Molecular Detection of Venous Thrombosis in Mouse Models Using SPECT/CT.

The efficacy of thrombolysis is inversely correlated with thrombus age. During early thrombogenesis, activated factor XIII (FXIIIa) cross-links α2-AP to fibrin to protect it from early lysis. This was exploited to develop an α2-AP-based imaging agent to detect early clot formation likely susceptible to thrombolysis treatment. In this study, this imaging probe was improved and validated using 111In SPECT/CT in a mouse thrombosis model. In vitro fluorescent- and 111In-labelled imaging probe-to-fibrin cross-linking assays were performed. Thrombus formation was induced in C57Bl/6 mice by endothelial damage (FeCl3) or by ligation (stenosis) of the infrarenal vena cava (IVC). Two or six hours post-surgery, mice were injected with 111In-DTPA-A16 and ExiTron Nano 12000, and binding of the imaging tracer to thrombi was assessed by SPECT/CT. Subsequently, ex vivo IVCs were subjected to autoradiography and histochemical analysis for platelets and fibrin. Efficient in vitro cross-linking of A16 imaging probe to fibrin was obtained. In vivo IVC thrombosis models yielded stable platelet-rich thrombi with FeCl3 and fibrin and red cell-rich thrombi with stenosis. In the stenosis model, clot formation in the vena cava corresponded with a SPECT hotspot using an A16 imaging probe as a molecular tracer. The fibrin-targeting A16 probe showed specific binding to mouse thrombi in in vitro assays and the in vivo DVT model. The use of specific and covalent fibrin-binding probes might enable the clinical non-invasive imaging of early and active thrombosis.

Synthesis and application of cNGR-containing imaging agents for detection of angiogenesis.

Angiogenesis is a multi-step process regulated by pro- and anti-angiogenic factors. Inhibition of angiogenesis is a potential anti cancer treatment strategy that is now investigated clinically. In addition, advances in the understanding of the angiogenic process have led to the development of new angiogenesis therapies for ischemic heart disease. Currently, researchers search for objective measures that indicate pharmacological responses to pro- and anti-angiogenic drugs and therefore, there is a great interest in techniques to visualize angiogenesis noninvasively. As CD13 is selectively expressed in angiogenic blood vessels, it can serve as a target for molecular imaging tracers to noninvasively visualize angiogenic processes in animal models and patients. Here, an overview on the currently used CD13 targeted molecular imaging probes for noninvasive visualization of angiogenesis is given.

Incorporation of disulfide containing protein modules into multivalent antigenic conjugates: generation of antibodies against the thrombin-sensitive region of murine protein S.

Antigenic peptide conjugates can be used as vaccines and for the production of antibodies for clinical and research use. A method is presented here for the construction of conjugates incorporating oxidatively folded protein domains in their native conformation. This method was used to prepare multiple antigenic peptide constructs of the thrombin-sensitive loop region of murine anticoagulant protein S.

Application of an omonasteine ligation strategy for the total chemical synthesis of the BRD7 bromodomain.

The use of omonasteine (Omo) in sequential peptide ligation strategies extends the scope of homocysteine (Hcy) ligation to longer, methionine-rich proteins. Hcy-to-Omo conversion can be performed on-resin, while the Omo-to-Hcy deprotection can be performed in situ after peptide ligation. This strategy was successfully applied in the synthesis of the BRD7 bromodomain.

Nepsilon-(thiaprolyl)-lysine as a handle for site-specific protein conjugation.

In this article, we introduce the use of a thiaproline-modified lysine side-chain [Lys(Thz)], as an unlockable handle that enables late-stage, site-selective modification of chemically synthesized proteins. The Lys(Thz) residue was incorporated into the murine chemokine RANTES to demonstrate its compatibility with Boc/Bzl solid phase peptide synthesis, native chemical ligation, and disulfide bond formation. After oxidative folding of the protein, the thiol was liberated under mild reaction conditions [0.2 M hydroxylamine (NH2OH) or O-methylhydroxylamine (MeONH2), pH 4] and was subsequently reacted with thiol-selective tags. This side chain protection strategy enables the use of readily available thiol-reactive probes for the modification of internally disulfide bonded proteins.

Synthetic microcin C analogs targeting different aminoacyl-tRNA synthetases.

Microcin C (McC) is a potent antibacterial agent produced by some strains of Escherichia coli. McC consists of a ribosomally synthesized heptapeptide with a modified AMP attached through a phosphoramidate linkage to the alpha-carboxyl group of the terminal aspartate. McC is a Trojan horse inhibitor: it is actively taken inside sensitive cells and processed there, and the product of processing, a nonhydrolyzable aspartyl-adenylate, inhibits translation by preventing aminoacylation of tRNA(Asp) by aspartyl-tRNA synthetase (AspRS). Changing the last residue of the McC peptide should result in antibacterial compounds with targets other than AspRS. However, mutations that introduce amino acid substitutions in the last position of the McC peptide abolish McC production. Here, we report total chemical synthesis of three McC-like compounds containing a terminal aspartate, glutamate, or leucine attached to adenosine through a nonhydrolyzable sulfamoyl bond. We show that all three compounds function in a manner similar to that of McC, but the first compound inhibits bacterial growth by targeting AspRS while the latter two inhibit, respectively, GluRS and LeuRS. Our approach opens a way for creation of new antibacterial Trojan horse agents that target any 1 of the 20 tRNA synthetases in the cell.

Aminoacyl-tRNA synthetase inhibitors as potent and synergistic immunosuppressants.

The aminoacyl-tRNA synthetase family of enzymes is the target of many antibacterials and inhibitors of eukaryotic hyperproliferation. In screening analogues of 5'-O-(N-L-aminoacyl)-sulfamoyladenosine containing all 20 proteinogenic amino acids, we found these compounds to have potent immunosuppressive activity. Also, we found that combinations of these compounds inhibited the immune response synergistically. Based on these data, analogues with modifications at the aminoacyl and ribose moieties were designed and evaluated, and several of these showed high immunosuppressive potency, with one compound having an IC50 of 80 nM, when tested in a cellular mixed lymphocyte reaction assay. Apart from showing the potential of aminoacyl-tRNA synthetase inhibitors as immunosuppressants, the current study also provides arguments for careful evaluation of the immunosuppressive activity of developmental antibacterials that target these enzymes.