Compactness of erythroid and nonerythroid spectrin under different conditions.

<p>Histograms show size distribution of spectrin species obtained by DLS measurements of erythroid spectrin in (A) native; (B) unfolded in 8M urea and (C) unfolded in 6M GuHCl and of non-erythroid spectrin in (D) native; (E) unfolded in 8M urea and (F) unfolded in 6M GuHCl.</p

Unfolding curves for erythroid (A) and nonerythroid spectrin (B) are followed by tryptophan fluorescence intensity.

<p>Insets show the linear free energy extrapolation curve with respect to increasing concentration of the denaturant concentrations. The ΔG_D^H20 was obtained from the intercept on Y-axis by using linear extrapolation. Similar unfolding curves are followed by CD spectroscopy, shown for (C) erythroid (D) non-erythroid spectrin. Insets show the linear free energy extrapolation curve with respect to increasing concentration of the denaturant concentrations. The ΔG_D^H20 was obtained from the intercept on Y-axis by using linear extrapolation.</p

Necrosis-Inducing High-Valent Oxo–Rhenium(V) Complexes with Potent Antitumor Activity: Synthesis, Aquation Chemistry, Cisplatin Cross-Resistance Profile, and Mechanism of Action

Chemotherapy with the cytotoxic platinum (Pt) drugs cisplatin, carboplatin, and oxaliplatin is the mainstay of anticancer therapy in the clinic. The antitumor activity of Pt drugs originates from their ability to induce apoptosis via covalent adduct formation with nuclear DNA. While the phenomenal clinical success is highly encouraging, resistance and adverse toxic side effects limit the wider applicability of Pt drugs. To circumvent these limitations, we embarked on an effort to explore the antitumor potential of a new class of oxo–rhenium­(V) complexes of the type [(N∧N)­(EG)­Re­(O)­Cl] (where EG = ethylene glycolate and N∧N = bipyridine, Bpy (1); phenanthroline, Phen (2); 3,4,7,8-tetramethyl-phenanthroline, Me4Phen (3)). Investigation of speciation chemistry in aqueous media revealed the formation of [(N∧N)­Re­(O)­(OH)3] as the biologically active species. Complex 3 was found to be the most potent among the three, with IC50 values ranging from 0.1 to 0.4 μM against a panel of cancer cells, which is 5–70-fold lower when compared with cisplatin. The higher potency of 3 is attributed to its higher lipophilicity, which enhanced cellular uptake. Importantly, complex 3 efficiently overcomes cisplatin resistance in ovarian, lung, and prostate cancer cells. In addition to reporting the aquation chemistry and identifying the active species in aqueous media, we performed in-depth in vitro mechanistic studies, which revealed that complex 3 preferentially accumulates in mitochondria, depletes mitochondrial membrane potential, and upregulates intracellular reactive oxygen species (ROS), leading to ER stress-mediated necrosis-mediated cancer cell death

Probing Conformational Stability and Dynamics of Erythroid and Nonerythroid Spectrin: Effects of Urea and Guanidine Hydrochloride

<div><p>We have studied the conformational stability of the two homologous membrane skeletal proteins, the erythroid and non-erythroid spectrins, in their dimeric and tetrameric forms respectively during unfolding in the presence of urea and guanidine hydrochloride (GuHCl). Fluorescence and circular dichroism (CD) spectroscopy have been used to study the changes of intrinsic tryptophan fluorescence, anisotropy, far UV-CD and extrinsic fluorescence of bound 1-anilinonapthalene-8-sulfonic acid (ANS). Chemical unfolding of both proteins were reversible and could be described as a two state transition. The folded erythroid spectrin and non-erythroid spectrin were directly converted to unfolded monomer without formation of any intermediate. Fluorescence quenching, anisotropy, ANS binding and dynamic light scattering data suggest that in presence of low concentrations of the denaturants (up-to 1M) hydrogen bonding network and van der Waals interaction play a role inducing changes in quaternary as well as tertiary structures without complete dissociation of the subunits. This is the first report of two large worm like, multi-domain proteins obeying twofold rule which is commonly found in small globular proteins. The free energy of stabilization (ΔG_u^H₂⁰) for the dimeric spectrin has been 20 kcal/mol lesser than the tetrameric from.</p></div

Schematic diagram on the mechanism of unfolding of dimeric and tetrameric spectrin.

<p>Schematic diagram on the mechanism of unfolding of dimeric and tetrameric spectrin.</p

Structural changes in erythroid, nonerythroid spectrin in presence of increasing concentrations of urea and GuHCl monitored by fluorescence spectroscopy at pH 8.0 and 25°C.

<p>Fluorescence emission spectra of (A) erythroid spectrin and (B) nonerythroid spectrin are shown for the native (a), the unfolded protein in 8M urea (b) and the unfolded protein in 6M GuHCl (c). Changes in the ratio of intensities at 337nm and 350nm of tryptophan fluorescence (F₃₃₇/F₃₅₀) against the denaturant concentrations are shown in (C) for erythroid spectrin and (D) for non-erythroid spectrin. The data are normalized by taking (F₃₃₇/F₃₅₀) of native protein as 100%. Urea and GuHCl induced unfolding are shown by changes in steady state fluorescence anisotropy (r) in (E) erythroid and (F) nonerythroid spectrin and by the changes in emission maxima (λ_max) in (G) erythroid and (H) nonerythroid spectrin respectively.</p

Changes in apparent hydrodynamic radius of the erythroid (A) and nonerythroid spectrin (B) with increasing concentrations of urea and GuHCl.

<p>Changes in apparent hydrodynamic radius of the erythroid (A) and nonerythroid spectrin (B) with increasing concentrations of urea and GuHCl.</p

Acrylamide quenching of spectrin tryptophans showing plots of Stern Volmer constant (K_SV) versus denaturant concentrations for (A) erythroid spectrin and (B) nonerythroid spectrin.

<p>Changes in bimolecular quenching rate constants (k_q) versus denaturant concentrations are shown in (C) for erythroid and (D) for nonerythroid spectrin.</p

Denaturation of erythroid and nonerythroid spectrin monitored by CD spectroscopy.

<p>CD spectra of (A) erythroid spectrin and (B) nonerythroid spectrin showing the native (a) and the unfolded proteins in 8M urea (b) and 6M GuHCl (c). Urea and GuHCl induced changes in secondary structure of (C) erythroid spectrin and (D) nonerythroid spectrin by changes in ellipticity at 222 nm with increasing concentrations of the denaturants. Effects of urea and GuHCl on erythroid, non-erythroid spectrin (0.2 μM each) are shown by changes in ANS fluorescence at 470nm in (E) erythroid and (F) nonerythroid spectrin respectively.</p

Chemical Approach to Positional Isomers of Glucose–Platinum Conjugates Reveals Specific Cancer Targeting through Glucose-Transporter-Mediated Uptake <i>in Vitro</i> and <i>in Vivo</i>

Glycoconjugation is a promising strategy for specific targeting of cancer. In this study, we investigated the effect of d-glucose substitution position on the biological activity of glucose–platinum conjugates (Glc-Pts). We synthesized and characterized all possible positional isomers (C1α, C1β, C2, C3, C4, and C6) of a Glc-Pt. The synthetic routes presented here could, in principle, be extended to prepare glucose conjugates with different active ingredients, other than platinum. The biological activities of the compounds were evaluated both <i>in vitro</i> and <i>in vivo</i>. We discovered that varying the position of substitution of d-glucose alters not only the cellular uptake and cytotoxicity profile but also the GLUT1 specificity of resulting glycoconjugates, where GLUT1 is glucose transporter 1. The C1α- and C2-substituted Glc-Pts (<b>1α</b> and <b>2</b>) accumulate in cancer cells most efficiently compared to the others, whereas the C3-Glc-Pt (<b>3</b>) is taken up least efficiently. Compounds <b>1α</b> and <b>2</b> are more potent compared to <b>3</b> in DU145 cells. The α- and β-anomers of the C1-Glc-Pt also differ significantly in their cellular uptake and activity profiles. No significant differences in uptake of the Glc-Pts were observed in non-cancerous RWPE2 cells. The GLUT1 specificity of the Glc-Pts was evaluated by determining the cellular uptake in the absence and in the presence of the GLUT1 inhibitor cytochalasin B, and by comparing their anticancer activity in DU145 cells and a GLUT1 knockdown cell line. The results reveal that C2-substituted Glc-Pt <b>2</b> has the highest GLUT1-specific internalization, which also reflects the best cancer-targeting ability. In a syngeneic breast cancer mouse model overexpressing GLUT1, compound <b>2</b> showed antitumor efficacy and selective uptake in tumors with no observable toxicity. This study thus reveals the synthesis of all positional isomers of d-glucose substitution for platinum warheads with detailed glycotargeting characterization in cancer