Casein kinase 2 (CK2) is a constitutively expressed serine/threonine kinase that has a large diversity of cellular substrates. Thus, CK2 has been associated with a plethora of regulatory functions and dysregulation of CK2 has been linked to disease development in particular to cancer. The broad implications in disease pathology makes CK2 an attractive target. To date, the most advanced CK2 inhibitor is silmitasertib, which has been investigated in clinical trials for treatment of various cancers, albeit several off-targets for silmitasertib have been described. To ascertain the role of CK2 inhibition in cancer, other disease and normal physiology the development of a selective CK2 inhibitor would be highly desirable. In this study we explored the pyrazolo[1,5-a]pyrimidine hinge-binding moiety for the development of selective CK2 inhibitors. Optimization of this scaffold, which included macrocyclization, led to IC20 (31) a compound that displayed high in vitro potency for CK2 (KD = 12 nM) and exclusive selectivity for CK2. X-ray analysis revealed a canonical type-I binding mode for IC20. However, the polar carboxylic acid moiety that is shared by many CK2 inhibitors including silmitasertib was required for potency and reduced somewhat cellular activity. In summary, IC20 represents a highly selective and potent inhibitor of CK2, which can be used as a tool compound to study CK2 biology and potential new applications for the treatment of diseases.
Phenotypical screening is a widely used approach in drug discovery for the identification of small molecules with cellular activities. However, functional annotation of identified hits often poses a challenge. The development of small molecules with narrow or exclusive target selectivity such as chemical probes and chemogenomic (CG) libraries, greatly diminishes this challenge, but non-specific effects caused by compound toxicity or interference with basic cellular functions still pose a problem to associate phenotypic readouts with molecular targets. Hence, each compound should ideally be comprehensively characterized regarding its effects on general cell functions. Here, we report an optimized live-cell multiplexed assay that classifies cells based on nuclear morphology, presenting an excellent indicator for cellular responses such as early apoptosis and necrosis. This basic readout in combination with the detection of other general cell damaging activities of small molecules such as changes in cytoskeletal morphology, cell cycle and mitochondrial health provides a comprehensive time-dependent characterization of the effect of small molecules on cellular health in a single experiment. The developed high-content assay offers multi-dimensional comprehensive characterization that can be used to delineate generic effects regarding cell functions and cell viability, allowing an assessment of compound suitability for subsequent detailed phenotypic and mechanistic studies.
Serine/threonine kinase 17A (death-associated
protein kinase-related
apoptosis-inducing protein kinase 1DRAK1) is a part of the
death-associated protein kinase (DAPK) family and belongs to the so-called
dark kinome. Thus, the current state of knowledge of the cellular
function of DRAK1 and its involvement in pathophysiological processes
is very limited. Recently, DRAK1 has been implicated in tumorigenesis
of glioblastoma multiforme (GBM) and other cancers, but no selective
inhibitors of DRAK1 are available yet. To this end, we optimized a
pyrazolo[1,5-a]pyrimidine-based macrocyclic scaffold.
Structure-guided optimization of this macrocyclic scaffold led to
the development of CK156 (34), which displayed high in
vitro potency (K
D = 21 nM) and selectivity
in kinomewide screens. Crystal structures demonstrated that CK156
(34) acts as a type I inhibitor. However, contrary to
studies using genetic knockdown of DRAK1, we have seen the inhibition
of cell growth of glioma cells in 2D and 3D culture only at low micromolar
concentrations.
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