Lastly haspin inhibitor was assessed against a panel
Lastly, haspin inhibitor was assessed against a panel of 292 kinases at 10μM. At this high concentration, the TNF-alpha, recombinant murine protein inhibited thirteen kinases, in addition to haspin, ⩾90%., These kinases were CaMK2b, CaMK2d, CDK7-CycH-Mat1, cGK2, CK1d, CLK1, CLK2, DYRK1A, DYRK1B, DYRK3, PASK, PIM1 and PKD3. Interestingly, many of these enzymes belong to the CMGC group of kinases, unlike haspin which is a divergent member of the ePK family. In addition, a comparison of the profiles of and suggested that only six kinases, including haspin, were inhibited by both compounds ⩾90% at 10μM (). Profiling of additional haspin kinase inhibitors, such as and , may further reduce the number of kinases, besides haspin, which are known to be potently inhibited by all the compounds. In addition, the collective use of , and potentially other haspin inhibitors in cell based assays may allow for more concrete conclusions to be reached with regard to haspin’s biological functions.
In conclusion, a structure–activity relationship study of the beta-carbolines and , identified utilizing a recently developed HTS assay for haspin kinase inhibitory activity, was performed guided by insights obtained from a previously optimized compound series combined with in silico docking and metadynamic calculations. Increased potency was accomplished by introduction of a tethered primary amine onto the N-position of the beta-carboline. Potency was further increased by replacing the methyl at the 1-position with a trifluoromethyl giving . In addition, this analog demonstrated excellent in vitro metabolic stability in pooled mouse liver microsomes. Kinase profiling of suggested that it was fairly selective and inhibited only six kinases (⩾90% at 10μM), including haspin, in common with the previously identified acridine inhibitor . The beta-carboline haspin inhibitor (LDN-211898) described herein, along with other structurally distinct inhibitors such as – provide valuable molecular probes to study the cellular functions of haspin kinase and may have potential therapeutic utility in treating cancer.
Haspin (ploid Germ Cell-pecific Nuclear rote Kinase), also know as Gsg2 (erm Cell-pecific ene-2), is a serine/threonine kinase expressed in a variety of tissues (e.g., testis, bone narrow, thymus, and spleen) and in proliferating cells. It is also highly expressed in a number of neoplasms, including Burkitt’s lymphoma and diffuse large B cell lymphomas. Haspin’s kinase activity functions during mitosis, where it has been shown to phosphorylate histone H3 at Thr-3 (H3T3)., This phosphorylation begins in G2/early prophase, becomes maximal during prometaphase/metaphase and then diminishes during anaphase., Depletion of haspin by RNA interference significantly reduces H3 Thr-3 phosphorylation in cells and prevents normal completion of mitosis., , Human haspin, consisting of 798-amino acids, contains a C-terminal kinase domain that is a divergent member of the eukaryotic protein kinase (ePK) superfamily., Sequence comparisons and recent crystal structures reveal that haspin contains a number of structural differences from other ePKs, particularly in the C-terminal lobe of the kinase domain. For example, the highly conserved DFG motif involved in ATP binding and the APE motif involved in stabilizing the activation loop in many ePKs are altered. Haspin adopts a constitutively active conformation and conserved serine, threonine and tyrosine residues are absent from its activation loop., DYRKs (ual-specificity Trosine-egulated inases) belong to the CMGC family of ePKs and contain a conserved kinase domain and adjacent N-terminal DYRK homology box. This group of kinases can be further divided into class 1 kinases (DYRK1A and 1B) that have an N-terminal nuclear localization signal and a C-terminal PEST region and class 2 kinases (DYRK2, 3, and 4), which lack these motifs and are predominantly cytosolic. Although DYRKs phosphorylate substrates on serine or threonine residues, their activity depends upon autophosphorylation of an essential activation loop tyrosine during synthesis. DYRKs appear to contribute to regulation of an array of signaling pathways, including NFAT signaling in the brain and immune system, Hedgehog signaling, caspase activity during apoptosis, cell cycle progression and mitosis, and p53 activation in response to DNA damage., , ,