Development of indazole mineralocorticoid receptor antagonists and investigation into their selective late-stage functionalization

The derivatization of pharmaceuticals is a core activity in the discovery and development of new medicines. Late-stage functionalization via modern CH functionalization chemistry has emerged as a powerful technique with which to diversify advanced pharmaceutical intermediates. We report herein a case study in late-stage functionalization towards the development of a new class of indazole-based mineralocorticoid receptor antagonists (MRA). An effort to modify the electronics of the core indazole heterocycle inspired the use of modern CH borylation chemistry. New reactivity patterns were revealed and studied computationally. Ultimately, a de novo synthesis delivered a key 6-fluoroindazole compound 26, a potent MRA with excellent metabolic stability.     

MRE11-RAD50-NBS1 and ATM function as co-mediators of TRF1 in telomere length control

Human telomeres are associated with ATM and the protein complex consisting of MRE11, RAD50 and NBS1 (MRN), which are central to maintaining genomic stability. Here we show that when targeted to telomeres, wild-type RAD50 downregulates telomeric association of TRF1, a negative regulator of telomere maintenance. TRF1 binding to telomeres is upregulated in cells deficient in NBS1 or under ATM inhibition. The TRF1 association with telomeres induced by ATM inhibition is abrogated in cells lacking MRE11 or NBS1, suggesting that MRN and ATM function in the same pathway controlling TRF1 binding to telomeres. The ability of TRF1 to interact with telomeric DNA in vitro is impaired by ATM-mediated phosphorylation. We propose that MRN is required for TRF1 phosphorylation by ATM and that such phosphorylation results in the release of TRF1 from telomeres, promoting telomerase access to the ends of telomeres.     

Design and synthesis of novel N-sulfonyl-2-indole carboxamides as potent PPAR-gamma binding agents with potential application to the treatment of osteoporosis

The synthesis and structure-activity relationships of a novel series of N-sulfonyl-2-indole carboxamides that bind to peroxisome proliferator-activated receptor gamma (PPAR-gamma) are reported. Chemical optimization of the series led to the identification of 4q (IC(50)=50 nM) as a potent binding agent of PPAR-gamma. Also reported is preliminary cell based data suggesting the use of these compounds in the treatment of osteoporosis.     

mRNA Translation Regulation by the Gly-Ala Repeat of Epstein-Barr Virus Nuclear Antigen 1

The glycine-alanine repeat (GAr) sequence of the Epstein-Barr virus-encoded EBNA-1 prevents presentation of antigenic peptides to major histocompatibility complex class I molecules. This has been attributed to its capacity to suppress mRNA translation in cis. However, the underlying mechanism of this function remains largely unknown. Here, we have further investigated the effect of the GAr as a regulator of mRNA translation. Introduction of silent mutations in each codon of a 30-amino-acid GAr sequence does not significantly affect the translation-inhibitory capacity, whereas minimal alterations in the amino acid composition have strong effects, which underscores the observation that the amino acid sequence and not the mRNA sequence mediates GAr-dependent translation suppression. The capacity of the GAr to repress translation is dose and position dependent and leads to a relative accumulation of preinitiation complexes on the mRNA. Taken together with the surprising observation that fusion of the 5′ untranslated region (UTR) of the c-myc mRNA to the 5′ UTR of GAr-carrying mRNAs specifically inactivates the effect of the GAr, these results indicate that the GAr targets components of the translation initiation process. We propose a model in which the nascent GAr peptide delays the assembly of the initiation complex on its own mRNA.Epstein-Barr Virus (EBV) nuclear antigen 1 (EBNA-1) and latency-associated nuclear antigen 1 (LANA-1), from Kaposi''s sarcoma-associated herpesvirus (KSHV), are major latency proteins of these two gammaherpesviruses that are essential for maintaining viral episomes in infected cells (21, 22). Independent studies suggest that both proteins have evolved mechanisms to remain largely invisible to the immune system, which could otherwise eliminate latently infected cells (8, 9, 19, 25). These mechanisms act in cis and are mediated via an internal repeat region. In the case of EBNA-1 this region consists of an N-terminal glycine-alanine repeat (GAr), and for LANA-1 the region consists of a glutamine-glutamate-aspartate central repeat (QED-CR). Although the two domains do not share amino acid homology, both retard their own synthesis to reduce the production of defective ribosomal products that can be processed for the major histocompatibility complex (MHC) class I-restricted antigen presentation pathway (23, 24), highlighting the importance of translation control in regulating MHC class I-restricted antigen presentation. To compensate for their low rates of synthesis, both proteins also have slow turnover rates (4, 8).Regulation of translation for most prokaryotic and eukaryotic mRNAs occurs at the level of initiation, but there are examples where regulation of protein synthesis depends on the elongation stage (17). The two main types of translation initiation are the classic cap-dependent and the less frequent cap-independent translation mechanisms (5, 7, 11, 14, 16). In the former, the preinitiation complex is formed around the cap structure in the 5′ untranslated region (UTR) of the message, whereas in the latter the 40S subunit is directed toward the mRNA via an internal ribosome entry site (IRES). The mechanism of GAr- and LANA-1-mediated control of translation seems different from other types of viral regulation in several aspects. The EBNA-1 GAr is 60 to 300 amino acids long, depending on virus isolate, and is positioned in the N-terminal part of the protein. The GAr message is GC rich but does not activate protein kinase R and eukaryotic initiation factor 2α phosphorylation (25). The fact that the GAr has to be encoded to suppress translation, coupled with the restricted use of GGG and GGA codons to express Gly and of GCA to express Ala in the GAr (GAT, GAG, and CAG for aspartic acid, glutamic acid, and glutamine, respectively, in the LANA sequence), could suggest that codon exhaustion might explain the effect of these repeats. However, manipulations of sequence order, orientation, and composition of the QED-CR and GAr domains and the observation that antibodies directed toward the GAr can stimulate translation in vitro instead favor a direct role for the amino acid sequence (8, 25).Here, we have studied GAr-mediated regulation of translation in vitro and in vivo. The results presented suggest that, once synthesized, the nascent GAr peptide sequence prevents the assembly of the following upstream ribosomes. This knowledge should further understanding of how amino acid repeat sequences can affect mRNA translation in cis and should shed light on a novel type of viral control of mRNA translation and its implications in regulating MHC class I-restricted antigen presentation.     

Development of a soluble PTD-HPV18E7 fusion protein and its functional characterization in eukaryotic cells

Though accumulated evidence has demonstrated the transformation capacity of human papillomavirus （HPV） type 18 protein E7, the underlying mechanism is still arguable. Developing a protein transduction domain （PTD）-linked E7 molecule is a suitable strategy for assessing the biological functions of the protein. In the present study, HPV18 E7 protein fused to an N-terminal PTD was expressed in the form of glutathione S-transferase fusion protein in Escherichia coli with pGEX-4T- 3 vector. After glutathione-Sepharose 4B bead affinity purification, immunoblot identification and thrombin cleavage, the PTD-18E7 protein showed structural and functional activity in that it potently transduced the ceils and localized into their nuclei. The PTD-18E7 protein transduced the NIH3T3 ceils in 30 min and remained stable for at least 24 h. In addition, the PTD-18E7 protein interacted with retinoblastoma protein （pRB） and caused pRB degradation in the transduced NIH3T3 cells. In contrast to the pRB level, p27 protein level was elevated in the transduced NIH3T3 cells. The PTD-18E7 protein gives us a new tool to study the biological functions of the HPV E7 protein.     

Influence of the Qinghai-Tibetan railway and highway on the activities of wild animals

The effect of traffic and railway construction on the activities of wild animals during the daytime along the Qinghai-Tibetan highway between Budongquan (35°17′ N; 93°16′ E) and Wudaliang (35°13′ N; 93°04′ E) was studied in August 2003 and August 2004. Furthermore, passageways cross the Qinghai-Tibetan railway were monitored to determine the relationship between the usage frequency of the passageways and the distance to the Qinghai-Tibetan highway and the dimension of the passageways. The results showed that the traffic during the daytime had some effects on the Tibetan antelope (Pantholops hodgsoni), Tibetan gazelle (Procapra picticaudata) and Kiang (Equus kiang) when they were crossing the road, and especially it is significant on the Tibetan antelope. At the same time, they could adapt themselves to the changes in the surroundings by learning and by adjusting their behavior. Most of their activities took place in the morning in order to avoid the effects of traffic, and they could also find and use the passageways cross the Qinghai-Tibetan railway. The dimensions of the passageways, the distance to the Qinghai-Tibetan highway, the surrounding habitat, and human activities could influence the efficiency of the passageways. Most passageways cross the Qinghai-Tibetan railway could not be effectively used by the wildlife because of the short length and low height or because of human activities in the contiguous areas of the passageways. However, the wildlife could adapt themselves to the changes in the surroundings caused by the construction of the Qinghai-Tibetan railway by learning and by adjusting their behavior.     

The expression of serum steroid sex hormones and steroidogenic enzymes following intraperitoneal administration of dehydroepiandrosterone (DHEA) in male rats

The adrenals of humans and primates could secrete large amounts of dehydroepiandrosterone (DHEA) and its sulphate ester (DHEA-S) in the circulation, which act as precursors of active steroid hormones in a long series of peripheral target intracrine tissues. The marked decline of serum DHEA and DHEA-S concentrations with age in humans has been incriminated in the development of various pathologies. Therefore, this study aims to provide detailed information on the effects of the intraperitoneal injection of DHEA on circulating steroid hormones and their metabolites and their trade-off relationship over 24 h in male rats. In this study, 100 healthy adult male Sprague-Dawley (SD) rats were randomly divided into three groups: control, 25 mg kg⁻¹ DHEA-treated and 100 mg kg⁻¹ DHEA-treated. The animals were sacrificed at 0, 1.5, 3, 6, 12 or 24 h, and the samples were collected for subsequent analysis. Total cholesterol (TC) markedly decreased 3 h after the administration of 100 mg kg⁻¹ DHEA, but markedly increased 12 h after administration. The DHEA-S, progesterone (P), testosterone (T), oestradiol (E₂), cortisol (Cor) and aldosterone (Ald) concentrations also markedly increased after DHEA administration, with serum DHEA-S, T, E₂ and Cor levels peaking at 1.5 h. Over time, steroid hormone levels were depressed, but serum Cor and Ald levels were markedly elevated relative to the control group at 24 h. Furthermore, DHEA treatment produced a significant increase in P450scc, 17β-HSDIII, CYP17α and 3β-HSD mRNA expression at 1.5 h, but a decided decrease in P450scc and StAR mRNA expression at 12 and 24 h, and CYP17α and 17β-HSDIII expression at 12 h in the 100 mg kg⁻¹ DHEA group. In total, the results of the present study indicate that DHEA at high pharmacological doses may affect steroid through an effect on steroidogenic enzymes.