We have analyzed the kinetics of assembly and elongation of the mammalian RNA polymerase I complex on endogenous ribosomal genes in the nuclei of living cells with the use of in vivo microscopy. We show that components of the RNA polymerase I machinery are brought to ribosomal genes as distinct subunits and that assembly occurs via metastable intermediates. With the use of computational modeling of imaging data, we have determined the in vivo elongation time of the polymerase, and measurements of recruitment and incorporation frequencies show that incorporation of components into the assembling polymerase is inefficient. Our data provide a kinetic and mechanistic framework for the function of a mammalian RNA polymerase in living cells.

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Cycloheximide inhibits ribosomal DNA (rDNA) transcription in vivo. The mouse homologue of yeast Rrn3, a polymerase-associated transcription initiation factor, can complement extracts from cycloheximide-treated mammalian cells. Cycloheximide inhibits the phosphorylation of Rrn3 and causes its dissociation from RNA polymerase I. Rrn3 interacts with the rpa43 subunit of RNA polymerase I, and treatment with cycloheximide inhibits the formation of a Rrn3⅐rpa43 complex in vivo. Rrn3 produced in Sf9 cells but not in bacteria interacts with rpa43 in vitro, and such interaction is dependent upon the phosphorylation state of Rrn3. Significantly, neither dephosphorylated Rrn3 nor Rrn3 produced in Escherichia coli can restore transcription by extracts from cycloheximide-treated cells. These results suggest that the phosphorylation state of Rrn3 regulates rDNA transcription by determining the steady-state concentration of the Rrn3⅐RNA polymerase I complex within the nucleolus.In the early 1970s Feigelson and colleagues (1-3) reported that cycloheximide caused a rapid cessation of nucleolar RNA synthesis (ribosomal DNA transcription) and concluded that a rapidly turning over protein was required for RNA polymerase I (pol I) 1 activity in vivo. Subsequent studies have demonstrated that transcription by RNA polymerase I is subject to regulation at many levels (4, 5). At least three, and possibly more, polymerase-associated proteins, TIF-IA, Factor C*, and TFIC (6 -8), have been demonstrated to contribute to the regulation of rDNA transcription. TIF-IA and Factor C* were identified as factors that were required for the complementation of extracts of quiescent or cycloheximide-treated cells. TFIC was identified as that activity required to reconstitute transcription by extracts of glucocorticoid-treated P1798 cells.This lymphosarcoma cell line exits the cell cycle in response to the synthetic glucocorticoid dexamethasone (DEX) (6). Interestingly, TIF-IA, Factor C*, and TFIC shared several properties, including a tight association with the core polymerase (8 -10). TIF-IA and TFIC were purified and consisted of different polypeptides (10, 11). However, the lack of immunological and molecular tools precluded a definitive statement that TIF-IA and TFIC were the same or different proteins (reviewed in Refs. 4 and 5).The formation of the stable preinitiation complex in yeast requires an interaction between the upstream activating factor bound to the upstream promoter element and core factor, bound to the core promoter element. This complex then recruits transcriptionally competent RNA polymerase I to the transcription initiation site (Ref. 12 and references therein). Mechanistically, Rrn3 appears to "bridge" the polymerase and transcription initiation complexes (13-15). Thus, only pol I molecules in complex with Rrn3 are able to recognize the preinitiation complex and initiate transcription.Studies comparing the state of RNA polymerase I in growing and stationary yeast cells demonstrated that ϳ2% of the pol I in whole cell extracts was...

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Human African trypanosomiasis is a devastating disease with only a few treatment options, including pentamidine. Diamidine compounds such as pentamidine, DB75, and DB820 are potent antitrypanosomal compounds. Previous investigations have shown that diamidines accumulate to high concentrations in trypanosomes. However, the mechanism of action of this class of compounds remains unknown. A long-hypothesized mechanism of action has been binding to DNA and interference with DNA-associated enzymes. The fluorescent diamidines, DB75 and DB820, have been shown to localize not only in the DNA-containing nucleus and kinetoplast of trypanosomes but also to the acidocalcisomes. Here we investigate two series of analogs of DB75 and DB820 with various levels of in vitro antitrypanosomal activity to determine whether any correlation exists between trypanosome accumulation, distribution, and in vitro activity. Despite wide ranges of in vitro antitrypanosomal activity, all of the compounds investigated accumulated to millimolar concentrations in trypanosomes over a period of 8 h. Interestingly, some of the less potent compounds accumulated to concentrations much higher than those of more potent compounds. All of the compounds were localized to the DNA-containing nucleus and/or kinetoplast, and many were also found in the acidocalcisomes. Accumulation in the nucleus and kinetoplast should be important to the mechanism of action of these compounds. The acidocalcisomes may also play a role in the mechanism of action of these compounds. This investigation suggests that the extent of accumulation alone is not responsible for killing trypanosomes and that organelle-specific accumulation may not predict in vitro activity.Diamidine compounds, such as pentamidine, propamidine, and diminazene, have been used for many years as chemotherapeutic agents for infections caused by a variety of microbes, including parasites and fungi. Pentamidine has been used for almost 60 years as a treatment for human African trypanosomiasis and is also used to treat leishmaniasis and the opportunistic infection Pneumocystis pneumonia (32). Diminazene has been used widely for treatment of animal trypanosomiasis (9) and has also been used in humans (25). Recently, pafuramidine, or DB289, a methamidoxime prodrug of the diamidine DB75 (furamidine), has been developed as an oral treatment for early-stage sleeping sickness caused by Trypanosoma brucei gambiense. DB289 is currently in phase III clinical trials in sub-Saharan Africa (6). In addition to DB75 and DB289, a library of diamidines and prodrugs has been synthesized, with various activities against many parasites (1-3, 14-18, 32).Although diamidines have been used therapeutically for over half a century, their mechanism of action is not well understood. Many mechanisms of action have been proposed (32), but one mechanism of action of diamidines that has often been hypothesized is binding to DNA in the nucleus or kinetoplast, leading to interference of DNA-associated enzymes, such as topoisomerase II (27, 34)....

Treatment of NIH 3T3 cells with trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC), resulted in a dose-dependent increase in transcription from a rDNA reporter and from endogenous rRNA genes. Chromatin immunoprecipitation using anti-acetyl-histone H4 antibodies demonstrated a direct effect of TSA on the acetylation state of the ribosomal chromatin. TSA did not reverse inhibition of transcription from the rDNA reporter by retinoblastoma (Rb) protein, suggesting that the main mechanism by which Rb blocks rDNA transcription may not involve recruitment of deacetylases to rDNA chromatin. Overexpression of histone transacetylases p300, CBP and PCAF stimulated transcription in transfected NIH 3T3 cells. Recombinant p300, but not PCAF, stimulated rDNA transcription in vitro in the absence of nucleosomes, suggesting that the stimulation of rDNA transcription by TSA might have a chromatin-independent component. We found that the rDNA transcription factor UBF was acetylated in vivo. Finally, we also demonstrated the nucleolar localization of CBP. Our results suggest that the organization of ribosomal chromatin of higher eukaryotes is not static and that acetylation may be involved in affecting these dynamic changes directly through histone acetylation and/or through acetylation of UBF or one of the other components of rDNA transcription.

N-{trans-3-[(5-Cyano-6-methylpyridin-2-yl)oxy]-2,2,4,4-tetramethylcyclobutyl}imidazo[1,2-a]pyrimidine-3-carboxamide (1) was recently identified as a full antagonist of the androgen receptor, demonstrating excellent in vivo tumor growth inhibition in castration-resistant prostate cancer (CRPC). However, the imidazo[1,2-a]pyrimidine moiety is rapidly metabolized by aldehyde oxidase (AO). The present paper describes a number of medicinal chemistry strategies taken to avoid the AO-mediated oxidation of this particular system. Guided by an AO protein structure-based model, our investigation revealed the most probable site of AO oxidation and the observation that altering the heterocycle or blocking the reactive site are two of the more effective strategies for reducing AO metabolism. These strategies may be useful for other drug discovery programs.

PI3K, AKT, and mTOR are key kinases from PI3K signaling pathway being extensively pursued to treat a variety of cancers in oncology. To search for a structurally differentiated back-up candidate to PF-04691502, which is currently in phase I/II clinical trials for treating solid tumors, a lead optimization effort was carried out with a tricyclic imidazo[1,5]naphthyridine series. Integration of structurebased drug design and physical properties-based optimization yielded a potent and selective PI3K/mTOR dual kinase inhibitor PF-04979064. This manuscript discusses the lead optimization for the tricyclic series, which both improved the in vitro potency and addressed a number of ADMET issues including high metabolic clearance mediated by both P450 and aldehyde oxidase (AO), poor permeability, and poor solubility. An empirical scaling tool was developed to predict human clearance from in vitro human liver S9 assay data for tricyclic derivatives that were AO substrates.