Exploration of the P1 SAR of aldehyde cathepsin K inhibitors

The synthesis and biological activity of a series of aldehyde inhibitors of cathepsin K are reported. Exploration of the properties of the S(1) subsite with a series of alpha-amino aldehyde derivatives substituted at the P(1) position afforded compounds with cathepsin K IC(50)s between 52 microM and 15 nM.     

Novel bisbenzamidines as potential drug candidates for the treatment of Pneumocystis carinii pneumonia

A series of pentamidine congeners has been synthesized and screened for their in vitro activity against Pneumocystis carinii. Among the tested compounds, bisbenzamidines linked by a flexible pentanediamide or hexanediamide chain (7 and 9) emerged as exceptionally potent agents that were more effective and less toxic than pentamidine in the assays described in this study.     

Unbalanced collagenases/TIMP-1 expression and epithelial apoptosis in experimental lung fibrosis

In this study, we examined the sequential expression of several matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and growth factors as well as the presence of apoptosis in a model of pulmonary fibrosis induced in rats with paraquat and hyperoxia. Animals showing neither clinical nor morphological changes with this double aggression were classified as "resistant". Rats were killed at 1, 2, 3, and 6 wk, and lungs were used for collagen content, gene expression by real-time PCR, gelatinolytic activity by zymography, apoptosis by in situ DNA fragmentation, and protein localization by immunohistochemistry. Our results showed a significant decrease of collagenases MMP-8 and MMP-13, with an increase of TIMP-1 and transforming growth factor-beta. Immunoreactive TIMP-1 was increased in experimental rats and primarily localized in alveolar macrophages. Expression of gelatinases MMP-2 and MMP-9 mRNAs was not affected, but lung zymography revealed an increase in progelatinase B, progelatinase A, and its active form. Epithelial apoptosis was evident from the first week, whereas at later periods, interstitial cell apoptosis was also noticed. Resistant animals behave as controls. These findings suggest that an imbalance between collagenases and TIMPs, excessive gelatinolytic activity, and epithelial apoptosis participate in the fibrotic response in this experimental model.     

Iron porphyrin treatment extends survival in a transgenic animal model of amyotrophic lateral sclerosis

Oxidative damage, produced by mutant Cu/Zn superoxide dismutase (SOD1), may play a role in the pathogenesis of amyotrophic lateral sclerosis (ALS), a devastating motor neuron degenerative disease. A novel approach to antioxidant therapy is the use of metalloporphyrins that catalytically scavenge a wide range of reactive oxygen and reactive nitrogen species. In this study, we examined the therapeutic potential of iron porphyrin (FeTCPP) in the G93A mutant SOD1 transgenic mouse model of ALS. We found that intraperitoneal injection of FeTCPP significantly improved motor function and extended survival in G93A mice. Similar results were seen with a second group of mice wherein treatment with FeTCPP was initiated at the onset of hindlimb weakness-roughly equivalent to the time at which treatment would begin in human patients. FeTCPP-treated mice also showed a significant reduction in levels of malondialdehyde (a marker of lipid peroxidation), in total content of protein carbonyls (a marker of protein oxidation), and increased neuronal survival in the spinal cord. These results therefore provide further evidence of oxidative damage in a mouse model of ALS, and suggest that FeTCPP could be beneficial for the treatment of ALS patients.     

Transgene expression of green fluorescent protein and germ line transmission in cloned calves derived from in vitro-transfected somatic cells

In vitro transfection of cultured cells combined with nuclear transfer currently is the most effective procedure to produce transgenic livestock. In the present study, bovine primary fetal fibroblasts were transfected with a green fluorescent protein (GFP)-reporter transgene and used as nuclear donor cells in oocyte reconstructions. Because cell synchronization protocols are less effective after transfection, activated oocytes may be more suitable as hosts for nuclear transfer. To examine the role of host cytoplasm on transgene expression and developmental outcome, GFP-expressing fibroblasts were fused to oocytes reconstructed either before (metaphase) or after (telophase) activation. Expression of GFP was examined during early embryogenesis, in tissues of cloned calves, and again during embryogenesis, after passage through germ line using semen from the transgenic cloned offspring. Regardless of the kind of host cytoplasm used, GFP became detectable at the 8- to 16-cell stage, approximately 80 h after reconstruction, and remained positive at all later stages. After birth, although cloned calves obtained through both procedures expressed GFP in all tissues examined, expression levels varied both between tissues and between cells within the same tissue, indicating a partial shutdown of GFP expression during cellular differentiation. Moreover, nonexpressing fibroblasts derived from transgenic offspring were unable to direct GFP expression after nuclear transfer and development to the blastocyst stage, suggesting an irreversible silencing of transgenes. Nonetheless, GFP was expressed in approximately half the blastocysts obtained with sperm from a transgenic clone, confirming transmission of the transgene through the germ line.     

Synthesis of protected 2-amino-2-deoxy-D-xylothionolactam derivatives and some aspects of their reactivity

The synthesis of polyfunctionalized delta-lactams as key intermediates of glycomimetics in the 2-acetamido-2-deoxy sugar series is presented. Starting from a chiral gamma-amino vinylic ester synthesized from Garner's aldehyde and after regioselective reduction, 1-azido-3-(N-tert-butyloxycarbonyl-2,2-dimethyloxazolidin-4-yl)-2-propene was obtained. Next, a cis-dihydroxylation reaction provided the protected D-xylitol and L-arabinitol azides. A simple protection-deprotection sequence, followed by an oxidation and a reductive cyclization, led to protected 2-amino-delta-lactams bearing a tert-butyloxycarbonyl group on the amine functionality. To explore the reactivity of such compounds, activation of the lactam into the corresponding thionolactam was performed. The resulting 2-amino-D-xylothionolactam derivative, a versatile intermediate, allowed access to a first generation of protected 2-amino-D-xylosamidoxime derivatives which are of interest as precursors of N-acetylhexosaminidase and N-acetylglucosaminyltransferase inhibitors. In this series of compounds, epimerization at C-2 was observed. AM(1) calculations performed on these analogs showed that they adopted a B(2,5) conformation and that the axial epimer was favored in the protected series whereas the equatorial epimer was preferred in the unprotected series.     

Protective effect of Pongamia pinnata flowers against cisplatin and gentamicin induced nephrotoxicity in rats

Ethanolic extract of flowers of Pongamia pinnata was studied for its protective effect against cisplatin and gentamicin induced renal injury in rats. When the extract (300 & 600 mg kg(-1)) was administered orally for 10 days following cisplatin (5 mg kg(-1) i.p.) on day 5, toxicity of cisplatin, as measured by loss of body weight, elevated blood urea and serum creatinine declined significantly. Similarly in gentamicin (40 mg kg(-1) s.c.) induced renal injury, the extract (600 mg kg(-1)) normalized the raised blood urea and serum creatinine levels. Reversal of cisplatin and gentamicin renal cell damage as induced by tubular necrosis ie, marked congestion of the glomeruli with glomerular atrophy, degeneration of tubular epithelial cells with casts in the tubular lumen and infiltration of inflammatory cells in the interstitium was confirmed on histopathological examination. In the preventive regimen, co-administration of the extract with gentamicin significantly prevented the renal injury both functionally and histologically. Ethanolic extract of flowers had a marked nitric oxide free radical scavenging effect, suggesting an antioxidative property. Two flavonoids, known for their antioxidant activity viz. kaempferol and 3, 5, 6, 7, 8-pentamethoxy flavone were isolated from the extract. The results suggested that the flowers of Pongamia pinnata had a protective effect against cisplatin and gentamicin induced renal injury through antioxidant property.     

A 4-AP-sensitive current is enhanced by chronic carbon monoxide exposure in coronary artery myocytes

A physiological role of carbon monoxide has been suggested for coronary myocytes; however, direct evidence is lacking. The objective of this study was to test the effect of chronic carbon monoxide exposure on the K(+) currents of the coronary myocytes. The effect of 3-wk chronic exposure to carbon monoxide was assessed on K(+) currents in isolated rat left coronary myocytes by the use of the patch-clamp technique in the whole cell configuration. Moreover, membrane potential studies were performed on coronary artery rings using intracellular microelectrodes, and coronary blood flow in isolated heart preparation was recorded. Carbon monoxide did not change the amplitude of global whole cell K(+) current, but it did increase the component sensitive to 1 mM 4-aminopyridine. Carbon monoxide exposure hyperpolarized coronary artery segments by approximately 10 mV and, therefore, increased their sensitivity to 4-aminopyridine. This effect was associated with an enhancement of coronary blood flow. We conclude that chronic carbon monoxide increases a 4-aminopyridine-sensitive current in isolated coronary myocytes. This mechanism could, in part, contribute to hyperpolarization and to increased coronary blood flow observed with carbon monoxide.