Development of orally active oxytocin antagonists: studies on 1-(1-[4-[1-(2-methyl-1-oxidopyridin-3-ylmethyl)piperidin-4-yloxy]-2- methoxybenzoyl]piperidin-4-yl)-1,4-dihydrobenz[d][1,3]oxazin-2-one (L-372,662) and related pyridines

The previously reported oxytocin antagonist L-371,257 (2) has been modified at its acetylpiperidine terminus to incorporate various pyridine N-oxide groups. This modification has led to the identification of compounds with improved pharmacokinetics and excellent oral bioavailability. The pyridine N-oxide series is exemplified by L-372,662 (30), which possessed good potency in vitro (Ki = 4.1 nM, cloned human oxytocin receptor) and in vivo (intravenous AD50 = 0.71 mg/kg in the rat), excellent oral bioavailability (90% in the rat, 96% in the dog), good aqueous solubility (>8.5 mg/mL at pH 5.2) which should facilitate formulation for iv administration, and excellent selectivity against the human arginine vasopressin receptors. Incorporation of a 5-fluoro substituent on the central benzoyl ring of this class of oxytocin antagonists enhanced in vitro and in vivo potency but was detrimental to the pharmacokinetic profiles of these compounds. Although lipophilic substitution around the pyridine ring of compound 30 gave higher affinity in vitro, such substituents were a metabolic liability and caused shortfalls in vivo. Two approaches to prevent this metabolism, addition of a cyclic constraint and incorporation of trifluoromethyl groups, were examined. The former approach was ineffective because of metabolic hydroxylation on the constrained ring system, whereas the latter showed improvement in plasma pharmacokinetics in some cases.     

Comparative evaluation of FUNGITEST and broth microdilution methods for antifungal drug susceptibility testing of Candida species and Cryptococcus neoformans

The FUNGITEST method (Sanofi Diagnostics Pasteur, Paris, France) is a microplate-based procedure for the breakpoint testing of six antifungal agents (amphotericin B, flucytosine, fluconazole, itraconazole, ketoconazole, and miconazole). We compared the FUNGITEST method with a broth microdilution test, performed according to National Committee for Clinical Laboratory Standards document M27-A guidelines, for determining the in vitro susceptibilities of 180 isolates of Candida spp. (50 C. albicans, 50 C. glabrata, 10 C. kefyr, 20 C. krusei, 10 C. lusitaniae, 20 C. parapsilosis, and 20 C. tropicalis isolates) and 20 isolates of Cryptococcus neoformans. Overall, there was 100% agreement between the methods for amphotericin B, 95% agreement for flucytosine, 84% agreement for miconazole, 83% agreement for itraconazole, 77% agreement for ketoconazole, and 76% agreement for fluconazole. The overall agreement between the methods exceeded 80% for all species tested with the exception of C. glabrata (71% agreement). The poorest agreement between the results for individual agents was seen with C. glabrata (38% for fluconazole, 44% for ketoconazole, and 56% for itraconazole) and C. tropicalis (50% for miconazole). The FUNGITEST method misclassified as susceptible 2 of 12 (16.6%) fluconazole-resistant isolates, 2 of 10 (20%) itraconazole-resistant isolates, and 4 of 8 (50%) ketoconazole-resistant isolates of several Candida spp. Further development of the FUNGITEST procedure will be required before it can be recommended as an alternative method for the susceptibility testing of Candida spp. or C. neoformans.     

On the protective mechanisms of nitric oxide in acute pancreatitis

Nucleosome dimers from chicken erythrocytes show an ionic strength dependence of sedimentation coefficient similar to that of trimers, and indicative of a degree of compaction over a range of low ionic strengths. This is not easily reconciled with straight linkers but is consistent with bending or kinking of the linker DNA.     

Cerebral emboli and serum S100beta during cardiac operations

BACKGROUND: The glial protein S100beta has been used to estimate cerebral damage in a number of clinical settings. The purpose of this investigation was to determine the correlation between cerebral microemboli and S100beta levels during cardiac operations. METHODS: Transcranial Doppler ultrasonography was used to measure emboli in the right middle cerebral artery. Emboli counts (n = 111) were divided into five time periods: (1) incision to aortic cannulation; (2) aortic cannulation to cross-clamp onset; (3) cross-clamp onset to cross-clamp release; (4) cross-clamp release to decannulation; and (5) decannulation to chest closure. The level of S100beta (n = 156) was measured at baseline, at the end of cardiopulmonary bypass, then 150 and 270 minutes after cross-clamp release. RESULTS: The level of S100beta correlated with age, cardiopulmonary bypass time, cross-clamp time, and number of emboli at time period 2. Although cardiopulmonary bypass time was univariately associated with S100beta level, it became nonsignificant in a multivariable model that included age and cross-clamp time. CONCLUSIONS: The correlation of S100beta level with emboli measured during cannulation (time period 2) supports the hypothesis that cannulation is a high-risk time period for cerebral injury.     

Characteristics of cation binding to the I domains of LFA-1 and MAC-1. The LFA-1 I domain contains a Ca2+-binding site

The crystal structures of the I domains of integrins MAC-1 (alphaM beta2; CD11b/CD18) and LFA-1 (alphaL beta2; CD11a/CD18) show that a single conserved cation-binding site is present in each protein. Purified recombinant I domains have intrinsic ligand binding activity, and in several systems this interaction has been demonstrated to be cation-dependent. It has been proposed that the I domain cation-binding site represents a general metal ion-dependent adhesion motif utilized for binding protein ligands. Here we show that the purified recombinant I domain of LFA-1 (alphaLI) binds cations, but with significantly different characteristics compared with the I domain of MAC-1 (alphaMI). Both alphaLI and alphaMI bind 54Mn2+ in a conformation-dependent manner, and in general, cations with charge and size characteristics similar to Mn2+ most effectively inhibit 54Mn2+ binding. Surprisingly, however, physiological levels of Ca2+ (1-2 mM) inhibited 54Mn2+ binding to purified alphaLI, but not to alphaMI. Using 45Ca2+ and 54Mn2+ in direct binding studies, the dissociation constants (KD) for the interactions between these cations and alphaLI were estimated to be 5-6 x 10(-5) and 1-2 x 10(-5) M, respectively. Together with the available structural information, the data suggest differential affinities for Mn2+ and Ca2+ binding to the single conserved site within alphaLI. Antagonism of LFA-1, but not MAC-1, -mediated cell adhesion by Ca2+ may be related to the Ca2+ binding activity of the LFA-1 I domain.     

Caspases cleave focal adhesion kinase during apoptosis to generate a FRNK-like polypeptide

Focal adhesion kinase (Fak) is a non-receptor protein-tyrosine kinase that stimulates cell spreading and motility by promoting the formation of contact sites between the cell and the extracellular matrix (focal adhesions). It suppresses apoptosis by transducing survival signals that emanate from focal adhesions via the clustering of transmembrane integrins by components of the extracellular matrix. We demonstrate that Fak is cleaved by caspases at two distinct sites during apoptosis. The sites were mapped to DQTD772, which was preferentially cleaved by caspase-3, and VSWD704, which was preferentially cleaved by caspase-6 and cytotoxic T lymphocyte-derived granzyme B. The cleavage of Fak during apoptosis separates the tyrosine kinase domain from the focal adhesion targeting (FAT) domain. The carboxyl-terminal fragments that are generated suppress phosphorylation of endogenous Fak and thus resemble a natural variant of Fak, FRNK, that inhibits Fak activity by preventing the localization of Fak to focal adhesions. The cleavage of Fak by caspases may thus play an important role in the execution of the suicide program by disabling the anti-apoptotic function of Fak. Interestingly, rodent Fak lacks an optimal caspase-3 consensus cleavage site although it is cleaved in murine cells undergoing apoptosis at an upstream site. This appears to be the first example of a caspase substrate where the cleavage sites are not conserved between species.     

Aggregation and motor neuron toxicity of an ALS-linked SOD1 mutant independent from wild-type SOD1

Analysis of transgenic mice expressing familial amyotrophic lateral sclerosis (ALS)-linked mutations in the enzyme superoxide dismutase (SOD1) have shown that motor neuron death arises from a mutant-mediated toxic property or properties. In testing the disease mechanism, both elimination and elevation of wild-type SOD1 were found to have no effect on mutant-mediated disease, which demonstrates that the use of SOD mimetics is unlikely to be an effective therapy and raises the question of whether toxicity arises from superoxide-mediated oxidative stress. Aggregates containing SOD1 were common to disease caused by different mutants, implying that coaggregation of an unidentified essential component or components or aberrant catalysis by misfolded mutants underlies a portion of mutant-mediated toxicity.     

Protein thermostability above 100 degreesC: a key role for ionic interactions

The discovery of hyperthermophilic microorganisms and the analysis of hyperthermostable enzymes has established the fact that multisubunit enzymes can survive for prolonged periods at temperatures above 100 degreesC. We have carried out homology-based modeling and direct structure comparison on the hexameric glutamate dehydrogenases from the hyperthermophiles Pyrococcus furiosus and Thermococcus litoralis whose optimal growth temperatures are 100 degreesC and 88 degreesC, respectively, to determine key stabilizing features. These enzymes, which are 87% homologous, differ 16-fold in thermal stability at 104 degreesC. We observed that an intersubunit ion-pair network was substantially reduced in the less stable enzyme from T. litoralis, and two residues were then altered to restore these interactions. The single mutations both had adverse effects on the thermostability of the protein. However, with both mutations in place, we observed a fourfold improvement of stability at 104 degreesC over the wild-type enzyme. The catalytic properties of the enzymes were unaffected by the mutations. These results suggest that extensive ion-pair networks may provide a general strategy for manipulating enzyme thermostability of multisubunit enzymes. However, this study emphasizes the importance of the exact local environment of a residue in determining its effects on stability.