Synthesis of 5,6-dihydro-4-hydroxy-2-pyrones as HIV-1 protease inhibitors: the profound effect of polarity on antiviral activity.
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Animals↗
Biomedical subjects
D Ferguson
Publications and source records attributed to D Ferguson.
4-hydroxy-5,6-dihydropyrones. 2. Potent non-peptide inhibitors of HIV protease.
The 4-hydroxy-5,6-dihydropyrone template was utilized as a flexible scaffolding from which to build potent active site inhibitors of HIV protease. Dihydropyrone 1c (5,6-dihydro-4-hydroxy-6-phenyl-3-[(2-phenylethyl)thio]-2H-pyran-2-one) was modeled in the active site of HIV protease utilizing a similar binding mode found for the previously reported 4-hydroxybenzopyran-2-ones. Our model led us to pursue the synthesis of 6,6-disubstituted dihydropyrones with the aim of filling S1 and S2 and thereby increasing the potency of the parent dihydropyrone 1c which did not fill S2. Toward this end we attached various hydrophobic and hydrophilic side chains at the 6-position of the dihydropyrone to mimic the natural and unnatural amino acids known to be effective substrates at P2 and P2'. Parent dihydropyrone 1c (IC50 = 2100 nM) was elaborated into compounds with greater than a 100-fold increase in potency [18c, IC50 = 5 nM, 5-(3,6-dihydro-4-hydroxy-6-oxo-2-phenyl-5-[2-phenylethyl)thio] -2H-pyran-2-yl)pentanoic acid and 12c, IC50 = 51 nM, 5,6-dihydro-4-hydroxy-6-phenyl-6-(2-phenylethyl)-3- [(2-phenyl-ethyl)thio]-2H-pyran-2-one]. Optimization of the 3-position fragment to fill S1' and S2' afforded potent HIV protease inhibitor 49 [IC50 = 10 nM, 3-[(2-tert-butyl-5-methylphenyl)sulfanyl]-5,6-dihydro-4 -hydroxy-6-phenyl-6-(2-phenylethyl)-2H-pyran-2-one]. The resulting low molecular weight compounds (< 475) have one or no chiral centers and are readily synthesized.
Anti-HIV Agents↗
Logistic analysis of shovel and Carabelli's tooth traits in a Caucasoid population.
Caucasoid populations differ from Mongoloids by having a high prevalence of Carabelli's trait and a low prevalence of shovel trait. This study was conducted to investigate the association between the shovel and the Carabelli's traits in a Caucasoid population. The research design sought a Caucasoid population at Milwaukee of Wisconsin in United States. The Caucasoid group selected for study was the European descendant. The effects of sex and age on Carabelli's trait were controlled in this investigation, as was the association between tooth size and Carabelli's trait. Results show that males were more likely to have Carabelli's trait expressed on teeth than females. The buccolingual diameter of Carabelli's trait teeth was larger than that of teeth without the trait. After adjusting for sex, age, and tooth size, the existence of the shovel trait decreased the likelihood of having Carabelli's trait which shows an significant effect.
Asian People↗
Phosphorylation and subcellular redistribution of pleckstrin in human neutrophils.
Pleckstrin, originally described as a major substrate of protein kinase C (PKC) in platelets, was found to be highly expressed in human neutrophils (intracellular concentration, approximately 15 microM). As PKC isoforms play an important role in mediating neutrophil antimicrobial responses, we studied the regulation of pleckstrin phosphorylation in response to inflammatory stimuli. Following treatment of neutrophils with FMLP, 12-O-tetradecanoylphorbol-13-acetate, or opsonized zymosan, pleckstrin was rapidly phosphorylated, which resulted in a shift in its electrophoretic mobility. Several lines of evidence suggest that pleckstrin is phosphorylated in part by a nonconventional PKC following stimulation by FMLP: 1) chelation of intracellular Ca2+ had only a partial inhibitory effect; 2) diacylglycerol kinase inhibitors shortened the duration of phosphorylation, while the phosphatidic acid phosphohydrolase antagonist propranolol extended it; and 3) wortmannin and erbstatin blocked the phosphorylation of pleckstrin. These results suggest that nonconventional PKC isoforms, possibly delta or zeta, mediate the phosphorylation of pleckstrin. Both PKCdelta and -zeta are expressed in human neutrophils. Increased association of pleckstrin with both microsomes and with the cytoskeleton was observed in stimulated cells. These findings suggest that phosphorylation by nonconventional PKC isoforms induces a conformational change in pleckstrin that promotes its interaction with membranes and/or with the cytoskeleton. Such a translocation may serve to target proteins or lipids recognized by pleckstrin homology domains to sites where they can contribute to the microbicidal response.
Androstadienes↗
Functional expression and apical localization of the cystic fibrosis transmembrane conductance regulator in MDCK I cells.
The gene product affected in cystic fibrosis, the cystic fibrosis transmembrane conductance regulator (CFTR), is a chlorideselective ion channel that is regulated by cAMP-dependent protein kinase-mediated phosphorylation, ATP binding and ATP hydrolysis. Mutations in the CFTR gene may result in cystic fibrosis characterized by severe pathology (e.g. recurrent pulmonary infection, male infertility and pancreatic insufficiency) involving organs expressing the CFTR. Interestingly, in the kidney, where expression of the CFTR has been reported, impaired ion transport in patients suffering from cystic fibrosis could not be observed. To understand the role of the CFTR in chloride transport in the kidney, we attempted to identify an epithelial cell line that can serve as a model. We demonstrate that the CFTR is expressed constitutively in Madine-Darby canine kidney (MDCK) type I cells, which are thought to have originated from the distal tubule of the dog nephron. We show expression at the mRNA level, using reverse transcriptase-PCR, and at the protein level, using Western blot analysis with three different monoclonal antibodies. Iodide efflux measurements indicate that CFTR expression confers a plasma membrane anion conductance that is responsive to stimulation by cAMP. The cAMP-stimulated iodide release is sensitive to glybenclamide, diphenylamine carboxylic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid, but not to 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid, an inhibitor profile characteristic of the CFTR chloride channel. Finally, the polarized localization of the CFTR to the apical plasma membrane was established by iodide efflux measurements and cell-surface biotinylation on MDCK I monolayers. Interestingly, MDCK type II cells, which are thought to have originated from the proximal tubule of the kidney, lack CFTR protein expression and cAMP-stimulated chloride conductance. In conclusion, we propose that MDCK type I and II cells can serve as convenient model systems to study the physiological role and differential expression of CFTR in the distal and proximal tubule respectively.
Animals↗
The effects of pentoxifylline infusion on plasma 6-keto-prostaglandin F1 alpha and ex vivo endotoxin-induced tumour necrosis factor activity in horses.
Pentoxifylline (7.5 mg/kg) was bolused intravenously to eight healthy horses and was immediately followed by infusion (1.5 mg/kg/h) for 3 h. Clinical parameters were recorded and blood samples were collected for 24 h. Plasma was separated and concentrations of pentoxifylline, its reduced metabolite I, and 6-keto-prostaglandin F1 alpha were determined. Heparinized whole blood was also incubated ex vivo with 1 ng Escherichi coli endotoxin/mL blood for 6 h before determination of plasma tumour necrosis factor activity. The peak plasma concentrations of pentoxifylline and metabolite I occurred at 15 min after bolus injection and were 9.2 +/- 1.4 and 7.8 +/- 4.3 micrograms/mL, respectively. The half-life of elimination (t1/2 beta) of pentoxifylline was 1.44 h and volume of distribution (Vdarea) was 0.94 L/kg. The mean plasma concentration of 6-keto-prostaglandin F1 alpha increased over time, with a significant increase occurring 30 min after the bolus administration. Ex vivo plasma endotoxin-induced tumour necrosis factor activity was significantly decreased at 1.5 and 3 h of infusion. These results indicate that infusion of pentoxifylline will increase 6-keto-prostaglandin F1 alpha and significantly suppress endotoxin-induced tumour necrosis factor activity in horses during the period of infusion.
6-Ketoprostaglandin F1 alpha↗
Physiologic effects of an external nasal dilator.
An external nasal dilator (Breathe Right, CNS, Inc., Bloomington, MN) has become popular with athletes. The dilator is an adhesive band with a central elastic strip. When applied across the nasal valve, the device theoretically increases nasal valve area. We used acoustic rhinometry (Hood Laboratories, Pembroke, MA) to measure the cross-sectional area at the nasal valve with and without the device in 53 athletes. Thirty athletes were exercised on a cycle ergometer with and without the device while measuring physiologic parameters, including oxygen consumption (VO2), heart rate, and respiratory rate. The study was conducted in a randomized, double-blind, placebo-controlled manner. At rest, the external nasal dilator was found to significantly increase nasal valve area in all demographic groups measured. The device was also found to significantly decrease submaximal exercise perceived exertion, heart rate, ventilation, and VO2 when compared with placebo.
Acoustics↗
Detection of herpes simplex virus in clinical specimens by cytospin-enhanced direct immunofluorescence.
For 275 samples tested for herpes simplex virus (HSV), cytospin-enhanced direct immunofluorescence using Chemicon HSV monoclonal antibodies identified 80 (95%) and culture identified 77 (92%) of 84 confirmed positive specimens. Cytospin-prepared slides contained a greater number of total cells than standard cell spots, resulting in fewer inadequate cell smears and a higher HSV detection rate.
Centrifugation↗
Race and clinical outcome in breast cancer in a series with long-term follow-up evaluation.
PURPOSE: To compare the outcome of African American (AA) and Caucasian (C) breast cancer patients who had equivalent disease extent and were similarly treated. PATIENTS AND METHODS: We compared prognostic characteristics, treatment, and outcome of 1,037 C and 481 AA breast cancer patients treated with mastectomy between 1946 and 1987. The median follow-up duration was 15.6 years. RESULTS: During the study period, there was a successive increase in the percent of patients who presented with early breast cancer. Between 1980 and 1987, 35.1% AA versus 47.6% C patients had < or = 2-cm tumors and 50.0% AA versus 61.9% C patients were node-negative, while between 1946 and 1959, 27.7% AA and 31.3% C had < or = 2-cm tumors and 41.5% AA versus 40.4% C patients were node-negative. The treatments were similar during the study period. The 20-year disease-free survival (DFS) rate of AA compared with C patients with node-negative < or = 2-cm, 2.1- to 4-cm, and greater than 4-cm tumors and of patients with one to three and > or = four positive nodes was not significantly different. Equal-size tumors had similar proportion of positive axillary nodes in AA compared with C patients. The DFS for AA patients compared with C patients was similar in the periods 1946 to 1959, 1960 to 1969, and 1970 to 1979, but was lower between 1980 and 1987 (P = .02). In multivariable analysis, race was not a significant variable. CONCLUSION: In this large group of uniformly treated breast cancer patients, race was not an independent factor that influenced outcome. The racial differences seen between 1980 and 1987 are likely because of a larger percent of greater than 2-cm and node-positive tumors in AA patients. Education and access to early diagnosis should reduce or eliminate the racial differences seen.
Actuarial Analysis↗
Pharmacokinetics of propranolol in healthy cats during euthyroid and hyperthyroid states.
OBJECTIVE: To examine the pharmacokinetic profile of propranolol in cats before and during experimentally induced hyperthyroidism. ANIMALS: 8 conditioned, random-source, young adult, female cats. PROCEDURE: Propranolol was administered i.v. as a single bolus and 72 hours later by mouth. Thereafter, the cats were dosed for 5 weeks with L-thyroxine (50 micrograms/kg of body weight, s.c., once daily) to induce hyperthyroidism (serum thyroxine concentration, 217 +/- 17 nmol/L). Blood samples were obtained at appropriate intervals before and during hyperthyroidism and were analyzed for plasma propranolol concentration by use of high-performance liquid chromatography. RESULTS: In all cats, a two-compartment model best described the control and hyperthyroid intravenous data. The change in thyroid status from euthyroid to hyperthyroid caused a significant (P < 0.05), but small reduction in propranolol area under the curve (19,932 +/- 7,900 min.micrograms/L vs 15,911 +/- 1,400 min.micrograms/L) after i.v. administration. In contrast, after oral administration during the hyperthyroid state, a twofold increase (P < 0.05) in propranolol area under the curve (105,430 +/- 57,600 min.micrograms/L vs 226,811 +/- 112,000 min.micrograms/L) and peak serum propranolol concentration (651 +/- 247 micrograms/L vs 1191 +/- 590 micrograms/L) were attributed to significant (P < 0.05) increase in propranolol bioavailability caused by increased fractional absorption (57 +/- 28% vs 137 +/- 73%) and decreased total body clearance (58 +/- 27 ml/min/kg vs 30 +/- 19 ml/min/kg). Mean arrival time after oral dosing was significantly lengthened by hyperthyroidism (100 +/- 38 minutes vs 157 +/- 71 minutes). CLINICAL RELEVANCE: Hyperthyroidism-induced changes in propranolol pharmacokinetics may signal the need to reduce doses of propranolol when they are orally administered to hyperthyroid cats.
Administration, Oral↗
The effect of shovel trait on Carabelli's trait in Taiwan Chinese and Aboriginal populations.
Chinese and other Mongoloid populations differ from Caucasoids by having a high prevalence of shovel trait and a low prevalence of Carabelli's trait. This study was conducted to compare the association between the shovel and the Carabelli's traits between Chinese and aboriginal Mongoloid populations. The research is designed to sample randomly a Chinese population and an aboriginal population having low admixture with neighboring populations. The Mongoloid aboriginal group was from the Bunun tribe who resides in an isolated alpine area in Taiwan. The effects of sex and age on Carabelli's trait were controlled in this study, as was the association between tooth size and Carabelli's trait. Our results show that males had more Carabelli's trait expressed on teeth than females in both of these two Mongoloid populations. The buccolingual diameter of Carabelli's trait teeth was larger than that of teeth without the trait. After controlling for sex, age, and tooth size, the existence of the shovel trait significantly increased the likelihood of having Carabelli's trait, especially in Chinese, which implies another significant ethnic feature for Mongoloid identification.
Adolescent↗
Angiogenesis as a predictor of long-term survival for patients with node-negative breast cancer.
BACKGROUND: Angiogenesis (the formation of new blood vessels) is necessary for tumor growth and metastasis. PURPOSE: We investigated whether angiogenesis as measured by microvessel count (MVC) predicts clinical outcome in a series of patients with axillary lymph node-negative breast cancer who received no adjuvant therapy and who were followed for a long period of time. Our long-term goal is to identify those patients who may or may not need adjuvant chemotherapy. METHODS: Pathologic archival material and clinical information were analyzed for 167 patients treated with mastectomy from 1941 through 1987; none received adjuvant treatment. The median follow-up time among living patients was 15.4 years (range, 2.6-35.8 years). Ninety-six (58%) patients had a tumor size of 2 cm or less, 52 (31%) had tumors of 2.1-3 cm, and 19 (11%) had tumors of larger than 3 cm. Paraffin-embedded tissue sections were stained for expression of CD34 antigen on microvessel-associated endothelial cells by use of a monoclonal anti-CD34 antibody. Vascularity was defined as the number of microvessels (average of the three highest counts) per high-power microscopic field (400 x magnification) in the area of highest vascular density. A high vascular count was defined as 15 or more microvessels per field. Actuarial survival curves were calculated according to the Kaplan-Meier method and comparisons were made with the logrank test. The Cox proportional hazards model was used for multivariate analysis. All P values were based on two-sided testing. RESULTS: The 20-year disease-free survival (DFS) for the 167 node-negative patients treated with mastectomy and no adjuvant therapy was 74.8% (95% confidence interval [CI] = 64.7%-82.0%). The 20-year DFS was 93.1% (95% CI = 79.9%-97.7%) if the MVC was low versus 68.9% (95% CI = 56.8%-78.0%) if the MVC was high (P = .018). This difference was maintained irrespective of tumor size: for tumor size of 2 cm or less (93.3% [95% CI = 75.3%-98.3%] versus versus 67.8% [95% CI = 50.1%-80.3%]) and for tumor size of larger than 2 cm (92.3% [95% CI = 56.6%-98.9%] versus 70.9% [95% CI = 54.6%-81.6%]). However, the likelihood of a high MVC was greater with large tumors (P = .05). The proportions of tumors with low and high MVC were 33% and 67%, respectively, if the tumor size was 2 cm or less, and 20% and 80%, respectively, if tumor size was larger than 2 cm. There was no significant difference in the 20-year DFS as a function of tumor grade (P = .2). After combining patients with tumors of nuclear grades 2 and 3 compared with those of nuclear grade 1, the 20-year DFS was 93.9% (95% CI = 77.2%-98.4%) for low MVC versus 66.9% (95% CI = 52.2%-78.0%) for high MVC (P = .02). In a multivariate analysis that included the variables tumor size, age, nuclear grade, estrogen receptor status, and MVC, only MVC appeared to be an independent prognostic indicator (P = .04). CONCLUSIONS: Angiogenesis as measured by MVC is a reliable independent prognostic marker of long-term survival in patients with node-negative breast cancer. The prognostic usefulness of this marker is maintained after more than 15 years of follow-up. A low MVC identifies a subgroup of patients with DFS of 92% or more, independent of tumor size or grade.
Adult↗
Nonpeptidic HIV protease inhibitors: 4-hydroxy-pyran-2-one inhibitors with functional tethers to P1 phenyl ring to reach S3 pocket of the enzyme.
A systematic study of tethering various groups on 6-phenyl ring of 4-hydroxy-6-phenyl-3-[(2-isopropylphenyl)thio]pyran-2-one was performed to increase the binding affinity with HIV protease. This tethering approach was aimed to fill S3 pocket of the enzyme. Thus, tethering hydrophilic groups resulted in more potent inhibitors. Similarly, various aromatic hydrophobic rings as well as heterocyclic rings were explored as tethering substituents to alter the physical properties as well as to enhance the binding affinity with HIV protease. Inhibitor 24, 4-hydroxy-3-[(2-isopropylphenyl)thio]-6-[4-(3-pyridinylmethoxy+ ++ ) phenyl]-2H-pyran-2-one, was evaluated as a prototypic lead structure to study various physical as well as pharmacological properties of this class of HIV protease inhibitors.
Cell Line↗
Developmental abnormalities of the reproductive tract associated with infertility in Highland heifers.
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Animals↗
Urinary bladder function and drug development.
Disorders of the bladder are extremely common and are becoming more so in an ageing population. Recently, our understanding of lower urinary tract physiology and pathology has also increased. Here, Douglas Ferguson and Nim Christopher summarize this new knowledge of lower urinary tract function, the changes in innervation that occur with age and the common disease states, and discuss how it is being used to develop new drug treatments for bladder disorders.
Animals↗
Discovery and optimization of nonpeptide HIV-1 protease inhibitors.
Several small, achiral nonpeptide inhibitors of HIV-1 protease with low micromolar activity were identified by mass screening of the Parke-Davis compound library. Two of the compounds, structurally similar, were both found to be competitive and reversible inhibitors [compound 1, 4-hydroxy-3-(3-phenoxypropyl)-1-benzopyran-2-one: Ki = 1.0 microM; compound 2, 4-hydroxy-6-phenyl-3-(phenylthio)-pyran-2-one: Ki = 1.1 microM]. These inhibitors were chosen as initial leads for optimization of in vitro inhibitory activity based on molecular modeling and X-ray crystallographic structural data. While improvements in inhibitory potency were small with analogues of compound 1, important X-ray crystallographic structural information of the enzyme-inhibitor complex was gained. When bound, 1 was found to displace H2O301 in the active site while hydrogen bonding to the catalytic Asps and Ile50 and Ile150. The pyranone group of compound 2 was found to bind at the active site in the same manner, with the 6-phenyl and the 3-phenylthio occupying P1 and P1', respectively. The structural information was used to develop design strategies to reach three or four of the internal pockets, P2-P2'. This work led to analogues of diverse structure with high potency (IC50 < 10 nM) that contain either one or no chiral centers and remain nonpeptide. The highly potent compounds possess less anti-HIV activity in cellular assays than expected, and current optimization now focuses on increasing cellular activity. The value of the HIV-1 protease inhibitors described is their potential as better pharmacological agents with a different pattern of viral resistance development, relative to the peptide inhibitors in human clinical trials.
Benzopyrans↗
Evaluation of CMV Brite kit for detection of cytomegalovirus pp65 antigenemia in peripheral blood leukocytes by immunofluorescence.
The CMV Brite antigenemia kit was compared with culture and an established cytomegalovirus pp65 antigenemia assay (CMV AG). Of 300 clinical specimens tested, 92 were positive by CMV Brite, 83 were positive by CMV AG, and 34 were positive by culture. Discrepancies could be attributed to anticytomegalovirus therapy or low-level antigenemia.
Antigens, Viral↗
An every other year cyclic epidemic of infants hospitalized with respiratory syncytial virus.
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Hospitalization↗