Randine L. Dowling

Matrix metalloproteinase–activated doxorubicin prodrugs inhibit HT1080 xenograft growth better than doxorubicin with less toxicity

Matrix metalloproteinase (MMP)–activated prodrugs were formed by coupling MMP-cleavable peptides to doxorubicin. The resulting conjugates were excellent in vitro substrates for MMP-2, -9, and -14. HT1080, a fibrosarcoma cell line, was used as a model system to test these prodrugs because these cells, like tumor stromal fibroblasts, expressed several MMPs. In cultured HT1080 cells, simple MMP-cleavable peptides were primarily metabolized by neprilysin, a membrane-bound metalloproteinase. MMP-selective metabolism in cultured HT1080 cells was obtained by designing conjugates that were good MMP substrates but poor neprilysin substrates. To determine how conjugates were metabolized in animals, MMP-selective conjugates were given to mice with HT1080 xenografts and the distribution of doxorubicin was determined. These studies showed that MMP-selective conjugates were preferentially metabolized in HT1080 xenografts, relative to heart and plasma, leading to 10-fold increases in the tumor/heart ratio of doxorubicin. The doxorubicin deposited by a MMP-selective prodrug, compound 6, was more effective than doxorubicin at reducing HT1080 xenograft growth. In particular, compound 6 cured 8 of 10 mice with HT1080 xenografts at doses below the maximum tolerated dose, whereas doxorubicin cured 2 of 20 mice at its maximum tolerated dose. Compound 6 was less toxic than doxorubicin at this efficacious dose because mice treated with compound 6 had no detectable changes in body weight or reticulocytes, a marker for marrow toxicity. Hence, MMP-activated doxorubicin prodrugs have a much higher therapeutic index than doxorubicin using HT1080 xenografts as a preclinical model.

Heteroatom- and carbon-linked biphenyl analogs of Brequinar as immunosuppressive agents

Structure-activity relationships were explored for some analogs of Brequinar having a linking atom between the 2-biphenyl substituent and the quinoline ring. Activities as inhibitors of dihydroorotate dehydrogenase and the mixed lymphocyte reaction were related to the overall shape and lipophilicity of the 2-substituent.

Comparative studies of active site–ligand interactions among various recombinant constructs of human β-amyloid precursor protein cleaving enzyme

Amyloid precursor protein (APP) cleaving enzyme (BACE) is the enzyme responsible for beta-site cleavage of APP, leading to the formation of the amyloid-beta peptide that is thought to be pathogenic in Alzheimers disease (AD). Hence, BACE is an attractive pharmacological target, and numerous research groups have begun searching for potent and selective inhibitors of this enzyme as a potential mechanism for therapeutic intervention in AD. The mature enzyme is composed of a globular catalytic domain that is N-linked glycosylated in mammalian cells, a single transmembrane helix that anchors the enzyme to an intracellular membrane, and a short C-terminal domain that extends outside the phospholipid bilayer of the membrane. Here we have compared the substrate and active site-directed inhibitor binding properties of several recombinant constructs of human BACE. The constructs studied here address the importance of catalytic domain glycosylation state, inclusion of domains other than the catalytic domain, and incorporation into a membrane bilayer on the interactions of the enzyme active site with peptidic ligands. We find no significant differences in ligand binding properties among these various constructs. These data demonstrate that the nonglycosylated, soluble catalytic domain of BACE faithfully reflects the ligand binding properties of the full-length mature enzyme in its natural membrane environment. Thus, the use of the nonglycosylated, soluble catalytic domain of BACE is appropriate for studies aimed at understanding the determinants of ligand recognition by the enzyme active site.

Steady-State Kinetic Analysis of Human Ubiquitin-Activating Enzyme (E1) Using a Fluorescently Labeled Ubiquitin Substrate

We report the synthesis of fluorescently labeled ubiquitin (Ub) and its use for following ubiquitin transfer to various proteins. Using Oregon green (Og) succinimidyl ester, we prepared a population of Ub mainly labeled by a single Og molecule; greater than 95% of the Og label is associated with Lys 6 of Ub. We demonstrate that Og-Ub is efficiently accepted by Ub-utilizing enzymes, such as the human ubiquitin-activating enzyme (E1). We used this fluorescent substrate to follow the steady-state kinetics of human E1-catalyzed Ub-transfer to the ubiquitin-carrier enzyme Ubc4. In this reaction, E1 uses three substrates: ATP, Ubc4, and Ub. The steady-state kinetics of Og-Ub utilization by E1 is presented. We have also used analytical ultracentrifugation methods to establish that E1 is monomeric under our assay condition (low salt) as well as under physiological condition (150 mM NaCl).

Immunosuppressive structure-activity relationships of Brequinar and related cinchoninic acid derivatives

Abstract The immunosuppressive structure-activity relationships of substituted cinchoninic acid derivatives related to Brequinar were explored. Activities as inhibitors of dihydroorotate dehydrogenase and the mixed lymphocyte reaction were related to benzo-ring substitution, replacement of the benzo-ring by heterocycles, and variation in the 2-biphenyl, 3-methyl, and 4-carboxy groups.

Structure-activity relationships (SAR) of some tetracyclic heterocycles related to the immunosuppressive agent brequinar sodium

The structure-activity relationships of some tetracyclic heterocycles related to Brequinar were explored. Activities as inhibitors of dihydroorotate dehydrogenase and the mixed lymphocyte reaction are related to ring system, heteroatom placement, and pendant ring substitution.

An ELISA assay for murine interleukin-1β

An ELISA assay was developed for murine IL-1 beta (mIL-1 beta) using a polyclonal antibody generated in rabbits. The antibody was purified by affinity chromatography on protein A coupled to Sepharose followed by chromatography on mIL-1 beta coupled to Sepharose. The protein A and affinity purified populations were compared using radiolabeled mIL-1 beta and the results used to develop the conditions for the ELISA. The assay developed is sensitive to pg/ml concentrations of mIL-1 beta, is comparable in sensitivity to one which uses a hamster monoclonal antibody as the capture antibody, and can be used to detect IL-1 beta in peritoneal washings or tissue lysates from either mouse or rat. There is no cross reaction with any cytokine tested. The use of ELISA enhancement kits can increase the resolution at the lower concentration ranges without affecting assay sensitivity. This assay should prove useful for defining the presence and potential role for IL-1 beta in animal models of disease.

Comparative effects of selective cyclooxygenase 1 and cyclooxygenase 2 inhibitors on myeloperoxidase and 3 alpha-hydroxysteroid dehydrogenase.

The clinical efficacy of non-steroidal anti-inflammatory drugs (NSAIDs) is believed to result from the ability of these compounds to inhibit the inducible isoform of the enzyme cyclooxygenase, COX2. The gastrointestinal and renal side effects of these drugs, in contrast, are thought to relate to their ability to inhibit the constitutive isozyme, COX1. There is structural and pharmacological evidence that suggests that NSAIDs may also inhibit two unrelated enzymes, myeloperoxidase (MP) and 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD), potentially with untoward consequences for the patient. Our laboratories have been investigating a new structural class of potential COX inhibitors, the tri-cyclic aromatics. In this study we have examined the inhibitory potency of selected compounds for the enzymes human COX1, human COX2, human MP, and rat liver 3 alpha-HSD. The compounds selected span a range of COX isoform selectivities, from specific for COX2 to selective for COX1 only, and include three representative tri-cyclic aromatics. We found that compounds within the tri-cyclic aromatic class do not act as potent inhibitors of either myeloperoxidase or 3 alpha-HSD. These results demonstrate the unique inhibitor selectivity that can be achieved with the tri-cyclic aromatics. Examples of COX1 selective, and COX2 selective inhibitors within this structural class are presented.