In vitro pharmacological profiling of R406 identifies molecular targets underlying the clinical effects of fostamatinib

Abstract

Off-target pharmacology may contribute to both adverse and beneficial effects of a new drug. In vitro pharmacological profiling is often applied early in drug discovery; there are fewer reports addressing the relevance of broad profiles to clinical adverse effects. Here, we have characterized the pharmacological profile of the active metabolite of fostamatinib, R406, linking an understanding of drug selectivity to the increase in blood pressure observed in clinical studies. R406 was profiled in a broad range of in vitro assays to generate a comprehensive pharmacological profile and key targets were further investigated using functional and cellular assay systems. A combination of traditional literature searches and text-mining approaches established potential mechanistic links between the profile of R406 and clinical side effects. R406 was selective outside the kinase domain, with only antagonist activity at the adenosine A3 receptor in the range relevant to clinical effects. R406 was less selective in the kinase domain, having activity at many protein kinases at therapeutically relevant concentrations when tested in multiple in vitro systems. Systematic literature analyses identified KDR as the probable target underlying the blood pressure increase observed in patients. While the in vitro pharmacological profile of R406 suggests a lack of selectivity among kinases, a combination of classical searching and text-mining approaches rationalized the complex profile establishing linkage between off-target pharmacology and clinically observed effects. These results demonstrate the utility of in vitro pharmacological profiling for a compound in late-stage clinical development.

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“…Off-target inhibition of VEGFR2 was found to increase vascular resistance and reduced VEGF-induced nitric oxide release in animal models [50], where effects were dose-dependent, but effectively managed by drug withdrawal or concomitant treatment with antihypertensive agents [51]. R406 also inhibited VEGF-dependent human endothelial cell tube formation and tube outgrowth, further supporting a possible role for VEGF signaling in blood pressure effects of fostamatinib [48].…”

“…R406 is a potent and selective inhibitor of Syk; it inhibits Syk kinase activity by binding the catalytic domain (K i = 30 nM) and acts as an ATP-competitive inhibitor (IC 50 = 41 nM) [37]. As with other kinase inhibitors, additional molecular targets of fostamatinib have been explored to investigate potential off-target effects that may contribute to its pharmacological effects [47,48]. Selectivity of R406 has been demonstrated in panels of in vitro and cell-based assays.…”

“…In an initial selectivity assessment of 90 in vitro kinase assays, R406 bound to other targets (i.e., Flt3, Lyn and Lck), but showed five-to 100-fold greater inhibition of Syk than other tyrosine kinases when tested in a phosphorylation assay [37,49]. More recently, Rolf et al presented a comprehensive pharmacological profile of fostamatinib/R406, using a broad range of in vitro assays, followed by functional and cellular assays to further understand the relevance of key targets at therapeutically relevant concentrations [48]. Among nonkinases, antagonist activity was found against adenosine A 3 receptor, inhibiting adenosine uptake with an IC 50 of 100 nmol/l.…”

“…In a kinase screen of 387 individual kinases, R406 bound and showed activity against 117 kinases, 100 of which had IC 50 within three-fold of Syk. Out of these, the authors noted prior association of FLT1, KDR, SRC and KIT with blood pressure, based on their analysis of published literature [48].…”

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“…The significance of this change is unknown, as ovarian follicles, unlike corpora lutea, are avascular and therefore not expected to be affected by angiogenic inhibition. R406 also has significant off-target inhibitor activity for the VEGF and FGF family of receptors, with in vitro isolated enzyme IC 50 values similar to the SYK potency (IC 50 4-60 nM; Rolf et al 2015). In vivo potency, however, is likely to be much lower since elevation of blood pressure (a recognized biomarker of VEGFR activity [Curwen et al 2008]) is much less compared to other more potent VEGFR inhibitors (fostamatinib < 5 mmHg vs. 25 mmHg for sunitinb; Rolf et al 2015).…”