The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior.

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a porous membrane in a process analogous to the initial steps of cancer cell metastasis. The tested cells can be altered for the gene expression or treated with inhibitors to test for changes in the invasion potential. This experiment tests the aggressive phenotype of the mouse mammary tumor cells to discover and characterize the potential oncogenes that promote cell invasion.

Regulation of the oncogenic phenotype by the nuclear body protein ZC3H8.

Background: The Zc3h8 gene encodes a protein with three zinc finger motifs in the C-terminal region. The protein has been identified as a component of the Little Elongation Complex, involved in transcription of small nuclear RNAs. ZC3H8 is overexpressed in a number of human and mouse breast cancer cell lines, and elevated mRNA levels are associated with a poorer prognosis for women with breast cancer.

The Role of Lysophospholipid Acyltransferases in the Golgi Complex.

Determining the abundance of phospholipids and neutral lipids in cellular membranes is paramount to understanding their biological functions. Many lipid-modifying enzymes have yet to be characterized due to limitations in substrate-product measurements and purification of membrane-bound enzymes. The method described here uses radiolabeled phospholipid substrates and cell-purified organelles to quantify phospholipid metabolism using thin-layer chromatography.

Patterns of Rib Growth in the Human Child.

Introduction: Whereas there is substantial information on the changes of the rib cage during childhood and asymmetry of the thorax in children with scoliosis, there are virtually no normative data on the growth of individual ribs throughout childhood.

Methods: The Hamann-Todd (HT) Osteological Collection provided the bones of 32 human specimens aged 1-18 years. A total of 6,226 individual photographs of all vertebral bodies and ribs were obtained from these specimens.

Differential roles of C-terminal Eps15 homology domain proteins as vesiculators and tubulators of recycling endosomes.

Endocytic recycling involves the return of membranes and receptors to the plasma membrane following their internalization into the cell. Recycling generally occurs from a series of vesicular and tubular membranes localized to the perinuclear region, collectively known as the endocytic recycling compartment. Within this compartment, receptors are sorted into tubular extensions that later undergo vesiculation, allowing transport vesicles to move along microtubules and return to the cell surface where they ultimately undergo fusion with the plasma membrane.

A role for phospholipase A2 activity in membrane tubule formation and TGN trafficking.

We have investigated the role of phospholipase A(2) (PLA(2) ) enzymes in generating membrane tubules at the trans-Golgi network (TGN). Constitutive TGN membrane tubules and those induced by over-expressing kinase dead protein kinase D were inhibited by the PLA(2) inhibitors ONO-RS-082 (ONO) and bromoenol lactone. These antagonists also inhibited secretory delivery of both soluble and transmembrane cargoes.

Membrane topology of human AGPAT3 (LPAAT3).

Integral membrane lysophospholipid acyltransferases (AT) are involved in many reactions that produce phospholipids and triglycerides. Enzymes that utilize lysophosphatidic acid (LPA) as an acceptor substrate have been termed LPAATs, and several are members of the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) gene family. Amino acid sequence comparisons with other acyltransferases reveal that AGPATs contain four conserved motifs (I-IV), whose invariant residues appear to be important for catalysis and/or substrate recognition.

Lysophosphatidic acid acyltransferase 3 regulates Golgi complex structure and function.

Recent studies have suggested that the functional organization of the Golgi complex is dependent on phospholipid remodeling enzymes. Here, we report the identification of an integral membrane lysophosphatidic acid-specific acyltransferase, LPAAT3, which regulates Golgi membrane tubule formation, trafficking, and structure by altering phospholipids and lysophospholipids. Overexpression of LPAAT3 significantly inhibited the formation of Golgi membrane tubules in vivo and in vitro.

Constraining the amide bond in N-sulfonylated dipeptide VLA-4 antagonists.

The integrin VLA-4 is implicated in several inflammatory disease states. In search of non-peptidic antagonists of VLA-4, rotational constraints were imposed on the amide bond of prototypical N-sulfonylated dipeptide VLA-4 antagonists. By judicious structural modification of the side chains, trisubstituted imidazoles with moderate binding potencies were obtained, for example, 19, VLA-4 IC(50)=237 nM.

Highly constrained bicyclic VLA-4 antagonists.

VLA-4 is implicated in several inflammatory and autoimmune disease states. A series of cyclic beta-amino acids (beta-aa) was studied as VLA-4 antagonists. Binding affinity was highly dependent on the dihedral angle (phi) between the amino and the carboxyl termini of the beta-aa.

Use of acyltransferase inhibitors to block vesicular traffic between the ER and Golgi complex.

This article describes the use of acyltransferase inhibitors as probes for studying the potential role of lysophospholipid acyltransferases (LPAT) in intracellular membrane trafficking in the secretory and endocytic pathways. The small molecule inhibitors that are described here were originally found as acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors. One of these, CI-976 (2,2-methyl-N-(2,4,6,-trimethoxyphenyl)dodecanamide), was also found to be a potent LPAT inhibitor.

Role of cathepsin S-dependent epithelial cell apoptosis in IFN-gamma-induced alveolar remodeling and pulmonary emphysema.

Th1/Tc1 inflammation and remodeling responses characterized by tissue atrophy and destruction frequently coexist in human diseases and disorders. However, the mechanisms that are used by Th1/Tc1 cytokines, like IFN-gamma, to induce these responses have not been defined. To elucidate the mechanism(s) of IFN-gamma-induced tissue remodeling and destruction, we characterized the pathway that lung-targeted, transgenic IFN-gamma uses to induce alveolar remodeling in a murine pulmonary emphysema modeling system.

Bioisosteric replacement of anilide with benzoxazole: potent and orally bioavailable antagonists of VLA-4.

We have designed and synthesized a series of heterocyclic bioisosteres for an anilide based on molecular modeling. Excellent potency was retained in the benzoxazole and the benzimidazole derivatives, where a hydrogen bond acceptor is appropriately positioned to mimic the amide bond oxygen. The deletion of the hydrogen bond donor (N-H) led to improved lipophilicity and bioavailability.

Amidines as amide bond replacements in VLA-4 antagonists.

VLA-4 (alpha(4)beta(1), very late activating antigen-4), a key cell surface integrin plays an important role in inflammation by promoting leukocyte attachment and extravasation from the vasculature into the peripheral tissues. As such, VLA-4 antagonists may be useful in the treatment, prevention, and suppression of diseases where cell adhesion and migration are important such as asthma, rheumatoid arthritis, and multiple sclerosis. Herein, we report on the discovery, synthesis, and biological evaluation of amidines as small molecule antagonists of VLA-4.

A small molecule alpha4beta1/alpha4beta7 antagonist differentiates between the low-affinity states of alpha4beta1 and alpha4beta7: characterization of divalent cation dependence.

An alpha4beta1/alpha4beta7 dual antagonist, 35S-compound 1, was used as a model ligand to study the effect of divalent cations on the activation state and ligand binding properties of alpha4 integrins. In the presence of 1 mM each Ca2+/Mg2+, 35S-compound 1 bound to several cell lines expressing both alpha4beta1 and alpha4beta7, but 2S-[(1-benzenesulfonyl-pyrrolidine-2S-carbonyl)-amino]-4-[4-methyl-2S-(methyl-[2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl]-amino) pentanoylamino]-butyric acid (BIO7662), a specific alpha4beta1 antagonist, completely inhibited 35S-compound 1 binding, suggesting that alpha4beta1 was responsible for the observed binding. 35S-Compound 1 bound RPMI-8866 cells expressing predominantly alpha4beta7 with a KD of 1.

Substituted 3-amino biaryl propionic acids as potent VLA-4 antagonists.

A series of substituted N-(3,5-dichlorobenzenesulfonyl)-(L)-prolyl- and (L)-azetidyl-beta-biaryl beta-alanine derivatives was prepared as selective and potent VLA-4 antagonists. The 2,6-dioxygenated biaryl substitution pattern is important for optimizing potency. Oral bioavailability was variable and may be a result of binding to circulating plasma proteins.

N-aryl-prolyl-dipeptides as potent antagonists of VLA-4.

The design, synthesis, and biological evaluation of N-arylprolyl-dipeptide derivatives as small molecule VLA-4 antagonists is described. Potency against VLA-4 and alpha(4)beta(7) and rat pharmacokinetic evaluation revealed some advantages over the related N-(arylsulfonyl)-prolyl-dipeptide analogues.

N-(arylacetyl)-biphenylalanines as potent VLA-4 antagonists.

A series of potent N-(aralkyl-, arylcycloalkyl-, and heteroaryl-acyl)-4-biphenylalanine VLA-4 antagonists was prepared by rapid analogue methods using solid-phase chemistry. Further optimization led to several highly potent compounds (IC(50) <1 nM). Evaluation of rat pharmacokinetic revealed generally high clearance.

Alpha(4)beta(7)/alpha(4)beta(1) dual integrin antagonists block alpha(4)beta(7)-dependent adhesion under shear flow.

The alpha(4) integrin, alpha(4)beta(7), plays an important role in recruiting circulating lymphocytes to the gastrointestinal tract, where its ligand mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is preferentially expressed on high endothelial venules (HEVs). Dual antagonists of alpha(4)beta(1) and alpha(4)beta(7), N-(2,6-dichlorobenzoyl)-(L)-4-(2',6'-bis-methoxyphenyl)phenylalanine (TR14035) and N-(N-[(3,5-dichlorobenzene)sulfonyl]-2-(R)-methylpropyl)-(D)-phenylalanine (compound 1), were tested for their ability to block the binding of alpha(4)beta(7)-expressing cells to soluble ligand in suspension and under in vitro and in vivo shear flow. Compound 1 and TR14035 blocked the binding of human alpha(4)beta(7) to an (125)I-MAdCAM-Ig fusion protein with IC(50) values of 2.

Substituted tetrahydrofuroyl-1-phenylalanine derivatives as potent and specific VLA-4 antagonists.

A series of substituted tetrahydrofuroyl-1-phenylalanine derivatives was prepared and evaluated as VLA-4 antagonists. Substitution of the alpha carbon of the tetrahydrofuran with aryl groups increased the specificity for VLA-4 versus alpha(4)beta(7) while amide substitution increased the potency of the series without increasing the specificity. Substitution of the beta carbon of the tetrahydrofuran with keto or amino groups slightly improved the specificity for VLA-4 versus alpha(4)beta(7) but with a significant loss in binding affinity for VLA-4.

N-Tetrahydrofuroyl-(L)-phenylalanine derivatives as potent VLA-4 antagonists.

Given the proposed involvement of VLA-4 in inflammatory processes, a program to identify orally active VLA-4 antagonists was initiated. Herein, we report the discovery of a N-tetrahydrofuroyl-(L)-phenylalanine derivative (17) and related analogues as potent VLA-4 antagonists with good oral bioavailability.

Substituted N-(3,5-dichlorobenzenesulfonyl)-L-prolyl-phenylalanine analogues as potent VLA-4 antagonists.

A series of substituted N-(3,5-dichlorobenzenesulfonyl)-L-prolyl- and alpha-methyl-L-prolyl-phenylalanine derivatives was prepared as VLA-4/VCAM antagonists. The compounds showed excellent potency with a wide variety of neutral, polar, electron withdrawing or donating groups on the phenylalanine ring (IC50 approximately 1 nM). Heteroaryl ring substitution for phenylalanine was also well tolerated.

The discovery of acylated beta-amino acids as potent and orally bioavailable VLA-4 antagonists.

Acylated beta-amino acids are described as potent, specific and orally bioavailable antagonists of VLA-4. The initial lead was identified from a combinatorial library. Subsequent optimization using a traditional medicinal chemistry approach led to significant improvement in potency (up to 8-fold) while maintaining good pharmacokinetic properties.

Effects of VLA-4 antagonists in rat whole embryo culture.

Background: Pharmacological antagonism of VLA-4 (Very Late Antigen 4, alpha(4)beta(1) integrin) has become an attractive target for the treatment of predominantly eosinophil mediated disease states such as asthma, allergic rhinitis, multiple sclerosis, rheumatoid arthritis, diabetes, and inflammatory bowel disease. Gene knockouts of the alpha(4)-integrin subunit of VLA-4 or its cell surface ligand, VCAM-1, however, have been shown to result in embryo-lethality in homozygous null mice due to defects in chorio-allantoic or epi-myocardial fusion. Although gene knockout phenotypes are not always manifested by pharmacological antagonism, those studies suggested that VLA-4 antagonists might cause embryo-lethality or drug-induced malformations.