Analysis of selective target engagement by small-molecule sphingosine kinase inhibitors using the Cellular Thermal Shift Assay (CETSA).

The recently renewed interest in scientific rigor and reproducibility is of critical importance for both scientists developing new targeted small-molecule inhibitors and those employing these molecule in cellular studies, alike. While off-target effects are commonly considered as limitations for any given small-molecule inhibitor, the ability of a given compound to distinguish between enzyme isoforms is often neglected when employing compounds in cellular studies. To call attention to this issue, we have compared the results of an assay for "direct target engagement", the Cellular Thermal Shift Assay (CETSA), to the published isoform selectivity of 12 commercially available sphingosine kinase 1 and 2 (SphK 1 and SphK2) inhibitors. Our results suggest that, at the concentrations commonly employed in cellular assay systems, none of the tested SKIs can be considered isoform selective. Thus, caution and complimentary assay strategies must be employed to fully discern isoform selectivity for the SphKs. Moreover, caution must be employed by the scientific community as a whole when designing experiments that aim to discern the effects of one enzyme isoform versus another to ensure that the concentration ranges used are able to distinguish isoform selectivity.

SKI-178: A Multitargeted Inhibitor of Sphingosine Kinase and Microtubule Dynamics Demonstrating Therapeutic Efficacy in Acute Myeloid Leukemia Models.

AIM: To further characterize the selectivity, mechanism-of-action and therapeutic efficacy of the novel small molecule inhibitor, SKI-178. METHODS: Using the state-of-the-art Cellular Thermal Shift Assay (CETSA) technique to detect "direct target engagement" of proteins intact cells, in vitro and in vivo assays, pharmacological assays and multiple mouse models of acute myeloid leukemia (AML). RESULTS: Herein, we demonstrate that SKI-178 directly target engages both Sphingosine Kinase 1 and 2. We also present evidence that, in addition to its actions as a Sphingosine Kinase Inhibitor, SKI-178 functions as a microtubule network disrupting agent both in vitro and in intact cells. Interestingly, we separately demonstrate that simultaneous SphK inhibition and microtubule disruption synergistically induces apoptosis in AML cell lines. Furthermore, we demonstrate that SKI-178 is well tolerated in normal healthy mice. Most importantly, we demonstrate that SKI-178 has therapeutic efficacy in several mouse models of AML. CONCLUSION: SKI-178 is a multi-targeted agent that functions both as an inhibitor of the SphKs as well as a disruptor of the microtubule network. SKI-178 induced apoptosis arises from a synergistic interaction of these two activities. SKI-178 is safe and effective in mouse models of AML, supporting its further development as a multi-targeted anti-cancer therapeutic agent.

The apoptotic mechanism of action of the sphingosine kinase 1 selective inhibitor SKI-178 in human acute myeloid leukemia cell lines.

We previously developed SKI-178 (N'-[(1E)-1-(3,4-dimethoxyphenyl)ethylidene]-3-(4-methoxxyphenyl)-1H-pyrazole-5-carbohydrazide) as a novel sphingosine kinase-1 (SphK1) selective inhibitor and, herein, sought to determine the mechanism-of-action of SKI-178-induced cell death. Using human acute myeloid leukemia (AML) cell lines as a model, we present evidence that SKI-178 induces prolonged mitosis followed by apoptotic cell death through the intrinsic apoptotic cascade. Further examination of the mechanism of action of SKI-178 implicated c-Jun NH2-terminal kinase (JNK) and cyclin-dependent protein kinase 1 (CDK1) as critical factors required for SKI-178-induced apoptosis. In cell cycle synchronized human AML cell lines, we demonstrate that entry into mitosis is required for apoptotic induction by SKI-178 and that CDK1, not JNK, is required for SKI-178-induced apoptosis. We further demonstrate that the sustained activation of CDK1 during prolonged mitosis, mediated by SKI-178, leads to the simultaneous phosphorylation of the prosurvival Bcl-2 family members, Bcl-2 and Bcl-xl, as well as the phosphorylation and subsequent degradation of Mcl-1. Moreover, multidrug resistance mediated by multidrug-resistant protein1 and/or prosurvival Bcl-2 family member overexpression did not affect the sensitivity of AML cells to SKI-178. Taken together, these findings highlight the therapeutic potential of SKI-178 targeting SphK1 as a novel therapeutic agent for the treatment of AML, including multidrug-resistant/recurrent AML subtypes.

A novel selective multikinase inhibitor of ROCK and MRCK effectively blocks cancer cell migration and invasion.

Two structurally related protein kinase families, the Rho kinases (ROCK) and the myotonic dystrophy kinase-related Cdc42-binding kinases (MRCK) are required for migration and invasion of cancer cells. We hypothesized that simultaneous targeting of these two kinase families might represent a novel therapeutic strategy to block the migration and invasion of metastatic cancers. To this end, we developed DJ4 as a novel small molecule inhibitor of these kinases. DJ4 potently inhibited activities of ROCK and MRCK in an ATP competitive manner. In cellular functional assays, DJ4 treatment significantly blocked stress fiber formation and inhibited migration and invasion of multiple cancer cell lines in a concentration dependent manner. Our results strongly indicate that DJ4 may be further developed as a novel anti-metastatic chemotherapeutic agent for multiple cancers.