Ribozyme 的结构及其化学修饰

本文对四种类型Ribozyme的结构进行了综述, 并着重讨论了锤头型Ribozyme的化学修饰及对其稳定性和催化活性的影响。     

Covalent‐Allosteric Kinase Inhibitors

Targeting and stabilizing distinct kinase conformations is an instrumental strategy for dissecting conformation‐dependent signaling of protein kinases. Herein the structure‐based design, synthesis, and evaluation of pleckstrin homology (PH) domain‐dependent covalent‐allosteric inhibitors (CAIs) of the kinase Akt is reported. These inhibitors bind covalently to a distinct cysteine of the kinase and thereby stabilize the inactive kinase conformation. These modulators exhibit high potency and selectivity, and represent an innovative approach for chemical biology and medicinal chemistry research.     

An Allosteric Receptor by Simultaneous “Casting” and “Molding” in a Dynamic Combinatorial Library

Allosteric synthetic receptors are difficult to access by design. Herein we report a dynamic combinatorial strategy towards such systems based on the simultaneous use of two different templates. Through a process of simultaneous casting (the assembly of a library member around a template) and molding (the assembly of a library member inside the binding pocket of a template), a Russian‐doll‐like termolecular complex was obtained with remarkable selectivity. Analysis of the stepwise formation of the complex indicates that binding of the two partners by the central macrocycle exhibits significant positive cooperativity. Such allosteric systems represent hubs that may have considerable potential in systems chemistry.     

Plug and play with RNA

Retooling RNA: RNA aptamers are high-affinity ligands that can be assembled with other structures to yield multivalent molecules. These properties have been addressed in two recent studies: One describes a GFP-like RNA reporter used to study the dynamics of endogenous RNA; the other study reports on an aptamer-templated assembly of multi-enzyme complexes in bacteria for the controlled production of secondary molecules (see picture).     

Identification of ligands for the Tau exon 10 splicing regulatory element RNA by using dynamic combinatorial chemistry

We describe the use of dynamic combinatorial chemistry (DCC) to identify ligands for the stem-loop structure located at the exon 10-5'-intron junction of Tau pre-mRNA, which is involved in the onset of several tauopathies including frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). A series of ligands that combine the small aminoglycoside neamine and heteroaromatic moieties (azaquinolone and two acridines) have been identified by using DCC. These compounds effectively bind the stem-loop RNA target (the concentration required for 50% RNA response (EC(50)): 2-58 μM), as determined by fluorescence titration experiments. Importantly, most of them are able to stabilize both the wild-type and the +3 and +14 mutated sequences associated with the development of FTDP-17 without producing a significant change in the overall structure of the RNA (as analyzed by circular dichroism (CD) spectroscopy), which is a key factor for recognition by the splicing regulatory machinery. A good correlation has been found between the affinity of the ligands for the target and their ability to stabilize the RNA secondary structure.     

Localized Template‐Driven Functionalization of Nanoparticles by Dynamic Combinatorial Chemistry

We have developed a method for the localized functionalization of gold nanoparticles using imine‐based dynamic combinatorial chemistry. By using DNA templates, amines were grafted on the aldehyde‐functionalized nanoparticles only if and where the nanoparticles interacted with the template molecules. Functionalization of the nanoparticles was controlled solely by the DNA template; only amines capable of interacting with DNA were bound to the surface. Interestingly, even though our libraries contained only a handful of simple amines, the DNA sequence influenced their attachment to the surface. Our method opens up new opportunities for the synthesis of multivalent, nanoparticle‐based receptors for biomacromolecules.     

New loop-loop tertiary interactions in self-splicing introns of subgroup IC and ID: a complete 3D model of the Tetrahymena thermophila ribozyme

Background: Group I introns self-splice via two consecutive trans-esterification reactions in the presence of guanosine cofactor and magnesium ions. Comparative sequence analysis has established that a catalytic core of about 120 nucleotides is conserved in all known group I introns. This core is generally not sufficient for activity, however, and most self-splicing group I introns require non-nonserved peripheral elements to stabilize the complete three-dimensional (3D) structure. The physico-chemical properties of group I introns make them excellent systems for unraveling the structural basis of the RNA-RNA interactions responsible for promoting the self-assembly of complex RNAs.Results: We present phylogenetic and experimental evidence for the existence of three additional tertiary base pairings between hairpin loops within peripheral components of subgroup IC1 and ID introns. Each of these new long range interactions, called P13, P14 and P16, involves a terminal loop located in domain 2. Although domains 2 of IC and ID introns share very strong sequence similarity, their terminal loops interact with domains 5 and 9 (subgroup IC1) and domain 6 (subgroup ID). Based on these tertiary contacts, comparative sequence analysis, and published experimental results such as Fe(II)-EDTA protection patterns, we propose 3D models for two entire group I introns, the subgroup IC1 intron in the large ribosomal precursor RNA of Tetrahymena thermophila and the SdCob.1 subgroup ID intron found in the cytochrome b gene of Saccharomyces douglasii.Conclusions: Three-dimensional models of group I introns belonging to four different subgroups are now available. They all emphasize the modular and hierarchical organization of the architecture of group I introns and the widespread use of base-pairings between terminal hairpin loops for stabilizing the folded and active structures of large and complex RNA molecules.     

Solution-phase combinatorial libraries: modulating cellular signaling by targeting protein-protein or protein-DNA interactions

The high-throughput synthesis and screening of compound libraries hold tremendous promise for drug discovery and powerful methods for both solid-phase and solution-phase library preparation have been introduced. The question of which approach (solution-phase versus solid-phase) is best for the preparation of chemical libraries has been replaced by which approach is most appropriate for a particular target or screen. Herein we highlight distinctions in the two approaches that might serve as useful considerations at the onset of new programs. This is followed by a more personal account of our own focus on solution-phase techniques for the preparation of libraries designed to modulate cellular signaling by targeting protein-protein or protein-DNA interactions. The screening of our libraries against a prototypical set of extracellular and intracellular targets, using a wide range of assay formats, provided the first small-molecule modulators of the protein-protein interactions studied, and a generalized approach for conducting such studies.     

Structural and Kinetic Profiling of Allosteric Modulation of Duplex DNA Induced by DNA-Binding Polyamide Analogues

A combined structural and quantitative biophysical profile of the DNA binding affinity, kinetics and sequence-selectivity of hairpin polyamide analogues is described. DNA duplexes containing either target polyamide binding sites or mismatch sequences are immobilized on a microelectrode surface. Quantitation of the DNA binding profile of polyamides containing N-terminal 1-alkylimidazole (Im) units exhibit picomolar binding affinities for their target sequences, whereas 5-alkylthiazole (Nt) units are an order of magnitude lower (low nanomolar). Comparative NMR structural analyses of the polyamide series shows that the steric bulk distal to the DNA-binding face of the hairpin iPr-Nt polyamide plays an influential role in the allosteric modulation of the overall DNA duplex structure. This combined kinetic and structural study provides a foundation to develop next-generation hairpin designs where the DNA-binding profile of polyamides is reconciled with their physicochemical properties.     

Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin: Fragment‐Based Drug Design Facilitated by Dynamic Combinatorial Chemistry

Fragment‐based drug design (FBDD) affords active compounds for biological targets. While there are numerous reports on FBDD by fragment growing/optimization, fragment linking has rarely been reported. Dynamic combinatorial chemistry (DCC) has become a powerful hit‐identification strategy for biological targets. We report the synergistic combination of fragment linking and DCC to identify inhibitors of the aspartic protease endothiapepsin. Based on X‐ray crystal structures of endothiapepsin in complex with fragments, we designed a library of bis‐acylhydrazones and used DCC to identify potent inhibitors. The most potent inhibitor exhibits an IC₅₀ value of 54 nm , which represents a 240‐fold improvement in potency compared to the parent hits. Subsequent X‐ray crystallography validated the predicted binding mode, thus demonstrating the efficiency of the combination of fragment linking and DCC as a hit‐identification strategy. This approach could be applied to a range of biological targets, and holds the potential to facilitate hit‐to‐lead optimization.     

The measurement of molecular diversity by receptor site interaction simulation

The assembly of large compound libraries for the purpose of screening against various receptor targets to identify chemical leads for drug discovery programs has created a need for methods to measure the molecular diversity of such libraries. The method described here, for which we propose the acronym RESIS (for Receptor Site Interaction Simulation), relates directly to this use. A database is built of three-dimensional representations of the compounds in the library and a set of three-point three-dimensional theoretical receptor sites is generated based on putative hydrophobic and polar interactions. A series of flexible, three-dimensional searches is then performed over the database, using each of the theoretical sites as the basis for one such search. The resulting pattern of hits across the grid of theoretical receptor sites provides a measure of the molecular diversity of the compound library. This can be conveniently displayed as a density map which provides a readily comprehensible visual impression of the library diversity characteristics. A library of 7500 drug compounds derived from the CIPSLINEPC databases was characterized with respect to molecular diversity using the RESIS method. Some specific uses for the information obtained from application of the method are discussed. A comparison was made of the results from the RESIS method with those from a recently published two-dimensional approach for assessing molecular diversity using sets of compounds from the Maybridge database (MAY).