Human lipoxygenase: developments in its structure, function, relevance to diseases and challenges in drug development.

Human lipoxygenases (LOXs) are the enzymes participating in the metabolism of the polyunsaturated fatty acids and catalyzing their oxidation to a variety of eicosanoids, which as the secondary signal transducers have a major impact on human homeostasis. They are involved in many diseases such as inflammatory responses, cancers, cardiovascular and kidney diseases, neurodegenerative disorders and metabolic syndrome. This review summarizes recent developments concerning human 12S-LOX and rabbit 15-LOX projected upon available structural data of LOX and COX oxidoreductases, with conclusions that might apply to LOX family of enzymes in general. Namely: (i) Human lipoxygenases might act as oligomers consisting of active and apo monomers. (ii) Sequential homodimers might act as structural heterodimers with the dimeric interface formed by the interactions resembling the leucine zipper in the coiled-coil superstructure. (iii) Two commonly recognized domains are not sufficient to explain LOX flexibility. Molecular architecture should contain assignment of another regulatory domain of alpha-beta character, possibly important in molecular signaling, which might provide another avenue for targeted drug development. (iv) Allosteric mechanism might involve orchestrated conformational changes and flexibility of the coils connecting the structured elements and ligands binding in more than one monomer.

Val17Ile single nucleotide polymorphisms similarly as Ala15Thr could be related to the lower secretory dynamics of PAI-1 secretion: theoretical evidence.

Plasminogen activator inhibitor (PAI-1) is a fast acting inhibitor of tissue and urokinase plasminogen activators (tPA and uPA). In that way PAI-1 regulates proteolytic activity of many physiological and pathological processes [1-3]. PAI-1 plays an important role in blood coagulation controlling clot lysis which is triggered by tPA activated plasminogen [4]. Only two types of mutations are reported to be associated with PAI-1; one is the frame-shift mutation in exon 4 of PAI-1 gene resulting in a truncated nonfunctional protein and in complete PAI-1 deficiency. The other SNP causes Ala15Thr mutation in the signal peptide. A literature search revealed five variants of polymorphisms during a study of over one thousand individuals. Two are associated with thrombophilia (765 4G/5G and -844 A>G, in the promoter), risk of myocardial infarction and postoperative deep venous thrombosis related to higher than normal levels of PAI-1. The other SNPs associated with PAI-1 deficiency are Ala15Thr, Val17Ile and they are located in the central hydrophobic core of the PAI-1 signal peptide of PAI-1 and Asn195Ile in the 'A' ß sheet of the PAI-1. We have analyzed two SNPs not reported to be associated with PAI-1 deficiency. Our analysis suggests that Val17Ile PAI-1 variant might cause slower PAI-1 secretion leading to the deficiency at time and place where it is needed in a similar way as for Ala15Thr SNP. The Asn195Ile mutant may be more stable only as latent form thus no PAI-1 deficiency is expected in this mutant.

Eicosanoids in prevention and management of diseases.

Eicosanoids, which originate from polyunsaturated fatty acids (PUFAs), have a major impact on homeostasis maintenance as secondary signal transducers. Signal cascade, which includes reception, processing and signal transduction coming from the environment into the cell, determines the type of response evoked. Signal distortion may take place on every level of this cascade and this in consequence could lead to the development of many diseases. Any intervention into PUFAs metabolism leads to quantitative and qualitative changes of synthesized eicosanoids. Some of them promote, whereas others inhibit carcinogenesis, some are pro- or anti-inflammatory and the overall result depends on the outcome of these contradictory effects. The type and amount of produced eicosanoids depends on substrates' availability and activity of enzymes catalyzing different stages of their transformation. A particularly negative role was assigned to the over expression of phospholipase A2, cyclooxygenase-2, 5- and 12-lipoxygenases, while the contribution of other oxygenases and their metabolites is considerably less clear. The information about their interplay is extremely sparse and inadequate to understand intricacies of the mechanisms involved. There are indications that utilization of selected eicosanoids (their analogs, agonists or antagonists) could be a better way of disease prevention and treatment, more effective than excessive dietary supplementation of fatty acids. This review presents a more global picture of oxygenases and their PUFA metabolites giving a brief summary of our current understanding of perspectives and pitfalls of their regulation and mediatory action in human diseases.

Effect of crystal freezing and small-molecule binding on internal cavity size in a large protein: X-ray and docking studies of lipoxygenase at ambient and low temperature at 2.0 A resolution.

Flash-freezing is a technique that is commonly used nowadays to collect diffraction data for X-ray structural analysis. It can affect both the crystal and molecular structure and the molecule's surface, as well as the internal cavities. X-ray structural data often serve as a template for the protein receptor in docking calculations. Thus, the size and shape of the binding site determines which small molecules could be found as potential ligands in silico, especially during high-throughput rigid docking. Data were analyzed for wild soybean lipoxygenase-3 (MW 97 kDa) at 293 and 93 K and compared with the results from studies of its molecular complexes with known inhibitors, structures published by others for a derivative of the same enzyme (98 K) or a topologically close isozyme lipoxygenase-1 (at ambient temperature and 100 K). Analysis of these data allows the following conclusions. (i) Very small changes in the relative orientation of the molecules in the crystal can cause major changes in the crystal reciprocal lattice. (ii) The volume of the internal cavities can ;shrink' by several percent upon freezing even when the unit-cell and the protein molecular volume show changes of only 1-2%. (iii) Using a receptor structure determined based on cryogenic data as a target for computational screening requires flexible docking to enable the expansion of the binding-site cavity and sampling of the alternative conformations of the crucial residues.

Aspirin blocks binding of photosensitizer SnET2 into human serum albumin: implications for photodynamic therapy.

Tin etiopurpurin dichloride (SnET2) is one of the photosensitizers under investigation to be used in photodynamic therapy of prostate cancer. The drug is delivered intravenously, transported in vivo by liposomes and plasma proteins and localized within the prostate. SnET2 exists in two tautomeric forms (I - closed ring, II - open ring) with I converting spontaneously into the more energetically stable form II at physiological pH. Up to approximately 50% of the drug can be carried by serum albumin, although this association can increase photo-bleaching and diminish the drug efficiency. Molecular modeling and force field calculations indicate that Sudlow Site I in human serum albumin (HSA) is the most probable binding site for both forms of SnET2, with the porphyrin moiety nestling between domains IIA and IB, and the esterolytic side group oriented toward domain IIIA of HSA. Other drugs, including aspirin, bind to the same part of HSA. SnET2 does not bind to HSA when pre-incubated with aspirin, which confirms that its place of binding to this protein must be located near Lys199. This observation could be exploited to improve photo-efficiency of SnET2 by finding drugs that could compete with the photosensitizer for binding into Sudlow Site I of HSA.

Plasminogen activator inhibitor type-1 controls the process of the in vitro sprout formation.

Plasminogen activator inhibitor type-1 (PAI-1), the primary regulator of plasminogen activator - urokinase (uPA) plays a crucial role in the cell adhesion and migration and in angiogenesis. We had previously demonstrated that PAI-1 - endothelial cell interplay is critical for the formation of new blood vessels and the process is mostly conducted via uPA- anti-proteinase interaction. In the present study we wished to further examine the role of PAI-1 in the sprout formation, representing the first step of new capillary vessels development by evaluating the effect of PAI-1 on the sprout area. We addressed the issue by assessing the influence of cysteine-mutated PAI-1 proteins characterized by a prolonged half-life time (hD beta T - T(1/2)= 63.59 h and beta T-T(1/2)= 6931.47 h), and therefore more stable anti-uPA activity, on the appearance of newly formed sprouts. We found that both CysPAI-1 proteins significantly diminished the mean sprout area in a concentration-dependent fashion. The inhibitory effect present in the two examined endothelial cells systems of different origin and functional characteristics - human umbilical vein endothelial cells (HUVEC) and human lung microvascular endothelial cells (HLMVEC) cultures - was noticeably greater for HLMVEC -high urokinase-producers. Moreover, the inhibition rate was significantly greater for the beta T mutant than that for the hD beta T PAI-1 mutant in all examined doses (P<0.002), proving a key role of anti-proteinase activity for this effect. We concluded that, apart from the total sprout length, as it had repeatedly been demonstrated before, also affects the sprout area in the in vitro sprout formation angiogenesis assay. This effect was achieved mainly via PAI-1's antiproteinase activity.

Three-dimensional structure of a purple lipoxygenase.

Polyunsaturated fatty acid metabolism is governed primarily by two enzymes, prostaglandin H synthase and lipoxygenase. The crystal structure of the metastable product-oxidized purple form of soybean lipoxygenase-3 was determined at 2.0 A resolution. The data reveal that the chromophore corresponds to an iron-peroxide complex, a potential intermediate in the catalyzed reaction. A significant alteration of the iron site accompanies the formation of the complex. The structure, the first for a fatty acid-lipoxygenase complex, also reveals an unexpected mode of binding, and identifies amino acid residues that may play significant roles in catalysis, regio- and stereoselectivity.

Binding site of amiloride to urokinase plasminogen activator depends on species.

A novel drug candidate is checked on its potency on animal models before it can advance to human phase of the research. Usually negative results on animal phase disqualify it. Targeting specific enzymes by small chemicals raises the question about the appropriateness of this approach. As an example, the urokinase (uPA) is recognized as an important enzyme responsible for cancer metastasis and angiogenesis. It is therefore important to ask the question if a small chemical will inhibit uPA of different species with the same or different potency. Using DNA sequence and known structure of uPA we have modeled 3D structures of uPAs for several different species. By theoretical calculations we have determined most probable structure of amiloride/uPAs complexes. Catalytic triad (B57, B102, B195) and specificity pocket (B187-B197, B212-B229) are highly conserved in all cases, and are the regions responsible for proteolytic activity and recognition of the substrate. Significant differences were observed in a different region (loop B93-B101), that we identified as binding site of amiloride to the tissue plasminogen activator (tPA). Although tPA shares the same function of activating plasminogen and it is structurally similar to uPA. Amiloride is a specific inhibitor of uPA but does not inhibit tPA. Our study shows that predicted position of amiloride depends on species and in some cases was located, as expected, in the specificity pocket, but in the other cases close to the loop B93-B101. This location could weaken affinity of binding or prevent inhibition of uPA. Therefore, drug screening and elimination process based solely on animal study, without careful structural analysis, could lead to the elimination of potential drugs for humans.

Recombinant PAI-1 inhibits angiogenesis and reduces size of LNCaP prostate cancer xenografts in SCID mice.

To understand the fundamental determinants of urokinase plasminogen activator (uPA) driven angiogenesis in cancer we studied how inhibition of uPA activity could reduce neovascularization and consequently reduce tumor size in experimental animals. Proteolytic enzymes are required to mediate tumor cell invasion to adjacent tissues and initiate the metastatic process. Many different human cancers commonly overexpress the urokinase plasminogen activator system, one of the proteolytic enzyme systems. Reduction of urokinase activity in cancer cells is evidently associated with diminished invasion and metastasis. However, it has been shown recently that inhibitors of uPA could reduce tumor size also. The mechanism of action leading to decline in tumor growth rate is not clear. Proteolysis is responsible for degradation of proteins, for invasion or metastasis, but not for the proliferate properties of the cancer cells. It is difficult to envision that diminishing the size of tumor is due to simply blocking of uPA activity of cancer cells. Instead, inhibitors of uPA may be interacting with the elements of the extracellular matrix, such the neovascular bed surrounding tumors that has been reported to contain high amounts of uPA and its receptor. Overall these data strongly suggest that inhibitors of urokinase limit cancer growth by inhibiting angiogenesis. However, it is possible also that uPA inhibitors could act on cancer cells directly or prevent angiogenesis by alternative mechanisms that are not related to uPA inhibition. Therefore, we examined if plasminogen activator inhibitor (PAI-1) could limit angiogenesis. If it does, it will provide definitive evidence of uPA/PAI-1 involvement in reduction of cancer growth. Indeed, our study demonstrates that exogenously applied 14-1b PAI-1 is a powerful inhibitor of angiogenesis in three different in vitro models and is a powerful anti-cancer agent in a SCID mice model inoculated with human LNCaP prostate cancer cells.

Two-dimensional molecular layers: interplay of H-bonding and van der waals interactions in the self-assembly of N,N'-dialkylsulfamides

N,N'-Dialkylsulfamide molecules assemble into solid-state structures consisting of 2D layers. The 2D layers are based on a hydrogen-bonded network of the sulfamide groups and the close-packing of alkyl groups on both sides of the 2D H-bonded network. The thickness of a 2D layer is proportional to the size of the alkyl substituents. The interplay of H-bonding and van der Waals interactions leads to stable 2D layers that pack into 3D structures.

Curcumin inhibits lipoxygenase by binding to its central cavity: theoretical and X-ray evidence.

Many lipoxygenase inhibitors including curcumin are currently being studied for their anti-carcinogenic properties. Curcumin is a naturally occurring polyphenolic phytochemical isolated from the powdered rhizome of the plant Curcuma longa that possesses anti-inflammatory properties and inhibits cancer formation in mice. Recently it was shown that the soybean lipoxygenase L1 catalyzed the oxygenation of curcumin and that curcumin can act as a lipoxygenase substrate. In the current study, we investigated the fate of curcumin when used as a soybean lipoxygenase L3 substrate. By use of X-ray diffraction and mass spectrometry, we found an unoccupied electron mass that appears to be an unusual degradation product of curcumin (4-hydroxyperoxy-2-methoxyphenol) located near the soybean L3 catalytic site. Understanding how curcumin inhibits lipoxygenase may help in the development of novel anti-cancer drugs used for treatment where lipoxygenases are involved.

Synthesis and characterization of the dimethyl-substituted bisbenzimidazole ligand and its manganese complex.

Macrocycles with unique properties provide new avenues for the design of novel catalysts and materials. Here, we report, for the first time, the synthesis and characterization of the dimethyl-substituted bisbenzimidazole ligand (Me2BBZ) and its manganese complex (Mn-Me2BBZ). The Me2BBZ ligand is similar to porphyrin and phthalocyanine macrocycles in terms of its cavity size and metal-binding mode, but owing to electronic and charge differences, it exhibits properties that make it distinct from its structural counterparts. For instance, the optical spectra of bisbenzimidazoles lack transitions in the 500-900 nm region. Perhaps the most significant feature of the Me2BBZ ligand, however, is its inherent nonplanarity. Geometric restraints within this nonplanar ligand give rise to two atropisomers, which, when separated, could have potential in chiral catalysis and recognition. In addition, here we show that this nonplanarity can help to promote unusual crystal-packing interactions. Within the structure of the Mn-Me2BBZ complex, intermolecular pi-stacking interactions of the phenyl and benzimidazole groups lead to the formation of a distinct two-dimensional "staircase" lattice comprised of alternating Mn-Me2BBZ atropisomers. The potential significance of this structural arrangement is revealed by temperature-dependent magnetic studies that indicate weak antiferromagnetic coupling between the metal ions in the crystal. Fine-tuning of these long-range electronic and magnetic interactions could be useful for the design of novel molecular materials.

Angiostatic activity of synthetic inhibitors of urokinase type plasminogen activator.

We hypothesize that tumor angiogenesis can be limited by the reduction of enzymatic activity of the urokinase type plasminogen activator. The proposed mechanism is elimination of proteolytic activity by the advancing tip of capillaries which utilize proteolysis to produce space needed for vessel expansion. To test our hypothesis, we have investigated the angiostatic activity of synthetic low molecular weight inhibitors of urokinase: amiloride, benzamidine, EGCG, B428, and B623 using the chicken embryo corioallantoic membrane (CAM) model. We found that all tested inhibitors of urokinase cause a significant reduction of angiogenesis.

Molecular basis of specific inhibition of urokinase plasminogen activator by amiloride.

The urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) are very similar serine proteases with the same physiological function, the activation of plasminogen. An increased amount or activity of uPA but not tPA has been detected in human cancers. The PAs are weak proteolytic enzymes, but they activate plasminogen to plasmin, a strong proteolytic enzyme largely responsible for the malignant properties of cancers. It has been shown recently that the administration of uPA inhibitors can reduce tumor size. Inhibitors of uPA could therefore be used as anti-cancer and anti-angiogenesis agents. It has been found that amiloride competitively inhibits the catalytic activity of uPA but not tPA. Modification of this chemical could therefore produce a new class of uPA specific inhibitors and a new class of anti-cancer agents. The X-ray structure of the uPA complex with amiloride is not known. There are structural differences in the specificity pocket of uPA and tPA. However, the potential energy of binding amiloride is lower outside this cavity in the case of tPA. A region responsible for binding amiloride to tPA has been proposed as the loop B93-B101, reached in negatively charged amino acids present in tPA but not uPA.

Structural and thermochemical characterization of lipoxygenase-catechol complexes.

A complex between native, iron(II) soybean lipoxygenase 3 and 4-nitrocatechol, a known inhibitor of the enzyme, has been detected by isothermal titration calorimetry and characterized by X-ray crystallography. The compound moors in the central cavity of the protein close to the essential iron atom, but not in a bonding arrangement with it. The iron ligands experience a significant rearrangement upon formation of the complex relative to their positions in the native enzyme; a water molecule becomes bound to iron in the complex, and one histidine ligand moves away from the iron to become involved in a hydrogen bonding interaction with the catechol. These changes in position result in a trigonal pyramid coordination geometry for iron in the complex. Molecular modeling and force field calculations predict more than one stable complex between 4-nitrocatechol and the central cavity of lipoxygenase 3, but the interaction having the small molecule in the same orientation as the one found in the crystal structure was the most favorable. These observations reveal specific details of the interaction between lipoxygenase and a small molecule and raise the possibility that changes in the ligand environment of the iron atom could be a feature of the product activation reaction or the catalytic mechanism.

Structure of soybean lipoxygenase L3 and a comparison with its L1 isoenzyme.

Soybean lipoxygenase isoenzyme L3 represents a second example (after L1) of the X-ray structure (R = 17% at 2.6 A resolution) for a member of the large family of lipoxygenases. L1 and L3 have different characteristics in catalysis, although they share 72% sequence identity (the changes impact 255 amino acids) and similar folding (average C alpha rms deviation of 1 A). The critical nonheme iron site has the same features as for L1:3O and 3N in pseudo C3v orientation, with two oxygen atoms (from Asn713 and water) at a nonbinding distance. Asn713 and His518 are strategically located at the junction of three cavities connecting the iron site with the molecule surface. The most visible differences between L1 and L3 isoenzymes occur in and near these cavities, affecting their accessibility and volume. Among the L1/L3 substitutions Glu256/ Thr274, Tyr409/His429, and Ser747/Asp766 affect the salt bridges (L1: Glu256...His248 and Asp490...Arg707) that in L1 restrict the access to the iron site from two opposite directions. The L3 molecule has a passage going through the whole length of the helical domain, starting at the interface with the Nt-domain (near 25-27 and 254-278) and going to the opposite end of the Ct-domain (near 367, 749). The substrate binding and the role of His513, His266, His776 (and other residues nearby) are illustrated and discussed by using models of linoleic acid binding. These hypotheses provide a possible explanation for a stringent stereo-specificity of catalytic products in L1 (that produces predominantly 13-hydroperoxide) versus the lack of such specificity in L3 (that turns out a mixture of 9- and 13-hydroperoxides and their diastereoisomers).

Inhibitors of urokinase reduce size of prostate cancer xenografts in severe combined immunodeficient mice.

Proteolytic enzymes are required to mediate tumor cell invasion and metastasis. The urokinase plasminogen activator (uPA) is commonly overexpressed by many human cancers. Therefore, uPA is a logical target to inhibit cancer invasion and metastasis. However, uPA inhibitors also reduce tumor growth. We used a mutated form of plasminogen activator inhibitor type 1 to conform a correlation between the inactivation of uPA and tumor size; we have compared these results with the action of p-aminobenzamidine and amiloride, known inhibitors of uPA. Our results show that blocking uPA by uPA inhibitors reduces tumor size in experimental animals. Our molecular simulation of docking inhibitors to the urokinase reveals that all tested small molecule inhibitors bind in proximity of uPA's specificity pocket, a critical site for future search of novel anticancer uPA inhibitors.

Flash-freezing causes a stress-induced modulation in a crystal structure of soybean lipoxygenase l3.

A dynamic conformational flexibility of a protein might be a source of non-covalent structural heterogeneity, causing diminished diffracting ability of crystals and disorder in a crystal structure of soybean lipoxygenase L3. Room-temperature data, space group C2, correspond to a structure with large channels lined mostly or in part by disordered fragments of the molecule or flexible loops with an increased thermal vibration. A rapid change in temperature of approximately 200 K creates a wave of a stress-induced modulation that propagates in the crystal changing its reciprocal space into a three-dimensional quilt-like mixture of C and P intertwined lattices. Low-temperature data indicate a transformation from the dynamic to static disorder, leading to a primitive unit cell with 10% reduced volume. The molecules, formerly related by a twofold axis are rotated by approximately 7 degrees and are shifted along the diagonal to be approximately 4 A, closer together. During a routine data collection for the flash-frozen crystals of similar properties such phenomena could easily go unnoticed leading to biased results because of such effects and possibly improper indexing of the data.