Human With No Lysine Kinase 3 (WNK3): A Target Enabling Package (TEP)

The Target Enabling Package (TEP) programme's foundation is built upon the recognition that genetic data is proving to be a powerful tool for target validation. As such, TEPs provide a critical mass of reagents and knowledge on a protein target to allow rapid biochemical and chemical exploration and characterisation of proteins with genetic linkage to key disease areas. TEPs provide an answer to the missing link between genomics and chemical biology, provide a starting point for chemical probe generation and therefore catalyse new biology and disease understanding with the ultimate aim of enabling translation collaborations and target/ drug discovery. We are committed to generating and making available 24 high-quality TEPs by June 2020.SUMMARY OF PROJECT Kinases WNK1-4 regulate cation-chloride cotransporters via phosphorylation of SPAK and OSR1 and thereby control salt homeostasis, cell volume and blood pressure. Gain of function mutations in WNK kinases are found in Gordon’s hypertension syndrome suggesting the WNK pathway as a therapeutic target. WNK3 inhibition in particular has also been shown to reduce cerebral injury after Ischemic stroke. Here we present assays and crystal structures that define (i) the molecular basis for disease mutations; (ii) the multiple functional domains of WNK kinases and their protein interactions; (iii) the binding of small molecule kinase inhibitors and a potential allosteric pocket.The work performed at the SGC has been funded by a grant from the Wellcome [106169/ZZ14/Z]

Cooperative structure of the heterotrimeric pre-mRNA retention and splicing complex

The precursor mRNA (pre-mRNA) retention and splicing (RES) complex is a spliceosomal complex that is present in yeast and humans and is important for RNA splicing and retention of unspliced pre-mRNA. Here, we present the solution NMR structure of the RES core complex from Saccharomyces cerevisiae. Complex formation leads to an intricate folding of three components-Snu17p, Bud13p and Pml1p-that stabilizes the RNA-recognition motif (RRM) fold of Snu17p and increases binding affinity in tertiary interactions between the components by more than 100-fold compared to that in binary interactions. RES interacts with pre-mRNA within the spliceosome, and through the assembly of the RES core complex RNA binding efficiency is increased. The three-dimensional structure of the RES core complex highlights the importance of cooperative folding and binding in the functional organization of the spliceosome

Recognition of distinct RNA motifs by the clustered CCCH zinc fingers of neuronal protein Unkempt

Unkempt is an evolutionarily conserved RNA-binding protein that regulates translation of its target genes and is required for the establishment of the early bipolar neuronal morphology. Here we determined the X-ray crystal structure of mouse Unkempt and show that its six CCCH zinc fingers (ZnFs) form two compact clusters, ZnF–3 and ZnF4–6, that recognize distinct trinucleotide RNA substrates. Both ZnF clusters adopt a similar overall topology and use distinct recognition principles to target specific RNA sequences. Structure-guided point mutations reduce the RNA binding affinity of Unkempt both in vitro and in vivo, ablate Unkempt’s translational control and impair the ability of Unkempt to induce a bipolar cellular morphology. Our study unravels a new mode of RNA sequence recognition by clusters of CCCH ZnFs that is critical for post-transcriptional control of neuronal morphology

Thermal profiling reveals phenylalanine hydroxylase as an off-target of panobinostat

We describe a two-dimensional thermal proteome profiling strategy that can be combined with an orthogonal chemoproteomics approach to enable comprehensive target profiling of the marketed histone deacetylase inhibitor panobinostat. The N-hydroxycinnamide moiety is identified as critical for potent and tetrahydrobiopterin-competitive inhibition of phenylalanine hydroxylase leading to increases in phenylalanine and decreases in tyrosine levels. These findings provide a rationale for adverse clinical observations and suggest repurposing of the drug for treatment of tyrosinemia