Joseph P. Taulane

Cancer-related bone pain is attenuated by a systemically available δ-opioid receptor agonist

Abstract Patients with bone cancer report severe pain and receive &mgr;‐opioids. We developed a family of peptidomimetic &dgr;‐agonists, one of which H2N‐Tyr‐dVal‐Gly‐Phe‐Ala‐OH Symbol binds with a 1700× affinity at the &dgr; versus &mgr; receptor. To examine the systemic analgesic efficacy of this &dgr;‐agonist versus morphine in osteosarcoma pain, osteosarcoma cells are injected into one femur of the anesthetized mouse. After 10–18 days, a decalcification of the injected femur occurs along with a pronounced tactile allodynia. IP morphine and Symbol produced a dose‐dependent reversal of allodynia with the respective ED50 values being 5.3 ± 1.9 mg/kg for morphine and 1.3 ± 0.3 mg/kg for [Symbol. Plotting peak effect versus area under the analgesic curve for doses of morphine and [Symbol revealed overlapping curves suggesting that for a given effect, [Symbol produced a similar duration of action as morphine. These effects were reversed by IP naloxone (3 mg/kg). IP naltrindole (1 mg/kg) preferentially reversed [Symbol. The upper dose effects of morphine but not [Symbol were limited by pronounced hyperactivity. No other effects were noted. These results show that IP [Symbol through a &dgr; receptor produces analgesia equal in efficacy to that of morphine but with a 4.5‐fold greater potency. Over the doses examined, morphine actions were side effect limited. The &dgr; side effects were not so limited, suggesting a favorable therapeutic ratio for &dgr;‐agonists in this pain model. These studies suggest that a systemically delivered &dgr;‐opioid agonist has pronounced analgesic properties on a preclinical cancer pain model. Figure. No Caption available. Figure. No Caption available. Figure. No Caption available. Figure. No Caption available. Figure. No Caption available. Figure. No Caption available. Figure. No Caption available. Figure. No Caption available.

Collagen-based structures containing the peptoid residue N-isobutylglycine (Nleu): Synthesis and biophysical studies of Gly-Pro-Nleu sequences by circular dichroism, ultraviolet absorbance, and optical rotation

A peptoid residue N-isobutylglycine (Nleu) was introduced as a proline surrogate in collagen-like triple helical structures. A series of single chain and template-assembled collagen-based peptide-peptoid structures composed of Gly-Pro-Nleu sequences were prepared by solid-phase segment condensation methods. Both a synthetic route in solution and a solid phase method were employed to couple the KTA (cis,cis-1,3,5-trimethylcyclohexane-1,3,5-tricarboxylic acid, also known as the Kemp triacid) based template, KTA-(Gly-OH)3, to peptide-peptoid chains. Biophysical studies using CD, uv absorbance, and optical rotation measurements demonstrated that these compounds form triple-helical structures when the chains are longer than critical lengths. Results from melting curve measurements indicated that the Gly-Pro-Nleu sequence is comparable to the Gly-Pro-Pro sequence in stabilizing a triple-helical conformation. The KTA-based template stabilized triple-helical structures as can be seen by the increased melting temperatures as compared to equivalent single chain molecules. In addition, the template reduced the minimum chain length necessary to form a triple helix from six to only three trimer repeats.

Structure-activity relationships of dynorphin a analogues modified in the address sequence.

The peptide [Pro3]Dyn A(1-11)-NH2 2 exhibits high affinity (K(i) = 2.4 nM) and over 2000-fold selectivity for the opioid receptor. Stepwise removal of the C-terminal residues from this ligand demonstrated that its positively charged Arg residues, particularly Arg6 and Arg7, were crucial for binding to the kappa receptor. Analogues shorter than seven amino acids lacked significant affinity for opioid receptors. Comparison with a series of truncated analogues of Dyn A showed that the relative losses in binding potency differed only slightly between the two series. The neutral residues Ile8 and Pro10 could be removed without significant loss in affinity for the kappa receptor. Their replacement, in the Pro3 analogue, with additional Arg residues led to analogues with improved kappa affinity (e.g., [Pro3,Arg8]Dyn A(1-11)-NH2 20: K(i)(kappa) = 0.44 nM). This type of modification did not compromise the high kappa selectivity of the Pro3 analogues. These findings support the view that a negatively charged domain in the putative second extracellular loop of the kappa receptor selectively recognizes residues 6-11 of dynorphin through electrostatic interactions. As with parent compound 2, analogue 20 and related compounds displayed kappa antagonist properties.

The immune response to Epstein-Barr nuclear antigen: Conformational and structural features of antibody binding to synthetic peptides☆

Naturally developing human antibodies to the Epstein-Barr nuclear antigen recognize synthetic peptides containing sequences from the unusual glycine-alanine region of this protein. We tested antibody binding to a series of peptides of from five to 20 amino acids in length. Peptides as small as seven amino acids could bind but optimal results required chain lengths of 15. Binding was extremely sensitive to small changes in the length and sequence of the peptide, and also to the temp of the reaction. The changes can be ascribed to two factors: (1) deletion of the site of antigen binding and (2) loss of peptide secondary structure.

Characteristics of Rabbit Muscle Adenylate Kinase Inhibition by Sulfur and Recovery by Dithiothreitol

Structure-function relationships of rabbit muscle adenylate kinase (RMAK) were studied by examining the characteristics of inhibitions by hydrophobic inhibitors and reactivations by sulfhydryl reagents. RMAK is inhibited by 1-butanol,N-ethylmaleimide (NEM) and elemental sulfur (S8) with increasing effectiveness in the order of increasing hydrophobicity. Characteristics of these hydrophobic inhibitors are compared with inhibitors forming covalent bonds or reversible complexes. A mechanism is proposed for hydrophobic inhibitors of RMAK that involves conformational changes promoted by interacting with hydrophobic regions. The reversal of RMAK inhibition by sulfhydryl compounds involves a conformational change that exposes hydrophobic regions and the inhibitor to water. Circular dichroism (CD) data show changes in the secondary structures of RMAK, indicating that the inhibitors and the sulfhydryl compounds promote conformational changes. The results of these studies show that the activity of a small enzyme can be controlled in a manner analogous to the allosteric control of larger enzymes.

A Gas-Phase Cell for Infrared Spectra of Sublimable Compounds

We have designed and constructed a novel gas cell for infrared spectroscopic studies. Detailed features of the cell are described. Gas-phase spectra have been obtained for N-methylacetamide, camphorolactam, and acetyl-L-alanine-N-methylamide at elevated temperatures and high vacuum. The results show highly resolved spectra for cis and trans amide bonds.

Comparative inhibition patterns of adenylate kinases from mammals, bird, fish and microorganisms

The S8 inhibitions of AKs from six different sources were studied in mammals, birds, fish, and a microorganism. All AKs tested were inhibited by S8. Except for carp, all inhibited AKs from those tested were reactivated by DTT. Inhibitions of AKs by other hydrophobic inhibitors, NEM, butanol and ethanol were also studied. The inhibitions by S8 suggest that the hydrophobic pockets in the AKs cover a wide phylogenetic range. All inhibitions by S8 are reactivated by DTT. Unlike the inhibitions by S8, the characteristics of inhibitions by the other hydrophobic inhibitors differed among the AK sources tested and none was the irreversible type. The data suggest that no covalent bonds were formed with NEM. Similarly, the ability to reactivate the inhibitions by DTT differed among the AK sources. The possibility that the hydrophobic domains in the AKs may serve as part of an enzyme activity control mechanism is discussed.