inhibit more than one P. falciparum protein kinase, or indeed other malaria targets.ResultsAs a result of this screen, 9 active compound series were identified; further validation was carried out on 4 of these series, with 3 being progressed into hits to lead chemistry. The detailed evaluation of one of these series is described.DiscussionThis screening approach proved to be an effective way to identify series for further optimisation against malaria. Compound optimisation was carried out in the absence of knowledge of the molecular target. Some of the series had to be halted for various reasons. Mode of action studies to find the molecular target may be useful when problems prevent further chemical optimisation.ConclusionsProgressible series were identified through phenotypic screening of a relatively small focused kinase scaffold chemical library.