Organoids in image-based phenotypic chemical screens

Image-based phenotypic screening relies on the extraction of multivariate information from cells cultured under a large variety of conditions. Technical advances in high-throughput microscopy enable screening in increasingly complex and biologically relevant model systems. To this end, organoids hold great potential for high-content screening because they recapitulate many aspects of parent tissues and can be derived from patient material. However, screening is substantially more difficult in organoids than in classical cell lines from both technical and analytical standpoints. In this review, we present an overview of studies employing organoids for screening applications. We discuss the promises and challenges of small-molecule treatments in organoids and give practical advice on designing, running, and analyzing high-content organoid-based phenotypic screens.Subject terms: High-throughput screening, Drug delivery     

Enzymatic profiling system in a small-molecule microarray

[reaction: see text] We have developed a microarray-based strategy for detection of three major classes of hydrolytic enzymes on the basis of their catalytic activities. This enables the sensitive detection of proteins not merely by their bindings but rather by their enzymatic activities. This may provide a valuable tool for screening, identification, and characterization of new enzymes in a high-throughput fashion.     

Ligand.Info small-molecule Meta-Database

Ligand.Info is a compilation of various publicly available databases of small molecules. The total size of the Meta-Database is over 1 million entries. The compound records contain calculated three-dimensional coordinates and sometimes information about biological activity. Some molecules have information about FDA drug approving status or about anti-HIV activity. Meta-Database can be downloaded from the http://Ligand.Info web page. The database can also be screened using a Java-based tool. The tool can interactively cluster sets of molecules on the user side and automatically download similar molecules from the server. The application requires the Java Runtime Environment 1.4 or higher, which can be automatically downloaded from Sun Microsystems or Apple Computer and installed during the first use of Ligand.Info on desktop systems, which support Java (Ms Windows, Mac OS, Solaris, and Linux). The Ligand.Info Meta-Database can be used for virtual high-throughput screening of new potential drugs. Presented examples showed that using a known antiviral drug as query the system was able to find others antiviral drugs and inhibitors.     

Ribosomal route to small-molecule diversity

The cyanobactin ribosomal peptide (RP) natural product pathway was manipulated to incorporate multiple tandem mutations and non-proteinogenic amino acids, using eight heterologous components simultaneously expressed in Escherichia coli . These studies reveal the potential of RPs for the rational synthesis of complex, new small molecules over multiple-step biosynthetic pathways using simple genetic engineering.     

Metastable States of small-molecule solutions

Metastable states such as gels and glasses that are commonly seen in nanoparticle suspensions have found application in a wide range of products including toothpaste, hand cream, paints, and car tires. The equilibrium and metastable state behavior of nanoparticle suspensions are often described by simple fluid models where particles are treated as having hard cores and interacting with short-range attractions. Here we explore similar models to describe the presence of metastable states of small-molecule solutions. We have recently shown that the equilibrium solubilities of small hydrogen-bonding molecules and nanoparticles fall onto a corresponding-states solubility curve suggesting that with similar average strengths of attraction these molecules have similar solubilities. This observation implies that metastable states in small-molecule solutions may be found under conditions similar to those where metastable states are observed in nanoparticle and colloidal suspensions. Here we seek confirmation of this concept by exploring the existence of metastable states in solutions of small molecules.     

Skeletal diversity in small-molecule synthesis using ligand-controlled catalysis

Two Pd-catalyzed reductive transformations of diynes tethered through a silyl ether linkage were developed, where the reaction outcomes were controlled solely by selection of phosphine ligand. We screened Pd precatalysts, ligands, and additives to optimize conditions selective either for reductive cyclization or hydrogenation of this substrate class. Sixteen silyl ether-tethered diynes were prepared and subjected to the best catalyst/ligand combinations for each pathway. Silacyclic dienes and silyl-tethered enyne products of these reactions were elaborated to densely substituted, stereochemically- and appendage-rich, bicyclic and tricyclic small molecules in 1-3 synthetic steps. These studies illustrate how small modifications to a transition-metal catalyst can be used to access a diverse set of small molecules, in a fashion analogous to biosynthetic pathways such as terpene biosynthesis, where minor changes to enzyme structure direct skeletal differentiation.     

Dynamics of small-molecule glass formers confined in nanopores

We report a comparative neutron scattering study of the molecular mobility and nonexponential relaxation of three structurally similar glass-forming liquids, isopropanol, propylene glycol, and glycerol, both in bulk and confined in porous Vycor glass. Confinement reduces molecular mobility in all three liquids, and suppresses crystallization in isopropanol. High-resolution quasielastic neutron scattering spectra were fit to Fourier transformed Kohlrausch functions exp[-(t∕τ)(β)], describing the α-relaxation processes in these liquids. The stretching parameter β is roughly constant with wavevector Q and over the temperature range explored in bulk glycerol and propylene glycol, but varies both with Q and temperature in confinement. Average relaxation times <τ(Q)> are longer at lower temperatures and in confinement. They obey a power law <τ(Q)> ∝ Q(-γ), where the exponent γ is modified by confinement. Comparison of the bulk and confined liquids lends support to the idea that structural and∕or dynamical heterogeneity underlies the nonexponential relaxation of glass formers, as widely hypothesized in the literature.     

Internet resources integrating many small-molecule databases

()New data, tools and services recently made available on the web server (http://cactus.nci.nih.gov) of the Computer-Aided Drug Design (CADD) Group, NCI, NIH, developed in the context of chemoinformatics and drug development work, are presented. These tools are designed for searching for structures in very large databases of small molecules. One of them is a web service-the Chemical Structure Look-up Service (CSLS)-for very rapid structure look-up in an aggregated collection of more than 80 databases comprising more than 27 million unique structures at the time of this writing. CSLS contains pointers to the entries in toxicology-related databases, catalogues of commercially available samples, drugs, assay results data sets, and databases in several other categories. CSLS allows the user to find out very rapidly in which one(s) of all these databases a given structure occurs independent of the representation of the input structure, by making use of InChIs as well as new CACTVS hashcode-based identifiers. These latter, calculable, identifiers are designed to take into account tautomerism, different resonance structures drawn for charged species, and presence of additional fragments. They make possible fine-tunable yet rapid compound identification and database overlap analyses in very large compound collections.     

A new small-molecule Stat3 inhibitor

In this issue of Chemistry & Biology, Schust et al. report the discovery of a small molecule (Stattic) that inhibits the binding of a high affinity phosphopeptide for the SH2 domain of Stat3. Stattic is a new tool for studying Stat3 signaling and demonstrates that the SH2 domain is not a dead target.     

A generic small-molecule microarray immobilization strategy

Small-molecule microarrays are often limited by the requirement for each compound undergoing immobilization to contain a common functional group or by the need to prepare glass slides containing photo-reactive groups. Herein, we present a generic strategy that allows any compound library to be immobilized. This was achieved by printing a fluorous-tagged photo-reactive 3-aryl-3-trifluoromethyldiazirine, which undergoes non-selective insertion into compounds following UV-activation, onto fluorous-functionalized glass slides. The arrays could be reused following aqueous stripping and re-assessment of the compounds with the same protein or another target of interest.     

RNA enzymes with two small-molecule substrates

Background: The ‘RNA world’ hypothesis posits ancient organisms employing versatile catalysis by RNAs. In particular, such a metabolism would have required RNA catalysts that join small molecules. Such anabolic reactions now occur very widely, for example in phospholipid, terpene, amino acid and nucleotide synthetic pathways in modern organisms. Present RNA systems, however, do not perform such reactions using substrates that do not base pair. Here we ask whether this lack is a methodological artifact due to the practice of selection-amplification, or a fundamental property of active sites reconstructed within RNA structures.Results: Three rationally modified RNA enzymes, Iso6-G, Iso6-2G and Iso6-3G, catalyze the formation of (5′→5′) polyphosphate-linked oligonucleotides in trans. One of these, Iso6-G RNA, has a specific substrate site for a guanosine triphosphate, GTP, dGTP or ddGTP, and one nonspecific substrate site for a terminal-phosphate-containing small molecule. This ribozyme catalyzes multiple turnovers, proceeding at a constant rate. Guanosine specificity is probably not attributable to Watson-Crick base pairing.Conclusions: Ribozymes can readily bind multiple small-molecule substrates simultaneously and catalyze reactions that build up larger products, apparently independent of substrate-RNA Watson-Crick base pairing. RNA enzymes therefore parallel proteins, which often overcome the entropic difficulties of positioning multiple small substrates for catalysis of anabolic reactions. These results support the idea of a complex ancestral metabolism based on RNA catalysis.     

An oligomer-based approach to skeletal diversity in small-molecule synthesis

Access to small molecules of widely varying molecular shapes has been identified as an enabling step in the discovery of biologically active materials. In this communication we introduce an approach to the systematic development of architecturally distinct chemical compounds based upon the assembly of reactive monomers into linear oligomers, each of which encodes a unique molecular framework under a common set of reaction conditions. Certain products of the initial chemical transformation (Ru-catalyzed metathesis reaction) encode additional skeletons upon treatment with a second common set of reagents (Diels-Alder dienophiles). Application of this oligomerization approach has led to the discovery of a previously unreported tandem ene-yne-yne metathesis-6pi-electrocyclization-1,5-hydride migration that converts a linear substrate into a complex tricyclic 1,3-diene in a single step. Thus, the reported strategy might serve not only as a generator of skeletally diverse small molecules but also as a discovery platform for the identification of novel chemical transformations.     

Invited Lecture. Defects in small-molecule and polymeric nematics

Abstract

After a reminder of the essential topological characters of defects in phases with nematic symmetries, a review is given of the observations of textures and defects in polymeric liquid crystals. Emphasis is placed on (a) the diversity of these observations according to the type and character of the polymer, and (b) the differences with small-molecule liquid crystals. However, the topological characters are common. The differences have to be explained in terms of molecular configurations. In this respect, we develop some physical consequences of the scarcity of chain ends, in particular a possible segregation and ordering process that relaxes strong splay deformations and that occurs in the cores of disclinations with a wedge character. After having discussed models of isolated disclinations, we investigate the possibility of the thermodynamical stability of sets of disclinations. We show that this problem has some analogies with the classical Flory-Huggins theory of the stability of polymers in solution, the disclinations playing here the role of the polymeric chains. Finally we indicate the existence of other features specific to polymeric nematics, apart from the question of chain ends, such as the correlations between chains and the related phenomena of frustration.     

Progress in small-molecule luminescent materials for organic light-emitting diodes

Organic light-emitting diodes(OLEDs) have been extensively studied since the first efficient device based on small molecular luminescent materials was reported by Tang. Organic electroluminescent material, one of the centerpieces of OLEDs, has been the focus of studies by many material scientists. To obtain high luminosity and to keep material costs low, a few remarkable design concepts have been developed. Aggregation-induced emission(AIE) materials were invented to overcome the common fluorescence-quenching problem, and cross-dipole stacking of fluorescent molecules was shown to be an effective method to get high solid-state luminescence. To exceed the limit of internal quantum efficiency of conventional fluorescent materials, phosphorescent materials were successfully applied in highly efficient electroluminescent devices. Most recently, delayed fluorescent materials via reverse-intersystem crossing(RISC) from triplet to singlet and the "hot exciton" materials based on hybridized local and charge-transfer(HLCT) states were developed to be a new generation of low-cost luminescent materials as efficient as phosphorescent materials. In terms of the device-fabrication process, solution-processible small molecular luminescent materials possess the advantages of high purity(vs. polymers) and low procession cost(vs. vacuum deposition), which are garnering them increasing attention. Herein, we review the progress of the development of small-molecule luminescent materials with different design concepts and features, and also briefly examine future development tendencies of luminescent materials.     

Recent advances in mitochondria-and lysosomes-targeted small-molecule two-photon fluorescent probes

This review summarized the recent advances in small-molecule two-photon fl uorescent probes for monitoring a wide variety of biomolecules and changes inside micro-environment in mitochondria and lysosomes, or served as mitotracker and lysotracker with the assistance of two-photon microscopy.     

Enhanced small-molecule assembly through directional intramolecular forces

[reaction: see text] Directional intramolecular interactions play a critical role in the self-assembly of donor-sigma-acceptor molecules in solution. Amide functions on the periphery of the tricyclic core stabilize a C3-symmetric monomer conformation by intramolecular hydrogen bonding and dipole-dipole interactions. The molecules are effective organogelators and show long-range ordering in the bulk.     

Arresting cancer proliferation by small-molecule gene regulation

A small library of pyrrole-imidazole polyamide-DNA alkylator (chlorambucil) conjugates was screened for effects on morphology and growth characteristics of a human colon carcinoma cell line, and a compound was identified that causes cells to arrest in the G2/M stage of the cell cycle. Microarray analysis indicates that the histone H4c gene is significantly downregulated by this polyamide. RT-PCR and Western blotting experiments confirm this result, and siRNA to H4c mRNA yields the same cellular response. Strikingly, reduction of H4 protein by >50% does not lead to widespread changes in global gene expression. Sequence-specific alkylation within the coding region of the H4c gene in cell culture was confirmed by LM-PCR. The compound is active in a wide range of cancer cell lines, and treated cells do not form tumors in nude mice. The compound is also active in vivo, blocking tumor growth in mice, without obvious animal toxicity.