The circadian clock controls daily oscillations of gene expression at the cellular level. We report the development of a high-throughput circadian functional assay system that consists of luminescent reporter cells, screening automation, and a data analysis pipeline. We applied this system to further dissect the molecular mechanisms underlying the mammalian circadian clock using a chemical biology approach. We analyzed the effect of 1,280 pharmacologically active compounds with diverse structures on the circadian period length that is indicative of the core clock mechanism. Our screening paradigm identified many compounds previously known to change the circadian period or phase, demonstrating the validity of the assay system. Furthermore, we found that small molecule inhibitors of glycogen synthase kinase 3 (GSK-3) consistently caused a strong short period phenotype in contrast to the well-known period lengthening by lithium, another presumed GSK-3 inhibitor. siRNA-mediated knockdown of GSK-3␤ also caused a short period, confirming the phenotype obtained with the small molecule inhibitors. These results clarify the role of GSK-3␤ in the period regulation of the mammalian clockworks and highlight the effectiveness of chemical biology in exploring unidentified mechanisms of the circadian clock.screening ͉ small molecule library ͉ kinase G enetic networks of regulated transcription and protein turnover lie at the core of circadian regulation in all organisms. Mutation in key nodes of the circadian networks causes changes in overt behavioral and physiological rhythms (1, 2). For example, familial advanced sleep phase syndrome with early sleep times and early-morning awakening is attributed to missense mutations of human PER2 and CSNK1D genes (3, 4). The clock genes constitute the transcription/translation-based negative feedback loop of the core oscillator; CLOCK/BMAL1 heterodimers activate transcription of Per and Cry genes, and PER and CRY proteins in turn inhibit their own transcription (5). In addition to transcriptional regulation, posttranslational modifications of clock proteins by phosphorylation, ubiquitination, and acetylation play essential roles in the oscillator mechanism (6, 7).The molecular clock machinery resides at the cellular level, and each single cell shows circadian rhythmicity in a cell-autonomous manner (8-10). At the organismal level, the cellular oscillators are organized in a hierarchy, in which the suprachiasmatic nucleus (SCN) constitutes the central circadian pacemaker (11). In the SCN, the cellular clocks are synchronized to form a coherent oscillator through intracellular coupling (12), making the SCN clock more robust against genetic and environmental perturbations than peripheral oscillators (13). Therefore, a cell-based assay system using cultured fibroblasts that lack intercellular coupling (9, 10) will provide a particularly responsive system to characterize the circadian clockwork through an unbiased, phenotype-driven screening (14, 15). Perturbations may be revealed in such a c...

HBV persistence and transmission require HBV replication, which depends on the assembly of a core particle composed of capsid protein (Cp), polymerase, and pregenomic RNA. Reverse transcription to produce infectious DNA-containing particles occurs solely within the core residing in the cytoplasm (9, 10). Thus, core assembly is likely to be a high value target for therapeutics (11).The capsid, the protein shell of the core, is built of 120 Cp dimers arranged with T ϭ 4 symmetry (12, 13). The dimer interfaces are evident as spikes (14-16) that are the major epitope of the capsid (17). Cp in low ionic strength solution is dimeric (18). We have studied ionic strength-dependent capsid assembly extensively in vitro by using the Cp assembly domain (Cp149) (residues 1-149) lacking the 34-residue C-terminal RNA-binding domain (19)(20)(21). Assembly is nucleated by a trimer of Cp dimers and proceeds without accumulating observable populations of intermediates (22). Interactions between dimers are weak but sum to give a globally stable capsid (23). These capsids persist, even under conditions where they are not thermodynamically favored, because of hysteresis to dissociation (24). Some Cp mutations lead to faster assembly and greater stability (25), indicating that wild-type Cp is suboptimal for assembly and suggesting that assembly is regulated in vivo, possibly by conformational change. In support of this assertion, we found that Zn 2ϩ alters the conformation of Cp dimers and enhances the rate of assembly, suggesting that capsid assembly is allosterically regulated (26).Recently, it was discovered that heteroaryldihydropyrimidines (HAPs) (Fig. 1) affect the accumulation of HBV capsids (27,28). HAP drugs decreased the yield of assembled core and HBV genomes from cells that constitutively produce HBV. Electron microscopy showed that Cp assembled in vitro in the presence of HAP drugs led to polymers that had abnormal morphology (29). Similarly, small molecules such as bis ANS {5,5-bis[8-(phenylamino)-1-naphthalenesulfonate]} alter Cp assembly in vitro (30). Recent reports suggest that other small molecules also inhibit normal HBV capsid assembly (31-33).Here, we describe the mechanism of a representative HAP compound, HAP-1 [methyl 4-(2-chloro-4-f luorophenyl)-6-methyl-2-(pyridin-2-yl)-1,4-dihydropyrimidine-5-carboxylate] (Fig. 1). In vitro, low concentrations of HAP-1 enhance both the rate and extent of assembly by favoring an assembly-active state; thus, HAP-1 acts like an allosteric effector. At higher concentrations, HAP-1 led to aberrant noncapsid polymers in vitro, even at the expense of preexisting capsids. We propose that both of these effects on assembly contribute to reducing HBV virion production. Materials and MethodsSynthesis of HAP-1. Preparation of racemic HAP-1 (Fig. 1) was adapted from the patent literature (27,28,34). Condensation of a pyridylamidine with a substituted ␣-carboxymethyl enone gave a 30% yield of Ͼ97% pure HAP-1 after chromatographic purification. HAP-1 was characterized by 1 H, 13 C, and 19 ...

Covalent bond formation to proteins is made difficult by their multiple unprotected functional groups and normally low concentrations. A water-soluble sulfonated bathophenanthroline ligand (2) was used to promote a highly efficient Cu(I)-mediated azide-alkyne cycloaddition (CuAAC) reaction for the chemoselective attachment of biologically relevant molecules to cowpea mosaic virus (CPMV). The ligated substrates included complex sugars, peptides, poly(ethylene oxide) polymers, and the iron carrier protein transferrin, with routine success even for cases that were previously resistant to azide-alkyne coupling using the conventional ligand tris(triazolyl)amine (1). The use of 4-6 equiv of substrate was sufficient to achieve loadings of 60-115 molecules/virion in yields of 60-85%. Although it is sensitive to oxygen, the reliably efficient performance of the Cu.2 system makes it a useful tool for demanding bioconjugation applications.

Copper-based catalysts for the 1,3-dipolar cycloaddition of azides and alkynes were screened in parallel fashion using a fluorescence quenching assay. The method was designed to identify systems able to accelerate the coupling of reactants at micromolar concentrations in aqueous mixtures and to obtain quantitative comparisons of their activities. In addition to the tris(triazolylamines) previously reported, two types of compounds (bipy/phen and 2-pyridyl Schiff bases) were found to exhibit significant ligand-accelerated catalysis, with one complex showing especially dramatic rate enhancements. Preliminary explorations of the dependence of reaction rate on pH, ligand:Cu ratio, and Cu concentration are described.