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Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia.

Lee-Sherick AB, Zhang W, Menachof KK, Hill AA, Rinella S, Kirkpatrick G, Page LS, Stashko MA, Jordan CT, Wei Q, Liu J, Zhang D, DeRyckere D, Wang X, Frye S, Earp HS, Graham DK - Oncotarget (2015)

Bottom Line: Treatment with UNC1666 mediated biochemical and functional effects in AML cell lines expressing Mer or Flt3 internal tandem duplication (ITD), including decreased phosphorylation of Mer, Flt3 and downstream effectors Stat, Akt and Erk, induction of apoptosis in up to 98% of cells, and reduction of colony formation by greater than 90%, compared to treatment with vehicle.These effects were dose-dependent, with inhibition of downstream signaling and functional effects correlating with the degree of Mer or Flt3 kinase inhibition.In summary, UNC1666 is a novel potent small molecule tyrosine kinase inhibitor that decreases oncogenic signaling and myeloblast survival, thereby validating dual Mer/Flt3 inhibition as an attractive treatment strategy for AML.

View Article: PubMed Central - PubMed

Affiliation: University of Colorado, Department of Pediatrics, Aurora, CO, USA.

ABSTRACT
Mer and Flt3 receptor tyrosine kinases have been implicated as therapeutic targets in acute myeloid leukemia (AML). In this manuscript we describe UNC1666, a novel ATP-competitive small molecule tyrosine kinase inhibitor, which potently diminishes Mer and Flt3 phosphorylation in AML. Treatment with UNC1666 mediated biochemical and functional effects in AML cell lines expressing Mer or Flt3 internal tandem duplication (ITD), including decreased phosphorylation of Mer, Flt3 and downstream effectors Stat, Akt and Erk, induction of apoptosis in up to 98% of cells, and reduction of colony formation by greater than 90%, compared to treatment with vehicle. These effects were dose-dependent, with inhibition of downstream signaling and functional effects correlating with the degree of Mer or Flt3 kinase inhibition. Treatment of primary AML patient samples expressing Mer and/or Flt3-ITD with UNC1666 also inhibited Mer and Flt3 intracellular signaling, induced apoptosis, and inhibited colony formation. In summary, UNC1666 is a novel potent small molecule tyrosine kinase inhibitor that decreases oncogenic signaling and myeloblast survival, thereby validating dual Mer/Flt3 inhibition as an attractive treatment strategy for AML.

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UNC1666 is a novel inhibitor of Mer and Flt3 tyrosine kinases(A) Chemical structure of UNC1666, with inhibition constant (Ki) of 0.16 nM for Mer (enzymatic IC50: 0.55 nM) and 0.67 nM for Flt3 (enzymatic IC50: 0.69 nM). (B) Chemical structure of UNC1653, which lacks significant activity against Mer (enzymatic IC50: 560 nM) and Flt3 (enzymatic IC50: 220 nM) and is used as a negative control in these studies. (C) Whole cell lysates from AML cell lines with known Flt3 mutation status were analyzed by immunoblot and demonstrate presence or absence of the Mer tyrosine kinase (above) and the Flt3 tyrosine kinase (middle). Actin is shown as an indicator of total protein (below).
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Figure 1: UNC1666 is a novel inhibitor of Mer and Flt3 tyrosine kinases(A) Chemical structure of UNC1666, with inhibition constant (Ki) of 0.16 nM for Mer (enzymatic IC50: 0.55 nM) and 0.67 nM for Flt3 (enzymatic IC50: 0.69 nM). (B) Chemical structure of UNC1653, which lacks significant activity against Mer (enzymatic IC50: 560 nM) and Flt3 (enzymatic IC50: 220 nM) and is used as a negative control in these studies. (C) Whole cell lysates from AML cell lines with known Flt3 mutation status were analyzed by immunoblot and demonstrate presence or absence of the Mer tyrosine kinase (above) and the Flt3 tyrosine kinase (middle). Actin is shown as an indicator of total protein (below).

Mentions: We previously reported UNC1062 [23], a selective ATP-competitive type I inhibitor of Mer. However, its low solubility and poor in vivo pharmacokinetic properties made UNC1062 unsuitable for in vivo studies. To develop further Mer inhibitors, a new pyrrolopyrimidine scaffold with better solubility was introduced using a structure-based design approach [24]. UNC1666, a pyrrolopyrimidine analogue with a structure similar to UNC1062, is also an ATP-competitive type I inhibitor (Figure 1A). Analysis of the inhibition constant (Ki) proved this compound to be more potent and selective for Mer (MCE IC50 0.55 nM; Ki 0.16 nM) compared to previously described Mer inhibitors [23, 25]. Additionally, UNC1666 inhibits Flt3 (MCE IC50 0.69 nM; Ki 0.67 nM) equipotently in enzymatic MCE assays. A comprehensive protein kinase profiling panel provided by Carna Biosciences was used to assess off-target kinase inhibition mediated by UNC1666 at a concentration of 46 nM, more than 50-fold higher than its MCE IC50 values against Mer and Flt3 (Supplemental Table 2). Only the Trk proteins were inhibited greater than 95% in response to treatment with UNC1666. Additional MCE assays were performed to determine inhibition of TrkA (as a surrogate for the Trk family kinases) and revealed similar potency (MCE IC50 0.57 nM) (Supplemental Table 2). Furthermore, we analyzed the effect of UNC1666 on both Tyro-3 and Axl (members of the TAM receptor tyrosine kinase family along with Mer), which demonstrated enzymatic MCE IC50 values of 29 nM and 37 nM, respectively.


Efficacy of a Mer and Flt3 tyrosine kinase small molecule inhibitor, UNC1666, in acute myeloid leukemia.

Lee-Sherick AB, Zhang W, Menachof KK, Hill AA, Rinella S, Kirkpatrick G, Page LS, Stashko MA, Jordan CT, Wei Q, Liu J, Zhang D, DeRyckere D, Wang X, Frye S, Earp HS, Graham DK - Oncotarget (2015)

UNC1666 is a novel inhibitor of Mer and Flt3 tyrosine kinases(A) Chemical structure of UNC1666, with inhibition constant (Ki) of 0.16 nM for Mer (enzymatic IC50: 0.55 nM) and 0.67 nM for Flt3 (enzymatic IC50: 0.69 nM). (B) Chemical structure of UNC1653, which lacks significant activity against Mer (enzymatic IC50: 560 nM) and Flt3 (enzymatic IC50: 220 nM) and is used as a negative control in these studies. (C) Whole cell lysates from AML cell lines with known Flt3 mutation status were analyzed by immunoblot and demonstrate presence or absence of the Mer tyrosine kinase (above) and the Flt3 tyrosine kinase (middle). Actin is shown as an indicator of total protein (below).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4466645&req=5

Figure 1: UNC1666 is a novel inhibitor of Mer and Flt3 tyrosine kinases(A) Chemical structure of UNC1666, with inhibition constant (Ki) of 0.16 nM for Mer (enzymatic IC50: 0.55 nM) and 0.67 nM for Flt3 (enzymatic IC50: 0.69 nM). (B) Chemical structure of UNC1653, which lacks significant activity against Mer (enzymatic IC50: 560 nM) and Flt3 (enzymatic IC50: 220 nM) and is used as a negative control in these studies. (C) Whole cell lysates from AML cell lines with known Flt3 mutation status were analyzed by immunoblot and demonstrate presence or absence of the Mer tyrosine kinase (above) and the Flt3 tyrosine kinase (middle). Actin is shown as an indicator of total protein (below).
Mentions: We previously reported UNC1062 [23], a selective ATP-competitive type I inhibitor of Mer. However, its low solubility and poor in vivo pharmacokinetic properties made UNC1062 unsuitable for in vivo studies. To develop further Mer inhibitors, a new pyrrolopyrimidine scaffold with better solubility was introduced using a structure-based design approach [24]. UNC1666, a pyrrolopyrimidine analogue with a structure similar to UNC1062, is also an ATP-competitive type I inhibitor (Figure 1A). Analysis of the inhibition constant (Ki) proved this compound to be more potent and selective for Mer (MCE IC50 0.55 nM; Ki 0.16 nM) compared to previously described Mer inhibitors [23, 25]. Additionally, UNC1666 inhibits Flt3 (MCE IC50 0.69 nM; Ki 0.67 nM) equipotently in enzymatic MCE assays. A comprehensive protein kinase profiling panel provided by Carna Biosciences was used to assess off-target kinase inhibition mediated by UNC1666 at a concentration of 46 nM, more than 50-fold higher than its MCE IC50 values against Mer and Flt3 (Supplemental Table 2). Only the Trk proteins were inhibited greater than 95% in response to treatment with UNC1666. Additional MCE assays were performed to determine inhibition of TrkA (as a surrogate for the Trk family kinases) and revealed similar potency (MCE IC50 0.57 nM) (Supplemental Table 2). Furthermore, we analyzed the effect of UNC1666 on both Tyro-3 and Axl (members of the TAM receptor tyrosine kinase family along with Mer), which demonstrated enzymatic MCE IC50 values of 29 nM and 37 nM, respectively.

Bottom Line: Treatment with UNC1666 mediated biochemical and functional effects in AML cell lines expressing Mer or Flt3 internal tandem duplication (ITD), including decreased phosphorylation of Mer, Flt3 and downstream effectors Stat, Akt and Erk, induction of apoptosis in up to 98% of cells, and reduction of colony formation by greater than 90%, compared to treatment with vehicle.These effects were dose-dependent, with inhibition of downstream signaling and functional effects correlating with the degree of Mer or Flt3 kinase inhibition.In summary, UNC1666 is a novel potent small molecule tyrosine kinase inhibitor that decreases oncogenic signaling and myeloblast survival, thereby validating dual Mer/Flt3 inhibition as an attractive treatment strategy for AML.

View Article: PubMed Central - PubMed

Affiliation: University of Colorado, Department of Pediatrics, Aurora, CO, USA.

ABSTRACT
Mer and Flt3 receptor tyrosine kinases have been implicated as therapeutic targets in acute myeloid leukemia (AML). In this manuscript we describe UNC1666, a novel ATP-competitive small molecule tyrosine kinase inhibitor, which potently diminishes Mer and Flt3 phosphorylation in AML. Treatment with UNC1666 mediated biochemical and functional effects in AML cell lines expressing Mer or Flt3 internal tandem duplication (ITD), including decreased phosphorylation of Mer, Flt3 and downstream effectors Stat, Akt and Erk, induction of apoptosis in up to 98% of cells, and reduction of colony formation by greater than 90%, compared to treatment with vehicle. These effects were dose-dependent, with inhibition of downstream signaling and functional effects correlating with the degree of Mer or Flt3 kinase inhibition. Treatment of primary AML patient samples expressing Mer and/or Flt3-ITD with UNC1666 also inhibited Mer and Flt3 intracellular signaling, induced apoptosis, and inhibited colony formation. In summary, UNC1666 is a novel potent small molecule tyrosine kinase inhibitor that decreases oncogenic signaling and myeloblast survival, thereby validating dual Mer/Flt3 inhibition as an attractive treatment strategy for AML.

Show MeSH
Related in: MedlinePlus