Small molecule receptor tyrosine kinase inhibitor of platelet-derived growth factor signaling (SU9518) modifies radiation response in fibroblasts and endothelial cells

BACKGROUND: Several small receptor tyrosine kinase inhibitors (RTKI) have entered clinical cancer trials alone and in combination with radiotherapy or chemotherapy. The inhibitory spectrum of these compounds is often not restricted to a single target. For example Imatinib/Gleevec (primarily a bcr/abl kinase inhibitor) or SU11248 (mainly a VEGFR inhibitor) are also potent inhibitors of PDGFR and other kinases. We showed previously that PDGF signaling inhibition attenuates radiation-induced lung fibrosis in a mouse model. Here we investigate effects of SU9518, a PDGFR inhibitor combined with ionizing radiation in human primary fibroblasts and endothelial cells in vitro, with a view on utilizing RTKI for antifibrotic therapy. METHODS: Protein levels of PDGFR-α/-β and phosphorylated PDGFR in fibroblasts were analyzed using western and immunocytochemistry assays. Functional proliferation and clonogenic assays were performed (i) to assess PDGFR-mediated survival and proliferation in fibroblasts and endothelial cells after SU9518 (small molecule inhibitor of PDGF receptor tyrosine kinase); (ii) to test the potency und selectivity of the PDGF RTK inhibitor after stimulation with PDGF isoforms (-AB, -AA, -BB) and VEGF+bFGF. In order to simulate in vivo conditions and to understand the role of radiation-induced paracrine PDGF secretion, co-culture models consisting of fibroblasts and endothelial cells were employed. RESULTS: In fibroblasts, radiation markedly activated PDGF signaling as detected by enhanced PDGFR phosphorylation which was potently inhibited by SU9518. In fibroblast clonogenic assay, SU9518 reduced PDGF stimulated fibroblast survival by 57%. Likewise, SU9518 potently inhibited fibroblast and endothelial cell proliferation. In the co-culture model, radiation of endothelial cells and fibroblast cells substantially stimulated proliferation of non irradiated fibroblasts and vice versa. Importantly, the RTK inhibitor significantly inhibited this paracrine radiation-induced fibroblast and endothelial cell activation. CONCLUSION: Radiation-induced autocrine and paracrine PDGF signaling plays an important role in fibroblast and endothelial cell proliferation. SU9518, a PDGFR tyrosine kinase inhibitor, reduces radiation-induced fibroblast and endothelial cell activation. This may explain therapeutic anticancer effects of Imatinib/Gleevec, and at the same time it could open a way of attenuating radiation-induced fibrosis

Light-induced structural changes and the site of O=O bond formation in PSII caught by XFEL

Photosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total molecular mass of 350 kDa for a monomer. It catalyses light-driven water oxidation at its catalytic centre, the oxygen-evolving complex (OEC). The structure of PSII has been analysed at 1.9 Å resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn4CaO5 cluster organized in an asymmetric, 'distorted-chair' form. This structure was further analysed with femtosecond X-ray free electron lasers (XFEL), providing the 'radiation damage-free' structure. The mechanism of O=O bond formation, however, remains obscure owing to the lack of intermediate-state structures. Here we describe the structural changes in PSII induced by two-flash illumination at room temperature at a resolution of 2.35 Å using time-resolved serial femtosecond crystallography with an XFEL provided by the SPring-8 ångström compact free-electron laser. An isomorphous difference Fourier map between the two-flash and dark-adapted states revealed two areas of apparent changes: around the QB/non-haem iron and the Mn4CaO5 cluster. The changes around the QB/non-haem iron region reflected the electron and proton transfers induced by the two-flash illumination. In the region around the OEC, a water molecule located 3.5 Å from the Mn4CaO5 cluster disappeared from the map upon two-flash illumination. This reduced the distance between another water molecule and the oxygen atom O4, suggesting that proton transfer also occurred. Importantly, the two-flash-minus-dark isomorphous difference Fourier map showed an apparent positive peak around O5, a unique μ4-oxo-bridge located in the quasi-centre of Mn1 and Mn4 (refs 4,5). This suggests the insertion of a new oxygen atom (O6) close to O5, providing an O=O distance of 1.5 Å between these two oxygen atoms. This provides a mechanism for the O=O bond formation consistent with that proposed previousl

Native structure of photosystem II at 1.95 Å resolution viewed by femtosecond X-ray pulses

Photosynthesis converts light energy into biologically useful chemical energy vital to life on Earth. The initial reaction of photosynthesis takes place in photosystem II (PSII), a 700-kilodalton homodimeric membrane protein complex which catalyses photo-oxidation of water into dioxygen through an S-state cycle of the oxygen evolving complex (OEC). The structure of PSII has been solved by X-ray diffraction (XRD) at 1.9-ångström (Å) resolution, which revealed that the OEC is a Mn4CaO5-cluster coordinated by a well-defined protein environment1. However, extended X-ray absorption fine structure (EXAFS) studies showed that the manganese cations in the OEC are easily reduced by X-ray irradiation2, and slight differences were found in the Mn–Mn distances between the results of XRD1, EXAFS3–7 and theoretical studies8–14. Here we report a ‘radiation-damage-free’ structure of PSII from Thermosynechococcus vulcanus in the S1 state at a resolution of 1.95 Å using femtosecond X-ray pulses of the SPring-8 ångström compact free-electron laser (SACLA) and a huge number of large, highly isomorphous PSII crystals. Compared with the structure from XRD, the OEC in the X-ray free electron laser structure has Mn–Mn distances that are shorter by 0.1–0.2 Å. The valences of each manganese atom were tentatively assigned as Mn1D(III), Mn2C(IV), Mn3B(IV) and Mn4A(III), based on the average Mn–ligand distances and analysis of the Jahn–Teller axis on Mn(III). One of the oxo-bridged oxygens, O5, has significantly longer Mn–O distances in contrast to the other oxo-oxygen atoms, suggesting that it is a hydroxide ion instead of a normal oxygen dianion and therefore may serve as one of the substrate oxygen atoms. These findings provide a structural basis for the mechanism of oxygen evolution, and we expect that this structure will provide a blueprint for design of artificial catalysts for water oxidation

Epimerization and desaturation by carbapenem synthase (CarC). A hybrid DFT study.

The mechanism of the unusual epimerization and desaturation reactions catalyzed by carbapenem synthase was investigated using the hybrid density functional method B3LYP. Several different models have been used in the calculations to study five component reactions. Both protonated and deprotonated models for the substrate have been explored so that the effects of hydrogen bonds could be characterized. Besides the iron site, it is proposed that a some tyrosine residue, possibly Tyr67, is involved in the hydrogen abstraction step. The calculated energetics and barrier heights support this hypothesis, and are consistent with the known experimental data concerning CarC and other 2-oxoglutarate dependent dioxygenases

Ethylene biosynthesis by 1-aminocyclopropane-1-carboxylic acid oxidase: a DFT study.

The reaction catalyzed by the plant enzyme 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO) was investigated by using hybrid density functional theory. ACCO belongs to the non-heme iron(II) enzyme superfamily and carries out the bicarbonate-dependent two-electron oxidation of its substrate ACC (1-aminocyclopropane-1-carboxylic acid) concomitant with the reduction of dioxygen and oxidation of a reducing agent probably ascorbate. The reaction gives ethylene, CO(2), cyanide and two water molecules. A model including the mononuclear iron complex with ACC in the first coordination sphere was used to study the details of O-O bond cleavage and cyclopropane ring opening. Calculations imply that this unusual and complex reaction is triggered by a hydrogen atom abstraction step generating a radical on the amino nitrogen of ACC. Subsequently, cyclopropane ring opening followed by O-O bond heterolysis leads to a very reactive iron(IV)-oxo intermediate, which decomposes to ethylene and cyanoformate with very low energy barriers. The reaction is assisted by bicarbonate located in the second coordination sphere of the metal

Mechanism for cyclization reaction by clavaminic acid synthase. Insights from modeling studies.

The mechanism of the oxidative cyclization reaction catalyzed by clavaminic acid synthase (CAS) was studied in silico. First, a classical molecular dynamics (MD) simulation was performed to obtain a realistic structure of the CAS-Fe(IV)=O-succinate-substrate complex; then potential of mean force (PMF) was calculated to assess the feasibility of the beta-lactam ring, more specifically its C4' corner, approaching the oxo atom. Based on the MD structure, a relatively large model of the active site region was selected and used in the B3LYP investigation of the reaction mechanism. The computational results suggest that once the oxoferryl species is formed, the oxidative cyclization catalyzed by CAS most likely involves either a mechanism involving C4'(S)-H bond cleavage of the monocyclic beta-lactam ring, or a biosynthetically unprecedented mechanism comprising (1) oxidation of the hydroxyl group of PCA to an O-radical, (2) retro-aldol-like decomposition of the O-radical to an aldehyde and a C-centered radical, which is stabilized by the captodative effect, (3) abstraction of a hydrogen atom from the C4'(S) position of the C-centered radical by the Fe(III)-OH species yielding an azomethine ylide, and (4) 1,3-dipolar cycloaddition to the ylide with aldehyde acting as a dipolarophile. Precedent for the new proposed mechanism comes from the reported synthesis of oxapenams via 1,3-dipolar cycloaddition reactions of aldehydes and ketones

Growth Differentiation Factor 15 Predicts AM-Cause Morbidity and Mortality in Stable Coronary Heart Disease

Background-—Growth differentiation factor-15 (GDF-15) is related to major bleeding when measured at initial presentation in patients with acute coronary syndromes (ACSs) treated with dual antiplatelet therapy. It is unknown whether follow-up measurements provide additional information. The objective of this study was to investigate whether GDF-15 measured 1 month after an ACS provides additional information beyond the baseline levels with regard to the risk of major bleeding. Methods and Results-—GDF-15 was measured at baseline and at 1 month after an ACS in 4049 patients included in the PLATelet inhibition and patient Outcomes (PLATO) trial. The association between 1-month GDF-15 level and non–coronary artery bypass grafting surgery-related major bleeding was assessed by a multivariable Cox model, adjusting for baseline GDF-15, age, anemia, impaired renal function, history of gastrointestinal bleeding, and sex. Elevated GDF-15 (>1800 ng/L) at 1 month was associated with an increased risk of non-coronary artery bypass grafting-related major bleeding (3.9% versus 1.2%; hazard ratio, 3.38; 95% CI, 1.89–6.06), independent of baseline GDF-15. Patients who had elevated GDF-15 levels at baseline and subsequent nonelevated GDF-15 at 1 month had a similar risk as patients who had nonelevated levels at both measurements. Conclusions-—GDF-15 at 1 month after an ACS is related to the risk of bleeding during DAPT and provides additional information on the bleeding risk beyond baseline GDF-15 levels. GDF-15 levels may therefore be useful as part of decision support concerning long-term antithrombotic treatment in patients post-ACS