Mass spectrometric techniques for label-free high-throughput screening in drug discovery

High-throughput screening (HTS) is an important tool for finding active compounds to initiate medicinal chemistry programs in pharmaceutical discovery research. Traditional HTS methods rely on fluorescent or radiolabeled reagents and/or coupling assays to permit quantitation of enzymatic target inhibition or activation. Mass spectrometry-based high-throughput screening (MS-HTS) is an alternative that is not susceptible to the limitations imposed by labeling and coupling enzymes. MS-HTS offers a selective and sensitive analytical method for unlabeled substrates and products. Furthermore, method development times are reduced without the need to incorporate labels or coupling assays. MS-HTS also permits screening of targets that are difficult or impossible to screen by other techniques. For example, enzymes that are challenging to purify can lead to the nonspecific detection of structurally similar components of the impure enzyme or matrix of membraneous enzymes. The high selectivity of tandem mass spectrometry (MS/MS) enables these screens to proceed with low levels of background noise to sensitively discover interesting hits even with relatively weak activity. In this article, we describe three techniques that we have adapted for large-scale (approximately 175,000 sample) compound library screening, including four-way parallel multiplexed electrospray liquid chromatography tandem mass spectrometry (MUX-LC/MS/MS), four-way parallel staggered gradient liquid chromatography tandem mass spectrometry (LC/MS/MS), and eight-way staggered flow injection MS/MS following 384-well plate solid-phase extraction (SPE). These methods are capable of analyzing a 384-well plate in 37 min, with typical analysis times of less than 2 h. The quality of the MS-HTS approach is demonstrated herein with screening data from two large-scale screens.     

Age alters regional distribution of blood flow during moderate-intensity exercise

During dynamic exercise in warm environments, requisite increases in skin and active muscle blood flows are supported by increasing cardiac output (Qc) and redistributing flow away from splanchnic and renal circulations. To examine the effect of age on these responses, six young (Y; 26 +/- 2 yr) and six older (O; 64 +/- 2 yr) men performed upright cycle exercise at 35 and 60% of peak O2 consumption (VO2peak) in 22 and 36 degrees C environments. To further isolate age, the two age groups were closely matched for VO2peak, weight, surface area, and body composition. Measurements included heart rate, Qc (CO2 rebreathing), skin blood flow (from increases in forearm blood flow (venous occlusion plethysmography), splanchnic blood flow (indocyanine green dilution), renal blood flow (p-amino-hippurate clearance), and plasma norepinephrine concentration. There were no significant age differences in Qc; however, in both environments the O group maintained Qc at a higher stroke volume and lower heart rate. At 60% VO2peak, forearm blood flow was significantly lower in the O subjects in each environment. Splanchnic blood flow fell (by 12-14% in both groups) at the lower intensity, then decreased to a greater extent at 60% VO2peak in Y than in O subjects (e.g., -45 +/- 2 vs. -33 +/- 3% for the hot environment, P < 0.01). Renal blood flow was lower at rest in the O group, remained relatively constant at 35% VO2peak, then decreased by 20-25% in both groups at 60% VO2peak. At 60% VO2peak, 27 and 37% more total blood flow was redistributed away from these two circulations in the Y than in the O group at 22 and 36 degrees, respectively. It was concluded that the greater increase in skin blood flow in Y subjects is partially supported by a greater redistribution of blood flow away from splanchnic and renal vascular beds.     

Spatial and temporal expression of angiogenic molecules during tumor growth and progression

The growth and metastasis of cancer directly correlates with tumor angiogenesis. A better understanding of the expression of regulatory factors controlling angiogenesis is important in exploiting this process therapeutically. Our present study demonstrates that small tumors (3-4 mm in diameter) express more basic fibroblast growth factor (bFGF) and interleukin 8 (IL-8) than large tumors (> 10 mm in diameter), whereas more vascular endothelial growth factor (VEGF) is expressed in large tumors. Immunostaining showed a heterogeneous distribution of angiogenic factors within the tumor; expression of bFGF and IL-8 was highest on the periphery of a large tumor, where cell division is maximum. VEGF expression was higher in the center of the tumor. In vitro studies demonstrated that sparse cultures of tumor cells expressed higher levels of bFGF and IL-8 than confluent cultures. In contrast, the expression of bFGF and IL-8 was not diminished in tumor cells growing on confluent monolayers of normal cells. VEGF expression was upregulated by cell density irrespective of contact with tumor cells or normal cells. These results demonstrate that the expression of different angiogenic factors in tumor cells can be regulated by their proximity to other tumor cells or host cells.     

Evidence of transmission of hepatitis B virus to spouses from sequence analysis of the viral genome

Despite advances in postnatal care, patients born with a congenital diaphragmatic hernia (CDH) suffer substantial morbidity and mortality. The present study was undertaken to determine the prognostic influence of prenatally-diagnosed liver herniation in the hemithorax in fetuses with CDH. The medical records of 48 patients evaluated for a prenatally-diagnosed left CDH were retrospectively reviewed. Patients were analysed according to the position of the liver by prenatal ultrasound; 32 fetuses had a major portion of the liver herniated into the left hemithorax ('liver up') and 16 had an intra-abdominal liver ('liver down'). Liver position was determined using colour-flow Doppler ultrasonography. There were two fetal deaths in the liver-up group and one in the liver-down group. The liver-up group more frequently required extracorporeal membrane oxygenation (ECMO) support (53 per cent) compared with the liver-down group (19 per cent). Postnatal survival was significantly less in the liver-up group (43 per cent) vs. the liver-down group (93 per cent). Fetuses with congenital diaphragmatic hernia and liver herniated into the hemithorax have a much worse prognosis than similarly afflicted fetuses without liver herniation. Prenatal ultrasonographic diagnosis of congenital diaphragmatic hernia allows for preparation for a critically ill newborn and aids in prenatal family counselling.     

Ambulatory treatment with ceftriaxone in febrile neutropenic children

We conducted a prospective nonrandomized study of outpatient therapy with ceftriaxone as a single agent in 50 episodes of fever and neutropenia in children treated with various myelosuppressive regimens for different malignancies. All patients underwent clinical and radiological evaluation and blood/urine cultures taken before starting therapy. Patients with dehydration, hypotension, rigor and clinical exit-site infection of indwelling right-sided catheters were excluded. Forty-one patients completed an antibiotic course of 7 days: in 12 patients fever returned to normal on day 2, in 10 patients on day 3, and in 8 patients on day 4. The duration of neutropenia following the initial febrile episode was 3-10 days. In some patients fever returned to normal after 2 days, but neutropenia persisted up to 10 days. Two patients were bacteremic--Escherichia coli in one, and Acinetobacter/Staphylococcus coagulase negative in another; all isolates were sensitive to ceftriaxone. In nine episodes, antimicrobial therapy was modified because of persistent fever > 39 degrees C in five patients, bacteremia in two, enterocolitis in one, breakthrough fever in two, and bronchopneumonia in one. The low incidence of bacterial isolation is probably attributed to the selection of patients with low risk features. Patients and parents complied with and favored outpatient therapy to hospitalization.     

Effect of acute hypoglycemia on visual information processing in adults with type 1 diabetes mellitus

Acute hypoglycemia in people with type 1 (insulin-dependent) diabetes mellitus causes general impairment in cognitive performance. The effects on more specific cognitive processes are less well defined. Acute hypoglycemia has been shown to impair visual information processing in nondiabetic human subjects and has now been examined in 16 adult subjects with type 1 diabetes. All subjects had normal visual acuity and no diabetic retinopathy, and their median (range) age was 24 (18-47) years with a median (range) duration of type 1 diabetes of 8 (2-18) years and a mean (SD) HbA1c of 8.5 (1.3)%. A hyperinsulinemic glucose clamp technique was used to maintain arterialized blood glucose at 5.0 mmol l(-1), and on separate test days, either euglycemia was continued or hypoglycemia (2.6 mmol l(-1)) was induced. During each condition subjects performed tests of visual processing and cognitive function. Hypoglycemia caused a significant disruption in general cognitive ability as assessed by digit symbol (p < 0.001) and trail-making B (p < 0.05) tasks. Conventional measures of visual acuity were unaffected by hypoglycemia, but visual information processing deteriorated significantly as indexed by inspection time (p < 0.005) and visual change detection (p < 0.01). Contrast sensitivity tended to deteriorate during hypoglycemia (p = 0.06). In conclusion, hypoglycemia impairs important aspects of early visual information processing and contrast sensitivity in adults with type 1 diabetes. Further research is needed to evaluate the functional relevance of such changes for everyday tasks that require the intake of visual information at speed and under conditions of low contrast.     

Effect on normal vaginal flora of three intravaginal microbicidal agents potentially active against human immunodeficiency virus type 1

The effect on normal vaginal flora of three intravaginal microbicides potentially active against human immunodeficiency virus type 1 was examined. Volunteers received dextrin sulfate (D2S), nonoxynol-9 (N-9), or docusate sodium in separate placebo-controlled studies. High vaginal swabs were obtained for bacterial culture before and after microbicide application. D2S did not affect the vaginal flora. However, lactobacilli decreased by > or = 10(2) cfu/mL in 9 (56%) of 16 women given N-9 and in 5 (63%) of 8 women given docusate sodium. Women using N-9 were also significantly more likely to become colonized abnormally (usually with aerobic gram-negative rods) than were those using placebo, as were women using docusate sodium. Women with reduced lactobacilli were less likely to regain normal flora than were those whose lactobacilli were unaffected. However, coliform colonization occurred whether lactobacilli produced H2O2 or not. Continuous use of N-9 could induce susceptibility to urinary and gynecological infection. It is essential that potential microbicides are examined for activity against normal vaginal flora.     

Platelet activation and turnover in the primary antiphospholipid syndrome

Recent advances in glaucoma genetics hold potential for dramatically changing the clinical care of glaucoma patients. To date, 5 primary open-angle glaucoma genes and 2 congenital glaucoma genes have been mapped. As more glaucoma genes are identified, earlier diagnosis for glaucoma should become more readily available. Progress in molecular genetics holds considerable promise for both current and future therapy of glaucoma. Glaucoma classification will be tailored to each individual based upon that person's family history, i.e. family glaucoma genotype. In the future, the optimum treatment for a specific glaucoma patient might rely on the knowledge of the phenotype of that person's causal gene, without having to resort to 'trial and error'. At this time, glaucoma treatment is restricted to lowering intraocular pressure. In the near future, with the knowledge of the pathophysiology caused by the defective glaucoma gene, more traditional drug treatments may be used to bypass the gene defect. Ultimately, gene therapy would replace the mutant gene with a normal one before visual loss has occurred as has been done with a model for retinitis pigmentosa, the retinal degeneration mouse.     

Selective block by glyceryl nonivamide of inwardly rectifying K+ current in rat anterior pituitary GH3 cells

The effects of glyceryl nonivamide (GLNVA) on ionic currents were compared and examined in rat pituitary GH3 cells. Hyperpolarization-activated K+ currents in GH3 cells bathed in high-K+ Ca2+-free external solution were studied to assess effects of GLNVA on the an inwardly rectifying K+ current (I(K(IR))). GLNVA is very potent in blocking I(K(IR)) in a concentration-dependent manner, with a half maximal concentrations of 0.1 microM. The complete block of I(K(IR)) achieved with concentrations > or = 1 microM revealed the presence of a non-inactivating current. We also found that GLNVA at a concentration above 30 microM inhibited L-type voltage-dependent Ca2+ current and two components of K+ outward currents, while GLNVA (< or = 3 microM) did not have any effect on them. This study shows that GLNVA, in addition to retaining the capability of eliciting peptidergic neurons, is a selective block of I(K(IR)) in GH3 cells and will provide a useful tool for characterizing I(K(IR)) and understanding its physiological function. In addition, the carefulness should be taken about the interpretation of GLNVA-mediated responses in vivo or in vitro.