Towards the Development of Small‐Molecule MO25 Binders as Potential Indirect SPAK/OSR1 Kinase Inhibitors

Abstract

The binding of the scaffolding protein MO25 to SPAK and OSR1 protein kinases, which regulate ion homeostasis, causes increases of up to 100-fold in their catalytic activity. Various animal models have shown that the inhibition of SPAK and OSR1 lowers blood pressure, and so here we present a new indirect approach to inhibiting SPAK and OSR1 kinases by targeting their protein partner MO25. To explore this approach, we developed a fluorescent polarisation assay and used it in screening of a small in-house library of ≈4000 compounds. This led to the identification of one compound-HK01-as the first small-molecule inhibitor of the MO25-dependent activation of SPAK and OSR1 in vitro. Our data confirm the feasibility of targeting this protein-protein interaction by small-molecule compounds and highlights their potential to modulate ion co-transporters and thus cellular electrolyte balance.

WNK Signaling Inhibitors as Potential Antihypertensive Drugs

“…Data were taken from Kikuchie tal. [26] ChemMedChem 2017, 12,1677 -1686 www.chemmedchem.org Chemical structure and in vitro OSR1 inhibitory activity of Rafoxanide, an allosteric SPAK/OSR1 kinase inhibitor.…”

“…Ta rgeting this protein-protein interaction has been pursued by the discovery of small molecules that bind MO25 and hence inhibit its ability to bind and activate SPAK and OSR1 kinases.F or this, af luorescence polarization that employed a 16-mer peptide, whichc ontains aW EW motif derived from SPAK that is known to mediate the binding to MO25, [5] was developed. [26] This assayw as used in screening al ibrary of~4000 compounds and this led to the identification of HK01 as a binder of MO25(K d = 127 AE 6 mm) ( Figure 9). [26] Notably,t his compound was able to inhibit the 16-mer WEW peptide binding to MO25 in vitro (IC 50 = 78 AE 4 mm).…”

“…[26] This assayw as used in screening al ibrary of~4000 compounds and this led to the identification of HK01 as a binder of MO25(K d = 127 AE 6 mm) ( Figure 9). [26] Notably,t his compound was able to inhibit the 16-mer WEW peptide binding to MO25 in vitro (IC 50 = 78 AE 4 mm). [26] Further characterization showedt hat HK01 wasa ble to inhibitt he MO25 dependent activation of OSR1 Thr185Glu in vitro and in cells.…”

Pharmacological targeting of SPAK kinase in disorders of impaired epithelial transport

“…To explore this approach, Kadri et al developed a fluorescent polarization assay and used it in screening of a small in-house library of ~4000 compounds. This led to the identification of one compound-HK01-as the first small-molecule inhibitor of the MO25-dependent activation of SPAK and OSR1 in vitro [105] (Figure 1). This data confirm the feasibility of targeting this protein-protein interaction by small-molecule compounds and highlights their potential to modulate ion co-transporters and thus cellular electrolyte balance.…”

“…OSR1 differs from SPAK in lacking the P/A rich (PAPA) domain. STOCK1S-50699 is a small-molecule inhibitor that blocks the interaction between SPAK/OSR1 and WNK by binding to the CCT domain [102]; Closantel is a small-molecule inhibitor that binds to constitutively active or WNK-sensitive(T233E)SPAK [98]; WNK463 inhibits of WNK1 catalytic activity [100]; and HK01 is an inhibitor of the M025 [105]. …”