Mechanism of Dimerization of a Recombinant Mature Vascular Endothelial Growth Factor C

The vascular endothelial growth factors (VEGFs) and their tyrosine kinase receptors play a pivotal role in angiogenesis and lymphangiogenesis during development and in pathologies such as tumor growth. The VEGFs function as disulfide-linked antiparallel homodimers. The lymphangiogenic factors, VEGF-C and VEGF-D, exist as monomers and dimers, and dimerization is regulated by a unique unpaired cysteine. In this study, we have characterized the redox state of this unpaired cysteine in a recombinant mature monomeric and dimeric VEGF-C by mass spectrometry. Our findings indicate that the unpaired cysteine regulates dimerization via thiol–disulfide exchange involving the interdimer disulfide bond

Direct Polymerization of the Arsenic Drug PENAO to Obtain Nanoparticles with High Thiol-Reactivity and Anti-Cancer Efficiency

PENAO (4-(<i>N</i>-(<i>S</i>-penicillaminylacetyl)­amino) phenylarsonous acid), which is a mitochondria inhibitor that reacts with adenine nucleotide translocator (ANT), is currently being trialed in patients with solid tumors. To increase the stability of the drug, the formation of nanoparticles has been proposed. Herein, the direct synthesis of polymeric micelles based on the anticancer drug PENAO is presented. PENAO is readily available for amidation reaction to form PENAO MA (4-(<i>N</i>-(<i>S</i>-penicillaminylacetyl) amino) phenylarsonous acid methacrylamide) which undergoes RAFT (reversible addition–fragmentation chain transfer) polymerization with poly­(ethylene glycol methyl ether methacrylate) as comonomer and poly­(methyl methacrylate) (pMMA) as chain transfer agent, resulting in p­(MMA)-<i>b</i>-p­(PEG-<i>co</i>-PENAO) block copolymers with 3–15 wt % of PENAO MA. The different block copolymers self-assembled into micelle structures, varying in size and stability (<i>D</i>_h = 84–234 nm, cmc = 0.5–82 μg mol^–1) depending on the hydrophilic to hydrophobic ratio of the polymer blocks and the amount of drug in the corona of the particle. The more stable micelle structures were investigated toward 143B human osteosarcoma cells, showing an enhanced cytotoxicity and cellular uptake compared to the free drug PENAO (IC₅₀ (PENAO) = 2.7 ± 0.3 μM; IC₅₀ (micelle M4) = 0.8 ± 0.02 μM). Furthermore, PENAOs arsonous acid residue remains active when incorporated into a polymer matrix and conjugates to small mono and closely spaced dithiols and is able to actively target the mitochondria, which is PENAO’s main target to introduce growth inhibition in cancer cells. As a result, no cleavable linker between drug and polymer was necessary for the delivery of PENAO to osteosarcoma cells. These findings provide a rationale for <i>in vivo</i> studies of micelle M4 versus PENAO in an osteosarcoma animal model

Table S2

Excel spreadsheet of labile disulphide bonds present in some molecules of a protein crystal but absent in others (same PDB, sheet 1), or present in some structures of a protein but absent in others (different PDB, sheet 2)

Table S1

Excel spreadsheet of unique disulphides in a culled set of X-ray structures described by G. Wang and R. Dunbrack, Jr. (file pdbaanr)

Supplementary material from Identification of allosteric disulfides from labile bonds in X-ray structures

Table S3, Figures S1-S

Table S2 from Identification of allosteric disulfides from labile bonds in X-ray structures

Excel spreadsheet of labile disulphide bonds present in some molecules of a protein crystal but absent in others (same PDB, sheet 1), or present in some structures of a protein but absent in others (different PDB, sheet 2)

Table S1 from Identification of allosteric disulfides from labile bonds in X-ray structures

Excel spreadsheet of unique disulphides in a culled set of X-ray structures described by G. Wang and R. Dunbrack, Jr. (file pdbaanr)

Employing Pancreatic Tumor γ‑Glutamyltransferase for Therapeutic Delivery

γ-Glutamyltransferase (γGT) is a cell surface enzyme that catalyzes hydrolysis of the bond linking the glutamate and cysteine residues of glutathione and glutathione-S-conjugates. We have observed that human pancreatic tumor cells and tumor-associated stellate cells express high levels of this enzyme when compared to normal pancreatic epithelial and stellate cells. Detection of the protein in tumor sections correlated with γGT activity on the surface of the cultured tumor and stellate cells. We tested whether the tumor γGT could be employed to deliver a therapeutic to the tumor endothelial cells. GSAO is a glutathione-S-conjugate of a trivalent arsenical that is activated to enter endothelial cells by γGT cleavage of the γ-glutamyl residue. The arsenical moiety triggers proliferation arrest and death of the endothelial cells by targeting the mitochondria. Human pancreatic tumor and stellate cell γGT activated GSAO in culture and γGT activity positively correlated with GSAO-mediated proliferation arrest and death of endothelial cells in Transwell and coculture systems. A soluble form of γGT is found in blood, and we measured the rate of activation of GSAO by this enzyme. We calculated that systemically administered GSAO would circulate through the pancreatic blood supply several times before appreciable activation by normal blood levels of γGT. In support of this finding, tumor γGT activity positively correlated with GSAO-mediated inhibition of pancreatic tumor angiogenesis and tumor growth in mice. Our findings indicate that pancreatic tumor γGT can be used to deliver a therapeutic to the tumor

Organic Arsenicals As Efficient and Highly Specific Linkers for Protein/Peptide–Polymer Conjugation

The entropy-driven affinity of trivalent (in)­organic arsenicals for closely spaced dithiols has been exploited to develop a novel route to peptide/protein–polymer conjugation. A trivalent arsenous acid (As­(III)) derivative (<b>1</b>) obtained from <i>p</i>-arsanilic acid (As­(V)) was shown to readily undergo conjugation to the therapeutic peptide salmon calcitonin (sCT) via bridging of the Cys¹-Cys⁷ disulfide, which was verified by RP-HPLC and MALDI-ToF-MS. Conjugation was shown to proceed rapidly (<i>t</i> < 2 min) <i>in situ</i> and stoichiometrically through sequential reduction–conjugation protocols, therefore exhibiting conjugation efficiencies equivalent to those reported for the current leading disulfide-bond targeting strategies. Furthermore, using bovine serum albumin as a model protein, the trivalent organic arsenical <b>1</b> was found to demonstrate enhanced specificity for disulfide-bond bridging in the presence of free cysteine residues relative to established maleimide functional reagents. This specificity represents a shift toward potential orthogonality, by clearly distinguishing between the reactivity of mono- and disulfide-derived (vicinal or neighbors-through-space) dithiols. Finally, <i>p</i>-arsanilic acid was transformed into an initiator for aqueous single electron-transfer living radical polymerization, allowing the synthesis of hydrophilic arsenic-functional polymers which were shown to exhibit negligible cytotoxicity relative to a small molecule organic arsenical, and an unfunctionalized polymer control. Poly­(poly­[ethylene glycol] methyl ether acrylate) (PPEGA₄₈₀, DP_<i>n</i> = 10, <i>M</i>_n,NMR = 4900 g·mol^–1, <i><i>Đ</i></i> = 1.07) possessing a pentavalent arsenic acid (As­(V)) α-chain end was transformed into trivalent As­(III) post-polymerization via initial reduction by biological reducing agent glutathione (GSH), followed by binding of GSH. Conjugation of the resulting As­(III)-functional polymer to sCT was realized within 35 min as indicated by RP-HPLC and verified later by thermodynamically driven release of sCT, from the conjugate, in the presence of strong chelating reagent ethanedithiol

Lone mothers in Ireland A local study

Available from British Library Document Supply Centre-DSC:7769.54283(21) / BLDSC - British Library Document Supply CentreSIGLEGBUnited Kingdo