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PubMed2005

Small molecule microarrays: recent advances and applications.

Directed or exploratory drug development programs constantly seek robust screening platforms for the high fidelity identification and validation of potential targets. Small-molecule microarrays (SMMs) have risen to this call by elegantly forging the capability of combinatorial chemistry in producing myriad compounds with the powerful throughput afforded by microarrays. This synergism offers scientists a versatile tool for rapid compound analysis and discovery. Microarrays of small molecules have already been successfully applied in important areas ranging from protein profiling to the discovery of therapeutic leads. Recent interesting developments towards improved immobilization strategies and library creation methods, together with novel advances herein described, have set the stage for SMMs to take on wider and more routine applications in academia and industry. As a rapidly maturing technology, SMMs pave the way forward in high-throughput exploration, both in the identification of biologically significant natural and synthetic small molecules and in harnessing their vast potential towards medicinal and diagnostic applications.

Combinatorial Chemistry Techniques↗
PubMed2004

Fluorous tagging strategy for solution-phase synthesis of small molecules, peptides and oligosaccharides.

The purification of reaction mixtures is a slow process in organic synthesis, especially during the production of large numbers of analogs and compound libraries. Phase-tag methods such as solid-phase synthesis and fluorous synthesis, provide efficient ways of addressing the separation issue. Fluorous synthesis employs functionalized perfluoroalkyl groups attached to substrates or reagents. The separation of the resulting fluorous molecules can be achieved using strong and selective fluorous liquid-liquid extraction, fluorous silica gel-based solid-phase extraction or high-performance liquid chromatography. Fluorous technology is a novel solution-phase method, which has the advantages of fast reaction times in homogeneous environments, being readily adaptable to literature conditions, having easy intermediate analysis, and having flexibility in reaction scale and scope. In principle, any synthetic methods that use a solid-support could be conducted in solution-phase by replacing the polymer linker with a corresponding fluorous tag. This review summarizes the progress of fluorous tags in solution-phase synthesis of small molecules, peptides and oligosaccharides.

Fluorides↗
PubMed1997

Accessing rare activities from random RNA sequences: the importance of the length of molecules in the starting pool.

BACKGROUND: In the past few years numerous binding and catalytic motifs have been isolated from pools of random nucleic acid sequences. To extend the utility of this approach it is important to learn how to design random-sequence pools that provide maximal access to rare activities. In an effort to better define the relative merits of longer and shorter pools (i.e. pools with longer or shorter random-sequence segments), we have examined the inhibitory effect of excess arbitrary sequence on ribozyme activity and have evaluated whether this inhibition overshadows the calculated advantage of longer pools. RESULTS: The calculated advantage of longer sequences was highly dependent on the size and complexity of the desired motif. Small, simple motifs were not much more abundant in longer molecules. In contrast, larger motifs, particularly the most complex (highly modular) motifs, were much more likely to be present in longer molecules. The experimentally determined inhibition of activity by excess sequence was moderate, with bulk effects among four libraries ranging from no effect to 18-fold inhibition. The median effect among 60 clones was fivefold inhibition. CONCLUSIONS: For accessing simple motifs (e.g. motifs at least as small and simple as the hammerhead ribozyme motif), longer pools have little if any advantage. For more complex motifs, the inhibitory effect of excess sequence does not approach the calculated advantage of pools of longer molecules. Thus, when seeking to access rare activities, the length of typical random-sequence pools (< or = 70 random positions) is shorter than optimal. As this conclusion holds over a range of incubation conditions, it may also be relevant when considering the emergence of new functional motifs during early evolution.

Base Composition↗
PubMed2006

Engineered protein inhibitors of proteases.

The control of proteolysis in an organism is achieved under normal circumstances through a balance of protease production, degradation and inactivation, via interaction with an endogenous inhibitor. When one of these mechanisms for control of proteolysis fails, it can result in the onset or progression of disease. Control of aberrant proteolysis is, therefore, a potetntial point of therapeutic intervention, and can be achieved either through the replacement of an absent endogenous inhibitor or by dosing with an inhibitor that is specific for a protease that is being over produced. Engineered protein inhibitors of proteases offer the potential to overcome the difficulties involved in identifying specific inhibitors via small-molecule-based approaches.

Animals↗
PubMed2003

Small molecule inhibitors of the RNA-dependent protein kinase.

The RNA-dependent protein kinase (PKR) is an interferon-induced serine/threonine protein kinase that phosphorylates the alpha subunit of the eukaryotic initiation factor 2 in response to viral infection. Classical genetic approaches for studying the role of PKR in cell signaling have their limitations due to overlapping but non-redundant pathways. Small molecule inhibitors of PKR will be useful in this regard. We report here, the discovery of a small molecule inhibitor of the kinase reaction of PKR. The inhibitor was discovered by screening a library of 26 different ATP-binding site directed inhibitors of varying structure. We also describe the development of a high-throughput assay for screening a large number of compounds for a PKR inhibitor using a rabbit reticulocyte lysate system and luciferase mRNA. The assay takes advantage of the fact that the reticulocyte lysate is rich in components of the translational machinery, of which PKR is an integral part. This assay can be carried out with added exogenous human PKR to study the effect of various compounds in their ability to rescue the translational block imposed by human PKR.

Enzyme Inhibitors↗
PubMed2002

[Advances in research on cytokine mimetic peptides/non-peptides stimulating hematopoiesis].

Cytokines such as erythropoietin (EPO) and thrombopoitein (TPO) and so on, which stimulate hematopoiesis, can regulate self-renewal, proliferation, differentiation, maturation and programmed cell death of hematopoietic cells through specifically binding to surface receptors. Recently random phage display peptide libraries and other screening methods have been used to isolate mimetic including small peptides and non-peptides molecules, which can mimic the same effects as cytokines, such as EPO and TPO, and demonstrate the similar potency and activity as EPO and TPO in a panel of in vitro biological assays and in animal experiments. These approaches are critical to further research of interactive mechanisms between cytokine and receptor, receptor activation and rational design of other desired cytokine mimetic. This review concisely introduced recent advances in research on mimetic of EPO, TPO and other cytokines and future directions.

Animals↗
PubMed2006

Selection of CC chemokine receptor 5-binding peptide from a phage display peptide library.

Human CC chemokine receptor (CCR) 5 is a G protein-coupled receptor involved in a broad range of human diseases that mediates HIV-1 viral entry into cells. Certain small molecule receptor antagonists to CCR5 have been useful in therapy for these diseases. In this study, CCR5-expressing CHO cells (CHO/CCR5 cells) were used to select CCR5-binding peptides from a phage-displayed 12-mers peptide library. All of the 30 clones selected from the library showed specific binding to CHO/CCR5 cells by enzyme linked immunosorbent assay (ELISA). Seventeen out of the 30 clones shared the amino acid motif AFDWTFVPSLIL. The motif-containing phages and synthetic peptide AFDWTFVPSLIL blocked the binding of mAb 2D7 to CHO/CCR5 cells and competitively inhibited the ability of chemokine regulated on activation normal T cell expressed and secreted (RANTES) binding to CHO/CCR5 cells. Furthermore, the peptide AFDWTFVPSLIL also inhibited RANTES induced increase in the intracellular Ca2+ level in CHO/CCR5 cells. These results suggest that the peptide AFDWTFVPSLIL was specific for CCR5 and that it might become a CCR5 antagonist.

Amino Acid Motifs↗
PubMed2003

Retroviral technology--applications for expressed peptide libraries.

The cell is an extremely complex network of interactions between large numbers of molecules. Understanding this entire network and the information arising from it is an overwhelming and challenging task. Reverse genetics has given us the possibility to discover unknown interactions and their related pathways. With the help of peptide libraries, interactions between biomolecules can be disrupted or distorted and the signaling pathways where these proteins are involved, altered. Consequently, novel biological pathways can be discerned. The peptide libraries become a pool of shapes, some of them might behave as dominant effectors. With the use of retroviral transfer vectors those libraries can be expressed in a stable manner in the mammalian cell. A strong selection and screening process can finally lead to specific peptides. Novel high-throughput approaches might allow for the rapid creation of small-molecule switches in protein-protein interactions. Reverse genetics and as such the expression of small molecules that will have a specific biological outcome, can become an answer to our queries.

Animals↗
PubMed2000

Determination of the refractive index increments of small molecules for correction of surface plasmon resonance data.

The refractive index increments (RIIs) of several important low-molecular-weight compounds that bind to DNA or RNA were determined with a differential refractometer for correction of data obtained on surface plasmon resonance (SPR) biosensors. Although the ability to investigate small molecule-macromolecule interactions by SPR is relatively new, the technique is rapidly becoming a primary method to screen focused combinatorial libraries and to quantitatively characterize the interactions between compounds identified as binders and target macromolecules. The most widely used SPR analysis software, BIAevaluation (Biacore, Inc.), assumes that the RIIs of ligand and macromolecule are identical. While the assumption is reasonable for studies involving like molecules such as protein-protein interactions, results presented here demonstrate that RII values for small molecules can be significantly different than those of protein or nucleic acid receptors. The results also show that the RII values can vary greatly depending on the structure of the small molecule. Indeed, the RIIs of the molecules investigated here differ by a factor of 2. Any difference in the RII of interacting molecules must be considered for complete analysis of SPR data. Failure to correct for RII differences can result in serious error in data interpretation, especially for systems involving a ligand:receptor stoichiometry greater than 1. The results serve as the beginning of an SPR correction database for the RIIs of small molecules. Additionally, the results can be used to approximate the RIIs of a variety of other small molecules.

Molecular Weight↗
PubMed2002

Evaluation of a novel shape-based computational filter for lead evolution: application to thrombin inhibitors.

A novel shape-feature-based computational method is described and used to rapidly filter compound libraries. The computational model, built using three-dimensional conformations of active and inactive molecules, consists of a collection of whole molecule shapes and chemical feature positions that are ranked according to their correlation with activity. A small ensemble of these shapes and features is used to filter virtual compound libraries. The method is applied to two thrombin data sets and is shown to be efficient in identifying novel scaffolds with enhanced hit rates.

Combinatorial Chemistry Techniques↗
PubMed2001

Discovery of a non-peptide small molecule that selectively mimics the biological actions of calcitonin.

Calcitonin (CT), a 32-amino acid peptide hormone secreted mainly from the thyroid gland, plays an important role in maintaining bone homeostasis. To discover non-peptide small molecules with biological actions similar to those of CT, a cell-based screening of an in-house chemical library was performed and a pyridone derivative (SUN B8155) was identified. Like CT, it elevated cyclic AMP (cAMP) levels in T47D and UMR106-06 cells which endogenously express human and rat CT receptor, respectively. SUN B8155 also stimulated cAMP formation in cells expressing recombinant human CT receptor, but not in those expressing human parathyroid hormone/parathyroid hormone-related peptide receptor. Accumulation of cAMP in T47D cells was blocked by a selective antagonist of CT receptor, salmon CT(8-32), whereas SUN B8155 did not displace the specific binding of [(125)I]CT to the receptor. Our results suggested that the compound selectively interacts with the CT receptor by a mechanism similar to but probably different from that of CT itself. In rats, intraperitoneal administration of SUN B8155 significantly lowered serum calcium levels, like CT. Our results demonstrate, for the first time, that the biological activities of the newly identified small molecule can mimic that of CT, acting via the CT receptor.

Animals↗
PubMed1990

Characterization of cDNA clones defining variant forms of human neural cell adhesion molecule N-CAM.

The neural cell adhesion molecule N-CAM has been identified in a number of species and comprises at least three major cell surface polypeptides of different molecular structures and tissue distributions. We report here the isolation and characterization of cDNA clones encoding two of the three major forms of N-CAM from a human neuroblastoma cDNA library. One of the clones, NII-6, provides the first complete sequence of a small cytoplasmic domain (140 kDa) form of the molecule in humans and differs in a number of respects from cDNA clones derived from human muscle. These differences include the presence of a 30-bp insert in the fourth immunoglobulin-like domain of N-CAM, a 3-bp insert in the extracellular portion of the molecule, and an additional 6 bp in the middle of the membrane-spanning segment. Based on the analysis of a genomic DNA clone spanning these regions of N-CAM, the first two differences arise by alternate splicing of RNA and occur in some, but not all clones; the additional 6 bp may reflect a genetic polymorphism. A second cDNA clone, NI-10, encodes the complete sequence of a segment that is specific to the large cytoplasmic domain (180 kDa) polypeptide of human N-CAM and is very similar to corresponding segments of mouse, chicken, and rat N-CAM. This sequence also arises by alternative splicing of RNA. In addition, we have identified a genomic DNA segment encoding sequences specific to the third, small surface domain (120 kDa) polypeptide of N-CAM. The data presented here and previously define the DNA sequences of the membrane-bound forms and known variants of human N-CAM. From these sequences, a wide variety of probes can be generated for investigating the expression of particular N-CAM polypeptides in normal and pathological tissues.

Amino Acid Sequence↗
PubMed2001

New technologies for chemical genetics.

Chemical genetics, in which small molecules are used in lieu of mutations to study biological processes, requires large and diverse chemical libraries to specifically perturb different biological pathways. Here we describe a suite of technologies that enable chemical libraries prepared by split-pool solid phase synthesis to be screened in a diverse range of chemical genetic assays. Compounds are synthesized on 500 micron high-capacity polystyrene beads, and arrayed into individual wells of 384-well plates using a hand-held bead arrayer. Compounds are cleaved from synthesis beads using a chemically-resistant ceramic dispensing system, producing individual stock solutions of single compounds. Nanoliter volumes of these solutions are then transferred into assay plates using an array of stainless steel pins mounted on a robotic arm. We have designed reusable 1536- and 6144-well assay plates made of silicone rubber that can be cast in the laboratory and filled by hand. This integrated technology platform enables hundreds of biological assays to be performed from the product of a single synthesis bead, enabling the results of different chemical genetic experiments to be directly compared.

Cell Extracts↗
PubMed1994

A fast and efficient method to generate biologically relevant conformations.

Mutual binding between a ligand of low molecular weight and its macromolecular receptor demands structural complementarity of both species at the recognition site. To predict binding properties of new molecules before synthesis, information about possible conformations of drug molecules at the active site is required, especially if the 3D structure of the receptor is not known. The statistical analysis of small-molecule crystal data allows one to elucidate conformational preferences of molecular fragments and accordingly to compile libraries of putative ligand conformations. A comparison of geometries adopted by corresponding fragments in ligands bound to proteins shows similar distributions in conformations space. We have developed an automatic procedure that generates different conformers of a given ligand. The entire molecule is decomposed into its individual ring and open-chain torsional fragments, each used in a variety of favorable conformations. The latter ones are produced according to the library information about conformational preferences. During this building process, an extensive energy ranking is applied. Conformers ranked as energetically favorable are subjected to an optimization in torsion angle space. During minimization, unfavorable van der Waals interactions are removed while keeping the open-chain torsion angles as close as possible to the experimentally most frequently observed values. In order to assess how well the generated conformers map conformation space, a comparison with experimental data has been performed. This comparison gives some confidence in the efficiency and completeness of this approach. For some ligands that had been structurally characterized by protein crystallography the program was used to generate sets of some 10 to 100 conformers. Among these, geometries are found that fall convincingly close to the conformations actually adopted by these ligands at the binding site.

Adenosine Monophosphate↗
PubMed2006

Proteomic profiling of metalloprotease activities with cocktails of active-site probes.

Metalloproteases are a large, diverse class of enzymes involved in many physiological and disease processes. Metalloproteases are regulated by post-translational mechanisms that diminish the effectiveness of conventional genomic and proteomic methods for their functional characterization. Chemical probes directed at active sites offer a potential way to measure metalloprotease activities in biological systems; however, large variations in structure limit the scope of any single small-molecule probe aimed at profiling this enzyme class. Here, we address this problem by creating a library of metalloprotease-directed probes that show complementary target selectivity. These probes were applied as a 'cocktail' to proteomes and their labeling profiles were analyzed collectively using an advanced liquid chromatography-mass spectrometry platform. More than 20 metalloproteases were identified, including members from nearly all of the major branches of this enzyme class. These findings suggest that chemical proteomic methods can serve as a universal strategy to profile the activity of the metalloprotease superfamily in complex biological systems.

Affinity Labels↗
PubMed2004

Discovery of an allosteric site in the caspases.

Allosteric regulation of proteins by conformational change is a primary means of biological control. Traditionally it has been difficult to identify and characterize novel allosteric sites and ligands that freeze these conformational states. We present a site-directed approach using Tethering for trapping inhibitory small molecules at sites away from the active site by reversible disulfide bond formation. We screened a library of 10,000 thiol-containing compounds against accessible cysteines of two members of the caspase family of proteases, caspase-3 and -7. We discovered a previously unreported and conserved allosteric site in a deep cavity at the dimer interface 14 A from the active site. This site contains a natural cysteine that, when disulfide-bonded with either of two specific compounds, inactivates these proteases. The allosteric site is functionally coupled to the active site, such that binding of the compounds at the allosteric site prevents peptide binding at the active site. The x-ray crystal structures of caspase-7 bound by either compound demonstrates that they inhibit caspase-7 by trapping a zymogen-like conformation. This approach may be useful to identify new allosteric sites from natural or engineered cysteines, to study allosteric transitions in proteins, and to nucleate drug discovery efforts.

Allosteric Regulation↗
PubMed2005

Small molecule intervention in microtubule-associated human disease.

Microtubules are essential for a number of cellular processes that include the transport of intracellular cargo or organelles across long distances and the assembly of the mitotic spindle. The identification of numerous microtubule-associated proteins and the progressive elucidation of the mechanisms of microtubule assembly and transport are beginning to have a profound impact on the study and treatment of human genetic disease. A number of seemingly unrelated phenotypes have now been linked to microtubular dysfunction, especially in systems dependent heavily on microtubule-based transport, such as neurons and ciliated cells. In parallel, the association of microtubule transport defects with human genetic disease has led to the realization that targeting various aspects of microtubular biology with small molecules might offer new therapeutic paradigms, including the development of new therapeutic utility for seemingly old drugs. In this review, we discuss the use of small molecules in the investigation of microtubule-associated processes and particularly the screens of chemical compound libraries for the identification of lead compounds with potential utility in microtubule-associated disease processes.

Animals↗
PubMed2002

Pathway development and pilot library realization in diversity-oriented synthesis: exploring Ferrier and Pauson-Khand reactions on a glycal template.

Through a correlation of the ability of small molecules to bind biological macromolecules and their ability to modulate cellular and organismal processes, chemistry can inform biology and vice versa. Diversity-oriented organic synthesis (DOS), which aims to provide structurally complex and diverse small molecules efficiently, can play a key role in such chemical genetic studies. Here we illustrate the trial-and-error experimentation that can refine an initial pathway-planning exercise and result eventually in an effective diversity pathway. By exploring Ferrier and Pauson-Khand reactions on a glycal template, we have developed efficient and stereoselective syntheses of tricyclic compounds. In this pathway, diversity results from the substituents and their spatial relationships about the tricyclic rings. A pilot split-pool library synthesis of 2500 tricyclic compounds highlights the use of planning considerations in DOS and a "one-bead, one-stock solution" technology platform. Additionally, it illustrates a promising synthetic pathway for future chemical genetic studies.

Carbohydrates↗