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Biomedical subjects

D W Green

Publications and source records attributed to D W Green.

At least 55 records · Page 3Linked to original sources

A check on rational drug design: crystal structure of a complex of human immunodeficiency virus type 1 protease with a novel gamma-turn mimetic inhibitor.

We have previously reported (Newlander et al., J. Med. Chem. 1993, 36, 2321-2331) the design of human immunodeficiency virus type 1 (HIV-1) protease inhibitors incorporating C7 mimetics that lock three amino acid residues of a peptide sequence into a gamma-turn. The design of one such compound, SB203238, was based on X-ray structures of reduced amide aspartyl protease inhibitors. It incorporates a gamma-turn mimetic in the P2-P1' position, where the carbonyl of the C7 ring is replaced with an sp3 methylene group yielding a constrained reduced amide. It shows competitive inhibition with Ki = 430 nM at pH 6.0. The three-dimensional structure of SB203238 bound to the active site of HIV-1 protease has been determined at 2.3 A resolution by X-ray diffraction and refined to a crystallographic R-factor (R = sigma magnitude of Fo magnitude of - magnitude of Fc magnitude of /sigma magnitude of Fo magnitude of, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively) of 0.177. The inhibitor lies in an extended conformation in the active site; however, because of the constrained geometry of the C7 ring, it maintains fewer hydrogen bonds with the protein than in most other HIV-1 protease-inhibitor complexes. More importantly, the inhibitor binds to the enzyme differently than predicted in its design, by binding with the P2-P1' alpha-carbon atoms shifted by approximately one-half a residue toward the N-terminus from their presumed positions. This study illustrates the importance of structural information in an approach to rational drug design.

Amino Acid Sequence↗

A numerical and experimental investigation of the flow acceleration region proximal to an orifice.

Attempts to quantify valvular regurgitation have recently been focused on the proximal orifice flow field. A complete description of the proximal orifice flow field is provided in this investigation. A steady state in vitro model accessible by both color Doppler ultrasound (CDU) and laser Doppler velocimetry (LDV) was utilized. Velocities for varying flow rates and orifices were calculated by finite element modeling (FEM), by LDV and by CDU. The steady flow model was composed of circular orifices of 3, 5 and 10 mm diameters at flow rates from 0.7 to 10 L/min. Regurgitant flow rates were calculated from the proximal CDU data by two separate methods. The first approach utilized angle corrected velocities while the second approach utilized only velocities which did not require angle correction (centerline velocities). Both methods correlated well with known flow rates (y = 0.97x -0.09, r = 0.98, SEE = 0.45, p < 0.0001; and y = 1.0x + 0.07, r = 0.99, SEE = 0.27, p < 0.0001, respectively) and were superior to results obtained by assuming a hemispherical geometry as is done in the aliasing technique. The methodology provides a complete analysis of the proximal flow field and involves fewer geometric assumptions than the aliasing approach. This may prove to be an advantage when analyzing in vivo flow fields with complex, uncertain geometry.

Acceleration↗

Nucleated erythrocyte count in newborn infants with left-sided congenital diaphragmatic hernia: relationship with the need for extracorporeal membrane oxygenation and survival.

Inadequate cardiac output in fetuses with left-sided congenital diaphragmatic hernia may cause chronic hypoxia and increased erythropoiesis. Postnatal nucleated erythrocyte counts were measured in 28 newborn infants with left-sided congenital diaphragmatic hernia who were eligible for extracorporeal membrane oxygenation (ECMO). Nucleated erythrocyte counts were lowest in infants who survived without ECMO, intermediate in survivors requiring ECMO, and highest in infants who died despite ECMO.

Analysis of Variance↗

Decreased platelet counts in infants of diabetic mothers.

Infants of diabetic mothers (IDMs) have hematologic indices consistent with increased fetal erythropoiesis, presumably in response to chronic intrauterine hypoxemia. We hypothesized that increased erythropoiesis, as is evident from increased nucleated erythrocyte counts, would be accompanied by interrelated changes in thrombopoiesis and would correlate with maternal glycemic control during pregnancy. We compared absolute nucleated erythrocyte counts and platelet counts obtained in the first 24 hours of life in 32 term, nonasphyxiated IDMs with 47 controls. The IDM group had higher absolute nucleated erythrocyte counts (1.0 +/- 1.3 x 10(9)/L versus 0.4 +/- 0.7 x 10(9)/L; p < 0.05), and lower platelet counts (235 +/- 77 x 10(9)/L versus 348 +/- 79 x 10(9)/L; p < 0.001) than controls. Absolute nucleated erythrocyte counts correlated inversely with platelet counts (r = -0.28; p < 0.02). These neonatal hematologic measurements did not correlate with various parameters of maternal glycemic control. We conclude that in IDMs, increased erythropoiesis is accompanied by decreased platelet counts. These data are consistent with the theory of an erythropoietin-induced shift of fetal multipotent stem cell differentiation toward erythropoiesis at the expense of thrombopoiesis.

Diabetes Mellitus, Type 1↗

Mivacurium-induced prolonged neuromuscular block.

We report a case of prolonged neuromuscular block after administration of mivacurium 0.2 mg kg-1 to a 16-yr-old patient where the duration of block was 2.5 h. The interesting points in this case were that the patient had homozygous atypical plasma cholinesterase deficiency (both parents had a normal phenotype) following liver transplantation. Investigations showed low plasma cholinesterase activity (343 iu litre-1; normal 600-1400) and dibucaine number was 25 (normal 76-83). Despite possessing atypical enzyme normally associated with markedly prolonged duration of suxamethonium, on two occasions the patient received suxamethonium and responded normally. This had not previously been reported. The patient demonstrated prolonged block with mivacurium as a result of atypical enzyme (despite normal metabolism of suxamethonium).

Adolescent↗

Cognitive structural models: the perception of risk and prevention in coronary heart disease.

This paper examines the relationship between causal models and the assessment of actions in a naturalistic domain. Two studies used a novel network diagram technique to elicit individual causal models of the factors affecting the risk of coronary heart disease. The same individuals also rated the effectiveness of different preventive actions. In their diagrams, individuals depicted the causal interrelationships between various factors and estimated the strength of the connecting paths. Total path strength accounted for two-thirds of the variance in ratings of preventive actions. A number of methodological issues were also explored, which supported the utility of the technique. Consensual models allowed a ready comparison between groups of subjects and showed the importance of distinguishing the presence of paths from the strengths of those paths. The studies support the value of the technique in analysing individual differences in the way people represent complex causal structures; various extensions and applications are also proposed.

Cognition↗

Nucleated erythrocytes and intraventricular hemorrhage in preterm neonates.

OBJECTIVES: Increased circulating nucleated erythrocytes (NRBCs) in the newborn period can be markers of chronic fetal hypoxia, which in turn may be a risk factor for intracranial hemorrhage (IVH). To evaluate the relation between chronic intrauterine hypoxia and IVH, we compared the courses of the absolute NRBC (ANRBC) count in preterm newborns with and without intracranial hemorrhage. METHODS: We measured ANRBC counts in the first 6 days of life in appropriate for gestational age newborns at 32 weeks' gestation or earlier with (n = 46) and without (n = 103) IVH, who were not at risk for altered erythropoiesis. RESULTS: The ANRBC counts at birth were higher in infants who developed severe IVH than in control infants without IVH (P < .03). The ANRBC counts peaked on day 2 or 3 in newborns with IVH, but declined continuously from a peak on day 1 in the control group. Stepwise regression analysis of multiple variables revealed that the grade of IVH had the greatest impact on ANRBC counts. An ANRBC count of at least 2.0 x 10(9)/L on day 1 of life had a sensitivity of 63% and a specificity of 79% in predicting grade III or IV IVH: CONCLUSION: An elevated or increasing ANRBC count in a preterm newborn is a potential marker for an impending or present severe IVH, respectively, and may reflect a state of altered prenatal or postnatal erythropoiesis.

Biomarkers↗

An orally bioavailable HIV-1 protease inhibitor containing an imidazole-derived peptide bond replacement: crystallographic and pharmacokinetic analysis.

(2R,4S,5S,1'S)-2-Phenylmethyl-4-hydroxy-5-(tert-butoxycarbonyl) amino-6-phenylhexanoyl-N-(1'-imidazo-2-yl)-2'-methylpropanamide (compound 2) is a tripeptide analogue inhibitor of HIV-1 protease in which a C-terminal imidazole substituent constitutes an isoelectronic, structural mimic of a carboxamide group. Compound 2 is a potent inhibitor of the protease (K(i) = 18 nM) and inhibits HIV-1 acute infectivity of CD4+ T-lymphocytes (IC50 = 570 nM). Crystallographic analysis of an HIV-1 protease-compound 2 complex demonstrates that the nitrogen atoms of the imidazole ring assume the same hydrogen-bonding interactions with the protease as amide linkages in other peptide analogue inhibitors. The sole substitution of the C-terminal carboxamide of a hydroxyethylene-containing tripeptide analogue with an imidazole group imparts greatly improved pharmacokinetic and oral bioavailability properties on the compound compared to its carboxamide-containing homologue (compound 1). While the oral bioavailability of compound 1 in rats was negligible, compound 2 displayed oral bioavailabilities of 30% and 14%, respectively, in rats and monkeys.

Administration, Oral↗

Rational design, synthesis, and crystallographic analysis of a hydroxyethylene-based HIV-1 protease inhibitor containing a heterocyclic P1'--P2' amide bond isostere.

The rational design and synthesis of a highly potent inhibitor of HIV-1 protease have been accomplished. The inhibitor, SB 206343, is based on a model derived from the structure of the MVT-101/HIV-1 protease complex and contains a 4(5)-acylimidazole ring as an isosteric replacement for the P1'--P2' amide bond. It is a competitive inhibitor with an apparent inhibition constant of 0.6 nM at pH 6.0. The three-dimensional structure of SB 206343 bound in the active site of HIV-1 protease has been determined at 2.3 A resolution by X-ray diffraction techniques and refined to a crystallographic discrepancy factor, R (= sigma parallel Fo magnitude of/Fc parallel/sigma magnitude of), of 0.194. The inhibitor is held in the enzyme by a set of hydrophobic and polar interactions. N-3 of the imidazole ring participates in a novel hydrogen-bonding interaction with the bound water molecule, demonstrating the effectiveness of the imidazole ring as an isosteric replacement for the P1'--P2' amide bond in hydroxyethylene-based HIV-1 protease inhibitors. Also present are hydrogen-bonding interactions between N-1 of the imidazole ring and the carbonyl of Gly-127 as well as between the imidazole acyl carbonyl oxygen and the amide nitrogen of Asp-129, exemplifying the peptidomimetic nature of the 4(5)-acylimidazole isostere. All of these interactions are in qualitative agreement with those predicted by the model.

Amides↗

Inversion of the substrate specificity of yeast alcohol dehydrogenase.

The relationship between the size of the substrate binding pocket and the catalytic reactivities with varied alcohols was studied with the Saccharomyces cerevisiae alcohol dehydrogenase I (ScADH) and compared with the liver enzymes from horse (EqADH, EE isoenzyme) and monkey (MmADH alpha, alpha-isoenzyme). The yeast enzyme is most active with ethanol, and its activity decreases as the size of the alcohol is increased, whereas the activities of the liver enzymes increase with larger alcohols. The substrate pocket in ScADH was enlarged by single substitutions of Thr-48 to Ser (T48S), Trp-57 to Met (W57M), and Trp-93 to Ala (W93A), and a double change, T48S:W93A, and a triple, T48S:W57M:W93A. The T48S enzyme has the same pattern of activity (V/K) as wild-type ScADH for linear primary alcohols. The W57M enzymes have lowered reactivity with primary and secondary alcohols. The W93A and T48S:W93A enzymes resemble MmADH alpha in having an inverted specificity pattern for primary alcohols, being 3- and 10-fold more active on hexanol and 350- and 540-fold less active on ethanol, and are as reactive as the liver enzymes with long chain primary alcohols. The three Ala-93 enzymes also acquired weak activity on branched chain alcohols and cyclohexanol.

Aldehyde Dehydrogenase↗

Biochemical characteristics and cellular regulation of phosphodiesterase IV.

Considerable interest has been generated in the potential utility of phosphodiesterase (PDE) IV inhibitors as a novel class of anti-asthmatic agents. Because a detailed understanding of the molecular and biochemical characteristics of any molecular target of interest provides a key ingredient for rational drug design, we cloned a cDNA encoding a PDE IV (hPDE IV) from a human monocyte library and expressed, purified and characterized the recombinant gene product. Purified hPDE IV has kinetic characteristics consistent with native PDE IV isolated from tissue sources. In addition, it is inhibited by rolipram (Ki = 60 nM) and other archetypical PDE IV-selective inhibitors. Purified hPDE IV also contains a high affinity binding site for rolipram (Kd = 2 nM), although the precise relationship between this site and the cAMP catalytic site is not clear. In other studies in which the regulation of PDE IV expression was examined in U937 cells, a human monocytic cell line, prolonged treatment with salbutamol was shown to induce an increase in the activity of PDE IV. This up-regulation of PDE IV activity appears to be mediated by cAMP and occurs at the transcriptional or pretranscriptional level. As a consequence of PDE IV up-regulation, the sensitivity of U937 cells to the inhibitory effects of adenylyl cyclase activators on cell function is greatly diminished. If such regulation of PDE IV occurs in inflammatory cells in vivo, it could have implications for the therapeutic use of beta-adrenoceptor agonists. Specifically, induction of PDE IV activity in asthmatics being treated with beta-adrenoceptor agonists could result in a heterologous desensitization of inflammatory cells to endogenous anti-inflammatory agents (e.g., epinephrine, prostaglandin E2).

3',5'-Cyclic-AMP Phosphodiesterases↗

Efficacy of postoperative pain treatment regimens using both buprenorphine and papaveretum sequentially after abdominal hysterectomy.

We have undertaken a double-blind, controlled study to test the hypothesis that the efficacy of standard postoperative analgesia by papaveretum or buprenorphine is not compromised by previous or subsequent standard doses of the other agent. After total abdominal hysterectomy under a standardized general anaesthetic, 120 patients (four groups of 30) were allocated randomly to receive, on demand, a single i.v. dose of buprenorphine 0.15 mg or papaveretum 10 mg, followed sequentially by a single dose of i.m. buprenorphine 0.3 mg or papaveretum 20 mg. Three hours after the i.m. dose, all patients received sublingual buprenorphine 0.4 mg. Pain was scored using both a 10-cm horizontal visual analogue scale (VAS) and a nominal scale. Pain intensity differences and patient and nurse satisfaction with the regimens were recorded. Observations were continued for 8 h after operation. The efficacy of papaveretum or buprenorphine was not compromised by previous or subsequent standard doses of the other agent. All four treatment regimens were similarly well tolerated and gave acceptable analgesia in the immediate postoperative period.

Adolescent↗

Alpha-isoenzyme of alcohol dehydrogenase from monkey liver. Cloning, expression, mechanism, coenzyme, and substrate specificity.

The cDNA for the alpha-isoenzyme from rhesus monkey (Macaca mulatta) liver was cloned and expressed in yeast. The alpha-isoenzymes of human and monkey liver alcohol dehydrogenase differ from the other human and horse liver enzymes in having Met57, Ala93, and Val116 instead of Leu57, Phe93, and Leu116 in the substrate binding pocket and Gly47 instead of Arg47 near the pyrophosphate moiety of the coenzyme. The effects of these differences on the kinetic mechanism, substrate specificity, and coenzyme binding were studied with the purified, recombinant monkey alpha-isoenzyme (MmADH alpha) and mutated enzymes with Gly47 substituted with His or Arg. The mechanism appears to be random for the binding of NAD+ and ethanol and ordered for NADH and acetaldehyde, with formation of a dead-end enzyme-NADH-ethanol complex. MmADH alpha reacts 130-fold slower (V/K) with ethanol and 3-25-fold slower with 2-methyl alcohols but 20-fold faster with cyclohexanol, as compared with horse (Equus caballus) liver EE isoenzyme (EqADH). MmADH alpha is stereoselective for the R isomer of 2-butanol, whereas EqADH favors the S isomer. Both enzymes have comparable reactivity with larger primary alcohols. MmADH alpha is more reactive with secondary alcohols and has highest activity with cyclohexanol. However, it does not react with steroids such as 5 beta-androstane-17 beta-ol-3-one. Molecular modeling suggests that the differences between MmADH alpha and EqADH are a result of the substitution of Ala for Phe93 and Thr for Ser48. MmADH alpha binds NAD+ most rapidly when a group with a pK of 7.4 is unprotonated, implicating His51 in this reaction. The G47R substitution decreased the dissociation constants for NAD+ and NADH and turnover numbers only about 2-fold, whereas the G47H substitution increased dissociation constants 7-14-fold and turnover numbers 4-fold. A basic residue at position 47 is not crucial for activity, as multiple interactions determine coenzyme affinity.

Alcohol Dehydrogenase↗

Neonatal hematocrit and maternal glycemic control in insulin-dependent diabetes.

We hypothesized that in infants of diabetic women neonatal hematocrit would correlate with third-trimester maternal glycemic control. Neonatal hematocrit correlated with the maternal total glycosylated hemoglobin level at delivery (r = 0.36; p less than 0.05) but not at 36 weeks of gestation in 32 mother-infant pairs. Improved maternal glycemic control during late gestation may decrease the incidence of neonatal polycythemia.

Blood Glucose↗

Dissolution of ionizable drugs into unbuffered solution: a comprehensive model for mass transport and reaction in the rotating disk geometry.

A model has been developed to describe the mass transport and reaction of ionizable compounds where mass transfer is caused by convection and diffusion from a rotating disk. Dissolution rates of benzoic acid, 2-naphthoic acid, and indomethacin in aqueous solutions of high ionic strength (I = 0.5 with potassium chloride) at 25 degrees C were investigated. The model includes the effects of diffusion, convection, and simultaneous acid/base reaction at all points in the region adjacent to the dissolving solid. The solution of the transport equations is obtained numerically with an iterative algorithm which uses (a) closure of all material balances and (b) equilibria at the solid/liquid surface as constraints. The model solution yields both the flux of the dissolving acid and the concentration profile of each component. Reduced values of all reaction rate constants are used in the region adjacent to the dissolving surface to allow convergence of the computation. Although nonequilibrium concentration values are calculated, it is shown that the theoretical dissolution rate determined as the solution of the model is insensitive to the magnitude of the rate constants as their maximum useable values are approached. Comparisons of the model results with experimentally determined fluxes show close agreement and confirm that the transport mechanisms in the model formulation are consistent with the measured values. Further, the inclusion of convection allows accurate calculations without utilization of an arbitrary boundary layer thickness. Accurate dissolution rates can be determined using this technique under a wide range of conditions, except at low pH.

Acids↗