Tyrosine kinase inhibitors. 6. Structure-activity relationships among N- and 3-substituted 2,2'-diselenobis(1H-indoles) for inhibition of protein tyrosine kinases and comparative in vitro and in vivo studies against selected sulfur congeners.

A small series of 2,2'-diselenobis(1H-indoles) was synthesized as redox-modified congeners of our earlier reported 2,2'-dithiobis(1H-indole) series. Utilizing chemistry similar to that developed earlier for the disulfur series, compounds were made from 2-halogeno-3-indolecarboxylic acid precursors bearing various polar functionality at the C-3 position and small alkyl substituents at the N-1 position of the indole nucleus. Additional compounds were derived from (R)- or (S)-tryptophan via a novel application of diselenium dichloride as an electrophilic source of diselenium, and a much improved process to a 2,2'-dithiobis(1H-indole) congener was developed utilizing disulfur dichloride as a source of disulfur. Against isolated epidermal growth factor receptor (EGFr), platelet-derived growth factor receptor (PDGFr), and v-src tyrosine kinases, compounds in this series displayed broad inhibitory activity with IC50 = 0.9 to > 100 microM vs EGFr, 3.4 to > 50 microM vs PDGFr, and 0.4-6.7 microM vs v-src. In general, compounds derived from tryptophan displayed the greatest potency against EGFr and those from 2-halogeno-3-indolecarboxylic acids greater potency against PDGFr and v-src. Enzyme kinetics studies showed that both classes of compounds display primarily noncompetitive inhibition with respect to either ATP or peptide substrate. The sulfhydryl reducing agent dithiothreitol (DTT) caused a general decrease in inhibition of the EGFr and v-src tyrosine kinases by both the diselenium and disulfur series with the reversal of enzyme inhibition occurring less readily within the diselenium series. In whole cell studies, compounds of this class were growth inhibitory against Swiss 3T3 mouse fibroblasts with IC50 values from 0.5 to 19.5 microM, and the observed SAR was different from that of the 2,2'-dithiobis(1H-indoles). A comparative study in the same cell line on the effects of the 2,2'-diselenobis(1H-indole) derived from (R)-tryptophan vs its disulfur congener on growth factor mediated tyrosine phosphorylation showed that this compound significantly inhibited EGFr and PDGFr (in response to its ligand) autophosphorylation with complete suppression at 25 and 5 microM, respectively. Tyrosine phosphorylation of an 85 kDa protein typically phosphorylated in response to bFGF was also exquisitely sensitive to this compound, and it displayed inhibitory effects on DNA, RNA, and protein synthesis at submicromolar concentrations. The disulfur congener exhibited a qualitatively similar pattern; however, its potency was 10-fold less. This same diselenium/disulfur pair was evaluated in vivo against the B16 melanoma, colon carcinoma 26, and M5076 sarcoma murine tumors, and the A431 epidermoid, and C6 glioma human tumor xenografts. At maximum tolerated doses (1.8 and 5.0 mg/kg/injection, respectively), neither the diselenium nor disulfur congener was effective against the C6 glioma when administered intraperitoneally on a d1-9 schedule. Studies were also carried out against the A431 epidermoid xenograft to evaluate the same pair of compounds via continuous subcutaneous infusion from Alzet miniosmotic pumps. The maximum dose that could be administered daily was limited by compound solubility. Neither compound produced an antitumor effect in a 7-day continuous infusion study. In the 27-day study, the disulfur compound was inactive whereas the diselenium compound produced a 10.8-day growth delay without appreciable treatment related weight loss. The in vitro and in vivo findings offer a mechanistic rationale as to why the 2,2'-diselenobis(1H-indoles) are more potent inhibitors than their disulfur congeners.

A specific inhibitor of the epidermal growth factor receptor tyrosine kinase.

A small molecule called PD 153035 inhibited the epidermal growth factor (EGF) receptor tyrosine kinase with a 5-pM inhibition constant. The inhibitor was specific for the EGF receptor tyrosine kinase and inhibited other purified tyrosine kinases only at micromolar or higher concentrations. PD 153035 rapidly suppressed autophosphorylation of the EGF receptor at low nanomolar concentrations in fibroblasts or in human epidermoid carcinoma cells and selectively blocked EGF-mediated cellular processes including mitogenesis, early gene expression, and oncogenic transformation. PD 153035 demonstrates an increase in potency over that of other tyrosine kinase inhibitors of four to five orders of magnitude for inhibition of isolated EGF receptor tyrosine kinase and three to four orders of magnitude for inhibition of cellular phosphorylation.

Expression of a 118 Kd protein in colon carcinoma cells that is immunologically related to PLC-gamma 1 and is augmented by serum-free conditions.

Immunoprecipitation of cell lysates from serum-starved HCT-8 colon carcinoma cells with monoclonal antibodies raised to PLC-gamma 1 precipitates proteins which can be detected by immunoblotting; the 148 Kd PLC-gamma 1 and a smaller protein of approximately 118 Kd (p118). Expression of p118 is suppressed in cells grown in serum but is present 8 to 12 h after cells are placed in serum-free medium. Conversely, serum-starved cells that express this protein suppress it 6 to 8 h after exposure to 10% fetal calf serum. Pulse chase experiments indicate that the protein is not a degradation product of PLC. This protein has thus far been detected only in human carcinoma cells and not in normal or transformed fibroblasts. Although the protein has not been identified, its expression pattern may suggest a role contributing to the serum-independent nature of human carcinoma cell lines.

Structure and evolution of the XcyI restriction-modification system.

The XcyI restriction-modification system from Xanthomonas cyanopsidis recognizes the sequence, CCCGGG. The XcyI endonuclease and methylase genes have been cloned and sequenced and were found to be aligned in a head to tail orientation with the methylase preceding and overlapping the endonuclease by one base pair. The nucleotide sequence codes for an N4 cytosine methyltransferase with a predicted molecular weight of 33,500 and an endonuclease comprised of 333 codons and a molecular weight of 36,600. Sequence comparisons revealed significant similarity between the XcyI, CfrI and SmaI methylisomers. In contrast, no similarity was detected between the primary structures of the XcyI and SmaI endonucleases. The XcyI restriction-modification system is highly homologous to the XmaI genes, although the DNA sequences flanking the genes rapidly diverge. The sequence of the XcyI endonuclease contains two motifs which have recently been identified as essential to the activity of the EcoRV endonuclease.