Douglas W. Morgan

Specific in vitro activation of Ca/Mg-ATPase by vitamin D-dependent rat renal calcium binding protein (calbindin D28K)

The vitamin D-dependent, calcium-binding protein from rat kidney, calbindin D28k (renal CaBP) specifically stimulates Ca,Mg-ATPase activity of human erythrocyte plasma membranes in a dose-dependent, calcium-sensitive manner. This stimulation was about two-fold compared to a three-fold stimulation by calmodulin. The effect was specific since other calcium-binding proteins and low molecular weight proteins did not stimulate Ca,Mg-ATPase activity. Renal CaBP did not stimulate cyclic nucleotide phosphodiesterase at concentrations greater than those which stimulated Ca,Mg-ATPase activity. This is the first report of a specific in vitro effect of renal CaBP on an enzyme system.

Interleukin-1β stimulates cytosolic phospholipase A2 in rheumatoid synovial fibroblasts

Phospholipase A2 (PLA2) activities in rheumatoid synovial fibroblasts (RSF) stimulated with interleukin-1 beta (IL-1 beta) were investigated. RSF incubated in the presence of IL-1 beta (120 pg/ml) for 18 h secreted 35 fold more PGE2 than did those incubated without IL-1 beta. IL-1 beta treatment did not increase the level of secretory PLA2 (sPLA2) activity or sPLA2 protein in the conditioned medium or subcellular fractions of lysed RSF. In contrast, the cell-associated PLA2 activity increased 3 to 4 fold in IL-1 beta stimulated RSF when compared with the control. The IL-1 beta stimulated, cell-associated PLA2 required submicromolar concentrations of calcium for activity, a characteristic consistent with the calcium sensitivity of cytosolic PLA2 (cPLA2) activity reported in other cell types, such as U937 cells. These findings demonstrate that an elevation in a cytosolic PLA2, rather than a sPLA2, is associated with increased PGE2 production in IL-1 beta stimulated RSF.

Discovery of selective hydroxamic acid inhibitors of tumor necrosis factor-α converting enzyme

Abstract Modification of the P1′ substituent of macrocyclic matrix metalloproteinase (MMP) inhibitors provided compounds that are selective for inhibition of tumor necrosis factor-α converting enzyme (TACE) over MMP-1 and MMP-2. Several analogues potently inhibited the release of TNF-α in a THP-1 cellular assay. Compounds containing a trimethoxyphenyl group in the P1′ substituent demonstrated TACE selectivity across several series of hydroxamate-based inhibitors.

Biaryl Ether Retrohydroxamates as Potent, Long-lived, Orally Bioavailable MMP Inhibitors

A novel series of biaryl ether reverse hydroxamate MMP inhibitors has been developed. These compounds are potent MMP-2 inhibitors with limited activity against MMP-1. Select members of this series exhibit excellent pharmacokinetic properties with long elimination half-lives (7 h) and high oral bioavailability (100%).

SYNTHETIC INHIBITOR OF MATRIX METALLOPROTEASES DECREASES TUMOR GROWTH AND METASTASES IN A SYNGENEIC MODEL OF RAT PROSTATE CANCER IN VIVO

Members of the matrix metalloprotease (MMP) family are implicated in the progression of several malignancies including prostate cancer due to their ability to break down extracellular matrix (ECM) components. In this study, we have evaluated the ability of a synthetic MMP inhibitor (A‐177430) to block tumor growth and metastases in a syngeneic model of rat prostate cancer. In an in vitro substrate assay, A‐177430 exhibited nanomolar potency (IC50 2–6 nM) against the enzymatic activity of several MMPs. For in vivo studies, male Copenhagen rats were injected s.c. with Mat Ly Lu rat prostate cancer cells (1 × 106 cells ) into the right flank and animals were administered i.p.with different doses (10–100 mg/kg per day) of A‐177430 for 16 days. Administration of A‐177430 resulted in a dose‐dependent decrease in tumor volume as compared to a control group of animals receiving vehicle alone. The maximum dose (100 mg/kg per day) of A‐177430 exhibited complete arrest in tumor growth and prevented the development of macroscopic tumor metastases to lungs without exhibiting any noticeable side effects. Histologic examination of primary tumors from experimental animals showed extensive tumor necrosis and decreased tumor angiogenesis as determined by factor VIII staining of primary tumors following A‐177430 treatment. These primary tumors from experimental animals also exhibited a significant increase in tumor cell DNA fragmentation as determined by TUNEL assay. Collectively, these results demonstrate the ability of MMP inhibitors to block tumor growth and metastases by blocking ECM degradation and by inhibiting tumor angiogenesis and promotion of prostate cancer cell apoptosis in vivo. Int. J. Cancer 87:276–282, 2000.

The design, synthesis, and structure-activity relationships of a series of macrocyclic MMP inhibitors.

A series of succinate-derived hydroxamic acids incorporating a macrocyclic ring were designed, synthesized, and evaluated as inhibitors of matrix metalloproteinases. The inhibitors were designed based on the published X-ray crystal structure of batimastat (1) complexed with human neutrophil collagenase (MMP-8). The synthesized compounds were shown to inhibit selected MMPs in vitro with low nanomolar potency.

Secretory phospholipase A2 inhibitors and calmodulin antagonists as inhibitors of cytosolic phospholipase A2

Human cytosolic phospholipase A2 (cPLA2, 85 kDa) appears to be pharmacologically distinct from human secretory phospholipase A2 (sPLA2, 14 kDa). Marine natural products and PLA2 substrate and product analogs were potent inhibitors of human recombinant sPLA2 (r-sPLA2), whereas these compounds stimulated, weakly inhibited, or had no effect on cPLA2 activity from the human monocytic cell line U937. In contrast, within a series of seven reported calmodulin (CaM) antagonists tested, significant correlations among the rank order of potencies of these compounds as inhibitors of cPLA2, r-sPLA2, and a CaM-dependent phosphodiesterase were observed. The correlated inhibitory effects of the hydrophobic CaM antagonists on cPLA2 and sPLA2 may reflect a common feature (possibly a hydrophobic domain) shared by these two types of enzymes.

Sodiumcalcium exchange in sarcolemmal vesicles from tracheal smooth muscle

Abstract Sarcolemmal vesicles prepared by a new procedure from bovine tracheal smooth muscle were found to have a NaCa exchange activity that is significantly higher than that reported for different preparations from other types of smooth muscle. The exchange process system co-purified with 5′-nucleotidase, a plasma membrane marker enzyme, and was significantly enriched (over 100-fold) compared to mitochondria ( cytochrome -c oxidase) but only slightly enriched (4-fold) compared to sarcoplasmic reticulum ( NADPH-cytochrome -c reductase). The Na+ dependence of Ca2+ transport was demonstrated through both uptake and efflux procedures. The uptake profile with respect to Ca2+ was monotonic with a linear ν o vs. ν o · S −1 plot. The resultant K m of Ca2+ from the airway sarcolemmal vesicles (20 μM) was similar in magnitude to the K m of cardiac sarcolemmal vesicles (30 μM). Tracheal vesicles demonstrated a V max of 0.3–0.5 nmol · mg −1 · s − which is significantly higher than that reported in preparations from other smooth muscle types. Furthermore, two processes found to stimulate cardiac Na-Ca exchange, pretreatment with either a mixture of dithiothreitol and Fe2+ or with chymotrypsin, were ineffective on the tracheal smooth muscle. Thus, the Na-Ca exchanger identified in tracheal smooth muscle appears to be different from that observed in cardiac muscle, implying that regulation of this activity may also be different.

Pharmacology of ABT-491, a highly potent platelet-activating factor receptor antagonist

ABT-491 (4-ethynyl-N, N-dimethyl-3-[3-fluoro-4-[(2-methyl-1H-imidazo-[4,5-c]pyridin-1-yl)methy l]benzoyl]-1H- indole-1-carboxamide hydrochloride) is a novel PAF (platelet-activating factor) receptor antagonist with a K(i) for inhibiting PAF binding to human platelets of 0.6 nM. Binding kinetics of ABT-491 to the PAF receptor is consistent with a relatively slow off-rate of the antagonist when compared to PAF. Inhibition of PAF binding is selective and is correlated with functional antagonism of PAF-mediated cellular responses (Ca2+ mobilization, priming, and degranulation). Administration of ABT-491 in vivo leads to potent inhibition of PAF-induced inflammatory responses (increased vascular permeability, hypotension, and edema) and PAF-induced lethality. Oral potency (ED50) was between 0.03 and 0.4 mg/kg in rat, mouse, and guinea-pig. When administered intravenously in these species, ABT-491 exhibited ED50 values between 0.005 and 0.016 mg/kg. An oral dose of 0.5 mg/kg in rat provided > 50% protection for 8 h against cutaneous PAF challenge. ABT-491 administered orally was also effective in inhibiting lipopolysaccharide-induced hypotension (ED50 = 0.04 mg/kg), gastrointestinal damage (0.05 mg/kg, 79% inhibition), and lethality (1 mg/kg, 85% vs. 57% survival). The potency of this novel antagonist suggests that ABT-491 will be useful in the treatment of PAF-mediated diseases.

Multiple pathways for signal transduction in the regulation of arachidonic acid metabolism in rat peritoneal macrophages

The roles of guanine nucleotide-binding proteins (G-proteins) and cyclic AMP (cAMP) in signal-transduction of inflammatory stimuli leading to arachidonic acid metabolism in resident rat peritoneal macrophages (RPM) were investigated. Opsonized zymosan, targeting multiple receptors and latex particles coated with IgG (latex-IgG), targeting Fc receptors, were used as models of in vivo inflammatory stimuli encountered by macrophages. A comparison of the patterns of eicosanoid products produced in response to these stimuli showed differences: opsonized zymosan stimulated production of more leukotriene B4 (LTB4) than prostaglandin E2 (PGE2), while latex-IgG stimulated production of more PGE2 than LTB4. Non-selective stimulation of G-proteins by GTP and non-hydrolyzable analogs of GTP also stimulated arachidonic acid metabolism; these agents were not selective for PGE2 or LTB4 production. Cholera toxin, however, selectively stimulated production of PGE2 rather than LTB4 and also increased intracellular cAMP concentrations. The increased cAMP did not appear to mediate cholera toxin stimulation since forskolin, which also increased cAMP, was inhibitory to PGE2 production. This suggests that latex-IgG and cholera toxin may activate arachidonic acid metabolism through a G-protein other than Gs to induce PGE2 production specifically. The effects of pertussis toxin were biphasic: a partial inhibitory effect was observed at a low concentration of pertussis toxin (1 ng/ml) on opsonized zymosan or latrix-IgG stimulated arachidonic acid metabolism, while a high concentration of pertussis toxin (100 ng/ml) augmented the stimuli. A pertussis toxin-sensitive G-protein, possibly Gi, may therefore mediate a portion of the stimulatory signals. We have concluded that multiple pathways probably exist for opsonized zymosan and latex-IgG stimulation of arachidonic acid metabolism potentially involving multiple G-proteins.