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The mitotic spindle assembly checkpoint (SAC) is an intricate cell signaling system that ensures the high fidelity and timely segregation of chromosomes during cell division. Mistakes in this process can lead to the loss, gain, or rearrangement of the genetic material. Gross chromosomal aberrations are usually lethal but can cause birth and development defects as well as cancer. Despite advances in the identification of SAC protein components, important details of the interactions underpinning chromosome segregation regulation remain to be established. This review discusses the current understanding of the function, structure, mode of regulation, and dynamics of the assembly and disassembly of SAC subcomplexes, which ultimately safeguard the accurate transmission of a stable genome to descendants. We also discuss how diverse oncoviruses take control of human cell division by exploiting the SAC and the potential of this signaling circuitry as a pool of drug targets to develop effective cancer therapies.

Background: Histone lysine demethylases (KDMs) are of interest as drug targets due to their regulatory roles in chromatin organization and their tight associations with diseases including cancer and mental disorders. The first KDM inhibitors for KDM1 have entered clinical trials, and efforts are ongoing to develop potent, selective and cell-active ‘probe’ molecules for this target class. Robust cellular assays to assess the specific engagement of KDM inhibitors in cells as well as their cellular selectivity are a prerequisite for the development of high-quality inhibitors. Here we describe the use of a high-content cellular immunofluorescence assay as a method for demonstrating target engagement in cells. Results: A panel of assays for the Jumonji C subfamily of KDMs was developed to encompass all major branches of the JmjC phylogenetic tree. These assays compare compound activity against wild-type KDM proteins to a catalytically inactive version of the KDM, in which residues involved in the active-site iron coordination are mutated to inactivate the enzyme activity. These mutants are critical for assessing the specific effect of KDM inhibitors and for revealing indirect effects on histone methylation status. The reported assays make use of ectopically expressed demethylases, and we demonstrate their use to profile several recently identified classes of KDM inhibitors and their structurally matched inactive controls. The generated data correlate well with assay results assessing endogenous KDM inhibition and confirm the selectivity observed in biochemical assays with isolated enzymes. We find that both cellular permeability and competition with 2-oxoglutarate affect the translation of biochemical activity to cellular inhibition. Conclusions: High-content-based immunofluorescence assays have been established for eight KDM members of the 2-oxoglutarate-dependent oxygenases covering all major branches of the JmjC-KDM phylogenetic tree. The usage of both full-length, wi

The enzyme deoxyuridine triphosphate nucleotidohydrolase (dUTPase) is a ubiquitous enzyme that can be considered as the first line of defence against misincorporation of uracil into DNA. Inhibition of dUTPase is lethal in E. coli, S. cervvisiae 'y) and is also likely to be lethal in many other organisms including Plasmodium spp. and Leishmania spp., which are the causative agents of malaria and leishmaniasis respectively. By taking advantage of the differences in the structures of the enzymes in human and in the protozoa, dUTPase was chosen as a chemotherapeutic target. The main aim of this research project was to develop selective inhibitors against leishmanial and plasmodium dUTPases. The research was focused on building a structure-activity relationship study making analogues of previously identified inhibitors within our research group. The work was also supported by molecular modelling studies on the P. falciparum enzyme. The synthesised analogues were nucleosides derivatives of dUMP, which is a proven inhibitor of the leishmanial dUTPase. The different series of compounds included 5'-tritylated nucleosides and acyclic derivatives of dUMP. Three acyclic derivatives showed particular promise against the enzyme with sub-microM Ki values against P. falciparum dUTPase. Some compounds also inhibited in vitro the P. falciparum growth with IC50 values in the microM range. Promising results were obtained also against L. donovani parasite with IC, values in the 10-50 microM range. An antimicrobial screening of compounds previously made in the group was performed by the author against five bacteria strains (E. coli, Ps. aeruginosa, E. faecalis, P. vulgaris and S. aureus) to evaluate the potential application as an antibacterial agent.

The enzyme deoxyuridine triphosphate nucleotidohydrolase (dUTPase) is a ubiquitous enzyme that can be considered as the first line of defence against misincorporation of uracil into DNA. Inhibition of dUTPase is lethal in E. coli, S. cervvisiae 'y) and is also likely to be lethal in many other organisms including Plasmodium spp. and Leishmania spp., which are the causative agents of malaria and leishmaniasis respectively. By taking advantage of the differences in the structures of the enzymes in human and in the protozoa, dUTPase was chosen as a chemotherapeutic target. The main aim of this research project was to develop selective inhibitors against leishmanial and plasmodium dUTPases. The research was focused on building a structure-activity relationship study making analogues of previously identified inhibitors within our research group. The work was also supported by molecular modelling studies on the P. falciparum enzyme. The synthesised analogues were nucleosides derivatives of dUMP, which is a proven inhibitor of the leishmanial dUTPase. The different series of compounds included 5'-tritylated nucleosides and acyclic derivatives of dUMP. Three acyclic derivatives showed particular promise against the enzyme with sub-microM Ki values against P. falciparum dUTPase. Some compounds also inhibited in vitro the P. falciparum growth with IC50 values in the microM range. Promising results were obtained also against L. donovani parasite with IC, values in the 10-50 microM range. An antimicrobial screening of compounds previously made in the group was performed by the author against five bacteria strains (E. coli, Ps. aeruginosa, E. faecalis, P. vulgaris and S. aureus) to evaluate the potential application as an antibacterial agent.