Small molecules,big impact: a history of chemical inhibitors and the cytoskeleton

Chemical inhibitors, whether natural products or synthetic, have had an enormous impact on the study of the eukaryotic cytoskeleton. Here we review the history of some of the most widely used cytoskeletal poisons and their influence on our understanding of cytoskeletal functions. We then highlight several new inhibitors and the targeted screens used to identify them and discuss why this approach has been successful.     

Phagemid encoded small molecules for high throughput screening of chemical libraries

A new strategy for monovalently displaying small molecules on phage surfaces was developed and applied to high throughput screening for molecules with high binding affinity to the target protein. Peptidyl carrier protein (PCP) excised from nonribosomal peptide synthetase was monovalently displayed on the surface of M13 phage as pIII fusion proteins. Small molecules of diverse structures were conjugated to coenzyme A (CoA) and then covalently attached to the phage displayed PCP by Sfp phosphopantetheinyl transferase. Because Sfp is broadly promiscuous for the transfer of small molecule linked phosphopantetheinyl moieties to apo PCP domains, this approach will enable displaying libraries of small molecules on phage surfaces. Unique 20-base-pair (bp) DNA sequences were also incorporated into the phagemid DNA so that each compound displayed on the phage surface was encoded by a DNA bar code encapsulated inside the phage coat protein. Single round selection of phage displayed small molecules achieved more than 2000-fold enrichment of small molecules with nM binding affinity to the target protein. The selection process is further accelerated by the use of DNA decoding arrays for identifying the selected small molecules.     

SAR by ILOEs: an NMR-based approach to reverse chemical genetics

Reverse chemical genetics is an emerging technique that makes use of small molecule inhibitors to characterize how a protein functions. In this regard, we have developed an NMR-based approach (SAR by ILOEs) that enables the identification of high affinity ligands for a given protein target without the need of a specific assay. Our approach is of general applicability and could result very powerful in reverse chemical-genetics studies, target validation, and lead discovery. We report a recent application on the design and synthesis of compounds that inhibit protein-membrane interactions.     

Forward chemical genetics: library scaffold design

With the unraveling of the entire human genome, it has become imperative to understand the function of the gene products, proteins. Within the past several years, chemical genetics has gained recognition as a powerful approach to study protein function by using small molecules as gene knock-out or knock-in mimics. Forward chemical genetics is a three-step process; the design and synthesis of a small molecule library represents the first step followed secondly by the search for novel phenotypes and then by isolation and identification of target protein(s). This review will focus on the first step, the design of the scaffold for small molecule libraries. It will also examine the connection between the choice of a scaffold and the propensity of that library to demonstrate enhanced biological activity when tested in certain cellular systems.     

Targeting apoptosis via chemical design: inhibition of bid-induced cell death by small organic molecules

Bid is a key member of the Bcl-2 family proteins involved in the control of the apoptotic cascade in cells, leading to cell death. Uncontrolled cell death is associated with several human pathologies, such as neurodegenerative diseases and ischemic injuries. Therefore, Bid represents a potential yet unexplored and challenging target for strategies aimed at the development of therapeutic agents. Here we show that a multidisciplinary NMR-based approach that we named SAR by ILOEs (structure activity relationships by interligand nuclear Overhauser effect) allowed us to rationally design a series of 4-phenylsulfanyl-phenylamine derivatives that are capable of occupying a deep hydrophobic crevice on the surface of Bid. These compounds represent the first antiapoptotic small molecules targeting a Bcl-2 protein as shown by their ability to inhibit tBid-induced SMAC release, caspase-3 activation, and cell death.     

Chemical dimerizers and three-hybrid systems: scanning the proteome for targets of organic small molecules

The integration of technological advances in areas as diverse as chemical biology, proteomics, genomics, automation, and bioinformatics has led to the emergence of novel screening paradigms for analyzing the molecular basis of drug action. This review summarizes recent advances in three-hybrid technologies and their application to the characterization of small molecule-protein interactions and proteome-wide identification of drug receptors.     

The Gaussian Generalized Born model: application to small molecules

This work presents a Generalized Born model for the computation of the electrostatic component of solvation energies which is based on volume integration. An analytic masking function is introduced to remove Coulombic singularities. This approach leads to analytic formulae for the computation of Born radii, which are differentiable to arbitrary order, and computationally straightforward to implement.     

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.     

A one-bead, one-stock solution approach to chemical genetics: part 2

BACKGROUND: Chemical genetics provides a systematic means to study biology using small molecules to effect spatial and temporal control over protein function. As complementary approaches, phenotypic and proteomic screens of structurally diverse and complex small molecules may yield not only interesting individual probes of biological function, but also global information about small molecule collections and the interactions of their members with biological systems. RESULTS: We report a general high-throughput method for converting high-capacity beads into arrayed stock solutions amenable to both phenotypic and proteomic assays. Polystyrene beads from diversity-oriented syntheses were arrayed individually into wells. Bound compounds were cleaved, eluted, and resuspended to generate 'mother plates' of stock solutions. The second phase of development of our technology platform includes optimized cleavage and elution conditions, a novel bead arraying method, and robotic distribution of stock solutions of small molecules into 'daughter plates' for direct use in chemical genetic assays. This library formatting strategy enables what we refer to as annotation screening, in which every member of a library is annotated with biological assay data. This phase was validated by arraying and screening 708 members of an encoded 4320-member library of structurally diverse and complex dihydropyrancarboxamides. CONCLUSIONS: Our 'one-bead, multiple-stock solution' library formatting strategy is a central element of a technology platform aimed at advancing chemical genetics. Annotation screening provides a means for biology to inform chemistry, complementary to the way that chemistry can inform biology in conventional ('investigator-initiated') small molecule screens.     

Application of gel chromatography to small molecules

Summary Liuid chromatographic separations of hydrocarbons, alcohols, triglycerides and surfactants with molecular weights ranging from 100–1,000 were accomplished, using columns packed with porous polymeric beads. The fractionation capability of the porous gels has been extended to smaller molecules through the development of small porosity gels. The gels separated according to molecular size and are therefore useful both for separation and identification purposes. Since they require no stationary liquid phase, they afford long column life without need of a saturated carrier.

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Zusammenfassung Flüssigkeits-chromatographische Trennungen von Kohlenwasserstoffen, Alkoholen, Triglyceriden und oberflächenaktiven Substanzen mit Molekulargewichten von 100–1000 wurden mit Hilfe von porösen Polymerkörnern erreicht. Das Trennvermögen der porösen Gele wurde durch Entwicklung kleinporiger Typen auf kleinere Moleküle ausgedehnt. Da die Moleküle entsprechend ihrer Größe getrennt werden, kann außer der Trennung auch eine Identifizierung erreicht werden. Da keine flüssige stationäre Phase erforderlich ist, ist eine lange Lebensdauer der Säulen gewährleistet.

     

Macrocycloadditions leading to conformationally restricted small molecules

[reaction: see text] The Cu(I)-catalyzed cycloaddition of alkynes and azides (click reaction) provides a robust method for the construction of macrocyclic small molecules via an intramolecular macrocycloaddition. A three-subunit system has been used to explore the tolerance of this macrocycloaddition to variations of stereochemistries and substituents.     

Self-organization of disc-like molecules: chemical aspects

The hierarchical self-assembly of disc-shaped molecules leads to the formation of discotic liquid crystals. These materials are of fundamental importance not only as models for the study of energy and charge migration in self-organized systems but also as functional materials for device applications such as, one-dimensional conductors, photoconductors, light emitting diodes, photovoltaic solar cells, field-effect transistors and gas sensors. The negative birefringence films formed by polymerized nematic discotic liquid crystals have been commercialized as compensation foils to enlarge the viewing angle of commonly used twisted nematic liquid crystal displays. To date the number of discotic liquid crystals derived from more than 50 different cores comes to about 3000. This critical review describes, after an in-depth introduction, recent advances in basic design principles and synthetic approaches towards the preparation of most frequently encountered discotic liquid crystals.     

Binding of small guest molecules to multivalent receptors

The complexation of phenol derivatives, aromatic carboxylic acids, and n-octylgalactopyranoside by hydrogen-bonded exo-receptors is described. The receptors are formed by self-assembly of differently functionalized calix[4]arene dimelamines with 5,5-diethyl barbiturate or butyl cyanurate. The multivalent complementary recognition site of the receptors is used very efficiently to complex multiple guests. A 1:6 binding mode was observed for phenol derivatives forming single hydrogen bonds with all six recognition sites of an ureido functionalized receptor assembly, while 1:3 complexation was observed for phenol derivatives which form two hydrogen bonds with two different ureido recognition sites of the same receptor. Aromatic carboxylic acids are complexed in a 1:6 ratio by receptors having six amino recognition sites. The complexation of n-octylgalactopyranoside by Gly-L-Ser functionalized receptors is also described, indicating that it is possible to use small peptidic fragments to complex biologically important molecules.     

Transition-metal-templated synthesis of rotaxanes and catenanes: From small molecules to polymers

This article provides an overview of the fundamental principles of the synthesis of metallocatenanes and metallorotaxanes. It also describes the synthesis and properties of electronic conducting polymers—polypyrrole and polythiophene—built around metallocatenanes and metallorotaxanes. The particular properties of this new class of polymers, including the possibility of transmetallation reactions being performed with them and the observation of electronic coupling between the metal centers and the conducting matrix, are discussed. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 3470–3477, 2003