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Guided by co-crystal structures of compounds 15, 22 and 30, an SBDD approach led to the discovery of the 6-methyl pyridone series as a novel class of GKAs that potently activate GK in enzyme and cell assays. Anti-diabetic OGTT efficacy was demonstrated with 54 in a mouse model of type 2 diabetes., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

PKC (protein kinase C) θ is predominantly expressed in T-cells and is critically involved in immunity. Design of PKCθ-selective molecules to manage autoimmune disorders by targeting its activator-binding C1 domain requires the knowledge of its structure and the activator-binding residues. The C1 domain consists of twin C1 domains, C1A and C1B, of which C1B plays a critical role in the membrane translocation and activation of PKCθ. In the present study we determined the crystal structure of PKCθC1B to 1.63 Å (1 Å=0.1 nm) resolution, which showed that Trp(253) at the rim of the activator-binding pocket was orientated towards the membrane, whereas in PKCδC1B the homologous tryptophan residue was orientated away from the membrane. This particular orientation of Trp(253) affects the size of the activator-binding pocket and the membrane affinity. To further probe the structural constraints on activator-binding, five residues lining the activator-binding site were mutated (Y239A, T243A, W253G, L255G and Q258G) and the binding affinities of the PKCθC1B mutants were measured. These mutants showed reduced binding affinities for phorbol ester [PDBu (phorbol 12,13-dibutyrate)] and diacylglycerol [DOG (sn-1,2-dioctanoylglycerol), SAG (sn-1-stearoyl 2-arachidonyl glycerol)]. All five full-length PKCθ mutants exhibited reduced phorbol-ester-induced membrane translocation compared with the wild-type. These results provide insights into the PKCθ activator-binding domain, which will aid in future design of PKCθ-selective molecules.

Glucokinase (GK) is a new target for the treatment of type II diabetes mellitus (T2DM). In order to find a structure-simplified small molecule GK activator, 19 salicylic acid derivatives were designed and synthesized based on new lead compound (1). Experimental results showed that the potency of compound 8h is superior to control RO-28-0450 in GK activation.

Rationale: The efficiency of Sirtuin1, a major target for the treatment of various metabolic disorders such as inflammation and type 2 diabetes mellitus, can be modulated via low molecular mass SIRT1 activators (e.g. resveratrol, SRT1720, and SRT2104).The administration of such compounds results in increased deacetylation of substrates including p53, FOXO1, and PGC1alpha, potentially leading to an improved physical performance. Consequently, proactive and preventive anti-doping measures are required and an assay dedicated to serum and plasma was desirable., Methods: Model substances of emerging SIRT1 drug candidates were obtained and synthesized and their mass spectrometric behavior following positive or negative electrospray ionization and collision-induced dissociation was elucidated using low and high resolution/high accuracy (tandem) mass spectrometry. Subsequently, a screening and confirmation procedure necessitating 100 μL of plasma was established employing liquid chromatography/tandem mass spectrometry (LC/MS/MS) based on diagnostic ion transitions recorded in multiple reaction monitoring mode. Sample preparation consisted of the addition of two deuterated internal standards (D(8)-SRT1720 and D(4)-resveratrol) to the plasma specimen and subsequent protein precipitation., Results: Characteristic product ions indicative of the core structures of the model analytes were characterized and utilized for the development of a multi-analyte LC/MS/MS detection method applicable to sports drug testing programs. The doping control assay was validated with regard to specificity, limits of detection (0.1-1 ng/mL), recoveries (90-98%), intraday and interday precisions (2-18%), and ion suppression/enhancement effects., Conclusions: The fragmentation pathways of SRT1720 and 4 SIRT1 activator models based on a common thiazole-imidazole nucleus as well as two different complementary activators (SIRT1 activator 3 and CAY10602), comprising a quinoxaline core, were studied. The resulting information was used to establish and validate a sports drug testing methodology relevant for an efficient and timely anti-doping procedure, targeting a new class of emerging therapeutics possessing significant potential for misuse in elite and amateur sport., (Copyright © 2012 John Wiley & Sons, Ltd.)

Background: Schizophrenia is a neurodegenerative disorder that occurs worldwide and can be difficult to diagnose. It is the foremost neurological disorder leading to suicide among patients in both developed and underdeveloped countries. D-amino acid oxidase activator (DAOA), also known as G72, is directly implicated in the glutamateric hypothesis of schizophrenia. It activates D-amino acid oxidase, which oxidizes D-serine, leading to modulation of the N-methyl-D-aspartate receptor., Methods: MODELLER (9v10) was utilized to generate three dimensional structures of the DAOA candidate gene. The HOPE server was used for mutational analysis. The Molecular Evolutionary Genetics Analysis (MEGA5) tool was utilized to reconstruct the evolutionary history of the candidate gene DAOA. AutoDock was used for protein-ligand docking and Gramm-X and PatchDock for protein-protein docking., Results: A suitable template (1ZCA) was selected by employing BLASTp on the basis of 33% query coverage, 27% identity and E-value 4.9. The Rampage evaluation tool showed 91.1% favored region, 4.9% allowed region and 4.1% outlier region in DAOA. ERRAT demonstrated that the predicted model had a 50.909% quality factor. Mutational analysis of DAOA revealed significant effects on hydrogen bonding and correct folding of the DAOA protein, which in turn affect protein conformation. Ciona was inferred as the outgroup. Tetrapods were in their appropriate clusters with bifurcations. Human amino acid sequences are conserved, with chimpanzee and gorilla showing more than 80% homology and bootstrap value based on 1000 replications. Molecular docking analysis was employed to elucidate the binding mode of the reported ligand complex for DAOA. The docking experiment demonstrated that DAOA is involved in major amino acid interactions: the residues that interact most strongly with the ligand C28H28N3O5PS2 are polar but uncharged (Gln36, Asn38, Thr 122) and non-polar hydrophobic (Ile119, Ser171, Ser21, Ala31). Protein-protein docking simulation demonstrated two ionic bonds and one hydrogen bond involving DAOA. Lys-7 of the receptor protein interacted with Lys-163 and Asp-2037. Tyr-03 interacted with Arg-286 of the ligand protein and formed a hydrogen bond., Conclusion: The predicted interactions might serve to inhibit the disease-related allele. It is assumed that current bioinformatics methods will contribute significantly to identifying, analyzing and curing schizophrenia. There is an urgent need to develop effective drugs for schizophrenia, and tools for examining candidate genes more accurately and efficiently are required.

The present review is intended to focus on naturally occurring cytoprotective agents such as resveratrol (trans-3,4',5-trihydroxystilbene) and other related compounds, probably with similar molecular mechanisms of action and high capacity to find applications in medical fields. Several physiological aspects have been ascribed to resveratrol and similar compounds. Resveratrol, among others, has been recently described as a silent information regulator T1 (SIRT1) activator that increases AMP-activated protein kinase (AMPK) phosphorylation and reduces the oxidative damage biomarkers during aging in laboratory settings. The reports on resveratrol and other SIRT1 activators from various sources are encouraging. The pharmacological strategies for modulation of sirtuins by small molecules through allosteric mechanisms should gain a greater momentum including human research. Resveratrol and resveratrol-like molecules seem to fulfill the requirement of a new horizon in drug research since these molecules cover a growing research means as antioxidants with allosteric mechanism in epigenetic drug targets. However, one should keep in mind the challenges of extrapolation of basic research into clinical results. Overall, the issue of sirtuins in biology and disease provides an insight on therapeutic potentials of sirtuin-based therapeutics and demonstrates the high complexity of drug-targeting these modalities for human applications.

Protein phosphatases of the type 2A family (PP2A) represent a major fraction of cellular Ser/Thr phosphatase activity in any given human tissue. In this review, we describe how the holoenzymic nature of PP2A and the existence of several distinct PP2A composing subunits allow for the generation of multiple structurally and functionally different PP2A complexes, explaining why PP2A is involved in the regulation of so many diverse cell biological and physiological processes. Moreover, in human disease, most notably in several cancers and Alzheimer's Disease, PP2A expression and/or activity have been found significantly decreased, underscoring its important functions as a major tumor suppressor and tau phosphatase. Hence, several recent preclinical studies have demonstrated that pharmacological restoration of PP2A activity, as well as pharmacological PP2A inhibition, under certain conditions, may be of significant future therapeutic value.

Soluble guanylate cyclase (sGC) is the primary mammalian nitric oxide (NO) sensor. Through the strong thermodynamic σ-trans effect of NO, binding of NO at the distal side of the ferrous heme induces cleavage of the proximal FeN(His) bond, activating the catalytic domain of the enzyme. It has been proposed that nitroxyl (HNO) is also capable of activating sGC, but the key question remains as to whether HNO can induce cleavage of the FeN(His) bond. Here we report calculated binding constants for 1-methylimidazole (MI) to [Fe(P)(X)] (P=porphine(2-)) where X=NO, HNO, CO, and MI to evaluate the trans interaction of these groups, X, with the proximal imidazole (histidine) in sGC. Systematic assessment of DFT methods suggests that the prediction of accurate MI binding constants is critically dependent on the inclusion of van der Waals interactions (-D functionals). Calculated (B3LYP-D/TZVP) MI binding constants for X=NO and MI are 110 and 5.6 × 10(5)M(-1), respectively, predicted only one order of magnitude higher than the corresponding experimentally determined values. MI binding constants where X=HNO and CO are consistently predicted to be essentially equal and ~six orders of magnitude larger than those of NO, indicating that CO and HNO mediate a weak thermodynamic trans effect in this system. Orbital analysis of the key σ-bonding orbital, π*(h)_d(z2), and comparison of FeN(MI) bond lengths support this prediction. This suggests that HNO does not induce a σ-trans effect strong enough to promote cleavage of the FeN(His) bond-a key step in the activation of sGC., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Small molecule modulators of phosphatases can lead to clinically useful drugs and serve as invaluable tools to study functional roles of various phosphatases in vivo. Here, we describe lead discovery strategies for identification of inhibitors and activators of intestinal alkaline phosphatases. To identify isozyme-selective inhibitors and activators of the human and mouse intestinal alkaline phosphatases, ultrahigh throughput chemiluminescent assays, utilizing CDP-Star as a substrate, were developed for murine intestinal alkaline phosphatase (mIAP), human intestinal alkaline phosphatase (hIAP), human placental alkaline phosphatase (PLAP), and human tissue-nonspecific alkaline phosphatase (TNAP) isozymes. Using these 1,536-well assays, concurrent HTS screens of the MLSMR library of 323,000 compounds were conducted for human and mouse IAP isozymes monitoring both inhibition and activation. This parallel screening approach led to identification of a novel inhibitory scaffold selective for murine intestinal alkaline phosphatase. SAR efforts based on parallel testing of analogs against different AP isozymes generated a potent inhibitor of the murine IAP with IC50 of 540 nM, at least 65-fold selectivity against human TNAP, and >185 selectivity against human PLAP.

Glucokinase activator is expectedly associated with a dual mechanism for lowering blood glucose concentration by the enhancement of glucose uptake in the liver and insulin secretion from pancreatic beta cell. Therefore, glucokinase has been an attractive target for anti-diabetic therapy. Novel benzamide derivatives were synthesized and tested using in vitro assays by measuring fold increase of glucokinase activity at 5.0 mM glucose concentration. Among the prepared compounds, YH-GKA was found to be an active glucokinase activator with EC(50) of 70 nM and glucose area under the curve reduction of 29.6% at 50 mg/kg in an oral glucose tolerance test. In a subchronic study with ob/ob mice, YH-GKA showed significant decrease in blood glucose levels and no adverse effects on serum lipids or body weight. Overall, YH-GKA is a promising candidate for the therapy of type 2 diabetes mellitus.

Modification on the catalytic core of 10-23 DNAzyme with protein-like functional groups is a potential approach to obtain its more efficient analogs. In our efforts for this purpose, a lead structure (DZ-2-9) with 8-aza-7-deaza-2'-deoxyadenosine at the A9 position in its catalytic core was obtained. Here we report our structure-activity relationship studies on this lead structure. Various functional groups of different chemical properties were introduced through the 7-substituents of 8-aza-7-deaza-2'-deoxyadenosine to DZ-2-9. The functional groups capable of forming hydrogen bonds, like amino and hydroxyl groups, are more favorable for catalytic rate enhancement than the large groups with spacial occupation, like phenyl and tert-butylphenyl groups, and the flexible alkyl linkage was the more preferred choice for optimizing their positive effect. Furthermore, they exerted positive effect cooperatively with the N8 atom. These results give us a clear hint in the design of compounds for A9 substitution of 10-23 DNAzyme for more efficient DNAzymes.

Various newly prepared and previously known vitamin B₁₂ derivatives have been studied as potential soluble guanylyl cyclase (sGC) activators. All compounds tested were found to activate the sGC enzyme, although to differing extents. The best results were obtained with the derivatives synthesized from c-lactone and possessing aliphatic amides in the c- and d-positions.

Sirtuins 1-7 (SIRT1-7) belong to the third class of deacetylase enzymes, which are dependent on NAD(+) for activity. Sirtuins activity is linked to gene repression, metabolic control, apoptosis and cell survival, DNA repair, development, inflammation, neuroprotection, and healthy aging. Because sirtuins modulation could have beneficial effects on human diseases there is a growing interest in the discovery of small molecules modifying their activities. We review here those compounds known to activate or inhibit sirtuins, discussing the data that support the use of sirtuin-based therapies. Almost all sirtuin activators have been described only for SIRT1. Resveratrol is a natural compound which activates SIRT1, and may help in the treatment or prevention of obesity, and in preventing tumorigenesis and the aging-related decline in heart function and neuronal loss. Due to its poor bioavailability, reformulated versions of resveratrol with improved bioavailability have been developed (resVida, Longevinex(®) , SRT501). Molecules that are structurally unrelated to resveratrol (SRT1720, SRT2104, SRT2379, among others) have been also developed to stimulate sirtuin activities more potently than resveratrol. Sirtuin inhibitors with a wide range of core structures have been identified for SIRT1, SIRT2, SIRT3 and SIRT5 (splitomicin, sirtinol, AGK2, cambinol, suramin, tenovin, salermide, among others). SIRT1 inhibition has been proposed in the treatment of cancer, immunodeficiency virus infections, Fragile X mental retardation syndrome and for preventing or treating parasitic diseases, whereas SIRT2 inhibitors might be useful for the treatment of cancer and neurodegenerative diseases., (Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.)

Members of the 2-hydroxyacyl-CoA dehydratase enzyme family catalyze the β,α-dehydration of various CoA-esters in the fermentation of amino acids by clostridia. Abstraction of the nonacidic β-proton of the 2-hydroxyacyl-CoA compounds is achieved by the reductive generation of ketyl radicals on the substrate, which is initiated by the transfer of an electron at low redox potentials. The highly energetic electron needed on the dehydratase is donated by a [4Fe-4S] cluster containing ATPase, termed activator. We investigated the activator of the 2-hydroxyisocaproyl-CoA dehydratase from Clostridium difficile. The activator is a homodimeric protein structurally related to acetate and sugar kinases, Hsc70 and actin, and has a [4Fe-4S] cluster bound in the dimer interface. The crystal structures of the Mg-ADP, Mg-ADPNP, and nucleotide-free states of the reduced activator have been solved at 1.6-3.0 Å resolution, allowing us to define the position of Mg(2+) and water molecules in the vicinity of the nucleotides and the [4Fe-4S] cluster. The structures reveal redox- and nucleotide dependent changes agreeing with the modulation of the reduction potential of the [4Fe-4S] cluster by conformational changes. We also investigated the propensity of the activator to form a complex with its cognate dehydratase in the presence of Mg-ADP and Mg-ADPNP and together with the structural data present a refined mechanistic scheme for the ATP-dependent electron transfer between activator and dehydratase.

Somatic missense mutations in PIK3CA, which encodes the p110α catalytic subunit of phosphoinositide 3-kinases, occur frequently in human cancers. Activating mutations spread across multiple domains, some of which are located at inhibitory contact sites formed with the regulatory subunit p85α. PIK3R1, which encodes p85α, also has activating somatic mutations. We find a strong correlation between lipid kinase and lipid-binding activities for both wild-type (WT) and a representative set of oncogenic mutant complexes of p110α/p85α. Lipid binding involves both electrostatic and hydrophobic interactions. Activation caused by a phosphorylated receptor tyrosine kinase (RTK) peptide binding to the p85α N-terminal SH2 domain (nSH2) induces lipid binding. This depends on the polybasic activation loop as well as a conserved hydrophobic motif in the C-terminal region of the kinase domain. The hotspot E545K mutant largely mimics the activated WT p110α. It shows the highest basal activity and lipid binding, and is not significantly activated by an RTK phosphopeptide. Both the hotspot H1047R mutant and rare mutations (C420R, M1043I, H1047L, G1049R and p85α-N564D) also show increased basal kinase activities and lipid binding. However, their activities are further enhanced by an RTK phosphopeptide to levels markedly exceeding that of activated WT p110α. Phosphopeptide binding to p110β/p85α and p110δ/p85α complexes also induces their lipid binding. We present a crystal structure of WT p110α complexed with the p85α inter-SH2 domain and the inhibitor PIK-108. Additional to the ATP-binding pocket, an unexpected, second PIK-108 binding site is observed in the kinase C-lobe. We show a global conformational change in p110α consistent with allosteric regulation of the kinase domain by nSH2. These findings broaden our understanding of the differential biological outputs exhibited by distinct types of mutations regarding growth factor dependence, and suggest a two-tier classification scheme relating p110α and p85α mutations with signalling potential.

A small series of benzisoxazole analogs that effectively activate glycogen synthase (GS) was prepared in WO2011057956. These novel GS activators are claimed to be beneficial for the treatment or prophylaxis of metabolic disease and disorders. The 1,2-benzisoxazole-3-ol moiety is utilized in the present patent as a bioisoster of benzoic acid, which has often been employed in prior examples of the GS activators.

Bacterial plasminogen (Pg) activators generate plasmin to degrade fibrin blood clots and other proteins that modulate the pathogenesis of infection, yet despite strong homology between mammalian Pgs, the activity of bacterial Pg activators is thought to be restricted to the Pg of their host mammalian species. Thus, we found that Streptococcus uberis Pg activator (SUPA), isolated from a Streptococcus species that infects cows but not humans, robustly activated bovine but not human Pg in purified systems and in plasma. Consistent with this, SUPA formed a higher avidity complex (118-fold) with bovine Pg than with human Pg and non-proteolytically activated bovine but not human Pg. Surprisingly, however, the presence of human fibrin overrides the species-restricted action of SUPA. First, human fibrin enhanced the binding avidity of SUPA for human Pg by 4-8-fold in the presence and absence of chloride ion (a negative regulator). Second, although SUPA did not protect plasmin from inactivation by α(2)-antiplasmin, fibrin did protect human plasmin, which formed a 31-fold higher avidity complex with SUPA than Pg. Third, fibrin significantly enhanced Pg activation by reducing the K(m) (4-fold) and improving the catalytic efficiency of the SUPA complex (6-fold). Taken together, these data suggest that indirect molecular interactions may override the species-restricted activity of bacterial Pg activators; this may affect the pathogenesis of infections or may be exploited to facilitate the design of new blood clot-dissolving drugs.

Nitric oxide (NO) signaling mediates many important physiological processes through the receptor soluble guanylate cyclase (sGC). Under disease conditions sGC heme can be oxidized resulting in NO insensitivity. Here, we show that the therapeutic compound cinaciguat (Cin) rescues dysfunctional sGC by direct displacement of the oxidized heme., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We investigated how glycerol, urea, glucose and a GKA influence kinetics and stability of wild-type and mutant GK. Glycerol and glucose stabilized GK additively. Glycerol barely affected the TF spectra of all GKs but decreased k(cat), glucose S(0.5) and K(D) values and ATP K(M) while leaving cooperativity unchanged. Glycerol sensitized all GKs to GKA as shown by TF. Glucose increased TF of GKs without influence of glycerol on the effect. Glycerol and GKA affected kinetics and binding additively. The activation energies for thermal denaturation of GK were a function of glucose with K(D)s of 3 and 1mM without and with glycerol, respectively. High urea denatured wild type GK reversibly at 20 and 60°C and urea treatment of irreversibly heat denatured GK allowed refolding as demonstrated by TF including glucose response. We concluded: Glycerol stabilizes GK indirectly without changing the folding structure of the apoenzyme, by restructuring the surface water of the protein, whereas glucose stabilizes GK directly by binding to its substrate site and inducing a compact conformation. Glucose or glycerol (alone or combined) is unable to prevent irreversible heat denaturation above 40°C. However, urea denatures GK reversibly even at 60°C by binding to the protein backbone and directly interacting with hydrophobic side chains. It prevents irreversible aggregation allowing complete refolding when urea is removed. This study establishes the foundation for exploring numerous instability mutants among the more than 600 variant GKs causing diabetes in animals and humans., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Introduction: The renin-angiotensin system (RAS) is a main therapeutic target for cardiovascular diseases. Within the last two decades, novel components of the RAS have been discovered, opening new opportunities to interfere with its activity. Angiotensin(Ang)-(1-7) is synthesized by angiotensin-converting enzyme 2 (ACE2), and interacts with the G-protein-coupled receptor Mas. The axis formed by ACE2/Ang-(1-7)/Mas represents an endogenous counter regulatory pathway within the RAS., Areas Covered: In this review, the authors discuss patents and recent initiatives to develop therapeutic strategies based on the ACE2/Ang-(1-7)/Mas axis., Expert Opinion: Many publications and patents support a strategy to interfere with the activity of the RAS by stimulating its counter-regulatory axis mainly in two different ways: i) To increase the activity of ACE2, which will impact the system by increasing the inactivation of Ang II and the production of Ang-(1-7); ii) To stimulate Mas, taking advantage of nanostructured formulations of the natural peptide or analogues of Ang-(1-7). Although the preclinical studies are compelling, the possible impact of these novel therapeutic tools for the treatment of cardiometabolic diseases will only be known after completion of the ongoing clinical studies.

Introduction: Reversible acetylation is a key post-translational modification of target proteins. Sirtuin deacetylases represent the homolog of the yeast silent information regulator (SIR2). Although seven sirtuins have been found in mammals, all sirtuin activators described to date act through SIRT1., Areas Covered: Areas covered in this paper include a review of the patent literature associated with SIRT1 activators, with a focus on therapeutic applications, primarily related to the use of pharmaceuticals and nutraceuticals containing resveratrol (RSV), and the development of second-generation activators unrelated to RSV. Also discussed is the current controversy over whether or not these molecules are actual SIRT1 activators., Expert Opinion: Developing effective strategies to protect against diet-induced metabolic imbalance is necessary to fight against current obesity rates. The hypothalamus is a candidate for developing drugs that suppress SIRT1 degradation, as a strategy for treating metabolic syndrome. Deciphering the basic mechanism of activators is essential to develop effective strategies to alter sirtuin activity. This is true regardless of the apparent controversy of whether in vitro activation of SIRT1 is direct or not, depending on the experimental design, and whether sirtuins may play a major role in longevity. The numerous studies on their positive effects against age-related diseases, obesity and other metabolic disorders are still valid, promising to positively influence the development of treatments to improve human health.

The paracaspase domain of MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a component of a gene translocation fused to the N-terminal domains of the cellular inhibitor of apoptosis protein 2. The paracaspase itself, commonly known as MALT1, participates in the NF-κB (nuclear factor κB) pathway, probably by driving survival signals downstream of the B-cell antigen receptor through MALT1 proteolytic activity. We have developed methods for the expression and purification of recombinant full-length MALT1 and its constituent catalytic domain alone. Both are activated by dimerization without cleavage, with a similar dimerization barrier to the distantly related cousins, the apical caspases. By using positional-scanning peptidyl substrate libraries we demonstrate that the activity and specificity of full-length MALT1 is recapitulated by the catalytic domain alone, showing a stringent requirement for cleaving after arginine, and with striking peptide length constraints for efficient hydrolysis. Rates of cleavage (kcat/Km values) of optimal peptidyl substrates are in the same order (10(3)-10(4) M(-1)·s(-1)) as for a putative target protein CYLD. Thus MALT1 has many similarities to caspase 8, even cleaving the putative target protein CYLD with comparable efficiencies, but with diametrically opposite primary substrate specificity.

A series of novel 1,4-diaryl-2-azetidinones were synthesized and evaluated for antiproliferative activity, cell cycle effects, and apoptosis induction. Strong cytotoxicity was observed with the best compounds (±)-trans-20, (±)-trans-21, and enantiomers (+)-trans-20 and (+)-trans-21, which exhibited IC(50) values of 3-13 nM against duodenal adenocarcinoma cells. They induced inhibition of tubulin polymerization and subsequent G2/M arrest. This effect was accompanied by activation of AMP-activated protein kinase (AMPK), activation of caspase-3, and induction of apoptosis. Additionally, the most potent compounds displayed antiproliferative activity against different colon cancer cell lines, opening the route to a new class of potential therapeutic agents against colon cancer.

Elevated circulating estrogen levels, as a result of increased peripheral aromatization of androgens by aromatase, have been indicated to underlie the association between obesity and a higher risk of breast cancer in postmenopausal women. Although aromatase inhibitors have been used as a first-line therapy for estrogen receptor-positive breast cancer in postmenopausal women, their potential as breast cancer chemopreventive agents has been limited due to toxicities and high costs. It is therefore imperative to develop new aromatase-inhibiting/suppressing agents with lower toxicities and lower costs for breast cancer chemoprevention, especially in obese postmenopausal women. The expression of the aromatase gene, CYP19, is controlled in a tissue-specific manner by the alternate use of different promoters. In obese postmenopausal women, increased peripheral aromatase is primarily attributed to the activity of the glucocorticoid-stimulated promoter, PI.4, and the cAMP-stimulated promoter, PII. In the present study, we show that methylseleninic acid (MSA), a second-generation selenium compound, can effectively suppress aromatase activation by dexamethasone, a synthetic glucocorticoid, and forskolin, a specific activator of adenylate cyclase. Unlike the action of aromatase inhibitors, MSA suppression of aromatase activation is not mediated via direct inhibition of aromatase enzymatic activity. Rather, it is attributable to a marked downregulation of promoters PI.4- and PII-specific aromatase mRNA expression, and thereby a reduction of aromatase protein. Considering the low-cost and low-toxicity nature of MSA, our findings provide a strong rationale for the further development of MSA as a breast cancer chemopreventive agent for obese postmenopausal women.

The effects of selenite and tellurite on the mammalian enzyme lipoamide dehydrogenase were compared. Selenite acts as a substrate of lipoamide dehydrogenase in a process requiring the presence of lipoamide. In contrast, tellurite is a potent inhibitor, effective in the low micromolar range. The inhibitory effect of tellurite on lipoamide dehydrogenase is partially reverted by dithiothreitol indicating the participation of the thiol groups of the enzyme. Tellurite, but not selenite, stimulates the diaphorase activity of lipoamide dehydrogenase. In a mitochondrial matrix protein preparation, which contains lipoamide dehydrogenase, an inhibitory action similar to that observed on the purified enzyme was also elicited by tellurite. Human embryonic kidney cells (HEK 293 T) treated with tellurite show a partial inhibition of lipoamide dehydrogenase. In addition to the toxicological implications of tellurium compounds, the reported results suggest that tellurite and its derivatives can be used as potential tools for studying biochemical reactions., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In this study we have investigated the effect of reactive oxygen species produced by some chemicals in aqueous solutions on activity of adenosine deaminase 2 (ADA2) purified from human blood plasma. An activating effect on ADA2 was observed in vitro with sodium nitroprusside (SNP), the source of NO (nitrosonium ions NO(-) in aqueous solutions). Not SH-groups of cysteine but other amino acid residues sensitive to NO were responsible for ADA2 activation. The SNP-derived activation was more pronounced when purified ADA2 was preincubated with heparin and different proteins as an experimental model of the protein environment in vivo. The most effective was heparin, which is known for its ability to regulate enzyme and protein functions in extracellular matrix. We conclude that ADA2 is a protein with flexible conformation that is affected by the protein environment, and it changes its activity under oxidative (nitrosative) stress.

Human cancers often contain genetic alterations that disable G1/S checkpoint control and loss of this checkpoint is thought to critically contribute to cancer generation by permitting inappropriate proliferation and distorting fate-driven cell cycle exit. The identification of cell permeable small molecules that activate the G1/S checkpoint may therefore represent a broadly applicable and clinically effective strategy for the treatment of cancer. Here we describe the identification of several novel small molecules that trigger G1/S checkpoint activation and characterise the mechanism of action for one, CCT020312, in detail. Transcriptional profiling by cDNA microarray combined with reverse genetics revealed phosphorylation of the eukaryotic initiation factor 2-alpha (EIF2A) through the eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3/PERK) as the mechanism of action of this compound. While EIF2AK3/PERK activation classically follows endoplasmic reticulum (ER) stress signalling that sets off a range of different cellular responses, CCT020312 does not trigger these other cellular responses but instead selectively elicits EIF2AK3/PERK signalling. Phosphorylation of EIF2A by EIF2A kinases is a known means to block protein translation and hence restriction point transit in G1, but further supports apoptosis in specific contexts. Significantly, EIF2AK3/PERK signalling has previously been linked to the resistance of cancer cells to multiple anticancer chemotherapeutic agents, including drugs that target the ubiquitin/proteasome pathway and taxanes. Consistent with such findings CCT020312 sensitizes cancer cells with defective taxane-induced EIF2A phosphorylation to paclitaxel treatment. Our work therefore identifies CCT020312 as a novel small molecule chemical tool for the selective activation of EIF2A-mediated translation control with utility for proof-of-concept applications in EIF2A-centered therapeutic approaches, and as a chemical starting point for pathway selective agent development. We demonstrate that consistent with its mode of action CCT020312 is capable of delivering potent, and EIF2AK3 selective, proliferation control and can act as a sensitizer to chemotherapy-associated stresses as elicited by taxanes.

Sirtuin (Sir2) proteins being key regulators of numerous cellular processes have been, over the recent past, the subject of intense study. Sirs have been implicated in diverse physiological processes ranging from aging and cancer to neurological dysfunctions. Studies on Sir2s using tools of genetics, molecular biology, biochemistry and structural biology have provided significant insight into the diverse functions of this class of deacetylases. This apart, medicinal chemistry approaches have enabled the discovery of modulators (both activators and inhibitors) of Sir2 activity of diverse chemical structures and properties. The availability of these small molecule modulators of Sir2 activity not only has pharmacological significance but also opens up the possibility of exploiting chemical genetic approaches in understanding the role of this multi-functional enzyme in cellular processes.

GM1 gangliosidosis, a neurodegenerative disorder, and Morquio B disease, a skeletal disorder, are lysosomal storage disorders caused by inherited defects in the enzyme β-galactosidase (GLB1; EC 3.1.2.23; MIM #611458). Enzyme replacement therapy (ERT), a standard of care for a number of non-neuronopathic lysosomal storage disorders, is not yet available for GLB1 deficiency. Although functionally active recombinant human and feline GLB1 precursors have been purified, ERT has not yet been demonstrated in GM1 gangliosidosis or Morquio B disease models. A major obstacle to developing effective therapy may be the stability of human GLB1. We show here that mouse GLB1 has greater stability when compared to human GLB1, and that human GLB1 activity is temperature and protective-dependent on protein cathepsin A, while that of mouse GLB1 is not. These findings may impact on the eventual development of ERT for GLB1 deficiency. Despite our attempts to improve the extracellular stability of human GLB1 through sequence modification and the use of chemical chaperone N-butyldeoxygalactonojirimycin, the specific enzyme activity remained well below that of mGLB1., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Scientific evaluation of a patent aiming for the development of indole and indazole derivatives from biaryloxymethylarene as glycogen synthase activators, a key enzyme involved in type 2 diabetes mellitus.

Background: Platelet activating factor (PAF) has been proposed as a key factor and initial trigger in atherosclerosis. Recently, a modulation of PAF metabolism by bioactive food constituents has been suggested. In this study we investigated the effect of fish polar lipid consumption on PAF metabolism., Results: The specific activities of four PAF metabolic enzymes; in leukocytes, platelets and plasma, and PAF concentration; either in blood cells or plasma were determined. Samples were acquired at the beginning and at the end of a previously conducted study in male New Zealand white rabbits that were fed for 45 days with atherogenic diet supplemented (group-B, n = 6) or not (group-A, n = 6) with gilthead sea bream (Sparus aurata) polar lipids.The specific activity of PAF-Acetylhydrolase (PAF-AH); a catabolic enzyme of PAF, was decreased in rabbits' platelets of both A and B groups and in rabbits' leukocytes of group A (p < 0.05). On the other hand the specific activity of Lipoprotein-associated Phospholipase A2 (Lp-PLA2); the catabolic enzyme of PAF in plasma was increased in both A and B groups in both leukocytes and platelets (p < 0.05). PAF-cholinephosphotransferase (PAF-CPT); a biosynthetic enzyme of PAF showed increased specific activity only in rabbits' leukocytes of group A (p < 0.05). Neither of the two groups showed any change in Lyso-PAF-acetyltransferase (Lyso-PAF-AT) specific activity (p > 0.05). Free and bound PAF levels increased in group A while decreased in group B (p < 0.05)., Conclusions: Gilthead sea bream (Sparus aurata) polar lipids modulate PAF metabolism upon atherosclerotic conditions in rabbits leading to lower PAF levels and activity in blood of rabbits with reduced early atherosclerotic lesions compared to control group.

Studies on the anti-proliferative activities of novel baicalein derivatives demonstrated that compounds 8 and 9 were able to activate AMPK by enhancing the levels of phosphorylated AMPKα, and showed more potent anti-proliferative effects than baicalein and AICAR in A431, SK-OV-3, DU 145 and HeLa cells, suggesting an alternative therapeutic approach for benzyl baicalein in cancer therapy.

Objective: Sirtuin 1 (SIRT1) and its activator resveratrol are emerging as major regulators of metabolic processes. We investigate the site of resveratrol action on glucose metabolism and the contribution of SIRT1 to these effects. Because the arcuate nucleus in the mediobasal hypothalamus (MBH) plays a pivotal role in integrating peripheral metabolic responses to nutrients and hormones, we examined whether the actions of resveratrol are mediated at the MBH., Research Design and Methods: Sprague Dawley (SD) male rats received acute central (MBH) or systemic injections of vehicle, resveratrol, or SIRT1 inhibitor during basal pancreatic insulin clamp studies. To delineate the pathway(s) by which MBH resveratrol modulates hepatic glucose production, we silenced hypothalamic SIRT1 expression using a short hairpin RNA (shRNA) inhibited the hypothalamic ATP-sensitive potassium (K(ATP)) channel with glibenclamide, or selectively transected the hepatic branch of the vagus nerve while infusing resveratrol centrally., Results: Our studies show that marked improvement in insulin sensitivity can be elicited by acute administration of resveratrol to the MBH or during acute systemic administration. Selective inhibition of hypothalamic SIRT1 using a cell-permeable SIRT1 inhibitor or SIRT1-shRNA negated the effect of central and peripheral resveratrol on glucose production. Blockade of the K(ATP) channel and hepatic vagotomy significantly attenuated the effect of central resveratrol on hepatic glucose production. In addition, we found no evidence for hypothalamic AMPK activation after MBH resveratrol administration., Conclusions: Taken together, these studies demonstrate that resveratrol improves glucose homeostasis mainly through a central SIRT1-dependent pathway and that the MBH is a major site of resveratrol action.

We report, for the first time, that certain N-acetylthiourea derivatives serve as highly potent and isozyme selective activators for the recombinant form of human histone deacetylase-8 in the assay system containing Fluor-de-Lys as a fluorescent substrate. The experimental data reveals that such activating feature is manifested via decrease in the K(m) value of the enzyme's substrate and increase in the catalytic turnover rate of the enzyme., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

AMP-activated kinase (AMPK) as a key controller in the regulation of whole-body energy homeostasis, plays an important role in protecting the body from metabolic diseases. Recently, improved glucose, lipid utility and increased insulin sensitivity were observed on several diabetic rodent models treated with crude mogrosides isolated from the fruit of Siraitia grosvenorii Swingle, but the precise active compounds responsible for the anti-diabetic activity of this plant have not been clearly identified. In our current work, acid hydrolysis of crude mogrosides provided five new cucurbitane triterpenoids (1-4, 8), along with three known ones (5-7). The main aglycone mogrol (7) and compounds 4 and 8 were found to be potent AMPK activators in the HepG2 cell line. This result suggested AMPK activation by the mogroside aglycones 7 and 8 was proved to contribute at least partially to the anti-hyperglycemic and anti-lipidemic properties in vivo of S. grosvenorii., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The EF1143 protein from Enterococcus faecalis is a distant homolog of deoxynucleotide triphosphate triphosphohydrolases (dNTPases) from Escherichia coli and Thermus thermophilus. These dNTPases are important components in the regulation of the dNTP pool in bacteria. Biochemical assays of the EF1143 dNTPase activity demonstrated nonspecific hydrolysis of all canonical dNTPs in the presence of Mn(2+). In contrast, with Mg(2+) hydrolysis required the presence of dGTP as an effector, activating the degradation of dATP and dCTP with dGTP also being consumed in the reaction with dATP. The crystal structure of EF1143 and dynamic light scattering measurements in solution revealed a tetrameric oligomer as the most probable biologically active unit. The tetramer contains four dGTP specific allosteric regulatory sites and four active sites. Examination of the active site with the dATP substrate suggests an in-line nucleophilic attack on the α-phosphate center as a possible mechanism of the hydrolysis and two highly conserved residues, His-129 and Glu-122, as an acid-base catalytic dyad. Structural differences between EF1143 apo and holo forms revealed mobility of the α3 helix that can regulate the size of the active site binding pocket and could be stabilized in the open conformation upon formation of the tetramer and dGTP effector binding.

We report on a hit generation and hit-to-lead program of a novel class of glucokinase activators (GKAs). Hit compounds, activators at low glucose concentration only were identified by vHTS. Scaffold modification reliably afforded activators also at high substrate level. Potency was increased by introduction of a hydrogen bond acceptor as proposed by molecular docking. Replacement of the initial alkylene linkers with a rigid 1,2-phenylene motif followed by further studies eventually furnished a series of potent lead compounds exhibiting steep SAR., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

AMP-activated protein kinase (AMPK) is a sensor and regulator of cellular energy metabolism potentially implicated in a broad range of conditions, including obesity and Alzheimer's disease. Its role in the control of key metabolic enzymes makes this kinase a central player in glucose and lipid homeostasis. Recently, by screening a library of synthetic small molecules selected for their structural similarity with the natural polyphenol resveratrol, we identified RSVA314 and RSVA405 as potent indirect activators of AMPK (half-maximal effective concentration [EC₅₀] = 1 μmol/L in cell-based assays). Here we show that RSVA314 and RSVA405 can significantly activate AMPK and inhibit acetyl-CoA carboxylase (ACC), one target of AMPK and a key regulator of fatty acid biogenesis, in nondifferentiated and proliferating 3T3-L1 adipocytes. We found that RSVA314 and RSVA405 treatments inhibited 3T3-L1 adipocyte differentiation by interfering with mitotic clonal expansion during preadipocyte proliferation (half-maximal inhibitory concentration [IC₅₀] = 0.5 μmol/L). RSVA314 and RSVA405 prevented the adipogenesis-dependent transcriptional changes of multiple gene products involved in the adipogenic process, including peroxisome proliferator-activated receptor (PPAR)-γ, CCAAT/enhancer-binding protein α (C/EBPα), fatty acid synthase, fatty acid binding protein 4 (aP2), RANTES or resistin. Furthermore, orally administered RSVA405 at 20 and 100 mg/kg/d significantly reduced the body weight gain of mice fed a high-fat diet. This work shows that the novel small-molecule activators of AMPK (RSVA314 and RSVA405) are potent inhibitors of adipogenesis and thus may have therapeutic potential against obesity.

Nrf2-mediated activation of antioxidant response element is a central part of molecular mechanisms governing the protective function of phase II detoxification and antioxidant enzymes against carcinogenesis, oxidative stress, and inflammation. Nrf2 is sequestered in the cytoplasm by its repressor, Keap1. We have designed and synthesized novel chalcone derivatives as Nrf2 activators. The potency of these compounds was measured by the expression of Nrf2 dependent antioxidant genes GCLM, NQO1, and HO1 in human lung epithelial cells, while the cytotoxicity was analyzed using MTT assay. In vivo potency of identified lead compounds to activate Nrf2 was evaluated using a mouse model. Our studies showed 2-trifluoromethyl-2'-methoxychalone (2b) to be a potent activator of Nrf2, both in vitro and in mice. Additional experiments showed that the activation of Nrf2 by this compound is independent of reactive oxygen species or redox changes. We have discussed a quantitative structure-activity relationship and proposed a possible mechanism of Nrf2 activation.

Novel types of PKCα activators based on isobenzofuranone bearing a myo-inositol moiety were designed and synthesized. The derivatives with bulky substituents on the myo-inositol moiety significantly activated PKCα, but their binding sites were not the same as that of phorbol ester., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: The essential purine salvage pathway of Trypanosoma brucei bears interesting catalytic enzymes for chemotherapeutic intervention of Human African Trypanosomiasis. Unlike mammalian cells, trypanosomes lack de novo purine synthesis and completely rely on salvage from their hosts. One of the key enzymes is adenosine kinase which catalyzes the phosphorylation of ingested adenosine to form adenosine monophosphate (AMP) utilizing adenosine triphosphate (ATP) as the preferred phosphoryl donor., Methods and Findings: Here, we present the first structures of Trypanosoma brucei rhodesiense adenosine kinase (TbrAK): the structure of TbrAK in complex with the bisubstrate inhibitor P(1),P(5)-di(adenosine-5')-pentaphosphate (AP5A) at 1.55 Å, and TbrAK complexed with the recently discovered activator 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine (compound 1) at 2.8 Å resolution., Conclusions: The structural details and their comparison give new insights into substrate and activator binding to TbrAK at the molecular level. Further structure-activity relationship analyses of a series of derivatives of compound 1 support the observed binding mode of the activator and provide a possible mechanism of action with respect to their activating effect towards TbrAK.

The 2,4,6-trimethylpyridinium derivative of histamine is an effective activator of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). However, unlike other CA activators, which bind at the entrance of the active site cavity, an X-ray crystal structure of hCA II in complex with the 1-[2-(1H-imidazol-4-yl)-ethyl]-2,4,6-trimethylpyridinium salt evidenced a binding mode never observed before either for activators or inhibitors of this enzyme, with the 2,4,6-trimethylpyridinium ring pointing towards the metal ion deep within the enzyme cavity, and several strong hydrophobic interactions stabilizing the adduct. Indeed, incubation of the activator with the enzyme for several days leads to potent inhibitory effects. This is the first example of a CA activator which after a longer contact with the enzyme behaves as an inhibitor., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Lipoic acid moieties were attached to amine or amino acids showing activating properties against the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). The obtained lipoic acid conjugates of histamine, L-histidine methyl ester, and L-carnosine methyl ester were attached to gold nanoparticles (NPs) by reaction with Au(III) salts in reducing conditions. The CA activators (CAAs)-coated NPs showed low nanomolar activation (K(A)s of 1-9 nM) of relevant cytosolic, membrane-bound, mitochondrial, and transmembrane CA isoforms, such as CA I, II, IV, VA, VII, and XIV. These NPs also effectively activated CAs ex vivo, in whole blood experiments, with an increase of 200-280% of the CA activity. This is the first example of enzyme activation with nanoparticles and may lead to biomedical applications for conditions in which the CA activity is diminished, such as aging, Alzheimer's disease, or CA deficiency syndrome.

The neutrophil formyl peptide receptors (FPR1 and FPR2) are G-protein coupled receptors that can induce pro-inflammatory as well as anti-inflammatory activities when activated. Accordingly, these receptors may become therapeutic targets for the development of novel drugs to be used for reducing the inflammation induced injuries in asthma, rheumatoid arthritis, Alzheimer's disease, cardiovascular diseases and traumatic shock. We screened a library of more then 50K small compounds for an ability of the compounds to induce a transient rise in intracellular Ca(2+) in cells transfected to express FPR2 (earlier called FPRL1 or the lipoxin A(4) receptor). Ten agonist hits were selected for further analysis representing different chemical series and five new together with five earlier described molecules were further profiled. Compounds 1-10 gave rise to a calcium response in the FPR2 transfectants with EC(50) values ranging from 4×10(-9)M to 2×10(-7)M. All 10 compounds activated human neutrophils to release superoxide, and based on the potency of their activity, the three most potent activators of the neutrophil NADPH-oxidase were further characterized. These three agonists were largely resistant to inactivation by neutrophil produced reactive oxygen species and shown to trigger the same functional repertoire in neutrophils as earlier described peptide agonists. Accordingly they induced chemotaxis, granule mobilization and secretion of superoxide. Interestingly, the oxidase activity was largely inhibited by cyclosporine H, an FPR1 selective antagonist, but not by PBP10, an FPR2 selective inhibitor, suggesting that FPR1 is the preferred receptor in neutrophils for all three agonists., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Human digestive carboxypeptidases CPA1, CPA2, and CPB1 are secreted by the pancreas as inactive proenzymes containing a 94-96-amino acid-long propeptide. Activation of procarboxypeptidases is initiated by proteolytic cleavage at the C-terminal end of the propeptide by trypsin. Here, we demonstrate that subsequent cleavage of the propeptide by chymotrypsin C (CTRC) induces a nearly 10-fold increase in the activity of trypsin-activated CPA1 and CPA2, whereas CPB1 activity is unaffected. Other human pancreatic proteases such as chymotrypsin B1, chymotrypsin B2, chymotrypsin-like enzyme-1, elastase 2A, elastase 3A, or elastase 3B are inactive or markedly less effective at promoting procarboxypeptidase activation. On the basis of these observations, we propose that CTRC is a physiological co-activator of proCPA1 and proCPA2. Furthermore, the results confirm and extend the notion that CTRC is a key regulator of digestive zymogen activation.

Proteasomes degrade a multitude of protein substrates in the cytosol and nucleus, and thereby are essential for many aspects of cellular function. Because the proteolytic sites are sequestered in a closed barrel-shaped structure, activators are required to facilitate substrate access. Structural and biochemical studies of two activator families, 11S and Blm10, have provided insights to proteasome activation mechanisms, although the biological functions of these factors remain obscure. Recent advances have improved our understanding of the third activator family, including the 19S activator, which targets polyubiquitylated proteins for degradation. Here we present a structural perspective on how proteasomes are activated and how substrates are delivered to the proteolytic sites., (Copyright © 2011 Elsevier Inc. All rights reserved.)

AMP-activated protein kinase (AMPK) is a metabolic sensor involved in intracellular energy metabolism through the control of several homeostatic mechanisms, which include autophagy and protein degradation. Recently, we reported that AMPK activation by resveratrol promotes autophagy-dependent degradation of the amyloid-β (Aβ) peptides, the core components of the cerebral senile plaques in Alzheimer's disease. To identify more potent enhancers of Aβ degradation, we screened a library of synthetic small molecules selected for their structural similarities with resveratrol. Here, we report the identification of a series of structurally related molecules, the RSVA series, which inhibited Aβ accumulation in cell lines nearly 40 times more potently than did resveratrol. Two of these molecules, RSVA314 and RSVA405, were further characterized and were found to facilitate CaMKKβ-dependent activation of AMPK, to inhibit mTOR (mammalian target of rapamycin), and to promote autophagy to increase Aβ degradation by the lysosomal system (apparent EC(50) ∼ 1 μM). This work identifies the RSVA compounds as promising lead molecules for the development of a new class of AMPK activating drugs controlling mTOR signaling, autophagy, and Aβ clearance.

Members of the sirtuin family including the founding protein Sir2 in Saccharomyces cerevisiae have been linked to lifespan extension in simple organisms. This finding prompted evaluation of the role of Sir2 orthologues in many aging-associated conditions including neurodegeneration, type II diabetes and cancer. These studies have demonstrated that genetic and pharmacologic manipulation of sirtuin activity have beneficial effects in a surprisingly broad spectrum of aging-associated conditions suggesting that the Sir2-family of enzymes presents an attractive target for the development of pharmacological agents. While the initial model favored pharmacological activators of sirtuins as calorie restriction mimetics, it now appears that either activation or inhibition of sirtuins may be desirable for ameliorating disease depending on the pathological condition and the target tissue. In this chapter we review the development of pharmacological small molecule activators and inhibitors of the sirtuin family of enzymes.

Background: SRT1720 and SRT2183 were described recently as activators of the NAD+-dependent deacetylase, SIRT1. These molecules enhanced metabolic function when administered to rodents at doses of 100-500 mg/kg/day, purportedly by activating SIRT1 enzymatic activity in various tissues; however, considerable controversy surrounds these claims., Results: We find that these molecules do not activate SIRT1 deacetylase activity when tested in a variety of enzymatic assay formats and conditions. The compounds effectively decrease acetylated p53 in cells treated with DNA damaging agents but do so in cells that lack SIRT1, calling into question their designation as direct activators of SIRT1. In contrast, we find that the compounds inhibit p300 histone acetyltransferase activity in vitro, suggesting a possible mechanism for their effects in vivo., Conclusion: Structural features of these molecules may account for false-positive activation using fluorescence-based assays.

Previous studies on the in vitro metabolism of 4-alkylsulfonyl-2-pyridone-based glucokinase activators revealed a facile, non-enzymatic displacement of the 4-alkylsulfonyl group by glutathione. In the present studies, a role for glutathione-S-transferases (GST) as catalysts in the desulfonylation reaction was demonstrated using a combination of human liver microsomes, human liver cytosol and human GSTs. The identification of a glutathione conjugate in circulation following intravenous administration of a candidate 4-methylsulfonyl-2-pyridone to rats confirmed the relevance of the in vitro findings., (Copyright © 2010 Elsevier Ltd. All rights reserved.)