Valosine containing protein (VCP), also known as p97, is a member of AAA ATPase family that is involved in several biological processes and plays a central role in the ubiquitin-mediated degradation of misfolded proteins. VCP is an ubiquitously expressed, highly abundant protein and has been found overexpressed in many tumor types, sometimes associated with poor prognosis. In this respect, VCP has recently received a great deal of attention as a potential new target for cancer therapy. In this paper, the discovery and structure-activity relationships of alkylsulfanyl-1,2,4-triazoles, a new class of potent, allosteric VCP inhibitors, are described. Medicinal chemistry manipulation of compound 1, identified via HTS, led to the discovery of potent and selective inhibitors with submicromolar activity in cells and clear mechanism of action at consistent doses. This represents a first step toward a new class of potential anticancer agents.
A novel ligand based on a pyridine-containing macrocycle bearing two acetic and one methylenephosphonic arms (PCP2A) has been synthesized. An efficient synthesis of PCP2A is based on the macrocyclization reaction between 2,6-bis(chloromethyl)pyridine and a 1,4, 7-triazaheptane derivative bearing a methylenephosphonate group on N-4. The Gd(III) complex of PCP2A displays characteristic properties which make it a very promising contrast agent for improved applications in magnetic resonance imaging. In fact it shows (i) a very high stability constant (log K(GdPCP2A) = 23.4) which should guarantee against the in vivo release of toxic free Gd(III) ions and free ligand molecules and (ii) a relaxivity that is about 2 times higher than the values reported for contrast agents currently used in the clinical practice. Its high relaxivity is the result of the presence of two water molecules in the inner coordination sphere and a significant contribution from water molecule(s) hydrogen bonded to the phosphonate group. Moreover, the inner sphere water molecules are involved in an exchange with the bulk water which is relatively fast. This property is important for the attainment of an even higher relaxivity once the molecular reorientation rate of the [GdPCP2A(H(2)O)(2)](-) moiety is lengthened by means of conjugation to a macromolecular substrate.
A generic LC/ESI(+)-oaTOFMS method has been developed for routine automated high accuracy mass determinations of different classes of substances. The system makes use of micro-high-performance liquid chromatography and a hybrid quadrupole/orthogonal acceleration time-of-flight (Q-oaTOF) mass spectrometer. Reproducible and accurate mass measurements were obtained using an electrospray dual sprayer with reserpine as reference compound, introduced into the mass spectrometer alternating with the samples. Experiments were performed to optimize analyte/reference response ratio, statistical algorithm correction setting, and analyte concentration. In these experiments, a clear dependence of the mass measurement error on the analyte/reference response ratio was observed. The dependence of average mass error versus different dead time correction algorithm settings (Np factors) was also explored. In the final automated procedure, verified for a statistically significant set of compounds ( approximately 550) obtained from a medicinal chemistry department, about 70% of the analyzed samples satisfied the acceptance criteria fixed at a maximum error of +/-5 ppm (mass range 150-800 Da).
The R132H mutation of cytosolic isocitrate dehydrogenase (IDH1) is present in the majority of low grade gliomas. Immunotherapy in these tumors has an interesting, still unexploited, therapeutic potential, as they are less immunosuppressive than glioblastomas.Using site-directed mutagenesis we introduced the R132H mutation into the murine glioma cell line GL261, creating mIDH1-GL261. Presence of the mutation was confirmed by immunoblotting and production of the oncometabolite 2-hydroxyglutarate (2HG), demonstrated by mass spectrometry (LC-MS/MS) performed on cell supernatant. In vitro mIDH1-GL261 had different morphology but similar growth rate than parental GL261 (p-GL261). After intracranial injection, MRI suggested that the initial growth rate was slower in mIDH1-GL261 than p-GL261 gliomas but overall survival was similar. mIDH1-GL261 gliomas showed evidence of R132H expression and of intratumoral 2HG production (evaluated by MRS and LC-MS/MS). Immunizations were performed nine days after intracranial implantation of mIDH1- or p-GL261 cells by three subcutaneous injections of five different peptides encompassing the IDH1 mutation site, all emulsified with Montanide ISA-51, in association with GM-CSF. Control mice were injected with four ovalbumin peptides or vehicle. Mice with mIDH1-GL261 but not p-GL261 gliomas treated with mIDH1 peptides survived longer than controls; 25% of them were cured. Immunized mice showed higher amounts of peripheral CD8+ T cells, higher production of IFN-γ, and evidence of anti-mIDH1 antibodies. Immunizations led to intratumoral up-regulation of IFN-γ, granzyme-b and perforin-1 and down-regulation of TGF-β2 and IL-10.These results support the translational potential of immunotherapeutic targeting of gliomas carrying IDH1 mutations.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-014-0180-0) contains supplementary material, which is available to authorized users.
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