Crystal structures of several urea- and thiourea-derived compounds in complex with the nicotinamide phosphoribosyltransferase (Nampt) protein were utilized to design a potent amide-containing ...inhibitor bearing an aza-indole moiety (7, Nampt BC IC50 = 9.0 nM, A2780 cell proliferation IC50 = 10 nM). The Nampt–7 cocrystal structure was subsequently obtained and enabled the design of additional amide-containing inhibitors which incorporated various other fused 6,5-heterocyclic moieties and biaryl sulfone or sulfonamide motifs. Additional modifications of these molecules afforded many potent biaryl sulfone-containing Nampt inhibitors which also exhibited favorable in vitro ADME properties (microsomal and hepatocyte stability, MDCK permeability, plasma protein binding). An optimized compound (58) was a potent inhibitor of multiple cancer cell lines (IC50 <10 nM vs U251, HT1080, PC3, MiaPaCa2, and HCT116 lines), displayed acceptable mouse PK properties (F = 41%, CL = 52.4 mL/min/kg), and exhibited robust efficacy in a U251 mouse xenograft model.
Nicotinamide phosphoribosyltransferase (Nampt) is a promising anticancer target. Virtual screening identified a thiourea analogue, compound 5, as a novel highly potent Nampt inhibitor. Guided by the ...cocrystal structure of 5, SAR exploration revealed that the corresponding urea compound 7 exhibited similar potency with an improved solubility profile. These studies also indicated that a 3-pyridyl group was the preferred substituent at one inhibitor terminus and also identified a urea moiety as the optimal linker to the remainder of the inhibitor structure. Further SAR optimization of the other inhibitor terminus ultimately yielded compound 50 as a urea-containing Nampt inhibitor which exhibited excellent biochemical and cellular potency (enzyme IC50 = 0.007 μM; A2780 IC50 = 0.032 μM). Compound 50 also showed excellent in vivo antitumor efficacy when dosed orally in an A2780 ovarian tumor xenograft model (TGI of 97% was observed on day 17).
Transcriptional activation of target genes represents an important component of the tumour-suppressor function of p53 and provides a functional link between p53 and various growth-regulatory ...processes, including cell cycle progression (p21/WAF1), DNA repair (GADD45) and apoptosis (bax). Here we use a differential cloning approach to identify the gene encoding insulin-like growth factor binding protein 3 (IGF-BP3) as a novel p53-regulated target gene. Induction of IGF-BP3 gene expression by wild-type but not mutant p53 is associated with enhanced secretion of an active form of IGF-BP3 capable of inhibiting mitogenic signalling by the insulin-like growth factor IGF-1. Our results indicate that IGF-BP3 may link p53 to potential novel autocrine/paracrine signalling pathways and to processes regulated by or dependent on IGF(s), such as cellular growth, transformation and survival.
A co-crystal structure of amide-containing compound (4) in complex with the nicotinamide phosphoribosyltransferase (Nampt) protein and molecular modeling were utilized to design and discover a potent ...novel cyanoguanidine-containing inhibitor bearing a sulfone moiety (5, Nampt Biochemical IC50=2.5nM, A2780 cell proliferation IC50=9.7nM). Further SAR exploration identified several additional cyanoguanidine-containing compounds with high potency and good microsomal stability. Among these, compound 15 was selected for in vivo profiling and demonstrated good oral exposure in mice. It also exhibited excellent in vivo antitumor efficacy when dosed orally in an A2780 ovarian tumor xenograft model. The co-crystal structure of this compound in complex with the NAMPT protein was also determined.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Exposure of mammalian cells to hypoxia, radiation and certain chemotherapeutic agents promotes cell cycle arrest and/or apoptosis. Activation of p53 responsive genes is believed to play an important ...role in mediating such responses. In this study we identified a novel gene, PA26, which maps to chromosome 6q21 and encodes at least three transcript isoforms, of which two are differentially induced by genotoxic stress (UV, gamma-irradiation and cytotoxic drugs) in a p53-dependent manner. A functional p53-responsive element was identified in the second intron of the PA26 gene, in consistance with a mechanism of transcriptional induction of the PA26 gene by p53. No clues to its functions were revealed by sequence analysis, although pronounced negative regulation by serum factors argues for a potential role of PA26 in growth regulation. Immunological analysis suggests that PA26 protein(s) is localized to the cell nucleus. Our results suggest that the PA26 gene is a novel p53 target gene with properties common to the GADD family of growth arrest and DNA damage-inducible stress-response genes, and, thus, a potential novel regulator of cellular growth.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The insulin-like growth factor-I receptor (IGF-IR) is involved in
tumorigenesis. The aim of the present study was to investigate whether
the IGF-IR is a physiological target for p53 in osteosarcoma ...cells. The
p53-induced regulation of IGF-IR levels was studied in a
tetracycline-regulated expression system. When expressed in Saos-2,
osteosarcoma cells that lack p53, wild-type p53 decreased, whereas
mutated p53 increased IGF-IR expression, and IGF-I-induced tyrosine
phosphorylation of the IGF-IR. Similarly, wild-type p53 decreased
IGF-I-induced tyrosine phosphorylation of IRS-1. A functional and
physical interaction between p53 and Sp1, in the regulation of the
IGF-R, was studied in osteosarcoma cells. Expression of p53 decreased
IGF-IR promoter activity, whereas no effect on promoter activity was
seen by Sp1 expressed alone. However, Sp1 counteracted the inhibitory
effect of p53 on IGF-IR promoter activity in a dose-dependent manner.
Furthermore, wild-type and mutated p53 were coimmunoprecipitated with
Sp1, indicating a physical interaction between p53 and Sp1.
In conclusion, p53 regulates IGF-IR expression, as reflected by a
reduction in IGF-IR protein and a parallel reduction in IGF-I-induced
tyrosine phosphorylation of the IGF-IR and IRS-1 in an osteosarcoma
cell line. These data indicate that the IGF-I receptor is a
physiological target for p53 in osteosarcoma cells. Furthermore, data
supporting an interaction between p53 and Sp1 in the regulation of the
promoter activity of IGF-IR are presented.
The p53 tumor suppressor protein induces cell cycle arrest or apoptosis in response to cellular stresses. We have identified PRG3 (
p
53-
r
esponsive
g
ene 3), which is induced specifically under ...p53-dependent apoptotic conditions in human colon cancer cells, and encodes a novel polypeptide of 373 amino acids with a predicted molecular mass of 40.5 kDa. PRG3 has significant homology to bacterial oxidoreductases and the apoptosis-inducing factor, AIF, and the gene was assigned to chromosome 10q21.3–q22.1. Expression of PRG3 was induced by the activation of endogenous p53 and it contains a p53-responsive element. Unlike AIF, PRG3 localizes in the cytoplasm and its ectopic expression induces apoptosis. An amino-terminal deletion mutant of PRG3 that lacks a putative oxidoreductase activity retains its apoptotic activity, suggesting that the oxidoreductase activity is dispensable for the apoptotic function of PRG3. The PRG3 gene is thus a novel p53 target gene in a p53-dependent apoptosis pathway.
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BFBNIB, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The p53 tumor suppressor protein is a transcription factor with sequence-specific DNA binding activity that is thought to be important for the growth-inhibitory function of p53. DNA binding appears ...to require activation of a cryptic form of p53 by allosteric mechanisms involving a negative regulatory domain at the carboxyl terminus of p53. The latent form of p53, reactive to the carboxyl-terminal antibody PAb421, is produced in a variety of eukaryotic cells, suggesting that activation of p53 is an important rate-limiting step in vivo. In this report we provide evidence that phosphorylation of serine 378 within the carboxyl-terminal negative regulatory domain of the human p53 protein by protein kinase C correlates with loss of PAb421 reactivity and a concomitant activation of sequence-specific DNA binding. These effects are reversed by subsequent dephosphorylation of the protein kinase C-reactive site by protein phosphatases 1 (PP1) and 2A (PP2A), which restore the reactivity of p53 to PAb421 and regenerate the latent form of p53 lacking significant DNA binding activity. Thus, p53 is subject to both positive and negative regulation by reversible enzymatic modifications affecting the latent or active state of the protein, suggesting a possible mechanism for the regulation of its tumor suppressor function.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The VHL tumor suppressor gene has previously been reported to encode a protein of 213 amino acid residues. Here we report the identification of a second major VHL gene product with an apparent ...molecular weight of 18 kD, pVHL18, which appears to arise from alternate translation initiation at a second AUG codon (codon 54) within the VHL open reading frame. In vitro and in vivo studies indicate that the internal codon in the VHL mRNA is necessary and sufficient for production of pVHL18. pVHL18 can bind to elongin B, elongin C, and Hs-CUL2. When reintroduced into renal carcinoma cells that lack a wild-type VHL allele, pVHL18 suppresses basal levels of VEGF expression, restores hypoxia-inducibility of VEGF expression, and inhibits tumor formation in nude mice. These data strongly support the existence of two distinct VHL gene products in VHL tumor suppression.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The genomics and proteomics sciences have fundamentally changed the ways in which drug targets are being identified, characterized and validated. Here we review how genomics and proteomics research ...is improving our understanding of genetic determinants of drug susceptibility and response and, conversely, how organic small molecules mediate their pharmacological effects by modulating genome and proteome activities. We also examine the effect this improved understanding has on the drug discovery and development process.
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