JP2010540892A - Method for screening compound having anti-inflammatory activity - Google Patents

Abstract

  A method for screening a compound that enhances the activity of a protein by binding to an extracellular portion of the protein Rac1 from the group consisting of a protein, a peptide, a peptidomimetic, an antibody, or a small organic molecule, Comprises the following steps: a) contacting a confluent layer of cultured endothelial cells grown on a porous membrane with at least one test compound alone or with a vascular permeable stimulant. B) Measuring vascular permeability using a suitable agent that can be detected by colorimetry.

Description

  The present invention relates to drug screening. More specifically, the present invention relates to proteins, peptides, peptidomimetics, antibodies, and small molecules that bind to vascular endothelium (VE) -cadherin and affect specific signal transduction processes mediated by VE-cadherin. It relates to methods for screening, identification and characterization of molecules. It also relates to the use of these compounds to prevent the opening of endothelial adhesive junctions between endothelial cells and to prevent morphological changes of certain endothelial cells as a result of these phenomena. Compounds with these characteristics are useful in the treatment of all diseases where inflammatory reactions, vascular leakage, and endothelial damage play a role. They can also prevent the formation of new capillaries and are therefore useful in the treatment of cancer.

  The endothelial layer that seamlessly covers the inside of all blood vessels has a very important barrier function, and prevents blood components such as blood-derived substances, cells, and serum from entering the underlying tissue. Barrier function is responsible for the interaction of homo- and heterotopic molecules on many adjacent endothelial cells and similar interactions with molecules on circulating blood cells. Through is tightly controlled. This failure of barrier function leads to serious physiological effects and injury to the underlying tissue. It is associated with the pathogenesis of inflammatory diseases, edema formation, and angiogenesis, including but not limited to ischemia-reperfusion injury, trauma / resuscitation caused by, for example, myocardial infarction or organ transplantation And systemic inflammatory response syndrome (SIRS) as a consequence of sepsis, macular degeneration of the eye, and associated with tumor growth. Therefore, it is preferable to identify compounds that can maintain the integrity of endothelial adhesive binding. These compounds can be used to treat or prevent these disease processes.

Endothelial barrier disorders Endothelial disorders can occur in a variety of ways, for example, as an imbalance between the release of relaxant and contractile factors and between the release of anticoagulant and clot mediators or as a decrease in barrier function. (Rubanyi, Journal of Cardiovascular Pharmacology 22, Sl-14.1993; McQuaid & Keenan Experimental Physiology 82, 369-376 (1997)). Such disorders are associated with a number of conditions, including hypercholesterolemia, hypertension, diabetes-related vascular disease, atherosclerosis, septic shock, and adult respiratory distress syndrome (Sinclair, Braude Haslam & Evans, Chest. 106: 535-539 (1994); Davies, Fulton & Hagen, Br J Surg. 82: 1598-610 (1995)).

  One of the major abnormalities associated with acute inflammatory diseases is a decrease in endothelial barrier function. Endothelial structural and functional integrity is necessary to maintain barrier function, and if either of these is compromised, solute and excess plasma leaks through the monolayer, causing tissue edema and inflammatory cell migration cause. Many drugs increase monolayer permeability by causing endothelial cell morphological changes, such as contraction or retraction, leading to the formation of intercellular gaps (Lum & Malik, Am. J. Physiol. 267: L223-L241 (1994)). These agents include, for example, thrombin, bradykinin, and vascular endothelial growth factor (VEGF). Endothelial cell contraction resembles the control of the actin-myosin interaction in smooth muscle but occurs over a longer period of time and is more precisely described as contracture. This mechanism of contraction is thought to be involved in increasing intracellular Ca 2+ concentration, activation of myosin light chain kinase, phosphorylation of myosin light chain, and reconstitution of F-actin microfilaments. Regression is a more passive process and is independent of myosin light chain kinase and is an actin-linking protein, protein kinase C, which is important for maintaining cell-cell and cell-matrix interactions. (PKC)-involved in stimulated phosphorylation (Lum & Malik, Am. J. Physiol. 267: L223-L241 (1994)).

  Increased permeability of the vessel wall allows excess fluid and protein to leak into the interstitial space. This acute inflammatory event is often associated with tissue ischemia and acute organ dysfunction. Thrombin formed at the site of activated endothelial cells (EC) causes this capillary barrier dysfunction by forming large paracellular holes between adjacent ECs (Carbajal et al, Am J Physiol Cell Physiol 279: C195-C204, 2000). This process characterizes changes in EC morphology by myosin light chain phosphorylation (MCLP) that triggers the development of F-actin-dependent cytoskeletal contractile tension (Garcia et al, J Cell Physiol. 1995; 163: 510 -522 Lum & Malik, Am J Physiol Heart Circ Physiol. 273 (5): H2442-H2451. (1997)).

  The signal transduction mechanism of this contraction process is involved in thrombin receptor protein cleavage and activation. This receptor is coupled to the Gq family heterotrimeric G protein, which stimulates phospholipase Gβ to mobilize Ca ions from intracellular stores, D-myoinositol 1,4,5- Releases triphosphate. The subsequent increase in intracellular Ca ion concentration activates Ca ion-calmodulin-dependent MLC kinase, which phosphorylates serine 19 and threonine 18 of MLC (Goeckeler & Wysolmerski, J. Cell Biol. 1995). ; 130: 613-627). MLCP elicits myosin Mg ion-ATPase activity that causes myosin binding to F-actin and subsequent actomyosin stress fiber formation (Ridley & Hall, Cell, 70, 389-399 (1992)). Phosphorylation of MLC converts the non-myocyte myosin II soluble folded 10S form to the insoluble unfolded 6S form. This process is characterized by the reconstruction of myosin from scattered intracellular motility to punctate spots and ribbons associated with large bundles of F-actin (Verkhovsky et al, The Journal of Cell Biology, 131, 989-1002 (1995)). The net result is a sustained morphological change of the endothelial cells and disruption of the barrier function.

Vascular endothelium (VE) -cadherin thrombin-induced hyperendothelial permeability can also be mediated by cell-cell adhesion (Dejana J. Clin. Invest. 98: 1949-1953 (1996)). Endothelial cell-cell adhesion is primarily determined by the function of vascular endothelium (VE) cadherin (cadherin 5), a Ca-dependent cell-cell adhesion molecule that forms an adhesive bond. The function of cadherin 5 is controlled from the cytoplasm side through the association with α-catenin (homologous to vinculin) and accessory proteins β-catenin, placoglobin (g-catenin) and p120, which are bound to the F-actin cytoskeleton in turn. The

  VE-cadherin has emerged as an adhesion molecule that plays a fundamental role in capillary permeability and in morphogenesis and proliferative phenomena associated with angiogenesis (Vincent et al, Am J Physiol Cell Physiol, 286 (5): C987-C997 (2004)). Like other cadherins, VE-cadherin mediates calcium-dependent, homophilic adhesion and functions as a plasma membrane binding site in the cytoskeleton. However, VE-cadherin is incorporated into signal transduction pathways and cellular systems that are uniquely important to the vascular endothelium. Recent advances in endothelial cell biology and physiology reveal the nature of VE-cadherin that may be unique within the cadherin family of adhesion molecules. For these reasons, VE-cadherin represents cadherins that are typical of the cadherin family but are unique in function and physiological relevance. There is accumulating evidence that VE-cadherin-mediated cell-cell adhesion is controlled by a dynamic balance between phosphorylation and dephosphorylation of junction proteins including cadherin and catenin. Increased tyrosine phosphorylation of β-catenin results in dissociation of catenin from cadherin and from the cytoskeleton, leading to weak adhesive bonds (AJ). Similarly, tyrosine phosphorylation of VE-cadherin and β-catenin occurred in loose AJ and was significantly reduced in a tightly confluent monolayer (Tinsley et al., J Biol Chem, 274, 24930-24934). (1999)).

  Also, correct clustering of VE-cadherin monomers in adhesive bonding is essential for the correct signaling activity of VE-cadherin. This is because cells with a chimeric variant (IL2-VE) containing a full-length VE-cadherin cytoplasmic end can bind β-catenin and p120, but fail to cause correct signaling (Lampugnani et al, MoI. Biol. of the Cell, 13, 1175-1189 (2002)).

Rho- and Rac-GTPases, and vascular permeability Rho GTPases are a family of small GTPases that have prominent effects on the actin cytoskeleton of cells. For vasculature function, they are involved in the control of cell morphology, cell contraction, cell motility, and cell adhesion. The three most famous family members of the Rho GTPase are RhoA, Rac and cdc42. Activation of RhoA induces the formation of f-actin stress fibers in cells, while Rac and cdc42 affect the actin cytoskeleton by inducing membrane raffles and microspikes, respectively (Hall, Science 279: 509-5l41998). Rac and cdc42 can affect MLCK activity to a limited extent through activation of the protein PAK (Goeckeler et al. J. Biol. Chem., 275, 24, 18366-18374 (2000 On the other hand, RhoA has a significant stimulatory effect on the actin-myosin interaction due to its ability to stabilize the phosphorylation state of MLC (Katoh et al., Am. J. Physiol. Cell. Physiol. 280, C1669-C1679 (2001)). This occurs by activation of Rho kinase which inhibits in its turn the phosphatase PP1M which hydrolyzes phosphorylated MLC. Rho kinase also inhibits the actin-cleaving activity of cofilin, thus stabilizing f-actin fibers (Toshima et al., Mol. Biol. Of the Cell. 12, 1131-1145 (2001)). Furthermore, Rho kinase can also be involved in the anchoring of the actin cytoskeleton to proteins in the cell membrane, thus potentially affecting the interaction between the binding protein and the actin cytoskeleton (Fukata et al. Cell Biol 145: 347-361 (1999)).

  Thrombin can activate RhoA via Gα12 / 13 and the so-called guanine nucleotide exchange factor (GEF) (Seasholtz et al; Mol: Pharmacol. 55, 949-956 (1999).). GEF exchanges RhoA-bound GDP for GTP, which activates RhoA. This activation causes RhoA to migrate to the cell membrane where it is bound by its lipophilic geranyl-geranyl-anchor.

  RhoA can be activated by a number of vasoactive agents including lysophosphatidic acid, thrombin, and endothelin. Membrane-bound RhoA is dissociated from the membrane by activation of guanine nucleotide dissociation inhibitor (GDI) or after the action of GTPase activating protein (GAP). Guanine nucleotide dissociation inhibitor (GDI) is a regulatory protein that binds to the carboxyl terminus of RhoA.

  GDI inhibits the activity of RhoA by delaying the dissociation of GDP and pulling activated RhoA away from the cell membrane. Thrombin activates RhoA directly in human endothelial cells and induces the transfer of RhoA to the cell membrane. Under the same conditions, the related GTPase Rac was not activated. Specific inhibition of RhoA by C3 transferase from Clostridium botulinum reduces thrombin-induced endothelial MLC phosphorylation and increased permeability, but transiently increases histamine-dependent permeability Had no effect (van Nieuw Amerongen et al. Circ Res. 1998; 83: 1115-11231 (1998)). Since the specific Rho kinase inhibitor Y27632 similarly reduces thrombin-induced endothelial permeability, the action of RhoA appears to be mediated through Rho kinase.

  Rac1 and RhoA have an antagonistic effect on endothelial barrier function. Acute hypoxia inhibits Rac1 and activates RhoA in normal adult pulmonary artery endothelial cells (PAEC), leading to disruption of barrier function (Wojciak-Stothard and Ridley, Vascul Pharmacol., 39: 187-99 ( 2002)). PAECs from piglets with chronic hypoxia-induced pulmonary hypertension have a stable and abnormal phenotype with a continuous decrease in Rac1 and an increase in RhoA activity. These activities correlate with changes in endothelial cytoskeleton, adhesive junctions, and permeability. Activation of Rac1 and inhibition of RhoA restored the abnormal phenotype and permeability to normal (Wojciak-Stothard et al., Am. J. Physiol, Lung Cell MoI. Physiol. 290, Ll173-Ll182 ( 2006)).

  Therefore, it is preferred to screen for substances that restore the physiological balance of Rac1 and RhoA activity to the levels observed in endothelial cells in normal steady state.

Adhesion plaque kinase and its role in endothelial permeability Adhesion plaque kinase is composed of a central catalytic domain that is flanked by large N- and C-terminal domains. The N-terminal region contains FERM homology that can bind integrins and growth factor receptors. The C-terminal non-catalytic domain, also called FRNK (FAK-related non-kinase), not only directs FAK to the adhesion complex for signal transduction, but also provides a binding site for other binding molecules. Has a FAT sequence that interacts with the cytoplasm. To date, at least five tyrosine residues have been identified in FAK.

  Phosphorylation of these tyrosine residues is directly correlated with kinase activity. This is supported by the correlation between FAK activity and monolayer permeability. Several models have been proposed to explain the effect of adhesion plaque formation on the barrier structure. Enhanced adhesion of endothelial cells to the extracellular matrix can help stabilize the monolayer against detachment due to lateral contractile forces produced by inflammatory mediators. Thus, adhesion plaque activation occurs in parallel with cell contraction and can offset reduced cell-cell binding during inflammatory stimulation. Another hypothesis is that FAK activation and adhesion plaque remodeling positively contributes to the opening of endothelial cell-cell junctions by providing a mechanical basis for endothelial cells to change contraction or morphology It has been proposed to do. The last (but not least) possibility is that the local complex acts as an aggregation point where multiple scaffold proteins or signaling molecules are integrated, affecting the barrier function.

  In addition to well-characterized activation of MAPK, PI3K, and eNOS, a potential signaling phenomenon downstream from FAK is the formation of myosin light chain phosphorylation that causes actin-myosin contraction and the formation of Rho-dependent stress fibers. Which are paracellular permeable properties (Wu, J Physiol 569., 359-366 (2005)). Therefore, it is preferred to inhibit FAK phosphorylation to promote endothelial integrity.

Inflammation and Endothelial Disorders: Pathological Outcomes and Treatment Outcomes Endothelial disorders and the ease of leakage of the endothelial barrier are important elements of a wide range of inflammatory diseases. Inflammatory responses are characterized by extravasation of blood components such as plasma proteins and serum, resulting in significant interstitial tissue edema.

  In addition, neutrophils, which are the main factors of inflammatory response, can translocate from the bloodstream into the underlying tissue.

  At the same time, these effects of the leakiness of the endothelial layer cause substantial damage to healthy organs and tissues. Adult respiratory distress syndrome (ARDS), acute lung injury (ALI), glomerulonephritis, acute and chronic allograft rejection, inflammatory skin diseases, rheumatoid arthritis, asthma, atherosclerosis, Systemic lupus erythematosus (SLE), collagen disease, vasculitis, and limb reattachment, myocardial infarction, crush wounds, shock, stroke, and is linked to a number of diseased organ disorders including ischemic reperfusion injury . It is also a prerequisite for new blood vessel formation by endothelial growth, and thus such angiogenesis, including but not limited to wet age-related macular degeneration, and cancer growth and metastasis, is a pathology Can be ill, which has been shown to play a central role.

BRIEF DESCRIPTION OF THE INVENTION The invention relates to the screening, identification, characterization of proteins, peptides, peptidomimetics, antibodies, and small molecules that modulate drug-mediated interactions and signaling events that cause endothelial hyperpermeability, And how to use. Agents identified by these screening methods bind to vascular endothelium (VE) -cadherin expressed during adhesion bonding of the endothelial cell layer and by modulating its conformation and / or phosphorylation status , Show effect. More specifically, these agents promote endothelial integrity by stabilizing VE-cadherin clustering at cell-cell junctions. Given the importance of disruption of endothelial barrier function for a wide range of diseases, these agents have broad applicability as therapeutic and / or prophylactic drugs.

According to one embodiment, the present invention is a method for screening a protein, peptide, peptidomimetic, antibody, or small organic molecule that enhances the activity of this protein by binding to the extracellular portion of the protein Rac1:
a. Contacting a confluent layer of cultured endothelial cells grown on a porous membrane with at least one test compound alone or with a vascular permeability stimulant;
b. The method comprises measuring endothelial permeability using a suitable agent that can be detected colorimetrically.

According to another embodiment, the present invention is a method of screening for a protein, peptide, peptidomimetic, antibody, or small organic molecule that enhances the activity of this protein by binding to the extracellular portion of the protein Rac1. :
a. Contacting a confluent layer of cultured endothelial cells with at least one test compound;
b. Lysing the endothelial cells in a lysation buffer;
c. The method is provided comprising measuring the amount of Rac1 activity using a specific assay.

In other embodiments, the present invention is a method of screening for proteins, peptides, peptidomimetics, antibodies, and small organic molecules that interfere with the activity of RhoA and consequently changes in the cytoskeletal structure of endothelial cells:
a. Contacting a confluent layer of cultured endothelial cells with thrombin in the presence of at least one test compound;
b. Lysing the endothelial cells in a lysation buffer;
c. The method is provided comprising the step of measuring RhoA activity using a specific assay.

In other embodiments, the invention is a method of screening for proteins, peptides, peptidomimetics, antibodies, and small organic molecules that prevent phosphorylation of adhesion plaque kinase:
a. Contacting a confluent layer of cultured endothelial cells with thrombin in the presence of at least one test compound;
b. Lysing the endothelial cells in a lysation buffer; and c. The method comprises measuring adhesion plaque kinase phosphorylation using a specific assay.

In other embodiments, the present invention is a method for screening proteins, peptides, peptidomimetics, antibodies, and small organic molecules that prevent vascular leakage that causes a systemic inflammatory response in warm-blooded animals:
a. Initiating a systemic inflammatory response by applying an appropriate amount of bacterial lipopolysaccharide (LPS);
b. Exposing the animal to at least one test compound;
c. Injecting the animal with an appropriate amount of an appropriately sized fluorescently labeled microbead;
d. Slaughtering the animal after an appropriate amount of time,
e. Excising and homogenizing animal organs or tissues; and f. The method is provided comprising measuring the amount of fluorescence in the homogenate.

A compound identified by the claimed method of screening. Permeability of confluent HUVEC layers after 45 minutes incubation with thrombin. Activation of Rac1 with compound 1A. This gel is the result of a pull-down assay, as described in Example 1. Lane 1: medium control, lane 2: 1 minute HUVEC activation with thrombin, lane 3: treatment with compound 1A alone for 1 minute; lane 4: HUVEC with thrombin and compound 1A, 1 minute, lane 5: Thrombin 5 minutes; Lane 6: Compound 1A, 5 minutes, Lane 7: Thrombin and Compound 1A, 5 minutes. β-actin was used as a control for total protein content. Inhibition of thrombin-induced activation of RhoA by compound 1A. This gel is the result of a pull-down assay, as described in Example 1. Lane 1: medium control, lane 2: 1 minute HUVEC activation with thrombin, lane 3: treatment with compound 1A alone for 1 minute; lane 4: HUVEC with thrombin and compound 1A, 1 minute, lane 5: Thrombin 5 minutes; Lane 6: Compound 1A, 5 minutes, Lane 7: Thrombin and Compound 1A, 5 minutes. β-actin was used as a control for total protein content. Activation of Rac1 with compound 1B. This gel is the result of a pull-down assay, as described in Example 1. Lane 1: medium control, lane 2: 1 minute HUVEC activation with thrombin, lane 3: treatment with compound 1B alone for 1 minute; lane 4: HUVEC with thrombin and compound 1B, 1 minute, lane 5: Thrombin 5 minutes; Lane 6: Compound 1B, 5 minutes, Lane 7: Thrombin and Compound 1B, 5 minutes. Inhibition of thrombin-induced activation of RhoA by compound 1B at time points 1, 5, and 10 minutes after stimulation with thrombin. Quantification and time dependence of Rac1 activation by compound 1B. Quantification and time dependence of the inhibition of thrombin-induced activation of RhoA by compound 1B. Inhibition of thrombin-induced phosphorylation of FAK by Compound 1A. This graph shows the time course of inhibition of FAK phosphorylation induced by thrombin. Inhibition of LPS-induced vascular leakage by Compound 1A. FIG. 5 is a fluorescent image of a lung section from a rat in which vascular leakage was induced by LPS treatment. Section a) is from a control animal and section b) is from an animal treated with Compound 1A.

  Preferentially, these assays are performed in the order described above, which constitutes a screening tree and selectively contains compounds having a particular physiological activity claimed by the present invention. Identify.

  A useful method for measuring the permeability of a confluent layer of endothelial cells is the dual chamber method, in which the endothelial cells are porous to separate the chambers. Grown on the membrane. Cells are preferentially incubated for 30 to 120 minutes with human umbilical vein endothelial cells (HUVEC) alone or with an endothelial permeable stimulant. Preferentially thrombin is used as such a stimulant. Permeability can be measured using a penetrating dye and its absorption can be measured colorimetrically. Preferentially, Evans Blue can be used as the dye, either itself or bound to albumin.

  A useful method for analyzing the activation status of Rac1 and RhoA is based on the principle of the so-called pull-down assay. In this format, the GTP binding activation state of each protein in the cell lysate binds to an immobilized binding partner, and a monoclonal antibody (MAb) made specifically for the protein in question. Is detected. Subsequently, the amount of GTP binding activation state can be quantified by suitable detection methods including, but not limited to, Western blotting or fluorescence detection.

  To measure Rac1 activation in human umbilical vein endothelial cells (HUVEC), cells are incubated with test compounds under suitable conditions for various times up to 30 minutes and then lysed. Lysate is added to the p21-binding domain (PBD) of appropriately immobilized p21-activated protein kinase (PAK) and the amount of activated Rac1 is quantified using a Rac1-specific MAb.

  To measure the inhibition of thrombin-induced activation of RhoA in HUVEC, cells are incubated with a suitable amount of thrombin with or without a test compound under suitable conditions for various times up to 10 minutes, Then it is dissolved. The lysate is added to an appropriately immobilized rhotekin binding domain (RBD) and the signal is measured using a suitable detection method.

  To measure the inhibition of thrombin-mediated FAK phosphorylation in HUVEC, cells are incubated with a suitable amount of thrombin with or without a test compound under suitable conditions for various times up to 60 minutes, and thereafter Dissolved. Lysates were subjected to Western blot analysis using site-specific antibodies raised against SDS-PAGE and FAK phosphorylated tyrosine residues.

  To measure inhibition of LPS-induced vascular leakage in rodents, animals are administered an amount of gram-negative lipopolysaccharide (LPS) optimal to achieve a systemic inflammatory response. After various time periods between 0 and 4 hours, the test substance was administered intravenously, followed by the appropriate amount of fluorescent microbeads. The animals are then sacrificed and the organs (lung, kidney, spleen, heart, brain) are excised, cut into thin slices suitable for microscopic analysis, and fixed with paraformaldehyde. The number of extravasated microparticles is counted using a fluorescence microscope.

  Alternatively, the organ is homogenized and the amount of microbeads trapped in the tissue is measured using a suitable fluorescence detector.

  The compounds identified using the screening methods according to the present invention are useful in the development of drugs for the prevention and / or treatment of diseases caused by inflammatory reactions and / or endothelial rupture and vascular leakage. Thus, in accordance with other aspects of the present invention, the compounds of the present invention include, but are not limited to, septic shock, wound-related sepsis, eg post-ischemic, such as after myocardial infarction / reperfusion or organ transplantation Reperfusion injury, frostbite wound or shock, acute inflammatory reaction-mediated lung injury such as respiratory distress syndrome, acute pancreatitis, cirrhosis, uveitis, asthma, traumatic brain injury, nephritis, atopic dermatitis, psoriasis, inflammatory bowel Administered for the treatment and / or prevention of disease, macular degeneration of the eye, diabetic retinopathy, neovascular glaucoma, retinal vein occlusion, and tumor growth.

  In order to achieve a therapeutic effect in these diseases, the compounds of the invention can be given orally or parenterally and are formulated into suitable pharmaceutical formulations using pharmaceutically acceptable excipients or carriers. be able to. The invention therefore also relates to a pharmaceutical composition comprising an active ingredient identified by the screening method according to the invention and further comprising a pharmaceutically acceptable excipient or carrier.

  Pharmaceutically acceptable excipients are for use in animals, particularly humans, approved by the federal or state government regulatory authorities or listed in the US pharmacopoeia or generally accepted pharmacopoeia. It is an excipient. Such pharmaceutical carriers can be sterile liquids, such as water and oils (including animals, vegetables and synthetic sources such as peanut oil, soybean oil, mineral oil and the like). The aqueous carrier may also contain, for example, glucose and glycerol.

  Suitable excipients include but are not limited to starch, glucose, lactose, sucrose, gelatin, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, glycerol, propylene glycol, ethanol, etc. Can be. The composition may also include wetting and / or emulsifying agents, or pH buffering agents.

  The composition can be in the form of a solution, suspension, emulsion, tablet, capsule, powder, or sustained release formulation. Such compositions comprise a therapeutically effective amount of the compound, together with a suitable amount of carrier so as to provide a form suitable for administration to the patient being treated and a form suitable for the therapeutic form.

Example 1
Compound screening method for identifying agents that reduce thrombin-induced permeability of endothelial monolayers through stabilization of VE-cadherin binding HUVEC is a standard condition of 12 mm transwell plates, polycarbonate membranes Grow on 3μm Corning to confluence. Two hours before the start of the experiment, the standard growth medium is replaced with growth medium without phenol red in the upper and lower chambers. To induce permeability, 5 U / ml thrombin (Calbiochem) or 5 U / ml plus test compound is added to the upper wells. HUVEC are incubated for 45 minutes. At the end of the incubation period, the medium in the upper chamber is replaced with growth medium without phenol red and containing 4% BSA and 0.67 mg / ml Evans Blue. Evans blue is allowed to diffuse into the lower chamber for 10 minutes, after which the upper chamber is removed and the absorbance of the medium recovered from the lower chamber is measured at a wavelength of 620 nm.

Example 2
Compound screening method for identifying substances that activate Rac1 through stabilization of VE-cadherin binding HUVECs are grown to confluence under standard conditions. Prior to inducing Rac1 activity, HUVECs were starved by using IMDM (Gibco) without growth factors and serum supplements for 4 hours. Rac1 activity is induced by adding 50 μg / ml test compound to the starvation medium for 1, 5, and 10 minutes. Activated Rac1 was isolated using Upstate's Rac1 / Cdc42 assay reagent according to product instructions. The isolate was separated on a 15% polyacrylamide gel and blotted onto a nitrocellulose membrane (Bio-Rad). Rac1 was detected using Upstate anti-Rac1 clone 23A8, anti-mouse (1: 250).

Example 3
Compound screening methods for identifying substances that inhibit thrombin-induced RhoA activation HUVECs are grown to confluence under standard conditions. Prior to inducing Rho activity, HUVECs were starved by using IMDM (Gibco) without growth factors and serum (serum supplements) for 4 hours. After the starvation time, 5 U / ml thrombin (Calbiochem) or 5 U thrombin and 50 μg / ml test compound are added to the starvation medium for 1, 5, and 10 minutes. Activated RhoA was isolated using Upstate's Rho assay reagent according to product instructions. The isolate was separated on a 15% polyacrylamide gel and blotted onto a nitrocellulose membrane (Bio-Rad). RhoA was detected using Upstate's anti-Rho (-A, -B, -C), clone 55 (1: 500).

Example 4
Compound screening method immunoprecipitation to identify substances that inhibit thrombin-induced FAK phosphorylation at the autophosphorylation site Tyr397:
HUVECs were incubated with FX06 (50 μg / ml), thrombin (1 U / ml, Sigma Aldrich), and thrombin / test compound for the times indicated. After washing with ice-cold PBS (GIBCO), cells were removed from the culture flask in Tris lysis buffer (+ 1% TritonX (Bio-Rad), NP40 (Sigma Aldrich) and protease and phosphatase inhibitor cocktail (Sigma Aldrich)). And was dissolved on ice for 20 minutes. The lysate was vortexed severely every 5 minutes. After lysis, the lysate was centrifuged (15,000 rpm / 10 min / 4 ° C.) and the supernatant was added to 50 μl Sepharose beads (Sigma Aldrich) preincubated with a total volume of 1 μg FAK antibody (BD Transduction Laboratories). The beads were agitated on wheels for 2 hours at 4 ° C. and then washed 3 times with ice-cold PBS. 2x sample buffer was added and incubated at 95 ° C for 5 minutes. Sample buffer was then removed from the beads and applied to Western blotting.

Western blot:
A 10% polyacrylamide gel was run to separate the precipitated protein. The gel was blotted onto a PVDF (Bio-Rad) membrane using a hoefer semi-drive blotting system. Next, the membrane was washed with TBS / 0.5% TWEEN (TBST) and blocked with 1% BSA / TBST for 1 hour at room temperature. Next, the p397FAK antibody (0.2 μg / ml; BD Transduction Laboratories) was incubated overnight at 4 ° C. with 1% BSA / TBST solution. For detection, the bound Ab was visualized by chemiluminescence using HRP-labeled goat anti-mouse Ab (1: 25000; Bio-Rad) TBST solution (ECL system, Amersham Corp., Arlington Heights, IL). Recorded on film.

Example 5
Compound screening method for identifying substances that inhibit LPS-induced vascular leakage in rodents Male HimOFA / SPF rats (Institute for Biomedical Research, Medical School Vienna) having a body weight of 260-320 g were Breeding at the Institute for Biomedical Research, Medical School Vienna. All experiments were approved by the Government of Vienna (Amt der Wiener Landesregierung), MA58. Rats are anesthetized with 100 mg / kg thiopentone (Sandoz). The trachea is cannulated to promote breathing. The right jugular vein is cannulated for drug administration. A catheter is placed in the right carotid artery to measure mean arterial pressure (MAP). After surgery, animals are randomly selected into treatment groups. All rats receive replacement fluid (600 μl 0.9% saline as an intravenous infusion) and are stabilized for 15 minutes. Throughout the experiment, body temperature is adjusted using a thermostatic blanket. After stabilization time, endotoxin shock is induced by rapid administration of 12 mg / kg LPS (E. coli serotype 0.127: B8; Sigma). Sixty minutes after LPS administration, animals receive a rapid dose of 3 mg / kg test compound or saline. Five hours and 50 minutes after LPS administration, rats receive rapid administration of fluorescent microparticles; 125 × 10 ⁶ beads / kg body weight (Fluo Spheres polystyrene microspheres; 1 μm yellow-green fluorescence (505/515) Invitrogen Molecular Probes).

  Six hours after LPS administration, animals are sacrificed and lungs are removed to assess vascular leakage. Lung vascular leakage is assessed by measuring fluorescence / g tissue. For these purposes, lung tissue is digested with ethanolic KOH and fluorescent particles are centrifuged as recommended by the "Manual for using Fluorescent Microspheres to measure organ perfusion" Fluorescent Microsphere Resource Center; University of Washington. ). Fluorescence is measured using a Spectra Max Gemini S Fluorometer.

Claims (11)

A method for screening compounds from the group consisting of proteins, peptides, peptidomimetics, antibodies, or small organic molecules that enhance the activity of this protein by binding to the extracellular portion of the protein Rac1:
a. Contacting a confluent layer of cultured endothelial cells grown on a porous membrane with at least one test compound alone or with a vascular permeable stimulant;
b. The method comprising measuring vascular permeability using a suitable agent that can be detected colorimetrically. A method for screening compounds from the group consisting of proteins, peptides, peptidomimetics, antibodies, and small organic molecules that enhance the activity of this protein by binding to the extracellular portion of the protein Rac1:
a. Contacting a confluent layer of cultured endothelial cells with at least one test compound;
b. Lysing the endothelial cells in a lysation buffer; and c. Measuring the amount of Rac1 activity. A method for screening compounds from the group consisting of proteins, peptides, peptidomimetics, antibodies, and small organic molecules that interfere with the activity of RhoA and consequently changes in the cytoskeletal structure of endothelial cells:
a. Contacting a confluent layer of cultured endothelial cells with thrombin in the presence of at least one test compound;
b. Lysing the endothelial cells in a lysation buffer; and c. The method comprising measuring RhoA activity. A method for screening a compound that prevents phosphorylation of adhesion plaque kinase from the group consisting of proteins, peptides, peptidomimetics, antibodies, and small organic molecules:
a. Contacting a confluent layer of cultured endothelial cells with thrombin in the presence of at least one test compound;
b. Lysing the endothelial cells in a lysation buffer; and c. Measuring the phosphorylation of adhesion plaque kinase. A method for screening a compound that prevents vascular leakage that causes a systemic inflammatory response in warm-blooded animals from the group consisting of proteins, peptides, peptidomimetics, antibodies, and small organic molecules:
a. Initiating a systemic inflammatory response by applying an appropriate amount of bacterial lipopolysaccharide (LPS);
b. Exposing the animal to at least one test compound;
c. Injecting the animal with an appropriate amount of an appropriately sized fluorescently labeled microbead;
d. Slaughtering the animal after an appropriate amount of time,
e. Excising and homogenizing animal organs or tissues; and f. Measuring the amount of fluorescence in the homogenate.   A method for screening a compound from the group consisting of proteins, peptides, peptidomimetics, antibodies, and small organic molecules, comprising one or more methods according to claims 1-5.   A compound from the group consisting of proteins, peptides, peptidomimetics, antibodies, and small organic molecules according to claim 6, characterized in that it comprises all five methods according to claims 1-5. How to screen.   A compound from the group consisting of proteins, peptides, peptidomimetics, antibodies, and small organic molecules according to claim 6, characterized in that it comprises all five methods according to claims 1-5. A method of screening, wherein the method starts with the method of claim 1 or 2 and then the method of claim 2, 3 and 4 is performed.   From the group consisting of proteins, peptides, peptidomimetics, antibodies, and small organic molecules, using the screening method according to any one of claims 1 to 8, inflammatory reaction and / or endothelial rupture and vascular leakage A compound identified for the development of a medicament for the prevention and / or treatment of diseases caused by.   A pharmaceutical composition comprising the compound according to claim 9.   Septic shock, wound associated sepsis, post-ischemic reperfusion injury such as myocardial infarction / reperfusion or organ transplantation, frostbite wound or shock, acute inflammatory response mediator such as respiratory distress syndrome Lung disorder, acute pancreatitis, cirrhosis, uveitis, asthma, traumatic brain disorder, nephritis, atopic dermatitis, psoriasis, inflammatory bowel disease, macular degeneration of the eyes, diabetic retinopathy, neovascular glaucoma, retinal vein occlusion 11. A pharmaceutical composition according to claim 10 for the treatment and / or prevention of diseases and tumor growth.

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