Molecular Interventions

Published by American Society for Pharmacology and Experimental Therapeutics
Print ISSN: 1534-0384
Publications
Apoptosis (programmed cell death) is a physiological process used to eliminate superfluous, damaged, infected, or aged cells in multicellular organisms. During apoptosis the cellular architecture is dismantled from within in a highly controlled fashion. Members of the caspase family of cysteine proteases are responsible for the destructive phase of apoptosis. One major pathway to caspase activation involves the formation of a multisubunit protease activation complex called the apoptosome. The apoptosome is assembled in response to signals that provoke mitochondrial outer membrane permeabilization and the release of cytochrome c into the cytosol. Recent studies indicate that the apoptosome is a wheel-like structure consisting of seven molecules of Apaf-1 and a similar number of caspase-9 dimers. Knowledge of the structure of the apoptosome will likely lead to the design of therapeutic modulators of apoptosis.
 
Pray for the welfare of government; since were it not for the fear of government; men would swallow each other alive.
 
The peptide Angiotensin II (Ang II), part of the renin-angiotensin system (RAS), participates in the control of systemic arterial pressure. Ang II participates in increasing smooth muscle tone, and its positive effects on smooth muscle cell DNA synthesis are inhibited by treatment with prazosin, an alpha(1)-adrenoceptor agonist. Ang II also induces the expression of alpha(1)-adrenoceptor, especially the alpha(1D) subtype. Other findings suggest that the molecular signals activated by Ang II and by alpha(1D)-adrenoceptor might interweave, thus leading to the augmentation of smooth muscle tone and hypertension.
 
The presence or absence of estrogen receptor (ER) expression in tumor cells affects prognosis and guides treatment choices. Kumar et al. suggest that a shortened form of the metastatic tumor antigen 1 (MTA1s) acts to sequester the estrogen receptor (ER) in the cytoplasm, inhibiting its ability to transactivate specific genes and, presumably, adding to the ER's ability to transduce non-genomic (cytoplasmic) signaling mechanisms. However, if the cancer is negative for ERalpha in the nucleus, but is positive for ERalpha in the cytoplasm, how does this sequestration affect the treatment of the patient or our understanding of the disease process? Cheng et al. caution that these results must be interpreted carefully with regard to what is known about estrogen-dependent and -independent tumor growth and chemotherapeutic strategies to destroy them.
 
A recent article suggests that the well known tumor suppressor PDCD4 also functions as a pro-inflammatory agent. The PDCD4 counteragent miR-21, a pro-oncogenic micro-RNA, is described as an anti-inflammatory agent. The authors of this research article provide evidence that mice lacking PDCD4 are protected from the lethal effects of lipopolysaccharide (LPS). This report also confirms miR-21 as a negative regulator of PDCD4 expression after LPS stimulation. Downstream mediators of the pro-inflammatory activity of PDCD4 include IL-10, an anti-inflammatory cytokine that is negatively regulated by PDCD4, and IL-6, a pro-inflammatory cytokine that appears to be upregulated in a PDCD4 dependent manner, possibly through an increase in NF-κB activity. Is it possible that a tumor-suppressor protein and an oncogenic micro-RNA can be oppositely targeted to control inflammatory disease?
 
The killer cell immunoglobulin-like receptors (KIR) are a recently discovered family of activating and inhibitory receptors that control natural killer (NK) cell function. KIR exist as a diverse family of receptors that have evolved rapidly by both gene duplication and recombination events. These findings were unexpected for a family of genes involved primarily in the innate immune response. These findings together with the observation that several of these genes have human leukocyte antigen (HLA) class I ligands, have led to a flurry of investigation into how KIR participate in viral infections, autoimmune diseases and malignancies. This review summarizes the major features of these genes and discusses how they may be involved in both disease pathogenesis and its amelioration.
 
Gene expression arrays allow researchers to profile the differences between cell lines or tissues and they may identify genetic markers of development, organ maturation, or tumor progression. Although a primary tumor that grows in a host and a tumor-cell-line derived from that primary tumor and grown in vitro share similar gene expression profiles, there are, not unexpectedly, some important differences. In fact, Stein and colleagues have found that genes that are differentially expressed in primary tumors as compared to the specific genes expressed in their cell-line derivatives are more reliably predictive of tumor tractability. Thus, sensitivity in vitro might not reflect sensitivity in vivo. Because anti-tumor compounds are largely evaluated in cell culture assays, these compounds' therapeutic utility must be judged in light of genes described by Stein et al. that better predict tractability.
 
Relationship of expression of Na + ,K + -ATPase α 3 subunit and binding of oleandrin to human pancreatic cancer cell membranes. 
The class of steroid-like compounds designated cardiac glycosides includes well-known drugs such as digoxin, digitoxin, and ouabain. Their continued efficacy in treatment of congestive heart failure and as anti-arrhythmic agents is well appreciated. Less well known, however, is the emerging role of this category of compounds in the prevention and/or treatment of proliferative diseases such as cancer. New findings within the past five years have revealed these compounds to be involved in complex cell-signal transduction mechanisms, resulting in selective control of human tumor but not normal cellular proliferation. As such, they represent a promising form of targeted cancer chemotherapy. New clinical studies of their anticancer potential as single or adjuvant treatments may provide insight into these potentially valuable therapeutic options. This review focuses on recent findings on cellular pharmacology of cardiac glycosides as they relate to treatment of human cancer and attempts to explain why these agents have been overlooked in the past.
 
Sensory experience affects quality of subsequent sleep. A. Early sensory deprivation reduces slow-wave activity. (Left) Representative SWS power spectra of light-reared (LR) and dark-reared (DR) cats. DR kittens were raised in complete darkness from birth for 4 months. (Center) Group data. Reduction of delta power after DR is restricted to visual cortex (VC) and not seen in somatosensory area (SS). (Right) Slow-wave plasticity is reversible. Recovery of delta activity after light re-exposure for one to two months. Adapted from Miyamoto et al. (53). B. Sensory stimulation during waking enhances interhemispheric asymmetry of EEG power density. Ordinate indicates EEG power density of hemisphere contralateral to intact whisker side as a percentage of ipsilateral hemisphere (whisker-cut side) during SWS. Lines above the abscissa indicate significant difference between hemispheres. Top line (total) represents statistics for all data, middle and bottom lines for stimulation of intact left or right side whiskers. Adapted from Vyazovski et al. (54).  
Modulation of auditory activity and song replay during sleep in vocal motor system of songbirds. A. Auditory response is gated by sleep. (Left): Rectified averaged multiunit response to multiple presentations of bird's own song (BOS) in the nucleus robustus archistriatalis (RA). Recording of 3 sites from one adult bird. W: during waking; S: during sleep. (Right): Raster plot of single RA unit activity. The response to BOS developed as the anesthetic (urethane: arrow) took effect. Adapted from Dave et al. (68). B. Neuronal replay during sleep. Simultaneous recordings from two neurons in RA. Color spectrograph of the song that was sung (Top). Raw traces of neuronal activity during sleep (Spon). Premotor activity during singing (Mot). Adapted from Dave and Margoliash (70).
Synaptic plasticity underlying learning and memory has been proposed, on the basis of several experimental approaches, to be intimately related with sleep: 1) The idea that sleep contributes to stabilization of acquired memory arises from numerous studies depriving subjects or animals of sleep. 2) Evidence from developing technologies supports "offline" reprocessing of recent experiences during sleep. 3) Recent analysis of the thalamocortical system establishes the reciprocal observation that sleep itself is a plastic process affected by waking experience. This overview synthesizes these converging perspectives across a variety of brain regions and species. We propose the developing visual pathway as a fruitful model for comprehensive understanding of sleep and synaptic plasticity.
 
Neuropeptide S (NPS) is a newly identified transmitter that modulates arousal and fear responses. NPS activates an orphan G protein-coupled receptor that is expressed throughout the central nervous system, including brain centers that regulate sleep/wakefulness and anxiety. In contrast, the NPS precursor mRNA is found only in a few discrete nuclei in the brainstem as well as in a few scattered cells in the hypothalamus and amygdala. The most prominent expression of NPS precursor is found in a previously uncharacterized cluster of neurons in the pontine area, located between the noradrenergic locus ceruleus and Barrington's nucleus. Central administration of NPS induces long-lasting arousal and suppresses all stages of sleep. In addition, NPS produces an anxiolytic profile in a variety of behavioral models. The unique pharmacological spectrum of NPS makes it an interesting target for pharmaceutical development. It also enhances our understanding of the neurobiological mechanisms of sleep/wakefulness regulation and the neuronal processing of stress.
 
Worldwide, more than one billion people are affected by CNS disorders. Despite the huge demand for treatments, existing drugs have limited or no efficacy for some neurological diseases, including brain cancer and certain epilepsies. Furthermore, no effective therapies are available at all for some common disorders of the central nervous system (CNS) such as Alzheimer's disease. ATP-binding cassette (ABC) transporters at the blood-brain barrier (BBB) have become increasingly important in the treatment and pathogenesis of CNS disorders. Here we highlight a novel strategy--targeting signaling pathways that control ABC transporters at the BBB--to protect the brain, improve brain drug delivery, and reduce CNS pathology.
 
The anti-alcoholism drug disulfiram (Antabuse), which is an inhibitor of aldehyde dehydrogenase, induces an aversive reaction to alcohol consumption and thereby helps patients reduce alcohol intake. Recent clinical trials, initiated to investigate whether disulfiram could be used to treat individuals who abuse both alcohol and cocaine, have indicated that disulfiram effectively decreases cocaine consumption. Yet the ability of disulfiram to curb cocaine intake cannot be explained by the disruption of ethanol metabolism. Here, we synthesize clinical and animal data that point to dopamine beta-hydroxylase inhibition as a mechanism underlying the efficacy of disulfiram in the treatment of cocaine dependence.
 
Demand curves describe the relationship between the cost and consumption of behavioral reinforcers. The cost incurred by a rhesus monkey working for either food (squares) or saccharin-sweetened water (triangles) is given as the number of responses (e.g., depression of a lever) required to elicit a unit of reward (e.g., a food pellet). This cost can be varied by the experimenter, whereby the ratio of responses per reward is fixed to a predetermined value [i.e., the fixed-ratio response (FR), plotted on the x axis]. Consumption can be given, on the y axis, as either the number of reinforcers given per day A. or the number of responses elicited per day B . See (21 ). 
Elasticity of demand. A. An organism’s demand for a given 
The behavioral economics of ketamine consumption. A. Despite differences in the doses of ketamine made available to rhesus monkeys (green, 0.03 mg/kg; blue, 0.1 mg/kg; yellow, 0.3 mg/kg), the demand curves and economic parameters (e.g., P max ) that describe usage of the drug are constant and thus descriptive for ketamine. B. Given that unit price P is equal 
Comparisons of demand for four distinct drugs. Normalization of demand parameters allows for the comparison of demand for four distinct drugs: alfentanil, nalbuphine, cocaine, and methohexital. [See text for details. Also see (23) .] 
Behavioral economics provides a quantitative means to describe the demand for alternative drugs by a single organism. The graphs show the demand for methadone (squares) in the presence of hydromorphone (triangles); the price of the latter is held constant (see text for details). The graph in A. represents a closed economy; the graph in B. represents an open economy. Data are from one human participant (R. Spiga, personal communication). 
Behavioral economic concepts have proven useful for an overall understanding of the regulation of behavior by environmental commodities and complements a pharmacological perspective on drug abuse in several ways. First, a quantitative assessment of drug demand, equated in terms of drug potency, allows meaningful comparisons to be made among drug reinforcers within and across pharmacological classes. Second, behavioral economics provides a conceptual framework for understanding key factors, both pharmacological and environmental, that contribute to reductions in consumption of illicit drugs. Finally, behavioral economics provides a basis for generalization from laboratory and clinical studies to the development of novel behavioral and pharmacological therapies.
 
Immunotherapy for treating illicit drug abuse is a rapidly advancing field. There are currently two major approaches to developing drug-specific immunotherapies: active and passive. Active immunotherapy involves conjugating a drug-like hapten to a carrier protein and using traditional immunization approaches to generate a drug-specific immune response in the patient. In contrast, passive immunotherapy utilizes preformed monoclonal antibodies. Whether generated by active immunization or delivered passively, antibodies act as pharmacokinetic antagonists by binding the drug in the blood-stream and reducing the amount and rate of drug delivery to receptors in the brain. A newly emerging technology in anti-drug immunotherapy is the use of antibody fragments, or scFvs, rather than intact immunoglobulin G (IgG). These scFvs can retain the same binding properties as the original mAbs, and are onethird the molecular weight, providing a scaffold for creating antibody treatments with more customizable properties. Another nascent area of research utilizing the scFv scaffold is in creating drug-specific scFv-nanoparticle conjugates. These conjugates could improve upon current drug-specific antibody paradigms by increasing multivalency and allowing pharmacokinetic customization, while avoiding interactions with endogenous antibody receptor pathways. These parallel approaches to immunotherapy are moving rapidly toward the clinic and may soon provide new therapies for treating drug abuse.
 
Drugs of abuse such as opioids and stimulants share a common dopaminergic reward pathway; however, in response to continual intermittent exposure to such drugs, there are neuronal alterations leading to changes in behavior. Regulators of G protein signaling (RGS) are proteins that negatively regulate G protein signaling and are expressed in brain areas important for the pharmacology of abused drugs. Moreover, the level of expression of several of these proteins is regulated by abused drugs. In this article, we discuss RGS proteins, their regulation by morphine and stimulants, and how altered levels of these proteins affect cell signaling to contribute to the pharmacology and behavioral consequence of abused drugs. Finally, we consider if RGS proteins represent viable targets for drug abuse medications.
 
The principal psychoactive component of marijuana, Δ(9)-tetrahydrocannabinol (THC), activates CB1 cannabinoid receptors (CB1Rs). Unfortunately, pharmacological research into the design of effective THC analogs has been hampered by psychiatric side effects. THC-based drug design of a less academic nature, however, has led to the marketing of "synthetic marijuana," labeled as K2 or "Spice," among other terms, which elicits psychotropic actions via CB1R activation. Because of structural dissimilarity to THC, the active ingredients of K2/Spice preparations are widely unregulated. The K2/Spice "phenomenon" provides a context for considering whether marijuana-based drugs will truly provide innovative therapeutics or merely perpetuate drug abuse.
 
Animal models for human diseases are highly valued for their utility in developing new therapies. Animals have long provided suitable platforms for the development of innovative surgical procedures and for the study of disease states that are relatively easy to produce in otherwise healthy animals, such as diabetes or hypertension. Increasingly, new strains of animals susceptible to common human illnesses are being introduced into medical research, promising new inroads into the treatment of a variety of organic disorders. Despite these advances in model development, psychiatric disorders, by and large, remain among the hardest to induce experimentally, and the search for reasonable animal procedures to study diseases of the mind is an ongoing challenge for experimental biologists. An exception to this limitation, however, comes in the study of drug abuse. Major developments in this area of research over the last several decades have steadily advanced our ability to identify pharmacological, genetic, and environmental determinants that contribute to the development of drug dependence and addictive behavior.
 
Perry Molinoff recognizes the distinctions between basic and applied science, between academic and industrial research, and between the preclinical and clinical realities of drug development. But he generally discusses these categories in fluid, practical terms, having throughout his career crossed the lines of distinction that have sometimes been rather heavily drawn among pharmacologists. As a third-year medical student at Harvard, he decided "to take a year off" to conduct laboratory research. After receiving his MD and pursuing further clinical and postdoctoral work, he enjoyed an academic career that included fourteen years as the A.N. Richards Professor and Chair of Pharmacology at the University of Pennsylvania School of Medicine. He has just completed six years as Vice President of Neuroscience and Genitourinary Drug Discovery for Bristol-Myers Squibb and will soon return to teaching, in the Departments of Psychiatry and Pharmacology at Yale University. Referring to himself as either pharmacologist or neuroscientist, depending on context, he has made fundamental discoveries in receptor biology, has overseen the discovery and development of drugs and their subsequent clinical trials, and has mentored a host of pharmacologists and neuroscientists who themselves have established careers in industry and academia. The pursuit of discovery as its own reward emerges as a theme that has marked his professional life (and is perhaps reflected also in the images displayed in his office of the Himalayan mountains, photographed by Molinoff himself from the Everest base camp last year).
 
Intracellular accessory proteins can be critical for G protein-coupled receptor (GPCR) biogenesis, including aspects of GPCR trafficking. Recent discoveries include the identification of multiple membrane-associated proteins that dictate not only the intracellular sequestration and/or transport of GPCRs, but also modulate-quite dramatically-GPCR ligand specificity subsequent to delivery to the cell surface. These exciting discoveries have shifted earlier paradigms of GPCR functionality.
 
Because the rates at which therapeutics are cleared from the body can affect their effectiveness, knowing and accounting for the variables that contribute to drug clearance is of utmost importance when designing a drug dosage regimen for patients. The activities of the manifold cytochrome P450 enzymes [(CYPs), the most clinically important of which is often CYP3A] must be considered as they are essential for modification and metabolism of compounds (i.e., therapeutic, xenobiotic, etc.) prior to their excretion. To this end, much research has been expended on trying to identify and develop drug probes that accurately predict the metabolism of CYP3A substrates in individuals. Recently, Benet has written on the futility of such an enterprise; however, other researchers believe the identification of valuable predictive probes is not only possible but crucial.
 
Blocking sperm motility has great appeal as a male contraceptive. A drug that targets sperm motility might have a very rapid onset of action, possibly allowing for administration immediately prior to intercourse and minimizing concerns about compliance. Promising sperm motility targets include transmembrane calcium channels, a unique adenylyl cyclase, and novel flagellar proteins. Future efforts directed towards effectively antagonizing the activities of these or other such targets will be required to completely impair sperm production, function, or both and create a usable male "pill."
 
They had said that it couldn't be done-the worldwide eradication of smallpox. To hear D.A. Henderson tell it, the job of leading the World Health Organization's initiative to conquer the disease in the 1960s and 1970s rather fell into his lap. In fact, he describes each of the posts that he has held with great modesty, beginning with his military service at the Centers for Disease Control and Prevention all the way through his assignments as Dean of Public Health at Johns Hopkins, Associate Director of the Office of Science and Technology Policy in the Executive Office of the President, and more recently as Director of the Office of Public Health Preparedness under Secretary Thompson at the Department of Health and Human Services. Confronted with enormous challenges in terms of public health initiatives, Henderson describes each assignment as a matter of communicating with the people he works with and the people that he serves, and drawing on their insights to devise strategies for accomplishing the task at hand. With bioterrorism posing one of the major public health concerns to face the United States and the world, it's gratifying to know that someone with Henderson's track record and wide-ranging expertise is paying attention and making sure that medical and government officials are preparing to respond to the threat. Again and again, Henderson appears to have the knack for showing up in the right place at the right time with just the right idea.
 
Functional domains of the human estrogen receptors. ER α and ER β share a highly conserved central 
Corepressors in ER Physiology and Pharmacology
Estrogens are key regulators of growth, differentiation, and the physiological functions of a wide range of target tissues, including the male and female reproductive tracts, breast, and skeletal, nervous, cardiovascular, digestive and immune systems. The majority of these biological activities of estrogens are mediated through two genetically distinct receptors, ERalpha and ERbeta, which function as hormone-inducible transcription factors. Over the past decade, it has become increasingly clear that the recruitment of coregulatory proteins to ERs is required for ER-mediated transcriptional and biological activities. These "coactivator" complexes enable the ERs to respond appropriately: 1) to hormones or pharmacological ligands, 2) interpret extra- and intra-cellular signals, 3) catalyze the process of chromatin condensation and 4) to communicate with the general transcription apparatus at target gene promoters. In addition to activating proteins, the existence of corepressors, proteins that function as negative regulators of ER activity in either physiological or pharmacological contexts, provides an additional level of complexity in ER action. This review also describes current efforts aimed at developing pharmaceutical agents that target ER-cofactor interactions as therapeutics for estrogen-associated pathologies.
 
Analysis of the human genome project tells us that there may be as few as 3000 genes that are likely to be good drug targets. Although the number of targets is still very large, these data have been interpreted by some to mean that the pharmaceutical industry may someday run out of novel drug targets. Despite the doom and gloom of such analysis, there is considerable reason for optimism. Drugs may exhibit selectivity of action beyond that predicted by target expression alone. Drugs that act at a single molecular target may have very different pharmacology and, as a result, different therapeutic uses. Three well-characterized model systems are highlighted to illustrate this point. The first model system is exemplified by nifedipine and verapamil, both of which act on L-type calcium channels. Both drugs are used to treat hypertension, but only verapamil can be used to produce atrioventricular block in patients with atrial fibrillation. The second model system describes the therapeutic exploitation of unusual conditions that occur in the ischemic myocardium to produce drugs that are more effective for suppressing ischemia-induced arrhythmias. The third model system discusses the mechanisms through which phosphodiesterase-5 (PDE5) inhibitors act selectively to facilitate penile erection while having little effect in the non-penile vasculature that also expresses PDE5.
 
Second-Order Rate Constants for Oxidants and Electrophiles Reaction with the Thiol of GSH. 
Chemical structures of electrophilic phytochemicals. 
Modulation of cellular signaling mechanisms by electrophiles. Electophiles (E) can act at many loci within the cell. A. At the cellular membrane, electrophiles can modulate ion channel activity (e.g., adduction to essential cysteine residues of the TRP channel, thereby activating TRP signaling). Some adducts can be sequestered by reaction with glutathione (GSH), and GS-electrophile adducts can undergo further thiol exchange reactions or exit the cell via a multidrug resistant protein (MDR) mechanism. B. Cytosolic proteins can react with electrophiles, inducing changes in protein structure, activity, subcellular localization, or stability. An example of these outcomes is the modulation of protein kinase signaling by electrophiles and subsequent modulation of protein activity or gene regulation (see panel C). Electrophiles also affect signaling by influencing mitochondrial respiration and redox status, for example by reacting with cytochrome c. C. In the nucleus, transcription factors can react with electrophiles to affect the transcription of genes involved in cell survival, metabolism, and differentiation. During severe oxidative conditions, electrophiles can also form adducts with DNA and promote miscoding. 
Over the past several years, research on biologically relevant electrophiles has been replete with new insights, expanding our understanding of the roles electrophiles play in vivo. Importantly, many electrophiles can form reversible covalent adducts with both proteins and small-molecule thiols in cells. This post-translational protein modification has important ramifications, including changes in protein enzymatic activity, the transduction of signals within and between cells, and alterations in gene expression. Electrophiles modulate a variety of cellular signaling processes that are involved in several major diseases with inflammatory components. The electrophilic fatty-acid derivatives discussed in this work are naturally occurring products of redox reactions and enzymatic activity. Furthermore, several of these electrophilic species and their derivatives represent potential therapeutic candidates.
 
In the adult brain, cyclin-dependent kinase 5 (Cdk5) can be beneficial by contributing to memory formation or can be detrimental by causing neurodegeneration, and it is of great interest to understand this dichotomy. Currently, it remains largely unknown which mechanisms are regulated by Cdk5. Recent studies by Hawasli et al. and Qu et al., however, are significant advances towards mechanistic insights. Hawasli et al. demonstrate that Cdk5 regulates protease-directed degradation of an important synaptic receptor, which impacts memory formation. Qu et al. show that Cdk5 inhibits the activity of an enzyme that metabolizes reactive oxygen species, which then leads to neurodegeneration. These two studies hold promise for establishing treatments to prevent cognitive dysfunction and neurodegeneration.
 
Gender-based differences in the incidence of hypertensive and coronary artery disease, the development of atherosclerosis, and myocardial remodeling after infarction are attributable to the indirect effect of estrogen on risk factor profiles, such as cholesterol levels, glucose metabolism, and insulin levels. More recent evidence, however, suggests that activated estrogen receptor (ER) mediates signaling cascades that culminate in direct protective effects such as vasodilation, inhibition of response to vessel injury, limiting myocardial injury after infarction, and attenuating cardiac hypertrophy. Although the ER is usually thought of as a ligand-dependent transcription factor, it can also rapidly mobilize signals at the plasma membrane and in the cytoplasm. Thus, a greater understanding of ER function and regulation may lead to the development of highly specific therapeutics that mediate the prevention and treatment of cardiovascular diseases.
 
Top-cited authors
Jan-Ake Gustafsson
  • Nova Scotia Museum
Jason Matthews
  • University of Oslo
William M. Pardridge
  • University of California, Los Angeles
Peiying Yang
  • University of Texas MD Anderson Cancer Center
Robert A Newman
  • University of Texas MD Anderson Cancer Center