The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase is a master checkpoint regulator safeguarding the genome. Upon DNA damage, the ATR-ATRIP complex is recruited to sites of DNA damage by RPA-coated single-stranded DNA and activated by an elusive process. Here, we show that ATR is transformed into a hyperphosphorylated state after DNA damage, and that a single autophosphorylation event at Thr 1989 is crucial for ATR activation. Phosphorylation of Thr 1989 relies on RPA, ATRIP, and ATR kinase activity, but unexpectedly not on the ATR stimulator TopBP1. Recruitment of ATR-ATRIP to RPA-ssDNA leads to congregation of ATR-ATRIP complexes and promotes Thr 1989 phosphorylation in trans. Phosphorylated Thr 1989 is directly recognized by TopBP1 via the BRCT domains 7 and 8, enabling TopBP1 to engage ATR-ATRIP, to stimulate the ATR kinase, and to facilitate ATR substrate recognition. Thus, ATR autophosphorylation on RPA-ssDNA is a molecular switch to launch robust checkpoint response.
Transmembrane anion transport modality is enjoying a renewed interest because of recent advances toward anticancer therapy. Here we show bis(sulfonamides) as efficient receptors for selective Cl(-) ion binding and transport across lipid bilayer membranes. Anion-binding studies by (1)H NMR indicate a logical correlation between the acidity of sulfonamide N-H proton and binding strength. Such recognition is influenced further by the lipophilicity of a receptor during the ion-transport process. The anion-binding and transport activity of a bis(sulfonamide) system are far superior compared to those of the corresponding bis(carboxylic amide) derivative. Fluorescent-based assays confirm the Cl(-)/anion antiport as the operational mechanism of the ion transport by bis(sulfonamides). Disruption of ionic homeostasis by the transported Cl(-) ion, via bis(sulfonamide), is found to impose cell death. Induction of a caspase-dependent intrinsic pathway of apoptosis is confirmed by monitoring the changes in mitrochondrial membrane potential, cytochrome c leakage, activation of family of caspases, and nuclear fragmentation studies.
Effective targeting of mitochondria has emerged as an alternative strategy in cancer chemotherapy. However, considering mitochondria's crucial role in cellular energetics, metabolism and signaling, targeting mitochondria with small molecules would lead to severe side effects in cancer patients. Moreover, mitochondrial functions are highly dependent on other cellular organelles like nucleus. Hence, simultaneous targeting of mitochondria and nucleus could lead to more effective anticancer strategy. To achieve this goal, we have developed sub 200 nm particles from dual drug conjugates derived from direct tethering of mitochondria damaging drug (α- tocopheryl succinate) and nucleus damaging drugs (cisplatin, doxorubicin and paclitaxel). These dual drug conjugated nanoparticles were internalized into the acidic lysosomal compartments of the HeLa cervical cancer cells through endocytosis and induced apoptosis through cell cycle arrest. These nanoparticles damaged mitochondrial morphology and triggered the release of cytochrome c. Furthermore, these nanoparticles target nucleus to induce DNA damage, fragment the nuclear morphology and damage the cytoskeletal protein tubulin. Therefore, these dual drug conjugated nanoparticles can be successfully used as a platform technology for simultaneous targeting of multiple subcellular organelles in cancer cells to improve the therapeutic efficacy of the free drugs.
Despite the great interest in artificial ion channel design, only a small number of channel-forming molecules are currently available for addressing challenging problems, particularly in the biological systems. Recent advances in chloride-mediated cell death, aided by synthetic ion carriers, encouraged us to develop chloride selective supramolecular ion channels. The present work describes vicinal diols, tethered to a rigid 1,3-diethynylbenzene core, as pivotal moieties for the barrel-rosette ion channel formation, and the activity of such channels was tuned by controlling the lipophilicity of designed monomers. Selective transport of chloride ions via an antiport mechanism and channel formation in the lipid bilayer membranes were confirmed for the most active molecule. A theoretical model of the supramolecular barrel-rosette, favored by a network of intermolecular hydrogen bonding, has been proposed. The artificial ion-channel-mediated transport of chloride into cells and subsequent disruption of cellular ionic homeostasis were evident. Perturbation of chloride homeostasis in cells instigates cell death by inducing the caspase-mediated intrinsic pathway of apoptosis.
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