Discovery of a nanomolar inhibitor of lung adenocarcinoma in vitro

Efficient methods for the preparation of 5'-substituted 5'-amino-5'-deoxy-N(6)-ureidoadenosine derivatives are described. Compounds were screened for antiproliferative activity against a panel of murine and human cell lines (L1210, CEM, and HeLa) and/or against the NCI-60. The most potent derivative inhibited the lung adenocarcinoma cell line NCI-H522 at low nanomolar concentrations (GI50=9.7nM).status: publishe

CCAAT-enhancer binding protein-β expression and elevation in Alzheimer's disease and microglial cell cultures.

CCAAT-enhancer binding proteins are transcription factors that help to regulate a wide range of inflammatory mediators, as well as several key elements of energy metabolism. Because C/EBPs are expressed by rodent astrocytes and microglia, and because they are induced by pro-inflammatory cytokines that are chronically upregulated in the Alzheimer's disease (AD) cortex, we have investigated whether C/EBPs are expressed and upregulated in the AD cortex. Here, we demonstrate for the first time that C/EBPβ can be detected by Western blots in AD and nondemented elderly (ND) cortex, and that it is significantly increased in AD cortical samples. In situ, C/EBPβ localizes immunohistochemically to microglia. In microglia cultured from rapid autopsies of elderly patient's brains and in the BV-2 murine microglia cell line, we have shown that C/EBPβ can be upregulated by C/EBP-inducing cytokines or lipopolysaccharide and exhibits nuclear translocation possibly indicating functional activity. Given the known co-regulatory role of C/EBPs in pivotal inflammatory mechanisms, many of which are present in AD, we propose that upregulation of C/EBPs in the AD brain could be an important orchestrator of pathogenic changes

C/EBPβ bright field immunohistochemistry across multiple brain regions.

<p>Comparisons of C/EBPβ immuno-labeling across six different brain regions demonstrates increased immunoreactivity for C/EBPβ on activated AD microglia in AD tissue as compared to ND tissue. As shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086617#pone-0086617-g005" target="_blank">Figure 5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086617#pone-0086617-g007" target="_blank">7</a>, clustered activated microglia in the AD brain are almost invariably associated with Aβ plaques. Percent area of C/EBPβ immunostaining by image analysis is shown in parentheses. (A) Superior parietal lobule (AD 0.54% vs. ND 0.3%, AD/ND ratio = 1.8). (B) Locus coeruleus (AD 2.2% vs. ND 0.29%, AD/ND ratio = 7.6). (C) Temporal lobe (AD 1.18% vs. ND 0.6%, AD/ND ratio = 2). (D) Visual cortex (AD 0.56% vs. ND 0.47%, AD/ND ratio = 1.2). (E) Superior frontal gyrus (AD 1.36% vs. ND 0.47%, AD/ND ratio = 2.9). (F) Mid-frontal gyrus (AD 0.63% vs. ND 0.55%, AD/ND ratio = 1.1). (All micrographs are at 200X, scale bars = 800 µm).</p

Synthesis and SAR of 2',3'-bis-O-substituted N(6), 5'-bis-ureidoadenosine derivatives: Implications for prodrug delivery and mechanism of action

A series of 2',3'-bis-O-silylated or -acylated derivatives of lead compound 3a (2',3'-bis-O-tert-butyldimethylsilyl-5'-deoxy-5'-(N-methylcarbamoyl)amino-N(6)-(N-phenylcarbamoyl)adenosine) were prepared and evaluated for antiproliferative activity against a panel of murine and human cancer cell lines (L1210, FM3A, CEM, and HeLa). 2',3'-O-Silyl groups investigated included triethylsilyl (10a), tert-butyldiphenylsilyl (10b), and triisopropylsilyl (10c). 2',3'-O-Acyl groups investigated included acetyl (13a), benzoyl (13b), isobutyryl (13c), butanoyl (13d), pivaloyl (13e), hexanoyl (13f), octanoyl (13g), decanoyl (13h), and hexadecanoyl (13i). IC(50) values ranged from 3.0±0.3 to >200μg/mL, with no improvement relative to lead compound 3a. Derivative 10a was approximately equipotent to 3a, while compounds 13e-g were from three to fivefold less potent, and all other compounds were significantly much less active. A desilylated derivative (5'-deoxy-5'-(N-methylcarbamoyl)amino-N(6)-(N-phenylcarbamoyl)adenosine; 5) and several representative derivatives from each subgroup (10a-10c, 13a-13c) were screened for binding affinity for bone morphogenetic protein receptor 1b (BMPR1b). Only compound 5 showed appreciable affinity (K(d)=11.7±0.5μM), consistent with the inference that 3a may act as a prodrug depot form of the biologically active derivative 5. Docking studies (Surflex Dock, Sybyl X 1.3) for compounds 3a and 5 support this conclusion.status: publishe

C/EBPβ in treated BV-2 cell Western blots.

<p>(A) BV-2 cells treated with 1 µg/ml LPS for differing times. Maximal expression of C/EBPβ (including the LIP isoform) is induced in nuclear fractions (NF) at 4 hours relative to 1, 24, or 48 hours, or 48 hour untreated controls. Cytoplasmic fractions (CF) show little to no expression of C/EBPβ. Lanes: (1) 1 hour NF (2) 1 hour CF (3) 4 hour NF (4) 4 hour CF (5) 24 hour NF (6) 24 hour CF (7) 48 hour NF (8) 48 hour CF (9) 48 hour Control NF (10) molecular weight marker–not visible (11) 48 hour Control CF (12) Blank. β-actin control shown in lower panel. (B) BV-2 cells treated with 1 µg/ml LPS or 10 ng/ml IL-6 and/or 1 or 5 µM aggregated Aβ(1–42). C/EBPβ expression was increased in cells treated with LPS and further increased by IL-6 treatment. Odd numbered lanes are corresponding cytoplasmic fractions for nuclear fractions (NF). Even numbered lanes are nuclear fractions: (2) Control untreated (4) LPS (6) IL-6 (8) Aβ 5 µM (10) Aβ 1 µM+IL-6 (12) Aβ 5 µM+IL-6.</p

C/EBPβ confocal fluorescent immunocytochemistry in human microglia cultures.

<p>Parallel wells received (A) no cytokine treatment or 50 ng/ml (B) IL-1β, (C) IL-6, or (D) TNF-α for 4 hours. Microglia treated with cytokines show increased expression and nuclear localization of C/EBPβ. All wells were imaged concurrently with the same photomultiplier tube intensity, gain, and offset settings using an Olympus Fluoview Confocal microscope–200X (scale bar = 100 µm).</p

C/EBPβ Western blots in AD and ND frontal lobe gray matter.

<p>When normalized to β-actin, the data show a significant increase in C/EBPβ protein expression in AD compared to ND samples (t₁₆ = 2.9, P<0.01). Summary densitometry data for the blots (mean ratios of C/EBPβ/β-actin integrated optical density values, IDV) are provided in the bottom panel.</p

C/EBPβ immunocytochemistry of cultured murine BV-2 cell line.

<p>C/EBPβ is increased both in numbers of immunopositive cells and in intensity of fluorescence in the nucleus of cells treated with (B) 1 µg/ml LPS as compared with (A) controls (scale bar = 10 µm). Some control cells were positive for C/EBPβ but the immunofluorescence was notably less intense. The average mean gray value of pixels in LPS treated cells (51.14±3.15, SEM) was significantly higher than the control (38.45±1.86, SEM) (P<0.05). Integrated density in LPS treated cells (6.87±0.42, SEM) was also significantly higher than the control (4.23±0.31, SEM) (P<0.01).</p

Synthesis, SAR, and preliminary mechanistic evaluation of novel antiproliferative N(6),5'-bis-ureido- and 5'-carbamoyl-N(6)-ureidoadenosine derivatives

We have developed efficient methods for the preparation of N(6),5'-bis-ureidoadenosine derivatives and their 5'-carbamoyl-N(6)-ureido congeners. Treatment of 5'-azido-5'-deoxy-N(6)-(N-alkyl or -arylurea)adenosine derivatives (6a-d) with H(2)/Pd-C or Ph(3)P/H(2)O, followed by N-methyl-p-nitrophenylcarbamate gave N(6),5'-bis-ureido products 7a-d in 49-78% yield. Analogous derivatives in the 5'-carbamoyl-N(6)-ureido series were prepared by treatment of 2',3'-bis-O-TBS-adenosine (11) with N-methyl-p-nitrophenylcarbamate followed by acylation with appropriate isocyanates which gave 13a-d in 45-69% yield. A more versatile route for obtaining potentially vast libraries of compounds from both series was achieved by treatment of 5'-N-methylureido- or 5'-N-methylcarbamoyladenosine derivatives with ethylchlorformate to give N(6)-ethoxycarbonyl derivatives (9 and 14) in 55-63% yields, respectively. Simple heating of 9 or 14 in the presence of primary alkyl- or arylamines gave the corresponding N(6),5'-bis-ureido- or 5'-carbamoyl-N(6)-ureidoadenosine derivatives in good yields (33-72% and 39-83%; 10a-e and 15a-e, respectively). Significant antiproliferative activities (IC(50)≈4-10μg/mL) were observed for a majority of the N(6),5'-bis-ureido derivatives, whereas the 5'-carbamoyl-N(6)-ureido derivatives were generally less active (IC(50) >100μg/mL). A 2',3'-O-desilylated derivative (5'-amino-5'-deoxy-5'-N-methylureido-N(6)-(N-phenylcarbamoyl)adenosine, 16) was shown to inhibit binding of 16 of 441 protein kinases to immobilized ATP-binding site ligands by 30-40% in a competitive binding assay at 10μM. Compound 16 was also shown to bind to bone morphogenetic protein receptor 1b (BMPR1b) with a Kd=11.5±0.7μM.status: publishe

C/EBPβ bright field immunohistochemistry.

<p>Single and dual-label immunohistochemistry demonstrates increased immunoreactivity for C/EBPβ on activated AD microglia invested in amyloid plaques in AD tissue as compared to ND tissue. C/EBPβ immunoreactivity in (A) ND SFG tissue, (B and C) AD SFG tissue, and (D) AD entorhinal cortex tissue. (A–B–200X, scale bars = 800 µm. C–D–400X, scale bars = 400 µm). Percent area of C/EBPβ immunostaining is higher in AD (B: 2.23%) vs. ND (A: 0.44%). (E) C/EBPβ immunoreactive cells (black) and 4G8-immunoreactive Aβ plaques (brown) in AD SFG tissue. (F) C/EBPβ immunoreactive cells (black) and thioflavine S-labeled Aβ plaques (green fluorescence) in AD SFG tissue. (E–F–200X, inset 400X). Significantly higher percentages of C/EBPβ immunopositive cells were associated with Aβ plaques (E: 96.9%, F: 89.4%, both Student’s <i>t</i>-test, P<0.01) than not associated with Aβ plaques.</p