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PubMed2004

In vitro screening for inhibitors of the human mitotic kinesin Eg5 with antimitotic and antitumor activities.

Human Eg5, a member of the kinesin superfamily, plays a key role in mitosis, as it is required for the formation of a bipolar spindle. We describe here the first in vitro microtubule-activated ATPase-based assay for the identification of small-molecule inhibitors of Eg5. We screened preselected libraries obtained from the National Cancer Institute and identified S-trityl-L-cysteine as the most effective Eg5 inhibitor with an IC50 of 1.0 micromol/L for the inhibition of basal ATPase activity and 140 nmol/L for the microtubule-activated ATPase activity. Subsequent cell-based assays revealed that S-trityl-L-cysteine induced mitotic arrest in HeLa cells (IC50, 700 nmol/L) with characteristic monoastral spindles. S-trityl-L-cysteine is 36 times more potent for inducing mitotic arrest than the well-studied inhibitor, monastrol. Gossypol, flexeril, and two phenothiazine analogues were also identified as Eg5 inhibitors, and we found that they all result in monoastral spindles in HeLa cells. It is notable that all the Eg5 inhibitors identified here have been shown previously to inhibit tumor cell line growth in the NCI 60 tumor cell line screen, and we conclude that their antitumor activity may at least in part be explained by their ability to inhibit Eg5 activity.

Adenosine Triphosphatases↗
PubMed1997

In vitro pharmacological characterization of PD 166285, a new nanomolar potent and broadly active protein tyrosine kinase inhibitor.

PD 166285, a novel protein tyrosine kinase inhibitor of a new structural class, the 6-aryl-pyrido[2,3-d]pyrimidines, was synthesized as the most potent and soluble analog of a series of small molecules originally identified by screening a compound library with assays that measured protein tyrosine kinase activity. PD 166285 was found to inhibit Src nonreceptor tyrosine kinase, fibroblast growth factor receptor-1, epidermal growth factor receptor and platelet-derived growth factor receptor beta subunit (PDGFR-beta), tyrosine kinases with half-maximal inhibitory potencies (IC50 values) of 8.4 +/- 2.3 nM (n = 6), 39.3 +/- 2.8 nM (n = 16), 87.5 +/- 13.7 nM (n = 6) and 98.3 +/- 7.9 nM (n = 16), respectively. PD 166285 also demonstrated inhibitory activity against mitogen-activated protein kinase (IC50 = 5 microM) and protein kinase C (IC50 = 22.7 microM). PD 166285 was further characterized as an ATP competitive inhibitor of Src nonreceptor tyrosine kinase, PDGFR-beta, fibroblast growth factor receptor-1 and epidermal growth factor receptor tyrosine kinases. In addition, PD 166285 inhibited PDGF- and EGF-stimulated receptor autophosphorylation in vascular smooth muscle cells (VSMCs) and A431 cells, respectively, and basic fibroblast growth factor-mediated tyrosine phosphorylation in Sf9 cells, with IC50 values of 6.5 nM, 1.6 microM and 97.3 nM, respectively, further establishing a tyrosine kinase mechanism of inhibition. The inhibition of PDGF receptor autophosphorylation in VSMCs by PD 166285 was long lasting and persisted for 4 days after a single 1-hr exposure followed by extensive washing. The PDGF-induced tyrosine phosphorylation of the 44- and 42-kDa mitogen-activated protein kinase isoforms was also blocked as a result of the inhibition of PDGF-stimulated receptor autophosphorylation by PD 166285 in VSMCs. The effects of PD 166285 were also demonstrated in functional assays of cell attachment, migration and proliferation, in which vascular cell adhesion to vitronectin, PDGF-directed chemotaxis and serum-stimulated cell growth were all potently inhibited with IC50 values of 80 yo 120 nM. Finally, PD 166285 uniquely demonstrated potent inhibition of phorbol ester-induced production of 92-kDa gelatinase A (MMP-9) in VSMC without affecting 72-kDa gelatinase B (MMP-2) as measured by gelatin zymography. These results highlight the biological characteristics of PD 166285 as a broadly active protein tyrosine kinase capable of potently inhibiting a number of kinase mediated cellular functions, including cell attachment, movement and replication. The potential therapeutic utility of this broadly acting inhibitor as an antiproliferative and antimigratory agent could extend to such diseases as cancer, atherosclerosis and restenosis, in which redundancies in protein kinase signaling pathways are known to exist.

Animals↗
PubMed2003

Discovery and optimization of non-steroidal FXR agonists from natural product-like libraries.

The efficient regulation of cholesterol biosynthesis, metabolism, acquisition, and transport is an essential component of lipid homeostasis. The farnesoid X receptor (FXR) is a transcriptional sensor for bile acids, the primary product of cholesterol metabolism. Accordingly, the development of potent, selective, small molecule agonists, partial agonists, and antagonists of FXR would be an important step in further deconvoluting FXR physiology. Herein, we describe the development of four novel classes of potent FXR activators originating from natural product-like libraries. Initial screening of a 10,000-membered, diversity-orientated library of benzopyran containing small molecules for FXR activation utilizing a cell-based reporter assay led to the identification of several lead compounds possessing low micromolar activity (EC50's = 5-10 microM). These compounds were systematically optimized employing parallel solution-phase synthesis and solid-phase synthesis to provide four classes of compounds that potently activate FXR. Two series of compounds, bearing stilbene or biaryl moieties, contain members that are the most potent FXR agonists reported to date in cell-based assays. These compounds may find future utility as chemical tools in studies aimed at further defining the physiological role of FXR and discovering potential therapeutic agents for the treatment of diseases linked to cholesterol and bile acid metabolism and homeostasis.

Benzopyrans↗
PubMedDate not supplied

A combinatorial approach toward the discovery of non-peptide, subtype-selective somatostatin receptor ligands.

The tetradecapeptide somatostatin is widely distributed throughout the body and is thought to be involved with a variety of regulatory functions. Recently, five human somatostatin receptors (hSSTR1-5) have been cloned and characterized. Several selective peptidal agonists of the hSSTR receptors are known, and we sought to apply this information to the design of novel non-peptide small molecule ligands for each receptor. Initial computational methods identified a 200 nM murine SSTR2 active compound via a database search of our sample collection. A combinatorial library was designed around the structural class of the compound with the goal of rapidly developing this initial lead into the desired subtype-selective small molecules in order to characterize the pharmacology of each of the receptor subtypes. The library was synthesized using the resin-archive, iterative deconvolution format. The total number of unique compounds in the library was expected to be 131,670, present in 79 mixtures of 1330 or 2660 compounds per mixture. Through sequences of screening and mixture deconvolution, the components of selective and highly active (Ki = 50 pM to 200 nM) non-peptide small molecule ligands for somatostatin subtypes 1, 2, 4, and 5 were identified. In addition to discovering compounds with the desired activity and selectivity, useful structure/activity information was generated which can be used in the design of new compounds and second-generation combinatorial libraries.

Combinatorial Chemistry Techniques↗
PubMed2005

1,4-Benzodiazepine-2,5-diones as small molecule antagonists of the HDM2-p53 interaction: discovery and SAR.

A library of 1,4-benzodiazepine-2,5-diones was screened for binding to the p53-binding domain of HDM2 using Thermofluor, a miniaturized thermal denaturation assay. The hits obtained were shown to bind to HDM2 in the p53-binding pocket using a fluorescence polarization (FP) peptide displacement assay. The potency of the series was optimized, leading to sub-micromolar antagonists of the p53-HDM2 interaction.

Benzodiazepines↗
PubMed1999

Small molecule antagonists of the bradykinin B1 receptor.

Screening Pharmacopeia's encoded combinatorial libraries has led to the identification of potent, selective, competitive antagonists at the bradykinin B1 receptor. Libraries were screened using a displacement assay of [3H]-des-Arglo-kallidin ([3H]-dAK) at IMR-90 cells expressing an endogenous human B1 receptor (Bmax = 20,000 receptors/cell, K(D) = 0.5+/-0.1 nM) or against membranes from 293E cells expressing a recombinant human B1 receptor (Bmax = 8,000 receptors/cell, K(D) = 0.5 +/- 0.3 nM). Compound PS020990, an optimized, representative member from the class of compounds, inhibits specific binding of 3H-dAK at IMR-90 cells with a KI of 6 +/- 1 nM. The compound inhibits dAK-induced phosphatidyl inositol turnover (K(Bapp) = 0.4 +/- 0.2 nM) and calcium mobilization (K(Bapp) = 17 +/- 2 nM) in IMR-90 cells. Compounds from the lead series are inactive at the B2 receptor and are > 1000-fold specific for B1 vs. a variety of other receptors, ion channels and enzymes. PS020990 and other related chemotypes therefore offer an excellent opportunity to explore further the role of B1 receptors in disease models and represent a potential therapeutic avenue.

Bradykinin↗
PubMed2003

Use of multicomponent, domino, and other one-pot syntheses on solid phase: powerful tools for the generation of libraries of diverse and complex compounds.

The availability of small organic molecules covering as much chemical space as possible is seen as the only means that guarantees potential modulation of the many biological targets that are ultimately being unveiled by genomics. Therefore diversity oriented organic synthesis is rapidly becoming one of the paradigms in the process of modern drug discovery. This has spurred research in those fields of chemical investigation that lead to the rapid assembly of not only molecular diversity, but also molecular complexity. As a consequence multi-component as well as domino or related reactions are witnessing a new spring. Coupling these one-pot processes with solid-phase synthesis offers new perspectives for the preparation of both primary and thematic libraries. The progresses recently made in this field that perfectly suits the needs of modern drug discovery are the subject of the present review.

Chemistry, Organic↗
PubMed2004

Induction of reversible hemolytic anemia in living zebrafish using a novel small molecule.

We used zebrafish to screen and identify small molecules that affect the process of vertebrate hematopoietic development. Zebrafish embryos were exposed to a library of 5000 synthetic compounds and screened for defects in primitive erythropoiesis. Here, we present the characterization of hemolytic anemia induced in zebrafish by the small molecule 5115318 (3-[5-methyl-furan 2-yl]-propionic acid N'-phenyl-hydrazide). This compound is capable of generating hemoglobin aggregates and Heinz bodies in red cells in vivo only. The induced anemia is reversible and treated fish recover in about 4 days. This study shows the feasibility of using zebrafish to screen for small molecules that can modulate the specific process of erythropoiesis.

Anemia, Hemolytic↗
PubMed1999

An antibody single-domain phage display library of a native heavy chain variable region: isolation of functional single-domain VH molecules with a unique interface.

To develop very small antibody-derived recognition units for experimental, medical, and drug design purposes, a heavy chain variable region (VH) single-domain phage-display library was designed and constructed. The scaffold that was used for library construction was a native sequence of a monoclonal antibody with a unique VH/VL interface. There was no need to modify any residues in the VL interface to avoid non-specific binding of VH domain. The library repertoire, consisting of 4x10(8)independent clones, was generated by the randomization of nine amino acid residues in complementary determining region 3. The library was screened by binding to protein antigens, and individual clones were isolated. The VH genes encoding for specific binding clones were rescued and large amounts of soluble and stable single-domain VH protein were made from insoluble inclusion bodies by in vitro refolding and purification. Biochemical and biophysical characterization of the VH protein revealed a highly specific, correctly folded, and stable monomeric molecule. Binding studies demonstrated an affinity of 20 nM. The properties of these molecules make them attractive for clinical, industrial, and research applications, as well as a step toward improvement in the design of small molecules that are based on the hypervariable loops of antibodies.

Amino Acid Sequence↗
PubMed1999

Inhibition of gene expression in human cells through small molecule-RNA interactions.

Small molecules that bind their biological receptors with high affinity and selectivity can be isolated from randomized pools of combinatorial libraries. RNA-protein interactions are important in many cellular functions, including transcription, RNA splicing, and translation. One example of such interactions is the mechanism of trans-activation of HIV-1 gene expression that requires the interaction of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5' end of all nascent HIV-1 transcripts. Here we demonstrate the isolation of small TAR RNA-binding molecules from an encoded combinatorial library. We have made an encoded combinatorial tripeptide library of 24,389 possible members from D-and L-alpha amino acids on TentaGel resin. Using on-bead screening we have identified a small family of mostly heterochiral tripeptides capable of structure-specific binding to the bulge loop of TAR RNA. In vitro binding studies reveal stereospecific discrimination when the best tripeptide ligand is compared with diastereomeric peptide sequences. In addition, the most strongly binding tripeptide was shown to suppress transcriptional activation by Tat protein in human cells with an IC(50) of approximately 50 nM. Our results indicate that tripeptide RNA ligands are cell permeable, nontoxic to cells, and capable of inhibiting expression of specific genes by interfering with RNA-protein interactions.

Base Sequence↗
PubMedDate not supplied

A solid-phase, library synthesis of natural-product-like derivatives from an enantiomerically pure tetrahydroquinoline scaffold.

With the goal of developing a library synthesis of tetrahydroquinoline-derived natural-product-like small molecules, a practical synthesis of enantiomerically pure tetrahydroquinoline scaffold was achieved. An asymmetric aminohydroxylation reaction was the key step in this strategy. This scaffold was further immobilized onto the solid support for the library generation. The library was obtained from three diversity sites: (i) acylation of the hydroxyl group (R(1)), (ii) coupling of the Fmoc-protected amino acid to the amino group (R(2)), and (iii) amidation of the N-terminal amine group (R(3)).

Biological Factors↗
PubMed2004

Creation and discovery of ligand-receptor pairs for transcriptional control with small molecules.

The nuclear receptor retinoid X receptor (RXR) is a ligand-activated transcription factor. To create receptors for a new ligand, a structure-based approach was used to generate a library of approximately 380,000 mutant RXR genes. To discover functional variants within the library, we used chemical complementation, a method of protein engineering that uses the power of genetic selection. Wild-type RXR has an EC50 of 500 nM for 9-cis retinoic acid (9cRA) and an EC50 of >10 microM for the synthetic retinoid-like compound LG335 in yeast. The library produced ligand-receptor pairs with LG335 that have a variety of EC50 values (40 nM to >2 microM) and activation levels (10-80% of wild-type RXR with 9cRA) in yeast. The variant I268V;A272V;I310L;F313M has an EC50 for LG335 of 40 nM and an EC50 for 9cRA of >10 microM in yeast. This variant has essentially the reverse ligand specificity of wild-type RXR and is transcriptionally active at a 10-fold-lower ligand concentration in yeast. This EC50 is 25-fold lower than the best receptor we have engineered through site-directed mutagenesis, Q275C;I310M;F313I. Furthermore, the variants' EC50 values and activation levels in yeast and mammalian cells correlate. This protein engineering method should be extendable to produce other functional ligand-receptor pairs, which can be selected and characterized from libraries within weeks. Coupling large library construction with chemical complementation could be used to engineer proteins that bind virtually any small molecule for conditional gene expression, applications in metabolic engineering, and biosensors and to engineer enzymes through genetic selection.

Amino Acid Substitution↗
PubMedDate not supplied

Design, microwave-assisted synthesis, and photophysical properties of small molecule organic antennas for luminescence resonance energy transfer.

A highly efficient microwave-assisted method was successfully developed for the synthesis of a library of carbostyril analogues. The reaction time for synthesis of carbostyril analogues was drastically reduced from a reported 18-58 h to only 80 min. Compounds obtained directly from each synthesis were more than 90% pure and did not require any further purification. On the basis of the fluorescence spectra of the compounds in the initial library, four carbostyril analogues were designed. Two of these analogues showed very favorable fluorescence profiles and have the potential to be used as small molecule organic antennas for LRET studies.

Combinatorial Chemistry Techniques↗
PubMed2003

Biological characterization of a novel, orally active small molecule gonadotropin-releasing hormone (GnRH) antagonist using castrated and intact rats.

Gonadotropin-releasing hormone (GnRH) receptor antagonists have potential in treating numerous hormone-dependent pathologies including cancers of the prostate, breast, and ovary, endometriosis, and fertility disorders. An unmet clinical need exists for an orally available GnRH receptor antagonist. Guided by structure-activity relationships, ligand-based targeted library designs, and biomarker measurements, our discovery efforts have yielded a novel, small molecule GnRH receptor antagonist, 5-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthalenyl)methyl]-N-(2,4,6-trimethoxyphenyl)-2-furamide (CMPD1). CMPD1 bound with low nanomolar affinities to human, rat, and mouse GnRH receptors (6.0, 3.8, and 2.2 nM, respectively). CMPD1 was more than 100-fold selective for GnRH receptors versus various G-protein-coupled receptors and other enzymes and ion channels. In cells expressing recombinant rat GnRH receptors, CMPD1 was a competitive antagonist of GnRH-stimulated increases in extracellular acidification rates in Cytosensor microphysiometer assays. In cells expressing recombinant human GnRH receptors, CMPD1 was a potent inhibitor of GnRH-stimulated total inositol phosphate accumulation. The effects of CMPD1 on circulating levels of luteinizing hormone (LH) and testosterone were studied in castrated and intact male rats, respectively. Intravenous and oral administration of CMPD1 dose dependently suppressed GnRH-mediated elevations of LH in castrated male rats and testosterone in gonad-intact male rats. Moreover, CMPD1, when given at 20 mg/kg i.v. to intact male rats, inhibited the elevations of LH and testosterone stimulated by the superagonist of GnRH, [d-Ala(6), des-Gly(10)]GnRH (GnRH-A). These data suggest that CMPD1 is a potent, selective, orally active GnRH receptor antagonist that may have potential application as a therapeutic agent for treating hormone-dependent cancers and diseases.

Anilides↗
PubMed2002

Synthesis of a group-selective antibody library against haptens.

A generic strategy is described for the generation of libraries comprising hapten-selective antibody genes against a group of structurally related low molecular weight target molecules. Hapten antibody libraries are frequently suffering from high background levels of irrelevant antibody genes as a consequence of the immunization, where the small non-immunogenic target molecule is coupled to a large immunogenic carrier protein. In order to elevate the percentage of hapten-specific genes in the library, B cells harboring antibody genes against the group of triazine herbicides were enriched from 21 individual splenocyte populations by means of immunomagnetic separation (IMS). IMS utilizes the specific binding of membrane-associated immunoglobulin receptors on the B cell surface to hapten-coated paramagnetic beads. The variable genes of the specifically enriched subpopulation were cloned into a phagemid vector. The corresponding library yielded up to 75% triazine binding antibody clones after three rounds of phage selection. At least half of these antibodies (abs) were displaceable by triazines resulting in quantitative assays with nanomolar sensitivities. In contrast, no displaceable clone was obtained at the same selection level in a control library, where IMS was omitted. Due to the elevated percentage of relevant antibody genes, the library can be utilized either for the direct isolation of functional antibodies against various triazine herbicides or as group-specific gene source for evolutionary antibody optimization.

Animals↗
PubMed2006

Development and comparison of nonradioactive in vitro kinase assays for NIMA-related kinase 2.

NIMA (never in mitosis arrest)-related kinase 2 (Nek2) is a serine/threonine kinase required for centrosome splitting and bipolar spindle formation during mitosis. Currently, two in vitro kinase assays are commercially available: (i) a radioactive assay from Upstate Biotechnology and (ii) a nonradioactive fluorescence resonance energy transfer (FRET) assay from Invitrogen. However, due to several limitations such as radioactive waste management and lower sensitivity, a need for more robust nonradioactive assays would be ideal. Accordingly, we have developed four quantitative and sensitive nonradioactive Nek2 in vitro kinase assays: (i) a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) using peptides identified from a physiologically relevant protein substrate, (ii) DELFIA using Nek2 itself, (iii) a homogeneous time-resolved FRET assay termed LANCE, and (iv) A method of detecting phosphorylated products by HPLC. The DELFIA and LANCE assays are robust in that they generated more than 10-fold and 20-fold increases in signal-to-noise ratios, respectively, and are amenable to robotic high-throughput screening platforms. Validation of all four assays was confirmed by identifying a panel of small molecule ATP competitive inhibitors from an internal corporate library. The most potent compounds consistently demonstrated less than 100 nM activity regardless of the assay format and therefore were complementary. In summary, the Nek2 in vitro time-resolved FRET kinase assays reported are sensitive, quantitative, reproducible and amenable to high-throughput screening with improved waste management over radioactive assays.

Animals↗
PubMed2003

Applications of one-bead one-compound combinatorial libraries and chemical microarrays in signal transduction research.

The "one-bead-one-compound" (OBOC) combinatorial library method synthesizes millions of random compounds such that each bead displays only one compound. Bead libraries are screened, and positive beads are isolated for structure analysis. Peptide substrates and inhibitors of protein kinases, and peptide ligands for cell surface receptors have been identified using this method. A novel encoding strategy for OBOC libraries has been developed to identify peptidomimetic and small-molecule ligands that specifically interact with cellular proteins. These ligands will be tested for their effects on cell signaling and used to construct chemical microarrays for further characterization of ligand-protein interactions.

Combinatorial Chemistry Techniques↗
PubMedDate not supplied

Development of nanoparticle libraries for biosensing.

Magnetic and magnetofluorescent nanoparticles have become important materials for biological applications especially for sensing, separation, and imaging. To achieve target specificity, these nanomaterials are often covalently modified with binding proteins such as antibodies or proteins. Here we report on the creation of nanoparticle libraries that achieve specificity through multivalent modification with small molecules. We explore different synthetic routes to attach small molecules with anhydride, amine, hydroxyl carboxyl, thiol, and epoxy handles. We show that the derived nanomaterials have unique biological functions, possess different behaviors in cell screens, and can be used as substrates for biological screens.

Amines↗