Most cancers are characterized by multiple molecular alterations, but identification of the key proteins involved in these signaling pathways is currently beyond reach. We show that the inhibitor PU-H71 preferentially targets tumor-enriched Hsp90 complexes and affinity captures Hsp90-dependent oncogenic client proteins. We have used PU-H71 affinity capture to design a proteomic approach that, when combined with bioinformatic pathway analysis, identifies dysregulated signaling networks and key oncoproteins in chronic myeloid leukemia. The identified interactome overlaps with the well-characterized altered proteome in this cancer, indicating that this method can provide global insights into the biology of individual tumors, including primary patient specimens. In addition, we show that this approach can be used to identify previously uncharacterized oncoproteins and mechanisms, potentially leading to new targeted therapies. We further show that the abundance of the PU-H71-enriched Hsp90 species, which is not dictated by Hsp90 expression alone, is predictive of the cell’s sensitivity to Hsp90 inhibition.

Introduction The present investigation focuses on the chemical and biological fate of 89Zr in mice. Electrophoreses of 89Zr solvated or chelated in different conditions are here presented. The biological fate of mice injected with [89Zr]Zr-oxalate, [89Zr]Zr-chloride, [89Zr]Zr-phosphate, [89Zr]Zr-desferrioxamine and [89Zr]Zr-citrate is studied with the biodistribution, the clearances and PET images. A special focus is also given regarding the quality of 89Zr bone accumulation. Methods Electrophoreses were carried out on chromatography paper and read by gamma counting. Then, the solutions were intravenously injected in mice, imaged at different time points and sacrificed. The bones, the epiphysis and the marrow substance were separated and evaluated with gamma counts. Results The clearances of [89Zr]Zr-chloride and [89Zr]Zr-oxalate reached 20% of ID after 6 days whereas [89Zr]Zr-phosphate was only 5% of ID. [89Zr]Zr-citrate and [89Zr]Zr-DFO were noticeably excreted after the first day p.i.. [89Zr]Zr-chloride and [89Zr]Zr-oxalate resulted in a respective bone uptake of ~15% ID/g and~20% ID/g at 8 h p.i. with minor losses after 6 days. [89Zr]Zr-citrate bone uptake was also observed, but [89Zr]Zr-phosphate was absorbed in high amounts in the liver and the spleen. The marrow cells were insignificantly radioactive in comparison to the calcified tissues. Conclusion Despite the complexity of Zr coordination, the electrophoretic analyses provided detailed evidences of Zr charges either as salts or as complexes. This study also shows that weakly chelated, 89Zr is a bone seeker and has a strong affinity for phosphate.

Highlights d Pathologic protein networks and their engagement in clinic are monitored by imaging d Real-time tumor pharmacometric data are obtained at the level of individual tumors d Theranostic and clinical assay combined provide quantitative tumor measurements d The platform provides dose and schedule information for epichaperome targeting

3-Pyrrolidineacetic acid (1a), certain piperidinecarboxylic acids--i.e., 3-piperidinecarboxylic acid (2a), 1,2,5,6-tetrahydro-3-pyridinecarboxylic acid (3a), and cis-4-hydroxy-3-piperidinecarboxylic acid (4a)--cis-3-aminocyclohexanecarboxylic acid (5a, cis-3-ACHC), and gamma-aminobutyric acid (6a, GABA) itself are among the most potent inhibitors of [3H]GABA uptake by neurons and glia in vitro. These hydrophilic amino acids, however, do not readily enter the central nervous system in pharmacologically significant amounts following peripheral administration. We now report that N-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid (2b) is a specific GABA-uptake inhibitor that is more potent, more lipophilic and, in limited testing, as selective as 2a. Similar results were obtained with the N-(4,4-diphenyl-3-butenyl) derivatives of 1a, 3a, and 4a. By contrast, N-(4,4-diphenyl-3-butenyl) derivatives of 5a and 6a were not more potent than the parent amino acids and appear to inhibit GABA uptake, at least in part, by a nonselective mechanism of action. The N-(4,4-diphenyl-3-butenyl)amino acids 1b-4b exhibit anticonvulsant activity in rodents following oral or intraperitoneal administration [Yunger, L.M.; et al. J. Pharmacol. Exp. Ther. 1984, 228, 109].

Pantothenate kinase (CoaA) catalyzes the first step of the coenzyme A biosynthetic pathway. Here we report the identification of the Staphylococcus aureus coaA gene and characterization of the enzyme. We have also identified a series of low-molecular-weight compounds which are effective inhibitors of S. aureus CoaA.Increasing reports of antibiotic resistance involving opportunistic gram-positive pathogens, including methicillin-resistant Staphylococcus aureus, have emphasized the critical need for the development of antimicrobial compounds with novel modes of action. Coenzyme A (CoA), an essential cofactor for maintaining life, is used in a multitude of biochemical reactions. In most bacteria, CoA is synthesized from pantothenic acid (vitamin B 5 ) in 5 steps (5), with the first step being the phosphorylation of pantothenate by pantothenate kinase (CoaA). Although this pathway also exists in eukaryotes, in most cases there is no sequence homology between the prokaryotic and eukaryotic CoA biosynthetic enzymes (7,9,12,18,24,27). Thus, there is the potential for developing highly specific inhibitors of bacterial CoA enyzmes.Unlike the case for other biosynthetic pathways of bacteria, the genes involved in CoA biosynthesis are not organized as operons. This has delayed the identification of the enzymes responsible for CoA synthesis, even though the intermediate chemical steps have been known since the 1960s (1). With the recent identification of the Escherichia coli genes encoding the enzymes CoaBC and CoaE, the entire pathway is now known for this organism (9,10,13,19,21). Interestingly, the gene coaA, which encodes the first enzyme in the pathway, has no homolog in the complete genome sequences of the S. aureus strains Mu50 and N315 (11).Cloning and purification of S. aureus CoaA. Initially, the coaA gene sequences in S. aureus strains Mu50 and N15 (GenBank accession numbers BA000017 and BA000018, respectively) were identified through searches of the ERGO comparative genomic database (previously WIT) (http://ergo .integratedgenomics.com/ERGO/) (8). We cloned the S. aureus RN4220 coaA gene and overexpressed it using standard techniques (4, 17). S. aureus RN4220 coaA was amplified by PCR, introducing an NdeI site at the start codon and an XhoI site after the stop codon, and cloned into pSTBlue1 using the Perfectly Blunt Cloning kit. The gene was excised by digestion with NdeI and XhoI and ligated into similarly digested pET28a. The final construct encoded the N-terminal six-His-tagged S. aureus CoaA.Tuner (DE3) cells were transformed with this construct and grown at 37°C in Luria-Bertani medium-50-g/ml kanamycin. Protein expression was induced by 500 M isopropylthio-␤-Dgalactoside, and cells were harvested 3 h postinduction. The cell pellet was resuspended and sonicated, and cell debris was removed by centrifugation. The supernatant was subjected to Ni-chelating column chromatography followed by a HiTrap Q Sepharose ion exchange column. Enzyme identity was confirmed through N-terminal sequencing and matrix-assisted...