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A selective ATP-competitive sphingosine kinase inhibitor demonstrates anti-cancer properties.

Pitman MR, Powell JA, Coolen C, Moretti PA, Zebol JR, Pham DH, Finnie JW, Don AS, Ebert LM, Bonder CS, Gliddon BL, Pitson SM - Oncotarget (2015)

Bottom Line: Sphingosine 1-phosphate is a signaling molecule with anti-apoptotic, pro-proliferative and pro-angiogenic effects, while conversely, ceramide and sphingosine are pro-apoptotic.Here we report a first-in-class ATP-binding site-directed small molecule SK inhibitor, MP-A08, discovered using an approach of structural homology modelling of the ATP-binding site of SK1 and in silico docking with small molecule libraries.MP-A08 blocks pro-proliferative signalling pathways, induces mitochondrial-associated apoptosis in a SK-dependent manner, and reduces the growth of human lung adenocarcinoma tumours in a mouse xenograft model by both inducing tumour cell apoptosis and inhibiting tumour angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cancer Biology, University of South Australia and SA Pathology, Adelaide, SA 5000, Australia.

ABSTRACT
The dynamic balance of cellular sphingolipids, the sphingolipid rheostat, is an important determinant of cell fate, and is commonly deregulated in cancer. Sphingosine 1-phosphate is a signaling molecule with anti-apoptotic, pro-proliferative and pro-angiogenic effects, while conversely, ceramide and sphingosine are pro-apoptotic. The sphingosine kinases (SKs) are key regulators of this sphingolipid rheostat, and are attractive targets for anti-cancer therapy. Here we report a first-in-class ATP-binding site-directed small molecule SK inhibitor, MP-A08, discovered using an approach of structural homology modelling of the ATP-binding site of SK1 and in silico docking with small molecule libraries. MP-A08 is a highly selective ATP competitive SK inhibitor that targets both SK1 and SK2. MP-A08 blocks pro-proliferative signalling pathways, induces mitochondrial-associated apoptosis in a SK-dependent manner, and reduces the growth of human lung adenocarcinoma tumours in a mouse xenograft model by both inducing tumour cell apoptosis and inhibiting tumour angiogenesis. Thus, this selective ATP competitive SK inhibitor provides a promising candidate for potential development as an anti-cancer therapy, and also, due to its different mode of inhibition to other known SK inhibitors, both validates the SKs as targets for anti-cancer therapy, and represents an important experimental tool to study these enzymes.

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MP-A08 is a novel ATP-competitive inhibitor for SK1 and SK2Lineweaver–Burke plots showing inhibition kinetics MP-A08 against recombinant SK1 (A) and SK2 (B) with varying ATP concentration. Data show MP-A08 employed at 50 μM (▲) or 25 μM (■), or with vehicle control (●), and are mean ± SD from four independent experiments. (C) The ATP-binding pocket of the recently solved SK1 crystal structure (3VZD) with MP-A08 docked. Each of the regions highly conserved in all SKs (see Supplementary Figure 1) that comprise the ATP-binding pocket are colored in the same scheme as in Figure 1A. SK1Arg185 and SK1Arg191 are colored in light pink. The atoms in MP-A08 are colored according to chemical elements; oxygen in red, nitrogen in blue, and sulphur in yellow. (D) The predicted SK2 model is represented with MP-A08 docked. Each of the conserved motifs that comprise the ATP-binding pocket are colored as in C. SK2Arg315 and SK2Arg321 are colored in light pink. (E) Assessment of residual SK2 activity in the ATP-binding pocket mutants of residues predicted to be important for SK2 binding to MP-A08. SK2 activities were determined after overexpression in HEK293T cells, and represented as % activity compared to wildtype SK2 (WT). Empty vector (EV) transfected cells show negligible contribution from endogenous SK to the activities displayed. The lower panel shows similar expression levels of all SK2 variants, but all activities were adjusted for slight variations in expression. All data shown are mean ± SD (n = 4). (F) Effect of MP-A08 (250 μM) on the activity of SK2 variants harboring mutations in the ATP-binding site. Values are represented as % activity compared to vehicle control, mean ± SD (n = 4). Significance from SK2WT was determined by student t-test (*p < 0.05, **p < 0.01 and ***p < 0.001)
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Figure 2: MP-A08 is a novel ATP-competitive inhibitor for SK1 and SK2Lineweaver–Burke plots showing inhibition kinetics MP-A08 against recombinant SK1 (A) and SK2 (B) with varying ATP concentration. Data show MP-A08 employed at 50 μM (▲) or 25 μM (■), or with vehicle control (●), and are mean ± SD from four independent experiments. (C) The ATP-binding pocket of the recently solved SK1 crystal structure (3VZD) with MP-A08 docked. Each of the regions highly conserved in all SKs (see Supplementary Figure 1) that comprise the ATP-binding pocket are colored in the same scheme as in Figure 1A. SK1Arg185 and SK1Arg191 are colored in light pink. The atoms in MP-A08 are colored according to chemical elements; oxygen in red, nitrogen in blue, and sulphur in yellow. (D) The predicted SK2 model is represented with MP-A08 docked. Each of the conserved motifs that comprise the ATP-binding pocket are colored as in C. SK2Arg315 and SK2Arg321 are colored in light pink. (E) Assessment of residual SK2 activity in the ATP-binding pocket mutants of residues predicted to be important for SK2 binding to MP-A08. SK2 activities were determined after overexpression in HEK293T cells, and represented as % activity compared to wildtype SK2 (WT). Empty vector (EV) transfected cells show negligible contribution from endogenous SK to the activities displayed. The lower panel shows similar expression levels of all SK2 variants, but all activities were adjusted for slight variations in expression. All data shown are mean ± SD (n = 4). (F) Effect of MP-A08 (250 μM) on the activity of SK2 variants harboring mutations in the ATP-binding site. Values are represented as % activity compared to vehicle control, mean ± SD (n = 4). Significance from SK2WT was determined by student t-test (*p < 0.05, **p < 0.01 and ***p < 0.001)

Mentions: Inhibition kinetics confirmed that MP-A08 was an ATP-competitive inhibitor of human SK1 and SK2 (Figure 2A–2B). Somewhat surprisingly, MP-A08 was a higher affinity inhibitor of SK2 than SK1, with Ki values of 6.9 ± 0.8 μM and 27 ± 3 μM, respectively. In order to analyze these differences in binding to SK1 and SK2 we produced a homology model of SK2 using the recent SK1 crystal structure, and then docked MP-A08 into the SK1 crystal structure (Figure 2C) and the predicted SK2 ATP-binding pocket (Figure 2D). Comparison of the SK1 crystal structure and the SK2 model revealed a highly conserved ATP pocket with the exception of the substitutions at SK2Phe154/SK1Arg24 and SK2Asn187/SK1Arg57 (Figure 2C–2D, Supplementary Figure 3A–3B). In the SK1 structure Arg24 and Thr54 appear to coordinate one of the central phenyl rings of MP-A08 and shield the amine groups from the basic side-chains of Arg24 and Arg57. The second central phenyl ring likely shields the internal amine group from the Arg185 and Arg191 side-chains, pointing the sulfonyl groups towards Asn22, Ser79 and Leu83 in SK1 (Figure 2C). One methyl substituted ring points out towards the outside of the pocket and the other is orientated towards the internal Ser112 and the Arg185 and Arg191 side-chains in SK1. The predicted orientation of MP-A08 in the ATP-binding pocket of SK2 is altered due to the substituted residues at Phe154 and Asn187 (Arg24 and Arg57 in SK1, respectively). The bulky aromatic side-chain of Phe154 and the smaller basic side-chain of Asn187 alter both the size and charge of the ATP pocket in SK2 (Figure 2D and Supplementary Figure 3A–3B). The terminal methylphenyl rings point towards the side-chain of Thr184 and Asn152. Compared to the SK1 pocket, the central phenyl rings of MP-A08 are shifted towards the bottom of the SK2 pocket, coordinated by Arg315 and Arg321. The sulfonyl groups are tethered via hydrogen bonding to Asn152 and Ser242 side-chains.


A selective ATP-competitive sphingosine kinase inhibitor demonstrates anti-cancer properties.

Pitman MR, Powell JA, Coolen C, Moretti PA, Zebol JR, Pham DH, Finnie JW, Don AS, Ebert LM, Bonder CS, Gliddon BL, Pitson SM - Oncotarget (2015)

MP-A08 is a novel ATP-competitive inhibitor for SK1 and SK2Lineweaver–Burke plots showing inhibition kinetics MP-A08 against recombinant SK1 (A) and SK2 (B) with varying ATP concentration. Data show MP-A08 employed at 50 μM (▲) or 25 μM (■), or with vehicle control (●), and are mean ± SD from four independent experiments. (C) The ATP-binding pocket of the recently solved SK1 crystal structure (3VZD) with MP-A08 docked. Each of the regions highly conserved in all SKs (see Supplementary Figure 1) that comprise the ATP-binding pocket are colored in the same scheme as in Figure 1A. SK1Arg185 and SK1Arg191 are colored in light pink. The atoms in MP-A08 are colored according to chemical elements; oxygen in red, nitrogen in blue, and sulphur in yellow. (D) The predicted SK2 model is represented with MP-A08 docked. Each of the conserved motifs that comprise the ATP-binding pocket are colored as in C. SK2Arg315 and SK2Arg321 are colored in light pink. (E) Assessment of residual SK2 activity in the ATP-binding pocket mutants of residues predicted to be important for SK2 binding to MP-A08. SK2 activities were determined after overexpression in HEK293T cells, and represented as % activity compared to wildtype SK2 (WT). Empty vector (EV) transfected cells show negligible contribution from endogenous SK to the activities displayed. The lower panel shows similar expression levels of all SK2 variants, but all activities were adjusted for slight variations in expression. All data shown are mean ± SD (n = 4). (F) Effect of MP-A08 (250 μM) on the activity of SK2 variants harboring mutations in the ATP-binding site. Values are represented as % activity compared to vehicle control, mean ± SD (n = 4). Significance from SK2WT was determined by student t-test (*p < 0.05, **p < 0.01 and ***p < 0.001)
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Related In: Results  -  Collection

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Show All Figures
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Figure 2: MP-A08 is a novel ATP-competitive inhibitor for SK1 and SK2Lineweaver–Burke plots showing inhibition kinetics MP-A08 against recombinant SK1 (A) and SK2 (B) with varying ATP concentration. Data show MP-A08 employed at 50 μM (▲) or 25 μM (■), or with vehicle control (●), and are mean ± SD from four independent experiments. (C) The ATP-binding pocket of the recently solved SK1 crystal structure (3VZD) with MP-A08 docked. Each of the regions highly conserved in all SKs (see Supplementary Figure 1) that comprise the ATP-binding pocket are colored in the same scheme as in Figure 1A. SK1Arg185 and SK1Arg191 are colored in light pink. The atoms in MP-A08 are colored according to chemical elements; oxygen in red, nitrogen in blue, and sulphur in yellow. (D) The predicted SK2 model is represented with MP-A08 docked. Each of the conserved motifs that comprise the ATP-binding pocket are colored as in C. SK2Arg315 and SK2Arg321 are colored in light pink. (E) Assessment of residual SK2 activity in the ATP-binding pocket mutants of residues predicted to be important for SK2 binding to MP-A08. SK2 activities were determined after overexpression in HEK293T cells, and represented as % activity compared to wildtype SK2 (WT). Empty vector (EV) transfected cells show negligible contribution from endogenous SK to the activities displayed. The lower panel shows similar expression levels of all SK2 variants, but all activities were adjusted for slight variations in expression. All data shown are mean ± SD (n = 4). (F) Effect of MP-A08 (250 μM) on the activity of SK2 variants harboring mutations in the ATP-binding site. Values are represented as % activity compared to vehicle control, mean ± SD (n = 4). Significance from SK2WT was determined by student t-test (*p < 0.05, **p < 0.01 and ***p < 0.001)
Mentions: Inhibition kinetics confirmed that MP-A08 was an ATP-competitive inhibitor of human SK1 and SK2 (Figure 2A–2B). Somewhat surprisingly, MP-A08 was a higher affinity inhibitor of SK2 than SK1, with Ki values of 6.9 ± 0.8 μM and 27 ± 3 μM, respectively. In order to analyze these differences in binding to SK1 and SK2 we produced a homology model of SK2 using the recent SK1 crystal structure, and then docked MP-A08 into the SK1 crystal structure (Figure 2C) and the predicted SK2 ATP-binding pocket (Figure 2D). Comparison of the SK1 crystal structure and the SK2 model revealed a highly conserved ATP pocket with the exception of the substitutions at SK2Phe154/SK1Arg24 and SK2Asn187/SK1Arg57 (Figure 2C–2D, Supplementary Figure 3A–3B). In the SK1 structure Arg24 and Thr54 appear to coordinate one of the central phenyl rings of MP-A08 and shield the amine groups from the basic side-chains of Arg24 and Arg57. The second central phenyl ring likely shields the internal amine group from the Arg185 and Arg191 side-chains, pointing the sulfonyl groups towards Asn22, Ser79 and Leu83 in SK1 (Figure 2C). One methyl substituted ring points out towards the outside of the pocket and the other is orientated towards the internal Ser112 and the Arg185 and Arg191 side-chains in SK1. The predicted orientation of MP-A08 in the ATP-binding pocket of SK2 is altered due to the substituted residues at Phe154 and Asn187 (Arg24 and Arg57 in SK1, respectively). The bulky aromatic side-chain of Phe154 and the smaller basic side-chain of Asn187 alter both the size and charge of the ATP pocket in SK2 (Figure 2D and Supplementary Figure 3A–3B). The terminal methylphenyl rings point towards the side-chain of Thr184 and Asn152. Compared to the SK1 pocket, the central phenyl rings of MP-A08 are shifted towards the bottom of the SK2 pocket, coordinated by Arg315 and Arg321. The sulfonyl groups are tethered via hydrogen bonding to Asn152 and Ser242 side-chains.

Bottom Line: Sphingosine 1-phosphate is a signaling molecule with anti-apoptotic, pro-proliferative and pro-angiogenic effects, while conversely, ceramide and sphingosine are pro-apoptotic.Here we report a first-in-class ATP-binding site-directed small molecule SK inhibitor, MP-A08, discovered using an approach of structural homology modelling of the ATP-binding site of SK1 and in silico docking with small molecule libraries.MP-A08 blocks pro-proliferative signalling pathways, induces mitochondrial-associated apoptosis in a SK-dependent manner, and reduces the growth of human lung adenocarcinoma tumours in a mouse xenograft model by both inducing tumour cell apoptosis and inhibiting tumour angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cancer Biology, University of South Australia and SA Pathology, Adelaide, SA 5000, Australia.

ABSTRACT
The dynamic balance of cellular sphingolipids, the sphingolipid rheostat, is an important determinant of cell fate, and is commonly deregulated in cancer. Sphingosine 1-phosphate is a signaling molecule with anti-apoptotic, pro-proliferative and pro-angiogenic effects, while conversely, ceramide and sphingosine are pro-apoptotic. The sphingosine kinases (SKs) are key regulators of this sphingolipid rheostat, and are attractive targets for anti-cancer therapy. Here we report a first-in-class ATP-binding site-directed small molecule SK inhibitor, MP-A08, discovered using an approach of structural homology modelling of the ATP-binding site of SK1 and in silico docking with small molecule libraries. MP-A08 is a highly selective ATP competitive SK inhibitor that targets both SK1 and SK2. MP-A08 blocks pro-proliferative signalling pathways, induces mitochondrial-associated apoptosis in a SK-dependent manner, and reduces the growth of human lung adenocarcinoma tumours in a mouse xenograft model by both inducing tumour cell apoptosis and inhibiting tumour angiogenesis. Thus, this selective ATP competitive SK inhibitor provides a promising candidate for potential development as an anti-cancer therapy, and also, due to its different mode of inhibition to other known SK inhibitors, both validates the SKs as targets for anti-cancer therapy, and represents an important experimental tool to study these enzymes.

Show MeSH
Related in: MedlinePlus