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Antipain Y, a new antipain analog that inhibits neurotransmitter release from rat dorsal root ganglion neurons

Decalpenic acid is a natural small molecule previously isolated from the fermentation broth of fungi that induces early osteoblastic markers in pluripotent mesenchymal cells. Treatment of mouse pluripotent mesenchymal C3H10T1/2 cells with decalpenic acid gave rise to a morphological change similar to that induced by the treatment with retinoic acid, i.e. the cells adopted a more elongated spindle shape. Using a retinoic acid response element reporter and receptor activity assays, we show that decalpenic acid is a new retinoid with selectivity towards retinoic acid receptors γ and α. The induction of early osteoblastic markers by decalpenic acid was significantly inhibited by treatment with the retinoid antagonist, LE540, or with small interfering RNA-mediated knockdown of retinoic acid receptor γ. These results demonstrated that decalpenic acid induces early osteoblastic markers in pluripotent mesenchymal cells through activation of retinoic acid receptor γ.

Osteoblasts are the cells responsible for bone formation during embryonic development and adult life. Small compounds that could induce osteoblast differentiation might be promising sources of therapies for bone diseases such as osteoporosis. During screening for inducers of osteoblast differentiation of mouse pluripotent mesenchymal C3H10T1/2 cells, we isolated a small compound from the fermentation broth of Penicillium verruculosum CR37010. This compound, named decalpenic acid, bears a decalin moiety with a tetraenoic acid side chain. Treatment of C3H10T1/2 cells with decalpenic acid alone induced the expression of early osteoblast markers, such as alkaline phosphatase activity and osteopontin mRNA, but did not induce the late osteoblast marker osteocalcin mRNA or adipocyte markers under our experimental conditions.

Cyclooctatin has been isolated from Streptomyces melanosporofaciens MI614-43F2 as part of a program designed to find microorganism-produced inhibitors of lysophospholipase. It was purified by chromatography on silica gel, Capcell Pak C18 (HPLC) and Sephadex LH-20 followed by solvent extraction and then isolated as a colorless powder. Cyclooctatin has the molecular formula of C20H34O3. It is competitive with the substrate, and the inhibition constant (Ki) was 4.8 x 10(-6) M.

Benastatins have been isolated as part of a program designed to find microorganism-produced inhibitors of glutathione S-transferase from Streptomyces sp. MI384-DF12. They were purified by chromatography of reversed-phase silica gel, silica gel and Capcell Pak C18 (HPLC) followed by solvent extraction and then isolated as yellow powders. Benastatins A and B have the molecular formulae, C30H28O7 and C30H30O7, respectively. They were competitive with 3,4-dichloronitrobenzene as the substrate, and the inhibition constants (Ki) of benastatins A and B were 5.0 x 10(-6) and 3.7 x 10(-6), respectively.

Poststatin, a new inhibitor of prolyl endopeptidase (PEP) was discovered in the fermentation broth of Streptomyces viridochromogenes MH534-30F3. It was purified by Diaion HP-20, Sephadex LH-20 and YMC-gel (ODS-A) column chromatography and then isolated as a colorless powder. Poststatin has the molecular formula C26H47N5O7. The IC50 value of poststatin against the PEP of partially purified porcine kidney was 0.03 microgram/ml. It has low acute toxicity. No deaths occured after iv injection of 250 mg/kg of this agent to mice.

The relationships between structures and inhibitory activities of glycosidase inhibitors of gem-diamine 1-N-iminosugars in media of enzyme assays have been investigated. It has been proved that gem-diamine 1-N-iminosugar smoothly undergoes a structural change to a hydrated ketone or its derivative via a hemiaminal in the media (pH 5.0-6.3), and that the products generated in the media as well as the parent gem-diamine 1-N-iminosugars potently inhibit glycosidases.

New benzo[a]naphthacenequinone metabolites, designated bequinostatins A and B, have been isolated from the culture broth of the benastatin-producing strain Streptomyces sp. MI384-DF12. The structures of bequinostatins A and B were determined by spectral analyses to be 5,6,8,13-tetrahydro-1,6,7,9,11-pentahydroxy-8,13-dioxo-3- pentylbenzo[a]naphthacene-2-carboxylic acid and 2-decarboxybequinostatin A, respectively. Bequinostatin A showed considerable inhibitory activity against human pi class glutathione S-transferase (GST pi).

Benastatin C, a new member of the benastatins, has been isolated from the culture broth of Streptomyces sp. MI384-DF12. The structure of benastatin C was elucidated as 2-decarboxy-benastatin A by NMR studies. Benastatin D, 2-decarboxybenastatin B, was derived from benastatin B. Benastatins C and D showed inhibitory activities against human pi class glutathione S-transferase (GST pi) and excellent stimulatory activities on the murine lymphocyte blastogenesis in vitro.

Nagstatin, a new inhibitor of N-acetyl-beta-D-glucosaminidase (NAG-ase) was discovered in the fermentation broth of Streptomyces amakusaensis MG846-fF3. It was purified by chromatography on Dowex 50W, Avicel and Sephadex LH-20 followed by the treatment of active carbon and then isolated as colorless powder. Nagstatin has the molecular formula of C12H17N3O6. It is competitive with the substrate, and the inhibition constant (Ki) was 1.7 x 10(-8) M.

Our previous studies suggested that the administration of exogenous gangliosides to the body modulates enzymatic networks in the brain. In the present study, we tested whether that is the case with another organ, spleen. By testing the dose response relationship, we found that there is a optimum dose for the effect of enzymatic modulation of GM1 (monosialoganglioside 1) administration. Although the optimum level varied depending on each of the examined hydrolytic enzymes, it usually fell in the range around 50 micrograms/kg body weight. The findings led us to conclude that the enzyme-modulating actions of gangliosides come not merely from the bizarre actions in vivo of high molecular exogenous substances.

Experimental allergic encephalomyelitis (EAE) is one of the experimental models of human demyelinating diseases and recently is regarded also as a useful model for studying cell-mediated autoimmune diseases. Because of the possibility of induction of systemic changes in this model, we investigated enzymatic changes in serum and main organs of the diseases animals, including brain, spinal cord, limb muscle, heart muscle, spleen, liver and kidney. The enzymes measured consisted of 7 aminopeptidases, 5 endopeptidases, 3 glycosidases, creatine kinase, phosphatase and esterase. Significant changes of many enzymatic activities occurred in all the organs tested in 1 to 2 weeks after the administration of EAE antigen, myelin basic protein (MBP). Interesting correlations of the pattern of enzymatic changes were seen among most of the organs tested. Those patterns changed in the course of the 2 weeks and there remained marked changes characteristic for each organ. This model may represent some type of systemic autoimmune diseases.

The present study was performed to investigate the enzymatic changes in dystrophic chickens compared to those of dystrophic mice. The activities of 14 kinds of aminopeptidases, 5 kinds of endopeptidase, 4 kinds of glycosidases, phosphatase, esterase, and ribonuclease were measured in muscles of control and dystrophic chickens. When the enzyme activities were expressed as specific activity per unit weight of organs, only some of them were found to be significantly elevated in dystrophic chickens; e.g., alanine aminopeptidase (Ala-AP), Gly-AP and cathepsin D. On the contrary, the activities of alpha-D-glycosidase, alpha-D-galactosidase and alpha-D-mannosidase were significantly decreased. Muscular protein contents of dystrophic chickens also tended to be lower than those of controls. These observations offer a striking contrast with the one obtained in the study on dystrophic mice. However, when expressed as specific activity per mg protein, many enzyme activities were found to be significantly elevated suggesting an extensive abnormality of metabolism in dystrophic chickens. Among 14 kinds of aminopeptidase activities, highly significant elevations were seen especially in AP-A, AP-B, Gly-AP, Ala-AP, Ser-AP, Pro-AP, Leu-AP, Met-AP and Trp-AP. Interestingly enough, a statistical approach suggested a significant correlation between the aminopeptidase changes of dystrophic chickens with those of dystrophic mice. In addition to aminopeptidases, there were highly significant increases in the activities of cathepsin D, alpha-D-glucosidase, beta-D-galactosidase, alpha-D-mannosidase, esterase and RNase. These results indicate that the intramuscular metabolic abnormality of dystrophic chickens are generally different from but partly resembled with those of dystrophic mice.

We investigated the role of intramuscular enzymatic changes in the development of muscular weakness in rats suffering from experimental allergic neuritis. At an initial stage without apparent clinical symptoms, enzymatic changes of similar types occurred in the muscles of the forelimbs and hind limbs. At a later stage when the weakness appeared in the hind limb but not in the forelimb, dissociation of the pattern of the enzymatic changes occurred between the two limbs. Comparison of the intramuscular enzymatic changes between the two stages and between the two limbs suggested that the increased activities of aminopeptidases and endopeptidases play some important roles in the development of muscular weakness in this experimental model. Low molecular weight protease inhibitors may thus be worthy of a trial in this disease condition.

In order to confirm the pathophysiological isgnificance of the dissociation between the plasma levels of aminopeptidases A and B in spontaneously hypertensive rats, we tested the effects of several enzyme inhibitors including inhibitors of aminopeptidase B on the course of hypertension in these rats. In addition to an ihibitor of angiotensin-converting enzyme, inhibitors of aminopeptidase B (arphamenine B and bestatin) significantly suppressed the development of hypertension in these animals. This finding may represent an important clue to the pathogenesis of essential hypertension.

The present study was undertaken to compare the peculiarity of enzymatic changes in dystrophic hamsters and mice. Various enzymatic activities in muscle, bone, heart, spleen, liver and kidney were measured. The enzymes tested include 7 aminopeptidases, 5 endopeptidases, 3 glycosidases, creatine kinase, phosphatase and esterase. In dystrophic mice, the enzymatic changes were chiefly confined to muscle and bone. In dystrophic hamsters, on the other hand, extensive and pronounced changes in enzymatic activities were seen not only in skeletal muscle but also in bone, heart muscle, spleen, liver and kidney. Furthermore, resemblance of pattern of enzymatic changes was seen among several organs including skeletal muscle, heart muscle, bone and spleen in the hamster model. Comparing the enzymatic changes in these two models, dystrophic mouse may be regarded as more specific a model for musculoskeletal diseases. Dystrophic hamster may be related more to multiorgan diseases possibly associated with immunological or other systemic diseases. These models may represent two different disease categories, respectively.

We examined the relation of hydrolytic enzymes in spleen to the aging process in mice over a period of 30 months. When the enzymatic activities were expressed as activities per milligrams protein, those of serine proteinases and dipeptidyl peptidase IV (Gly-Pro-AP) significantly decreased with age, whereas that of L-leucine aminopeptidase (Leu-AP) increased significantly. However, when expressed as total activities, the enzymatic activities in spleen generally tended to increase with age, except in the case of serine proteinases, because of the age-related increase in spleen weight. The results were taken to indicate that the activities of serine proteinases become relatively more deficient in the spleen as age increases. The results of a multivariate study maintained this peculiarity of serine proteinases in comparison with other enzymes. The relative deficiency of serine proteinases in spleen may be somehow related to immunodeficiency in aged animals, as judged from dimilar findings in animal models of systemic erythematodes.

1. 1. Amastatin, a specific inhibitor of aminopeptidase A (L-α-aspartyl(l-α-aspartyl(l-α-glutamyl)-peptide hydrolase, EC 3.4.11.7), was linked to an agarose matrix and by this affinity chromatography aminopeptidase A of pig kidneys was purified as a single protein shown by acrylamide gel electrophoresis. 2. 2. Aminopeptidase A which was purified 710-fold, hydrolyzed only acidic amino acid β-naphthylamide. The optimum pH and the optimum temperature was 7.5 and 45–50°C, respectively. 3. 3. The molecular weight was approx. 300 000 as determined by Sephadex G-200 gel filtration. 4. 4. The activity of aminopeptidase A was not affected by sulfhydryl agents, S-S dissociating agents and serine proteinase inhibitor, but was inhibited strongly by metal chelating agents, and enhanced by alkaline earth metals. 5. 5. Amastatin inhibited aminopeptidase A in a competitive manner with l-glutamic acid β-naphthylamide, and the Ki value was calculated to be 2.5· 10−7 M. The inhibitory effect of amastatin on aminopeptidase A was not reversed by addition of Ca2+.

We compared the therapeutic effects of various low-molecular-weight enzyme inhibitors on dystrophic mice. Leupeptin, bestatin, forphenicinol and forphenicine significantly affected the enzymatic activities in the dystrophic muscles. The pattern of enzymatic changes in the muscles of forelimb and hindlimb caused by these inhibitors were similar in spite of the variety of their inhibitory spectra in vitro. However, comparing the pattern of enzymatic changes in spleen, forphenicinol differed from the other inhibitors tested. This may be related to the peculiar effects of this inhibitor on immunologically responsive cells.

Leupeptins, leupeptin Pr and leupeptin Ac, strongly inhibit proteolysis by plasmin, trypsin and papain, but do not inhibit proteolysis by α-chymotrypsin. The inhibition is competitive with substrates. The inhibitory effect on esterolysis by plasmin and trypsin is weaker than on proteolysis. The results with derivatives of leupeptins which contain carboxyl or alcohol instead of aldehyde and of di-n-butyl acetals of leupeptins indicate that the free aldehyde group plays a role in the activities. Leupeptins are absorbed orally and at least about 25 % is excreted in urine. Oral administration of leupeptins exhibited an anti-inflammatory effect on edema. Leupeptins inhibited thrombokinase reaction and coagulation of blood of human and rabbit. Type of inhibition was different from heparin. Coagulation of blood of rats and dogs are not inhibited. The effects of leupeptins on thrombokinase, thrombin, plasmin, trypsin, papain, kallikrein and α-chymotrypsin were compared with those of e-aminocaproic acid, trans-4-aminomethylcyclohexanecarboxylic acid, soybean trypsin inhibitor and trasylol.