Ansari M. Aleem

Synthetic curcuminoids modulate the arachidonic acid metabolism of human platelet 12-lipoxygenase and reduce sprout formation of human endothelial cells

Platelet 12-lipoxygenase (P-12-LOX) is overexpressed in different types of cancers, including prostate cancer, and the level of expression is correlated with the grade of this cancer. Arachidonic acid is metabolized by 12-LOX to 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], and this biologically active metabolite is involved in prostate cancer progression by modulating cell proliferation in multiple cancer-related pathways inducing angiogenesis and metastasis. Thus, inhibition of P-12-LOX can reduce these two processes. Several lipoxygenase inhibitors are known, including plant and mammalian lipoxygenases, but only a few of them are known inhibitors of P-12-LOX. Curcumin is one of these lipoxygenase inhibitors. Using a homology model of the three-dimensional structure of human P-12-LOX, we did computational docking of synthetic curcuminoids (curcumin derivatives) to identify inhibitors superior to curcumin. Docking of the known inhibitors curcumin and NDGA to P-12-LOX was used to optimize the docking protocol for the system in study. Over 75% of the compounds of interest were successfully docked into the active site of P-12-LOX, many of them sharing similar binding modes. Curcuminoids that did not dock into the active site did not inhibit P-12-LOX. From a set of the curcuminoids that were successfully docked and selected for testing, two were found to inhibit human lipoxygenase better than curcumin. False-positive curcuminoids showed high LogP (theoretical) values, indicating poor water solubility, a possible reason for lack of inhibitory activity or/and nonrealistic binding. Additionally, the curcuminoids inhibiting P-12-LOX were tested for their ability to reduce sprout formation of endothelial cells (in vitro model of angiogenesis). We found that only curcuminoids inhibiting human P-12-LOX and the known inhibitor NDGA reduced sprout formation. Only limited inhibition of sprout formation at ∼IC50 concentrations has been seen. At IC50, a substantial amount of 12-HETE can be produced by lipoxygenase, providing a stimulus for angiogenic sprouting of endothelial cells. Increasing the concentration of lipoxygenase inhibitors above IC50, thus decreasing the concentration of 12(S)-HETE produced, greatly reduced sprout formation for all inhibitors tested. This universal event for all tested lipoxygenase inhibitors suggests that the inhibition of sprout formation was most likely due to the inhibition of human P-12-LOX but not other cancer-related pathways. [Mol Cancer Ther 2006;5(5):1371–82]

Probing Dimerization and Structural Flexibility of Mammalian Lipoxygenases by Small-Angle X-ray Scattering

Human lipoxygenases (LOXs) and their metabolites have a great impact on human homeostasis and are of interest for targeted drug design. This goal requires detailed knowledge of their structures and an understanding of structure-function relationship. At the moment, there are two complete crystal structures for mammalian LOX [rabbit 12/15LOX (r-12/15LOX) and human 5LOX (h-5LOX)] and a fragment of human 12LOX. The low-resolution structures in solution for various LOX isoforms have brought about controversial results. Here we explored the behavior of r-12/15LOX in aqueous solution under different conditions (salt and pH) by small-angle X-ray scattering (SAXS) and compared it with human platelet-type 12S-LOX (hp-12LOX) and h-5LOX. Thermodynamic calculations concerning the stability of molecular assemblies, thermal motion analysis [TLSMD (translation, libration, and screw rotation motion detection based on crystallographic temperature factor B(j))], and results of SAXS analyses brought about the following conclusions: (i) in contrast to its crystal structure, r-12/15LOX functions as a monomer that dominates in solution; (ii) it dimerizes at higher protein concentrations in the presence of salt and with increasing degree of motional freedom of the N-terminal PLAT domain, as suggested by the Y98,614→R double mutant; (iii) in aqueous solutions, hp-12LOX is stable as a dimer, in contrast to h-5LOX and r-12/15LOX, which are monomeric; and (iv) all three mammalian isozymes show a high level of flexibility not only for the PLAT domain but also for other subdomains of the catalytic part in TLS (translation, libration, and screw rotation) analysis and hp-12LOX in SAXS.

Accelerated thrombus lysis in the blood of plasminogen activator inhibitor deficient mice is inhibited by PAI-1 with a very long half-life

Patients with defective plasminogen activator inhibitor protein (PAI-1) or with PAI-1 deficiency can experience hemorrhage as a result of a hyperfibrinolysis. In these patients, a normal thrombus forms, but endogenous lysis is unchecked as tissue plasminogen activator is unopposed. Treatment includes anti-fibrinolytic agents, including oral tranexamic acid. Another treatment option is the administration of PAI-1, but this serpin rapidly inactivates itself. We have developed a mutant plasminogen activator inhibitor with a very long half life (VLHL PAI-1, t1/2>700 h). Here we investigate VLHL PAI-1 effects in the blood of PAI-1 deficient mice, as a model of human disease. Using a thrombelastograph, we found that blood clots of PAI-1 knockout mice were lysed much more quickly than wild type mice. Additionally, blood clots had less shear elastic modulus strength than clots of normal animals. VLHL PAI-1 treatment of PAI-1 deficient mice extended or prevented thrombus lysis and increased clot strength in a concentration dependent fashion. These two parameters determine the extent of thrombus growth and regression; thus, further testing is anticipated to confirm the effectiveness of plasminogen activator inhibitor with a very long half life in an in vivo model and we hope that this protein can be effective in human PAI-1 deficiency disorder.

Do Human Lipoxygenases have a PDZ Regulatory Domain

Human lipoxygenases and products of their catalytic reaction have a well established connection to many human diseases. Despite their importance in inflammation, cancer, cardiorenal and other ailments the drug development is impaired by the lack of structural details to understand their intricate specificity and function in molecular and cellular signaling. The major effort so far has been directed towards understanding the determinants of their specificity and inhibition of their active site with the iron cofactor. Their structure is believed to consist of only two domains: one regulatory - a beta-sandwich, important for membrane binding, and one, mostly helical, catalytic domain. Although recently published cohort studies on single nucleotide polymorphism and occurrence of diseases, SAXS analysis and new biochemical data throw new light on lipoxygenase suggesting symbiosis of regulatory functions with an allosteric mechanism and more flexible structure than anticipated. The goal of this brief review is to direct an attention to the structural features of an anticipated topology and stimulate discussion/research to prove or disapprove our hypothesis that lipoxygenases may possess about approximately 110 amino acids PDZ-like fragments of functional importance. If they do have a second regulatory domain, it might help to explain their association with other molecules, role in signaling pathways and present a new avenue to explore the regulation of their behavior, and thus intervention in the course of diseases.