Genetic validation of Aspergillus fumigatus phosphoglucomutase as a viable therapeutic target in invasive aspergillosis

Aspergillus fumigatus is the causative agent of invasive aspergillosis, an infection with mortality rates of up to 50%. The glucan-rich cell wall of A. fumigatus is a protective structure that is absent from human cells and is a potential target for antifungal treatments. Glucan is synthesized from the donor uridine diphosphate glucose, with the conversion of glucose-6-phosphate to glucose-1-phosphate by the enzyme phosphoglucomutase (PGM) representing a key step in its biosynthesis. Here, we explore the possibility of selectively targeting A. fumigatus PGM (AfPGM) as an antifungal treatment strategy. Using a promoter replacement strategy, we constructed a conditional pgm mutant and revealed that pgm is required for A. fumigatus growth and cell wall integrity. In addition, using a fragment screen, we identified the thiol-reactive compound isothiazolone fragment of PGM as targeting a cysteine residue not conserved in the human ortholog. Furthermore, through scaffold exploration, we synthesized a para-aryl derivative (ISFP10) and demonstrated that it inhibits AfPGM with an IC(50) of 2 μM and exhibits 50-fold selectivity over the human enzyme. Taken together, our data provide genetic validation of PGM as a therapeutic target and suggest new avenues for inhibiting AfPGM using covalent inhibitors that could serve as tools for chemical validation

Toxicants in Water: Hydrochemical Appraisal of Toxic Metals Concentration and Seasonal Variation in Drinking Water Quality in Oil and Gas Field Area of Rivers State, Nigeria

Groundwater pollution is a major issue in many tropical countries. Governments at all levels are doing little or nothing to supply inhabitants with clean and accessible water, particularly in Nigeria’s Niger Delta region. This study compares differences in water quality parameters in the study area (determine the level of pollutions in the different sites). The investigation made use of standard analytical methodologies. All sampling, conservation, transportation as well as analysis followed the usual APHA procedures (2012). To prevent degradation of the organic substances, all obtained samples were transferred to the laboratory, while keeping in an icebox. Result shows that during wet season, the mean values obtained for water quality parameters were significantly lower in site 9 compared with that obtained in other sites (p  0.05) and both alkalinity and SO4 which were significantly higher in site 9 than site 1 (p  0.05) while other water quality parameters were significantly lower in site 9 than other sites excluding Cl and Mg which were both significantly higher in site 9 than site 8 (p < 0.05). Extra efforts must be made to completely understand the hydrogeochemical properties and appropriateness of groundwater in Nigeria’s core Niger Delta region in order to ensure quality groundwater supply for varied applications. As a result, this research will contribute to the establishment of a quantitative understanding of the effects of many causes on groundwater level changes in every aquifer worldwide. This analysis also reinforces a useful resource for scholars, activists, and public officials looking to improve community awareness, planning, and performance. The verdicts will serve as a valuable guideline for policymakers, the Ministry of Water Resources, and development practitioners, as they highlight the need for appropriate approaches to mitigating toxic elements of water resource contamination in the core Niger Delta in order to protect public health from carcinogenic and non-carcinogenic risks

Dual functionality of <i>O</i>-GlcNAc transferase is required for <i>Drosophila </i>development

Post-translational modification of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc) catalysed by O-GlcNAc transferase (OGT) has been linked to regulation of diverse cellular functions. OGT possesses a C-terminal glycosyltransferase catalytic domain and N-terminal tetratricopeptide repeats that are implicated in protein–protein interactions. Drosophila OGT (DmOGT) is encoded by super sex combs (sxc), mutants of which are pupal lethal. However, it is not clear if this phenotype is caused by reduction of O-GlcNAcylation. Here we use a genetic approach to demonstrate that post-pupal Drosophila development can proceed with negligible OGT catalysis, while early embryonic development is OGT activity-dependent. Structural and enzymatic comparison between human OGT (hOGT) and DmOGT informed the rational design of DmOGT point mutants with a range of reduced catalytic activities. Strikingly, a severely hypomorphic OGT mutant complements sxc pupal lethality. However, the hypomorphic OGT mutant-rescued progeny do not produce F2 adults, because a set of Hox genes is de-repressed in F2 embryos, resulting in homeotic phenotypes. Thus, OGT catalytic activity is required up to late pupal stages, while further development proceeds with severely reduced OGT activity

Targeting a critical step in fungal hexosamine biosynthesis

Acknowledgements We wish to thank the Dundee Drug Discovery Unit for access to the Fragment library and the European Synchrotron Radiation Facility, Grenoble and Diamond Light Source, Oxford for time at the beamline. The assistance from Mr Martin Kierans, School of Life Sciences, University of Dundee with the Electron Microscopy is gratefully acknowledged. Funding and additional information This work was supported by a Wellcome Trust Postdoctoral Research Training Fellowship for Clinicians (WT105772/A/14/Z) to DL and an MRC Programme Grant (MR/M004139/1) to DMFvA. DB was funded by a University of Aberdeen Summer Research Scholarship.Peer reviewedPostprintPublisher PD

Mechanism of human PINK1 activation at the TOM complex by reconstitution

Loss of function mutations in PTEN-induced kinase 1 (PINK1) are a frequent cause of earlyonset Parkinson’s disease (PD). Stabilisation of PINK1 at the Translocase of Outer Membrane (TOM) complex of damaged mitochondria is a critical step for its activation. To date the mechanism of how PINK1 is activated in the TOM complex is unclear. Herein we report coexpression of human PINK1 and all seven TOM subunits in Saccharomyces cerevisiae is sufficient for PINK1 activation. We use this reconstitution system to systematically assess the role of each TOM subunit towards PINK1 activation. We unambiguously demonstrate that the TOM20 and TOM70 receptor subunits are required for optimal PINK1 activation and map their sites of interaction with PINK1 using AlphaFold structural modelling and mutagenesis. We also demonstrate an essential role of the pore-containing subunit TOM40 and its structurally associated subunits TOM7 and TOM22 for PINK1 activation. These molecular findings will aid in the development of small molecule activators of PINK1 as a therapeutic strategy for PD

Evidence for substrate assisted catalysis in <i>N</i>-acetylphosphoglucosamine mutase

11 pags, 4 figs, 2 tabs . -- Supplementary data is available at the Publisher's web pageN-acetylphosphoglucosamine mutase (AGM1) is a key component of the hexosamine biosynthetic pathway that produces UDP-GlcNAc, an essential precursor for a wide range of glycans in eukaryotes. AGM belongs to the α-D-phosphohexomutase metalloenzyme superfamily and catalyzes the interconversion of N-acetylglucosamine-6-phosphate (GlcNAc-6P) to N-acetylglucosamine-1-phosphate (GlcNAc-1P) through N-acetylglucosa-mine-1,6-bisphosphate (GlcNAc-1,6-bisP) as the catalytic intermediate. Although there is an understanding of the phosphoserine-dependent catalytic mechanism at enzymatic and structural level, the identity of the requisite catalytic base in AGM1/phosphoglucomu-tases is as yet unknown. Here, we present crystal structures of a Michaelis complex of AGM1 with GlcNAc-6P and Mg, and a complex of the inactive Ser69Ala mutant together with glucose-1,6-bisphosphate (Glc-1,6-bisP) that represents key snapshots along the reaction co-ordinate. Together with mutagenesis, these structures reveal that the phosphate group of the hexose-1,6-bisP intermediate may act as the catalytic base.This work was funded by the MRC Programme Grant M004139