ELR510444, a novel microtubule disruptor with multiple mechanisms of action.

Although several microtubule-targeting drugs are in clinical use, there remains a need to identify novel agents that can overcome the limitations of current therapies, including acquired and innate drug resistance and undesired side effects. In this study, we show that ELR510444 has potent microtubule-disrupting activity, causing a loss of cellular microtubules and the formation of aberrant mitotic spindles and leading to mitotic arrest and apoptosis of cancer cells. ELR510444 potently inhibited cell proliferation with an IC(50) value of 30.9 nM in MDA-MB-231 cells, inhibited the rate and extent of purified tubulin assembly, and displaced colchicine from tubulin, indicating that the drug directly interacts with tubulin at the colchicine-binding site. ELR510444 is not a substrate for the P-glycoprotein drug transporter and retains activity in ßIII-tubulin-overexpressing cell lines, suggesting that it circumvents both clinically relevant mechanisms of drug resistance to this class of agents. Our data show a close correlation between the concentration of ELR510444 required for inhibition of cellular proliferation and that required to cause significant loss of cellular microtubule density, consistent with its activity as a microtubule depolymerizer. ELR510444 also shows potent antitumor activity in the MDA-MB-231 xenograft model with at least a 2-fold therapeutic window. Studies in tumor endothelial cells show that a low concentration of ELR510444 (30 nM) rapidly alters endothelial cell shape, similar to the effect of the vascular disrupting agent combretastatin A4. These results suggest that ELR510444 is a novel microtubule-disrupting agent with potential antivascular effects and in vivo antitumor efficacy.

Theory of growth by differential sedimentation, with application to snowflake formation.

A simple model of irreversible aggregation under differential sedimentation of particles in a fluid is presented. The structure of the aggregates produced by this process is found to feed back on the dynamics in such a way as to stabilize both the exponents controlling the growth rate, and the fractal dimension of the clusters produced at readily predictable values. The aggregation of ice crystals to form snowflakes is considered as a potential application of the model.

Potassium currents in chick sensory neurons change with development.

We used the whole-cell configuration of the giga-seal voltage-clamp to study voltage-gated potassium currents in sensory neurons dissociated from dorsal root ganglia from embryonic and hatched chicks. Neurons from 8-, 10-, 14-, and 18-day-old embryos (E8, E10, E14, E18) and 1- to 5-day-old chicks were studied under conditions which inhibited inward currents and calcium-activated currents (tetrodotoxin, no added calcium, intracellular EGTA). At all ages, potassium currents were activated by depolarizations to potentials positive to -40 mV. At a given age the amount of inactivation of outward current during 50- to 100-ms steps varied from cell to cell; some cells showed no inactivation while in others the outward current declined to about half of the peak current. On average, the amount of inactivation was fairly stable at E8, E10, E18, and in hatched chicks but showed a transient increase at E14. In contrast, currents elicited by 50-ms test steps following 2-s conditioning steps showed an age dependent change. In E8 neurons, shifting the conditioning voltage from -100 to -90 mV had little or no effect on the current at the end of the test step while earlier outward current was reduced. In cells from older embryos or hatched chicks, similar conditioning voltages caused reductions of both early and late currents during the test step. The relative amount of late current inactivated by this protocol increased as the age of the chicks increased. In addition, the amount of variation in the inactivation properties was larger in cells from older embryos and hatched birds. The changes in outward current occur during a period in which new neurons are formed and existing neurons mature and establish function.

Transient potassium currents in avian sensory neurons.

1. We used the patch-clamp technique to study voltage-activated transient potassium currents in freshly dispersed and cultured chick dorsal root ganglion (DRG) cells. Whole-cell and cell-attached patch currents were recorded under conditions appropriate for recording potassium currents. 2. In whole-cell experiments, 100-ms depolarizations from normal resting potentials (-50 to -70 mV) elicited sustained outward currents that inactivated over a time scale of seconds. We attribute this behavior to a component of delayed rectifier current. After conditioning hyperpolarizations to potentials negative to -80 mV, depolarizations elicited transient outward current components that inactivated with time constants in the range of 8-26 ms. We attribute this behavior to a transient outward current component. 3. Conditioning hyperpolarizations increased the rate of activation of the net outward current implying that the removal of inactivation of the transient outward current allows it to contribute to early outward current during depolarizations from negative potentials. 4. Transient current was more prominent on the day the cells were dispersed and decreased with time in culture. 5. In cell-attached patches, single channels mediating outward currents were observed that were inactive at resting potentials but were active transiently during depolarizations to potentials positive to -30 mV. The probability of channels being open increased rapidly (peaking within approximately 6 ms) and then declined with a time constant in the range of 13-30 ms. With sodium as the main extracellular cation, single-channel conductances ranged from 18 to 32 pS. With potassium as the main extracellular cation, the single-channel conductance was approximately 43 pS, and the channel current reversed near 0 mV, as expected for a potassium current. 6. We conclude that the transient potassium channels mediate the component of transient outward current seen in the whole-cell experiments. This current is a relatively small component of the net current during depolarizations from normal resting potentials, but it can contribute significant outward current early in depolarizations from hyperpolarized potentials.