In silico drug design and molecular docking of novel amidophosphonates and sulfamidophosphonates as inhibitors of urokinase-type plasminogen activator

Cancer is one of the most serious health problems worldwide, affecting individuals from different sexes, ages, and races. However, the most frequent cancer types in the world are lung, prostate, stomach, colorectal, and esophagus in men; and breast, lung, stomach, colorectal and cervical in women. Currently, the search for new active substances used in oral targeted therapies are legitimate and opens up the possibility of an "ambulatory shift" in cancer treatment. In order to design anti-tumor drug candidates endowed with oral bioavailability, we studied trough an in silico approach the oral bioavailability of newly synthesized biomolecules; α-sulfamidophosphonates and α-amidophosphonates as well as their mechanism of action on the new target urokinase-type plasminogen activator (uPA). The studied compounds have been found to meet the five criteria of. Lipinski's rule. The Osiris, Molinspiration and SWISS/ADME calculations related to the compounds (1d, 2a) have shown that these compounds could be good candidates for interacting with the different targets, they have convincing characteristics in relation to the standard drug used. It can be concluded that these compounds are biologically important and possessing molecular properties desirable for being a drug candidate for oral use.The molecular docking results of the studied compounds revealed a good ligand-target interactions, the compounds (1d, 2a) presented a possibility of interacting as an inhibitor of the anticancer target: urokinase-type plasminogen activator (uPA).     

Structure-activity relationships of new N-acylanthranilic acid derivatives as plasminogen activator inhibitor-1 inhibitors

Novel anthranilic acid derivatives having substituted N-acyl side chains were designed and synthesized for evaluation as plasminogen activator inhibitor-1 (PAI-1) inhibitors. Compounds with a 4-diphenylmethyl-1-piperazinyl moiety on the acyl side chains in general exhibited potent in vitro PAI-1 inhibitory activity and good pharmacokinetic profiles after oral administration in rats. Compound 16f (TM5275) was identified as a promising candidate for further pharmacological evaluation.     

Structural basis for selectivity of a small molecule, S1-binding, submicromolar inhibitor of urokinase-type plasminogen activator

BACKGROUND: Urokinase-type plasminogen activator (uPA) is a protease associated with tumor metastasis and invasion. Inhibitors of uPA may have potential as drugs for prostate, breast and other cancers. Therapeutically useful inhibitors must be selective for uPA and not appreciably inhibit the related, and structurally and functionally similar enzyme, tissue-type plasminogen activator (tPA), involved in the vital blood-clotting cascade. RESULTS: We produced mutagenically deglycosylated low molecular weight uPA and determined the crystal structure of its complex with 4-iodobenzo[b]thiophene 2-carboxamidine (K(i) = 0.21 +/- 0.02 microM). To probe the structural determinants of the affinity and selectivity of this inhibitor for uPA we also determined the structures of its trypsin and thrombin complexes, of apo-trypsin, apo-thrombin and apo-factor Xa, and of uPA, trypsin and thrombin bound by compounds that are less effective uPA inhibitors, benzo[b]thiophene-2-carboxamidine, thieno[2,3-b]-pyridine-2-carboxamidine and benzamidine. The K(i) values of each inhibitor toward uPA, tPA, trypsin, tryptase, thrombin and factor Xa were determined and compared. One selectivity determinant of the benzo[b]thiophene-2-carboxamidines for uPA involves a hydrogen bond at the S1 site to Ogamma(Ser190) that is absent in the Ala190 proteases, tPA, thrombin and factor Xa. Other subtle differences in the architecture of the S1 site also influence inhibitor affinity and enzyme-bound structure. CONCLUSIONS: Subtle structural differences in the S1 site of uPA compared with that of related proteases, which result in part from the presence of a serine residue at position 190, account for the selectivity of small thiophene-2-carboxamidines for uPA, and afford a framework for structure-based design of small, potent, selective uPA inhibitors.     

Purification of a hybrid plasminogen activator protein

Recombinant DNA technology has been employed to produce a hybrid gene in which the kringle and serine protease domains of tissue plasminogen activator are linked to the heavy-chain Fd region of a fibrin-specific antibody. The hybrid gene is co-expressed with antibody light chains. This communication describes a purification procedure for the hybrid protein, involving affinity and ion-exchange chromatography. The purified hybrid protein has been used in vivo and in vitro clot lysis experiments and has been shown to be effective at clot dissolution.     

High-performance chromatographic method for the purification of tissue-type plasminogen activator

High-performance affinity chromatography was performed on five ligand-bound columns in an attempt to purify tissue-type plasminogen activator (t-PA), which is a glycoprotein with a high affinity for fibrin and also has two Kringle structures and finger-domain in its molecule. The five columns were concanavalin A-5PW, p-aminobenzamidine-5PW, imidinodiacetic acid-5PW, boric acid-5PW and lysine-5PW. All five were able to rapidly separate t-PA from contaminating proteins, with high resolution and recovery.     

Partial purification and characterization of epidermal plasminogen activator and their inhibitor

Plasminogen activator (PA) and PA inhibitor were partially purified from 2-d-old rat epidermis and characterized in order to elucidate the enzyme-inhibitor interaction in epidermis. PA extracted with buffer containing KSCN was first purified by Blue-Sepharose affinity chromatography. Separation of two PAs, with relative molecular mass (Mr) of 66000 and 44000, was accomplished by Con A-Sepharose column chromatography. The Mr 66000 enzyme had the properties of tissue-type PA (t-PA), while the Mr 44000 enzyme showed those of urokinase-type PA (u-PA) as determined by immunological and fibrin-binding studies. PA inhibitor was extracted in 1,4-piperazinediethanesulphonic acid buffer and purified by (NH4)2SO4 precipitation, gel filtration followed by a Mono Q column in an FPLC system. This inhibitor showed Mr 60000 and inhibited human u-PA activity in a dose- and time-dependent manner, but did not inhibit the activity of t-PA from human and murine melanoma cells or plasmin. It inhibited epidermal PA, Mr 44000, more effectively than it did the Mr 66000 epidermal PA. It was stable at 60 degrees C for 60 min or between pH 5 and 11. This study indicates that both u-PA and t-PA function in normal rat epidermis. On the other hand, an inhibitor which preferentially acts against u-PA exists, but inhibitor to t-PA does not appear to operate under normal epidermal functions.     

Application of in-gel protease assay in a biological sample: characterization and identification of urokinase-type plasminogen activator (uPA) in secreted proteins from a prostate cancer cell line PC-3

A considerable problem in proteomics is to separate and identify functional proteins that participate in specific biological processes. To expedite the analysis of active proteases, we have developed a substrate-specific, sensitive in-gel trypsin activity assay after two-dimensional (2-D) separation in a sodium dodecyl sulphate (SDS)-polyacrylamide gel [22]. Using this method, we detected and characterized Arg-specific protease activity in the secreted protein sample of a prostate cancer cell line, PC-3, in 1-D and 2-D gels. Mass spectrometry (MS) identified the protease as urokinase-type plasminogen activator (uPA). Western blotting using anti-uPA antibody and protease inhibition tests confirmed the identification. Since no antibody was involved in the procedure, the result clearly demonstrates the feasibility of this method for identifying novel proteases in biological samples.     

Regulation of calcium to tissue plasminogen activator secretion in mouse epidermal keratinocytes

The amounts of tissue plasminogen activator (tPA) of culture supernatants, cell surfaces as well as intracellular spaces of the mouse keratinocytes cultured in calcium-free medium or in the presence of low or high calcium concentration medium were tested using enzyme-linked immunosorbent assay (ELISA). The results indicated that cultured keratinocytes could secrete tPA into extracellular space and respond to the addition of high calcium concentration with a time-dependent increase of tPA secretion, and the peak value of tPA secretion appeared after 24 h of keratinocytes culture. Furthermore, comparing the amount of tPA in culture supernatants with that in cell surfaces, it was found that after culture for 24 h, the secreted tPA could bind to the surfaces of the keratinocytes through the lysine sites within its molecule, and this process was enhanced by high calcium concentration. On the basis of these data we assumed that in mouse keratinocytes, tPA firstly secretes into extracellular space and then binds to the keratinocytes surface, and calcium regulates tPA secretion and its following binding to the surface of keratinocytes, which may correlate with the differentiation of keratinocytes.     

A factor from granulosa cells can stimulate oocyte tissue plasminogen activator activity

Several studies indicate that substances synthesized by granulosa cells are capable of regulating oocyte activity. We have studied the effect of factors synthesized by granulosa cells on tPA activity of denuded oocytes using a co-culture system. The results show that an FSH-dependent factor(s) synthesized by granulosa cells (but not by theca-interstitial cells) is capable of stimulating tPA activity of denuded oocytes. This finding is important for understanding hormonal regulation of oocyte tPA activity by mediators synthesized in granulosa cells.     

Venous thrombolysis by tissue-type plasminogen activator in conjunction with the urokinase-fibrinogen covalent conjugate

Conjunctive administration of the tissue-type plasminogen activator (t-PA) and the urokinase-fibrinogen covalent conjugate (UK-Fbg) was studied by the example of venous thrombosis in dogs. Comparing the effect of separate use of the two components, we observed the potentiation of thrombolytic effect induced by an iv bolus infusion administration of the tissue-type plasminogen activator (1 and 4 mg, respectively) combined with a bolus administration 15 min after the first injection of the 25,000 IU UK-Fbg. Faster-action and potentiation effects of thromboysis were observed with the same administration scheme when the t-PA was used as bolus infusion (1 and 1 mg, respectively) combined wiht a bolus of the 250,000 IU fibrinogen-modified urokinase. The findings indicate an approach to the development of efficient thrombolytic compositions.     

Fibrinolytic effect of tissue plasminogen activator on cerebral embolism in stroke-prone spontaneously hypertensive rats.

To study the thrombolytic effect of tissue plasminogen activator (t-PA) on cerebral emboli, we characterized cerebral embolization in stroke-prone spontaneously hypertensive rats (SHRSPs) and Wistar Kyoto rats (WKYs). [125I]Fibrin clot particles (20-100 microns diameter) were injected twice at an interval of 90 min into the left internal carotid artery of WKYs and SHRSPs. After each injection, spontaneous embolus dissolution was monitored with a gamma-ray detector placed on the head of the embolic rats. Embolus dissolution was spontaneously generated in 15 min after the injection of fibrin clots. In WKYs, 21% and 42% of the clots were dissolved 30 and 90 min after the second embolization, respectively. On the other hand, the spontaneous embolus dissolution in SHRSPs was significantly lower than that of WKYs, indicating that the endogenous fibrinolytic ability of SHRSPs is less potent than that of normotensive rats. The intravenous administration of t-PA at doses of 75, 250 and 750 micrograms/kg caused a dose-dependent embolus dissolution in SHRSPs. Furthermore, systematically applied t-PA produced embolus dissolution without causing systemic plasminogen activation, fibrinogen breakdown or bleeding. In conclusion, the intravenous administration of t-PA produces selective embolus dissolution without systemic fibrino(geno)lysis in a cerebral embolic SHRSP.     

Distal hinge of plasminogen activator inhibitor-1 involves its latency transition and specificities toward serine proteases

Background  

The plasminogen activator inhibitor-1 (PAI-1) spontaneously converts from an inhibitory into a latent form. Specificity of PAI-1 is mainly determined by its reactive site (Arg346-Met347), which interacts with serine residue of tissue-type plasminogen activator (tPA) with concomitant formation of SDS-stable complex. Other sites may also play roles in determining the specificity of PAI-1 toward serine proteases.     

Depletion Interactions Produced by Nonadsorbing Charged and Uncharged Spheroids

The effect of macromolecule shape on the depletion attraction between two hard spherical particles in a solution with nonadsorbing hard spheroidal macromolecules of arbitrary size and aspect ratio was investigated using a modified form of the force-balance model of J. Y. Walz and A. Sharma (1994, J. Colloid Interface Sci. 168, 495). The macromolecules were represented as general spheroids, which could be either charged or uncharged. For the uncharged case, a set of analytical expressions describing the depletion attraction, valid for particles much larger than the characteristic macromolecule size, was developed. Comparisons with the case of spherical macromolecules were made under the condition of either constant macromolecule number density, rho(b), or constant volume fraction, phi. It was found that increasing the spheroidal macromolecule aspect ratio (major axis length/minor axis length) decreases the depletion attraction at constant rho(b), but increases the interaction at constant phi. In the latter case, the interaction produced by prolate macromolecules is greater than that produced by oblate macromolecules of equal axis lengths, while the opposite is true at constant rho(b). A simple scaling analysis is used to explain these trends. Surface charge is found to increase both the range and the magnitude of the depletion attraction; however, the general trends are the same as those found in the uncharged systems. Finally, the effect of the depletion attraction produced by spherical and spheroidal macromolecules on the stability of a dispersion of charged particles was examined. It was found that charged spheroids at concentrations of order 1% volume can produce secondary energy wells of sufficient magnitude to induce flocculation in a dispersion of charged spherical particles. Copyright 2000 Academic Press.     

Study of the primary structure of recombinant tissue plasminogen activator by reversed-phase high-performance liquid chromatographic tryptic mapping

Two high-resolution tryptic maps have been developed for recombinant tissue plasminogen activator (rt-PA) that separate the expected 51 tryptic peptides. The trypsin digestion was performed after reduction and S-carboxymethylation of the protein. The high-performance liquid chromatographic separation of the tryptic peptides used a Nova-Pak C18 (5 microns) column with a mobile phase that contained 0.1% aqueous trifluoroacetic acid (TFA) or 50 mM sodium phosphate (pH 2.85) and a linear gradient of acetonitrile. A TFA solvent system was also used for re-purification and for characterization of the peptides isolated from the phosphate-based separation. All of the isolated peptides had compositions consistent with the sequence proposed for rt-PA. The identities of the glycopeptides were confirmed by lectin chromatography on concanavalin A-Sepharose. The mixture of tryptic peptides was also treated with endo-beta-N-acetylglucosaminidase H and peptide:N-glycosidase F to locate the position of either high mannose or complex oligosaccharides. These studies demonstrated that a high mannose oligosaccharide is attached to Asn-117 while complex carbohydrate side-chains are attached to Asn-184 and Asn-448. The residue Asn-184 is the site of optional glycosylation that results in the formation of two rt-PA variants that contain either two or three oligosaccharides.     

Fast quantitation of recombinant plasminogen activator inhibitor type 1 in bacterial lysates by micropellicular reversed-phase liquid chromatography

A rapid reversed-phase HPLC assay is described for quantitating recombinant plasminogen activator inhibitor type 1 (PAI-1) in cultures of Escherichia coli. The assay utilized a short nonporous micropellicular C18 column with an acetonitrile gradient in 0.1% trifluoroacetic acid. The selective resolution of recombinant PAI-1 from major host proteins occurred within 1.3 min. Regeneration of initial chromatography conditions was fast and allowed successive injections every 3 min. The assay's limit of detection for recombinant PAI-1 was 0.5 microg in 5-microl injections of bacterial lysates and the assay was linear to 20 microg. The assay's apparent recovery was between 94 and 108% throughout the quantitative range. In a direct comparison to gel electrophoresis the reversed-phase assay proved superior in monitoring recombinant PAI-1 titers in cultures of E. coli.     

Uncharged Lewis bases yield polydimethylsiloxane ionomers with amidinium alkyldithiocarbamate side chains

Ionomers exhibiting good thermal stability have been prepared by mixing three uncharged components—a poly(dimethyl)siloxane (PDMS) with randomly substituted amidine side chains, an alkylamine, and carbon disulfide. In essence, the three uncharged components produce charged materials, polyelectrolytes with amidinium dithiocarbamate ion pairs. The preparation, structures, and properties of the ionomers at the microscopic and macroscopic levels are reported as a function of PDMS chain length, degree of amidine substitution, and length of the alky group of the amine component. Whereas ionomers with hexyldithiocarbamate anions are viscous liquids at room temperature, those with octadecyldithiocarbamate groups are semi-crystalline solids, exhibiting multiple thermal transitions between 0 °C and the temperature at which they become isotropic phases. The structural natures of those transitions have been characterized from analyses of data from differential scanning calorimetry, X-ray diffraction, and rheology measurements. Among the interesting properties exhibited by the ionomers are strong adhesion to different surfaces, and high ultimate shear stress considering their relatively low amidinium contents.     

非电荷型毛细管电色谱原位柱的色谱行为研究──阴离子表面活性剂的动态改性

 采用原位聚合的方法在毛细管中合成了非电荷型连续床电色谱原位柱 ,通过在电色谱流动相中加入阴离子表面活性剂十二烷基硫酸钠 (SDS)进行动态改性使其产生电渗流 ,考察了SDS浓度及有机改性剂浓度等因素对电渗流的影响。此类连续床柱制备容易 ,柱效可达 14万理论塔板 /m ,在不同的操作条件下有良好的稳定性 ,连续 10次运行 ,其死时间t0 与保留时间的精密度分别为 0 .2 2 %和 <0 .5 6 %。