During the past decade, the basophilic atypical kinase Haspin has emerged as a key player in mitosis responsible for phosphorylation of Thr3 residue of histone H3. Here, we report the construction of conjugates comprising an aromatic fragment targeted to the ATP-site of Haspin and a peptide mimicking the N-terminus of histone H3. The combination of effective solid phase synthesis procedures and a high throughput binding/displacement assay with fluorescence anisotropy readout afforded the development of inhibitors with remarkable subnanomolar affinity toward Haspin. The selectivity profiles of novel conjugates were established by affinity studies with a model basophilic kinase (catalytic subunit of cAMP-dependent protein kinase) and by a commercial 1-point inhibition assay with 43 protein kinases.

The chemical space of Haspin-targeting low-molecular-weight-compounds has not yet been widely explored, but several scaffolds (e.g., derivatives of acridine, β-carboline or 5-iodotubercidin) have emerged as promising inhibitors. The inclusion of Haspin into protein kinase panels for profiling of low-molecular-weight-compounds in several recent studies has provided valuable information about the structure-affinity or structure-activity relationship of well-known or novel inhibitors towards Haspin.

The atypical protein kinase haspin is a key player in mitosis by catalysing the phosphorylation of Thr3 in histone H3, and thus ensuring the normal function of the chromosomal passenger complex. Here, we report the development of bisubstrate-analogue inhibitors targeting haspin. The compounds were constructed by linking 5-iodotubercidin to the N terminus of histone H3 peptide. The new conjugates show high affinity (sub-nanomolar K ) towards haspin as well as slow kinetics of association and dissociation (residence time of several hours). This reflects a unique binding mode and translated into improved selectivity. The latter was confirmed in a biochemical binding/displacement assay with a panel of ten protein kinases, in a thermal shift assay with off-targets of 5-iodotubercidin (adenosine kinase and the Cdc2-like kinase family) and in assay with spiked HeLa cell lysate.

We performed an X-ray crystallographic study of complexes of protein kinase PIM-1 with three inhibitors comprising an adenosine mimetic moiety, a linker, and a peptide-mimetic (d-Arg)6 fragment. Guided by the structural models, simplified chemical structures with a reduced number of polar groups and chiral centers were designed. The developed inhibitors retained low-nanomolar potency and possessed remarkable selectivity toward the PIM kinases. The new inhibitors were derivatized with biotin or fluorescent dye Cy5 and then applied for the detection of PIM kinases in biochemical solutions and in complex biological samples. The sandwich assay utilizing a PIM-2-selective detection antibody featured a low limit of quantification (44 pg of active recombinant PIM-2). Fluorescent probes were efficiently taken up by U2OS cells and showed a high extent of co-localization with PIM-1 fused with a fluorescent protein. Overall, the developed inhibitors and derivatives represent versatile chemical tools for studying PIM function in cellular systems in normal and disease physiology.

Haspin is a mitotic protein kinase that is responsible for the phosphorylation of Thr3 of histone H3, thereby creating a recognition motif for docking of the chromosomal passenger complex that is crucial for the progression of cell division. Here, two high-resolution models of haspin with previously reported inhibitors consisting of an ATP analogue and a histone H3(1-7) peptide analogue are presented. The structures of the complexes confirm the bisubstrate character of the inhibitors by revealing the signature binding modes of the moieties targeting the ATP-binding site and the protein substrate-binding site of the kinase. This is the first structural model of a bisubstrate inhibitor targeting haspin. The presented structural data represent a model for the future development of more specific haspin inhibitors.