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Toward high-throughput synthesis of complex natural product-like compounds in the genomics and proteomics age.

In the age of high-throughput biology, novel genes and proteins are emerging quickly. The need for developing organic synthesis-derived methods that allow rapid access to polyfunctional, complex natural product-like compounds is growing constantly, largely because these small-molecule-based compounds serve as smart, powerful tools both in understanding the roles and functions of emerging biological targets and in validating their biological responses. Developing asymmetric synthesis-derived organic reactions on solid phase allows the synthesis of complex natural product-like compounds in a high-throughput manner. Solid phase organic synthesis is now commonly utilized in the library synthesis of rather simple compounds (i.e., compounds with no multiple stereogenic centers). With few exceptions, the synthesis of complex natural product-like derivatives is still in its infancy. Some recent efforts made in this area indicate opportunities yet to be explored.

Biological Factors↗

Synthesis, characterization and development of a high-throughput methodology for the discovery of botulinum neurotoxin a inhibitors.

Botulinum neurotoxins (BoNTs), etiological agents of the deadly food poisoning disease botulism, are the most toxic proteins currently known. Although only a few hundred cases of botulism are reported in the United States annually, there is growing interest in BoNTs attributable to their potential use as biological warfare agents. Neurotoxicity results from cleavage of the soluble NSF-attachment protein receptor complex proteins of the presynaptic vesicles by the BoNT light chain subunit, a Zn endopeptidase. Few effective inhibitors of BoNT/A LC (light chain) activity are known, and the discovery process is hampered by the lack of an efficient high-throughput assay for screening compound libraries. To alleviate this bottleneck, we have synthesized the peptide SNAPtide and have developed a robust assay for the high-throughput evaluation of BoNT/A LC inhibitors. Key aspects for the development of this optimized assay include the addition of a series of detergents, cosolvents, and salts, including 0.01% w/v Tween 20 to increase BoNT/A LC catalysis, stability, and ease of small molecule screening. To evaluate the effectiveness of the assay, a series of hydroxamate-based small molecules were synthesized and examined with BoNT/A LC. The methodology described is superior to other assays reported to date for the high-throughput identification of BoNT/A inhibitors.

Botulinum Toxins, Type A↗

AF12198, a novel low molecular weight antagonist, selectively binds the human type I interleukin (IL)-1 receptor and blocks in vivo responses to IL-1.

Interleukin-1 (IL-1) -alpha and -beta are potent regulators of inflammatory responses. The naturally occurring interleukin-1 receptor antagonist (IL-1ra) is effective in vitro and in vivo in modulating biological responses to IL-1. We have previously reported the discovery of IL-1 antagonist peptides from the search of phage display libraries. Further characterization of this group of peptides has led to a 15-mer, AF12198, Ac-FEWTPGWYQJYALPL-NH2 (J represents the unnatural amino acid, 2-azetidine-1-carboxylic acid), with both in vitro and in vivo IL-1 antagonist activity. AF12198 selectively binds the human type I IL-1 receptor but not the human type II receptor or the murine type I receptor. In vitro, AF12198 inhibits IL-1-induced IL-8 production by human dermal fibroblasts with a half-maximal inhibition concentration or IC50 of 25 nM and IL-1-induced intercellular adhesion molecule-1 (ICAM-1) expression by endothelial cells with an IC50 of 9 nM. When given as an intravenous infusion to cynomolgus monkeys, AF12198 blocks ex vivo IL-1 induction of IL-6 and down modulates in vivo induction of IL-6. This is the first small molecule to show IL-1 receptor antagonist activity in vivo.

Amino Acid Sequence↗

Identification of novel small-molecule inhibitors for human transketolase by high-throughput screening with fluorescent intensity (FLINT) assay.

The metabolic enzyme transketolase (TK) plays a crucial role in tumor cell nucleic acid synthesis, using glucose through the elevated nonoxidative pentose phosphate pathway (PPP). Identification of inhibitors specifically targeting TK and preventing the nonoxidative PPP from generating the RNA ribose precursor, ribose-5-phosphate, provides a novel approach for developing effective anticancer therapeutic agents. The full-length human transketolase gene was cloned and expressed in Escherichia coli and the recombinant human transketolase protein purified to homogeneity. A fluorescent intensity (FLINT) assay was developed and optimized. Library compounds were screened in a high-throughput screening (HTS) campaign using the FLINT assay. Fifty-four initial hits were identified. Among them, 2 scaffolds with high selectivity, ideal physiochemical properties, and low molecular weight were selected for lead optimization studies. These compounds specifically inhibited in vitro TK enzyme activity and suppressed tumor cell proliferation in at least 3 cancer cell lines: SW620, LS174T, and MIA PaCa-2. Identification of these active scaffolds represents a good starting point for development of drugs specifically targeting TK and the nonoxidative PPP for cancer therapy.

Cell Line, Tumor↗

Development of IL-5 receptor antagonists.

A wealth of both clinical and pre-clinical data has strongly implicated the eosinophil in the pathogenesis of asthma, highlighting this cell type as a potential target for novel anti-inflammatory approaches to asthma therapy. The Th2 lymphocyte derived cytokine Interleukin-5 (IL-5) has emerged as the key regulator of eosinophil production, thus identifying IL-5 as the principal molecular target for therapeutic intervention. This review highlights both the pharmaceutical approaches, and the major challenges, to the identification of small molecule and protein antagonists of the IL-5 receptor. Using examples of known inhibitors we discuss their current status and highlight the major development hurdles in progressing these molecules into the market place.

Animals↗

Streptavidin-binding and -dimerizing ligands discovered by phage display, topochemistry, and structure-based design.

Structural and mechanistic determinants of affinity of streptavidin-binding peptide ligands discovered by phage display are reviewed along with the use of streptavidin as a paradigm for structure-based design. A novel way of producing protein-dimerizing ligands in the streptavidin model system is discussed, in which crystal packing topochemically mediates or even catalyzes dimerization of adjacent bound ligands whose reactive ligating groups are presented toward one another in productive orientations in the crystal lattice. Finally, through crystallography on a set of streptavidin complexes with small molecule and peptide ligands at multiple pHs in two space groups, the mechanism by which ligands enhance intersubunit stabilization of the streptavidin tetramer is probed.

Amino Acid Sequence↗

A one-bead, one-stock solution approach to chemical genetics: part 1.

BACKGROUND: In chemical genetics, small molecules instead of genetic mutations are used to modulate the functions of proteins rapidly and conditionally, thereby allowing many biological processes to be explored. This approach requires the identification of compounds that regulate pathways and bind to proteins with high specificity. Structurally complex and diverse small molecules can be prepared using diversity-oriented synthesis, and the split-pool strategy allows their spatial segregation on individual polymer beads, but typically in quantities that limit their usefulness. RESULTS: We report full details of the first phase of our platform development, including the synthesis of a high-capacity solid-phase bead/linker system, the development of a reliable library encoding strategy, and the design of compound decoding methods both from macrobeads and stock solutions. This phase was validated by the analysis of an enantioselective, diversity-oriented synthesis resulting in an encoded 4320-member library of structurally complex dihydropyrancarboxamides. CONCLUSIONS: An efficient and accessible approach to split-pool, diversity-oriented synthesis using high-capacity macrobeads as individual microreactors has been developed. Each macrobead contains sufficient compound to generate a stock solution amenable to many biological assays, and reliable library encoding allows for rapid compound structure elucidation post-synthesis. This 'one-bead, one-stock solution' strategy is a central element of a technology platform aimed at advancing chemical genetics.

Combinatorial Chemistry Techniques↗

Natural combinatorial peptide libraries in cyanobacterial symbionts of marine ascidians.

A large family of cytotoxic cyclic peptides exemplified by the patellamides has been isolated from ascidians harboring the obligate cyanobacterial symbionts Prochloron spp.. Genome sequence analysis of these symbionts has revealed that Prochloron spp. synthesize patellamides by a ribosomal pathway. To understand how this pathway evolved to produce a suite of related metabolites, we analyzed 46 prochloron-containing ascidians from the tropical Pacific Ocean for the presence of patellamide biosynthetic genes and taxonomic markers. Here, we show that Prochloron spp. generate a diverse library of patellamides using small, hypervariable cassettes within a conserved genetic background. Each symbiont strain contains a single pathway, and mixtures of symbionts within ascidians lead to the accumulation of chemical libraries. We used this information to engineer the production of a new cyclic peptide in Escherichia coli, thereby demonstrating the power of comparative analysis of closely related symbiotic pathways to direct the genetic synthesis of new molecules.

Amino Acid Sequence↗

Development of a p38 Kinase Binding Assay for High Throughput Screening.

p38 is a member of the mitogen-activated protein kinase (MAPK) family of serine/threonine kinases, which is activated by cellular stressors and has been shown to be a critical enzyme in the synthesis and action of proinflammatory cytokines, tumor necrosis factor-a (TNF-alpha) and interleukin-1beta (IL-1beta). A group of pyridinyl imidazole compounds such as SB202190 have been identified as selective inhibitors of p38 that bind directly to the ATP pocket of the enzyme. These compounds inhibit the p38 kinase activity, block TNF-alpha and IL-1beta secretion both in vivo and in vitro and are found to be effective in animal models of arthritis, bone resorption, and endotoxin shock. We postulated that other classes of compounds capable of competing the binding of pyridinyl imidazole with p38 enzyme could have efficacy in the treatment of inflammatory diseases. Therefore, a simple and robust assay was developed to measure the ability of small molecules to inhibit the binding of tritium-labeled pyridinyl imidazole, SB202190, to recombinant p38 kinase. For assay development, the human p38 gene was cloned in baculovirus and then expressed in insect cells. Tritiated SB202190 was synthesized and used as the p38 ligand for a competitive filter binding assay. This assay has been used successfully to screen both synthetic and combinatorial chemical libraries for other classes of p38 kinase inhibitors.

Journal Article↗

Short interfering RNA (siRNA) as a novel therapeutic.

1. RNA interference (RNAi) is a robust method of post-transcriptional silencing of genes using double-stranded RNA (dsRNA) with sequence homology driven specificity. The dsRNA can be between 21 and 23 nucleotides long: this is converted to small interfering RNA (siRNA), which then mediates gene silencing by degradation/blocking of translation of the target mRNA. 2. RNA interference provides a simple, fast and cost-effective alternative to existing gene targeting approaches both in vitro and in vivo. The discovery of siRNAs that cause RNAi in mammalian cells opened the door to the therapeutic use of siRNAs. Highly intense research efforts are now aimed at developing siRNAs for therapeutic purposes. 3. Recent advances in the design and delivery of targeting molecules now allow efficient and highly specific gene silencing in mammalian systems. Synthetic siRNA libraries targeting thousands of mammalian genes are publicly available for high-throughput genetic screens for target discovery and validation. Recent studies have demonstrated the clinical potential of aptly designed siRNAs in various types of viral infections, cancer and renal and neurodegenerative disorders. 4. The present review provides insight into the novel therapeutic strategies of siRNA technology, which is the latest development in nucleic acid-based tools for knocking down gene expression, and its potential for silencing genes associated with various human diseases.

Amyloid Precursor Protein Secretases↗

Tendamistat as a scaffold for conformationally constrained phage peptide libraries.

The alpha-amylase inhibitor Tendamistat (Hoe-467), a 74 amino acid beta-sheet protein from Streptomyces tendae has been expressed on the surface of the filamentous bacteriophage M13. Phage displaying Tendamistat inhibit the hydrolysis of starch by alpha-amylase, indicating that the displayed protein is functional. The displayed Tendamistat has been used as a molecular scaffold for the presentation of constrained random peptides. Two loops, comprising residues 38 to 40 and 60 to 65 of Tendamistat, were randomized using PCR mutagenesis. Libraries of approximately 10(8) different mutant Tendamistat molecules were tested for binding to monoclonal antibody A8, which recognizes endothelin. After three cycles of biopanning, phage were isolated that specifically bound the monoclonal antibody. Loop swapping and alanine replacement mutagenesis indicated that residues in the 60 to 65 loop are responsible for binding to the monoclonal antibody. This work demonstrates the use of relatively small non-antibody protein scaffolds for the presentation of constrained random peptide sequences to select for novel binding molecules.

Alanine↗

Small molecule inhibitors of the CCR2b receptor. Part 1: Discovery and optimization of homopiperazine derivatives.

N,N'-Disubstituted homopiperazine derivatives have been discovered as CC-chemokine receptor 2b (CCR2b) inhibitors with submicromolar activity in the CCR2b binding assay. A 4-substituted benzyl group on one homopiperazine nitrogen was an important moiety for binding affinity to the CCR2b receptor. The SAR for CCR2b binding affinity correlated inversely with the sigma factor of the functional group on this benzyl moiety. Introduction of hydroxy groups to appropriate positions in the 3,3-diphenylpropyl group on the other homopiperazine nitrogen increased CCR2b binding activity. The synthesis of an informer library to search for alternative substructures is also described.

Cell Line↗

Discovery of selective, small-molecule inhibitors of RNA complexes--I. The Tat protein/TAR RNA complexes required for HIV-1 transcription.

We have developed a therapeutic program focusing on the inhibition of a human immunodeficiency virus-1 specific protein-RNA interaction. This program begins with a search for small organic molecules that would interfere with the binding of Tat protein to TAR RNA. The methodologies chosen to study the HIV-1 Tat-TAR interaction and inhibition include gel mobility shift assays, scintillation proximity assays, filtration assays, and mass spectrometry. These methods helped establish in vitro high-throughput screening assays which rapidly identified Tat-TAR inhibitors from our corporate compound library. Tat-activated reporter gene assays were then used to investigate the cellular activities of the Tat-TAR inhibitors. The cellular activity, selectivity, and toxicity data for select Tat-TAR inhibitors were determined. Evaluation of both the cellular data and the Tat-TAR inhibition results led to further testing in anti-HIV-1 infection assays.

Amino Acid Sequence↗

Identity between the novel integrin beta 7 subunit and an antigen found highly expressed on intraepithelial lymphocytes in the small intestine.

A cDNA clone encoding the N-terminal sequence of the murine integrin beta 7 subunit, a novel member of the leukocyte cell adhesion molecule subset (Leu-CAM), has been isolated. An N-terminal region of 13 contiguous amino acids deduced from the cDNA shows complete identity with the N-terminus of the 120 kDa subunit of the M290 antigen, a surface molecule found highly expressed on mouse intestinal intraepithelial lymphocytes (IEL). This unexpected result focuses two previously unconnected areas of research and suggests that integrins may have a special role to play in the defence of the gut mucosa.

Amino Acid Sequence↗

Cloning and sequence analysis of brain cDNA encoding a Xenopus D2 dopamine receptor.

A D2 dopamine receptor pharmacologically different from the mammalian D2 receptor has previously been characterized in the amphibian Xenopus laevis. Here we report the cloning of a Xenopus D2 receptor which revealed about 75% amino acid sequence identity with its mammalian counterpart and the presence of an additional 33 amino acid sequence in the 3rd cytoplasmic loop instead of the additional 29 residues in the large form of the mammalian D2 receptor. All 7 predicted transmembrane domains are highly conserved between the Xenopus and mammalian D2 receptors, as are the 1st and 2nd intracellular loop, the 1st and 3rd extracellular loop and the carboxy-terminal portion of the receptors. The amino-terminal portion, the 2nd extracellular loop and the middle portion of the 3rd intracellular loop of these receptors, however, differ considerably. Knowledge of the locations of these regions of conservation and divergence within the D2 receptors of Xenopus and mammals will help to delineate portions of the receptor molecule that are functionally important. Interestingly, the 5'-untranslated region of the Xenopus D2 receptor mRNA contains 4 small open reading frames which may affect translational efficiency.

Amino Acid Sequence↗

Domain antibodies: proteins for therapy.

Occurring naturally in "heavy chain" immunoglobulins from camels, and now produced in fully human form, domain antibodies (dAbs) are the smallest known antigen-binding fragments of antibodies, ranging from 11 kDa to 15 kDa. dAbs are the robust variable regions of the heavy and light chains of immunoglobulins (VH and VL respectively). They are highly expressed in microbial cell culture, show favourable biophysical properties including solubility and temperature stability, and are well suited to selection and affinity maturation by in vitro selection systems such as phage display. dAbs are bioactive as monomers and, owing to their small size and inherent stability, can be formatted into larger molecules to create drugs with prolonged serum half-lives or other pharmacological activities.

Animals↗

Sterol-dependent transactivation of the ABC1 promoter by the liver X receptor/retinoid X receptor.

Tangier disease, a condition characterized by low levels of high density lipoprotein and cholesterol accumulation in macrophages, is caused by mutations in the ATP-binding cassette transporter ABC1. In cultured macrophages, ABC1 mRNA was induced in an additive fashion by 22(R)-hydroxycholesterol and 9-cis-retinoic acid (9CRA), suggesting induction by nuclear hormone receptors of the liver X receptor (LXR) and retinoid X receptor (RXR) family. We cloned the 5'-end of the human ABC1 transcript from cholesterol-loaded THP1 macrophages. When transfected into RAW macrophages, the upstream promoter was induced 7-fold by 22(R)-hydroxycholesterol, 8-fold by 9CRA, and 37-fold by 9CRA and 22(R)-hydroxycholesterol. Furthermore, promoter activity was increased in a sterol-responsive fashion when cotransfected with LXRalpha/RXR or LXRbeta/RXR. Further experiments identified a direct repeat spaced by four nucleotides (from -70 to -55 base pairs) as a binding site for LXRalpha/RXR or LXRbeta/RXR. Mutations in this element abolished the sterol-mediated activation of the promoter. The results show sterol-dependent transactivation of the ABC1 promoter by LXR/RXR and suggest that small molecule agonists of LXR could be useful drugs to reverse foam cell formation and atherogenesis.

ATP Binding Cassette Transporter 1↗

Expression of Thy-1 on human hematopoietic progenitor cells.

Expression of Thy-1 on hematopoietic cells from human fetal liver (FL), cord blood (CB), and bone marrow (BM) was studied with a novel anti-Thy-1 antibody, 5E10. Specificity of 5E10 for human Thy-1 was demonstrated by immunoprecipitation of a 25-35-kD molecule, and the sequence of a cDNA that was cloned by immunoselection of COS cells transfected with a cDNA library derived from a 5E10+ cell line. Two- and three-color immunofluorescence staining experiments revealed that the Thy-1 expression is restricted to, an average, 1-4% of FL, CB, and BM cells, and binding to these cell types is essentially restricted to a very small subset of lymphoid cells and approximately 25% of CD34+ cells. Thy-1+ CD34+ cells were further characterized as CD38lo/CD45RO+/CD45RA-/CD71lo/c-kit(lo) and rhodamine 123dull. When CD34+ cells were sorted on the basis of Thy-1 expression, the majority of clonogenic cells were recovered in the CD34+Thy-1- fraction, whereas the majority of cells capable of producing myeloid colonies after 5-8 wk of long-term culture (long-term culture initiating cells) were recovered in the Thy-1+CD34+ fraction. In addition to CD34+ cells, Thy-1 was found to be expressed on a variable, very small number (< 1%) of CD34- mononuclear cells in BM, CB, and peripheral blood that were further characterized as CD3+ CD4+ lymphocytes. The restricted expression of Thy-1 on primitive hematopoietic cells is in agreement with a previous report (Baum et al., 1992. Proc. Natl. Acad. Sci. USA. 89:2804) in which Thy-1 expression was used to enrich for primitive hematopoietic cells from fetal tissue. Compared with those previous studies, we found Thy-1 expression on a larger proportion of CD34+ cells (25% in our study vs. 5% in Baum et al.) and furthermore performed studies on Thy-1 expression on CD34+ cells from CB, FL, and BM in relation to markers that are known to be differentially expressed on hematopoietic cells. Taken together our results indicate that Thy-1-specific antibody 5E10 is an attractive tool for further studies on the biology and purification of human stem cells.

Adult↗