Dennis Murphy

Identification of gene targets against dormant phase Mycobacterium tuberculosis infections

BackgroundMycobacterium tuberculosis, the causative agent of tuberculosis (TB), infects approximately 2 billion people worldwide and is the leading cause of mortality due to infectious disease. Current TB therapy involves a regimen of four antibiotics taken over a six month period. Patient compliance, cost of drugs and increasing incidence of drug resistant M. tuberculosis strains have added urgency to the development of novel TB therapies. Eradication of TB is affected by the ability of the bacterium to survive up to decades in a dormant state primarily in hypoxic granulomas in the lung and to cause recurrent infections.MethodsThe availability of M. tuberculosis genome-wide DNA microarrays has lead to the publication of several gene expression studies under simulated dormancy conditions. However, no single model best replicates the conditions of human pathogenicity. In order to identify novel TB drug targets, we performed a meta-analysis of multiple published datasets from gene expression DNA microarray experiments that modeled infection leading to and including the dormant state, along with data from genome-wide insertional mutagenesis that examined gene essentiality.ResultsBased on the analysis of these data sets following normalization, several genome wide trends were identified and used to guide the selection of targets for therapeutic development. The trends included the significant up-regulation of genes controlled by devR, down-regulation of protein and ATP synthesis, and the adaptation of two-carbon metabolism to the hypoxic and nutrient limited environment of the granuloma. Promising targets for drug discovery were several regulatory elements (devR/devS, relA, mprAB), enzymes involved in redox balance and respiration, sulfur transport and fixation, pantothenate, isoprene, and NAD biosynthesis. The advantages and liabilities of each target are discussed in the context of enzymology, bacterial pathways, target tractability, and drug development.ConclusionBased on our bioinformatics analysis and additional discussion of in-depth biological rationale, several novel anti-TB targets have been proposed as potential opportunities to improve present therapeutic treatments for this disease.

The evolution of core proteins involved in microRNA biogenesis

BackgroundMicroRNAs (miRNAs) are a recently discovered class of non-coding RNAs (ncRNAs) which play important roles in eukaryotic gene regulation. miRNA biogenesis and activation is a complex process involving multiple protein catalysts and involves the large macromolecular RNAi Silencing Complex or RISC. While phylogenetic analyses of miRNA genes have been previously published, the evolution of miRNA biogenesis itself has been little studied. In order to better understand the origin of miRNA processing in animals and plants, we determined the phyletic occurrences and evolutionary relationships of four major miRNA pathway protein components; Dicer, Argonaute, RISC RNA-binding proteins, and Exportin-5.ResultsPhylogenetic analyses show that all four miRNA pathway proteins were derived from large multiple protein families. As an example, vertebrate and invertebrate Argonaute (Ago) proteins diverged from a larger family of PIWI/Argonaute proteins found throughout eukaryotes. Further gene duplications among vertebrates after the evolution of chordates from urochordates but prior to the emergence of fishes lead to the evolution of four Ago paralogues. Invertebrate RISC RNA-binding proteins R2D2 and Loquacious are related to other RNA-binding protein families such as Staufens as well as vertebrate-specific TAR (HIV trans-activator RNA) RNA-binding protein (TRBP) and protein kinase R-activating protein (PACT). Export of small RNAs from the nucleus, including miRNA, is facilitated by three closely related karyopherin-related nuclear transporters, Exportin-5, Exportin-1 and Exportin-T. While all three exportins have direct orthologues in deutrostomes, missing exportins in arthropods (Exportin-T) and nematodes (Exportin-5) are likely compensated by dual specificities of one of the other exportin paralogues.ConclusionCo-opting particular isoforms from large, diverse protein families seems to be a common theme in the evolution of miRNA biogenesis. Human miRNA biogenesis proteins have direct, orthologues in cold-blooded fishes and, in some cases, urochordates and deutrostomes. However, lineage specific expansions of Dicer in plants and invertebrates as well as Argonaute and RNA-binding proteins in vertebrates suggests that novel ncRNA regulatory mechanisms can evolve in relatively short evolutionary timeframes. The occurrence of multiple homologues to RNA-binding and Argonaute/PIWI proteins also suggests the possible existence of further pathways for additional types of ncRNAs.

A novel method for chronic measurement of pleural pressure in conscious rats

Pleural pressures are used to evaluate lung function and are generally measured acutely in anesthetized animals. Previous attempts to measure pleural pressure chronically in conscious animals have involved surgical implantation of pressure-sensitive catheters directly into the pleural cavity. The success of these techniques has been limited by lung damage and/or tissue growth and encapsulation of the pressure-sensitive catheter with damping or loss of the signal. These problems have been eliminated by developing a novel surgical procedure for placement of a pressure-sensitive catheter beneath the pleural surface. The catheter (attached to a radiotelemetry transmitter) is surgically implanted beneath the serosal layer of the esophagus within the thoracic cavity. This is accomplished by making a small incision in the serosal layer of the esophagus caudal to the diaphragm and advancing the catheter cranially into the thoracic cavity until pressure changes are maximal. The accuracy of these measurements was verified by comparison with direct pleural pressure measurements over the range of -3 to -34 cm H2O. The pleural pressure changes remained constant for at least 14 weeks following surgery, and there was no evidence of tissue damage or growth around the catheter. This novel method for measuring pleural pressure chronically in conscious rats will facilitate evaluation of the effects of drugs, environmental agents, or disease on respiratory function by allowing repeated and simultaneous measurements of both ventilatory (breathing) patterns and lung function in conscious animals.

Discovery of potent, selective sulfonylfuran urea endothelial lipase inhibitors.

Endothelial lipase (EL) activity has been implicated in HDL catabolism, vascular inflammation, and atherogenesis, and inhibitors are therefore expected to be useful for the treatment of cardiovascular disease. Sulfonylfuran urea 1 was identified in a high-throughput screening campaign as a potent and non-selective EL inhibitor. A lead optimization effort was undertaken to improve potency and selectivity, and modifications leading to improved LPL selectivity were identified. Radiolabeling studies were undertaken to establish the mechanism of action for these inhibitors, which were ultimately demonstrated to be irreversible inhibitors.

Novel drug target strategies against Mycobacterium tuberculosis

The resurgence of drug resistant tuberculosis (TB) is a significant global healthcare challenge. Mycobacterium tuberculosis (MTB), TBs causative agent, evades the host immune system and drug regimes by entering prolonged periods of non-proliferation or dormancy. In infected individuals, the immune system sequesters MTB into structures called granulomas where the bacterium survives by shifting into a non-replicative state. Although still not well understood, progress has been made in characterizing the genetic program of MTB, activated by DosR (DevR) signal transduction that allows adaptation to the hypoxic, nutrient limiting granuloma microenvironment. Recent work, especially the identification genes involved in regulatory networks and the Enduring Hypoxic Response (EHR), hold promise for developing new drugs targeting dormancy phase MTB.

A novel method for chronic measurement of respiratory function in the conscious monkey.

INTRODUCTION Chronic measurement of respiratory function in the conscious monkey has been limited by the ability to monitor pleural pressure. Previous attempts to measure pleural pressure chronically in conscious animals have involved surgical implantation of pressure-sensitive catheters directly into the pleural cavity. The success of these techniques has been limited by lung damage and/or tissue growth and encapsulation of the pressure-sensitive catheter with damping or loss of the signal. These problems have been eliminated by developing a novel surgical procedure for placement of a pressure-sensitive catheter beneath the pleural surface. METHODS A pressure-sensitive catheter (attached to a radiotelemetry transmitter) was surgically implanted beneath the serosal layer of the esophagus within the thoracic cavity. This was accomplished by performing a midline laparotomy, making a small incision in the serosal layer of the esophagus caudal to the diaphragm, and advancing the catheter cranially along the esophagus and beneath the serosal layer into the thoracic cavity. The catheter was secured to the esophageal wall and the telemetry transmitter was sutured to the inner abdominal wall. Respiratory airflow measurements were obtained using a restraint chair equipped with a clear plastic helmet (plethysmograph chamber) that seals around the neck and encloses the head. RESULTS The decreases in pleural pressure during inspiration ranged from -5 to -15 mmHg and remained constant for at least 36 weeks following surgery. Intravenous administration of the respiratory depressant morphine, exposure to the respiratory stimulant 8% CO2, and intravenous administration of the bronchoconstrictive agent methacholine verified that this technique was able to detect and quantify ventilatory changes and an increase in airway resistance. DISCUSSION This novel method for chronic measurement of pleural pressure will facilitate evaluation of the effects of drugs, environmental agents, or disease on respiratory function by allowing repeated measurements of both ventilatory (breathing) patterns and the mechanical properties of the lung in a conscious nonhuman primate.

Effects of adenosine agonists and antagonists on pulmonary ventilation in conscious rats

1. The effects of intravenous injections of adenosine agonists and antagonists on pulmonary ventilation were investigated in conscious male Sprague-Dawley rats. 2. Adenosine agonists reduced pulmonary ventilation, whereas an adenosine antagonist increased pulmonary ventilation. 3. The adenosine-induced decrease in ventilation could be partially blocked by pretreatment with either an adenosine or opioid antagonist and completely blocked by a combined pretreatment with adenosine and opioid antagonists. 4. Thus, endogenous adenosine appears to have an inhibitory role in the control of pulmonary ventilation in conscious rats, and the mechanism of action appears to involve both adenosine and opioid receptors.

A non-invasive method for distinguishing central from peripheral nervous system effects of respiratory depressant drugs in conscious rats

1. A simple, non-invasive method for distinguishing the central from peripheral effects of respiratory depressant drugs was developed in conscious rats. 2. The procedure involves exposing rats for 5 min to an air mixture containing 8% CO2 (central stimulant) followed by an i.v. bolus injection of 300 micrograms/kg sodium cyanide (peripheral stimulant) and comparing the changes in minute volume and mean inspiratory flow (respiratory drive) before and after drug treatment. 3. The central depressant drugs morphine, xylazine, L-2-phenylisopropyladenosine (L-PIA) and gamma-hydroxybutyric acid. (GHBA) inhibited the CO2-induced increase in minute volume and enhanced the sodium cyanide-induced increase in mean inspiratory flow. 4. Peripheral depression produced by carotid body denervation had no effect on the CO2-induced increase in minute volume and inhibited the sodium cyanide-induced increase in mean inspiratory flow.

Microcapnometry: A Noninvasive Method for Monitoring Arterial CO2 Tension in Conscious Rats

Characterizing disorders in the control of pulmonary ventilation requires measuring changes in both ventilatory parameters and the partial pressure of arterial CO2. Although the conscious rat has been used as a model for evaluating the effects of drugs, environmental agents, and disease on the control of pulmonary ventilation, obtaining samples of arterial blood for CO2 analysis by needle puncture or arterial catheterization during ventilatory measurements is not practical in the conscious rat and has limited the use of this model. The objective of this study was to develop a noninvasive method for monitoring arterial CO2 tension in conscious rats during ventilatory measurements using end-tidal CO2 measurements. Progressive increases in end-tidal CO2 (125–400% of baseline) caused by progressive decreases in pulmonary ventilation (75–25% of baseline) following intravenous doses of the opioid analgesic morphine were measured in conscious rats using this noninvasive method. Changes in end-tidal CO2 were comp...

Respiratory Function Assessment in Safety Pharmacology

The known effects of drugs from a variety of pharmacological/therapeutic classes on the respiratory system, the life‐threatening consequences of respiratory dysfunction, and world‐wide regulatory safety guidelines all support the need for conducting respiratory evaluations in safety pharmacology. This unit provides an overview of the functional disorders of the respiratory system and presents the techniques and strategies considered to be most appropriate for detecting and characterizing drug‐induced respiratory disorders in safety pharmacology studies.