Kinetics and Ligand-Binding Preferences of Mycobacterium tuberculosis Thymidylate Synthases, ThyA and ThyX

Mycobacterium tuberculosis kills approximately 2 million people each year and presents an urgent need to identify new targets and new antitubercular drugs. Thymidylate synthase (TS) enzymes from other species offer good targets for drug development and the M. tuberculosis genome contains two putative TS enzymes, a conventional ThyA and a flavin-based ThyX. In M. tuberculosis, both TS enzymes have been implicated as essential for growth, either based on drug-resistance studies or genome-wide mutagenesis screens. To facilitate future small molecule inhibitors against these proteins, a detailed enzymatic characterization was necessary.After cloning, overexpression, and purification, the thymidylate-synthesizing ability of ThyA and ThyX gene products were directly confirmed by HPLC analysis of reaction products and substrate saturation kinetics were established. 5-Fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) was a potent inhibitor of both ThyA and ThyX, offering important clues to double-targeting strategies. In contrast, the folate-based 1843U89 was a potent inhibitor of ThyA but not ThyX suggesting that it should be possible to find ThyX-specific antifolates. A turnover-dependent kinetic assay, combined with the active-site titration approach of Ackermann and Potter, revealed that both M. tuberculosis enzymes had very low k(cat) values. One possible explanation for the low catalytic activity of M. tuberculosis ThyX is that its true biological substrates remain to be identified. Alternatively, this slow-growing pathogen, with low demands for TMP, may have evolved to down-regulate TS activities by altering the turnover rate of individual enzyme molecules, perhaps to preserve total protein quantities for other purposes. In many organisms, TS is often used as a part of larger complexes of macromolecules that control replication and DNA repair.Thus, the present enzymatic characterization of ThyA and ThyX from M. tuberculosis provides a framework for future development of cell-active inhibitors and the biological roles of these TS enzymes in M. tuberculosis

Expression of thymidylate synthase in human cells is an early G1 event regulated by CDK4 and p16INK4A but not E2F

Thymidylate synthase (TS) is the enzyme that catalyses the last step in de novo thymidylate synthesis. It is of interest clinically because it is an effective target for drugs such as 5-fluorouracil, often used in combination therapy. Despite a number of earlier reports indicating that TS is a cell cycle-dependent enzyme, this remains equivocal. Here, we show that in HCT116 cells synchronised by serum starvation, there is a clear dissociation between the expression of cyclin E (a well-characterised cell-cycle protein) and TS. Although both cyclin E and TS mRNA and protein increased during G1, TS upregulation was delayed. Moreover, TS levels did not decrease following S-phase completion while cyclin E decreased sharply. Similarly, clear differences were seen between cyclin E and TS as asynchronously growing HCT116 cells were growth-inhibited by low-serum treatment. In contrast to previous reports using rodent cells, adenovirus-mediated over-expression of E2F1 and cyclin E in three human cell lines had no effect on TS. Cell-cycle progression was blocked by treatment of cells with pharmacological inhibitors of CDK2 and CDK4 and by ectopic expression of p16INK4A. Whereas CDK2 inhibition had no effect on TS levels, inhibition of CDK4 was associated with decreased TS protein levels. These results provide the first evidence that drugs targeting CDK4 may be useful with anti-TS drugs as combination therapy for cancer

<i>Trypanosoma brucei</i> DHRF-TS revisited:characterisation of a bifunctional and highly unstable recombinant dihydrofolate reductase-thymidylate synthase

<div><p>Bifunctional dihydrofolate reductase–thymidylate synthase (DHFR-TS) is a chemically and genetically validated target in African trypanosomes, causative agents of sleeping sickness in humans and nagana in cattle. Here we report the kinetic properties and sensitivity of recombinant enzyme to a range of lipophilic and classical antifolate drugs. The purified recombinant enzyme, expressed as a fusion protein with elongation factor Ts (Tsf) in ThyA^- <i>Escherichia coli</i>, retains DHFR activity, but lacks any TS activity. TS activity was found to be extremely unstable (half-life of 28 s) following desalting of clarified bacterial lysates to remove small molecules. Stability could be improved 700-fold by inclusion of dUMP, but not by other pyrimidine or purine (deoxy)-nucleosides or nucleotides. Inclusion of dUMP during purification proved insufficient to prevent inactivation during the purification procedure. Methotrexate and trimetrexate were the most potent inhibitors of DHFR (<i>K</i>_i 0.1 and 0.6 nM, respectively) and FdUMP and nolatrexed of TS (<i>K</i>_i 14 and 39 nM, respectively). All inhibitors showed a marked drop-off in potency of 100- to 1,000-fold against trypanosomes grown in low folate medium lacking thymidine. The most potent inhibitors possessed a terminal glutamate moiety suggesting that transport or subsequent retention by polyglutamylation was important for biological activity. Supplementation of culture medium with folate markedly antagonised the potency of these folate-like inhibitors, as did thymidine in the case of the TS inhibitors raltitrexed and pemetrexed.</p></div

Contesting the financialization of urban space: Community organizations and the struggle to preserve affordable rental housing in New York City

As cities have become both site and object of capital accumulation in a neoliberal political economy, the challenges to community practice aimed at creating, preserving, and improving affordable housing and neighborhoods have grown. Financial markets and actors are increasingly central to the workings of capitalism, transforming the meaning and significance of mortgage capital in local communities and redrawing the relationship between housing and urban inequality. This article addresses the integration of housing and financial markets through the case of "predatory equity," a wave of aggressive private equity investment in New York City's affordable rental sector during the mid-2000s real estate boom. I consider the potential for community organizations to develop innovative, effective, and progressive practices to contest the impact of predatory equity on affordable housing. Highlighting how organizations employed discursive and empirical tactics as well as tactics that reworked the sites, spaces, and structures of finance, this research speaks to the political possibility of contemporary community practice

Developmental changes in growth factors released by the embryonic inner ear

Recent studies have demonstrated a role for neurotrophins in regulating the survival of developing auditory and vestibular neurons. However, the developmental time-course for neurotrophin production and release by inner ear tissues has not been defined. In the present study, neurotrophin-like activity was evaluated from culture medium conditioned by early- or midembryonic stage inner ears. Examination of the proteinaceous properties of conditioned medium revealed a developmental change in growth factor release by the inner ear. Neurotrophin-like molecules were not detected in medium conditioned by early stage otocysts. In contrast, neurotrophin-like bioactivity was detected in medium conditioned by middevelopmental stage inner ears. Western blot analysis revealed that NT-3 was released by the rat inner ear at midstages of inner ear development. ELISA measurements revealed that both NT-3 and BDNF are produced by the middevelopmental stage inner ear, and that NT-3 protein levels are higher than BDNF levels. These results suggest that there are developmental changes in the release of growth factors by the inner ear