Unconventional Secondary Structure Mimics: Ladder‐Rungs

Secondary structures tend to be recognizable because they have repeating structural motifs, but mimicry of these does not have to follow such well‐defined patterns. Bioinformatics studies to match side‐chain orientations of a novel hydantoin triazole chemotype ( 1 ) to protein‐protein interfaces revealed it tends to align well across parallel and antiparallel sheets, like rungs on a ladder. One set of these overlays was observed for the protein‐protein interaction uPA?uPAR. Consequently, chemotype 1 was made with appropriate side‐chains to mimic uPA at this interface. Biophysical assays indicate these compounds did in fact bind uPAR, and elicit cellular responses that affected invasion, migration, and wound healing.     

Differential inhibition of endothelial cell proliferation and migration by urokinase subdomains: amino-terminal fragment and kringle domain

The serine protease urokinase-type plasminogen activator (uPA) is implicated in pericellular proteolysis in a variety of physiological and pathological processes including angiogenesis and tumor metastasis. The kringle domain of uPA (UK1) has proven to be an anti-angiogenic molecule with unknown mechanism and amino terminal fragment of uPA (u-ATF) with additional growth factor-like domain can be used for blocking interaction of uPA and uPA receptor. Here, we compared anti-angiogenic activities of these two molecules in vitro and in vivo. The recombinant u-ATF from E. coli and refolded in vitro was found to bind to uPAR with high affinity, whereas E. coli-derived UK1 showed no binding by Biacore analysis. In contrast to UK1 having potent inhibitory effect, u-ATF exhibited low inhibitory effect on bovine capillary endothelial cell growth (ED(50)>320 nM). Furthermore, u-ATF inhibition of VEGF-induced migration of human umbilical vein endothelial cell was far less sensitive (IC(50) = 600 nM) than those observed with UK1, and angiogenesis inhibition was marginal in chorioallantoic membrane. These results suggest that kringle domain alone is sufficient for potent anti- angiogenic activity and additional growth factor-like domain diverts this molecule in undergoing different mechanism such as inhibition of uPA/uPAR interaction rather than undergoing distinct anti- angiogenic mechanism driven by kringle domain.     

Involvement of MAPK pathway in hypoxia-induced up-regulation of urokinase plasminogen activator receptor in a human prostatic cancer cell line, PC3MLN4

Clinical studies have shown that tumor hypoxia is associated with invasive growth and metastasis and may be an important prognostic factor adversely influencing survival in patients with tumors. To investigate the mechanisms involved in hypoxia-induced invasive growth and metastasis, hypoxia-mediated urokinase plasmalogen activator receptor (uPAR) expression, cellular invasiveness, and mitogen activated protein kinase (MAPK) activation were measured in a prostate cancer cell line, PC3MLN4. The levels of uPAR expression and cellular invasiveness were increased in hypoxic cells. Hypoxia-induced cellular invasiveness was blocked by an anti-uPAR monoclonal antibody. Phosphorylations of ERK and p38 kinases were also more extensive in hypoxic cells than in normoxic cells. Hypoxia-induced uPAR up-regulation was inhibited by pre-treatments with a specific inhibitor of MEK, PD98059 and a specific inhibitor of p38 MAP kinase, SB203580. Cell growth also increased in hypoxic cells. From these results, hypoxia increased tumor cell invasion by up-regulating uPAR expression, which might be mediated through ERK and p38 kinase signaling pathways in PC3MLN4 prostate cancer cell line.     

Streamlined Protein-Protein Interface Loop Mimicry

Cyclic peptides comprising endocyclic organic fragments, “cyclo-organopeptides”, can be probes for perturbing protein-protein interactions (PPIs). Finding loop mimics is difficult because of high conformational variability amongst targets. Backbone Matching (BM), introduced here, helps solve this problem in the illustrative cases by facilitating efficient evaluation of virtual cyclo-organopeptide core-structure libraries. Thus, 86 rigid organic fragments were selected to build a library of 602 cyclo-organopeptides comprising Ala and organic parts: “cyclo-{-(Ala)_n-organo-}”. The central hypothesis is “hit” library members have accessible low energy conformers corresponding to backbone structures of target protein loops, while library members which cannot attain this conformation are probably unworthy of further evaluation. BM thereby prioritizes candidate loop mimics, so that less than 10 cyclo-organopeptides are needed to be prepared to find leads for two illustrative PPIs: iNOS ⋅ SPSB2, and uPA ⋅ uPAR.     

Crystal Structures of Urokinase-type Plasminogen Activator in Complex with 4-（Aminomethyl） Benzoic Acid and 4-（Aminomethyl-phenyl）-methanol

Urokinase-type plasminogen activator （uPA） is a trypsin-like serine protease and plays a key role in several biological processes, including tissue remodeling, cell migration, and matrix degradation. The inhibitors of uPA have been shown to prevent the spread of metastasis and tumor growth, and accordingly uPA is widely recognized as a target for the treatment of cancer. In this work, we report the crystal structures of the complexes of uPA with its inhibitors： 4- （aminomethyl）-benzoic acid （AMBA） and 4-（aminomethyl-phenyl）-methanol （AMPM）, both at a resolution of 2.35 А. The inhibitory constants of these two inhibitors were measured by a chromogenic competitive assay, and it was found that AMBA is a better inhibitor for uPA （Ki = 2.68 mM） than AMPM （Ki = 13.99 mM）. The structural study shows that the binding mode of inhibitor AMBA on uPA is similar to that of AMPM on uPA, both docked into the active site S1 pocket of uPA. Structural details of these complexes are provided to explain the difference of inhibitory constants.     

Crystal Structures of 2-Aminobenzothiazole-based Inhibitors in Complexes with Urokinase-type Plasminogen Activator

Urokinase-type plasminogen activator （uPA） plays a crucial role in the regulation of plasminogen activation, tumor cell adhesion and migration. The inhibition of uPA activity is a promising mechanism for anti-cancer therapy. Most current uPA inhibitors employ a highly basic group （either amidine or guanidine group） to target the S1 pocket of uPA active site, which leads to poor oral bioavailability. Here we study the possibility of using less basic 2-aminobenzothiazole （ABT） as S1 pocket binding group. We report the crystal structures of uPA complexes with ABT or 2-amino-benzothiazole-6-carboxylic acid ethyl ester （ABTCE）. The inhibitory constants of these two inhibitors were measured by a chromogenic competitive assay, and it was found that ABTCE is a better inhibitor for uPA （Ki = 656 μM） than ABT （Ki = 5.03 mM）. This work shows that 2-amniobenzothiazole can be used as P1 group which may have better oral bioavailability than the commonly used amidine or guanidine group. We also found the ethyl ester group occupies the characteristic oxyanion hole and contacts to uPA 37- and 60-loops. Such work provides structural information for further improvements of potency and selectivity of this new class of uPA inhibitor.     

Urokinase activity in corneal fibroblasts may be modulated by DNA damage and secreted proteins

Proteases like urokinase-type plasminogen activator (uPA) play an important role in tumor invasion. Cells derived from ultraviolet radiation (UVR)-induced corneal sarcomas of Monodelphis domestica produce relatively high levels of uPA compared to the untransformed keratocytes suggesting a mechanism for their invasiveness. Because UVR is known to stimulate uPA production in many cell types, UVR exposure may further increase uPA expression in corneal tumor cells, thus enhancing their ability to infiltrate. We investigated control of basal uPA levels and the induction of uPA by UVR in transformed and untransformed corneal keratocytes from Monodelphis. These studies took advantage of the fact that Monodelphis possesses an active photolyase that can be stimulated to remove UVR-induced pyrimidine dimers by exposure to long-wavelength visible photoreactivating light (PRL). Our studies showed that significant induction of uPA occurred in response to 200 J/m2 UVR. This induction was partially blocked by treatment with PRL, indicating that DNA damage, the pyrimidine dimer in particular, played a role in uPA induction. In untransformed cultured corneal fibroblasts, the heparin-binding protein inhibitor, suramin, reduced basal uPA levels, UVR-induced uPA production and cell proliferation. Basic fibroblast growth factor, a heparin-binding growth factor known to be UVR-inducible in mesenchymal cells, stimulated uPA production and cell proliferation; however, anti-bFGF antibodies did not significantly decrease proliferation or basal uPA production. These findings suggested that basal levels of uPA secretion were modulated in response to heparin-binding growth factors and that these growth factors may also have mediated the effect of UVR on uPA levels.     

Essential oil polymorphism of wild growing Hungarian thyme (Thymus pannonicus) populations in the Carpathian Basin

The volatile oil compositions of Thymus pannonicus All. from nineteen different localities of Hungary were analyzed by GC/MS. The essential oil content of the Hungarian thyme samples varied between very low (0.14 mL/100 g DW) and fairly high (1.9 mL/100 g DW) values. Significant essential oil polymorphism was found: altogether twelve chemovarieties may have been determined, representing a way of adaptation to different habitat conditions. The main volatile compound of chemotype 1 was thymol (24.6-67.5%), while in the case of chemotype 2, thymol (36.5-63.7%) and p-cymene (11.5-27.3%) predominated. Thymol (28.4-63.7%), p-cymene (11.5-31.8%) and gamma-terpinene (9.7-20.9%) were identified as the chief monoterpenes of chemotype 3, while chemotype 4 contained thymol (36.5%), p-cymene (27.3%) and neral (11.2%). Chemotype 5 accumulated thymol (38.5%), p-cymene (20.6%), gamma-terpinene (12.0%) and beta-bisabolene (10.3%) as its main volatiles. The oil of chemotype 6 can be characterized by thymol (41.9%), p-cymene (20.2%), isoborneol (10.3%) and gamma-terpinene (9.9%), while that of chemotype 7 consisted of thymol (27.7%), linalyl acetate (18.8%), gamma-terpinene (18.6%) and alpha-cubebene (13.9%). In the oil of chemotype 8, p-cymene (45.0%), geraniol (13.6%) and linalyl acetate (9.9%) were found in higher percentages, while chemotype 9 mainly produced linalyl acetate (36.2%) and geranyl acetate (20.2%). Chemotype 10 accumulated germacrene-D (43.4) and beta-caryophyllene (15.0%), while the oil of chemotype 11 contained caryophyllene oxide (45.2%), alpha-cubebene (15.7%) and linalool (13.8%) in high proportions. Germacrene-D (29.7%), beta-caryophyllene (22.0%) and farnesol (10.4%) were identified as main essential oil compounds of chemotype 12. The last nine chemotypes were new for the literature, while the first seven contained thymol as their chief compound. The role of certain sesquiterpenes was found to be considerable.     

Modulation of protein-protein interactions with small organic molecules

Many proteins exert their biological roles as components of complexes, and the functions of proteins are often determined by their specific interactions with other proteins. Because of the central importance of protein-protein interactions for cellular processes, the ability to interfere with specific protein-protein interactions provides a powerful means of influencing the function of selected proteins within the cell. Cell-permeable small organic modulators of protein-protein interactions are thus highly desirable tools both for the study of physiological cellular processes and for the treatment of numerous diseased states. Herein a number of protein-protein interactions that are considered to be pharmaceutical targets are presented, which will familiarize the reader with the strategies that have been employed for the successful identification of small molecule modulators of these protein-protein interactions. These encouraging examples suggest that combined research efforts in the areas of functional proteomics, assay development, and organic synthesis will open up novel possibilities for the treatment of human diseases in the future.     

Uncharged isocoumarin-based inhibitors of urokinase-type plasminogen activator

Background  

Urokinase-type plasminogen activator (uPA) plays a major role in extracellular proteolytic events associated with tumor cell growth, migration and angiogenesis. Consequently, uPA is an attractive target for the development of small molecule active site inhibitors. Most of the recent drug development programs aimed at nonpeptidic inhibitors targeted at uPA have focused on arginino mimetics containing amidine or guanidine functional groups attached to aromatic or heterocyclic scaffolds. There is a general problem of limited bioavailability of these charged inhibitors. In the present study, uPA inhibitors were designed on an isocoumarin scaffold containing uncharged substituents.     

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.     

High-affinity chelator thiols for switchable and oriented immobilization of histidine-tagged proteins: a generic platform for protein chip technologies

Protein micro-/nanoarrays are becoming increasingly important in systematic approaches for the exploration of protein-protein interactions and dynamic protein networks, so there is a high demand for specific, generic, stable, uniform, and locally addressable protein immobilization on solid supports. Here we present multivalent metal-chelating thiols that are suitable for stable binding of histidine-tagged proteins on biocompatible self-assembled monolayers (SAMs). The architectures and physicochemical properties of these SAMs have been probed by various surface-sensitive techniques such as contact angle goniometry, ellipsometry, and infrared reflection-absorption spectroscopy. The specific molecular organization of proteins and protein complexes was demonstrated by surface plasmon resonance, confocal laser scanning, and atomic force microscopy. In contrast to the mono-NTA/His6 tag interaction, which has major drawbacks because of its low affinity and fast dissociation, drastically improved stability of protein binding by these multivalent chelator surfaces was observed. The immobilized histidine-tagged proteins are uniformly oriented and retain their function. At the same time, proteins can be removed from the chip surface under mild conditions (switchability). This new platform for switchable and oriented immobilization should assist proteome-wide wide analyses of protein-protein interactions as well as structural and single-molecule studies.     

7-(2-Anilinopyrimidin-4-yl)-1-benzazepin-2-ones Designed by a “Cut and Glue” Strategy Are Dual Aurora A/VEGF-R Kinase Inhibitors

Although overexpression and hyperactivity of protein kinases are causative for a wide range of human cancers, protein kinase inhibitors currently approved as cancer drugs address only a limited number of these enzymes. To identify new chemotypes addressing alternative protein kinases, the basic structure of a known PLK1/VEGF-R2 inhibitor class was formally dissected and reassembled. The resulting 7-(2-anilinopyrimidin-4-yl)-1-benzazepin-2-ones were synthesized and proved to be dual inhibitors of Aurora A kinase and VEGF receptor kinases. Crystal structures of two representatives of the new chemotype in complex with Aurora A showed the ligand orientation in the ATP binding pocket and provided the basis for rational structural modifications. Congeners with attached sulfamide substituents retained Aurora A inhibitory activity. In vitro screening of two members of the new kinase inhibitor family against the cancer cell line panel of the National Cancer Institute (NCI) showed antiproliferative activity in the single-digit micromolar concentration range in the majority of the cell lines.     

Design, synthesis, and characterization of urokinase plasminogen-activator-sensitive near-infrared reporter

The urokinase-type plasminogen activator (uPA) plays a critical role in malignancies, and its overexpression has been linked to poor clinical prognosis in breast cancer. The ability to noninvasively and serially map uPA expression as a biomarker would thus have significant potential in improving novel cancer therapies. Here, we describe the development of a selective uPA activatable near-infrared (NIR) fluorescent imaging probe. The probe consists of multiple peptide motifs, GGSGRSANAKC-NH2, terminally capped with different NIR fluorochromes (Cy5.5 or Cy7) and a pegylated poly-L-lysine graft copolymer. Upon addition of recombinant human uPA to the probe, significant fluorescence amplification was observed, up to 680% with the optimized preparation. No activation with negative control compounds and uPA inhibitors could be measured. These data indicate that the optimized preparation should be useful for imaging uPA in cancer.     

Down-regulation of survivin suppresses uro-plasminogen activator through transcription factor JunB

Survivin, a member of the inhibitors of apoptosis protein family, is expressed during development and in various human cancers. However, the clinical relevance of survivin in cancer is still a matter of debate. Genes induced by hepatocyte growth factor (HGF) were screened using cDNA microarray technology in the stomach cancer cell lines, NUGC3 and MKN28. The levels of JunB, survivin, and uro-plasminogen activator (uPA) were up-regulated in cells treated with HGF in a dose-dependent manner. HGF-induced up regulation of JunB, survivin, and uPA was inhibited by pre-treatment with a MEK inhibitor (PD 98059). HGF-induced up-regulation of uPA was repressed by survivin knockdown. HGF enhanced the binding activity of JunB to the survivin promoter in control cells, but not in the JunB-shRNA cells. Transfection with survivin- shRNA resulted in a decrement of cell proliferation, as determined with MTT assays. In an in vitro invasion assay, significantly fewer cells transfected with survivin shRNA than control cells were able to invade across a Matrigel membrane barrier. In conclusion, survivin appeared to play an important role in the up-regulation of uPA induced by HGF via JunB and might contribute to HGF-mediated tumor invasion and metastasis, which may serve as a promising target for gastric cancer therapy.     

Is inhibition process better described with MD(QM/MM) simulations? The case of urokinase type plasminogen activator inhibitors

Urokinase plasminogen activator (uPA) is an enzyme involved in cancer growth and metastasis. Therefore, the design of inhibitors of uPA is of high therapeutic value, and several chemical families have been explored, even if none has still emerged, emphasizing the need of a rationalized approach. This work represents a complete computational study of uPA complexed with five inhibitors, which present weak similarities. Molecular dynamics simulations in explicit solvent were conducted, and structural analyses, along with molecular mechanics (MM)/Poisson-Boltzmann surface area free energies estimations, yield precious structure-activity relationships of these inhibitors. Besides, we realized supplemental QM/MM computations that improved drastically the quality of our models providing original information on the hydrogen bonds and charge transfer effects, which are, most often, neglected in other studies. We suggest that these simulations and analyses could be reproduced for other systems involving protein/ligand molecular recognitions.     

Quantitative computer simulations of biomolecules: a snapshot

A recent workshop titled "Quantitative Computational Biophysics" at Florida State University provided an overview of the state of the art in quantitative modeling of biomolecular systems. The presentations covered a wide range of interrelated topics, including the development and validation of force fields, the modeling of protein-protein interactions, the sampling of conformational space, and the assessment of equilibration and statistical errors. Substantial progress in all these areas was reported.     

Preparation of Porous Protein-Based Hydogel for Highly Sensitive Protein Chips

Summary: Protein chips are important tools for high-throughput analysis of biological events. We have developed a novel method to prepare a protein-based hydrogel, that is, a “Three-Dimensional Nano-structured Protein Hydrogel” (3-D NPH), which is composed of protein and polymer nano-particles. The 3-D NPH could be easily prepared by dispensing a protein and polymer mixture on a substrate. Surprisingly, gold particles conjugated with protein A diffused into the 3-D NPH which was made of mouse IgG through the pores. We have shown that the protein chips made with our 3-D NPH method has tremendously improved sensitivity in detecting protein-protein interactions compared with that of direct protein immobilization methods.