Interaction of letrozole and its degradation products with aromatase: chemometric assessment of kinetics and structure-based binding validation

Letrozole is one of the most prescribed drugs for the treatment of breast cancer in post-menopausal women, and it is endowed with selective peripheral aromatase inhibitory activity. The efficacy of this drug is also a consequence of its long-lasting activity, likely due to its metabolic stability. The reactivity of cyano groups in the letrozole structure could, however, lead to chemical derivatives still endowed with residual biological activity. Herein, the chemical degradation process of the drug was studied by coupling multivariate curve resolution and spectrophotometric methodologies in order to assess a detailed kinetic profile. Three main derivatives were identified after drug exposure to different degradation conditions, consisting of acid-base and oxidative environments and stressing light. Molecular docking confirmed the capability of these compounds to accommodate into the active site of the enzyme, suggesting that the sustained inhibitory activity of letrozole may be at least in part attributed to the degradation compounds.     

S-Adenosylmethionine synthetase in bloodstream Trypanosoma brucei

S-adenosylmethionine synthetase was studied from bloodstream forms of Trypanosoma brucei brucei, the agent of African sleeping sickness. Two isoforms of the enzyme were evident from Eadie Hofstee and Hanes-Woolf plots of varying ATP or methionine concentrations. In the range 10–250 μM the K_m for methionine was 20 μM, and this changed to 200 μM for the range 0.5–5.0 mM. In the range 10–250 μM the K_m for ATP was 53 μM, and this changed to 1.75 mM for the range 0.5–5.0 mM. The trypanosome enzyme had a molecular weight of 145 kDa determined by agarose gel filtration. Methionine analogs including selenomethionine, L-2-amino-4-methoxy-cis but-3-enoic acid and ethionine acted as competitive inhibitors of methionine and as weak substrates when tested in the absence of methionine with [¹⁴C]ATP. The enzyme was not inducible in procyclic trypomastigotes in vitro, and the enzyme half-life was > 6 h. T. b. brucei AdoMet synthetase was inhibited by AdoMet (K_i 240 μM). The relative insensitivity of the trypanosome enzyme to control by product inhibition indicates it is markedly different from mammalian isoforms of the enzyme which are highly sensitive to AdoMet. Since trypanosomes treated with the ornithine decarboxylase antagonist DL-α-difluoromethylornithine accumulate AdoMet and dcAdoMet (final concentration ≈ 5 mM), this enzyme may be the critical drug target linking inhibition of polyamine synthesis to disruption of AdoMet metabolism.     

Characterization of the C2 subdomain of yeast mitochondrial initiation factor 2

The COOH-terminal part of the yeast mitochondrial initiation factor 2 (ymIF2), containing the C2 subdomain, was expressed and purified as a histidine-tagged polypeptide of 137 amino acids. Like the recombinant full-length protein, the C2 subdomain binds both formyl-Met-tRNA(f)(Met) and unformylated Met-tRNA(f)(Met) with only a small preference for the former species. Formation of a binary complex between the C2 subdomain or the full-length ymIF2 and initiator tRNA was also assessed by fluorescence measurements. The binding of coumarin-Met-tRNA(f) to either protein caused a blue shift of the coumarin emission spectrum and an increase in anisotropy. Full-length ymIF2 is functionally competent in forming an initiation complex and supporting formation of the first peptide bond on Escherichia coli ribosomes. The results demonstrate that ymIF2 has the same domain structure and biochemical properties of a typical IF2 species as found in bacteria or mammalian mitochondria--but with enhanced ability to bind unformylated initiator Met-tRNA.     

Prosaposin: a new player in cell death prevention of U937 monocytic cells

We report that prosaposin binds to U937 and is active as a protective factor on tumor necrosis factor alpha (TNFalpha)-induced cell death. The prosaposin-derived saposin C binds to U937 cells in a concentration-dependent manner, suggesting that prosaposin behaves similarly. Prosaposin binding induces U937 cell death prevention, reducing both necrosis and apoptosis. This effect was inhibited by mitogen-activated protein ERK kinase (MEK) and sphingosine kinase (SK) inhibitors, indicating that prosaposin prevents cell apoptosis by activation of extracellular signal-regulated kinases (ERKs) and sphingosine kinase. Prosaposin led to rapid ERK phosphorylation in U937 cells as detected by anti-phospho-p44/42 mitogen-activated protein (MAP) kinase and anti-phosphotyrosine reactivity on ERK immunoprecipitates. It was partially prevented by apo B-100 and pertussis toxin (PT), suggesting that both lipoprotein receptor-related protein (LRP) receptor and Go-coupled receptor may play a role in the prosaposin-triggered pathway. Moreover, sphingosine kinase activity was increased by prosaposin treatment as demonstrated by the enhanced intracellular formation of sphingosine-1-phosphate (S-1-P). The observation that the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin prevented the prosaposin effect on cell apoptosis suggests that sphingosine kinase exerts its anti-apoptotic activity by the PI3K-Akt pathway. Thus, cell apoptosis prevention by prosaposin occurs through ERK phosphorylation and sphingosine kinase. The biological effect triggered by prosaposin might be extended to primary cells because it triggers Erk phosphorylation in peripheral blood mononuclear cells (PBMCs). This is the first evidence of a biological effect consequent to a signal transduction pathway triggered by prosaposin in cells of non-neurological origin.     

Prion protein is a component of the multimolecular signaling complex involved in T cell activation

In this study we analyzed the interaction of prion protein PrP^C with components of glycosphingolipid-enriched microdomains in lymphoblastoid T cells. PrP^C was distributed in small clusters on the plasma membrane, as revealed by immunoelectron microscopy. PrP^C is present in microdomains, since it coimmunoprecipitates with GM3 and the raft marker GM1. A strict association between PrP^C and Fyn was revealed by scanning confocal microscopy and coimmunoprecipitation experiments. The phosphorylation protein ZAP-70 was immunoprecipitated by anti-PrP after T cell activation. These results demonstrate that PrP^C interacts with ZAP-70, suggesting that PrP^C is a component of the multimolecular signaling complex within microdomains involved in T cell activation.     

Trophic factor effects on cholinergic innervation in the cerebral cortex of the adult rat brain

The cholinergic pathway ascending from the nucleus basalis magnocellularis (NBM) to the cortex has been implicated in several important higher brain functions such as learning and memory. Following infarction of the frontoparietal cortical area in the rat, a retrograde atrophy of cholinergic cell bodies and fiber networks occurs in the basalocortical cholinergic system. We have observed that neuronal atrophy in the NBM induced by this lesion can be prevented by intracerebroventricular administration of exogenous nerve growth factor (NGF) or the monosialoganglioside GM₁. In addition, these agents can upregulate levels of cortical choline acetyltransferase (ChAT) activity in the remaining cortex adjacent to the lesion site. Furthermore, an enhancement in cortical high-affinity³H-choline uptake and a sustained in vivo release of cortical acetylcholine (ACh) after K⁺ stimulation are also observed after the application of neurotrophic agents. Moreover, these biochemical changes in the cortex are accompanied by an anatomical remodeling of cortical ChAT-immunoreactive fibers and their synaptic boutons.