Biomedical subjects

Heather L Handl

Publications and source records attributed to Heather L Handl.

8 recordsLinked to original sources

PubMed2005

Development of a lanthanide-based assay for detection of receptor-ligand interactions at the delta-opioid receptor.

A lanthanide-based assay for ligand-receptor interactions provides an attractive alternative to the traditional radiolabeled determinations in terms of sensitivity, throughput, and biohazards. We designed and tested five peptide ligands for the delta-opioid receptor that were modified with a europium (Eu)-containing chelate. These labeled ligands were tested for their binding affinities and compared with the unlabeled parental ligands. The Eu-diethylenetriaminepentaacetic acid (DTPA)-[D-Pen(2),l-Cys(5)] enkephalin (DPLCE) ligand bound to Chinese hamster ovary (CHO) cells overexpressing the human delta-opioid receptor with affinity similar to the unlabeled ligand. This ligand was used in competitive binding assays with results comparable to those obtained using the traditional radiolabeled binding assays. These lanthanide-based assays provide superior results with higher throughput and eliminate the need for radioactive waste disposal; hence, they are appropriate for high-throughput screening of ligand libraries.

Animals↗

PubMed2005

Sequence specific recognition of DNA by tailor-made hairpin conjugates of achiral seco-cyclopropaneindoline-2-benzofurancarboxamide and pyrrole-imidazole polyamides.

Hairpin conjugates of achiral seco-cyclopropaneindoline-2-benzofurancarboxamide (achiral seco-CI-Bf) and three diamides (ImPy 1, PyIm 2, and PyPy 3, where Py is pyrrole, and Im is imidazole), linked by a gamma-aminobutyrate group, were synthesized. The sequence-specific covalent alkylation of the achiral CI moiety with adenine-N3 in the minor groove was ascertained by thermally induced DNA cleavage experiments. The results provide evidence that hairpin conjugates of achiral seco-CI-Bf-gamma-polyamides could be tailored to target specific DNA sequences according to a set of general rules: the achiral CI moiety selectively reacts with adenine-N3, a stacked pair of imidazole/benzofuran prefers a G/C base pair, and a pyrrole/benzofuran prefers an A/T or T/A base pair. Models for the binding of hairpin conjugates 1-3 with sequences 5'-TCA(888)G-3', 5'-CAA(857)C-3', and 5'-TTA(843)C-3' are proposed.

AT Rich Sequence↗

PubMed2005

Lanthanide-based luminescent assays for ligand-receptor interactions.

The evaluation of receptor ligand interactions is important in the field of drug discovery and development. Currently these interactions are typically measured with cumbersome (low throughput) radiolabels. Higher throughput screens are available such as fluorescent measurements of G-protein coupled receptor-induced Ca2+ increases or fluorescence anisotropy, yet these have limited applicability and/or low signal to noise. Hence, there is a need to develop more widely applicable and more sensitive labels that can be used to monitor ligand-receptor interactions. Lanthanides provide an attractive alternative to the traditional labels used for monitoring ligand-receptor interactions. The incorporation of lanthanide labels into traditional assays used to assess receptor-ligand interactions can make these assays more affordable, less time consuming and amenable to automation. Lanthanides can be coupled to ligands and provide strong luminescent signals that can be detected using time-resolved fluorescence (TRF) methods. This approach takes advantage of the long fluorescence lifetime of the lanthanide and can detect less than one attomole of europium in a multiwell plate sample. This short review provides a basic introduction into lanthanides and TRF and describes some of the recent assays which have utilized lanthanides as labels to assess ligand-receptor interactions.

Affinity Labels↗

PubMed2004

Lanthanide-based time-resolved fluorescence of in cyto ligand-receptor interactions.

A lanthanide-based assay for ligand-receptor interactions provides an attractive alternative to the traditional radiolabeled determinations in terms of sensitivity, throughput, and biohazards. We designed and tested peptide ligands modified with an Eu-DTPA chelate. These labeled ligands were used in competitive binding assays with results comparable to those obtained using the traditional radiolabeled binding assays. The sensitivity of time-resolved fluorescence is sufficient to detect attomoles of europium, allowing assays in 96-well plates, compared with 30-mm dishes for (125)I binding assays to whole cells. We verified binding of Eu-DTPA-NDP-alpha-MSH to cells overexpressing the human melanocortin-4 receptor. The Eu-labeled ligand bound to these cells with an affinity similar to that of unlabeled NDP-alpha-MSH and was used to optimize a competitive binding assay. The lanthanide-based assays provided superior results with higher throughput and eliminated the need for radioactive waste disposal. This assay is appropriate for high-throughput screening of ligand libraries.

Binding, Competitive↗

PubMed2004

Novel targeting strategy based on multimeric ligands for drug delivery and molecular imaging: homooligomers of alpha-MSH.

Homooligomers constructed with 4- and 6-amino acid fragments of melanocortin (alpha-MSH) bind with higher affinity and with apparent cooperativity to melanocortin receptor, compared to their constituent monomers. Individual ligands were tethered with various spacers of different length and rigidity and the influence of spacers on binding was studied. Binding assays were performed on cells transfected with the melanocortin receptor, hMC4R. There is a 5-7-fold decrease in the EC(50) with the addition of each subunit, going from monomer to trimer. The Hill coefficient increases from 0.76 for the monomer to 1.12 for the dimer and 1.35 for the trimer. These data show a general trend of increasing avidity with increasing number of ligands in oligomers.

Cell Line↗

PubMed2004

Hitting multiple targets with multimeric ligands.

Multimeric ligands consist of multiple monomeric ligands attached to a single backbone molecule, creating a multimer that can bind to multiple receptors or targets simultaneously. Numerous examples of multimeric binding exist within nature. Due to the multiple and simultaneous binding events, multimeric ligands bind with an increased affinity compared to their corresponding monomers. Multimeric ligands may provide opportunities in the field of drug discovery by providing enhanced selectivity and affinity of binding interactions, thus providing molecular-based targeted therapies. However, gaps in our knowledge currently exist regarding the quantitative measures for important design characteristics, such as flexibility, length and orientation of the inter-ligand linkers, receptor density and ligand sequence. In this review, multimeric ligand binding in two separate phases is examined. The prerecruitment phase describes the binding of one ligand of a multimer to its corresponding receptor, an event similar to monomeric ligand binding. This results in transient increases in the local concentration of the other ligands, leading to apparent cooperativity. The postrecruitment phase only occurs once all receptors have been aligned and bound by their corresponding ligand. This phase is analogous to DNA-DNA interactions in that the stability of the complex is derived from physical orientation. Multiple factors influence the kinetics and thermodynamics of multimeric binding, and these are discussed.

Antineoplastic Agents↗

PubMed2002

Sequence selective recognition of DNA by hairpin conjugates of a racemic seco-cyclopropaneindoline-2-benzofurancarboxamide and polyamides.

Conjugates of racemic seco-cyclopropaneindoline-2-benzofurancarboxamide (CI-Bf) and four diamides (ImIm 1, ImPy 2, PyIm 3, and PyPy 4, where Py is pyrrole, and Im is imidazole), linked by a gamma-aminobutyrate group were synthesized. In addition to alkylating at adenine-N3 positions within an A(5) sequence, the imidazole-containing compounds 1 and 2 were found to also alkylate purine-N3 positions within a sequence 3'-GGGGGGA(888)CTGCTC(894)-5'. A model for the binding of hairpin conjugates 1 and 2 with the 3'-GACT-5' sequence is proposed.

Alkylation↗

PubMed2002

Novel furano analogues of duocarmycin C1 and C2: design, synthesis, and biological evaluation of seco-iso-cyclopropylfurano[2,3-e]indoline (seco-iso-CFI) and seco-cyclopropyltetrahydrofurano[2,3-f]quinoline (seco-CFQ) analogues.

The design, synthesis and biological evaluation of novel seco-iso-cyclopropylfurano[2,3-e]indoline (seco-iso-CFI) and the seco-cyclopropyltetrahydrofurano[2,3-f]quinoline (seco-CFQ) analogues of the duocarmycins are described. These novel analogues (4-7) were designed on the premise that the lone pair of electrons on the furano-oxygen atom could enter into conjugation with the isocyclopropylfurano[e]indolone (iso-CFI) alkylating moiety, formed from the loss of HCl in compounds 4-7. The seco-iso-CFI DNA alkylating pharmacophore was synthesized through a well precedented approach of 5-exo-trig aryl radical cyclization with a vinyl chloride. In our studies, in addition to the formation of the seco-iso-CFI product, an equal amount of an unexpected seco-CFQ product was also generated during the radical cyclization reaction. Like CC-1065 and adozelesin, using Taq DNA polymerase stop and thermal cleavage assays, the seco-iso-CFI compounds (4 and 6) and the seco-CFQ compounds (5 and 7) were shown to preferentially alkylate the adenine-N3 position within the minor groove of long stretches of A residues. A MM2 energy optimized molecular model of a 1:1 complex of compound 6 with DNA reveals that the iso-CFI compound fits snugly within the minor groove. Using a MTT based experiment, the cytotoxicity of compounds 4-7 were determined against the growth of murine leukemia (L1210), mastocytoma (P815) and melanoma (B16) cell lines. The concentrations of compounds required to inhibit the growth of these tumor cells by 50% is in the range of 10(-8)M. These compounds were also tested against a panel of human cancer cells by the National Cancer Institute, demonstrating that the compounds exhibited a high level of activity against selected solid tumors. At a concentration of 0.0084 microM (based on the IC(50) of compound 17 (seco-CBI-TMI) against the growth L1210 cells), while compounds 4 and 17 were toxic against murine bone marrow cells as judged by a colony forming study of freshly isolated murine progenitor hematopoeitic cells, compound 5, a seco-CFQ compound, was significantly less toxic. Flow cytometric analysis of P815 cells that had been incubated for 24h with compounds 4 and 5 at their cytotoxic IC(50) concentrations indicated the induction of apoptosis in a large percentage of cells, thereby suggesting that this might be the mechanism by which the iso-CFI compounds kill cells.

Animals↗