Chemical proteomic analysis reveals alternative modes of action for pyrido[2,3-d]pyrimidine kinase inhibitors

Small molecule inhibitors belonging to the pyrido[2,3-d]pyrimidine class of compounds were developed as antagonists of protein tyrosine kinases implicated in cancer progression. Derivatives from this compound class are effective against most of the imatinib mesylate-resistant BCR-ABL mutants isolated from advanced chronic myeloid leukemia patients. Here, we established an efficient proteomics method employing an immobilized pyrido[2,3-d]pyrimidine ligand as an affinity probe and identified more than 30 human protein kinases affected by this class of compounds. Remarkably, in vitro kinase assays revealed that the serine/threonine kinases Rip-like interacting caspase-like apoptosis-regulatory protein kinase (RICK) and p38alpha were among the most potently inhibited kinase targets. Thus, pyrido[2,3-d]pyrimidines did not discriminate between tyrosine and serine/threonine kinases. Instead, we found that these inhibitors are quite selective for protein kinases possessing a conserved small amino acid residue such as threonine at a critical site of the ATP binding pocket. We further demonstrated inhibition of both p38 and RICK kinase activities in intact cells upon pyrido[2,3-d]pyrimidine inhibitor treatment. Moreover, the established functions of these two kinases as signal transducers of inflammatory responses could be correlated with a potent in vivo inhibition of cytokine production by a pyrido[2,3-d]pyrimidine compound. Thus, our data demonstrate the utility of proteomic methods employing immobilized kinase inhibitors for identifying new targets linked to previously unrecognized therapeutic applications.     

Regulation of sister chromatid cohesion between chromosome arms

Sister chromatid separation in anaphase depends on the removal of cohesin complexes from chromosomes. In vertebrates, the bulk of cohesin is already removed from chromosome arms during prophase and prometaphase, whereas cohesin remains at centromeres until metaphase, when cohesin is cleaved by the protease separase. In unperturbed mitoses, arm cohesion nevertheless persists throughout metaphase and is principally sufficient to maintain sister chromatid cohesion. How arm cohesion is maintained until metaphase is unknown. Here we show that small amounts of cohesin can be detected in the interchromatid region of metaphase chromosome arms. If prometaphase is prolonged by treatment of cells with microtubule poisons, these cohesin complexes dissociate from chromosome arms, and arm cohesion is dissolved. If cohesin dissociation in prometaphase-arrested cells is prevented by depletion of Plk1 or inhibition of Aurora B, arm cohesion is maintained. These observations imply that, in unperturbed mitoses, small amounts of cohesin maintain arm cohesion until metaphase. When cells lacking Plk1 and Aurora B activity enter anaphase, chromatids lose cohesin. This loss is prevented by proteasome inhibitors, implying that it depends on separase activation. Separase may therefore be able to cleave cohesin at centromeres and on chromosome arms.     

Mechanistic studies of human molybdopterin synthase reaction and characterization of mutants identified in group B patients of molybdenum cofactor deficiency

Biosynthesis of the molybdenum cofactor involves the initial formation of precursor Z, its subsequent conversion to molybdopterin (MPT) by MPT synthase, and attachment of molybdenum to the dithiolene moiety of MPT. The sulfur used for the formation of the dithiolene group of MPT exists in the form of a thiocarboxylate group at the C terminus of the smaller subunit of MPT synthase. Human MPT synthase contains the MOCS2A and MOCS2B proteins that display homology to the Escherichia coli proteins MoaD and MoaE, respectively. MOCS2A and MOCS2B were purified after heterologous expression in E. coli, and the separately purified subunits readily assemble into a functional MPT synthase tetramer. The rate of conversion of precursor Z to MPT by the human enzyme is slower than that of the eubacterial homologue. To obtain insights into the molecular mechanism leading to human molybdenum cofactor deficiency, site-specific mutations identified in patients showing symptoms of molybdenum cofactor deficiency were generated. Characterization of a V7F substitution in MOCS2A, identified in a patient with an unusual mild form of the disease, showed that the mutation weakens the interaction between MOCS2A and MOCS2B, whereas a MOCS2B-E168K mutation identified in a severely affected patient attenuates binding of precursor Z.     

In vivo processed fragments of IGF binding protein-2 copurified with bioactive IGF-II

Proteolysis of insulin-like growth factor binding proteins (IGFBPs), the major carrier of insulin-like growth factors (IGFs) in the circulation, is an essential mechanism to regulate the bioavailability and half-live of IGFs. Screening for peptides in human hemofiltrate, stimulating the survival of PC-12 cells, resulted in the isolation of C-terminal IGFBP-2 fragments and intact IGF-II co-eluting during the chromatographic purification procedure. The IGFBP-2 fragments exhibited molecular masses of 12.7 and 12.9kDa and started with Gly169 and Gly167, respectively. The fragments were able to bind both IGFs. The stimulatory effect of the purified fraction on the survival of the PC-12 cells could be assigned exclusively to IGF-II, since it was abolished by the addition of neutralizing IGF-II antibodies. We suggest that in the circulation IGF-II is not only complexed with intact IGFBP but also with processed IGFBP-2 fragments not impairing the biological activity of IGF-II.     

NMR studies of the interaction of tryparedoxin with redox-inactive substrate homologues

Tryparedoxins (TXNs) are trypanothione-dependent peroxiredoxin oxidoreductases involved in hydroperoxide detoxification that have been shown to determine virulence in trypanosomatids. The structure of (15)N,(13)C-doubly-labeled, C-terminally-His-tagged tryparedoxin 1 from Crithidia fasciculata (Cf TXN1) was elucidated by three-dimensional NMR spectroscopy. Global folding was found to be similar to the crystal structure, but regions near the active site, especially the onset of helix alpha1 with the redox-active Cys 43 and helix alpha2 relevant to substrate binding, were less well defined in solution. The redox-inactive inhibitory substrate analogue N(1),N(8)-bis(ophthalmyl)spermidine was used to study the substrate/TXN interaction by two-dimensional (1)H,(15)N NMR spectroscopy. The NMR data complemented by molecular modeling revealed several alternative modes of ligand binding. The results confirm and extend the concept of TXN action and specificity derived from X-ray analysis and site-directed mutagenesis and thus improve the rational basis for inhibitor design.     

Analysis of candidate antagonists of IAP-mediated caspase inhibition using yeast reconstituted with the mammalian Apaf-1-activated apoptosis mechanism

We have reconstituted the Apaf-1-activated apoptosis mechanism in Sacchromyces cerevisiae such that the presence of a constitutively active form of Apaf-1 together with both Caspase-9 and Caspase-3 results in yeast death. This system is a good model of the Apaf-1-activated pathway in mammalian cells: MIHA (XIAP/hILP), and to a lesser degree MIHB (c-IAP1/HIAP2) and MIHC (c-IAP-2/HIAP1) can inhibit caspases in this system, and protection by IAPs (inhibitor of apoptosis) can be abrogated by coexpression of the Drosophila pro-apoptotic proteins HID and GRIM or the mammalian protein DIABLO/Smac. Using this system we demonstrate that unlike DIABLO/Smac, other proteins which interact with mammalian IAPs (TAB-1, Zap-1, Traf-1 and Traf-2) do not act to antagonise IAP- mediated caspase inhibition.     

Procathepsin D interacts with prosaposin in cancer cells but its internalization is not mediated by LDL receptor-related protein

The cell surface binding, endocytosis, and lysosomal routing of procathepsin D (procath-D) in cancer cells are mostly independent of the mannose-6-phosphate (M6P) receptors. In an attempt to define the receptor involved, we intracellularly cross-linked procath-D with a 68-kDa protein that we identified with specific antibodies as prosaposin in human breast and ovarian cancer cell lines. In cancer cells, this protein-protein interaction was resistant to ammonium chloride or M6P treatment, indicating that it was independent of the M6P receptors. A similar interaction also occurred in the breast cancer cell culture medium between the secreted prosaposin and procath-D. Since these two precursors can be endocytosed, we then determined whether they were interacting with the same cell surface receptor. In fibroblasts, we confirmed that the endocytosis of these two proteins was different since it was generally mediated by the M6P receptors for procath-D and mostly by LRP (LDL receptor-related protein) for prosaposin. In breast cancer cells, prosaposin endocytosis was not detected, in contrast to procath-D endocytosis, suggesting that the majority of procath-D is not internalized as a complex with prosaposin. Moreover, RAP (receptor-associated protein), a ligand inhibiting LRP-mediated endocytosis, prevented internalization of prosaposin in 49-F rat fibroblasts, but did not affect procath-D M6P-independent internalization in MDA-MB231 cells. We conclude that in breast cancer cells, even though procath-D interacts intracellularly and extracellarly with prosaposin, it is endocytosed independent of prosaposin by a receptor different from the M6P receptors and the LRP.     

Engineered CD4- and CXCR4-using simian immunodeficiency virus from African green monkeys is neutralization sensitive and replicates in nonstimulated lymphocytes

During human immunodeficiency virus type 1 (HIV-1) infection, disease progression correlates with the occurrence of variants using the coreceptor CXCR4 for cell entry. In contrast, apathogenic simian immunodeficiency virus (SIV) from African green monkeys (SIVagm), specifically the molecular virus clone SIVagm3mc, uses CCR5, Bob, and Bonzo as coreceptors throughout the course of infection. The influence of an altered coreceptor usage on SIVagm3mc replication was studied in vitro and in vivo. The putative coreceptor binding domain, the V3 region of the surface envelope (SU) glycoprotein, was replaced by the V3 loop of a CD4- and CXCR4-tropic HIV-1 strain. The resulting virus, termed SIVagm3-X4mc, exclusively used CD4 and CXCR4 for cell entry. Consequently, its in vitro replication was inhibited by SDF-1, the natural ligand of CXCR4. Surprisingly, SIVagm3-X4mc was able to replicate in vitro not only in interleukin-2- and phytohemagglutinin-stimulated but also in nonstimulated peripheral blood mononuclear cells (PBMCs) from nonhuman primates. After experimental infection of two pig-tailed macaques with either SIVagm3-X4mc or SIVagm3mc, the coreceptor usage was maintained during in vivo replication. Cell-associated and plasma viral loads, as well as viral DNA copy numbers, were found to be comparable between SIVagm3mc and SIVagm 3-X4mc infections, and no pathological changes were observed up to 14 months postinfection. Interestingly, the V3 loop exchange rendered SIVagm3-X4mc susceptible to neutralizing antibodies present in the sera of SIVagm3-X4mc- and SIVagm3mc-infected pig-tailed macaques. Our study describes for the first time a successful exchange of a V3 loop in nonpathogenic SIVagm resulting in CD4 and CXCR4 usage and modulation of virus replication in nonstimulated PBMCs as well as sensitivity toward neutralization.