Current Medicinal Chemistry - Volume 11, Issue 7, 2004

The Sir2 family of enzymes is a recently described class of NAD+-dependent protein deacetylases that use NAD+ as a reactant to deacetylate acetyllysine residues of protein substrates to form the aminolysine sidechain and a novel product 2’-O-acetyl-ADP-ribose. The founding member of the Sir2 proteins, the yeast Sir2p, has been identified as a key member of SIR complexes responsible for the long-term silencing of genes in the yeast Saccharomyces cerevisiae. Increase of Sir2 activity by caloric restriction or osmotic stress increases genome stability and lifespan in this organism. The Sir2 reaction mechanism couples ADPribosyltransfer and hydrolysis reactions via the formation of a stabilized ADPR-peptidyl intermediate. Principles of the chemistry of stabilized ADPR intermediates are examined for Sir2 and the mechanistically related ADP-ribosylcyclase CD38. An examination of the crystal structures of Sir2 family members is presented with a view to the chemical requirements of the Sir2 reaction. The present review describes the current knowledge of the Sir2 reaction, the reaction mechanism and the regulation of Sir2.

Cyclic ADP-ribose (cADPR), a general mediator involved in Ca2+ signaling, has the characteristic 18-membered ring consisting of an adenine, two riboses and a pyrophosphate, in which the two primary hydroxyl groups of the riboses are linked by a pyrophosphate unit. This review focuses on the chemical synthetic studies of cADPR analogs. These analogs have been used quite effectively in proving the mechanism of cADPR-mediated Ca2+ signaling pathways. These analogs are also expected to be lead structures for the development of drugs. Although cADPR analogs can be synthesized by enzymatic and chemo-enzymatic methods using ADP-ribosyl cyclase, the analogs obtained by these methods are limited due to the substrate-specificity of the enzymes. Consequently, chemical synthetic methods providing a greater variety of cADPR analogs are required. Chemical synthetic studies have demonstrated that the construction of the large 18-membered ring structure is quite difficult. Another problem encountered in the synthesis is the construction of the N1-substituted purine nucleoside structure. The N1- substituted inosine derivatives were prepared by condensation between the N1-(2,4-dinitrophenyl)inosine derivatives and the appropriate amines. For the preparation of the N1-substituted adenosine structures, condensation of the 4-cyano-5-(alkoxymethyleneamino)imidazole nucleosides with the appropriate amines was found to be effective. The first chemical construction of the 18-membered ring was achieved using a bisphosphate-type substrate conformationally restricted in the cyclized product-like syn-form around the N9- glycosyl linkage; however, the yield was inadequate. The key 18-membereding construction was significantly improved by employing the phenylthiophosphate-type substrates. When the substrates were activated by AgNO3 or I2 in the presence of molecular sieves in pyridine, the corresponding 18-membered ring products were obtained in high yields. Using this method as the key step, the chemically and biologically stable cADPR mimic, cADP-carbocyclic-ribose (cADPcR), was synthesized. This method has been applied subsequently to the synthesis of various cADPR analogs.

Cyclic adenosine diphosphoribose (cADPR) is an endogenous Ca2+ mobilizing nucleotide in many cell types and different species covering protozoa, plants and animals, including humans. cADPR is formed by ADP-ribosyl cyclases from nicotinamide adenine dinucleotide (NAD). Since at least some of the ADP-ribosyl cyclases are under the control of receptors for exogenous ligands, cADPR is regarded as a second messenger for Ca2+ signaling. The main intracellular target for cADPR is the ryanodine receptor, but it is unclear whether cADPR elicits Ca2+ release by direct binding or via a binding protein. Derivatives of NAD and cADPR are potent ADP-ribosyl cyclase inhibitors and cADPR antagonists. Since Ca2+ ions are regulators of many diverse cell functions, e.g. muscle contraction, secretion of neurotransmitters, hormones and enzymes, fertilization of oocytes, and lymphocyte activation and proliferation, the cADPR signaling pathway may become a valuable target for pharmaceutical intervention.

Mammalian ecto ADP-ribosyltransferases (ARTs) constitute a family of structurally related proteins expressed on the cell surface or secreted in the extracellular compartment. Using NAD+ as substrate, they transfer ADP-ribose groups onto target proteins. In contrast to intracellular poly(ADP-ribosyl)transferases (PARPs), these enzymes transfer a single ADPR and are thus mono-ARTs. Five paralogs (ART1-5) have been cloned but only four of them are expressed in human due to a defective ART2 gene, and six in the mouse as the result of ART2 gene duplication. The recent determination of the crystal structure of rat ART2 reveals homologies with bacterial ART toxins and provides a molecular basis for understanding the specificity of ARTs for their targets. A combination of different technological approaches reveals that ecto-ARTs are expressed in different tissues with privileged sites such as heart and skeletal muscles for ART1, T lymphocytes for ART2 or testis for ART5. It also indicates that ART expression is highly regulated. ADP-ribosylation of target proteins on cell surfaces or circulating in body fluids leads to reversible post-translational modifications which can inhibit the targets, as known for bacterial ARTs, or activate them, as in the crosstalk between mouse ART2 and the cytolytic P2X7 receptor on T lymphocytes. ART activity in the extracellular compartment provides sophisticated regulatory mechanisms for cell communication. This designates ecto- ARTs as new candidates for drug targeting.

The enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT), a member of the nucleotidyltransferase α / β phosphodiesterase superfamily, catalyzes the reaction NMN + ATP = NAD + PPi, representing the final step in the biosynthesis of NAD, a molecule playing a fundamental role as a cofactor in cellular redox reactions. NAD also serves as the substrate for reactions involved in important regulatory roles, such as protein covalent modifications, like ADP-ribosylation reactions, as well as Sir2 histone deacetylase, a recently discovered class of enzymes involved in the regulation of gene silencing. This overview describes the most recent findings on NMNATs from bacteria, archaea, yeast, animal and human sources, with detailed consideration of their major kinetic, molecular and structural features. On this regard, the different characteristics exhibited by the enzyme from the various species are highlighted. The possibility that NMNAT may represent an interesting candidate as a target for the rational design of selective chemotherapic agents has been suggested.

IMP dehydrogenase, the key enzyme in de novo synthesis of purine nucleotides, is an important therapeutic target. Three inhibitors of IMP dehydrogenase reached the market; ribavirin (Rebetol) a broadspectrum antiviral agent, which in combination with interferon-α is now used for treatment of hepatitis C virus infections, mizoribine (Bredinin) and mycophenolic mofetil (CellCept) have been introduced as immunosuppressants. Numerous novel inhibitors are under development. This review describes recent progress in the development of new drugs based on inhibition of IMP dehydrogenase.

Nutrient excess is associated with reduced insulin sensitivity (insulin resistance) and plays a central role in the pathogenesis of type 2 diabetes. Recently, free fatty acids as well as amino acids were shown to induce insulin resistance by decreasing glucose transport / phosphorylation with subsequent impairment of glycogen synthesis in human skeletal muscle. These results do not support the traditional concept of direct substrate competition with glucose for mitochondrial oxidation but indicate that the cellular mechanisms of such lipotoxicity and “proteotoxicity” might primarily affect the insulin signaling cascade. The signaling pathways involved in nutrient dependent modulation of insulin action include protein kinase C isoforms and IκB kinase. Therefore, pharmacological modulation of these enzymes might represent a promising target for future treatment of insulin resistance. Finally, hyperglycemia which occurs late in the insulin resistance syndrome further augments insulin resistance by mechanisms summarized as glucose toxicity. Chronic hyperglycemia might lead to inhibition of lipid oxidation and thereby to accumulation of intracellular lipid metabolites. Therefore, glucotoxicity might be in part indirectly caused by lipotoxicity (glucolipotoxicity). In conclusion, different nutrients affect common metabolic pathways and thereby induce insulin resistance in humans.

It has been strongly suggested that androgens and the cognate receptor (AR) may play important roles in the development and progression of prostate cancer. The AR is a transcription factor consisting of three major domains, i.e., N-terminal transactivation, middle DNA binding, and C-terminal steroid binding domains. Molecular events of androgen induced activation of the AR include conformation change, phosphorylation, acetylation, genomic DNA binding, and co-regulator recruiting. Many of these events can be manipulated in certain prostate cancer cells in favor of their progression. Dietary compounds and certain herbs have recently drawn a great deal of attention because of their relevance to development of several cancers including prostate cancer. We discuss in depth the findings from our and other laboratories of effects of dietary factors or herbs on the function of the AR and potential mechanisms on expression of the AR and AR regulated genes. We further discuss the potential implication of these dietary chemicals on prevention of development and progression of prostate cancer.

Since the introduction of tricyclic antidepressants (TCAs) and monoamine oxidase inhibitors (MAOIs) in mid-1950’s, treatment of depression has been dominated by monoamine hypotheses. The wellestablished clinical efficacy of TCAs and MAOIs is due, at least in part, to the enhancement of noradrenergic or serotonergic mechanisms, or to both. Unfortunately, their very broad mechanisms of action also include many unwanted effects related to their potent activity on cholinergic, adrenergic and histaminergic receptors. The introduction of selective serotonin reuptake inhibitors (SSRIs) over twenty years ago had been the next major step in the evolution of antidepressants to develop drugs as effective as the TCAs but of higher safety and tolerability profile. During the past two decades SSRIs (fluoxetine, fluvoxamine, paroxetine, sertraline, citalopram) gained incredible popularity and have become the most widely prescribed medication in the psychiatric practice. The evolution of antidepressants continued resulting in introduction of selective and reversible monoamine oxidase inhibitors (eg. moclobemid), selective noradrenaline (eg. reboxetine), dual noradrenaline and serotonin reuptake inhibitors (milnacipram, venlafaxin, duloxetin) and drugs with distinct neurochemical profiles such as mirtazapine, nefazadone and tianeptine. Different novel serotonin receptor ligands have also been intensively investigated. In spite of the remarkable structural diversity, most currently introduced antidepressants are ‘monoamine based’. Furthermore, these newer agents are neither more efficacious nor rapid acting than their predecessors and approximately 30% of the population do not respond to current therapies. By the turn of the new millennium, we are all witnessing a result of innovative developmental strategies based on the better understanding of pathophysiology of depressive disorder. Several truly novel concepts have emerged suggesting that the modulation of neuropeptide (substance P, corticotrophin-releasing factor, neuropeptide Y, vasopressin V1b, melanin-concentrating hormone-1), N-methyl-D-aspartate, nicotinic acetylcholine, dopaminergic, glucocorticoid, ä-opioid, cannabinoid and cytokine receptors, gamma-amino butyric acid (GABA) and intracellular messenger systems, transcription, neuroprotective and neurogenic factors, may provide an entirely new set of potential therapeutic targets, giving hope that further major advances might be anticipated in the treatment of depressive disorder soon. The goal of this review is to give a brief overview of the major advances from monoamine-based treatment strategies, and particularly focus on the new emerging approaches in the treatment of depression.