NMR Spectra of Glycine Isotopomers in Anisotropic Media: Subtle Chiral Interactions

NMR spectra of deuterated glycine-2-¹³C revealed interactions between chiral anisotropic gelatin and κ-carrageenan gels and the prochiral and chiral isotopomers. The ¹H, ²H and ¹³C NMR spectra of mixtures of racemic mono- and prochiral bis-deuterated glycine-2-¹³C were resolved and well simulated using distinct dipolar coupling constants <i>D</i>_CαH and <i>D</i>_CαD for the enantiomers and also for the -¹³C_αD₂- group (<i>D</i>_C,DA, and <i>D</i>_C,DB). The orientation of the proton or deuteron on the ¹³C_α-atom of glycine was assigned by analogy with alanine and lactate assuming that the molecular orientation of glycine isotopomers is the same. The assignment of the prochiral sites was derived from chiral analogues

Model based analysis of observed rate constants and steady-state activation.

<p>A. Three state transition model, which includes a low voltage sensitivity step (C1 – C2) and a high voltage sensitivity step (C2 – O). The rate constants used in the model fitting are: <i>k</i>₁ = A1(0).exp^0.0125V; <i>k</i>₂ = A2(0).exp^0.055V; <i>k</i>₋₁ = B1(0).exp^−0.025V; <i>k</i>₋₂ = B2(0).exp^−0.05V. The values of A1(0), A2(0), B1(0) and B2(0) for WT and all mutants are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031640#pone.0031640.s007" target="_blank">Table S5</a>. (i) The simulated unidirectional rates of activation/deactivation at low voltage sensitivity (dashed lines) and high voltage sensitivity (solid lines) compared to the observed rates (filled circles) for WT channels. (ii) The modelled steady-state activation (line) closely fits the observed steady-state activation of WT hERG (filled circles). B (i) Çomparison of the simulated rate constants of activation/deactivation (solid and dashed lines) to the observed rates (filled circles) for V549A. Simulated rate constants were obtained by scaling the WT rate parameters using the ratio of the observed rate constants for V549A compared to WT at <i>V</i>_0.5+40 mV (<i>k</i>₁), <i>V</i>_0.5+130 mV (<i>k</i>₂), <i>V</i>_0.5−40 mV (<i>k</i>₋₁) and <i>V</i>_0.5−180 mV (<i>k</i>₋₂) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031640#pone.0031640.s007" target="_blank">Table S5</a>). (ii) The modelled steady-state activation (line) is similar to the observed state-steady activation (filled circles) of V549A. C. (i) Comparison of the simulated (solid and dashed lines) and observed rates of activation/deactivation (filled circles) for Y545A. (ii) In the case of Y545A, the modelled steady-state activation (line) is very different to that for the observed steady-state activation (filled circles).</p

Activation of hERG S4–S5 linker mutants.

<p>A. Examples of tail currents recorded at −70 mV from (i) WT and (ii) A548V channels after varying the duration of steps to +20 mV. Dashed lines show single exponential fits to the envelope of peak tail currents. B. Plot of peak tail currents, from experiments shown in panel A, for WT and A548V channels. The fitted single exponential functions had time constants of 74 ms for WT and 37 ms for A548V. C. Plots of time constant of activation for WT (filled squares) and A548V (open squares) channels at voltages between 0 and +160 mV. D. Plots of time constants of activation for WT and A548V versus voltage after correcting for differences in the <i>V</i>_0.5 of activation. Note that the time constants of activation for A548V are no longer markedly different from WT after correcting for differences in the <i>V</i>_0.5 of activation. The dashed lines indicate the voltages (<i>V</i> = <i>V</i>_0.5+40 mV and <i>V</i> = <i>V</i>_0.5+180 mV) at which comparisons were made for time constants of activation for all mutants.</p

Summary of perturbations to rates of activation.

<p>Summary of time constants of activation for all mutants at A, <i>V</i> = <i>V</i>_0.5+40 mV and B, <i>V</i> = <i>V</i>_0.5+180 mV. Filled bars indicate values that are statistically significantly different to WT (* p<0.05, *** p<0.001).</p

Deactivation of hERG S4–S5 linker mutants.

<p>A. Typical examples of families of current traces recorded from (i) WT and (ii) Y542A channels during the voltage protocol shown in the inset. B. Expanded version of tail currents recorded at −110 mV to highlight the much faster deactivation of Y542A compared to WT. C. Plots of time constants of deactivation for WT (filled symbols) and Y542A (open symbols) versus voltage after correcting for differences in the <i>V</i>_0.5 of activation. The dashed lines indicate the voltages (<i>V</i> = <i>V</i>_0.5−40 mV and <i>V</i> = <i>V</i>_0.5−130 mV) at which comparisons were made for time constants of deactivation for all mutants.</p

Summary of perturbations to rates of deactivation.

<p>Summary of time constants of deactivation for all mutants at <i>V</i> = <i>V</i>_0.5−40 mV (A) and at <i>V</i> = <i>V</i>_0.5−130 mV (B). Filled bars indicate values that are statistically significantly different to WT (* p<0.05, *** p<0.001). † indicates the mutants where values are estimated values based on extrapolation more than 10 mV from the last measured data point (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031640#pone.0031640.s002" target="_blank">Fig. S2</a>).</p

Steady-state activation of hERG S4–S5 linker mutants.

<p>A. Typical examples of current traces recorded from (i) WT and (ii) A548V hERG channels using voltage protocol shown in inset. B. Plots of normalised peak tail currents versus test voltage for WT channels, where tail currents were recorded at −120 mV (open squares) or −70 mV (open circles) and for A548V recorded at −120 mV (closed squares) and Y542A recorded at −70 mV (closed circles). In each case the data have been fitted with a Boltzmann function. C. Summary of perturbations to Δ<i>G</i>⁰ of steady-state activation caused by each mutant compared to WT. Filled blocks indicate mutants where ΔΔ<i>G</i>⁰ was >4.2 kJ mol⁻¹.</p

Surfaces of hERG S4–S5 linker.

<p>A. NMR structure of hERG S4–S5 linker, from Asp540 to Leu550, superimposed on to the hERG homology model generated using Kv1.2 crystal structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031640#pone.0031640-Long2" target="_blank">[36]</a> as the template. Only one subunit is shown here for clarity. Each segment is labelled accordingly from S4 to S6. Residues from Asp540–Leue550 are coloured-coded based on their sidechains as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031640#pone-0031640-g001" target="_blank">Fig. 1C</a>. View (ii) shows the proximity of the S4–S5 linker to the residues from the S6 in the open state. B. Surface representation of residues Asp540–Phe550. Views shown are: (i) and (ii) S4–S5 linker parallel to the membrane with S4 and S5 helices at either end; (iii) membrane buried and (iv) solvent exposed surfaces. C. The surface electrostatic potential was calculated using APBS software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031640#pone.0031640-Baker1" target="_blank">[50]</a>. The membrane-buried surface (i) is neutral whereas the solvent exposed surface (ii) has an overall small negative charge.</p

Energy perturbation of S4–S5 linker mutants.

<p>The energy perturbation caused by mutations for steady-state activation, rates of activation and deactivation are shown as absolute values and mapped onto the S4–S5 linker viewed parallel to the membrane. The colour scale in steady-state activation A, was normalised to the value corresponding to the largest perturbation (−13.33 kJ mol⁻¹) whereas in the case of activation B, and deactivation C, the colour scale spans 0 to 4.2 kJ mol⁻¹. B. Perturbations to rates of activation measured at (i) a low voltage gradient: <i>V</i> = <i>V</i>_0.5+40 mV and (ii) a high voltage gradient: <i>V</i> = <i>V</i>_0.5+180 mV. C. Perturbations to rates of deactivation (fast component) measured at (i) a low voltage gradient: <i>V</i> = <i>V</i>_0.5−40 mV and (ii) a high voltage gradient: <i>V</i> = <i>V</i>_0.5−130 mV.</p

Hypoxia-Responsive Cobalt Complexes in Tumor Spheroids: Laser Ablation Inductively Coupled Plasma Mass Spectrometry and Magnetic Resonance Imaging Studies

Dense tumors are resistant to conventional chemotherapies due to the unique tumor microenvironment characterized by hypoxic regions that promote cellular dormancy. Bioreductive drugs that are activated in response to this hypoxic environment are an attractive strategy for therapy with anticipated lower harmful side effects in normoxic healthy tissue. Cobalt bioreductive pro-drugs that selectively release toxic payloads upon reduction in hypoxic cells have shown great promise as anticancer agents. However, the bioreductive response in the tumor microenvironment must be better understood, as current techniques for monitoring bioreduction to Co­(II) such as X-ray absorption near-edge structure and extended X-ray absorption fine structure provide limited information on speciation and require synchrotron radiation sources. Here, we present magnetic resonance imaging (MRI) as an accessible and powerful technique to monitor bioreduction by treating the cobalt complex as an MRI contrast agent and monitoring the change in water signal induced by reduction from diamagnetic Co­(III) to paramagnetic Co­(II). Cobalt pro-drugs built upon the tris­(2-pyridylmethyl)­amine ligand scaffold with varying charge were investigated for distribution and activity in a 3D tumor spheroid model by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and MRI. In addition, paramagnetic ¹H NMR spectroscopy of spheroids enabled determination of the speciation of activated Co­(II)­TPAx complexes. This study demonstrates the utility of MRI and associated spectroscopy techniques for understanding bioreductive cobalt pro-drugs in the tumor microenvironment and has broader implications for monitoring paramagnetic metal-based therapies