Structure of the SARS-CoV-2 Nsp1/5'-Untranslated Region Complex and Implications for Potential Therapeutic Targets, a Vaccine, and Virulence.

SARS-CoV-2 is the cause of the ongoing Coronavirus disease 19 (COVID-19) pandemic around the world causing pneumonia and lower respiratory tract infections. In understanding the SARS-CoV-2 pathogenicity and mechanism of action, it is essential to depict the full repertoire of expressed viral proteins. The recent biological studies have highlighted the leader protein Nsp1 of SARS-CoV-2 importance in shutting down the host protein production. Besides, it still enigmatic how Nsp1 regulates for translation. Here we report the novel structure of Nsp1 from SARS-CoV-2 in complex with the SL1 region of 5'UTR of SARS-CoV-2, and its factual interaction is corroborated with enzyme kinetics and experimental binding affinity studies. The studies also address how leader protein Nsp1 of SARS-CoV-2 recognizes its self RNA toward translational regulation by further recruitment of the 40S ribosome. With the aid of molecular dynamics and simulations, we also demonstrated the real-time stability and functional dynamics of the Nsp1/SL1 complex. The studies also report the potential inhibitors and their mode of action to block viral protein/RNA complex formation. This enhance our understanding of the mechanism of the first viral protein Nsp1 synthesized in the human cell to regulate the translation of self and host. Understanding the structure and mechanism of SARS-CoV-2 Nsp1 and its interplay with the viral RNA and ribosome will open the arena for exploring the development of live attenuated vaccines and effective therapeutic targets for this disease.

3,20-Dihydroxy-13α-19-norpregna-1,3,5(10)-trienes. Synthesis, structures, and cytotoxic, estrogenic, and antiestrogenic effects.

New 3,20-dihydroxy-13α-19-norpregna-1,3,5(10)-trienes were synthesized. The effects of these compounds on breast cancer cells and ERα activation were investigated. The scaffold of compounds containing the six-membered ring D' annulated at 16α,17α-positions was constructed via the Lewis acid catalyzed Diels-Alder reaction of butadiene with 3-methoxy-13α-19-norpregna-1,3,5(10),16-tetraen-20-one 5 under a pressure of 600 MPa. The hydrogenation of primary cyclohexene adduct 6 followed by the one-pot reduction-demethylation (DIBAH) gave target epimeric 3,20-dihydroxy steroids 8a and 8b. The Corey-Chaykovsky reaction of the same conjugated ketone 5 gave a 16α,17α-methylene-substituted compound. The reaction of the latter with DIBAH yielded 3,20(R,S)-dihydroxy-16α,17α-methyleno-13α-19-norpregna-1,3,5(10)-triene 10. The hydrogenation of the 16,17-double bond of compound 5 produced a mixture of 17α- and 17ß-epimeric ketones, reduction-demethylation of which gave 3,20(S)-dihydroxy-13α,17α-19-norpregna-1,3,5(10)-triene 12a and 3,20(R)-dihydroxy-13α,17ß-19-norpregna-1,3,5(10)-triene 12b. All compounds were fully characterized by 1D and 2D NMR, HRMS, and X-ray diffraction. All target compounds showed pronounced cytotoxic effect against MCF-7 breast cancer cells and NCI/ADR-RES doxorubicin-resistant cells at micromolar concentrations. The ERα-mediated luciferase reporter gene assay demonstrated that all compounds, except for compound 10, are ERα inhibitors, while cyclopropane compound 10 proved to be an ERα activator. Docking experiments showed that all compounds are well accommodated to LBD ERα but have some differences in the binding mode.

UPLC-MS/MS assay of 21-aminosteroid (lazaroid U74389G) for application in pharmacokinetic study.

Lazaroids are potent inhibitors of lipid peroxidation, both in vitro and in vivo. Additionally, a member of the lazaroid family, U-74389G (LAZ) has been shown to have specific radio-protective and anti-proliferative effects. However, there is no quantitative analytical method developed for measuring the therapeutic levels of LAZ for the aforementioned effects. This article highlights the development and validation of a sensitive UPLC-MS/MS method for the quantification of LAZ, and its subsequent application in pharmacokinetic studies in rats with the lower limit of quantification (LLOQ) of 1.95 ng/mL. LAZ and internal standard diadzein (IS) were separated using ACQUITY UPLC(®) BEH C18 column. Gradient elution was used at a flow rate of 0.45 mL/min with mobile phases consisting of 0.1% formic acid in water and 0.1% formic in acetonitrile. LAZ (m/z 612→260) and IS (m/z 255→199) were detected by electrospray ionization (ESI) using multiple reaction monitoring (MRM) in a positive mode on QTRAP(®) 5500 System. The UPLC-MS/MS method was validated as per the US FDA Guidelines for Bio-analytical Validation. LAZ was extracted from rat plasma (100 µL) using protein precipitation by acetonitrile with mean recovery and matrix factor in range of 47.7-56.1%, and 85.6-89.4%, respectively. The calibration curve for LAZ was linear in the range of 1.95-250 ng/mL. The inter-day and intra-day accuracy and precision values for LLOQ, low, medium, high and very high concentration QC samples were within ±15%. LAZ was tested under different storage conditions, for short-term bench-top stability (1h and 3h at 25°C), long-term stability (1 month at -80°C), freeze-thaw cycle stability (1 cycle and 3 cycles) and stability of processed samples in auto-sampler (24h at 10°C) with stability values within ±15% range of nominal concentrations. The validated UPLC-MS/MS method was further applied to a pharmacokinetic study in rats after a single intravenous dose of LAZ at 5 mg/kg.

Effects of lazaroids on intestinal ischemia and reperfusion injury in experimental models.

Mesenteric ischemia occurs in a number of clinically relevant pathophysiologic processes, including sepsis, hemorrhage, intestinal transplantation, severe burns, and mesenteric thrombosis. The readmission of molecular oxygen into an ischemic tissue promotes the oxidation of resuscitated tissue with certain pathophysiologic mechanisms. Depending on the duration and the intensity of ischemia, reoxygenation of the intestine that has been reperfused may further induce tissue injury. Intestinal ischemia and reperfusion injury can accelerate complex processes between the endothelium and different cell types leading to microvascular injury, cellular necrosis, and apoptosis. The injury due to reperfusion is found predominantly in the intestinal mucosa and submucosa, causing endothelial detachment. The 21-aminosteroids (lazaroids) are a family of compounds that inhibit lipid membrane peroxidation. Many of the performed studies show conflicting results, which reflect differences in experimental design, evolving time that (I/R) is induced, total or partial vascular occlusion, dosage of the lazaroid, and the exact period of time that the lazaroid is administered.

Ligand binding domain mutations of the glucocorticoid receptor selectively modify the effects with, but not binding of, cofactors.

We previously reported that several point mutations in the ligand binding domain (LBD) of glucocorticoid receptors (GRs) marginally affect the binding affinity of the synthetic glucocorticoids dexamethasone (Dex) and deacylcortivazol (DAC). However, these mutations dramatically alter the efficacy (A(max)) and potency (EC(50)) of agonists, along with the partial agonist activity (PAA) of the antisteroid Dex-mesylate (DM), for gene induction and repression in a steroid-dependent manner. This was proposed to result, in part, from altered protein-protein interactions in the complex of GR with the coactivator TIF2 despite normal TIF2 binding. To explore the generality of this phenomenon, we now ask whether these mutations also affect the transactivation properties, but not binding, of other GR-bound factors. We find that an elevated concentration of GR, to probe unidentified cofactors, or of the comodulator Ubc9 does not reverse the effects of GR LBD mutations that increase the EC(50) and lower the PAA with the GREtkLUC reporter in both CV-1 and U2OS cells. This behavior is more dramatic with Ubc9 and the isolated GR LBD fused to the GAL4 DNA binding domain, despite normal binding of Ubc9 to the mutant GRs. Similar effects, albeit gene, steroid, and transcriptional property-specific, are seen with full-length GRs and three endogenous genes in U2OS cells. Thus, changes in simple steady-state binding capacities of mutant receptors for factors cannot account for the modified transcriptional properties. In all cases, the nuclear translocation of Dex- and DAC-bound wild-type and mutant receptors is the same. These results are consistent with the earlier results with TIF2 and support the hypothesis that small changes in the GR LBD can alter the activities of the bound cofactor without modifying cofactor binding. We propose that this separation of binding and the modulation of transactivation parameters occurs for a wide variety of GR-associated cofactors.

Sequential metabolism of 7-dehydrocholesterol to steroidal 5,7-dienes in adrenal glands and its biological implication in the skin.

Since P450scc transforms 7-dehydrocholesterol (7DHC) to 7-dehydropregnenolone (7DHP) in vitro, we investigated sequential 7DHC metabolism by adrenal glands ex vivo. There was a rapid, time- and dose-dependent metabolism of 7DHC by adrenals from rats, pigs, rabbits and dogs with production of more polar 5,7-dienes as detected by RP-HPLC. Based on retention time (RT), UV spectra and mass spectrometry, we identified the major products common to all tested species as 7DHP, 22-hydroxy-7DHC and 20,22-dihydroxy-7DHC. The involvement of P450scc in adrenal metabolic transformation was confirmed by the inhibition of this process by DL-aminoglutethimide. The metabolism of 7DHC with subsequent production of 7DHP was stimulated by forscolin indicating involvement of cAMP dependent pathways. Additional minor products of 7DHC metabolism that were more polar than 7DHP were identified as 17-hydroxy-7DHP (in pig adrenals but not those of rats) and as pregna-4,7-diene-3,20-dione (7-dehydroprogesterone). Both products represented the major identifiable products of 7DHP metabolism in adrenal glands. Studies with purified enzymes show that StAR protein likely transports 7DHC to the inner mitochondrial membrane, that 7DHC can compete effectively with cholesterol for the substrate binding site on P450scc and that the catalytic efficiency of 3betaHSD for 7DHP (V(m)/K(m)) is 40% of that for pregnenolone. Skin mitochondria are capable of transforming 7DHC to 7DHP and the 7DHP is metabolized further by skin extracts. Finally, 7DHP, its photoderivative 20-oxopregnacalciferol, and pregnenolone exhibited biological activity in skin cells including inhibition of proliferation of epidermal keratinocytes and melanocytes, and melanoma cells. These findings define a novel steroidogenic pathway: 7DHC-->22(OH)7DHC-->20,22(OH)(2)7DHC-->7DHP, with potential further metabolism of 7DHP mediated by 3betaHSD or CYP17, depending on mammalian species. The 5-7 dienal intermediates of the pathway can be a source of biologically active vitamin D3 derivatives after delivery to or production in the skin, an organ intermittently exposed to solar radiation.

An efficient one-pot synthesis of 5, 9-cyclo-1, 11-oxido-pregn-16-ene-3, 20-dione from 9-bromide-11-hydroxypregna-1, 4, 16-trien-3, 20-dione by two annulation reactions.

An efficient method to prepare 5, 9-cyclo-1, 11-oxido-pregn-16-ene-3, 20-dione in one pot was reported. Treatment of 9-bromide-11-hydroxypregna-1, 4, 16-trien-3, 20-dione with Raney Ni in absolute ethanol afforded 5, 9-cyclo-1, 11-oxido-pregn-16-ene-3, 20-dione by two annulation reactions in reasonable yield. The absolute configuration was also confirmed by X-ray crystal analysis.

Mutations of glucocorticoid receptor differentially affect AF2 domain activity in a steroid-selective manner to alter the potency and efficacy of gene induction and repression.

The transcriptional activity of steroid hormones is intimately associated with their structure. Deacylcortivazol (DAC) contains several features that were predicted to make it an inactive glucocorticoid. Nevertheless, gene induction and repression by complexes of glucocorticoid receptor (GR) with DAC occur with potency (lower EC 50) greater than and efficacy (maximal activity, or A max) equal to those of the very active and smaller synthetic glucocorticoid dexamethasone (Dex). Guided by a recent X-ray structure of DAC bound to the GR ligand binding domain (LBD), we now report that several point mutants in the LBD have little effect on the binding of either agonist steroid. However, these same mutations dramatically alter the A max and/or EC 50 of exogenous and endogenous genes in a manner that depends on steroid structure. In some cases, Dex is no longer a full agonist. These properties appear to result from a preferential inactivation of the AF2 activation domain in the GR LBD of Dex-bound, but not DAC-bound, receptors. The Dex-bound receptors display normal binding to, but a greatly reduced response to, the coactivator TIF2, thus indicating a defect in the transmission efficiency of GR-steroid complex information to the coactivator TIF2. In addition, all GR mutants that are active in gene induction with either Dex or DAC have greatly reduced activity in gene repression. This contrasts with the reports of GR mutations preferentially suppressing GR-mediated induction. The properties of these GR mutants in gene induction support the hypothesis that the A max and EC 50 of GR-controlled gene expression can be independently modified, indicate that the receptor can be modified to favor activity with a specific agonist steroid, and suggest that new ligands with suitable substituents may be able to affect the same LBD conformational changes and thereby broaden the therapeutic applications of glucocorticoid steroids.

Comparison of two structurally diverse glucocorticoid receptor agonists: cortivazol selectively regulates a distinct set of genes separate from dexamethasone in CEM cells.

One goal of steroid research is precise differential regulation of gene expression by steroid hormone receptors through use of distinct ligands which modulate defined sets of cellular effects. Such "selective modulator" ligands are known for several receptors. Potent pyrazolo-glucocorticoid (11beta,16alpha)-21-(Acetyloxy)-11,17-dihydroxy-6,16-dimethyl-2'-phenyl-2'H-pregna-2,4,6-trieno[3,2-c]pyrazol-20-one) cortivazol activates the glucocorticoid receptor to regulate gene expression and can bring about apoptosis of leukemic CEM cells resistant to (9-fluoro-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13,16-trimethyl-6,7,8,11,12,14,15,16-octahydrocyclopenta[a]phenanthren-3-one) dexamethasone. We therefore tested the hypothesis that cortivazol and dexamethasone regulate non-identical sets of genes in CEM cells. We found that while cortivazol and dexamethasone overlap in regulation of most genes, each steroid regulates an exclusive set of transcripts in clone CEM-C7-14 (sensitive to apoptosis by both dexamethasone and cortivazol) and clone CEM-C1-15 (dexamethasone-resistant but cortivazol-sensitive). Fifty-seven genes were regulated uniquely to a statistically significant extent by cortivazol in both clones. Many of the cortivazol specific genes are key components of various signal transduction pathways. Our data clearly show cortivazol to be a selective modulator of GR action.

Free radical trapping as a therapeutic approach to neuroprotection in stroke: experimental and clinical studies with NXY-059 and free radical scavengers.

There is substantial experimental evidence that free radicals are produced in the brain during ischemia, during reperfusion and during intracranial hemorrhage. Removal of pathologically produced free radicals is therefore a viable approach to neuroprotection. Four compounds with free radical scavenging activity (tirilazad, ebselen, edaravone) or free radical trapping properties (NXY-059) have been examined in experimental models of stroke and evaluated clinically as neuroprotective agents. Both experimental and clinical results are reviewed in this article. Ebselen was a modestly effective neuroprotectant in a rat transient middle cerebral artery occlusion (MCAO) model when given before the start of ischemia, but not when the insult was severe. Data from the permanent MCAO model and an embolic stroke model suggested a bell shaped dose-response curve. The weak preclinical profile may explain the lack of success in clinical trials. Preclinical data on tirilazad in animal models of acute ischemic stroke are neither comprehensive nor consistent. There was little evidence of efficacy in permanent MCAO or when the drug was given several hours post-occlusion. This may explain the negative clinical trials as these did not target patients likely to reperfuse and treatment started several hours after stroke onset. While preclinical data on subarachnoid hemorrhage demonstrated an attenuation of vasospasm the clinical data were inconsistent. There is very limited published preclinical data on edaravone but it has been approved in Japan as a neuroprotectant for the treatment of stroke. Evidence is based on a single placebo controlled trial in a relatively small number of patients. The status of possible development of edaravone outside of Japan is not known. NXY-059 has been found to be a very effective agent in transient and permanent MCAO and thromboembolic models of acute ischemic stroke. Its preclinical development has been governed by adherence with the recommendations of the Stroke Therapy Academic Industry Roundtable (STAIR) group and is now being investigated in Phase III clinical trials using a therapeutic time window and plasma concentrations that are effective in rat and primate models of stroke.

Pregnane-type steroids from the inedible mushroom Thelephora terrestris.

Chromatographic fractionation of the methanol extract of fruiting bodies of the inedible Japanese mushroom Thelephora terrestris (Thelephoraceae) has led to the isolation and characterization of two unusual new pregnane-type steroids, 2beta,3alpha-dihydroxypregna-4,7,16-trien-12,20-dione (2) and 1alpha,2beta,3alpha-trihydroxypregna-4,7,16-trien-12,20-dione (3) named terresterones A and B (2, 3), as well as the previously known compound stizophyllin, now assigned as 2beta,3alpha,12beta-trihydroxypregna-4,7,16-trien-20-one (1). Elucidation of their structures and the revision of the stereochemical assignment of stizophyllin were achieved by means of extensive 1D and 2D NMR, UV, CD, IR, MS and molecular modeling experiments. This paper presents the first report on the isolation of true pregnane-type steroids from the Fungi kingdom.

21-Acetoxy-pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione conversion by Nocardioides simplex VKM Ac-2033D.

The conversion of 21-acetoxy-pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione (I) by Nocardioides simplex VKM Ac-2033D was studied purposed selective production of its 1(2)-dehydroanalogues-value precursors in the synthesis of modern glucocorticoids starting from 9alpha-hydroxyandrostenes. 21-Acetoxy-pregna-1(2),4(5),9(11),16(17)-tetraene-21-ol-3,20-dione (II), pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione (III) and pregna-1(2),4(5),9(11),16(17)-tetraene-21-ol-3,20-dione (IV) were revealed as metabolites, and the structures were confirmed by mass spectrometry and (1)H nuclear magnetic resonance (NMR) spectroscopy. The metabolic pathways of I by N. simplex included 1(2)-dehydrogenation and deacetylation. The sequence of the reactions was shown to depend on the transformation conditions. The presence of both soluble and membrane associated steroid esterases in N. simplex was demonstrated using cell fractionation. Unlike inducible 1(2)-dehydrogenase, steroid esterase was shown to be constitutive. The conditions providing selective accumulation of II from I by whole N. simplex cells were determined.

Distinct interaction of cortivazol with the ligand binding domain confers glucocorticoid receptor specificity: cortivazol is a specific ligand for the glucocorticoid receptor.

Ligand-receptor coupling is one of the important constituents of signal transduction and is essential for physiological transmission of actions of endogenous substances including steroid hormones. However, molecular mechanisms of the redundancy between glucocorticoid and mineralocorticoid actions remain unknown because of complicated cross-talk among, for example, these adrenal steroids, their cognate receptors, and target genes. Receptor-specific ligand that can distinctly modulate target gene expression should be developed to overcome this issue. In this report, we showed that a pyrazolosteroid cortivazol (CVZ) does not induce either nuclear translocation or transactivation function of the mineralocorticoid receptor (MR) but does both for the glucocorticoid receptor (GR). Moreover, deletion analysis of the C-terminal end of the GR has revealed that CVZ interacts with the distinct portion of the ligand binding domain (LBD) and differentially modulates the ligand-dependent interaction between transcription intermediary factor 2 and the LBD when compared with cortisol, dexamethasone, and aldosterone. Thus, it is indicated that CVZ may not be only a molecular probe for the analysis of the redundancy between the GR and MR in vivo but also a useful reagent to clarify structure-function relationship of the GR LBD.

Combination pharmacotherapy with delayed countershock vs standard advanced cardiac life support after prolonged ventricular fibrillation.

OBJECTIVE: To test the hypothesis that combination pharmacotherapy with delayed countershock would produce higher rates of return of spontaneous circulation (ROSC) and one-hour survival when compared with standard Advanced Cardiac Life Support (ACLS) therapy. METHODS: A prospective, block-randomized, blinded, laboratory experiment was conducted in an established swine model of prolonged ventricular fibrillation (VF). Fifty-six female domestic swine were anesthetized, instrumented, and shocked into VF with a bipolar pacing catheter. The VF was untreated for 8 minutes, then basic CPR was done mechanically for 1 minute. At 9 minutes of VF, the animals were randomized to treatment with one of seven therapies: group 1, combination pharmacotherapy with epinephrine (0.20 mg/kg), lidocaine (1.0 mg/kg), bretylium (5.0 mg/kg), propranolol (1.0 mg), and U-74389G (3.0 mg/kg); group 2, epinephrine (0.20 mg/kg); group 3, lidocaine (1.0 mg/kg) and bretylium (5.0 mg/kg); group 4, propranolol (1.0 mg); group 5, U-74389G (3.0 mg/kg); group 6, normal saline solution (volume equal to that for group 1); and group 7, standard ACLS (first countershock at 9 minutes of VF). Initial countershocks for groups 1-6 were given after 11 minutes of VF. Data were analyzed with two-tailed Fisher's exact test, with alpha set at 0.05. RESULTS: Return of spontaneous circulation occurred in group 1 = 8/8 (100%); group 2 = 7/8 (88%); group 3 = 3/8 (38%); group 4 = 3/8 (38%); group 5 = 5/8 (63%); group 6 = 4/8 (50%); and group 7 = 3/8 (38%). One-hour survival occurred in group 1 = 8/8 (100%); group 2 = 5/8 (63%); group 3 = 2/8 (25%); group 4 = 2/8 (25%); group 5 = 3/8 (38%); group 6 = 2/8 (25%); and group 7 = 1/8 (13%). CONCLUSIONS: Combination pharmacotherapy with delayed countershock (group 1) produced significantly higher rates of ROSC (p = 0.03) and one-hour survival (p = 0.001) when compared with standard ACLS in this porcine model of prolonged VF.

A novel stable supersaturated submicron lipid emulsion of tirilazad.

This paper describes a novel supersaturated submicron lipid emulsion for parenteral drug delivery. Tirilazad was used as an amphiphilic model drug for the preparation and characterization of the supersaturated emulsions with concentrations more than two times higher than the drug solubility in the emulsion. Analysis of particle size distribution using photon correlation spectroscopy indicated that the mean particle diameters of the supersaturated 10 and 20% lipid emulsions of tirilazad were approximately 210 and 240 nm, respectively, and that the mean particle size and size distribution of the supersaturated tirilazad emulsions were not different from those of the non-supersaturated tirilazad emulsions. The apparent viscosity of the supersaturated tirilazad emulsions, as measured by continuous rheometry, was similar to that of the non-supersaturated tirilazad emulsions. However, the zeta potential of the supersaturated tirilazad emulsions, as determined by the electrophoretic light-scattering technique, was found to be dependent on drug load and was lower than that of the non-supersaturated tirilazad emulsion. Fractionation by ultracentrifugation showed that at low drug loads, the amount of drug associated with the emulsion particles and the infranatant fraction appeared to increase linearly with increasing concentration of the drug. By contrast, the amount of drug associated with the emulsion particles increased rapidly, whereas the amount of drug associated with the infranatant fraction remained constant for emulsions with a high drug load. These results suggest that the excess drug in the supersaturated emulsions is largely associated with the lipid phase. Accelerated stability studies under stress conditions (i.e., autoclaving and shaking) and long-term storage stability tests demonstrated that the supersaturated tirilazad emulsions had excellent physical stability over the study period of 16 months.

A 21-aminosteroid, U-74006F, attenuates endotoxin-induced lung injury in awake sheep.

The purpose of the present study was to examine the efficacy of U-74006F, a 21-aminosteroid, on lung dysfunction induced by endotoxaemia in awake sheep with lung lymph fistula and haemodynamic monitoring. We measured pulmonary haemodynamics, lung lymph balance, circulating leucocyte count, arterial blood gas tensions, and levels of thromboxane (Tx) B2 and 6-keto-prostaglandin (PG) F1 alpha in plasma and lung lymph. We performed two experiments. In experiment 1 (n = 6), we intravenously infused Escherichia coli lipopolysaccharide endotoxin (1 microgram/kg) over 30 min and observed the parameters over 5 h. In experiment 2 (n = 6), we pretreated sheep with an intravenous bolus of U-74006F (2 mg/kg) 30 min before the infusion of endotoxin in the same manner of experiment 1, and continuously infused U-74006F (0.5 mg/kg per h) over 5 h after the bolus during the experiment. The U-74006F significantly suppressed the early pulmonary hypertension, the late increase in pulmonary permeability and the elevations of TxB2 and 6-keto-PGF1 alpha levels in plasma and lung lymph during the early period following endotoxaemia, although the compound did not change the time course of leucocytopenia and hypoxaemia. These findings suggest that the administration of U-74006F attenuates the lung dysfunction induced by endotoxaemia in awake sheep.

Potential therapeutic value of lazaroids in endotoxemia and other forms of sepsis.

Lazaroids are developed nonglucocorticoid analogues of methylprednisolone with multiple actions, including the scavenging of reactive oxygen species, the attenuation of inflammation, and the stabilization of biological membranes. In various experimental models, lazaroids were shown to enhance recovery from ischemia-reperfusion injury, central nervous system inflammation, oxidant stress, and panendothelial activation and injury; sepsis might be another important indication for the use of lazaroids. Administration of different lazaroids in experimental endotoxemia and other forms of sepsis indeed showed improvement of biological and hemodynamic parameters, attenuation of inflammation, preservation of oxygenation, and protection of endothelial cell integrity. Further research is needed to determine the effects of these drugs on organ function, mediator synthesis and release, and survival, before appropriate therapeutic protocols for their use in clinical sepsis can be developed.

Steroids 54. Amino acylamidosteroids.

Aminosteroids were prepared and acylated with protected amino acids by means of the mixed anhydride or the active ester method. The tert-butyloxycarbonyl- (BOC) protecting group was eliminated by acidolysis, and the benzyloxycarbonyl- (Z) group by catalytic hydrogenation. 3 beta- and 6 beta-Glycylamidosteroids were prepared by indirect amination of chloroacetamido derivatives, formed by the Ritter reaction on the corresponding 3 alpha,5 alpha-cyclo and 5 alpha,6 alpha-epoxy steroids. Water-soluble double salts were produced from the compounds for pharmacological investigations.

Reperfusion injury of postischemic skeletal muscle is attenuated by the 21-aminosteroids U-74389F and U-74500A independent of iron binding.

BACKGROUND: Lazaroids (21-aminosteroids) are a novel class of compounds that have been shown to limit experimental ischemic injury of varied causes. The mechanism of action is uncertain but may include scavenging of lipid peroxy radicals, iron binding, or direct membrane interaction. The purpose of these experiments was to evaluate the capacity of the lazaroids U-74500A and U-74389F to modify ischemia/reperfusion injury of skeletal muscle in a well-characterized model of high-grade partial ischemia. METHODS: Nonfasted male Sprague-Dawley rats were anesthetized, a tracheostomy tube was placed, and the carotid artery and jugular vein were cannulated. Animals received heparin (1 unit/gm) and crystalloid (1 ml/hr) intravenously. The baseline group (n = 6) was allowed a 30-minute equilibration period, after which resting transmembrane potential (Em) was measured in a hindlimb muscle. Muscle biopsy specimen was obtained; conjugated diene and thiobarbituric acid reactive substances were measured as indexes of lipid peroxidation. Spectrophotometric determination of plasma iron and unsaturated iron-binding capacity were performed (total iron-binding capacity and percent saturation were calculated). Animals received U-74389F (2 mg/kg, n = 7), U-74500A (2 mg/kg, n = 6), or vehicle only (0.02 mol/L citrate acid/citrate; n = 7) intraarterially before infrarenal aortic clamping was performed for 120 minutes. An additional group of animals received U-74389F (2 mg/kg, n = 7), U-74500A (2 mg/kg, n = 7), or vehicle (n = 11) intraarterially before infrarenal aortic clamping was performed for 120 minutes, followed by reperfusion for 30 minutes. RESULTS: Depolarization of resting Em was noted during ischemia, with partial repolarization on reperfusion, which was enhanced by either lazaroid. As expected, iron delocalization occurred during ischemia and persisted on reperfusion, with U-74500A effectively binding iron, whereas U-74389 did not. Vehicle but not the 21-aminosteroids inhibited lipid peroxidation. CONCLUSIONS: High-grade partial ischemia of skeletal muscle is associated with iron delocalization, which persists on reperfusion. Each lazaroid achieved a similar "membranoprotective" effect during reperfusion only despite lack of iron binding by U-74389F, suggesting a direct interaction with the cell membrane. These data support the concept that ischemic injury and reperfusion injury occur through fundamentally different mechanisms.