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Etoposide and other topoisomerase II-targeted drugs are important anticancer therapeutics. Unfortunately, the safe usage of these agents is limited by their indiscriminate induction of topoisomerase II-mediated DNA cleavage throughout the genome and by a lack of specificity toward cancer cells. Therefore, as a first step toward constraining the distribution of etoposide-induced DNA cleavage sites and developing sequence-specific topoisomerase II-targeted anticancer agents, we covalently coupled the core of etoposide to oligonucleotides centered on a topoisomerase II cleavage site in the PML gene. The initial sequence used for this ‘oligonucleotide-linked topoisomerase inhibitor’ (OTI) was identified as part of the translocation breakpoint of a patient with acute promyelocytic leukemia (APL). Subsequent OTI sequences were derived from the observed APL breakpoint between PML and RARA. Results indicate that OTIs can be used to direct the sites of etoposide-induced DNA cleavage mediated by topoisomerase IIα and topoisomerase IIβ. OTIs increased levels of enzyme-mediated cleavage by inhibiting DNA ligation, and cleavage complexes induced by OTIs were as stable as those induced by free etoposide. Finally, OTIs directed against the PML-RARA breakpoint displayed cleavage specificity for oligonucleotides with the translocation sequence over those with sequences matching either parental gene. These studies demonstrate the feasibility of using oligonucleotides to direct topoisomerase II-mediated DNA cleavage to specific sites in the genome.

Using mass spectrometry to detect enzymatic activity offers several advantages over fluorescence-based methods. Automation of sample handling and analysis using platforms such as the RapidFire (Agilent Technologies, Lexington, MA) has made these assays amenable to medium-throughput screening (of the order of 100,000 wells). However, true high-throughput screens (HTS) of large compound collections (1 million) are still considered too time-consuming to be feasible. Here we propose a simple multiplexing strategy that can be used to increase the throughput of RapidFire, making it viable for HTS. The method relies on the ability to analyze pooled samples from several reactions simultaneously and to deconvolute their origin using "mass-tagged" substrates. Using the JmjD2d H3K9me3 demethylase as a model system, we demonstrate the practicality of this method to achieve a 4-fold increase in throughput. This was achieved without any loss of assay quality. This multiplex strategy could easily be scaled to give even greater reductions in analysis time.

Two new bifunctional chelators that are derivatives of the bis(thiosemicarbazone) ATSMH(2) proligand have been prepared, one with two phenyl carboxylate substituents on the exocyclic nitrogens (L(1)H(2)) and one with a single phenyl carboxylate (L(2)H(2)). The new ligands have been characterised by NMR spectroscopy, mass spectrometry and in the case of L(1)H(2) by X-ray crystallography. The copper, nickel and zinc complexes of the new ligands have been synthesised and characterised. Electrochemical measurements show that the copper(II) complexes undergo a reversible reduction attributable to a Cu(II)/Cu(I) process. The new proligands have been tethered to the N-alpha-Boc-protected amino acids lysine and ornithine using solution and solid phase methods. The new amino acid conjugates form copper complexes and the complexes have been characterised by mass spectrometry and electronic spectroscopy. The bifunctional chelator L(2)H(2) has been conjugated to the tumour targeting peptide octreotide and the new ATSMH(2)-octreotide conjugate and its copper complex have been characterized by mass spectrometry. These new systems have the potential to be used for new targeted copper radiopharmaceuticals for imaging and therapy.

Identification of peptide substrates for proteases can be a major undertaking. To overcome issues such as feasibility and deconvolution, associated with large peptide libraries, a 'small but smart' generic fluorescence resonance energy transfer rapid endopeptidase profiling library (REPLi) was synthesised as a tool for rapidly identifying protease substrates. Within a tripeptide core, flanked by Gly residues, similar amino acids were paired giving rise to a relatively small library of 3375 peptides divided into 512 distinct pools each containing only 8 peptides. The REPLi was validated with trypsin, pepsin, the matrix metalloprotease (MMP)-12 and MMP-13 and calpains-1 and -2. In the case of calpain-2, a single iteration step involving LC-MS, provided the definitive residue specificity from which a highly sensitive fluorogenic substrate, (FAM)-Gly-Gly-Gly-Gln-Leu-Tyr-Gly-Gly-DPA-Arg-Arg-Lys-(TAMRA), was then designed. The thorough validation of this 'small but smart' peptide library with representatives from each of the four mechanistic protease classes indicates that the REPLi will be useful for the rapid identification of substrates for multiple proteases.

GPR10 is a novel G-protein coupled receptor that is the human orthologue of rat Unknown Hypothalamic Receptor-1 (UHR-1). Human prolactin-releasing peptide (PrRP) has been identified as an endogenous ligand for GPR10, and occurs as 31 and 20 amino acid forms. The present study characterizes the binding of [125I]-PrRP-20 to HEK293 cells stably expressing GPR10 receptors. Specific binding of [125I]-PrRP-20 was saturable, and analysis suggested evidence of both high and low affinity sites, with KD values of 0.026±0.006 and 0.57±0.14 nM respectively, and Bmax values of 3010±400 and 8570±2240 fmol mg protein−1 respectively. Kinetic studies were unable to distinguish two sites, but single site analysis of association and dissociation data produced a KD of 0.012 nM. Competition studies revealed that human and rat PrRP-20 and PrRP-31 all display high affinity for GPR10. A range of other drugs which are known ligands at receptors which share limited homology with GPR10 were also tested. None of the drugs tested, including the RF-amide neuropeptide FF, demonstrated any affinity for GPR10. Human PrRP-20 failed to alter basal or forskolin-stimulated levels of intracellular cyclic AMP in HEK293-GPR10 cells, suggesting that GPR10 does not couple via either Gs or Gi. Functional studies using measurements of intracellular calcium confirmed that human and rat PrRP-20 and PrRP-31 are all potent, full agonists at the GPR10 receptor. The response was blocked both by thapsigargin, indicating mobilization of intracellular Ca2+ stores. These studies indicate that [125I]-PrRP-20 is a specific, high affinity radioligand for GPR10. The availability of this radioligand binding assay will be a valuable tool for the investigation of the key features involved in PrRP binding and studies on the localization and function of GPR10. British Journal of Pharmacology (2000) 131, 683–688; doi:10.1038/sj.bjp.0703617

Epoxyeicosatrienoic acids (EETs) play important protective functions in cardiovascular and renal systems. Under physiological conditions, EETs are quickly converted by the soluble epoxide hydrolase (sEH) to diols which do not have the beneficiary roles. Inhibition of sEH with small molecules to increase the concentration of EETs therefore provides an attractive therapeutic strategy for cardiovascular diseases. We describe here the development of a high throughput cell-based assay to measure sEH activity and screen small molecular compounds as sEH inhibitors. This assay is based on the technology of fluorescence polarization (FP), utilizing a Cy3B labeled 14,15-DHET ligand and a rabbit anti-14,15-DHET antibody. With the optimized assay, we measured the cellular sEH activity of several cell lines expressing endogenous sEH as well as sEH BacMam transduced HEK-293 cells. The inhibitory effect of several known sEH inhibitors was evaluated in sEH BacMam transduced HEK-293 cells. Our data show that there is good agreement of pIC(50) values obtained between the FP format and a commercially available ELISA kit. To our knowledge, this is the first report of a high throughput cell-based assay for screening sEH inhibitors.

Most of the kinase inhibitors that are approved for therapeutic uses or that are undergoing clinical trials are directed toward the adenosine triphosphate (ATP) binding site of protein kinases. 5'-Fluorosulfonylbenzoyl 5'-adenosine (FSBA) is an activitybased probe (ABP) that covalently modifies a conserved lysine present in the nucleotide binding site of most kinases. Here the authors describe synthesis of FSBA derivatives, 2'-biotinyl-FSBA and 3'-biotinyl-FSBA as kinase ABPs, and delineate a Western blot method to screen and validate ATP competitive protein kinase inhibitors using biotinyl-FSBA as a nonselective activity-based probe for protein kinases.

0-4-Dimethylcarbamoylbenzyl-L-tyrosine, N (epsilon)-4-dimethylcarbamoylbenzyloxycarbonyl-L-lysine, and simple derivatives, have been prepared. The protecting groups are markedly more stable to trifluoroacetic acid than are the unsubstituted benzylanalogues. N-t-Butoxycarbonyl-O-4-dimethylcarbamoylbenzyl-L-serine (cyclohexylammonium salt) is also described.

The new α-amino acid, 2-amino-4-(3-pyridyl)butyric acid (4)[abbreviation, Ala(3 Pm)], has been synthesized and resolved. The N-t-butoxycarbonyl derivative of the L-amino-acid has been used in the picolyl ester ‘handle’ method to synthesize Boc-Ala(3 Pm)-Ala-Ala-Ala-Lys(Z)-OPic (8)(Pit = 4-picolyl) and its N-acetyl analogue (9), Boc-Ala(3 Pm)-Ala-Ala-D-Ala-Lys(Z)-OPic (10), and Boc-Ala(3 Pm)-Ala-Ala-Val-Lys(Z)-OPic (11). Compounds (8), (9), and (11) are weak inhibitors of human leucocytic elastase.