Putative phosphodiesterase inhibitors as potential new chemotherapies against African Trypanosomiasis

African trypanosomiasis is a disease caused by the Kinetoplastida parasites Trypanosoma brucei rhodesiense and T. b. gambiense. The distribution of the disease is split geographically with T. b. rhodesiense found in eastern sub-Saharan Africa and T. b. gambiense in the west of the continent. Current treatment for this fatal disease is wholly unsatisfactory with problems such as extreme toxicity, affordability and the emergence of resistance. The case for the generation of new potential chemotherapies is compelling and urgent. Phosphodiesterase (PDE) enzymes degrade the secondary signalling molecule cyclic adenosine monophosphate (cAMP) to AMP by hydrolysis, thereby modulating and regulating the signal transduction to the effector proteins. The phosphodiesterase enzymes in the PDEB family in T. brucei were shown to be essential to the host-infective bloodstream forms and validated as good drug targets using RNA-interference (Zoraghi, R. and Seebeck, T., 2002; Oberholzer, M., 2007). Prompted by these findings, two series of putative trypanosomal PDE inhibitors, from different sources, were thoroughly assessed in this project for their anti-trypanosomal activity and their intracellular effects on the trypanosome. The whole-cell in vitro efficacy for each compound, against T. brucei wildtype and the drug-resistant strain TbAT1 knockout, was established by the standard resazurin reduction assay. 25 compounds from Series 1 had EC50 values below 0.5 µM, with 7 under 100 nM and the most active having an EC50 value of 5.8 ± 3.4 nM. For the much smaller Series 2 (GJS Compounds), the most active compound was GJS-128 with an EC50 value of 79.4 ± 10.3 nM. This demonstrates that a number of compounds from both series have potent in vitro activity against trypanosomes that is better than or equal to the current chemotherapeutic compound diminazene, and some Series 1 compounds are on a par with pentamidine and melarsoprol. No major cross-resistance was displayed by the TbAT1 knockout strain to either Series 1 or the GJS series. Similarly, a panel of Series 1 compounds tested against the B48 strain (resistant to pentamidine and melaminophenyl arsenical drugs), and also against Trypanosoma equiperdum wildtype and diminazene resistant (PBR) strains, showed no major cross-resistance displayed by the other resistant strains. This suggests that there would also be little or no cross-resistance from refractory strains in the field, and also that the compounds are active against multiple Trypanosoma species. A small panel of Series 1 compounds were also tested for efficacy against trypanosomes in infected mice. 4 daily doses of 20 mg/kg bodyweight of Compound 48 significantly reduced parasitaemia by approximately 60% compared to untreated controls, however higher concentrations were not tolerated by the mice so a cure could not be demonstrated. A high-throughput method for monitoring the speed of action of test compounds on trypanosomes in real time was developed, based on the fluorescence of propidium iodide when bound with DNA. Optimisation of the protocol to 96-well plates and low cell densities provided higher resolution and accurate traces of the lysis of trypanosomes in a cell suspension compared to previously used methods, as well as a greatly increased capacity. The propidium iodide assay could also be converted to provide end-point EC50 values that were directly comparable to those established by the standard resazurin reduction assay. The majority of Series 1 compounds did not increase the intracellular concentration of cAMP on incubation with bloodstream form trypanosomes; those that did only induced a minor elevation of the intracellular concentration of the signalling molecule. Since genetic disruption to phosphodiesterase enzymes resulted in large increases in cAMP levels (Oberholzer, M. et al, 2007; Zoraghi, R. and Seebeck, T., 2002), the lack of increase in cAMP by the Series 1 compounds strongly suggest that they do not sufficiently inhibit the PDEs in live trypanosomes and kill the cells via an alternative pathway. In contrast, incubation with the GJS compounds did result in significant increases in intracellular cAMP concentration with the most active being GJS-128 recording an approximately 3-fold increase in cAMP over 3 hours at just 30 nM. The concentrations that begin to increase cAMP level are consistent with the EC50 values for trypanosomes cultured in vitro (this study), and is also in line with inhibition data of recombinant TbrPDEB enzymes (work conducted by Dr. Herrmann Tenor, ALTANA Pharma, and Prof. Thomas Seebeck, University of Bern). This gives a clear and consistent link between the cause of cAMP rise (inhibition of PDEB by GJS compounds) and the effect of that concentration increase on bloodstream form trypanosomes (cell death), demonstrating that the GJS series are inhibitors of trypanosomal PDEs and chemically validate PDEs as drug targets for potential new chemotherapies against African trypanosomiasis. The effect of PDE inhibition on the physiology of the bloodstream form trypanosomes was also investigated. Flow cytometry analysis and the assessment of DNA configuration by fluorescence microscopy after DAPI staining determined that PDE inhibition by GJS-128 resulted in a precise block of the cell cycle in cytokinesis. The replicating trypanosome synthesized and segregated its DNA into two nuclei and kinetoplasts as normal and proceeded to initiate the physical separation of mother and daughter cells. The cleavage furrow between the old and new flagella progressed normally until the point of abscission, at which point division was halted with only a small section of plasma membrane connecting the two almost separated cells. Both cells appeared viable and underwent subsequent rounds of DNA replication, segregation and attempted physical separation that was always blocked near completion. This indicates cAMP signalling plays an important role in the correct physical separation of the replicating bloodstream form trypanosomes. A trypanosome cell line resistant to GJS-128 was developed by chemical mutagenesis and continuous culture with gradually increasing, but sub-lethal concentrations of the PDE inhibitor. This cell line, termed R0.8, was >15-fold less sensitive to GJS-128 and displayed significant cross-resistance to the other GJS compounds, as well as to stable, membrane permeable cAMP analogues. The mode of resistance was investigated by comparing the cAMP profile of the R0.8 and parental wildtype strains on incubation with GJS-128. No major differences were observed suggesting that both the adenylyl cyclase and phosphodiesterase activities remained unchanged in the PDE inhibitor-resistant strain. In support of this, the sequencing of TbrPDEB1 and TbrPDEB2 in both strains, while uncovering the loss of heterozygosity in the R0.8 line, revealed no mutations that would impact on enzyme function or inhibitor binding in the resistant cell line. These data strongly suggest that the adaptation resulting in resistance to PDE inhibitors is located in the effector proteins downstream of the PDEs and adenylyl cyclases in the cAMP signalling pathway. Identifying a compound that inhibits phosphodiesterases in trypanosomes and elevates cAMP concentrations, along with the generation of a PDE inhibitor-resistant cell line will allow more detailed examination of all aspects of the cAMP signalling pathway in T. brucei and across the Kinetoplastida. Phosphodiesterases have also been demonstrated to be chemically inhibitable in trypanosomes and could prove to be the target of a new generation of chemotherapies against African trypanosomiasis

Molecular ecology and risk factors for third-generation cephalosporin-resistant Escherichia coli carriage by dogs living in urban and nearby rural settings

OBJECTIVES: To compare faecal third-generation cephalosporin-resistant (3GC-R) Escherichia coli isolates from dogs living in a city and in a rural area ∼30 km away; to compare isolates from dogs, cattle and humans in these regions; and to determine risk factors associated with 3GC-R E. coli carriage in these two cohorts of dogs. METHODS: Six hundred dogs were included, with faecal samples processed to recover 3GC-R E. coli using 2 mg/L cefotaxime. WGS was by Illumina and risk factor analyses were by multivariable linear regression using the results of an owner-completed survey. RESULTS: 3GC-R E. coli were excreted by 20/303 rural and 31/297 urban dogs. The dominant canine 3GC-R ST was ST963 (bla(CMY-2)), which also accounted for 25% of CMY-2-producing E. coli in humans. Phylogenetic overlap between cattle and rural dog CTX-M-14-producing E. coli ST117 was observed as well as acquisition of pMOO-32-positive E. coli ST10 by a rural dog, a plasmid common on cattle farms in the area. Feeding raw meat was associated with carrying 3GC-R E. coli in rural dogs, but not in urban dogs, where swimming in rivers was a weak risk factor. CONCLUSIONS: Given clear zoonotic potential for resistant canine E. coli, our work suggests interventions that may reduce this threat. In rural dogs, carriage of 3GC-R E. coli, particularly CTX-M producers, was phylogenetically associated with interaction with local cattle and epidemiologically associated with feeding raw meat. In urban dogs, sources of 3GC-R E. coli appear to be more varied and include environments such as rivers

Mitochondrial DNA is critical for longevity and metabolism of transmission stage Trypanosoma brucei.

The sleeping sickness parasite Trypanosoma brucei has a complex life cycle, alternating between a mammalian host and the tsetse fly vector. A tightly controlled developmental programme ensures parasite transmission between hosts as well as survival within them and involves strict regulation of mitochondrial activities. In the glucose-rich bloodstream, the replicative 'slender' stage is thought to produce ATP exclusively via glycolysis and uses the mitochondrial F1FO-ATP synthase as an ATP hydrolysis-driven proton pump to generate the mitochondrial membrane potential (ΔΨm). The 'procyclic' stage in the glucose-poor tsetse midgut depends on mitochondrial catabolism of amino acids for energy production, which involves oxidative phosphorylation with ATP production via the F1FO-ATP synthase. Both modes of the F1FO enzyme critically depend on FO subunit a, which is encoded in the parasite's mitochondrial DNA (kinetoplast or kDNA). Comparatively little is known about mitochondrial function and the role of kDNA in non-replicative 'stumpy' bloodstream forms, a developmental stage essential for disease transmission. Here we show that the L262P mutation in the nuclear-encoded F1 subunit γ that permits survival of 'slender' bloodstream forms lacking kDNA ('akinetoplastic' forms), via FO-independent generation of ΔΨm, also permits their differentiation into stumpy forms. However, these akinetoplastic stumpy cells lack a ΔΨm and have a reduced lifespan in vitro and in mice, which significantly alters the within-host dynamics of the parasite. We further show that generation of ΔΨm in stumpy parasites and their ability to use α-ketoglutarate to sustain viability depend on F1-ATPase activity. Surprisingly, however, loss of ΔΨm does not reduce stumpy life span. We conclude that the L262P γ subunit mutation does not enable FO-independent generation of ΔΨm in stumpy cells, most likely as a consequence of mitochondrial ATP production in these cells. In addition, kDNA-encoded genes other than FO subunit a are important for stumpy form viability

Pharmacological Validation of Trypanosoma brucei Phosphodiesterases as Novel Drug Targets

The development of drugs for neglected infectious diseases often uses parasite-specific enzymes as targets. We here demonstrate that parasite enzymes with highly conserved human homologs may represent a promising reservoir of new potential drug targets. The cyclic nucleotide-specific phosphodiesterases (PDEs) of Trypanosoma brucei, causative agent of the fatal human sleeping sickness, are essential for the parasite. The highly conserved human homologs are well-established drug targets. We here describe what is to our knowledge the first pharmacological validation of trypanosomal PDEs as drug targets. High-throughput screening of a proprietary compound library identified a number of potent hits. One compound, the tetrahydrophthalazinone compound A (Cpd A), was further characterized. It causes a dramatic increase of intracellular cyclic adenosine monophosphate (cAMP). Short-term cell viability is not affected, but cell proliferation is inhibited immediately, and cell death occurs within 3 days. Cpd A prevents cytokinesis, resulting in multinucleated, multiflagellated cells that eventually lyse. These observations pharmacologically validate the highly conserved trypanosomal PDEs as potential drug target

NASA ExoPAG Study Analysis Group 11: Preparing for the WFIRST Microlensing Survey

NASA's proposed WFIRST-AFTA mission will discover thousands of exoplanets with separations from the habitable zone out to unbound planets, using the technique of gravitational microlensing. The Study Analysis Group 11 of the NASA Exoplanet Program Analysis Group was convened to explore scientific programs that can be undertaken now, and in the years leading up to WFIRST's launch, in order to maximize the mission's scientific return and to reduce technical and scientific risk. This report presents those findings, which include suggested precursor Hubble Space Telescope observations, a ground-based, NIR microlensing survey, and other programs to develop and deepen community scientific expertise prior to the mission.Comment: 35 pages, 5 Figures. A brief overview of the findings is presented in the Executive Summary (2 pages

KMT-2016-BLG-2052L: Microlensing Binary Composed of M Dwarfs Revealed from a Very Long Time-scale Event

We present the analysis of a binary microlensing event KMT-2016-BLG-2052, for which the lensing-induced brightening of the source star lasted for 2 seasons. We determine the lens mass from the combined measurements of the microlens parallax \pie and angular Einstein radius \thetae. The measured mass indicates that the lens is a binary composed of M dwarfs with masses of $M_1\sim 0.34~M_\odot$ and $M_2\sim 0.17~M_\odot$. The measured relative lens-source proper motion of $\mu\sim 3.9~{\rm mas}~{\rm yr}^{-1}$ is smaller than $\sim 5~{\rm mas}~{\rm yr}^{-1}$ of typical Galactic lensing events, while the estimated angular Einstein radius of \thetae\sim 1.2~{\rm mas} is substantially greater than the typical value of $\sim 0.5~{\rm mas}$. Therefore, it turns out that the long time scale of the event is caused by the combination of the slow $\mu$ and large \thetae rather than the heavy mass of the lens. From the simulation of Galactic lensing events with very long time scales ($t_{\rm E}\gtrsim 100$ days), we find that the probabilities that long time-scale events are produced by lenses with masses $\geq 1.0~M_\odot$ and $\geq 3.0~M_\odot$ are $\sim 19\%$ and 2.6\%, respectively, indicating that events produced by heavy lenses comprise a minor fraction of long time-scale events. The results indicate that it is essential to determine lens masses by measuring both \pie and \thetae in order to firmly identify heavy stellar remnants such as neutron stars and black holes.Comment: 9 pages, 11 figure

Molecular Epidemiology of Escherichia coli Producing CTX-M and pAmpC β-Lactamases from Dairy Farms Identifies a Dominant Plasmid Encoding CTX-M-32 but No Evidence for Transmission to Humans in the Same Geographical Region

Third-generation cephalosporin resistance (3GC-R) in Escherichia coli is a rising problem in human and farmed-animal populations. We conducted whole-genome sequencing analysis of 138 representative 3GC-R isolates previously collected from dairy farms in southwest England and confirmed by PCR to carry acquired 3GC-R genes. This analysis identified bla(CTX-M) (131 isolates encoding CTX-M-1, -14, -15, -and 32 and the novel variant CTX-M-214), bla(CMY-2) (6 isolates), and bla(DHA-1) (1 isolate). A highly conserved plasmid was identified in 73 isolates, representing 27 E. coli sequence types. This novel ∼220-kb IncHI2 plasmid carrying bla(CTX-M-32) was sequenced to closure and designated pMOO-32. It was found experimentally to be stable in cattle and human transconjugant E. coli even in the absence of selective pressure and was found by multiplex PCR to be present on 26 study farms representing a remarkable range of transmission over 1,500 square kilometers. However, the plasmid was not found among human urinary E. coli isolates we recently characterized from people living in the same geographical location, collected in parallel with farm sampling. There were close relatives of two bla(CTX-M) plasmids circulating among eight human and two cattle isolates, and a closely related bla(CMY-2) plasmid was found in one cattle and one human isolate. However, phylogenetic evidence of recent sharing of 3GC-R strains between farms and humans in the same region was not found. IMPORTANCE Third-generation cephalosporins (3GCs) are critically important antibacterials, and 3GC resistance (3GC-R) threatens human health, particularly in the context of opportunistic pathogens such as Escherichia coli. There is some evidence for zoonotic transmission of 3GC-R E. coli through food, but little work has been done examining possible transmission via interaction of people with the local near-farm environment. We characterized acquired 3GC-R E. coli found on dairy farms in a geographically restricted region of the United Kingdom and compared these with E. coli from people living in the same region, collected in parallel. While there is strong evidence for recent farm-to-farm transmission of 3GC-R strains and plasmids—including one epidemic plasmid that has a remarkable capacity to be transmitted—there was no evidence that 3GC-R E. coli found on study farms had a significant impact on circulating 3GC-R E. coli strains or plasmids in the local human population