Insight into the strong inhibitory action of salt on activity of neocarzinostatin.

Enediyne anticancer drugs belong to one of the most potent category in inducing DNA damage. We report 85+/-5% inhibition on activity of neocarzinostatin by salt. As high sodium ion concentration is a known tumor cell feature, we explored the dynamic mechanism of inhibition. Using various analytical tools, we examined parameters involved in the four consecutive steps of the drug action, namely, drug releasing from carrier protein, drug-DNA binding, drug activating, and DNA damaging. Neither protein stability, nor drug release rate, was altered by salt. The salt inhibition level was similar in between the protein-bound and unbound enediyne chromophore. Salt did not quench the thiol-induced drug activation. The inhibition was independent of DNA lesion types and irrelevant with thiol structures. Collectively, no salt interaction was found in the releasing, activating, and DNA damaging step of the drug action. However, binding with DNA decreased linearly with salt and corresponded well with the salt-induced inhibition on the drug activity. Salt interference on the affinity of DNA binding was the main and sole cause of the severe salt inhibition. The inhibition factor should be carefully considered for all agents with similar DNA binding mode.

Studies on the mechanism of inhibition of bacterial ribonuclease P by aminoglycoside derivatives.

Ribonuclease P (RNase P) is a Mg2+-dependent endoribonuclease responsible for the 5'-maturation of transfer RNAs. It is a ribonucleoprotein complex containing an essential RNA and a varying number of protein subunits depending on the source: at least one, four and nine in Bacteria, Archaea and Eukarya, respectively. Since bacterial RNase P is required for viability and differs in structure/subunit composition from its eukaryal counterpart, it is a potential antibacterial target. To elucidate the basis for our previous finding that the hexa-arginine derivative of neomycin B is 500-fold more potent than neomycin B in inhibiting bacterial RNase P, we synthesized hexa-guanidinium and -lysyl conjugates of neomycin B and compared their inhibitory potential. Our studies indicate that side-chain length, flexibility and composition cumulatively account for the inhibitory potency of the aminoglycoside-arginine conjugates (AACs). We also demonstrate that AACs interfere with RNase P function by displacing Mg2+ ions. Moreover, our finding that an AAC can discriminate between a bacterial and archaeal (an experimental surrogate for eukaryal) RNase P holoenzyme lends promise to the design of aminoglycoside conjugates as selective inhibitors of bacterial RNase P, especially once the structural differences in RNase P from the three domains of life have been established.

Synthesis and convenient functionalization of azide-labeled diacylglycerol analogues for modular access to biologically active lipid probes.

Cell membrane lipids have been identified as key participants in cell signaling activities. One important role is their involvement as site-specific ligands in protein-membrane binding interactions, which result in the anchoring of peripheral proteins onto cellular membranes. These events generally regulate protein function and localization and have been implicated in both normal physiological processes and those pertaining to disease state onset. Thus, it is important to elucidate the details of interactions at the molecular level, such as lipid-binding specificities and affinities, the location of receptor binding domains and multivalency in binding. For this purpose, we have designed and developed azido-tagged lipid analogues as conveniently functionalizable lipid probe scaffolds. Herein, we report the design and synthesis of the initial structure of this type, diacylglycerol analogue 2, which contains an azide tag at the sn-1 position of the lipid headgroup. Direct functionalization of this compound with a range of reporter groups has been performed to illustrate the facile access to probes of use for characterizing binding. Quantitative lipid-binding studies using protein kinase C, a known DAG-binding receptor, demonstrate that these probes are active mimetics of natural DAG. Thus, these DAG probes will serve as robust sensors for studies aimed at understanding binding interactions and as precursors for the development of analogous probes of more complex phospholipids and glycolipids.

Enrichment for chemoresistant ovarian cancer stem cells from human cell lines.

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 µM cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Modular synthesis of biologically active phosphatidic acid probes using click chemistry.

Phosphatidic acid (PA) is an important signaling lipid that plays roles in a range of biological processes including both physiological and pathophysiological events. PA is one of a number of signaling lipids that can act as site-specific ligands for protein receptors in binding events that enforce membrane association and generally regulate both receptor function and subcellular localization. However, elucidation of the full scope of PA activities has proven problematic, primarily due to the lack of a consensus sequence among PA-binding receptors. Thus, experimental approaches, such as those employing lipid probes, are necessary for characterizing interactions at the molecular level. Herein, we describe an efficient modular approach to the synthesis of a range of PA probes that employs a late stage introduction of reporter groups. This strategy was exploited in the synthesis of PA probes bearing fluorescent and photoaffinity tags as well as a bifunctional probe containing both a photoaffinity moiety and an azide as a secondary handle for purification purposes. To discern the ability of these PA analogs to mimic the natural lipid in protein-binding properties, each compound was incorporated into vesicles for binding studies using a known PA receptor, the C2 domain of PKCalpha. In these studies, each compound exhibited binding properties that were comparable to those of synthetic PA, indicating their viability as probes for effectively studying the activities of PA in cellular processes.

Aponeocarzinostatin--a superior drug carrier exhibiting unusually high endurance against denaturants.

The enediyne antitumor antibiotic chromoproteins are very potent in causing DNA damages. During the drug delivery time course, the stability of the carrier protein becomes an important concern. To simulate conceivably offensive environment in biological contexts, such as cell membrane, we studied structural endurance of aponeocarzinostatin against several denaturants by circular dichroism and nuclear magnetic resonance spectroscopy. For comparison, we also examined proteins known to be stable and similar in size to aponeocarzinostatin. The results highlight the unusual structural stability of aponeocarzinostatin against chemical denaturants, suggesting the potential of aponeocarzinostatin as an inherently superior carrier in drug delivery systems.