3-Arylpiperidines as potentiators of existing antibacterial agents.

Important resistance patterns in Gram-negative pathogens include active efflux of antibiotics out of the cell via a cellular pump and decreased membrane permeability. A 3-arylpiperidine derivative (1) has been identified by high-throughput assay as a potentiator with an IC(50) approximately 90 microM. This report details the evaluation of the tether length, aryl substitution and the importance of the fluorine on antibiotic accumulation. Evaluation of various tether lengths demonstrated that the two-carbon tethered analogues are optimal. Removal of the fluorine has a modest effect on antibiotic accumulation and the defluorinated analogue 17 is equally potent to the original lead 1.

Identification of novel potent hydroxamic acid inhibitors of peptidyl deformylase and the importance of the hydroxamic acid functionality on inhibition.

Peptidyl deformylase (PDF) is a metallo protease that catalyzes the removal of a formyl group from the N-termini of prokaryotic prepared polypeptides, an essential step in bacterial protein synthesis. Screening of our compound collection using Staphylococcus aureus PDF afforded a very potent inhibitor with an IC(50) in the low nanomolar range. Unfortunately, the compound that contains a hydroxamic acid did not exhibit antibacterial activity (MIC). In order to address the lack of activity in the MIC assay and to determine what portion of the molecule was responsible for binding to PDF, we prepared several analogues. This paper describes our findings that the hydroxamic acid functionality found in 1 is mainly responsible for the high affinity to PDF. In addition, we identified an alternative class of PDF inhibitors, the N-hydroxy urea 18, which has both PDF and antibacterial activity.

The ligand affinity of proteins measured by isothermal denaturation kinetics.

An isothermal denaturation kinetic method was developed for identifying potential ligands of proteins and measuring their affinity. The method is suitable for finding ligands specific toward proteins of unknown function and for large-scale drug screening. It consists of analyzing the kinetics of isothermal denaturation of the protein-with and without the presence of potential specific ligands-as measured by long-wavelength fluorescent dyes whose quantum yield increases when bound to hydrophobic regions exposed upon unfolding of the proteins. The experimental procedure was developed using thymidylate kinase and stromelysin as target proteins. The kinetics of thermal unfolding of both of these enzymes were consistent with a pathway of two consecutive first-order rate-limiting steps. Reflecting the stabilizing effect of protein/ligand complexes, the presence of specific ligands decreased the value of the rate constants of both steps in a dose-dependent manner. The dependence of the rate constants on ligand concentration obeyed a simple binding isotherm, the analysis of which yielded an accurate equilibrium constant for ligand binding. The method was validated by comparing its results with those obtained under the same conditions by steady-state fluorescence spectroscopy, circular dichroism, and uv spectrophotometry: The corresponding rate constants were comparable for each of the analytical detection methods.

Emotion regulation as a mediator of associations between mother-child attachment and peer relationships in middle childhood.

Although a link between attachment and peer relationships has been established, the mechanisms that account for this link have not been identified. The 1st goal of this study was to test emotion regulation as a mediator of this link in middle childhood. The 2nd goal was to examine how different aspects of emotion regulation relate to peer competence. Fifth graders completed self-report and semiprojective measures to index mother-child attachment, mothers reported on children's emotionality and coping strategies, and teachers reported on children's peer competence. Constructive coping was related to both attachment and peer competence, and mediated the association between attachment and peer competence, suggesting that emotion regulation is one of the mechanisms accounting for attachment-peer links. Constructive coping was more strongly associated with peer competence for children high on negative emotionality than for children low on negative emotionality.

Calciuria in symptom-free primigravid women remote from term: is the response to an oral calcium challenge predictable?

OBJECTIVE: This study was undertaken to compare the calciuric response in symptom-free primigravid women to an oral calcium load between those with normal urinary calcium excretions and those with relatively low urinary calcium excretions. STUDY DESIGN: This was a prospective clinical trial. Eligible primigravid women between 16 and 20 weeks' gestation provided a 24-hour urine sample for determination of urinary calcium/urinary creatinine ratio. On the basis of these results the patients were divided into 2 groups: a relatively hypocalciuric group, in which the urinary calcium excretion was 3.4 mg. kg-1. 24 h-1. All participants undertook a 3-day low calcium dietary regimen. On the fourth day women underwent an oral calcium challenge. A 2-hour urine sample was collected before ingestion of 1 g calcium carbonate (preload). One hour after ingestion the women again collected a 2-hour urine sample (postload). The urinary calcium/urinary creatinine ratios in the preload and postload samples were determined and compared within and between the groups. RESULTS: The mean change (+/-SD) between the preload and postload urinary calcium/urinary creatinine ratios in the relatively hypocalciuric group was 0.60 +/- 1.44 (P =.04); that in the normocalciuric group was 3.09 +/- 2.26 (P =.11 ). There was a 5-fold difference in the response to calcium load between the hypocalciuric women and the normocalciuric women (0.60 vs 3.09), although this difference was not statistically significant (P =.20). CONCLUSIONS: Both hypocalciuric and normocalciuric women responded to an oral calcium challenge by an increase in the calcium excretion. The cause of the hypocalciuria in women at increased risk for preeclampsia is therefore not simply poor absorption of calcium.

Tipranavir (PNU-140690): a potent, orally bioavailable nonpeptidic HIV protease inhibitor of the 5,6-dihydro-4-hydroxy-2-pyrone sulfonamide class.

A broad screening program previously identified phenprocoumon (1) as a small molecule template for inhibition of HIV protease. Subsequent modification of this lead through iterative cycles of structure-based design led to the activity enhancements of pyrone and dihydropyrone ring systems (II and V) and amide-based substitution (III). Incorporation of sulfonamide substitution within the dihydropyrone template provided a series of highly potent HIV protease inhibitors, with structure-activity relationships described in this paper. Crystallographic studies provided further information on important binding interactions responsible for high enzymatic binding. These studies culminated in compound VI, which inhibits HIV protease with a Ki value of 8 pM and shows an IC90 value of 100 nM in antiviral cell culture. Clinical trials of this compound (PNU-140690, Tipranavir) for treatment of HIV infection are currently underway.

The psychosocial and physiological experiences of patients with an implantable cardioverter defibrillator.

This article describes the relationships between anxiety, self-efficacy, as well as the psychosocial and physiological experiences of patients with an internal cardioverter defibrillator (ICD). Although survival rates with ICDs are impressive, patients experience psychological and physiological responses to their lifesaving devices. Thirty-nine patients completed questionnaires during outpatient clinic visits. Patients' anxiety levels were correlated with fears about ICD malfunction, fear of death, fear of being shocked, loss of control, trouble related to sleeping, inability to concentrate, overprotective family members, and depression. Patients with physical symptoms had higher anxiety levels than those without physical symptoms. There was not a significant relationship between anxiety and discomfort associated with discharge, the number of discharges, or ejection fractions. Low self-efficacy was significantly related to fears about ICD malfunction as well as patients' inability to work, engage in hobbies, and drive a car. Patients with low self-efficacy had more physical symptoms and lower ejection fractions. There were no significant relationships between self-efficacy and frequency of ICD discharge or patients' discomfort during ICD discharge.

Non-peptidic HIV protease inhibitors: C2-symmetry-based design of bis-sulfonamide dihydropyrones.

Potent, non-peptidic, dihydropyrone sulfonamide HIV protease inhibitors have been previously described. Crystallographic analysis of dihydropyrone sulfonamide inhibitor/HIV protease complexes suggested incorporation of a second, C2 symmetry-related sulfonamide group. Selected bis-sulfonamide dihydropyrone analogues display high HIV protease inhibitory activity.

Trauma in pregnancy: normal Revised Trauma Score in relation to other markers of maternofetal status--a preliminary study.

OBJECTIVE: Our goal was to examine whether a correlation exists between the Revised Trauma Score assigned on admission and pregnancy outcome, as well as whether the Revised Trauma Score has any predictive value for optimal duration of cardiotocographic monitoring necessary to detect immediate adverse pregnancy outcome. STUDY DESIGN: A retrospective chart review was performed of 30 pregnant trauma patients admitted during a 1-year period. Evaluation of cardiotocographic data for either contractions or decelerations or both was performed without knowledge of Revised Trauma Score or maternofetal outcome at discharge. RESULTS: Review of uterine activity and fetal decelerations did not detect useful predictive patterns unless the tracing was immediately ominous, although uterine activity did initially decrease over time. CONCLUSIONS: The Revised Trauma Score lacks predictive value for both risk of adverse pregnancy outcome and need for prolonged cardiotocographic monitoring. A larger patient population needs to be studied for an accurate determination of whether the Revised Trauma Score has potential as a predictive tool.

Structure-based design of nonpeptidic HIV protease inhibitors: the sulfonamide-substituted cyclooctylpyramones.

Recently, cyclooctylpyranone derivatives with m-carboxamide substituents (e.g. 2c) were identified as potent, nonpeptidic HIV protease inhibitors, but these compounds lacked significant antiviral activity in cell culture. Substitution of a sulfonamide group at the meta position, however, produces compounds with excellent HIV protease binding affinity and antiviral activity. Guided by an iterative structure-based drug design process, we have prepared and evaluated a number of these derivatives, which are readily available via a seven-step synthesis. A few of the most potent compounds were further evaluated for such characteristics as pharmacokinetics and toxicity in rats and dogs. From this work, the p-cyanophenyl sulfonamide derivative 35k emerged as a promising inhibitor, was selected for further development, and entered phase I clinical trials.

Structure-based design of HIV protease inhibitors: 5,6-dihydro-4-hydroxy-2-pyrones as effective, nonpeptidic inhibitors.

From a broad screening program, the 4-hydroxycoumarin phenprocoumon (I) was previously identified as a lead template with HIV protease inhibitory activity. The crystal structure of phenprocoumon/HIV protease complex initiated a structure-based design effort that initially identified the 4-hydroxy-2-pyrone U-96988 (II) as a first-generation clinical candidate for the potential treatment of HIV infection. Based upon the crystal structure of the 4-hydroxy-2-pyrone III/HIV protease complex, a series of analogues incorporating a 5,6-dihydro-4-hydroxy-2-pyrone template were studied. It was recognized that in addition to having the required pharmacophore (the 4-hydroxy group with hydrogen-bonding interaction with the two catalytic aspartic acid residues and the lactone moiety replacing the ubiquitous water molecule in the active site), these 5,6-dihydro-4-hydroxy-2-pyrones incorporated side chains at the C-6 position that appropriately extended into the S1' and S2' subsites of the enzyme active site. The crystal structures of a number of representative 5,6-dihydro-4-hydroxy-2-pyrones complexed with the HIV protease were also determined to provide better understanding of the interaction between the enzyme and these inhibitors to aid the structure-based drug design effort. The crystal structures of the ligands in the enzyme active site did not always agree with the conformations expected from experience with previous pyrone inhibitors. This is likely due to the increased flexibility of the dihydropyrone ring. From this study, compound XIX exhibited reasonably high enzyme inhibitory activity (Ki = 15 nM) and showed antiviral activity (IC50 = 5 microM) in the cell-culture assay. This result provided a research direction which led to the discovery of active 5,6-dihydro-4-hydroxy-2-pyrones as potential agents for the treatment of HIV infection.

Effect of chronic tocolytic therapy on maternal ventricular function in pregnant rabbits.

OBJECTIVE: Our purpose was to determine whether peripartum cardiomyopathy may be associated with chronic beta-mimetic tocolytic therapy. STUDY DESIGN: On gestational day 20 (term 31 days), two 200 microliter Alzet miniosmotic pumps were implanted in the subcutaneous tissue of pregnant New Zealand White rabbits. Each pump was filled with terbutaline (20 micrograms/microliter, n = 7) or saline solution (0.9%, n = 7) and infused continuously for 7 days. The rabbits were killed on the twenty-eighth gestational day. Maternal hearts were placed on a Langendorff (nonejecting) perfusion apparatus for assessment of cardiac function. At a constant perfusion pressure and heart rate left ventricular diastolic pressure was varied while left ventricular developed pressure and left ventricular +/- rate of pressure rise, index values of left ventricular contractility and relaxation, were continuously recorded. Comparisons between groups at each preload were made by analysis of variance. RESULTS: Hearts taken from terbutaline-treated rabbits exhibited periodic arrhythmias and mechanical alternans in five of seven hearts versus one of seven in the saline solution group. At a preload of 0 mm Hg both left ventricular developed pressure (88.0 vs 48.4 mm Hg, p < 0.001) and left ventricular rate of pressure rise (1406 vs 653 mm Hg/sec, p < 0.001) were less in terbutaline-treated rabbits. At a preload of 10 mm Hg left ventricular developed pressure (104.4 vs 56.7 mm Hg, p < 0.01) and rate of pressure rise (1424 vs 694 mm Hg/sec, p < 0.001) were also significantly less in terbutaline-treated rabbits. Left ventricular relaxation was also impaired at all preloads. CONCLUSIONS: In this model chronic administration of terbutaline during late pregnancy significantly depresses global maternal cardiac function.

Effect of dual tocolysis on the incidence of severe intraventricular hemorrhage among extremely low-birth-weight infants.

OBJECTIVE: Our purpose was to evaluate the null hypothesis that dual tocolysis with magnesium sulfate and indomethacin does not alter the rate of grade III or IV intraventricular hemorrhage. STUDY DESIGN: Fifty-six neonates weighing 500 to 800 gm from mothers who received tocolytic therapy with magnesium sulfate alone or in combination with indomethacin were the subjects of this retrospective study. Demographic variables were evaluated with a Student t test, chi(2) analysis, Fisher exact test, or Mantel-Haenszel chi(2) as appropriate. RESULTS: There was an increased incidence of grade III to IV intraventricular hemorrhage among patients treated with dual therapy (p = 0.02). Logistic regression showed that fetal age and dual tocolysis with indomethacin were the only independent prognostic factors for severe intraventricular hemorrhage. CONCLUSION: The results indicate that dual tocolysis with indomethacin may place extremely low-birth-weight infants at increased risk for grade III to IV intraventricular hemorrhage.

Cycloalkylpyranones and cycloalkyldihydropyrones as HIV protease inhibitors: exploring the impact of ring size on structure-activity relationships.

Previously, 3-substituted cycloalkylpyranones, such as 2d, have proven to be effective inhibitors of HIV protease. In an initial series of 3-(1-phenylpropyl) derivatives with various cycloalkyl ring sizes, the cyclooctyl analog was the most potent. We became interested in exploring the influence of other structural changes, such as substitution on the phenyl ring and saturation of the 5,6-double bond, on the cycloalkyl ring size structure-activity relationship (SAR). Saturation of the 5,6-double bond in the pyrone ring significantly impacts the SAR, altering the optimal ring size from eight to six. Substitution of a sulfonamide at the meta position of the phenyl ring dramatically increases the potency of these inhibitors, but it does not change the optimal ring size in either the cycloalkylpyranone or the cycloalkyldihydropyrone series. This work has led to the identification of compounds with superb binding affinity for the HIV protease (Ki values in the 10-50 pM range). In addition, the cycloalkyldihydropyrones showed excellent antiviral activity in cell culture, with ED50 values as low as 1 microM.

The ethics of caring for conjoined twins. The Lakeberg twins.

In June 1993, conjoined twin Amy and Angela Lakeberg became the focus of national attention. They shared a complex six-chambered heart and one liver; only one could survive separation surgery; and even her chances were slim. The medical challenge was great and the ethical challenges were even greater.

Structure-based design of novel HIV protease inhibitors: sulfonamide-containing 4-hydroxycoumarins and 4-hydroxy-2-pyrones as potent non-peptidic inhibitors.

The low oral bioavailability and rapid biliary excretion of peptide-derived HIV protease inhibitors have limited their utility as potential therapeutic agents. Our broad screening program to discover non-peptidic HIV protease inhibitors previously identified compound I (phenprocoumon, Ki = 1 microM) as a lead template. Structure-based design of potent non-peptidic inhibitors, utilizing crystal structures of HIV protease/inhibitor complexes, provided a rational basis for the previously reported carboxamide-containing 4-hydroxycoumarins and 4-hydroxy-2-pyrones. The amino acid containing compound V (Ki = 4 nM) provided an example of a promising new series of HIV protease inhibitors with significantly improved enzymatic binding affinity. In this report, further structure-activity relationship studies, in which the carboxamide is replaced by a sulfonamide functionality, led to the identification of another series of nonamino acid containing promising inhibitors with significantly enhanced enzyme binding affinity and in vitro antiviral activity. The most active diastereomer of the sulfonamide-containing pyrone XVIII (Ki = 0.5 nM) shows improved antiviral activity (IC50 = 0.6 nM) and represents an example of a new design direction for the discovery of more potent non-peptidic HIV protease inhibitors as potential therapeutic agents for the treatment of HIV infection.

Etiology and outcome of extremely low-birth-weight infants.

OBJECTIVE: Our purpose was to determine whether the reason for delivery of extremely low-birth-weight infants influenced the immediate neonatal outcome. STUDY DESIGN: At a regional perinatal center a retrospective analysis of 111 neonates with birth weights between 500 and 800 gm and their respective mothers was performed. The mother-infant pairs were grouped according to the reason for delivery. Group 1 included those with idiopathic preterm labor. Group 2 included mothers with preterm rupture of membranes. Group 3 included those delivered for maternal or fetal indications. Group 4 included all multiple gestations. Maternal, intrapartum, and neonatal outcome variables were then evaluated for statistical significance by analysis of variance and chi2 methods and a p value of 0.05. RESULTS: The neonatal outcome variables (survival and incidence of major intraventricular hemorrhage, hyaline membrane disease, and fetal sepsis) were not found to be significantly different among the four groups tested. CONCLUSION: The reason for the delivery of extremely low-birth-weight infants does not have an impact on the immediate neonatal outcome in these neonates.