The challenges of developing novel antiparasitic drugs

Only a few novel classes of antiparasitic drugs have emerged over the last few decades, reflecting the difficulties associated with bringing a safe, effective molecule to market. In recent years, the screening paradigm has shifted from empirical whole parasite screening towards mechanism-based high throughput screening. This approach requires investment in molecular parasitology and in understanding the basic biology of parasites, as well as requiring considerable investment in an infrastructure for screening. Add to this the fact that the drug discovery process is iterative with high attrition, the Animal Health industry by necessity must focus on discovering medicines for diseases, which will deliver a return on investment. In recent years the rapid progression of genomics has unlocked a plethora of tools for target identification, validation and screening, revolutionising mechanism-based screening for antiparasitic drug discovery. The challenge still remains; however, to identify novel chemical entities with the properties required to deliver a safe, effective antiparasitic drug.     

Identification of hairs found in leopard seal (<Emphasis Type="Italic">Hydrurga leptonyx</Emphasis>) scats

The leopard seal is a top-order predator in the Southern Ocean ecosystem and preys on a wide variety of vertebrate species including seals and penguins. We assessed the use of hairs found in leopard seal scats to identify the species of pinniped consumed. A reference collection of hairs was obtained from four potential leopard seal prey species including crabeater, Weddell, Ross, and Southern elephant seals. Discrimination techniques applied to terrestrial mammals did not allow for identification of the seal hairs. Instead, a 2-dimensional (2-D) and 6-dimensional (6-D) analysis technique utilising Mahalanobis distances (D ²) was used. The smallest Mahalanobis distance together with the largest value of p(F) positively identified hairs from each species. The 6-D analysis was more accurate and applied to hairs found in the leopard seal scats. The majority of prey species were identified as crabeater seals, which are a known prey item of the leopard seal.     

Genome-wide analysis of signatures of selection in populations of African honey bees (Apis mellifera) using new web-based tools

Background

With the development of inexpensive, high-throughput sequencing technologies, it has become feasible to examine questions related to population genetics and molecular evolution of non-model species in their ecological contexts on a genome-wide scale. Here, we employed a newly developed suite of integrated, web-based programs to examine population dynamics and signatures of selection across the genome using several well-established tests, including F_ST, pN/pS, and McDonald-Kreitman. We applied these techniques to study populations of honey bees (Apis mellifera) in East Africa. In Kenya, there are several described A. mellifera subspecies, which are thought to be localized to distinct ecological regions.

Results

We performed whole genome sequencing of 11 worker honey bees from apiaries distributed throughout Kenya and identified 3.6 million putative single-nucleotide polymorphisms. The dense coverage allowed us to apply several computational procedures to study population structure and the evolutionary relationships among the populations, and to detect signs of adaptive evolution across the genome. While there is considerable gene flow among the sampled populations, there are clear distinctions between populations from the northern desert region and those from the temperate, savannah region. We identified several genes showing population genetic patterns consistent with positive selection within African bee populations, and between these populations and European A. mellifera or Asian Apis florea.

Conclusions

These results lay the groundwork for future studies of adaptive ecological evolution in honey bees, and demonstrate the use of new, freely available web-based tools and workflows (http://usegalaxy.org/r/kenyanbee) that can be applied to any model system with genomic information.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1712-0) contains supplementary material, which is available to authorized users.     

Structure of Mycobacterium tuberculosis thioredoxin in complex with quinol inhibitor PMX464

Thioredoxin (Trx) plays a critical role in the regulation of cellular redox homeostasis. Many disease causing pathogens rely on the Trx redox system for survival in conditions of environmental stress. The Trx redox system has been implicated in the resistance of Mycobacterium tuberculosis (Mtb) to phagocytosis. Trx is able to reduce a variety of target substrates and reactive oxygen species (ROS) through the cyclization of its active site dithiol to the oxidized disulphide Cys37-Cys40. Here we report the crystal structure of the Mtb Trx C active site mutant C40S (MtbTrxCC40S) in isolation and in complex with the hydroxycyclohexadienone inhibitor PMX464. We observe PMX464 is covalently bound to the active site residue Cys37 through Michael addition of the cyclohexadienone ring and also forms noncovalent contacts which mimic the binding of natural Trx ligands. In comparison with the ligand free MtbTrxCC40S structure a conformational change occurs in the PMX464 complex involving movement of helix α2 and the active site loop. These changes are almost identical to those observed for helix α2 in human Trx ligand complexes. Whereas the ligand free structure forms a homodimer the inhibitor complex unexpectedly forms a different dimer with one PMX464 molecule bound at the interface. This 2:1 MtbTrxCC40S-PMX464 complex is also observed using mass spectrometry measurements. This structure provides an unexpected scaffold for the design of improved Trx inhibitors targeted at developing treatments for tuberculosis.     

The K+ battery-regulating Arabidopsis K+ channel AKT2 is under the control of multiple post-translational steps

Potassium (K⁺) is an important nutrient for plants. It serves as a cofactor of various enzymes and as the major inorganic solute maintaining plant cell turgor. In a recent study, an as yet unknown role of K⁺ in plant homeostasis was shown. It was demonstrated that K⁺ gradients in vascular tissues can serve as an energy source for phloem (re)loading processes and that the voltage-gated K⁺ channels of the AKT2-type play a unique role in this process. The AKT2 channel can be converted by phosphorylation of specific serine residues (S210 and S329) into a non-rectifying channel that allows a rapid efflux of K⁺ from the sieve element/companion cells (SE/CC) complex. The energy of this flux is used by other transporters for phloem (re)loading processes. Nonetheless, the results do indicate that post-translational modifications at S210 and S329 alone cannot explain AKT2 regulation. Here, we discuss the existence of multiple post-translational modification steps that work in concert to convert AKT2 from an inward-rectifying into a non-rectifying K⁺ channel.Key words: potassium, channel, potassium channel, AKT2, phloem (re)loading, post-translational modifications, potassium batteryPotassium (K⁺) is the most abundant mineral element in plants, and together with nitrogen and phosphorous, is limiting for plant production in many natural and agricultural habitats. Voltage-gated K⁺ channels are key players in the acquisition of K⁺ ions from the soil and in its redistribution within the plant.¹ Structurally, these channels result from the assembly of four so-called α-subunits. The subunits are encoded by nine genes in Arabidopsis and both homo- and hetero-tetramers are expressed.^2,3 The K⁺ channel α-subunits can be categorized into four different subfamilies, based on the voltage-gating characteristics of the exogenous K⁺ conductance when expressed in an appropriate heterologous expression system. K_in α-subunits form hyperpolarization-activated channels that mediate K⁺ uptake.^4–7 K_out α-subunits form depolarization-activated channels that mediate K⁺ release from cells.^8–10 K_silent subunits appear unable to yield functional homomeric channels, but can combine with K_in subunits and fine-tune the K⁺-uptake properties of the resulting heteromeric channels.^11–14 Finally, K_weak α-subunits form channels with complex voltage-gating; they allow both K⁺ uptake and release.^15–19 In Arabidopsis, a single member is found in this subfamily, AKT2, and this channel can assemble in heteromeric channels with the K_in subunit KAT2.²⁰To date, only scarce and speculative information has been obtained for the function of K_weak channels. When expressed in heterologous expression systems, two different subpopulations of AKT2 channels differing in their sensitivity to voltage were found.²¹ Channels of the first type showed gating properties and currents analogous to that of K_in channels, while the other sort enabled a non-rectified (leak-like) current; they were open over the entire physiological voltage range.A given channel can be converted from one type to the other by post-translational modifications.²¹ A voltage-dependent phosphorylation was found to be an essential step for this switch,^22,23 although the kinase responsible for this conversion still needs to be uncovered.²⁴ In biophysical studies, mutant versions of the Arabidopsis K_weak channel subunit AKT2 have been created that showed impaired gating mode settings.^22,23 Recently, Gajdanowicz et al. generated transgenic Arabidopsis thaliana plants that express these mutant AKT2 channels in the background of the akt2-1 null-allele plant.²⁵ The major conclusion from analyses of these mutants is that the status switching of AKT2 from an inward-rectifying to a non-rectifying channel is crucial for plants to overcome energy-limiting conditions. This function of AKT2 could be correlated to its expression in phloem tissues. Selective expression of AKT2 under the control of the phloem companion cell-specific AtSUC2 promoter rescued the akt2-1 line, but conversely, selective expression of AKT2 under the control of the guard cell-specific GC1 promoter,²⁶ resulted in further impairment of plant growth (Fig. 1). By combining diverse experimental approaches with mathematical simulation methods, an existing model for phloem (re)loading^18,27 was fundamentally improved. This allowed the uncovering of a novel and interesting role of K⁺ in phloem physiology: K⁺ gradients present between the sieve element/companion cell (SE/CC) complex and the apoplast can serve as an energy source in phloem (re)loading processes. This “potassium battery” can be tapped by means of AKT2 regulation. This clarifies the observation of Deeken et al.²⁸ that in AKT2 loss-of-function mutant plants, assimilates leaking away from the sieve tube were not efficiently reloaded into the main phloem stream.Open in a separate windowFigure 1AKT2 expressed only in guard cells delays plant development. (A–C) Representative wild-type, akt2-1 and akt2-1+pGC1:AKT2 complementation plants grown for 7 weeks (A), 9 weeks (B) and 12 weeks (C) under 12-h day/12-h night conditions at normal light intensity (150 µmol m⁻² s⁻¹). (D) akt2-1+pGC1:AKT2 developed a similar number of leaves as the akt2-1 knock out plants, but bolting-time was delayed. (B and E) After 9 weeks, wild-type plants were at an advanced bolting stage, akt2-1 plants had started bolting, but only initial signs of bolting were visible in akt2-1+pGC1:AKT2 plants. (C and F) At 12 weeks, akt2-1 plants had caught up with the wild-type and akt2-1+pGC1:AKT2 was just starting to bolt, although rosette-leaves were showing clear signs of senescence. For the generation of akt2-1+pGC1:AKT2, the AKT2 cDNA was fused to the guard cell-specific GC1 promoter²⁶ kindly provided by J.I. Schroeder, San Diego. The pGC1:AKT2 construct was cloned into pGreen0229-35S by replacing the 35S promoter and then transformed into the akt2-1 knockout plant. All seeds were cold-treated for 24 h at 4°C. Plants were grown on artificial substrate (type GS-90, Einheitserde). After 2 weeks, seedlings were transferred to single pots. Plants were grown in 60% relative humidity at 21°C during the day and 18°C at night. Phenotypical analyses were done in the middle of the day. Data are shown as means ± SD of n ≥ 9 plants. Statistical analyses using Student''s t test: (D, WT/akt2-1: p < 2e-08; D, WT/pGC-AKT2: p < 2e-08; D, akt2-1/pGC-AKT2: p < 5e-03; E, WT/akt2-1: p < 4e-06; E, WT/pGC-AKT2: p < 1e-10; E, akt2-1/pGC-AKT2: p < 5e-04; F, WT/akt2-1: p = 0.51; F, WT/pGC-AKT2: p < 1e-10; F, akt2-1/pGC-AKT2: p < 1e-10).AKT2 expression is especially abundant in phloem tissues and the root stele, both of which are characterized by a poor availability of oxygen.^29,30 This local internal hypoxia impairs respiratory activity of the vascular tissue and concomitantly, respiratory ATP production is reduced.³¹ As a consequence, phloem transport is very susceptible to decreasing oxygen supply to the plant.^29,32 It is therefore comprehensible that the above mentioned support by the K⁺ driving force for sucrose retrieval is especially relevant in the phloem. Indeed Gajdanowicz et al.²⁵ showed that transgenic plants lacking the AKT2 K⁺ channel were severely impaired in growth when exposed to mild hypoxia (10% v:v), whereas growth of wild-type plants was unaffected by this treatment. These observations illustrate the importance of biochemical flexibility in plant cells to cope with the energetic consequences of the steep oxygen concentration gradients that generally occur in plant stems and roots.In fact, the role of K⁺ gradients in driving sugar, amino acid and organic acid transport across plant cell membranes was first suggested several decades ago.^33,34 Experimental evidence for this concept was provided by various tests in which pieces of plant tissue were incubated in solutions with different K⁺ concentrations and pH levels.^33,34 Unfortunately, at that time the lack of genetic information to support this hypothesis (e.g., identifying transporter proteins that could provide a molecular mechanism to explain the working mechanism of substrate transport driven by a K⁺-motive force) resulted in this idea falling into oblivion. Indeed, the unequivocal experimental observation of this new role of K⁺ gradients in phloem reloading is extremely challenging. Under normal experimental conditions, K⁺ fluxes and sucrose fluxes are coupled during phloem loading in source tissues and unloading in sink tissues. Nonetheless, computational simulations predict that under certain conditions, a local K⁺/Suc antiport is also thermodynamically possible. In this antiport system, the energy from the K⁺ gradient is used to transport Suc into the phloem. This process is only transient; flooding the apoplast with K⁺ will decrease the K⁺ gradient. However, the gradient can be maintained for longer if surrounding cells take up the apoplastic K⁺ for their own use. A K⁺/Suc antiport will not occur in obvious sink or source tissues since the energy balances in such cells are fundamentally different. Consequently, in these tissues only the coupled symport of K⁺ and Suc can be observed. However, the computational predictions allowed the identification of the experimental conditions under which the effect of the K⁺/Suc antiport system is empirically observable at the whole plant level.An essential role in the regulation of AKT2 is played by (de)phosphorylation events of serine residues at positions S210 and S329. The replacement of both serines by asparagine (AKT2-S210N-S329N) resulted in a K⁺-selective leak that is locked in a continuously open mode when the channels are expressed in Xenopus oocytes. Under certain conditions, plants expressing the AKT2-S210N-S329N mutation showed growth benefits over wild-type plants; akt2-1+AKT2-S210N-S329N plants reach the generative state faster, possess an increased number of leaves and increased fresh weight (Fig. 2). Intuitively, one would expect a continuously open channel to cause severe problems for the plant, not a benefit as was observed here. We therefore have to postulate that phosphorylation at residues AKT2-S210 and AKT2-S329 is insufficient for converting AKT2 from an inward-rectifying into a non-rectifying channel; other, as yet unknown mechanisms, must contribute to the switch in the AKT2 gating mode. Such a concept would correspond to results that would otherwise be hard to explain. For instance, when both serine residues were replaced by glutamate, the mutant AKT2-S210E-S329E still showed wild-type characteristics.²² The S to E substitution is expected to mimic the phosphorylated state better than the S to N replacement. Furthermore, position AKT2-K197 has a fundamental influence on the AKT2 gating mode.²³ AKT2 mutants with that particular lysine substituted with a serine are far less sensitive towards (de)phosphorylation; they display the characteristics of a pure inward-rectifying K⁺ channel,²³ and transgenic Arabidopsis plants expressing AKT2 channels with this substitution showed the characteristics of akt2-1 knock-out plants.²⁵ Initially, it was proposed that the positive charge is important for sensitizing AKT2 to phosphorylation. However, the charge-conserving mutant AKT2-K197R is similar to the charge inverting mutant AKT2-K197D,²³ a purely inward-rectifying channel (Fig. 3). We therefore need to take into account that in plants, K197 may also be a target of post-translational modification.³⁵ At present, we can explain the beneficial effect of the AKT2-S210N-S329N mutant on plant growth only by a multiple step regulation of AKT2 (Fig. 4). The double-N mutation would then bypass the phosphorylation step, but AKT2-S210N-S329N could still be deregulated into an inward-rectifying channel. Thus, AKT2 can be considered as a highly specialized K_in channel that can be converted into a leak-like channel by a cascade of post-translational modification steps.Open in a separate windowFigure 2Plants expressing the AKT2-S210N-S329N mutant reach the generative state faster than wild-type plants. The mutant channel AKT2-S210N-S329N was expressed under the control of the native AKT2 promoter in the akt2-1 knock-out background. (A) Photos of representative Arabidopsis thaliana plants grown 7 weeks under short day conditions (12-h day/12-h night, light intensity = 150 µE m⁻²s⁻¹). Seven weeks after sowing, plants expressing only AKT2-S210N-S329N mutant channels (n = 22) differed significantly (Student''s t test, p < 4e-05) from wild-type plants (n = 20) in the height of the main inflorescent stalk (B) and fresh weight (C). At later time points, these differences decrease.²⁵Open in a separate windowFigure 3The mutant AKT2-K197R channel is inward-rectifying. Steady-state current-voltage characteristics measured at the end of activation voltage steps. Currents were normalized to the current values measured at −145 mV in 10 mM K⁺ and are shown as means ± SD (n = 6).Open in a separate windowFigure 4Minimal model for AKT2 gating-mode regulation. To switch AKT2 from an inward-rectifying into a non-rectifying channel, at least two post-translational steps are postulated. (1) Phosphorylation at residues AKT2-S210 and AKT2-S329 (transitions [1]→[2] and [3]→[4]) and (2) a yet unknown modification that most likely involves the residue AKT2-K197 (transitions [1]→[3] and [2]→[4]). Only after both modifications will AKT2 allow the efflux of K⁺ (state [4]).     

Induction of autophagy by drug-resistant esophageal cancer cells promotes their survival and recovery following treatment with chemotherapeutics

We investigated the cell-death mechanisms induced in esophageal cancer cells in response to the chemotherapeutic drugs, 5-fluorouracil (5-FU) and cisplatin. Chemosensitive cell lines exhibited apoptosis whereas chemoresistant populations exhibited autophagy and a morphology resembling type II programmed cell death (PCD). Cell populations that respond with autophagy are more resistant and will recover following withdrawal of the chemotherapeutic agents. Specific inhibition of early autophagy induction with siRNA targeted to Beclin 1 and ATG7 significantly enhanced the effect of 5-FU and reduced the recovery of drug-treated cells. Pharmacological inhibitors of autophagy were evaluated for their ability to improve chemotherapeutic effect. The PtdIns 3-kinase inhibitor 3-methyladenine did not enhance the cytotoxicity of 5-FU. Disruption of lysosomal activity with bafilomycin A 1 or chloroquine caused extensive vesicular accumulation but did not improve chemotherapeutic effect. These observations suggest that an autophagic response to chemotherapy is a survival mechanism that promotes chemoresistance and recovery and that selective inhibition of autophagy regulators has the potential to improve chemotherapeutic regimes. Currently available indirect inhibitors of autophagy are, however, ineffective at modulating chemosensitivity in these esophageal cancer cell lines.