A G358S mutation in the Plasmodium falciparum Na+ pump PfATP4 confers clinically-relevant resistance to cipargamin.

Diverse compounds target the Plasmodium falciparum Na+ pump PfATP4, with cipargamin and (+)-SJ733 the most clinically-advanced. In a recent clinical trial for cipargamin, recrudescent parasites emerged, with most having a G358S mutation in PfATP4. Here, we show that PfATP4G358S parasites can withstand micromolar concentrations of cipargamin and (+)-SJ733, while remaining susceptible to antimalarials that do not target PfATP4. The G358S mutation in PfATP4, and the equivalent mutation in Toxoplasma gondii ATP4, decrease the sensitivity of ATP4 to inhibition by cipargamin and (+)-SJ733, thereby protecting parasites from disruption of Na+ regulation. The G358S mutation reduces the affinity of PfATP4 for Na+ and is associated with an increase in the parasite's resting cytosolic [Na+]. However, no defect in parasite growth or transmissibility is observed. Our findings suggest that PfATP4 inhibitors in clinical development should be tested against PfATP4G358S parasites, and that their combination with unrelated antimalarials may mitigate against resistance development.

Natural products as probes in pharmaceutical research.

From the start of the pharmaceutical research natural products played a key role in drug discovery and development. Over time many discoveries of fundamental new biology were triggered by the unique biological activity of natural products. Unprecedented chemical structures, novel chemotypes, often pave the way to investigate new biology and to explore new pathways and targets. This review summarizes the recent results in the area with a focus on research done in the laboratories of Novartis Institutes for BioMedical Research. We aim to put the technological advances in target identification techniques in the context to the current revival of phenotypic screening and the increasingly complex biological questions related to drug discovery.

Gift from Nature: Cyclomarin†A Kills Mycobacteria and Malaria Parasites by Distinct Modes of Action.

Malaria continues to be one of the most devastating human diseases despite many efforts to limit its spread by prevention of infection or by pharmaceutical treatment of patients. We have conducted a screen for antiplasmodial compounds by using a natural product library. Here we report on cyclomarin A as a potent growth inhibitor of Plasmodium falciparum and the identification of its molecular target, diadenosine triphosphate hydrolase (PfAp3Aase), by chemical proteomics. Using a biochemical assay, we could show that cyclomarin A is a specific inhibitor of the plasmodial enzyme but not of the closest human homologue hFHIT. Co-crystallisation experiments demonstrate a unique binding mode of the inhibitor. One molecule of cyclomarin A binds a dimeric PfAp3Aase and prevents the formation of the enzyme⋅substrate complex. These results validate PfAp3Aase as a new drug target for the treatment of malaria. We have previously elucidated the structurally unrelated regulatory subunit ClpC1 of the ClpP protease as the molecular target of cyclomarin A in Mycobacterium tuberculosis. Thus, cyclomarin A is a rare example of a natural product with two distinct and specific modes of action.

Luteipulveratus halotolerans sp. nov., an actinobacterium (Dermacoccaceae) from forest soil.

The taxonomic position of an actinobacterium strain, C296001T, isolated from a soil sample collected in Sarawak, Malaysia, was established using a polyphasic approach. Phylogenetically, strain C296001T was closely associated with the genus Luteipulveratus and formed a distinct monophyletic clade with the only described species, Luteipulveratus mongoliensis NBRC 105296T. The 16S rRNA gene sequence similarity between strain C296001T and L. mongoliensis was 98.7 %. DNA-DNA hybridization results showed that the relatedness of strain C296001T to L. mongoliensis was only 21.5 %. The DNA G+C content of strain C296001T was 71.7 mol%. Using a PacBio RS II system, whole genome sequences for strains C296001T and NBRC 105296T were obtained. The genome sizes of 4.5 Mbp and 5.4 Mbp determined were similar to those of other members of the family Dermacoccaceae. The cell-wall peptidoglycan contained lysine, alanine, aspartic acid, glutamic acid and serine, representing the peptidoglycan type A4α l-Lys-l-Ser-d-Asp. The major menaquinones were MK-8(H4), MK-8 and MK-8(H2). Phosphatidylglycerol, phosphatidylinositol, diphosphatidylglycerol and phosphoglycolipid were the polar lipids, while the whole-cell sugars were glucose, fucose and lesser amounts of ribose and galactose. The major fatty acids were iso-C16 : 0, anteiso-C17 : 0, iso-C16 : 1 H, anteiso-C17 : 1ω9c, iso-C18 : 0 and 10-methyl C17 : 0. Chemotaxonomic analyses showed that C296001T had typical characteristics of members of the genus Luteipulveratus, with the main differences occurring in phenotypic characteristics. On the basis of the phenotypic and chemotaxonomic evidence, it is proposed that strain C296001T be classified as a representative of a novel species in the genus Luteipulveratus, for which the name Luteipulveratus halotolerans sp. nov. is recommended. The type strain is C296001T ( = ATCC TSD-4T = JCM 30660T).

Nannocystin†A: an Elongation Factor 1 Inhibitor from Myxobacteria with Differential Anti-Cancer Properties.

Cultivation of myxobacteria of the Nannocystis genus led to the isolation and structure elucidation of a class of novel cyclic lactone inhibitors of elongation factor 1. Whole genome sequence analysis and annotation enabled identification of the putative biosynthetic cluster and synthesis process. In biological assays the compounds displayed anti-fungal and cytotoxic activity. Combined genetic and proteomic approaches identified the eukaryotic translation elongation factor 1α (EF-1α) as the primary target for this compound class. Nannocystin A (1) displayed differential activity across various cancer cell lines and EEF1A1 expression levels appear to be the main differentiating factor. Biochemical and genetic evidence support an overlapping binding site of 1 with the anti-cancer compound didemnin B on EF-1α. This myxobacterial chemotype thus offers an interesting starting point for further investigations of the potential of therapeutics targeting elongation factor 1.

The novolactone natural product disrupts the allosteric regulation of Hsp70.

The highly conserved 70 kDa heat shock proteins (Hsp70) play an integral role in proteostasis such that dysregulation has been implicated in numerous diseases. Elucidating the precise role of Hsp70 family members in the cellular context, however, has been hampered by the redundancy and intricate regulation of the chaperone network, and relatively few selective and potent tools. We have characterized a natural product, novolactone, that targets cytosolic and ER-localized isoforms of Hsp70 through a highly conserved covalent interaction at the interface between the substrate-binding and ATPase domains. Biochemical and structural analyses indicate that novolactone disrupts interdomain communication by allosterically inducing a conformational change in the Hsp70 protein to block ATP-induced substrate release and inhibit refolding activities. Thus, novolactone is a valuable tool for exploring the requirements of Hsp70 chaperones in diverse cellular contexts.

High-resolution chemical dissection of a model eukaryote reveals targets, pathways and gene functions.

Due to evolutionary conservation of biology, experimental knowledge captured from genetic studies in eukaryotic model organisms provides insight into human cellular pathways and ultimately physiology. Yeast chemogenomic profiling is a powerful approach for annotating cellular responses to small molecules. Using an optimized platform, we provide the relative sensitivities of the heterozygous and homozygous deletion collections for nearly 1800 biologically active compounds. The data quality enables unique insights into pathways that are sensitive and resistant to a given perturbation, as demonstrated with both known and novel compounds. We present examples of novel compounds that inhibit the therapeutically relevant fatty acid synthase and desaturase (Fas1p and Ole1p), and demonstrate how the individual profiles facilitate hypothesis-driven experiments to delineate compound mechanism of action. Importantly, the scale and diversity of tested compounds yields a dataset where the number of modulated pathways approaches saturation. This resource can be used to map novel biological connections, and also identify functions for unannotated genes. We validated hypotheses generated by global two-way hierarchical clustering of profiles for (i) novel compounds with a similar mechanism of action acting upon microtubules or vacuolar ATPases, and (ii) an un-annotated ORF, YIL060w, that plays a role in respiration in the mitochondria. Finally, we identify and characterize background mutations in the widely used yeast deletion collection which should improve the interpretation of past and future screens throughout the community. This comprehensive resource of cellular responses enables the expansion of our understanding of eukaryotic pathway biology.

Spiroindolones, a potent compound class for the treatment of malaria.

Recent reports of increased tolerance to artemisinin derivatives--the most recently adopted class of antimalarials--have prompted a need for new treatments. The spirotetrahydro-beta-carbolines, or spiroindolones, are potent drugs that kill the blood stages of Plasmodium falciparum and Plasmodium vivax clinical isolates at low nanomolar concentration. Spiroindolones rapidly inhibit protein synthesis in P. falciparum, an effect that is ablated in parasites bearing nonsynonymous mutations in the gene encoding the P-type cation-transporter ATPase4 (PfATP4). The optimized spiroindolone NITD609 shows pharmacokinetic properties compatible with once-daily oral dosing and has single-dose efficacy in a rodent malaria model.

Ribosomally synthesized thiopeptide antibiotics targeting elongation factor Tu.

We identified the thiomuracins, a novel family of thiopeptides produced by a rare-actinomycete bacterium typed as a Nonomuraea species, via a screen for inhibition of growth of the bacterial pathogen Staphylococcus aureus. Thiopeptides are a class of macrocyclic, highly modified peptides that are decorated by thiazoles and defined by a central six-membered heterocyclic ring system. Mining the genomes of thiopeptide-producing strains revealed the elusive biosynthetic route for this class of antibiotics. The thiopeptides are chromosomally encoded, ribosomally synthesized proteins, and isolation of gene clusters for production of thiomuracin and the related thiopeptide GE2270A revealed the post-translational machinery required for maturation. The target of the thiomuracins was identified as bacterial Elongation Factor Tu (EF-Tu). In addition to potently inhibiting a target that is unexploited by marketed human therapeutics, the thiomuracins have a low propensity for selecting for antibiotic resistance and confer no measurable cross-resistance to antibiotics in clinical use.

Winged helix transcription factor CPCR1 is involved in regulation of beta-lactam biosynthesis in the fungus Acremonium chrysogenum.

Winged helix transcription factors, including members of the forkhead and the RFX subclasses, are characteristic for the eukaryotic domains in animals and fungi but seem to be missing in plants. In this study, in vitro and in vivo approaches were used to determine the functional role of the RFX transcription factor CPCR1 from the filamentous fungus Acremonium chrysogenum in cephalosporin C biosynthesis. Gel retardation analyses were applied to identify new binding sites of the transcription factor in an intergenic promoter region of cephalosporin C biosynthesis genes. Here, we illustrate that CPCR1 recognizes and binds at least two sequences in the intergenic region between the pcbAB and pcbC genes. The in vivo relevance of the two sequences for gene activation was demonstrated by using pcbC promoter-lacZ fusions in A. chrysogenum. The deletion of both CPCR1 binding sites resulted in an extensive reduction of reporter gene activity in transgenic strains (to 12% of the activity level of the control). Furthermore, Acremonium transformants with multiple copies of the cpcR1 gene and knockout strains support the idea of CPCR1 being a regulator of cephalosporin C biosynthesis gene expression. Significant differences in pcbC gene transcript levels were obtained with the knockout transformants. More-than-twofold increases in the pcbC transcript level at 24 and 36 h of cultivation were followed by a reduction to approximately 80% from 48 to 96 h in the knockout strain. The overall levels of the production of cephalosporin C were identical in transformed and nontransformed strains; however, the knockout strains showed a striking reduction in the level of the biosynthesis of intermediate penicillin N to less than 20% of that of the recipient strain. We were able to show that the complementation of the cpcR1 gene in the knockout strains reverses pcbC transcript and penicillin N amounts to levels comparable to those in the control. These results clearly indicate the involvement of CPCR1 in the regulation of cephalosporin C biosynthesis. However, the complexity of the data points to a well-controlled or even functional redundant network of transcription factors, with CPCR1 being only one player within this process.