A Systematic Evaluation of the Bleomycin A₂ l-Threonine Side Chain:  Its Role in Preorganization of a Compact Conformation Implicated in Sequence-Selective DNA Cleavage

Abstract

The preparation and examination of 3−7 are detailed and constitute analogues of deglycobleomycin A2 (2) containing systematic modifications in the l-threonine side chain. The studies revealed a substantial impact of the substituent on the DNA cleavage efficiency and ratio of double strand/single strand (ds/ss) cleavage without affecting the characteristic 5‘-GPy selectivity. The results of the comparisons proved consistent with conformational effects of the substituent within the linker domain which restrict the number of accessible conformations (Φ ≅ −120°, Ψ = 60−180°) and favor adoption of a compact conformation (Φ ≅ −120°, Ψ ≅ 180°) implicated in sequence-selective DNA cleavage. The studies also identify one potential site that may adopt two nearly equivalent turn conformations. This site may constitute one swivel point in the structure that permits access to a class of related bound structures (Ψ = 60−180°) adaptable to variable conformational characteristics required of multiple cleavage sites, including access to both strands of duplex DNA from a single intercalation site important for both primary and secondary DNA cleavage.

DNA Cleavage/Repair and Signal Transduction Pathways in Irradiated Breast Tumor Cells

“…However, this shift was considerably more pronounced in the U937/p21AS/F4 line, a finding that is distinctly different from that observed previously in p21 antisense-expressing cells treated with PMA (16). Results obtained using the G99-549 murine antibody, which primarily recognizes underphosphorylated pRb (28), supported the notion that p 2l WAF1/clpl dysregulation promotes ara-C-mediated pRb dephosphorylation (Fig. 3S).…”

“…We failed to observe any direct evidence of cell killing in MCF-7 cells with either l,25-(OH) 2 -D 3 orEB 1089 with exposure times as long as 96 h, although it is possible that the reduction in the number of cells over time reflects both growth inhibition and (a limited degree of) cell death. While other laboratories have reported apoptosis in breast tumor cells (including MCF-7 cells) treated with vitamin D 3 compounds (26)(27)(28)(29), the susceptibility to apoptosis appears to be limited to a relatively small fraction of the target cell population in these studies (i.e. between 5 and 15%) (26,27).…”

“…The promotion of apoptosis could b< tumor cell proliferation (28)(29)(30)(31)(32)(33)(34). Similarly, studies indicate mediated through a reduction in levels of the anti-apoptosi that the primary response to radiation in both TP53 wild-(^proteins such as BCL2 in the face of an increase in TP5 fb mi* type (MCF-7) and TP53-mutated breast tumor cells (MDA MB 231) is a prolonged growth arrest (4,$), although it is well established that many irradiated tumor cells ultimately die through reproductive cell death.…”

“…Finally, since (i) bleomycin-induced double-strand cleavage exhibits strictly single-hit kinetics, (ii) the cleavages in both strands are based on C-4' abstraction, (iii) the two abstraction Ä sites are 15-18 Ä apart in B-DNA and (iv) there is no evidence that the drug is birunctional, it has been concluded that the two breaks are probably made in succession by a single bleomycin molecule that is reactivated in the process of forming the first break [26][27][28]. There is some evidence that the presence of the first break may direct the putative repositioning of bleomycin molecule for attack at a specific site in the complementary strand [29].…”

“…Although the chemical synthesis of bleomycin was first reported in 1982, recent technical improvements in the synthesis have recently allowed creation of a much wider array of analogues for basic and clinical investigation [28,31].…”