Wolfgang Sadee - US grants

This proposal focuses on the biological consequence of purine starvation in order to elucidate the mechanism of the antitumor effects of purine antimetabolites. These agents cause either general purine starvation or selective depletion of adenine or guanine alone. We have found that each class of purine depleting agents produces distinct biological effects, most notably the dramatic inhibition of DNA synthesis that occurs with guanine starvation. One portion of the project proposal deals with the interaction among the cellular toxicities caused by combined or individual adenine and guanine starvation in order to provide a biochemical rationale for the selection of purine antimetabolites as anticancer agents. The other portion addresses specifically the effects of guanine depletion alone. Inhibitors of de novo guanine nucleotide formation (via IMP dehydrogenase) rank among the most potent antiviral and antitumor agents. Yet it remains unknown why ribavirin for example is a potent antiviral agent with rather poor antitumor efficacy, while tiazofurine is a potent antitumor drug, at least in animal models. The toxicity of both agents can be reversed by the addition of a guanine source via the salvage bypass, which suggests that guanine starvation is indeed the major mode of action. Howeve, we have observed differences in the biochemical mechanism of action of these agents that may result from differential effect on guanine nucleotide metabolism. Indeed, GMP, GDP and GTP each serve different cellular functions, with GDP possibly representing the functional DNA precursor pool that feeds into the "replitase" system. The replitase is thought to be a multienzyme complex that utilizes distant DNA precursors and channels substrates directly to the site of replication forks. The proposal thus presents a novel approach to study the mechanism of the cellular toxicity of guanine depleting agents, that takes into account the possible compartmentation of guanine nucleotides (mono-, di and tri-phosphates) into general cellular pools and functional pools for specific purposes.

The binding of opiate ligands to the opiate receptor system is investigated in vivo in intact rat brain, in order to determine the relationships among opiate pharmacokinetics and metabolism, in vivo receptor binding and pharmacological effects. Current results demonstrate that there are significant qualitative and quantitative differences in opiate receptor binding in vivo and in vitro, including a partial loss of binding sensitivity to guanyl nucleotides which are thought to mediate receptor-effector coupling. Because of these in vivo - in vitro differences, new labeling techniques are being developed to study the opiate receptor multiplicity in vivo. At least three and possibly four distinct binding site populations can now be differentiated with our methods. Most strikingly, the in vivo labeling method has revealed the existence of novel type of binding sites with high selectivity for 4,5-epoxymorphinans (e.g., naloxone). These unique binding sites rapidly decay in vitro to a state of low affinity. Since receptor binding occurs in vivo, it can be correlated to the pharmacological effects; this approach may, therefore, yield information on the functions of individual receptor types. For example, results with buprenorphine suggest non-competitive auto-inhibition among the opiate receptor types. Recently, we have studied the interaction of a series of opiate agonists, mixed agonist antagonists, and antagonists to a type of in vivo binding site that resembles the Mu site characterized in vitro. This site appears to mediate the analgesic action of sufentanil and etorphine, but alone is insufficient to account for the action of mixed agonist-antagonists. Therefore, the proposed studies will include further development of the in vivo receptor labeling method and will address the question of the pharmacological functions of each type of opiate binding site. Moreover, changes of opiate receptor binding will be investigated that occur immediately after sacrifice of the aminals and homogenization of the brain tissues.

The mechanism of action of a variety of antimetabolite drug classes, most prominently those with anticancer activity, involves purine starvation; however, the mechanism of their biological cellular effects remains largely unknown. We have recently shown that purine biosynthesis inhibition results in a drastic reduction of DNA synthesis which is primarily caused by guanine, rather than adenine starvation. Consistent with this finding is the recent development of effective anticancer drugs that selectively inhibit IMP dehydrogenase and, thereby deplete only guanine. Moreover, DNA synthesis inhibition was paradoxically correlated with the depletion of GTP, rather than dGTP. Preliminary experiments aimed at resolving this paradox show that GTP depletion and DNA synthesis inhibition are associated with a selective reduction of the phosphorylation of histone H1. Modification of phosphorylation of this spacer histone is thought to affect DNA function and replication. Furthermore, labeling of histones in isolated cell nuclei with Gamma-32P-ATP and -GTP revealed that H1 is more efficiently phosphorylated with GTP than ATP. This proposal, therefore, tests the following hypotheses. i. GTP depletion causes a selective reduction of the phosphorylation of one or several sites on histone H1 or one of its variants. ii. The reduction of H1 phosphorylation by GTP depletion is linked to the inhibition of DNA synthesis. iii. A protein phosphokinase that utilized GTP may be involved in such regulation of DNA synthesis. Methods will include cell cultures, 32P labeling, enzyme isolation, peptide mapping, affinity labeling and cell mutant selection. One significant aspect of this proposed work would be the elucidation of a specific mechanism for a drug class that was formerly thought to act by a summation of multiple effects. This raises the possibility that cellular resistance to purine antimetabolites may occur through mutations affecting this specific mechanism. Moreover, this study could reveal a potential control mechanism of DNA synthesis that may normally serve to prevent cell division under unfavorable conditions.

Opioid drugs and peptides act through multiple receptors and second messenger systems, but the classical narcotic analgesics, such as morphine, interact preferentially with the mu opioid receptor. We have identified a human neuroblastoma line (SK-N-SH) that expresses abundant mu opioid receptors (50,000 sites per cell) and delta receptors (10,000 sites per cell). It represents the only mu receptor cell line currently available that can be grown continuously in tissue culture. Differentiation with retinoic acid (RA) of the neuroblastic subclone. SH-SY5Y, causes neuronal differentiation and greatly enhances opioid sensitivity, while treatment with a combination of RA and herbimycin-A yields a homogeneous neuronal culture and increases mu opioid receptor content. Optimal cell culture conditions will be defined to provide a unique model for studying the opioid receptor and its interaction with narcotic analgesics. The fully developed in vitro model will serve to determine the mechanism of opioid receptor interaction with GTP binding proteins (Gi and Go) and two second messenger systems that are opioid responsive in this line (adenylate cyclase and Ca++) important questions include the relative contributions of mu and delta receptors in regulating these processes, possible interactions between mu and delta opioid receptors, mechanisms of tolerance, such as receptor down-regulation, receptor desensitization and phosphorylation, loss of G proteins, and biochemical correlates of dependence, i.e., the cAMP overshoot. Opioid receptors will be purified from high yield cultures to obtain partial sequences from peptide fragments and to determine their post-translational modification, e.g., by phosphorylation as a potential mechanism of desensitization. Novel techniques of high molecular weight tandem mass spectrometry will be employed for peptide analysis, which also permit detailed analysis of phosphorylation, glycocylation, and acylation of the receptor protein. Further, tolerance and cross-tolerance will be studied between the opioid receptors and the alpha-2 adrenergic receptor after transfection of the alpha-2 R gene into SH-SY5Y cells. The results will address the question of homologous and heterologous desensitization of adenylate cyclase inhibition by morphine and clonidine which suppresses opioid withdrawal symptoms. In order to gain further insights into the molecular nature of the opioid receptor, we will also attempt to identify the genes encoding the opioid receptors, either from partial peptide sequences or with the use of a mammalian expression vector cloning system. Upon availability of opioid receptor genes in suitable vectors, stably transfected cell lines will be established to study receptor mechanisms under selected conditions with specified opioid receptor content, and mutations will be introduced to determine the functional domains of the opioid receptor.

The meeting has become the most important and prestigious annual meeting in the area of bio-medical research on opioid peptides, especially with respect to such topics as opioid receptors, endogenous opioid ligands and their processing as well as on mechanisms of opiate action, tolerance and physical dependence.

The overall objective of this project is to determine the molecular mechanism of activation, signal transduction, and regulation of a prototype G protein coupled receptor (GPCR), i.e., the human muscarinic cholinergic Hm1 receptor. Cholinergic deficits are a hallmark of senile dementias such as Alzheimer, and a fundamental understanding of cholinergic neurotransmission is needed in development of therapeutic strategies. This project employees a genetic approach to determine the functional domains of the Hm1 receptor by extensive mutational analysis, whit focus on three key processes that remain poorly understood for Hm1 specifically and for GPCRs in general. The first includes the molecular mechanisms of receptor activation and signal transduction via G proteins and second messenger pathways. Second, as a result of activation, the receptors undergo rapid cellular trafficking, i.e., sequestration, internalization, and recycling. The third and slower process involves the destruction of functional receptor (down-regulation_. Rapid receptor desensitization is not detectable for Hm1 in several tissue tested, and it is therefore not studied here. This laboratory has constructed numerous Hm1 mutants that permit ne for the first time to dissect these pathways and define the location of several requisite receptor domains. These include the central portion of the second intracellular loop (i2) which was unexpectedly found to play a major role in G protein activation and internalization, an S/T rich domain in the middle of the i3 loop which regulation internalization and subsequent downregulation, and a domain of the i3 loop which mediates a distinct second pathway of downregulation. As such domains have not been identified for any of the GPCRs, their complete characterization will add significantly to our understanding of GPCR regulation. Similar receptor domains exist in most GPCRs, and the general significance of these novel domains will therefore be determine d by mutational analysis of selected additional receptor genes within this large family. Because the regulatory i3 loop domain includes several serine and threonine residues, the involvement of protein kinase mediated phosphorylation in the internalization process will be tested. These studies will be extended to the analysis of similar domains in Hm2, Hm3, beta-2 adrenoceptor, and the thyrotropin releasing hormone (TRH) receptor to test the general validity of the functional domains for GPCRS. The results from this study will clarify the molecular mechanisms and the functional significance of Hm1 activation and cellular trafficking. The responsible receptor domains will then serve as a tool to isolate the target protein mediating receptor functions. Knowledge of Hm1 receptor regulation will benefit therapy of Alzheimer disease with muscarinic agonist.

pain; analgesia; neurotransmitter receptor; drug addiction; opioid receptor; travel;

The molecular mechanisms underlying narcotic tolerance and dependence remain poorly understood. Narcotic drugs primarily interact with the mu opioid receptor to produce acute effects, such as analgesia, and chronic effects, such as tolerance and dependence. A central goal of opioid research is to separate the acute from the undesirable chronic effects, in order to permit the safe use of narcotic analgesics. However, it remains to be shown whether the chronic effects result from the same signal transduction pathway via G proteins and second messengers, or from a distinct pathway. This project focusses on mu receptor regulation and cAMP signalling. We propose the hypothesis that upon agonist stimulation, the mu opioid receptor is slowly converted to a constitutively active state termed mu* (i.e., no longer requiring the presence of agonist) as a novel regulatory mechanism which had not been previously considered for neurotransmitter and hormone receptors. Further, constitutive activation to mu* is proposed to result from phosphorylation by a receptor kinase, a pathway which is independent of mu receptor coupling to second messengers. Since G protein coupled receptor (GPCR) kinases selectively phosphorylate activated receptors, the receptor can be trapped in the activated state mu* for prolonged time periods, thereby, driving narcotic tolerance and dependence. Initial results in SH-SYSY tissue culture, in U293 human kidney cells transfected with the mu receptor gene, and in an acute in vivo model of morphine tolerance and dependence in mice provide strong evidence in support of mu* playing a key role in tolerance and dependence. Kinase inhibitors were identified that prevent and reverse mu* formation in vitro and morphine tolerance and dependence in vivo, without affecting acute morphine effects. This proposal will focus on five main objectives. First, the biochemical regulation of mu receptor signal transduction via cAMP will be investigated in cells transfected with the mu receptor gene. Second, phosphorylation of the mu receptor will be studied in detail. Third, mu receptor kinases and phosphatases, and their regulation, will be studied as potential key factors determining narcotic tolerance and dependence. Fourth, kinase inhibitors will be identified as potential antiaddictive drugs. Fourth, all narcotic drugs will be reclassified according to their ability to affect C protein coupling and phosphorylation of mu and mu* for example, naloxone represents a mu antagonist with negative intrinsic activity, whereas the selective mu antagonist CTAP does not affect mu* activity (neutral antagonist) and was found to cause little withdrawal in morphine dependent mice. Neutral antagonists could serve in the treatment of narcotic dependence and overdose. The proposed studies will provide novel insights into narcotic addiction with broad potential therapeutic benefits.

9417001 Sadee This award supports a three-year cooperative research project between Dr. Wolfgang Sadee, School of Pharmacy, University of California, San Francisco and Professor Tatsuya Haga of Tokyo University. The first objective of this joint proposal is to identify those receptor domains responsible for activating the receptor kinases to initiate phosphorylation. The second objective focuses on the role of phosphorylation in protein coupling and cellular trafficking. The results are expected to reveal diverse effects of phosphorylation including desensitization of signal transduction. This proposal brings together research scientists from two countries working in the area of neuron function. Dr. Sadee and his graduate students will have the opportunity to become familiar with research techniques and practices at Tokyo University. Therefore, this proposal fulfills the objectives of the Program in its exchange and transfer of scientific knowledge through an international collaboration. ***

Bile acids and bile alcohols are to be identified in a large number of different vertebrate species. Bile acids are the water soluble end products of cholesterol metabolism, and their structure has evolved as vertebrates have evolved. The hypothesis to be tested is that bile acid structure will provide novel and useful information on the evolutionary relationships of vertebrates.

DESCRIPTION (provided by applicant): Drug addiction is a complex disorder thought to involve the interaction of environmental factors with multiple genetic factors. Combinations of genetic variations in several genes likely cooperate to increase the susceptibility of an individual to addiction. Candidate genes are those encoding drug metabolizing enzymes and transporters, neurotransmitter receptors, signaling proteins, and proteins affecting neurobiological functions, such as synaptic remodeling and memory. Polymorphisms that alter protein structure have been studied extensively, but cis-acting variations affecting gene expression and mRNA processing are likely to be more prevalent. Therefore, cis-regulatory polymorphisms might account for much of the diversity between individuals and their susceptibility to addiction. Yet, it has proven difficult to detect such cis-regulatory variants across a large panel of genes. Here, a novel approach to screen broadly for polymorphisms that affect gene expression and mRNA processing is introduced, taking advantage of single nucleotide polymorphisms (SNPs) in the transcribed region of a candidate gene. By precisely measuring the ratios of the two SNP alleles in both genomic DNA and mRNA in heterozygous carriers, one detects variations in mRNA levels, comparing one allele against the other in a relevant target tissue obtained by autopsy from multiple individuals. Genes showing significant allelic differences in mRNA levels are scanned for functional polymorphisms by haplotype and association analysis. The specific aims of this study are to: 1) Identify candidate genes with suitable marker SNPs located in transcribed regions that exhibit allele-specific mRNA differences in relevant target tissues (e.g., brain regions, liver). 2) Isolate haplotype blocks that are associated with differential allelic expression. 3) Discover functional genetic lesions associated with differential allelic expression. The results will permit us to assign functional significance to cis-regulatory polymorphisms as a guide in clinical association studies of drug addiction.

Drug addiction is a complex disorder with a strong genetic component, while the role of epigenetic factors[unreadable] remains unresolved. We propose that the interplay between genetic polymorphisms and epigenetic changes[unreadable] determines gene expression and possibly mRNA processing, serving a critical role in drug addiction. A large[unreadable] portion of suspected addiction susceptibility genes harbors CpG islands, methylation of which represents a[unreadable] main epigenetic mechanism. Both genetic and epigenetic factors likely contribute to addiction susceptibility[unreadable] and physiological changes occurring as a result of substance abuse. We will study these factors in autopsy[unreadable] tissues from the Miami Brain Endowment Bank, containing ~approximately 500 samples from cocaine and other drug[unreadable] abusers and age-matched controls. This repository enables genetic and epigenetic studies in relevant brain[unreadable] regions involved in addiction. CpG methylation can occur randomly between the two allele of a gene[unreadable] (represented in overall expression level), or in an allele-selective fashion. The latter causes an allelic[unreadable] expression imbalance (AEI), which represents a precise and quantitative phenotype for both genetic and[unreadable] epigenetic cis-acting factors. This permits us to address several questions. How does CpG island[unreadable] methylation vary across brain regions, and what is the variability among individuals? Does methylation affect[unreadable] gene expression, alternate promoter usage, or alternative splicing? What is the effect of substance abuse[unreadable] on CpG island methylation in candidate genes, in relevant brain regions? Do epigentic and genetic factors[unreadable] contribute to clinical status (addiction)? In this project, we target genes harboring CpG islands that are[unreadable] implicated in addiction, focusing on biogenic amine pathways, encoding synthetic and catabolic enzymes,[unreadable] vesicular and synaptic reuptake transporters, and receptors (MAOA, MAOB, COMT, TH, DAT, NET, VMAT2,[unreadable] DRD2, CHRNA4). We have developed high-throughput tools for measuring the genetic and epigenetic[unreadable] contribution to mRNA and protein expression, and alternative splicing. Our assays are allele-specific,[unreadable] enabling the evaluation of genetic and epigenetic factors in allelic expression, a powerful tool for assessing[unreadable] the quantitative impact of each factor. This novel approach, applied to anatomically defined brain tissues[unreadable] from drug addicts and controls, has the potential to yield significant insight into the role of and interplay[unreadable] between genetic and epigenetic factors, and add to our understanding of susceptibility to addiction.[unreadable]

[unreadable] DESCRIPTION (provided by applicant): Summary The neurotransmitter serotonin (5-hydroxytryptamine; 5-HT) plays a crucial role in developing and adult brain. Disruption of serotonergic activity has been proposed to contribute to major mental and neurodevelopmental disorders, including autism, major depression, anxiety disorders and schizophrenia. How serotoninergic activity is affected by genetic differences among individuals, however, is poorly understood. We hypothesize that genetic variants causing exceptionally high- or low-levels of gene expression alter the development of the brain in ways that predispose certain individuals to develop serious disorders as children or adults. The goal of this study is to identify common genetic variants (polymorphisms) that regulate the expression of key serotonin-related genes in developing and adult human brain. Using autopsy tissue samples, we will quantify the expression of mRNAs for 15 serotonin-related genes in developing and adult pons, hippocampus and frontal cortex in an allele-specific manner. Quantifying levels of mRNA produced from two alleles within each brain sample allows allele-specific differences in expression to be detected without making comparisons between samples. This novel approach greatly increases the accuracy and reproducibility of the measurements. Based upon our previous experience, we expect 3-4 of our selected genes to show significant and frequent allelic expression imbalance (AEI) in developing and/or adult brain. AEI is caused by regulatory polymorphisms located within or near the corresponding gene. Since expression is context-dependent, it is possible that the extent of AEI differs between brain regions and/or between developing and adult brain. One of the major objectives of this project is to determine whether AEI for certain genes occurs only in the developing brain, as has been proposed for the serotonin transporter (SERT). For genes that show AEI, we will search the neighborhood of the gene for single nucleotide polymorphisms (SNPs) for which heterozygosity correlates with AEI. Highly correlated SNPs will be further studied to determine whether they play a direct role in mRNA expression. We will first focus on AEI mechanisms for tryptophan hydroxylase 2 (TPH2) for which we have previously identified several highly correlated SNPs. Validated regulatory polymorphisms in TPH2 and other serotonin-related genes will be valuable biomarkers for diagnosing and analyzing major mental and neurodevelopmental disorders. -Relevance The goal of this study is to identify common regulatory variants in genes related to serotonin, a neurotransmitter that plays important roles in the developing and adult brain. We hypothesize that genetic variants that significantly alter the expression of key serotonin-related genes contribute to mental and neurodevelopmental disorders, including autism, major depression, anxiety disorders and schizophrenia. Identifying genetic variants that regulate key serotonin-related genes will be a significant step toward developing new methods for the diagnosis, treatment and, possibly, prevention of these disorders. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Understanding the genetic basis of disease and drug response has the potential to improve therapy and enable early intervention or prevention. However, the main genetic factors remain only partially understood, even while the number of candidate genes is rapidly growing, as a result of genome-wide association studies. Polymorphisms that alter protein sequence are readily detectable, but growing evidence indicates that regulatory polymorphisms are more prevalent, affecting mRNA expression, processing, and translation. Yet, regulatory variants are difficult to detect, and moreover, their functions depend on tissue context and environment, so that a majority remains hidden. The central goal of this proposal is a comprehensive discovery of regulatory polymorphisms in -200 pharmacotherapeutic candidate genes, followed by molecular studies to understand the underlying mechanisms, and clinical evaluation in drug therapy - the first such systematic study in pharmacogenomics. We have developed a comprehensive approach to the discovery of regulatory polymorphisms, measuring allelic mRNA expression, processing, and translation in relevant human target tissues. This approach has already revealed unexpected and frequent regulatory variants in genes encoding drug metabolizing enzymes and receptors, gaining a powerful link between genotype of proven function and clinical outcomes (examples: DRD2, TPH2, ACE, VK0RC1, CETP, and CYP3A4). These results support a critical role for regulatory polymorphisms in drug response. The main focus in this proposal is on drug metabolism genes and impact on pharmacokinetics-pharmacodynamics. In addition, building on other ongoing studies, the project includes genes encoding drug receptors/targets, with focus on CNS disorders (schizophrenia) and cardiovascular diseases (myocardial infarction, lipid metabolism), to be tested in association studies led by experienced clinical scientists. Driven by the motto 'from clinic to laboratory', new genetic studies have been initiated on estrogen and glucocorticoid receptors, the latter to be tested in glucocorticoid-resistant nephrotic syndrome in children. The long-term goal is to develop and validate genetic biomarker panels for optimizing personalized drug therapy. RELEVANCE: Advances in genomic sciences have raised expectations that drug therapy can be tailored to the individual patient. However, a large portion of genetic variability remains to be discovered. The 'Expression Genetics in Drug Therapy'program aims to fill an important gap, through a systematic study of gene regulation of drug metabolizing enzymes and receptors. We expect to develop genetic biomarkers for optimizing drug therapy.

Adult; Adverse effects; Affect; Area; Behavior; Biological Availability; Birth; Birth Weight; Blood; Blood - brain barrier anatomy; Blood Circulation; Brain; Buprenorphine; Cavia; Clinic; Clinical; Data; Development; dosage; Dose; Drug Kinetics; Emergency Care; exposed human population; Exposure to; Female; fetal; Fetal Development; Fetus; Goals; Hand; Human; human subject; Impairment; in utero; Infant Care; Liver; Maintece; Measures; Mediating; meetings; Methadone; Modeling; Mothers; Mus; Naloxone; naltrexol; Naltrexone; Neonatal; Neonatal Abstinence Syndrome; neonate; Newborn Infant; novel strategies; Opiate Addiction; Opioid; Opioid Antagonist; Opioid replacement therapy; opioid withdrawal; Oral; Oral Administration; palliative; patient population; Penetration; Perinatal; Peripheral; pharmacokinetic model; Phase; Physiology; Placenta; Plasma; postnatal; preempt; Pregcy; pregt; Pregt Women; Premature Birth; prenatal therapy; Preparation; prevent; Prevention; Procedures; Publishing; pup; Regimen; Risk; Route; Safety; Second Pregcy Trimester; Testing; Third Pregcy Trimester; Time; Toxic effect; Toxicology; Treatment Protocols; treatment strategy; Withdrawal; Withdrawal Symptom; Woman;