We developed a high-throughput HTRF (homogeneous time-resolved fluorescence) assay for Akt kinase activity and screened approx. 270000 compounds for their ability to inhibit the three isoforms of Akt. Two Akt inhibitors were identified that exhibited isoenzyme specificity. The first compound (Akt-I-1) inhibited only Akt1 (IC50 4.6 microM) while the second compound (Akt-I-1,2) inhibited both Akt1 and Akt2 with IC50 values of 2.7 and 21 microM respectively. Neither compound inhibited Akt3 nor mutants lacking the PH (pleckstrin homology) domain at concentrations up to 250 microM. These compounds were reversible inhibitors, and exhibited a linear mixed-type inhibition against ATP and peptide substrate. In addition to inhibiting kinase activity of individual Akt isoforms, both inhibitors blocked the phosphorylation and activation of the corresponding Akt isoforms by PDK1 (phosphoinositide-dependent kinase 1). A model is proposed in which these inhibitors bind to a site formed only in the presence of the PH domain. Binding of the inhibitor is postulated to promote the formation of an inactive conformation. In support of this model, antibodies to the Akt PH domain or hinge region blocked the inhibition of Akt by Akt-I-1 and Akt-I-1,2. These inhibitors were found to be cell-active and to block phosphorylation of Akt at Thr308 and Ser473, reduce the levels of active Akt in cells, block the phosphorylation of known Akt substrates and promote TRAIL (tumour-necrosis-factor-related apoptosis-inducing ligand)-induced apoptosis in LNCap prostate cancer cells.
We covalently linked doxorubicin with a peptide that is hydrolyzable by prostate-specific antigen. In the presence of prostate tumor cells secreting prostate-specific antigen, the peptide moiety of this conjugate, L-377,202, was hydrolyzed, resulting in the release of leucine-doxorubicin and doxorubicin, which are both very cytotoxic to cancer cells. However, L-377,202 was much less cytotoxic than conventional doxorubicin to cells in culture that do not secrete prostate-specific antigen. L-377,202 was approximately 15 times more effective than was conventional doxorubicin at inhibiting the growth of human prostate cancer tumors in nude mice when both drugs were used at their maximally tolerated doses. Nude mice inoculated with human prostate tumor cells secreting prostate-specific antigen showed considerable reductions in tumor burden with minimal total body weight loss when treated with L-377, 202. This improvement in therapeutic index correlated with the selective localization of leucine-doxorubicin and free doxorubicin in tissues secreting prostate-specific antigen after exposure to L-377,202.
Using three-dimensional (3D) telomeric analysis of buccal cells of 82 Alzheimer's disease (AD) patients and cognitively normal age and gender-matched controls, we have for the first time examined changes in the 3D nuclear telomeric architecture of buccal cells among levels of AD severity based on five 3D parameters: i) telomere length, ii) telomere number, iii) telomere aggregation, iv) nuclear volume, and v) a/c ratio, a measure of spatial telomere distribution. Our data indicate that matched controls have significantly different 3D telomere profiles compared to mild, moderate, and severe AD patients (p < 0.0001). Distinct profiles were also evident for each AD severity group. An increase in telomere number and aggregation concomitant with a decrease in telomere length from normal to severe AD defines the individual stages of the disease (p < 0.0001).
Human papillomaviruses (HPVs) are the etiologic agents responsible for benign epithelial proliferative disorders including genital warts and are a contributory factor in the pathogenesis of cervical cancer. HPVs demonstrate strict species and cell-type specificity, which is manifested by the inability of these viruses to induce disease in any species other than humans. The natural history of HPV infection in humans is closely mimicked by cottontail rabbit papillomavirus (CRPV) infection in domestic laboratory rabbits. The CRPV E7 gene is known to play an essential role in virus-mediated induction of papillomas. We now show by mutational analysis that the CRPV E7 protein's biochemical and biological properties, including binding to the retinoblastoma suppressor protein (pRB), transcription factor E2F transactivation of the adenovirus E2 promoter, disruption of pRB-E2F complexes, and cellular transformation as measured by growth in soft agar, mimic those of the
The preparation of a series of quinazoline-2,4-diones, 1-3, and pyrrolo[3,4-d]pyrimidine-2,4-diones, 4-8 is described. A small number of quinazolinedione analogs were identified from random screening to possess low micromolar (1.3-4.4 microM) potency in the nuclear factor of activated T cells-1-regulated beta-galactosidase expression assay. An expanded analog search resulted in identifying pyrrolopyrimidinedione 4b which is 5-10-fold (0.26 microM) more potent than the quinazolinediones. Replacement of the benzyl group with naphthyl led to greater potency and conformationally restricted analogs 4u-w. The naphthyl and acenaphthyl analogs are 10-100 times more potent inhibitors of beta-galactosidase expression than 4b. Binding affinity data for displacement of radiolabeled 4s from Jurkat cell membranes reflected an excellent correlation with the IC50 value for inhibition of beta-galactosidase activity. These products, whose structure-activity relationships are discussed, are of interest as potential agents for preventing interleukin-2 gene transcription.
The human papillomavirus E7 gene can transform murine fibroblasts and cooperate with other viral oncogenes in transforming primary cell cultures. One biochemical property associated with the E7 protein is binding to the retinoblastoma tumor suppressor gene product (pRB). Biochemical properties associated with pRB include binding to viral transforming proteins (ElA, large T, and E7), binding to cellular proteins (E2F and Myc), and binding to DNA. The mechanism by which E7 stimulates cell growth is uncertain. However, E7 binding to pRB inhibits binding of cellular proteins to pRB and appears to block the growth-suppressive activity of pRB. We have found that E7 also inhibits binding of pRB to DNA. A 60-kDa version of pRB (pRB60) produced in reticulocyte translation reactions or in bacteria bound quantitatively to DNA-cellulose. Recombinant E7 protein used at a 1:1 or 10:1 molar ratio with pRB60 blocked 50 or >95% of pRB60 DNA-binding activity, respectively. A mutant E7 protein (E7-Ala-24) with reduced pRB60-binding activity exhibited a parallel reduction in its blocking of pRB60 binding to DNA. An E7(20-29) peptide that blocks binding of E7 protein to pRB60 restored the DNA-binding activity of pRB60 in the presence of E7. Peptide E7(2-32) did not block pRB60 binding to DNA, while peptide E7(20-57) and an E7 fragment containing residues 1 to 60 partially blocked DNA binding. E7 species containing residues 3 to 75 were fully effective at blocking pRB60 binding to DNA. These studies indicate that E7 protein specifically blocks pRB60 binding to DNA and suggest that the E7 region responsible for this property lies between residues 32 and 75. The functional significance of these observations is unclear. However, we have found that a point mutation in pRB60 that impairs DNA-binding activity also blocks the ability of pRB60 to inhibit cell growth. This correlation suggests that the DNA-binding activity of retinoblastoma proteins contributes to their biological properties.Human papillomaviruses (HPV) are the etiologic agents responsible for warts and are believed to contribute to the pathogenesis of cervical cancer (16,55). The most commonly studied isolates of HPV associated with cervical cancer include 54). The HPV E6 and E7 genes are believed to participate in cell growth regulation. These genes are generally found in cervical cancer cells and cell lines associated with HPV infections, while the remainder of the HPV genome is commonly absent (3,41,43). Moreover, the E6 and E7 genes together are capable of immortalizing primary human keratinocytes (37). The E7 gene by itself is capable of transforming NIH 3T3 cells and can cooperate with ras in the transformation of primary baby rat kidney cells (39,46). E6 alone does not transform murine fibroblasts or cooperate with ras in transforming rat cells (5, 46). Since the E7 gene appears to be the most potent transforming activity in the HPV-16 genome, our laboratory has focused on the role of the E7 gene product in the pathogenesis of HPV-induced disease.
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