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PubMed2003

Microtiter cell-based assay for detection of agents that alter cellular levels of Her2 and EGFR.

Overexpression of the transmembrane tyrosine kinases Her2 and EGFR is associated with aggressive malignancies, and several therapeutic strategies targeting the two receptors are now in various stages of clinical development. Most of the known agents block the activation or inhibit the activity of the kinases; however, a more significant therapeutic outcome may result from degrading these oncoproteins. Here, we report the development of a microtiter cell-based assay that sensitively detects cellular levels of Her2 and EGFR. The assay is useful in identifying small molecules that alter cellular levels of these kinases and in quantifying their effect. The method gives results comparable to Western blot, but it is faster, less labor intensive, and amenable to high throughput.

Actins↗

PubMed2002

Multiple forms of U2 snRNA coexist in the silk moth Bombyx mori.

Eight U2 snRNA variants were isolated from several Bombyx mori U2-specific RT-PCR libraries. U2 sequences and secondary structures were generated and examined in terms of potential RNA and protein interactions. Analysis indicated that nucleotide changes occurred in both stem/loop and single-stranded areas. Changes in the double stranded areas were either compensatory, single substitutions (e.g. C <--> U) or prevented the double-stranded formation of one or two base pairs. The polymorphisms were clustered in moderately conserved regions. Some of the changes observed generated stronger base pairing. Inter-species conserved protein or RNA-binding sites were relatively unaffected. No polymorphic sites were found in known functional sequences. Bombyx mori and Drosophila melanogaster U2 sequences are 95% and 70% similar at the 5'- and the 3'-ends of the molecule, respectively. Phylogenetic analysis of the U2 sequences demonstrates remarkable conservation across species.

Animals↗

PubMed1998

Identification and characterization of two thrombin-activatable fibrinolysis inhibitor isoforms.

Thrombin-activatable fibrinolysis inhibitor (TAFI) is synthesized by the liver and is thought to circulate in plasma as a plasminogen-bound zymogen. When it is activated by the thrombin/thrombomodulin complex, activated TAFI exhibits carboxypeptidase B-like activity. To study the structure-function relationship of TAFI, we expressed recombinant human TAFI in insect cells. During the cloning of TAFI cDNA from several human liver cDNA libraries, we identified a second TAFI cDNA which differed from the published sequence at 2 positions. One of these sequences resulted in a substitution of alanine for threonine at residue 147, the other was a silent mutation. These substitutions were found in several cDNA libraries from different sources. Using Southern blot analysis, we confirmed the existence of this TAFI polymorphism in the population. In order to compare the activation and activity of TAFI isoforms, we expressed both isoforms in the baculovirus expression system, and compared the enzyme kinetics of the purified proteins. The molecular weight of recombinant TAFI is lower than plasma TAFI due to differences in glycosylation. The two recombinant TAFI isoforms had similar activation kinetics and the activated enzymes had similar carboxypeptidase B-like activity towards small molecule substrates. Their ability to retard clot lysis was found to be similar in a plate clot lysis assay.

Animals↗

PubMed2000

Decreased expression of a member of the Rho GTPase family, Cdc42Hs, in cells from Tangier disease - the small G protein may play a role in cholesterol efflux.

Cholesterol efflux (CE) is the initial and important step of reverse cholesterol transport (RCT), a major protective system against atherosclerosis. However, most of the molecular mechanism for CE still remains to be clarified. In the present study, cDNA subtraction revealed that the expression of a member of the Rho GTPase family, Cdc42Hs, was markedly decreased in both passaged fibroblasts and macrophages (Mφ) from patients with Tangier disease (TD), a rare lipoprotein disorder with reduced CE. This small G protein is known to have many cell biological activities such as rearrangement of actin cytoskeleton and vesicular transport, however the association between this molecule and lipid transport has never been reported. We demonstrate that MDCK cells expressing the dominant negative form of Cdc42Hs had reduced CE, inversely ones expressing the dominant active form had increased CE. From these observations, we would like to raise a novel hypothesis that this type of small G protein may play a role in some steps of CE. To our knowledge, the present study is the first demonstration that the expression of this molecule is altered in cells from human disease.

Animals↗

PubMed1998

Protein tyrosine kinases: structure, substrate specificity, and drug discovery.

Protein tyrosine kinases (PTKs) play a crucial role in many cell regulatory processes. It is therefore not surprising to see that functional perturbation of PTKs results in many diseases. Despite the diverse primary structure organization of various PTKs, the catalytic or kinase domains of various PTKs as well as that of Ser/Thr kinases are generally conserved. The high resolution crystal structure of a few PTKs has been solved in the last few years. In contrast to the well-defined linear peptide substrate motifs recognized by specific Ser/Thr kinases, the identification of specific substrate motifs for PTK has been slow. It is not until recently that through the use of combinatorial peptide library methods that specific recognition motifs for specific PTKs have begun to emerge. Efficient and specific peptide substrates for some PTKs with Km at the mid microM range have been identified. Based on these peptide substrates, relatively potent (IC50 at the low microM range) and highly selective pseudosubstrate-based peptide inhibitors have been developed. There has been enormous effort in the development of PTK inhibitors for diseases such as cancer, psoriasis, and osteoporosis. Several new high-throughput PTK assay technologies have recently been described. Small molecules against specific PTK have been developed. Most of them are competitive inhibitors at the ATP binding site. Some of these inhibitors have already been in clinical trial.

Amino Acid Sequence↗

PubMed1984

Localization of small nuclear polymerase I RNA sequences at the 5' end of the human rDNA transcription unit.

A human ribosomal DNA clone isolated from a genomic library was used to localize the DNA sequences coding for HeLa cell small nuclear polymerase I RNA (snPI RNA). By using a subcloned 1.2-kb EcoRI-SalI fragment, including the initiation region of the 45S rRNA transcription unit, it was shown that the snPI RNA sequences are located within the first 600 nucleotides of the 5' end of the external transcribed spacer. Strand-specific hybridization following exonuclease III digestion of the plasmid containing the 1.2-kb Eco-Sal fragment demonstrated that the snPI RNA molecules and the 45S pre-rRNA are transcribed from the same coding strand. A detailed mapping of individual snPI RNA molecules showed that most of these small RNA species span the putative early processing site at position 415 of the external transcribed spacer of the human rRNA precursor.

Base Sequence↗

PubMed1993

Molecular cloning of human von Ebner's gland protein, a member of the lipocalin superfamily highly expressed in lingual salivary glands.

Von Ebner's glands (VEG) are small lingual salivary glands. Their ducts open into trenches of circumvallate and foliate papillae, thus influencing the milieu where the interaction between taste receptor cells and sapid molecules takes place. The major secretions of human VEG is a protein with a molecular mass of 18 kDa. The human VEG protein crossreacts with antibodies raised against the rat VEG protein, indicating sequence similarity between the rat and human VEG proteins. This was subsequently confirmed by N-terminal protein sequencing. A cDNA clone, isolated from a human VEG library, contained an insert of 735 bp including an open reading frame that encodes the human VEG protein of 176 amino acids. Comparison of the human and rat VEG proteins revealed an overall identity of 60%. Immunocytochemistry, in situ hybridization and in vitro translation studies demonstrated the human VEG protein to be highly and exclusively expressed in VEG. The VEG proteins are members of the lipocalin protein superfamily and, together with the rat odorant binding protein II, they constitute a new subfamily. Sequence similarity to proteins such as the retinol binding protein and the odorant binding protein which are lipophilic ligand carriers, suggests a possible function for the human VEG protein in taste perception.

Amino Acid Sequence↗

PubMed2002

Combinatorial informatics in the post-genomics ERA.

The multitude of potential drug targets emerging from genome sequencing demands new approaches to drug discovery. A chemogenomics strategy, which involves the generation of small-molecule compounds that can be used both as tools to probe biological mechanisms and as leads for drug-property optimization, provides a highly parallel, industrialized solution. Key to the success of this strategy is an integrated suite of chemi-informatics applications that can allow the rapid and directed optimization of chemical compounds with drug-like properties using 'just-in-time' combinatorial chemical synthesis. An effective embodiment of this process requires new computational and data-mining tools that cover all aspects of library generation, compound selection and experimental design, and work effectively on a massive scale.

Combinatorial Chemistry Techniques↗

PubMed1995

Selection of peptide inhibitors of interactions involved in complex protein assemblies: association of the core and surface antigens of hepatitis B virus.

As an example for studies of contacts involved in complex biological systems, peptide ligands that bind to the core antigen of hepatitis B virus (HBcAg) have been selected from a random hexapeptide library displayed on filamentous phage. Affinity-purified phage bearing aa sequence LLGRMK, or some related sequences, bound full-length or truncated HBcAg but did not bind denatured HBcAg. The long (L), but not the short (S), hepatitis B virus envelope polypeptide, when synthesized in an in vitro system, bound firmly to HBcAg, indicating that interaction between HBcAg and the pre-S region of the L polypeptide is critical for virus morphogenesis. This interaction was inhibited by peptide ALLGRMKG, suggesting that this and related small molecules may inhibit viral assembly.

Amino Acid Sequence↗

PubMed2004

Overcoming compound interference in fluorescence polarization-based kinase assays using far-red tracers.

Kinase-mediated phosphorylation of proteins is critical to the regulation of many biological processes, including cell growth, apoptosis, and differentiation. Because of the central role that kinases play in processes that can lead to disease states, the targeting of kinases with small-molecule inhibitors is a validated strategy for therapeutic intervention. Classic methods for assaying kinases include nonhomogenous enzyme-linked immunosorbent assays or scintillation-based formats using [gamma-(32)P]ATP. However, homogenous fluorescence-based assays have gained in popularity in recent years due to decreased costs in reagent usage through miniaturization, increased throughput, and avoidance of regulatory costs associated with the use of radiation. Whereas the readout signal from a nonhomogenous or radioactive assay is largely impervious to interferences from matrix components (such as library compounds), all homogenous fluorescent assay formats are subject to such interferences. Interference from intrinsically fluorescent compounds or from scattered light due to precipitated compounds can interfere with assays that depend on a fluorescence intensity (or fluorescence quenching), fluorescence resonance energy transfer, or fluorescence polarization-based readout. Because these interfering factors show a greater effect at lower wavelengths, one strategy to overcome such interferences is to develop fluorescent assays using longer wavelength (red-shifted) fluorescent probes. In this article, we describe the PanVera PolarScreen far-red fluorescence polarization assay format, which mitigates assay interference from autofluorescent compounds or scattered light through the use of a far-red tracer. The tracer shows substantially less interference from light scatter or autofluorescent library compounds than do fluorescein-based tracers, and gives rise to a larger assay window than the popular far-red fluorophore Cy5.

Cyclic AMP-Dependent Protein Kinases↗

PubMed1995

Molecular cloning and characterization of an inner ear-specific structural protein.

Molecular biological studies of the mammalian inner ear have been limited by the relatively small size of the sensory endorgans contained within. The saccular otolithic organ in teleostian fish is structurally similar to its mammalian counterpart but can contain an order of magnitude more sensory cells. The prospect of the evolutionary conservation of proteins utilized in the vertebrate inner ear and the relative abundance of teleostian saccular sensory tissue made this an attractive system for molecular biological studies. A complementary DNA obtained by differential screening of a saccular complementary DNA library was identified that encodes an inner ear-specific collagen molecule.

Amino Acid Sequence↗

PubMed2005

Homogeneous high-throughput screening assays for HIV-1 integrase 3beta-processing and strand transfer activities.

HIV-1 integrase (HIV-IN) is a well-validated antiviral drug target catalyzing a multistep reaction to incorporate the HIV-1 provirus into the genome of the host cell. Small molecule inhibitors of HIV-1 integrase that specifically target the strand transfer step have demonstrated efficacy in the suppression of virus propagation. However, only few specific strand transfer inhibitors have been identified to date, and the need to screen for novel compound scaffolds persists. Here, the authors describe 2 homogeneous time-resolved fluorescent resonance energy transfer-based assays for the measurement of HIV-1 integrase 3'-processing and strand transfer activities. Both assays were optimized for high-throughput screening formats, and a diverse library containing more than 1 million compounds was screened in 1536-well plates for HIV-IN strand transfer inhibitors. As a result, compounds were found that selectively affect the enzymatic strand transfer reaction over 3beta processing. Moreover, several bioactive molecules were identified that inhibited HIV-1 reporter virus infection in cellular model systems. In conclusion, the assays presented herein have proven their utility for the identification of mechanistically interesting and biologically active inhibitors of HIV-1 integrase that hold potential for further development into potent antiviral drugs.

Antiviral Agents↗

PubMed2003

Non-peptidic small-molecule antagonists of the human platelet thrombin receptor PAR-1.

The thrombin receptor on human platelets is the first member identified of a new family of G-protein coupled receptors referred to as protease activated receptors (PARs). These receptors are activated by a unique mechanism involving proteolytic cleavage of a portion of the extracellular domain to generate a new N-terminus which then acts as a tethered or intramolecular ligand (agonist) for the receptor. The hexapeptide SFLLRN-NH2 comprising the new N-terminus is referred to as the Thrombin Receptor Activating Peptide, or "TRAP" Thrombin is the most potent agonist for platelet aggregation and selective blockade of the intramolecular activation step without effecting the proteolytic activity of thrombin should result in moderation of platelet activation and aggregation without interfering with the other coagulation cascade effects of thrombin. Screening of combinatorial libraries identified a novel, non-peptide PAR-1 thrombin receptor antagonist. Examination of structure-activity relationships revealed that portions of the molecule could be replaced resulting in simpler molecules of lower molecular weight that were at the same time more potent. Molecules in this series were effective antagonists of TRAP-stimulated platelet activation, but had limited activity when thrombin was the agonist. Additional directed screening and subsequent lead refinement resulted in a second series of isoxazole based compounds. Some of the resultant molecules were potent PAR-1 antagonists that were effective against both TRAP- and thrombin-stimulated receptor activation. These compounds do not inhibit the proteolytic effects of thrombin but rather interfere with the intramolecular binding of the tethered ligand (SFLLRN) to the transmembrane portion of the thrombin receptor. They represent promising leads for future explorations of antithrombotic activity of thrombin receptor antagonists.

Blood Platelets↗

PubMed1999

CMS: an adapter molecule involved in cytoskeletal rearrangements.

Cas ligand with multiple Src homology (SH) 3 domains (CMS) is an ubiquitously expressed signal transduction molecule that interacts with the focal adhesion protein p130(Cas). CMS contains three SH3 in its NH2 terminus and proline-rich sequences in its center region. The latter sequences mediate the binding to the SH3 domains of p130(Cas), Src-family kinases, p85 subunit of phosphatidylinositol 3-kinase, and Grb2. The COOH-terminal region contains putative actin binding sites and a coiled-coil domain that mediates homodimerization of CMS. CMS is a cytoplasmic protein that colocalizes with F-actin and p130(Cas) to membrane ruffles and leading edges of cells. Ectopic expression of CMS in COS-7 cells resulted in alteration in arrangement of the actin cytoskeleton. We observed a diffuse distribution of actin in small dots and less actin fiber formation. Altogether, these features suggest that CMS functions as a scaffolding molecule with a specialized role in regulation of the actin cytoskeleton.

Adaptor Proteins, Signal Transducing↗

PubMed2006

The mitochondrial effects of small organic ligands of BCL-2: sensitization of BCL-2-overexpressing cells to apoptosis by a pyrimidine-2,4,6-trione derivative.

We have investigated the mitochondrial effects of BH3I-2', Chelerythrine, and HA14-1, small organic molecules that share the ability to bind the BH3 domain of BCL-2. All compounds displayed a biphasic effect on mitochondrial respiration with uncoupling at low concentrations and respiratory inhibition at higher concentrations, the relative uncoupling potency being BH3I-2' (half-maximal uncoupling at about 80 nm) > Chelerythrine (half-maximal uncoupling at about 2 microm) > HA14-1 (half-maximal uncoupling at about 20 microm). At concentrations lower than required for uncoupling all compounds sensitized the permeability transition pore (PTP) to opening both in isolated mitochondria and intact cells. To assess whether the effects on BCL-2 binding, PTP induction and respiration could be due to different structural determinants we have tested a set of HA14-1 analogs from the Hoffmann-La Roche chemical library. We have identified 5-(6-chloro-2,4-dioxo-1,3,4,10-tetrahydro-2H-9-oxa-1,3-diaza-anthracen-10-yl)-pyrimidine-2,4,6-trione (EM20-25) as a molecule devoid of effects on respiration that is able to induce PTP opening, to disrupt the BCL-2/BAX interactions in situ and to activate caspase-9 in BCL-2-overexpressing cells. EM20-25 neutralized the antiapoptotic activity of overexpressed BCL-2 toward staurosporine and sensitized BCL-2-expressing cells from leukemic patients to the killing effects of staurosporine, chlorambucil, and fludarabine. These results provide a proof of principle that the potentially toxic effects of BCL-2 ligands on mitochondrial respiration are not essential for their antiapoptotic activity and represent an important step forward in the development of tumor-selective drugs acting on BCL-2.

Alkaloids↗

PubMed2004

Dissection of the dislocation pathway for type I membrane proteins with a new small molecule inhibitor, eeyarestatin.

The mammalian endoplasmic reticulum (ER)-to-cytosol degradation pathway for disposal of misfolded proteins is an attractive target for therapeutic intervention in diseases that are characterized by impaired protein degradation. The ability to do so is hampered by the small number of specific inhibitors available and by our limited understanding of the individual steps involved in this pathway. Cells that express a class I major histocompatibility complex (MHC) heavy chain-enhanced green fluorescent protein (EGFP) fusion protein and the human cytomegalovirus protein US11, which catalyzes dislocation of the class I MHC EGFP reporter, show only little fluorescence. Treatment with proteasome inhibitors increases their fluorescence by stabilizing EGFP-tagged MHC class I molecules. We used this change in signal intensity as a readout to screen a chemical library of 16,320 compounds and identified two structurally related compounds (eeyarestatin I and II) that interfered with the degradation of both EGFP-heavy chain and its endogenous unmodified class I MHC heavy chain counterpart. Eeyarestatin I also inhibited degradation of a second misfolded type I membrane protein, T-cell receptor alpha. Both compounds stabilize these dislocation substrates in the ER membrane, without preventing proteasomal turnover of cytosolic substrates. The new inhibitors must therefore interfere with a step that precedes proteasomal degradation. The use of eeyarestatin I thus allows the definition of a new intermediate in dislocation.

Catalysis↗

PubMed1987

Complementary deoxyribonucleic acid cloning of bovine transforming growth factor-beta 1.

Transforming growth factor-beta 1 (TGF beta 1) has been purified from a number of different sources and has a broad species specificity. To deduce the complete amino acid sequence of bovine TGF beta 1 we have isolated cDNA clones encoding the protein from a bovine fibropapilloma library using a human cDNA probe. Sequence analysis of two independent cDNA clones revealed that the 112 amino acids corresponding to bovine TGF beta 1 are identical to those of the human and porcine proteins. This unusually high degree of conservation in the primary structure of the human and bovine proteins reflects the strong evolutionary constraints for maintenance of structure and function of the molecule. As in the human, murine, and porcine systems, the mature form of TGF beta 1 is derived by proteolytic cleavage of a larger precursor. Small differences in amino acid sequence were observed in the portion of the precursor that does not include mature TGF beta 1, although 92% of the residues are still conserved. A 2.25 kilobase (kb) mRNA was identified in total bovine wart and bone RNA, whereas no message was detected in polyadenylated spleen or brain RNA. In addition to the major 2.25 kb message, we observed a 1.9 kb transcript in poly(A+) RNA from wart tissue.

Amino Acid Sequence↗

PubMed2004

Photo-affinity labeling strategies in identifying the protein ligands of bioactive small molecules: examples of targeted synthesis of drug analog photoprobes.

The discovery of many new targets by chemical genetics has frequently exploited the fact that their biologically active chemical ligands were reactive and thus could covalently bind to their protein target(s). When experimental compounds or therapeutic agents with unidentified mechanisms of action do not contain reactive groups that can covalently label the putative site of molecular action, it may be possible to create a reactive photo-affinity probe if there is sufficient knowledge of the structure-activity relationship of the chemical series. Two specific examples are presented. These include the use of photo-affinity probes in the identification of the mechanism of action of synthetic oxazolidinones, a class of novel acting antibiotics and in the identification of a novel target for the insulin-sensitizing thiazolidinediones. Developments in photo-affinity labeling and combinatorial library design now imply that the parallel incorporation of photo-probes into screening library design could, at least in principle, greatly facilitate reverse pharmacological and chemical genetics approaches to protein target discovery.

Animals↗