Resting skeletal muscle PNPLA2 (ATGL) and CPT1B are associated with peak fat oxidation rates in men and women but do not explain observed sex differences.

NEW FINDINGS: What is the central question of this study? What is the relationship between proteins in skeletal muscle and adipose tissue determined at rest and at peak rates of fat oxidation in men and women? What is the main finding and its importance? The resting contents of proteins in skeletal muscle involved in triglyceride hydrolysis and mitochondrial lipid transport were more strongly associated with peak fat oxidation rates than proteins related to lipid transport or hydrolysis in adipose tissue. Although females displayed higher relative rates of fat oxidation than males, this was not explained by the proteins measured in this study, suggesting that other factors determine sex differences in fat metabolism. ABSTRACT: We explored key proteins involved in fat metabolism that might be associated with peak fat oxidation (PFO) and account for sexual dimorphism in fuel metabolism during exercise. Thirty-six healthy adults [15 women; 40 ± 11 years of age; peak oxygen consumption 42.5 ± 9.5 ml (kg body mass)-1  min-1 ; mean ± SD] completed two exercise tests to determine PFO via indirect calorimetry. Resting adipose tissue and/or skeletal muscle biopsies were obtained to determine the adipose tissue protein content of PLIN1, ABHD5 (CGI-58), LIPE (HSL), PNPLA2 (ATGL), ACSL1, CPT1B and oestrogen receptor α (ERα) and the skeletal muscle protein content of FABP 3 (FABPpm), PNPLA2 (ATGL), ACSL1, CTP1B and ESR1 (ERα). Moderate strength correlations were found between PFO [in milligrams per kilogram of fat-free mass (FFM) per minute] and the protein content of PNPLA2 (ATGL) [rs  = 0.41 (0.03-0.68), P < 0.05] and CPT1B [rs  = 0.45 (0.09-0.71), P < 0.05] in skeletal muscle. No other statistically significant bivariate correlations were found consistently. Females had a greater relative PFO than males [7.1 ± 1.9 vs. 4.5 ± 1.3 and 7.3 ± 1.7 vs. 4.8 ± 1.2 mg (kg FFM)-1  min-1 in the adipose tissue (n = 14) and skeletal muscle (n = 12) subgroups, respectively (P < 0.05)]. No statistically significant sex differences were found in the content of these proteins. The regulation of PFO might involve processes relating to intramyocellular triglyceride hydrolysis and mitochondrial fatty acid transport, and adipose tissue is likely to play a more minor role than muscle. Sex differences in fat metabolism are likely to be attributable to factors other than the resting content of proteins in skeletal muscle and adipose tissue relating to triglyceride hydrolysis and fatty acid transport.

Determinants of Peak Fat Oxidation Rates During Cycling in Healthy Men and Women.

This study explored lifestyle and biological determinants of peak fat oxidation (PFO) during cycle ergometry, using duplicate measures to account for day-to-day variation. Seventy-three healthy adults (age range: 19-63 years; peak oxygen consumption [V˙O2peak]: 42.4 [10.1] ml·kg BM-1·min-1; n = 32 women]) completed trials 7-28 days apart that assessed resting metabolic rate, a resting venous blood sample, and PFO by indirect calorimetry during an incremental cycling test. Habitual physical activity (combined heart rate accelerometer) and dietary intake (weighed record) were assessed before the first trial. Body composition was assessed 2-7 days after the second identical trial by dual-energy X-ray absorptiometry scan. Multiple linear regressions were performed to identify determinants of PFO (mean of two cycle tests). A total variance of 79% in absolute PFO (g·min-1) was explained with positive coefficients for V˙O2peak (strongest predictor), FATmax (i.e the % of V˙O2peak that PFO occurred at), and resting fat oxidation rate (g·min-1), and negative coefficients for body fat mass (kg) and habitual physical activity level. When expressed relative to fat-free mass, 64% of variance in PFO was explained: positive coefficients for FATmax (strongest predictor), V˙O2peak, and resting fat oxidation rate, and negative coefficients for male sex and fat mass. This duplicate design revealed that biological and lifestyle factors explain a large proportion of variance in PFO during incremental cycling. After accounting for day-to-day variation in PFO, V˙O2peak and FATmax were strong and consistent predictors of PFO.

The day-to-day reliability of peak fat oxidation and FATMAX.

PURPOSE: Prior studies exploring the reliability of peak fat oxidation (PFO) and the intensity that elicits PFO (FATMAX) are often limited by small samples. This study characterised the reliability of PFO and FATMAX in a large cohort of healthy men and women. METHODS: Ninety-nine adults [49 women; age: 35 (11) years; [Formula: see text]O2peak: 42.2 (10.3) mL·kg BM-1·min-1; mean (SD)] completed two identical exercise tests (7-28 days apart) to determine PFO (g·min-1) and FATMAX (%[Formula: see text]O2peak) by indirect calorimetry. Systematic bias and the absolute and relative reliability of PFO and FATMAX were explored in the whole sample and sub-categories of: cardiorespiratory fitness, biological sex, objectively measured physical activity levels, fat mass index (derived by dual-energy X-ray absorptiometry) and menstrual cycle status. RESULTS: No systematic bias in PFO or FATMAX was found between exercise tests in the entire sample (- 0.01 g·min-1 and 0%[Formula: see text]O2peak, respectively; p > 0.05). Absolute reliability was poor [within-subject coefficient of variation: 21% and 26%; typical errors: ± 0.06 g·min-1 and × / ÷ 1.26%[Formula: see text]O2peak; 95% limits of agreement: ± 0.17 g·min-1 and × / ÷ 1.90%[Formula: see text]O2peak, respectively), despite high (r = 0.75) and moderate (r = 0.45) relative reliability for PFO and FATMAX, respectively. These findings were consistent across all sub-groups. CONCLUSION: Repeated assessments are required to more accurately determine PFO and FATMAX.

Dopaminergic D1 receptor effects on commissural inputs targeting layer V pyramidal subtypes of the mouse medial prefrontal cortex.

In humans, prefrontal cortical areas are known to support goal-directed behaviors, mediating a variety of functions that render behavior more flexible in the face of changing environmental demands. In mice, these functions are mediated by homologous regions within medial prefrontal cortex (mPFC) and rely heavily on proper dopaminergic tone. Comprised of two major subtypes, pyramidal tract (PT) and intratelencephalic (IT), layer V pyramidal cells serve as the major outputs of the mPFC, targeting brainstem nuclei and the contralateral hemisphere, respectively. However, it remains relatively unknown how cortical inputs targeting these subtypes are integrated. We explored how layer V pyramidal cell subtypes integrate commissural inputs, which integrate information flow between the hemispheres. An optogenetic approach was used to elicit commissural fiber activation onto PT and IT cells and the effects of D1 receptor activation on elicited EPSPs were explored. We showed that commissural inputs into PT and IT cells elicit facilitating and depressing EPSP patterns, respectively. D1 receptor activation increased the initial EPSP amplitude, enhanced EPSP facilitation, and prolonged EPSP decay time constant in PT cells. In IT cells, D1 receptor activation increased commissural-evoked initial EPSP amplitude but did not affect facilitation or EPSP shape. Furthermore, D1 receptor activation elicited burst firing in a subset of PT cells in response to commissural fiber activation. Combined, these results lend insight into the role of dopamine in promoting persistent firing and temporal integration in PT and IT cells, respectively, that in turn may contribute to working memory functions.

Synthesis, Biological Evaluation, and SAR Studies of 14ß-phenylacetyl Substituted 17-cyclopropylmethyl-7, 8-dihydronoroxymorphinones Derivatives: Ligands With Mixed NOP and Opioid Receptor Profile.

A series of 14ß-acyl substituted 17-cyclopropylmethyl-7,8-dihydronoroxymorphinone compounds has been synthesized and evaluated for affinity and efficacy for mu (MOP), kappa (KOP), and delta (DOP) opioid receptors and nociceptin/orphanin FQ peptide (NOP) receptors. The majority of the new ligands displayed high binding affinities for the three opioid receptors, and moderate affinity for NOP receptors. The affinities for NOP receptors are of particular interest as most classical opioid ligands do not bind to NOP receptors. The predominant activity in the [35S]GTPγS assay was partial agonism at each receptor. The results are consistent with our prediction that an appropriate 14ß side chain would access a binding site within the NOP receptor and result in substantially higher affinity than displayed by the parent compound naltrexone. Molecular modeling studies, utilizing the recently reported structure of the NOP receptor, are also consistent with this interpretation.

C-3- and C-4-Substituted Bicyclic Coumarin Sulfamates as Potent Steroid Sulfatase Inhibitors.

Synthetic routes to potent bicyclic nonsteroidal sulfamate-based active-site-directed inhibitors of the enzyme steroid sulfatase (STS), an emerging target in the treatment of postmenopausal hormone-dependent diseases, including breast cancer, are described. Sulfamate analogs 9-27 and 28-46 of the core in vivo active two-ring coumarin template, modified at the 4- and 3-positions, respectively, were synthesized to expand structure-activity relationships. α-Alkylacetoacetates were used to synthesize coumarin sulfamate derivatives with 3-position modifications, and the bicyclic ring of other parent coumarins was primarily constructed via the Pechmann synthesis of hydroxyl coumarins. Compounds were examined for STS inhibition in intact MCF-7 breast cancer cells and in placental microsomes. Low nanomolar potency STS inhibitors were achieved, and some were found to inhibit the enzyme in MCF-7 cells ca. 100-500 more potently than the parent 4-methylcoumarin-7-O-sulfamate 3, with the best compounds close in potency to the tricyclic clinical drug Irosustat. 3-Hexyl-4-methylcoumarin-7-O-sulfamate 29 and 3-benzyl-4-methylcoumarin-7-O-sulfamate 41 were particularly effective inhibitors with IC50 values of 0.68 and 1 nM in intact MCF-7 cells and 8 and 32 nM for placental microsomal STS, respectively. They were docked into the STS active site for comparison with estrone 3-O-sulfamate and Irosustat, showing their sulfamate group close to the catalytic hydrated formylglycine residue and their pendant group lying between the hydrophobic sidechains of L103, F178, and F488. Such highly potent STS inhibitors expand the structure-activity relationship for these coumarin sulfamate-based agents that possess therapeutic potential and may be worthy of further development.

Synthetic cADPR analogues may form only one of two possible conformational diastereoisomers.

Cyclic adenosine 5'-diphosphate ribose (cADPR) is an emerging Ca2+-mobilising second messenger. cADPR analogues have been generated as chemical biology tools via both chemo-enzymatic and total synthetic routes. Both routes rely on the cyclisation of a linear precursor to close an 18-membered macrocyclic ring. We show here that, after cyclisation, there are two possible macrocyclic product conformers that may be formed, depending on whether cyclisation occurs to the "right" or the "left" of the adenine base (as viewed along the H-8 → C-8 base axis). Molecular modelling demonstrates that these two conformers are distinct and cannot interconvert. The two conformers would present a different spatial layout of binding partners to the cADPR receptor/binding site. For chemo-enzymatically generated analogues Aplysia californica ADP-ribosyl cyclase acts as a template to generate solely the "right-handed" conformer and this corresponds to that of the natural messenger, as originally explored using crystallography. However, for a total synthetic analogue it is theoretically possible to generate either product, or a mixture, from a given linear precursor. Cyclisation on either face of the adenine base is broadly illustrated by the first chemical synthesis of the two enantiomers of a "southern" ribose-simplified cIDPR analogue 8-Br-N9-butyl-cIDPR, a cADPR analogue containing only one chiral sugar in the "northern" ribose, i.e. 8-Br-D- and its mirror image 8-Br-L-N9-butyl-cIDPR. By replacing the D-ribose with the unnatural L-ribose sugar, cyclisation of the linear precursor with pyrophosphate closure generates a cyclised product spectroscopically identical, but displaying equal and opposite specific rotation. These findings have implications for cADPR analogue design, synthesis and activity.

Layer-specific effects of dopaminergic D1 receptor activation on excitatory synaptic trains in layer V mouse prefrontal cortical pyramidal cells.

In humans, executive functions (e.g., working memory [WM]) are mediated in part by prefrontal cortical areas (PFC), where ventromedial areas may be homologous to ventromedial areas (mPFC) in rodents. Many executive functions are critically dependent on optimal dopamine levels within the PFC; however, our understanding of the role of dopamine in modulating PFC-mediated tasks is incomplete. Stable patterns of neuronal activity have been associated with WM processes, and recurrent excitatory synaptic activity has been proposed to play a role in this sustained activity. This excitatory activity may be regulated in a frequency-dependent manner. Thus, we examined the effects of dopamine D1-like receptor (D1R) activation on short-term excitatory postsynaptic potential (EPSP) dynamics in two subtypes of mouse layer V mPFC pyramidal neurons by varying evoked train frequency from 10 to 50 Hz. We isolated non-NMDA receptor (non-NMDAR) and NMDA receptor (NMDAR)-mediated components of EPSP trains, which were evoked by stimulating fibers located either within layer V or layer I of the mPFC. Interestingly, no differences in the effects of D1R activation were observed between subcortically projecting (PT or pyramidal tract) and contralaterally projecting (IT or intratelencephalic) layer V pyramidal cells. However, we found that D1R activation had layer-specific effects on NMDAR- and non-NMDAR-mediated EPSP trains: while D1R activation increased the amplitude of both components with layer V stimulation, with layer I stimulation D1R activation had no effect on non-NMDAR-mediated EPSP trains but decreased the amplitude of NMDAR-mediated EPSP trains. Our results suggest that dopamine, acting at D1-like receptors, increases the influence of local inputs from other layer V pyramidal cells, but may restrict the influence of layer I (tuft) inputs. Our demonstration of differential D1R regulation of excitatory synaptic dynamics in distinct compartments of mPFC layer V neurons may provide another important aspect linking cellular mechanisms of dopaminergic modulation to PFC network functioning, and ultimately to executive functions such as working memory.

Quinazolinone-Based Anticancer Agents: Synthesis, Antiproliferative SAR, Antitubulin Activity, and Tubulin Co-crystal Structure.

Quinazolinone-based anticancer agents were designed, decorated with functional groups from a 2-methoxyestradiol-based microtubule disruptor series, incorporating the aryl sulfamate motif of steroid sulfatase (STS) inhibitors. The steroidal AB-ring system was mimicked, favoring conformations with an N-2 substituent occupying D-ring space. Evaluation against breast and prostate tumor cell lines identified 7b with DU-145 antiproliferative activity (GI50 300 nM). A preliminary structure-activity relationship afforded compounds (e.g., 7j GI50 50 nM) with activity exceeding that of the parent. Both 7b and 7j inhibit tubulin assembly in vitro and colchicine binding, and 7j was successfully co-crystallized with the αß-tubulin heterodimer as the first of its class, its sulfamate group interacting positively at the colchicine binding site. Microtubule destabilization by 7j is likely achieved by preventing the curved-to-straight conformational transition in αß-tubulin. Quinazolinone sulfamates surprisingly showed weak STS inhibition. Preliminary in vivo studies in a multiple myeloma xenograft model for 7b showed oral activity, confirming the promise of this template.

Subtype-specific effects of dopaminergic D2 receptor activation on synaptic trains in layer V pyramidal neurons in the mouse prefrontal cortex.

In humans, prefrontal cortical areas are known to support executive functions. In mice, these functions are mediated by homologous regions in the medial prefrontal cortex (mPFC). Executive processes are critically dependent on optimal levels of dopamine (DA), but the cellular mechanisms of DA modulation are incompletely understood. Stable patterns of neuronal activity may be sensitive to frequency-dependent changes in synaptic transmission. We characterized the effects of D2 receptor (D2R) activation on short-term excitatory postsynaptic potential (EPSP) dynamics evoked at varying frequencies in the two subtypes of layer V pyramidal neurons in mouse mPFC We isolated NMDA receptor and non-NMDA receptor-mediated components of EPSP trains evoked by stimulating fibers within layer V or layer I. All significant effects of D2 receptor activation were confined to type I (corticopontine) cells. First, we found that with layer I stimulation, D2R activation reduces the amplitude of NMDAR-mediated EPSPs, with no effect on facilitation or depression of these responses at lower frequencies, but leading to facilitation with high frequency stimulation. Further, the non-NMDA component also underwent synaptic depression at low frequencies. Second, with layer V stimulation, D2R activation had no effect on NMDA or non-NMDA receptor-mediated EPSP components. Overall, our results suggest that D2R activation may modulate memory functions by inhibiting 'top-down' influences from apical tuft inputs activated at low frequencies, while promoting 'top-down' influences from inputs activated at higher frequencies. These data provide further insight into mechanisms of dopamine's modulation of executive functions.

Second messenger analogues highlight unexpected substrate sensitivity of CD38: total synthesis of the hybrid "L-cyclic inosine 5'-diphosphate ribose".

The multifunctional, transmembrane glycoprotein human CD38 catalyses the synthesis of three key Ca2+-mobilising messengers, including cyclic adenosine 5'-diphosphate ribose (cADPR), and CD38 knockout studies have revealed the relevance of the related signalling pathways to disease. To generate inhibitors of CD38 by total synthesis, analogues based on the cyclic inosine 5'-diphosphate ribose (cIDPR) template were synthesised. In the first example of a sugar hybrid cIDPR analogue, "L-cIDPR", the natural "northern" N1-linked D-ribose of cADPR was replaced by L-ribose. L-cIDPR is surprisingly still hydrolysed by CD38, whereas 8-Br-L-cIDPR is not cleaved, even at high enzyme concentrations. Thus, the inhibitory activity of L-cIDPR analogues appears to depend upon substitution of the base at C-8; 8-Br-L-cIDPR and 8-NH2-L-cIDPR inhibit CD38-mediated cADPR hydrolysis (IC50 7 µM and 21 µM respectively) with 8-Br-L-cIDPR over 20-fold more potent than 8-Br-cIDPR. In contrast, L-cIDPR displays a comparative 75-fold reduction in activity, but is only ca 2-fold less potent than cIDPR itself. Molecular modelling was used to explore the interaction of the CD38 catalytic residue Glu-226 with the "northern" ribose. We propose that Glu226 still acts as the catalytic residue even for an L-sugar substrate. 8-Br-L-cIDPR potentially binds non-productively in an upside-down fashion. Results highlight the key role of the "northern" ribose in the interaction of cADPR with CD38.

Ligand-induced activation of human TRPM2 requires the terminal ribose of ADPR and involves Arg1433 and Tyr1349.

TRPM2 (transient receptor potential channel, subfamily melastatin, member 2) is a Ca2+-permeable non-selective cation channel activated by the binding of adenosine 5'-diphosphoribose (ADPR) to its cytoplasmic NUDT9H domain (NUDT9 homology domain). Activation of TRPM2 by ADPR downstream of oxidative stress has been implicated in the pathogenesis of many human diseases, rendering TRPM2 an attractive novel target for pharmacological intervention. However, the structural basis underlying this activation is largely unknown. Since ADP (adenosine 5'-diphosphate) alone did not activate or antagonize the channel, we used a chemical biology approach employing synthetic analogues to focus on the role of the ADPR terminal ribose. All novel ADPR derivatives modified in the terminal ribose, including that with the seemingly minor change of methylating the anomeric-OH, abolished agonist activity at TRPM2. Antagonist activity improved as the terminal substituent increasingly resembled the natural ribose, indicating that gating by ADPR might require specific interactions between hydroxyl groups of the terminal ribose and the NUDT9H domain. By mutating amino acid residues of the NUDT9H domain, predicted by modelling and docking to interact with the terminal ribose, we demonstrate that abrogating hydrogen bonding of the amino acids Arg1433 and Tyr1349 interferes with activation of the channel by ADPR. Taken together, using the complementary experimental approaches of chemical modification of the ligand and site-directed mutagenesis of TRPM2, we demonstrate that channel activation critically depends on hydrogen bonding of Arg1433 and Tyr1349 with the terminal ribose. Our findings allow for a more rational design of novel TRPM2 antagonists that may ultimately lead to compounds of therapeutic potential.

SHIP2: Structure, Function and Inhibition.

SHIP2 is a phosphatase that acts at the 5-position of phosphatidylinositol 3,4,5-trisphosphate. It is one of several enzymes that catalyse dephosphorylation at the 5-position of phosphoinositides or inositol phosphates. SHIP2 has a confirmed role in opsismodysplasia, a disease of bone development, but also interacts with proteins involved in insulin signalling, cytoskeletal function (thus having an impact on endocytosis, adhesion, proliferation and apoptosis) and immune system function. The structure of three domains (constituting about 38 % of the protein) is known. Inhibitors of SHIP2 activity have been designed to interact with the catalytic domain with sub-micromolar IC50 values: these come from a range of structural classes and have been shown to have in vivo effects consistent with SHIP2 inhibition. Much remains unknown about the roles of SHIP2, and possible future directions for research are indicated.

The "Other" Inositols and Their Phosphates: Synthesis, Biology, and Medicine (with Recent Advances in myo-Inositol Chemistry).

Cell signaling via inositol phosphates, in particular via the second messenger myo-inositol 1,4,5-trisphosphate, and phosphoinositides comprises a huge field of biology. Of the nine 1,2,3,4,5,6-cyclohexanehexol isomers, myo-inositol is pre-eminent, with "other" inositols (cis-, epi-, allo-, muco-, neo-, L-chiro-, D-chiro-, and scyllo-) and derivatives rarer or thought not to exist in nature. However, neo- and d-chiro-inositol hexakisphosphates were recently revealed in both terrestrial and aquatic ecosystems, thus highlighting the paucity of knowledge of the origins and potential biological functions of such stereoisomers, a prevalent group of environmental organic phosphates, and their parent inositols. Some "other" inositols are medically relevant, for example, scyllo-inositol (neurodegenerative diseases) and d-chiro-inositol (diabetes). It is timely to consider exploration of the roles and applications of the "other" isomers and their derivatives, likely by exploiting techniques now well developed for the myo series.

The In Vitro and In Vivo Activity of the Microtubule Disruptor STX140 Is Mediated by Hif-1 Alpha and CAIX Expression.

Tumor neo-angiogenesis is regulated, in part, by the hypoxia-inducible gene HIF1. Evidence suggests HIF1 associates with polymerized microtubules and traffics to the nucleus. This study investigated the role of HIF1 in mediating the antitumor activity of two steroid-based sulfamate ester microtubule disruptors, STX140 and STX243, in vitro and in vivo. The effects of STX140, STX243 and the parental compound 2-methoxyestradiol (STX66) on HIF1α and HIF2α protein expression were assessed in vitro in MCF-7 and MDA-MB-231 cells cultured under hypoxia. More pertinently, their effects were examined on HIF1-regulated genes in vivo in mice bearing MCF-7 or MDA-MB-231 tumors. The level of mRNA expression of vascular endothelial growth factor (VEGF), glucose transporter 1 (GLUTI), phosphoglycerate kinase (PGK), ATP-binding cassette sub-family B member 1 (ABCB1) and carbonic anhydrase IX (CAIX) was quantified by Real-time Polymerase Chain Reaction (RT-PCR). Despite inhibiting nuclear HIF1α protein accumulation under hypoxia in vitro, STX140 and STX243 did not significantly regulate the expression of four out of five HIF1α-regulated genes in vitro and in vivo. Only CAIX mRNA expression was down-regulated both in vitro and in vivo. Immunoblot analysis showed that STX140 and STX243 reduced CAIX protein expression in vitro. These compounds had no effect on HIF2α translocation. The potential for inhibition of CAIX by STX140 and STX243 was examined by docking the ligands to the active site in comparison with a known sulfamate-based inhibitor. Microtubule disruption and antitumor activity of STX140 and STX243 is most likely HIF1-independent and may, at least in part, be mediated by inhibition of CAIX expression and activity.

Discovery and Development of the Aryl O-Sulfamate Pharmacophore for Oncology and Women's Health.

In 1994, following work from this laboratory, it was reported that estrone-3-O-sulfamate irreversibly inhibits a new potential hormone-dependent cancer target steroid sulfatase (STS). Subsequent drug discovery projects were initiated to develop the core aryl O-sulfamate pharmacophore that, over some 20 years, have led to steroidal and nonsteroidal drugs in numerous preclinical and clinical trials, with promising results in oncology and women's health, including endometriosis. Drugs have been designed to inhibit STS, e.g., Irosustat, as innovative dual-targeting aromatase-steroid sulfatase inhibitors (DASIs) and as multitargeting agents for hormone-independent tumors, such as the steroidal STX140 and nonsteroidal counterparts, acting inter alia through microtubule disruption. The aryl sulfamate pharmacophore is highly versatile, operating via three distinct mechanisms of action, and imbues attractive pharmaceutical properties. This Perspective gives a personal view of the work leading both to the therapeutic concepts and these drugs, their current status, and how they might develop in the future.

Estrogen O-sulfamates and their analogues: Clinical steroid sulfatase inhibitors with broad potential.

Estrogen sulfamate derivatives were the first irreversible active-site-directed inhibitors of steroid sulfatase (STS), an emerging drug target for endocrine therapy of hormone dependent diseases that catalyzes inter alia the hydrolysis of estrone sulfate to estrone. In recent years this has stimulated clinical investigation of the estradiol derivative both as an oral prodrug and its currently ongoing exploration in endometriosis. 2-Substituted steroid sulfamate derivatives show considerable potential as multi-targeting agents for hormone-independent disease, but are also potent STS inhibitors. The steroidal template has spawned nonsteroidal STS inhibitors one of which, Irosustat, has been evaluated clinically in breast cancer, endometrial cancer and prostate cancer and there is potential for innovative dual-targeting approaches. This review surveys the role of estrogen sulfamates, their analogues and current status.

Cyclic adenosine 5'-diphosphate ribose analogs without a "southern" ribose inhibit ADP-ribosyl cyclase-hydrolase CD38.

Cyclic adenosine 5'-diphosphate ribose (cADPR) analogs based on the cyclic inosine 5'-diphosphate ribose (cIDPR) template were synthesized by recently developed stereo- and regioselective N1-ribosylation. Replacing the base N9-ribose with a butyl chain generates inhibitors of cADPR hydrolysis by the human ADP-ribosyl cyclase CD38 catalytic domain (shCD38), illustrating the nonessential nature of the "southern" ribose for binding. Butyl substitution generally improves potency relative to the parent cIDPRs, and 8-amino-N9-butyl-cIDPR is comparable to the best noncovalent CD38 inhibitors to date (IC50 = 3.3 µM). Crystallographic analysis of the shCD38:8-amino-N9-butyl-cIDPR complex to a 2.05 Å resolution unexpectedly reveals an N1-hydrolyzed ligand in the active site, suggesting that it is the N6-imino form of cADPR that is hydrolyzed by CD38. While HPLC studies confirm ligand cleavage at very high protein concentrations, they indicate that hydrolysis does not occur under physiological concentrations. Taken together, these analogs confirm that the "northern" ribose is critical for CD38 activity and inhibition, provide new insight into the mechanism of cADPR hydrolysis by CD38, and may aid future inhibitor design.