Anti-DYRK1A monoclonal antibody, clone 8E20

Introduction: Congenital cytomegalovirus (HCMV) infection may cause significant fetal malformation and in severe cases fetal and neonatal death. Fetal injury may be caused indirectly by the placental response to infection. Dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) have recently been identified as critical kinases for HCMV replication. In this study we provide first evidence that DYRK1A and DYRK1B are utilised during HCMV placental replication. Methods: DYRK expression was investigated in AD169-and Merlin-infected TEV-1 trophoblast cells, ex vivo placental explants and naturally infected clinical placentae by immunofluorescence, western blot, co-immunoprecipitation and RT-qPCR. Results: HCMV-infected placental cells showed accumulation and re-localisation of DYRK1A and DYRK1B protein to areas of cytoplasmic virion assembly complexes and nuclear viral replication compartments, respectively. This accumulation was a result of upregulated DYRK1A/B protein expression with HCMV inducing up to a 5.3fold increase in DYRK1A and up to a 4.7-fold increase in DYRK1B protein, relative to mock-infected TEV-1 cells (p < 0.0001). Increased DYRK protein expression was correlated with DYRK1A/B mRNA upregulation, with HCMV-infected cells showing up to a 3.7-fold increase and 2.9-fold increase in DYRK1A and DYRK1B mRNA levels respectively (p < 0.05). Protein-protein interactions were detected between DYRK1A/1B complexes and HCMV immediate early IE2p86, early pp65 and pUL44 and late pp150 proteins. Treatment of HCMV-infected TEV-1 cells and placental explants with DYRK inhibitors significantly inhibited HCMV replication (p < 0.05) indicating these cellular kinases are required during HCMV placental replication. Conclusion: HCMV modulates cellular DYRKs during placental replication which may have implications for congenital HCMV pathogenesis and represent promising antiviral targets.

The discovery of potent kinase inhibitors with tunable selectivity and specificity has gained significant impetus in the last few decades. In accordance with this development, herein, we report the design and synthesis of a series of new heterocyclic derivatives comprising a 1,2,4 triazole nucleus, tethered to two flexible linkers on either side, utilizing a simple Schiff base approach (compounds 1-16). The compounds were characterised by various spectroscopic techniques and their biological evaluation was conducted using kinase inhibition assay on two targets, namely CLK1 and DYRK1A. Our investigation reveals that compounds 3-5 bearing hydroxy/methoxy substituents in the aromatic ring display excellent potency towards the receptors in the micromolar range. Indeed, compound 5 bearing a methoxy substitution at the meta position possesses the added advantage of being highly specific towards CLK1 among the targets. To reinforce this observation, we performed molecular modelling studies with the kinase inhibitors hymenialdisine (HMD) cocrystallized with CLK1 and harmine (HA) cocrystallized with DYRK1A kinases as references. We speculate that in either target, the cause of such high potency and selectivity might be attributed to the involvement of flexible linkers on either side of the triazole core, which might have compelled the ligands to enter into the active site of the receptor and maximize non-covalent interactions. Interestingly, we consider the selectivity of compound 5 to be due to the involvement of a H-bond between SH of triazole and Glu 169 carbonyl in the receptor, which is absent in the other derivatives for both targets. Thus, our efforts focus on how the variation/position of substituents in the inhibitors has a profound impact on potency and selectivity. Indeed, this work establishes innovative design principles towards the development of a therapeutic agent for the treatment of neurodegenerative diseases like that of Alzheimers.