Activation of SIRT6 Deacetylation by DNA Strand Breaks.

SIRT6 is an emerging regulator of longevity. Overexpression of SIRT6 extends the lifespan of mice. Conversely, SIRT6 knockout mice demonstrate severe metabolic defects and a shortened lifespan. The discrepancy between SIRT6's weak in vitro activity and robust in vivo activity has led to the hypothesis that this enzyme can be activated in response to DNA damage in cells. Here, we demonstrate that the deacetylase activity of SIRT6 can be stimulated by DNA strand breaks for synthetic peptide and histone substrates. The mechanism of activation is further explored by using an integrative chemical biology approach. SIRT6 can be preferentially activated by DNA lesions harboring a 5'-phosphate. The N- and C-termini of SIRT6 are strictly required for DNA break-induced activation. Additionally, the defatty-acylase activity of SIRT6 is also sensitive to DNA breaks, although the physiological significance needs further investigation. Collectively, our study sheds important light on the cellular regulation of diverse SIRT6 activities and suggests possible strategies for effective SIRT6 activation.

Alternative Pre-mRNA Splicing of the Mu Opioid Receptor Gene, OPRM1: Insight into Complex Mu Opioid Actions.

Most opioid analgesics used clinically, including morphine and fentanyl, as well as the recreational drug heroin, act primarily through the mu opioid receptor, a class A Rhodopsin-like G protein-coupled receptor (GPCR). The single-copy mu opioid receptor gene, OPRM1, undergoes extensive alternative splicing, creating multiple splice variants or isoforms via a variety of alternative splicing events. These OPRM1 splice variants can be categorized into three major types based on the receptor structure: (1) full-length 7 transmembrane (TM) C-terminal variants; (2) truncated 6TM variants; and (3) single TM variants. Increasing evidence suggests that these OPRM1 splice variants are pharmacologically important in mediating the distinct actions of various mu opioids. More importantly, the OPRM1 variants can be targeted for development of novel opioid analgesics that are potent against multiple types of pain, but devoid of many side-effects associated with traditional opiates. In this review, we provide an overview of OPRM1 alternative splicing and its functional relevance in opioid pharmacology.

Substrate-Dependent Sensitivity of SIRT1 to Nicotinamide Inhibition.

SIRT1 is the most extensively studied human sirtuin with a broad spectrum of endogenous targets. It has been implicated in the regulation of a myriad of cellular events, such as gene transcription, mitochondria biogenesis, insulin secretion as well as glucose and lipid metabolism. From a mechanistic perspective, nicotinamide (NAM), a byproduct of a sirtuin-catalyzed reaction, reverses a reaction intermediate to regenerate NAD+ through "base exchange", leading to the inhibition of the forward deacetylation. NAM has been suggested as a universal sirtuin negative regulator. Sirtuins have evolved different strategies in response to NAM regulation. Here, we report the detailed kinetic analysis of SIRT1-catalyzed reactions using endogenous substrate-based synthetic peptides. A novel substrate-dependent sensitivity of SIRT1 to NAM inhibition was observed. Additionally, SIRT1 demonstrated pH-dependent deacetylation with normal solvent isotope effects (SIEs), consistent with proton transfer in the rate-limiting step. Base exchange, in contrast, was insensitive to pH changes with no apparent SIEs, indicative of lack of proton transfer in the rate-limiting step. Consequently, NAM inhibition was attenuated at a high pH in proteated buffers. Our study provides new evidence for "activation by de-repression" as an effective sirtuin activation strategy.

Development of Second Generation Activity-Based Chemical Probes for Sirtuins.

NAD+ (nicotinamide adenine dinucleotide)-dependent protein deacylases, namely, the sirtuins, are important cell adaptor proteins that alter cell physiology in response to low calorie conditions. They are thought to mediate the beneficial effects of calorie restriction to extend longevity and improve health profiles. Novel chemical probes are highly desired for a better understanding of sirtuin's roles in various biological processes. We developed a group of remarkably simple activity-based chemical probes for the investigation of active sirtuin content in complex native proteomes. These probes harbor a thioacyllysine warhead, a diazirine photoaffinity tag, as well as a terminal alkyne bioorthogonal functional group. Compared to their benzophenone-containing counterparts, these new probes demonstrated improved labeling efficiency and sensitivity, shortened irradiation time, and reduced background signal. They were applied to the labeling of individual recombinant proteins, protein mixtures, and whole cell lysate. These cell permeable small molecule probes also enabled the cellular imaging of sirtuin activity change. Taken together, our study provides new chemical biology tools and future drug discovery strategies for perturbing the activity of different sirtuin isoforms.

Characterization of Miharamycin Biosynthesis Reveals a Hybrid NRPS-PKS to Synthesize High-Carbon Sugar from a Complex Nucleoside.

Miharamycins are peptidyl nucleoside antibiotics with a unique branched C9 pyranosyl amino acid core and a rare 2-aminopurine moiety. Inactivation of 19 genes in the biosynthetic gene cluster and identification of several unexpected intermediates suggest an alternative biosynthetic pathway, which is further supported by feeding experiments and in vitro characterization of an unusual adenylation domain recognizing a complex nucleoside derivative as the substrate. These results thereby provide an unprecedented biosynthetic route of high-carbon sugar catalyzed by atypical hybrid nonribosomal peptide synthetase-polyketide synthase.

The Miharamycins and Amipurimycin: their Structural Revision and the Total Synthesis of the Latter.

The structural puzzle of amipurimycin, a peptidyl nucleoside antibiotic, is solved by total synthesis and X-ray diffraction analysis, with the originally proposed configurations at C3' and C8' inverted and those at C6', C2'', and C3'' corrected. A similar structural revision of the relevant miharamycins is proposed via chemical transformations and then validated by X-ray diffraction analysis. The miharamycins bear an unusual trans-fused dioxabicyclo[4.3.0]nonane sugar scaffold, which was previously assigned as being in the cis configuration.

Identification of the Amipurimycin Gene Cluster Yields Insight into the Biosynthesis of C9 Sugar Nucleoside Antibiotics.

Feeding studies indicate a possible synthetic pattern for the N-terminal cis-aminocyclopentane carboxylic acid (ACPC) and suggest an unusual source of the high-carbon sugar skeleton of amipurimycin (APM). The biosynthetic gene cluster of APM was identified and confirmed by in vivo experiments. A C9 core intermediate was discovered from null mutants of ACPC pathway, and an ATP-grasp enzyme (ApmA8) was reconstituted in vitro for ACPC loading. Our observations allow a first proposal of the APM biosynthetic pathway.

New benzyl-aporphine alkaloids from Thalictrum cultratum.

Two new rare benzyl-aporphine alkaloids, thaliculine (1) and 6a,7-dehydrothaliculine (2), were isolated from the roots of Thalictrum cultratum Wall. Their structures were determined based on spectroscopic analysis including 1D, 2D NMR, and HR-ESI-MS. The stereochemistry of 1 was assigned by ECD spectroscopy. Compound 1 exhibited weak cytotoxicity against HL-60 cell line with IC50 value of 31.40 µM.

New chalcone and pterocarpoid derivatives from the roots of Flemingia philippinensis with antiproliferative activity and apoptosis-inducing property.

Investigation of the roots of Flemingia philippinensis resulted in the isolation of two new chalcones, flemiphilippinones B (1) and C (2), and one new pterocarpoid, demethylwedelolactone-11-methyl ether (3), together with 12 known compounds (4-15). The antiproliferative activity against PC-3 cells was evaluated and most compounds showed cytotoxicity, among which, compound 2 exhibited GI50 value of 14.12µM. The antiproliferative activity of 2 against Bel-7402 and CaEs-17 cells was also measured, with GI50 values of 1.91 and 2.58µM, respectively. Intensive mechanism study showed that 2 caused cell-cycle arrest at S/G2 phase and induced apoptosis in Bel-7402 cells through mitochondria-related pathway.