Sequence specific assignment and determination of OSR1 C-terminal domain structure by NMR

The binding of SPAK and OSR1 kinases to their upstream WNK kinases is mediated by the interaction of their highly conserved SPAK and OSR1 C-terminal domain (CTD) to RFx [V/I] peptide sequences from WNK kinases. A SPAK CTD knock-in mouse, where SPAK was unable to bind WNK kinases, exhibited low blood pressure. This highlighted the inhibition of SPAK and OSR1 kinases binding to their upstream WNK kinases as a plausible strategy in the discovery of new antihypertensive agents. To facilitate such endeavour, we herein report the optimisation and expression of isotopically labelled OSR1 CTD in E.coli and a structural model based on the sequence specific NMR assignments giving insights into the structure of apo OSR1 CTD. Additionally, we identified the OSR1 CTD amino acid residues that are important for the binding of an 18-mer RFQV peptide derived from human WNK4. Collectively, the NMR backbone assignments and the generated OSR1 CTD 3D model reported in this work will be a powerful resource for the NMR-based discovery of small molecule OSR1 (and SPAK) kinase inhibitors as potential antihypertensive agents

Parkinson's disease: are PINK1 activators inching closer to the clinic?

The activation of PINK1 by small molecules has emerged as a promising strategy in treating Parkinson’s disease (PD). Recent progress in this area has raised excitement around PINK1 activators as PD treatments, and herein we offer insight into these developments and their potential to deliver much needed novel PD treatments

SPAK and OSR1 kinases bind and phosphorylate the β2-Adrenergic receptor

SPAK and OSR1 are two cytoplasmic serine/threonine protein kinases that regulate the function of a series of sodium, potassium and chloride co-transporters via phosphorylation. Over recent years, it has emerged that these two kinases may have diverse function beyond the regulation of ion co-transporters. Inspired by this, we explored whether SPAK and OSR1 kinases impact physically and phosphorylate the β2-adrenergic receptor (β2ADR). Herein, we report that the amino acid sequence of the human β2ADR displays a SPAK/OSR1 consensus binding motif and using a series of pulldown and in vitro kinase assays we show that SPAK and OSR1 bind the β2ADR and phosphorylate it in vitro. This work provides a notable example of SPAK and OSR1 kinases binding to a G-protein coupled receptor and taps into the potential of these protein kinases in regulating membrane receptors beyond ion co-transporters

Chemical strategies for activating PINK1, a protein kinase mutated in Parkinson's Disease

PINK1 is a ubiquitously expressed mitochondrial serine/threonine protein kinase that has emerged as a key player in mitochondrial quality control. This protein kinase came to prominence in the mid‐2000s, when PINK1 mutations were found to cause early onset Parkinson's disease (PD). As most of the PD‐related mutations occurred in the kinase domain and impaired PINK1′s catalytic activity, it was suggested that small molecules that activated PINK1 would maintain mitochondrial quality control and, as a result, have neuroprotective effects. Working on this hypothesis, a few small‐molecule PINK1 activators that offer critical insights and distinct approaches for activating PINK1 have been discovered. Herein, we briefly highlight the discovery of these small molecules and offer insight into the future development of small‐molecule PINK1 activators as potential treatments for PD

The Cul4‐DDB1‐WDR3/WDR6 complex binds SPAK and OSR1 kinases in a phosphorylation‐dependent manner

SPAK and OSR1 are two protein kinases that play critical roles in regulating ion homeostasis. They are activated under osmotic stress through phosphorylation by their upstream WNK kinases at a conserved threonine site on their T‐loops. Additionally, WNK kinases phosphorylate SPAK and OSR1 at a highly conserved serine residue on their S‐motif, the function of which remains elusive. Using affinity pull down and mass spectrometry, we identified the E3 ubiquitin ligase complex Cullin 4‐DDB1‐WDR3/WDR6 as a binder to OSR1 kinase in a SPAK/OSR1 S‐motif phosphorylation‐dependent manner. This binding was found to be compromised by S‐motif phosphorylation following osmotic stress. Using proteasomal and neddylation inhibitors, we subsequently showed that OSR1 ubiquitylation was abolished under osmotic stress when its S‐motif is phosphorylated. These results provide the first example of an E3 ubiquitin ligase system that binds the OSR1 kinase and, thus, links the CRL4 complex to ion homeostasis

Aryloxy pivaloyloxymethyl prodrugs as nucleoside monophosphate prodrugs

Intracellular phosphorylation of therapeutic nucleoside analogues into their active triphosphate metabolites is a prerequisite for their pharmacological activity. However, the initial phosphorylation of these unnatural nucleosides into their monophosphate derivatives can be a rate-limiting step in their activation. To address this, we herein report the development of the aryloxy pivaloyloxymethyl prodrugs (POMtides) as a novel and effective nucleoside monophosphate prodrug technology and its successful application to the anticancer nucleoside analogue 5-fluoro-2′-deoxyuridine (FdUR)

Phosphotyrosine prodrugs: design, synthesis and anti-STAT3 activity of ISS-610 aryloxy triester phosphoramidate prodrugs

Unmasked phohate groups of phosphotyrosine-containing molecules carry two negative charges at physiological pH, which compromise their (passive) celular uptake. Also, these phosphate groups are often cleaved off by phosphatases. Together, these ultimately limit the pharmacological efficacy of the phosphotyrosine-containing compounds. To address these drawbacks, we herein present the application of the aryloxy triester phosphoramidate prodrug technology, a monophosphate prodrug technology, to the phosphotyrosine-containing compound ISS-610-Met, an analogue of the anticancer STAT3 dimerization inhibitor ISS-610. Our data shows that the generated ISS-610-Met prodrugs exhibited enhanced pharmacological activity and inhibition of STAT3 downstream signaling compared to the parent compound ISS-610-Met and the known STAT3 dimerization inhibitor ISS-610. These encouraging results provide a compelling proof of concept for the potential of the aryloxy triester phosphoramidate prodrug technology in the discovery of novel therapeutics that contain phosphotyrosine and its phospho mimics

The ProTide prodrug technology: from the concept to the clinic

The ProTide technology is a prodrug approach developed for the efficient intracellular delivery of nucleoside analogue monophosphates and monophosphonates. In this approach, the hydroxyls of the monophosphate or monophosphonate groups are masked by an aromatic group and an amino acid ester moiety, which are enzymatically cleaved-off inside cells to release the free nucleoside monophosphate and monophosphonate species. Structurally, this represents the current end-point of an extensive medicinal chemistry endeavor that spans almost three decades. It started from the masking of nucleoside monophosphate and monophosphonate groups by simple alkyl groups and evolved into the sophisticated ProTide system as known today. This technology has been extensively employed in drug discovery, and it has already led to the discovery of two FDA-approved (antiviral) ProTides. In this work, we will review the development of the ProTide technology, its application in drug discovery, and its role in the improvement of drug delivery and efficacy

The protide prodrug technology: Where next?

The ProTide prodrug technology has proved very useful in the discovery of nucleotide therapeutics and has successfully led to two FDA-approved drugs. However, with the extensive application of this prodrug approach to nucleotides for nearly three decades, the intellectual property (IP) landscape is becoming congested and, to overcome this, new inventive applications of the ProTide prodrug technology are emerging