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Lipid nanoparticles (LNPs) are the most extensively validated clinical delivery vehicles for mRNA therapeutics, exemplified by their widespread use in the mRNA COVID-19 vaccines. The pace of lipid nanoparticle (LNP) development for mRNA therapeutics is restricted by the limitations of existing methods for large-scale LNP screening. To address this challenge, we developed Quantitative Analysis of Reverse Transcribed Barcodes (QuART), a novel nucleic-acid-based system for measuring LNP functional delivery in vivo. QuART uses a bacterial retron reverse transcription system to couple functional mRNA delivery into the cytoplasm with a cDNA barcode readout. Our results demonstrate that QuART can be used to identify functional mRNA delivery both in vitro in cell culture and in vivo in mice. Multiplexing of QuART could enable high-throughput screening of LNP formulations, facilitating the rapid discovery of promising LNP candidates for mRNA therapeutics., Competing Interests: The authors declare no competing financial interest., (© 2025 The Authors. Published by American Chemical Society.)

Equilibrative nucleoside transporters (ENTs) mediate the transmembrane flux of endogenous nucleosides and nucleoside analogs used clinically. The predominant subtype, ENT1, has been well characterized. However, the other subtype, ENT2, has been less well characterized in its native milieu due to its relatively low expression and the confounding influence of coexpressed ENT1. We created a cell model where ENT1 was removed from human embryonic kidney (HEK293) cells using CRISPR/cas9 [ENT1 knockout (KO) cells]; this cell line has ENT2 as the only functional purine transporter. Transporter function was assessed through measurement of [ 3 H]2-chloroadenosine uptake. ENT1 protein was quantified based on the binding of [ 3 H]nitrobenzylthioinosine, and ENT1/ENT2 protein was detected by immunoblotting. Changes in expression of relevant transporters and enzymes involved in purine metabolism were examined by quantitative polymerase chain reaction. Wild-type HEK293 cells and ENT1KO cells had a similar expression of SLC29A2 /ENT2 transcript/protein and ENT2-mediated [ 3 H]2-chloroadenosine transport activity (V max values of 1.02 ± 0.06 and 1.50 ± 0.22 pmol/ μ l/s, respectively). Of the endogenous nucleosides/nucleobases tested, adenosine had the highest affinity (K i ) for ENT2 (2.6 μ M), while hypoxanthine was the only nucleobase with a submillimolar affinity (320 μ M). A range of nucleoside/nucleobase analogs were also tested for their affinity for ENT2 in this model, with affinities (K i ) ranging from 8.6 μ M for ticagrelor to 2,300 μ M for 6-mercaptopurine. Our data suggest that the removal of endogenous ENT1 from these cells does not change the expression or function of ENT2. This cell line should prove useful for the analysis of novel drugs acting via ENT2 and to study ENT2 regulation. SIGNIFICANCE STATEMENT: We have created a cell line whereby endogenous ENT2 can be studied in detail in the absence of the confounding influence of ENT1. Loss of ENT1 has no impact on the expression and function of ENT2. This novel cell line will provide an ideal model for studying drug interactions with ENT2 as well as the cellular regulation of ENT2 expression and function., (Copyright © 2024 by The Author(s).)

Background: In humans, two ubiquitously expressed N-myristoyltransferases, NMT1 and NMT2, catalyze myristate transfer to proteins to facilitate membrane targeting and signaling. We investigated the expression of NMTs in numerous cancers and found that NMT2 levels are dysregulated by epigenetic suppression, particularly so in hematologic malignancies. This suggests that pharmacological inhibition of the remaining NMT1 could allow for the selective killing of these cells, sparing normal cells with both NMTs., Methods and Results: Transcriptomic analysis of 1200 NMT inhibitor (NMTI)-treated cancer cell lines revealed that NMTI sensitivity relates not only to NMT2 loss or NMT1 dependency, but also correlates with a myristoylation inhibition sensitivity signature comprising 54 genes (MISS-54) enriched in hematologic cancers as well as testis, brain, lung, ovary, and colon cancers. Because non-myristoylated proteins are degraded by a glycine-specific N-degron, differential proteomics revealed the major impact of abrogating NMT1 genetically using CRISPR/Cas9 in cancer cells was surprisingly to reduce mitochondrial respiratory complex I proteins rather than cell signaling proteins, some of which were also reduced, albeit to a lesser extent. Cancer cell treatments with the first-in-class NMTI PCLX-001 (zelenirstat), which is undergoing human phase 1/2a trials in advanced lymphoma and solid tumors, recapitulated these effects. The most downregulated myristoylated mitochondrial protein was NDUFAF4, a complex I assembly factor. Knockout of NDUFAF4 or in vitro cell treatment with zelenirstat resulted in loss of complex I, oxidative phosphorylation and respiration, which impacted metabolomes., Conclusions: Targeting of both, oxidative phosphorylation and cell signaling partly explains the lethal effects of zelenirstat in select cancer types. While the prognostic value of the sensitivity score MISS-54 remains to be validated in patients, our findings continue to warrant the clinical development of zelenirstat as cancer treatment., (© 2024. The Author(s).)

Background: Doxorubicin, a first-line anticancer drug for osteosarcoma treatment, has been the subject of recent research exploring the mechanisms behind its chemoresistance and its ability to enhance cell migration at sublethal concentrations. Matrix metalloproteinase-2 (MMP-2), a type IV collagenase and zinc-dependent endopeptidase, is well-known for degrading the extracellular matrix and promoting cancer metastasis. Our previous work demonstrated that nuclear MMP-2 regulates ribosomal RNA transcription via histone clipping, thereby controlling gene expression. Additionally, MMP-2 activity is regulated by the non-receptor tyrosine kinase and oncogene, Src, which plays a crucial role in cell adhesion, invasion, and metastasis. Src kinase is primarily regulated by two endogenous inhibitors: C-terminal Src kinase (Csk) and Csk homologous kinase (CHK/MATK)., Aim: In this study, we reveal that the MMP-2 gene acts as an upstream regulator of Src kinase activity by suppressing its endogenous inhibitor, CHK/MATK, in osteosarcoma cells., Methods and Results: We show that enhanced osteosarcoma cell migration which is induced by sublethal concentrations of doxorubicin can be overcome by inactivating the MMP-2 gene or overexpressing CHK/MATK. Our findings highlight the MMP-2 gene as a promising additional target for combating cancer cell migration and metastasis. This is due to its role in suppressing on the gene and protein expression of the tumor suppressor CHK/MATK in osteosarcoma., Conclusion: By targeting the MMP-2 gene, we can potentially enhance the effectiveness of doxorubicin treatment and reduce chemoresistance in osteosarcoma., (© 2023 The Authors. Cancer Reports published by Wiley Periodicals LLC.)

The emergence of severe acute respiratory syndrome coronavirus 2, the causative agent of coronavirus disease 2019, has resulted in the largest pandemic in recent history. Current therapeutic strategies to mitigate this disease have focused on the development of vaccines and on drugs that inhibit the viral 3CL protease or RNA-dependent RNA polymerase enzymes. A less-explored and potentially complementary drug target is Nsp15, a uracil-specific RNA endonuclease that shields coronaviruses and other nidoviruses from mammalian innate immune defenses. Here, we perform a high-throughput screen of over 100,000 small molecules to identify Nsp15 inhibitors. We characterize the potency, mechanism, selectivity, and predicted binding mode of five lead compounds. We show that one of these, IPA-3, is an irreversible inhibitor that might act via covalent modification of Cys residues within Nsp15. Moreover, we demonstrate that three of these inhibitors (hexachlorophene, IPA-3, and CID5675221) block severe acute respiratory syndrome coronavirus 2 replication in cells at subtoxic doses. This study provides a pipeline for the identification of Nsp15 inhibitors and pinpoints lead compounds for further development against coronavirus disease 2019 and related coronavirus infections., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Immunomodulatory Siglecs are controlled by their glycoprotein and glycolipid ligands. Siglec-glycolipid interactions are often studied outside the context of a lipid bilayer, missing the complex behaviors of glycolipids in a membrane. Through optimizing a liposomal formulation to dissect Siglec-glycolipid interactions, it is shown that Siglec-6 can recognize glycolipids independent of its canonical binding pocket, suggesting that Siglec-6 possesses a secondary binding pocket tailored for recognizing glycolipids in a bilayer. A panel of synthetic neoglycolipids is used to probe the specificity of this glycolipid binding pocket on Siglec-6, leading to the development of a neoglycolipid with higher avidity for Siglec-6 compared to natural glycolipids. This neoglycolipid facilitates the delivery of liposomes to Siglec-6 on human mast cells, memory B-cells and placental syncytiotrophoblasts. A physiological relevance for glycolipid recognition by Siglec-6 is revealed for the binding and internalization of extracellular vesicles. These results demonstrate a unique and physiologically relevant ability of Siglec-6 to recognize glycolipids in a membrane., (© 2023. The Author(s).)

LPP2 is one of three enzymes in the lipid phosphate phosphatase family (LPP1-3) that dephosphorylate extracellular and intracellular bioactive lipid phosphates and pyrophosphates. LPP2 increases cell growth and LPP2 expression is elevated in a variety of malignancies, implying that LPP2 is a pro-tumorigenic factor. Methods: LPP2 expression in human breast tumors and normal breast tissue was measured by qPCR. To understand the role of LPP2, we knocked out its expression in multiple cell lines using CRISPR/Cas9. Cell proliferation and migration were compared between wild type and LPP2 knockout cells. Cell cycle was measured by flow cytometry, and cell cycle proteins were determined by western blotting. Effects of LPP2 on tumor growth were investigated using syngeneic and xenograft mouse breast cancer models. Results: LPP2 mRNA levels were higher in ER/PR positive, ER/HER2 positive, and triple negative human breast tumors, relative to normal breast tissue. Higher levels of LPP2 in breast tumors, hepatocellular carcinoma, pancreatic adenocarcinoma, and melanomas were prognostic of poorer survival. LPP2 mRNA expression is also increased in Hs-578T, MDA-MB-231, MCF7 and MDA-MB-468 breast cancer cell lines, relative to non-malignant Hs-578Bst, MCF10A and MCF-12A cells. LPP2 knockout in breast cancer cells decreased cell growth by inhibiting G1/S transition, whereas, increasing LPP2 levels in Hs-578Bst and MCF10A cells promoted proliferation. The effects of LPP2 on cell cycle were associated with changes in cyclin A2, cyclin B1, and cell cycle inhibitors, p27 or p21. The level of c-Myc was downregulated by knocking out LPP2, and it was partly restored by re-expressing LPP2. The positive correlation between the expression of LPP2 and c-Myc exists in multiple cancer cell lines including breast, lung, upper aerodigestive tract and urinary tract cancer. LPP2 knockout in MDA-MB-231 or 4T1 cells suppressed tumor formation in mouse breast cancer models, and decreased the in vivo expression of Ki67 and c-Myc of the cancer cells. Conclusion: Targeting LPP2 could provide a new strategy for decreasing c-Myc expression and tumor growth., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Gene-editing systems such as CRISPR-Cas9 readily enable individual gene phenotypes to be studied through loss of function. However, in certain instances, gene compensation can obfuscate the results of these studies, necessitating the editing of multiple genes to properly identify biological pathways and protein function. Performing multiple genetic modifications in cells remains difficult due to the requirement for multiple rounds of gene editing. While fluorescently labeled guide RNAs (gRNAs) are routinely used in laboratories for targeting CRISPR-Cas9 to disrupt individual loci, technical limitations in single gRNA (sgRNA) synthesis hinder the expansion of this approach to multicolor cell sorting. Here, we describe a modular strategy for synthesizing sgRNAs where each target sequence is conjugated to a unique fluorescent label, which enables fluorescence-activated cell sorting (FACS) to isolate cells that incorporate the desired combination of gene-editing constructs. We demonstrate that three short strands of RNA functionalized with strategically placed 5'-azide and 3'-alkyne terminal deoxyribonucleotides can be assembled in a one-step, template-assisted, copper-catalyzed alkyne-azide cycloaddition to generate fully functional, fluorophore-modified sgRNAs. Using these synthetic sgRNAs in combination with FACS, we achieved selective cleavage of two targeted genes, either separately as a single-color experiment or in combination as a dual-color experiment. These data indicate that our strategy for generating double-clicked sgRNA allows for Cas9 activity in cells. By minimizing the size of each RNA fragment to 41 nucleotides or less, this strategy is well suited for custom, scalable synthesis of sgRNAs.

CRISPR/Cas complexes enable precise gene editing in a wide variety of organisms. While the rigid identification of DNA sequences by these systems minimizes the potential for off-target effects, it consequently poses a problem for the recognition of sequences containing naturally occurring polymorphisms. The presence of genetic variance such as single nucleotide polymorphisms (SNPs) in a gene sequence can compromise the on-target activity of CRISPR systems. Thus, when attempting to target multiple variants of a human gene, or evolved variants of a pathogen gene using a single guide RNA, more flexibility is desirable. Here, we demonstrate that Cas9 can tolerate the inclusion of universal bases in individual guide RNAs, enabling simultaneous targeting of polymorphic sequences. Crucially, we find that specificity is selectively degenerate at the site of universal base incorporation, and remains otherwise preserved. We demonstrate the applicability of this technology to targeting multiple naturally occurring human SNPs with individual guide RNAs and to the design of Cas12a/Cpf1-based DETECTR probes capable of identifying multiple evolved variants of the HIV protease gene. Our findings extend the targeting capabilities of CRISPR/Cas systems beyond their canonical spacer sequences and highlight a use of natural and synthetic universal bases., (© 2022. The Author(s).)

An epithelial-to-mesenchymal transition (EMT) phenotype with cancer stem cell-like properties is a critical feature of aggressive/metastatic tumors, but the mechanism(s) that promote it and its relation to metabolic stress remain unknown. Here we show that Collapsin Response Mediator Protein 2A (CRMP2A) is unexpectedly and reversibly induced in cancer cells in response to multiple metabolic stresses, including low glucose and hypoxia, and inhibits EMT/stemness. Loss of CRMP2A, when metabolic stress decreases (e.g., around blood vessels in vivo) or by gene deletion, induces extensive microtubule remodeling, increased glutamine utilization toward pyrimidine synthesis, and an EMT/stemness phenotype with increased migration, chemoresistance, tumor initiation capacity/growth, and metastatic potential. In a cohort of 27 prostate cancer patients with biopsies from primary tumors and distant metastases, CRMP2A expression decreases in the metastatic versus primary tumors. CRMP2A is an endogenous molecular brake on cancer EMT/stemness and its loss increases the aggressiveness and metastatic potential of tumors., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Cell proliferation and survival require continuous ribosome biogenesis and protein synthesis. Genes encoding ribosomal RNA are physically located in a specialized substructure within the nucleus known as the nucleolus, which has a central role in the biogenesis of ribosomes. Matrix metalloproteinase-2 was previously detected in the nucleus, however, its role there is elusive. Herein we report that matrix metalloproteinase-2 resides within the nucleolus to regulate ribosomal RNA transcription. Matrix metalloproteinase-2 is enriched at the promoter region of ribosomal RNA gene repeats, and its inhibition downregulates preribosomal RNA transcription. The N-terminal tail of histone H3 is clipped by matrix metalloproteinase-2 in the nucleolus, which is associated with increased ribosomal RNA transcription. Knocking down/out matrix metalloproteinase-2, or inhibiting its activity, prevents histone H3 cleavage and reduces both ribosomal RNA transcription and cell proliferation. In addition to the known extracellular roles of matrix metalloproteinase-2 in tumor growth, our data reveal an epigenetic mechanism whereby intranucleolar matrix metalloproteinase-2 regulates cell proliferation through histone clipping and facilitation of ribosomal RNA transcription., (© 2021 Federation of European Biochemical Societies.)

Nicotinamide adenine dinucleotide (NAD + ) plays a vital role in cellular processes that govern human health and disease. Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme in NAD + biosynthesis. Thus, boosting NAD + level via an increase in NAMPT levels is an attractive approach for countering the effects of aging and metabolic disease. This study aimed to establish IRW (Ile-Arg-Trp), a small tripeptide derived from ovotransferrin, as a booster of NAMPT levels. Treatment of muscle (L6) cells with IRW increased intracellular NAMPT protein levels (2.2-fold, p < 0.05) and boosted NAD + ( p < 0.01). Both immunoprecipitation and recombinant NAMPT assays indicated the possible NAMPT-activating ability of IRW ( p < 0.01). Similarly, IRW increased NAMPT mRNA and protein levels in the liver (2.6-fold, p < 0.01) and muscle tissues (2.3-fold, p < 0.05) of C57BL/6J mice fed with a high-fat diet (HFD). A significantly increased level of circulating NAD + was also observed following IRW treatment (4.7 fold, p < 0.0001). Dosing of Drosophila melanogaster with IRW elevated both D-NAAM (fly NAMPT) and NAD + in vivo ( p < 0.05). However, IRW treatment did not boost NAMPT levels in SIRT1 KO cells, indicating a possible SIRT1 dependency for the pharmacological effect. Overall, these data indicate that IRW is a novel small peptide booster of the NAMPT pool.

In addition to advancing the development of gene-editing therapeutics, CRISPR/Cas9 is transforming how functional genetic studies are carried out in the lab. By increasing the ease with which genetic information can be inserted, deleted, or edited in cell and organism models, it facilitates genotype-phenotype analysis. Moreover, CRISPR/Cas9 has revolutionized the speed at which new genes underlying a particular phenotype can be identified through its application in genomic screens. Arrayed high-throughput and pooled lentiviral-based CRISPR/Cas9 screens have now been used in a wide variety of contexts, including the identification of essential genes, genes involved in cancer metastasis and tumor growth, and even genes involved in viral response. This technology has also been successfully used to identify drug targets and drug resistance mechanisms. Here, we provide a detailed protocol for performing a genome-wide pooled lentiviral CRISPR/Cas9 knockout screen to identify genetic modulators of a small-molecule drug. While we exemplify how to identify genes involved in resistance to a cytotoxic histone deacetylase inhibitor, Trichostatin A (TSA), the workflow we present can easily be adapted to different types of selections and other types of exogenous ligands or drugs., (© 2021. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Tubby-like proteins (TULPs) are characterized by a conserved C-terminal domain that binds phosphoinositides. Collectively, mammalian TULP1-4 proteins play essential roles in intracellular transport, cell differentiation, signaling, and motility. Yet, little is known about how the function of these proteins is regulated in cells. Here, we present the protein-protein interaction network of TULP3, a protein that is responsible for the trafficking of G-protein-coupled receptors to cilia and whose aberrant expression is associated with severe developmental disorders and polycystic kidney disease. We identify several protein interaction nodes linked to TULP3 that include enzymes involved in acetylation and ubiquitination. We show that acetylation of two key lysine residues on TULP3 by p300 increases TULP3 protein abundance and that deacetylation of these sites by HDAC1 decreases protein levels. Furthermore, we show that one of these sites is ubiquitinated in the absence of acetylation and that acetylation inversely correlates with ubiquitination of TULP3. This mechanism is evidently conserved across species and is active in zebrafish during development. Finally, we identify this same regulatory module in TULP1, TULP2, and TULP4 and demonstrate that the stability of these proteins is similarly modulated by an acetylation switch. This study unveils a signaling pathway that links nuclear enzymes to ciliary membrane receptors via TULP3, describes a dynamic mechanism for the regulation of all tubby-like proteins, and explores how to exploit it pharmacologically using drugs., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The CRISPR/Cas9 system has transformed how gene knockout and knock-in studies are performed in the lab, and it is poised to revolutionize medicine. However, one of the present limitations of this technology is its imperfect specificity. While CRISPR/Cas9 can be programmed to cut a specific DNA target sequence with relative precision, off-target sequence cleavage can occur in large genomes. Importantly, several techniques have recently been developed to measure CRISPR/Cas9 on- and off-target DNA cleavage in cells. Here, we present detailed protocols for evaluating the specificity of CRISPR/Cas9 and related systems in cells using both targeted-approaches, in which off-target sites are known a priori, and unbiased approaches which are able to identify off-target cleavage events throughout an entire genome. Together, these techniques can be used to assess the reliability of experimental models generated using CRISPR/Cas9 as well as the safety of therapeutics employing this technology.

CRISPR/Cas9 has revolutionized the ability to edit cellular DNA and is poised to transform the treatment of genetic diseases. One of the major concerns regarding its therapeutic use is the potential for off-target DNA cleavage, which could have detrimental consequences in vivo. To circumvent this, a number of strategies have been employed to develop next-generation CRISPR/Cas9 systems with improved specificity. These include the development of new protein variants of Cas9, as well as chemically modified guide RNA molecules. Here, we provide detailed protocols for two in vitro methods that enable the specificity of first- and next-generation CRISPR/Cas9 systems to be compared, and we demonstrate their applicability to evaluating chemically modified guide RNAs. One of these assays allows the specificity of different guide RNA/Cas9 complexes to be compared on a set of known off-target DNA sequences, while the second provides a broad specificity profile based on cleavage of a massive library of potential off-target DNA sequences. Collectively, these assays may be used to evaluate the specificity of different CRISPR/Cas9 systems on any DNA target sequence in a time- and cost-effective manner.

Resveratrol (3,4,5-Trihydroxy-trans-stilbene) is a naturally occurring polyphenol that exhibits beneficial pleiotropic health effects. It is one of the most promising natural molecules in the prevention and treatment of chronic diseases and autoimmune disorders. One of the key limitations in the clinical use of resveratrol is its extensive metabolic processing to its glucuronides and sulfates. It has been estimated that around 75% of this polyphenol is excreted via feces and urine. To possibly alleviate the extensive metabolic processing and improve bioavailability, we have added segments of acetylsalicylic acid to resveratrol in an attempt to maintain the functional properties of both. We initially characterized resveratrol-aspirin derivatives as products that can inhibit cytochrome P450 Family 1 Subfamily A Member 1 (CYP1A1) activity, DNA methyltransferase (DNMT) activity, and cyclooxygenase (COX) activity. In this study, we provide a detailed analysis of how resveratrol and its aspirin derivatives can inhibit nuclear factor kappa B (NFκB) activation, cytokine production, the growth rate of cancer cells, and in vivo alleviate intestinal inflammation and tumor growth. We identified resveratrol derivatives C3 and C11 as closely preserving resveratrol bioactivities of growth inhibition of cancer cells, inhibition of NFκB activation, activation of sirtuin, and 5' adenosine monophosphate-activated protein kinase (AMPK) activity. We speculate that the aspirin derivatives of resveratrol would be more metabolically stable, resulting in increased efficacy for treating immune disorders and as an anti-cancer agent.

In this issue of Cell Chemical Biology, Erdogan et al. (2020) describe a new CRISPR/Cas9-based strategy for performing directed evolution of mammalian proteins in situ. Using this technique to select functional mRuby3 variants within lysosomes, they identify mCRISPRed, a fluorescent protein that displays robust stability and activity at low pH., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Osteoblasts maintain the structural integrity of bone via differentiation and mineralization; therefore, their malfunction or reduced activity can cause serious bone disorders. Although studies have demonstrated the association between nutrients and bone, research on food-derived bioactive peptides and bone health are scanty., Methods: Osteoblasts MC3T3-E1 were treated with IRW (50 and 25 μM). Cell proliferation, cell cycle, osteoblastic differentiation, and mineralization were tested to evaluate the effects of IRW on osteogenesis promotion. The activation of PI3K-Akt-RUNX2 pathway and collagen synthesis were investigated to better understand the functions of IRW., Results: IRW treatment (50 and 25 μM) in MC3T3-E1 cells caused a significant increase in cell proliferation by increasing the percentage of S and G2/M phase. Furthermore, IRW promoted mineralization in MC3T3-E1 cells. Mechanistically, we found that IRW treatment resulted in a 4-fold increase of Akt serine phosphorylation and a 2-fold increase of its downstream target RUNX2. Expression levels of RUNX2 associated proteins were concomitantly altered: ALP (2-fold increase), Col1A2 (2-fold increase), RANKL (2-fold decrease), and OPG (2-fold increase). Meanwhile, a parallel collagen synthesis pathway was found to contribute to IRW-stimulated osteogenesis., Conclusions: IRW, an egg-derived small bioactive peptide enhances osteoblastic activity and stimulates osteogenesis. The stimulation is primarily due to the activation of PI3K-Akt-RUNX2 pathway and its downstream effectors, accompanied by a secondary collagen synthesis pathway., General Significance: Our results revealed the positive effects of tripeptide IRW on regulating osteogenesis and collagen synthesis, indicating its potential for the prevention or treatment of osteoporosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Receptor-interacting protein kinase 2 (RIP2 or RICK, herein referred to as RIPK2) is linked to the pathogen pathway that activates nuclear factor κ -light-chain-enhancer of activated B cells (NF κ B) and autophagic activation. Using molecular modeling (docking) and chemoinformatics analyses, we used the RIPK2/ponatinib crystal structure and searched in chemical databases for small molecules exerting binding interactions similar to those exerted by ponatinib. The identified RIPK2 inhibitors potently inhibited the proliferation of cancer cells by > 70% and also inhibited NF κ B activity. More importantly, in vivo inhibition of intestinal and lung inflammation rodent models suggests effectiveness to resolve inflammation with low toxicity to the animals. Thus, our identified RIPK2 inhibitor may offer possible therapeutic control of inflammation in diseases such as inflammatory bowel disease, asthma, cystic fibrosis, primary sclerosing cholangitis, and pancreatitis., (Copyright © 2018 by The Author(s).)

Off-target DNA cleavage is a paramount concern when applying CRISPR-Cas9 gene-editing technology to functional genetics and human therapeutic applications. Here, we show that incorporation of next-generation bridged nucleic acids (2',4'-BNA NC [N-Me]) as well as locked nucleic acids (LNA) at specific locations in CRISPR-RNAs (crRNAs) broadly reduces off-target DNA cleavage by Cas9 in vitro and in cells by several orders of magnitude. Using single-molecule FRET experiments we show that BNA NC incorporation slows Cas9 kinetics and improves specificity by inducing a highly dynamic crRNA-DNA duplex for off-target sequences, which shortens dwell time in the cleavage-competent, "zipped" conformation. In addition to describing a robust technique for improving the precision of CRISPR/Cas9-based gene editing, this study illuminates an application of synthetic nucleic acids.

The human genome encodes 538 protein kinases that transfer a γ-phosphate group from ATP to serine, threonine, or tyrosine residues. Many of these kinases are associated with human cancer initiation and progression. The recent development of small-molecule kinase inhibitors for the treatment of diverse types of cancer has proven successful in clinical therapy. Significantly, protein kinases are the second most targeted group of drug targets, after the G-protein-coupled receptors. Since the development of the first protein kinase inhibitor, in the early 1980s, 37 kinase inhibitors have received FDA approval for treatment of malignancies such as breast and lung cancer. Furthermore, about 150 kinase-targeted drugs are in clinical phase trials, and many kinase-specific inhibitors are in the preclinical stage of drug development. Nevertheless, many factors confound the clinical efficacy of these molecules. Specific tumor genetics, tumor microenvironment, drug resistance, and pharmacogenomics determine how useful a compound will be in the treatment of a given cancer. This review provides an overview of kinase-targeted drug discovery and development in relation to oncology and highlights the challenges and future potential for kinase-targeted cancer therapies.

DNA repair is essential for life, yet its efficiency declines with age for reasons that are unclear. Numerous proteins possess Nudix homology domains (NHDs) that have no known function. We show that NHDs are NAD + (oxidized form of nicotinamide adenine dinucleotide) binding domains that regulate protein-protein interactions. The binding of NAD + to the NHD domain of DBC1 (deleted in breast cancer 1) prevents it from inhibiting PARP1 [poly(adenosine diphosphate-ribose) polymerase], a critical DNA repair protein. As mice age and NAD + concentrations decline, DBC1 is increasingly bound to PARP1, causing DNA damage to accumulate, a process rapidly reversed by restoring the abundance of NAD + Thus, NAD + directly regulates protein-protein interactions, the modulation of which may protect against cancer, radiation, and aging., (Copyright © 2017, American Association for the Advancement of Science.)

The accumulation of damage caused by oxidative stress has been linked to aging and to the etiology of numerous age-related diseases. The longevity gene, sirtuin 6 (SIRT6), promotes genome stability by facilitating DNA repair, especially under oxidative stress conditions. Here we uncover the mechanism by which SIRT6 is activated by oxidative stress to promote DNA double-strand break (DSB) repair. We show that the stress-activated protein kinase, c-Jun N-terminal kinase (JNK), phosphorylates SIRT6 on serine 10 in response to oxidative stress. This post-translational modification facilitates the mobilization of SIRT6 to DNA damage sites and is required for efficient recruitment of poly (ADP-ribose) polymerase 1 (PARP1) to DNA break sites and for efficient repair of DSBs. Our results demonstrate a post-translational mechanism regulating SIRT6, and they provide the link between oxidative stress signaling and DNA repair pathways that may be critical for hormetic response and longevity assurance., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

The NAD(+)-dependent deacetylase SIRT1 plays key roles in numerous cellular processes including DNA repair, gene transcription, cell differentiation, and metabolism. Overexpression of SIRT1 protects against a number of age-related diseases including diabetes, cancer, and Alzheimer's disease. Moreover, overexpression of SIRT1 in the murine brain extends lifespan. A number of small-molecule sirtuin-activating compounds (STACs) that increase SIRT1 activity in vitro and in cells have been developed. While the mechanism for how these compounds act on SIRT1 was once controversial, it is becoming increasingly clear that they directly interact with SIRT1 and enhance its activity through an allosteric mechanism. Here, we present detailed chemical syntheses for four STACs, each from a distinct structural class. Also, we provide a general protocol for purifying active SIRT1 enzyme and outline two complementary enzymatic assays for characterizing the effects of STACs and similar compounds on SIRT1 activity., (© 2016 Elsevier Inc. All rights reserved.)

Nucleases containing programmable DNA-binding domains can alter the genomes of model organisms and have the potential to become human therapeutics. Here we present DNA-binding phage-assisted continuous evolution (DB-PACE) as a general approach for the laboratory evolution of DNA-binding activity and specificity. We used this system to generate transcription activator-like effectors nucleases (TALENs) with broadly improved DNA cleavage specificity, establishing DB-PACE as a versatile approach for improving the accuracy of genome-editing agents.

A number of small molecules with the ability to extend the lifespan of multiple organisms have recently been discovered. Resveratrol, amongst the most prominent of these, has gained widespread attention due to its ability to extend the lifespan of yeast, worms, and flies, and its ability to protect against age-related diseases such as cancer, Alzheimer's, and diabetes in mammals. In this review, we discuss the origins and molecular targets of resveratrol and provide an overview of its effects on the lifespan of simple model organisms and mammals. We also examine the unique ability of resveratrol to extend the healthy years, or healthspan, of mammals and its potential to counteract the symptoms of age-related disease. Finally, we explore the many scientific, medical, and economic challenges faced when translating these findings to the clinic, and examine potential approaches for realizing the possibility of human lifespan extension. This article is part of a Special Issue entitled: Resveratrol: Challenges in translating pre-clinical findings to improved patient outcomes., (Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.)

Recent studies in mice have identified single molecules that can delay multiple diseases of aging and extend lifespan. In theory, such molecules could prevent dozens of diseases simultaneously, potentially extending healthy years of life. In this review, we discuss recent advances, controversies, opportunities, and challenges surrounding the development of SIRT1 activators, molecules with the potential to delay aging and age-related diseases. Sirtuins comprise a family of NAD⁺-dependent deacylases that are central to the body's response to diet and exercise. New studies indicate that both natural and synthetic sirtuin activating compounds (STACs) work via a common allosteric mechanism to stimulate sirtuin activity, thereby conferring broad health benefits in rodents, primates, and possibly humans. The fact that two-thirds of people in the USA who consume multiple dietary supplements consume resveratrol, a SIRT1 activator, underscores the importance of understanding the biochemical mechanism, physiological effects, and safety of STACs., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Ever since eukaryotes subsumed the bacterial ancestor of mitochondria, the nuclear and mitochondrial genomes have had to closely coordinate their activities, as each encode different subunits of the oxidative phosphorylation (OXPHOS) system. Mitochondrial dysfunction is a hallmark of aging, but its causes are debated. We show that, during aging, there is a specific loss of mitochondrial, but not nuclear, encoded OXPHOS subunits. We trace the cause to an alternate PGC-1α/β-independent pathway of nuclear-mitochondrial communication that is induced by a decline in nuclear NAD(+) and the accumulation of HIF-1α under normoxic conditions, with parallels to Warburg reprogramming. Deleting SIRT1 accelerates this process, whereas raising NAD(+) levels in old mice restores mitochondrial function to that of a young mouse in a SIRT1-dependent manner. Thus, a pseudohypoxic state that disrupts PGC-1α/β-independent nuclear-mitochondrial communication contributes to the decline in mitochondrial function with age, a process that is apparently reversible., (Copyright © 2013 Elsevier Inc. All rights reserved.)

SIRT1 is an NAD (+) -dependent deacetylase that counteracts multiple disease states associated with aging and may underlie some of the health benefits of calorie restriction. Understanding how SIRT1 is regulated in vivo could therefore lead to new strategies to treat age-related diseases. SIRT1 forms a stable complex with DBC1, an endogenous inhibitor. Little is known regarding the biochemical nature of SIRT1-DBC1 complex formation, how it is regulated and whether or not it is possible to block this interaction pharmacologically. In this study, we show that critical residues within the catalytic core of SIRT1 mediate binding to DBC1 via its N-terminal region, and that several carboxamide SIRT1 inhibitors, including EX-527, can completely block this interaction. We identify two acetylation sites on DBC1 that regulate its ability to bind SIRT1 and suppress its activity. Furthermore, we show that DBC1 itself is a substrate for SIRT1. Surprisingly, the effect of EX-527 on SIRT1-DBC1 binding is independent of DBC1 acetylation. Together, these data show that protein acetylation serves as an endogenous regulatory mechanism for SIRT1-DBC1 binding and illuminate a new path to developing small-molecule modulators of SIRT1.

A molecule that treats multiple age-related diseases would have a major impact on global health and economics. The SIRT1 deacetylase has drawn attention in this regard as a target for drug design. Yet controversy exists around the mechanism of sirtuin-activating compounds (STACs). We found that specific hydrophobic motifs found in SIRT1 substrates such as PGC-1α and FOXO3a facilitate SIRT1 activation by STACs. A single amino acid in SIRT1, Glu(230), located in a structured N-terminal domain, was critical for activation by all previously reported STAC scaffolds and a new class of chemically distinct activators. In primary cells reconstituted with activation-defective SIRT1, the metabolic effects of STACs were blocked. Thus, SIRT1 can be directly activated through an allosteric mechanism common to chemically diverse STACs.

Type 1 diabetes is caused by autoimmune-mediated β cell destruction leading to insulin deficiency. The histone deacetylase SIRT1 plays an essential role in modulating several age-related diseases. Here we describe a family carrying a mutation in the SIRT1 gene, in which all five affected members developed an autoimmune disorder: four developed type 1 diabetes, and one developed ulcerative colitis. Initially, a 26-year-old man was diagnosed with the typical features of type 1 diabetes, including lean body mass, autoantibodies, T cell reactivity to β cell antigens, and a rapid dependence on insulin. Direct and exome sequencing identified the presence of a T-to-C exchange in exon 1 of SIRT1, corresponding to a leucine-to-proline mutation at residue 107. Expression of SIRT1-L107P in insulin-producing cells resulted in overproduction of nitric oxide, cytokines, and chemokines. These observations identify a role for SIRT1 in human autoimmunity and unveil a monogenic form of type 1 diabetes., (Copyright © 2013 Elsevier Inc. All rights reserved.)

SIRT1 is an evolutionarily conserved protein deacetylase that modulates stress response, cellular metabolism and aging in model organisms. While SIRT1 exerts beneficial effects in protecting against age-related diseases, the role of SIRT1 in cancer has been controversial. SIRT1 promotes cell survival by deacetylating, and thereby negatively regulating the activity of important tumor suppressors such as p53. In this regard, SIRT1 has been considered to be a potential oncogene, and SIRT1 inhibitors have been studied for possible anticancer therapeutic effects. In contrast, it has been shown that SIRT1 deficiency leads to increased genomic instability and tumorigenesis, and that overexpression of SIRT1 attenuates cancer formation in mice, suggesting it may also act as a tumor suppressor. Based on this evidence, SIRT1-activating molecules could act as candidate chemotherapeutic drugs. In order to gain insight into the role of SIRT1 in cancer, we performed a comprehensive resequencing analysis of the SIRT1 gene in 41 tumor cell lines and found an unusually excessive homozygosity, which was confirmed to be allelic loss by microsatellite analysis. Furthermore, we found two novel SIRT1 mutations (D739Y and R65&#95;A72del) in addition to the known, rare non-synonymous variation resulting in I731V. In vitro assays using purified SIRT1 protein showed that these mutations do not alter SIRT1 deacetylase activity or telomerase activity, which was shown to be regulated by SIRT1. We conclude that allelic loss or mutations in the SIRT1 gene occur prevalently during tumorigenesis, supporting the assertion that SIRT1 may serve as a tumor suppressor.

The sirtuins are NAD(+)-dependent, multifunctional lysine deacylases that play key roles in cellular homeostasis. They are increasingly being found to target a variety of substrates including acetyl-, butyryl-, malonyl-, and succinyl-lysines. Early assays for measuring sirtuin activity in vitro were criticized for their use of fluorophores on the peptide substrates used, which may alter the results obtained and not be representative of the in vivo situation. We describe a new protocol for the measurement of sirtuin activity by detecting the production of nicotinamide (NAM). The assay is amenable to any substrate and any modification removed by sirtuins. The assay may also be used to measure glycohydrolase (e.g., CD38) and ADP-ribosyltransferase activity (e.g., mARTs and PARPs).

Our interest in healthy aging and in evolutionarily conserved mechanisms of lifespan extension prompted us to investigate whether features of age-related decline in the honey bee could be attenuated with resveratrol. Resveratrol is regarded as a caloric restriction mimetic known to extend lifespan in some but not all model species. The current, prevailing view is that resveratrol works largely by activating signaling pathways. It has also been suggested that resveratrol may act as an antioxidant and confer protection against nervous system impairment and oxidative stress. To test whether honey bee lifespan, learning performance, and food perception could be altered by resveratrol, we supplemented the diets of honey bees and measured lifespan, olfactory learning, and gustatory responsiveness to sucrose. Furthermore, to test the effects of resveratrol under metabolic challenge, we used hyperoxic environments to generate oxidative stress. Under normal oxygen conditions, two resveratrol treatments-30 and 130 μM-lengthened average lifespan in wild-type honey bees by 38% and 33%, respectively. Both resveratrol treatments also lengthened maximum and median lifespan. In contrast, hyperoxic stress abolished the resveratrol life-extension response. Furthermore, resveratrol did not affect learning performance, but did alter gustation. Honey bees that were not fed resveratrol exhibited greater responsiveness to sugar, while those supplemented with resveratrol were less responsive to sugar. We also discovered that individuals fed a high dose of resveratrol-compared to controls-ingested fewer quantities of food under ad libitum feeding conditions.

Resveratrol induces mitochondrial biogenesis and protects against metabolic decline, but whether SIRT1 mediates these benefits is the subject of debate. To circumvent the developmental defects of germline SIRT1 knockouts, we have developed an inducible system that permits whole-body deletion of SIRT1 in adult mice. Mice treated with a moderate dose of resveratrol showed increased mitochondrial biogenesis and function, AMPK activation, and increased NAD(+) levels in skeletal muscle, whereas SIRT1 knockouts displayed none of these benefits. A mouse overexpressing SIRT1 mimicked these effects. A high dose of resveratrol activated AMPK in a SIRT1-independent manner, demonstrating that resveratrol dosage is a critical factor. Importantly, at both doses of resveratrol no improvements in mitochondrial function were observed in animals lacking SIRT1. Together these data indicate that SIRT1 plays an essential role in the ability of moderate doses of resveratrol to stimulate AMPK and improve mitochondrial function both in vitro and in vivo., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Berberine (BBR) has recently been shown to improve insulin sensitivity in rodent models of insulin resistance. Although this effect was explained partly through an observed activation of AMP-activated protein kinase (AMPK), the upstream and downstream mediators of this phenotype were not explored. Here, we show that BBR supplementation reverts mitochondrial dysfunction induced by High Fat Diet (HFD) and hyperglycemia in skeletal muscle, in part due to an increase in mitochondrial biogenesis. Furthermore, we observe that the prevention of mitochondrial dysfunction by BBR, the increase in mitochondrial biogenesis, as well as BBR-induced AMPK activation, are blocked in cells in which SIRT1 has been knocked-down. Taken together, these data reveal an important role for SIRT1 and mitochondrial biogenesis in the preventive effects of BBR on diet-induced insulin resistance., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In mammals, the transcriptional activity of signal transducer and activator of transcription 3 (STAT3) is regulated by the deacetylase SIRT1. However, whether the newly described nongenomic actions of STAT3 toward mitochondrial oxidative phosphorylation are dependent on SIRT1 is unclear. In this study, Sirt1 gene knock-out murine embryonic fibroblast (MEF) cells were used to delineate the role of SIRT1 in the regulation of STAT3 mitochondrial function. Here, we show that STAT3 mRNA and protein levels and the accumulation of serine-phosphorylated STAT3 in mitochondria were increased significantly in Sirt1-KO cells as compared with wild-type MEFs. Various mitochondrial bioenergetic parameters, such as the oxygen consumption rate in cell cultures, enzyme activities of the electron transport chain complexes in isolated mitochondria, and production of ATP and lactate, indicated that Sirt1-KO cells exhibited higher mitochondrial respiration as compared with wild-type MEFs. Two independent approaches, including ectopic expression of SIRT1 and siRNA-mediated knockdown of STAT3, led to reduction in intracellular ATP levels and increased lactate production in Sirt1-KO cells that were approaching those of wild-type controls. Comparison of profiles of phospho-antibody array data indicated that the deletion of SirT1 was accompanied by constitutive activation of the pro-inflammatory NF-κB pathway, which is key for STAT3 induction and increased cellular respiration in Sirt1-KO cells. Thus, SIRT1 appears to be a functional regulator of NF-κB-dependent STAT3 expression that induces mitochondrial biogenesis. These results have implications for understanding the interplay between STAT3 and SIRT1 in pro-inflammatory conditions.

Sirt1 is an NAD(+)-dependent deacetylase that extends lifespan in lower organisms and improves metabolism and delays the onset of age-related diseases in mammals. Here we show that SRT1720, a synthetic compound that was identified for its ability to activate Sirt1 in vitro, extends both mean and maximum lifespan of adult mice fed a high-fat diet. This lifespan extension is accompanied by health benefits including reduced liver steatosis, increased insulin sensitivity, enhanced locomotor activity and normalization of gene expression profiles and markers of inflammation and apoptosis, all in the absence of any observable toxicity. Using a conditional SIRT1 knockout mouse and specific gene knockdowns we show SRT1720 affects mitochondrial respiration in a Sirt1- and PGC-1α-dependent manner. These findings indicate that SRT1720 has long-term benefits and demonstrate for the first time the feasibility of designing novel molecules that are safe and effective in promoting longevity and preventing multiple age-related diseases in mammals.

SIRT3 is one of the seven mammalian sirtuin homologs of the yeast SIR2 gene. SIRT3 possesses NAD(+)-dependent protein deacetylase activity. Recent studies indicate that the murine SIRT3 gene expresses different transcript variants, resulting in three possible SIRT3 protein isoforms with various lengths at the N-terminus: M1 (aa 1-334), M2 (aa 15-334), and M3 (aa 78-334). The transcript variants 1 and 3 can only produce M3 protein, while M1 and M2 proteins are translationally initiated from different in-frame ATG sites in transcript 2. Here we report that three transcript variants of the mouse SIRT3 gene are broadly expressed in various mouse tissues. By expressing these SIRT3 isoforms in HEK293 cells through transient transfection, we confirmed recent reports that two longer murine SIRT3 proteins (M1 and M2) are targeted to mitochondria with higher efficiency than the shorter M3 isoform. Additionally, the M1 and M2 proteins are processed into a mature form. Using Edman degradation we identify Ile38 (majority) or Val42 as the N-terminal amino acid of the mature M1 isoform, and Met78 or Val79 as the N-terminal amino acid of the M3 isoform. Interestingly, we found that even upon mutation of the M2 ATG site in the M1 cDNA, a processed mature protein could still be produced. In terms of deacetylase activity, we found that although only the mature protein derived from M1 or M2 proteins were active against acetylated peptide substrates, all three forms had equal deacetylase activity towards a full-length native protein substrate, acetyl CoA synthetase 2., (© 2010 Wiley-Liss, Inc.)

SIRT1 is a protein deacetylase that has emerged as a therapeutic target for the development of activators to treat diseases of aging. SIRT1-activating compounds (STACs) have been developed that produce biological effects consistent with direct SIRT1 activation. At the molecular level, the mechanism by which STACs activate SIRT1 remains elusive. In the studies reported herein, the mechanism of SIRT1 activation is examined using representative compounds chosen from a collection of STACs. These studies reveal that activation of SIRT1 by STACs is strongly dependent on structural features of the peptide substrate. Significantly, and in contrast to studies reporting that peptides must bear a fluorophore for their deacetylation to be accelerated, we find that some STACs can accelerate the SIRT1-catalyzed deacetylation of specific unlabeled peptides composed only of natural amino acids. These results, together with others of this study, are at odds with a recent claim that complex formation between STACs and fluorophore-labeled peptides plays a role in the activation of SIRT1 (Pacholec, M., Chrunyk, B., Cunningham, D., Flynn, D., Griffith, D., Griffor, M., Loulakis, P., Pabst, B., Qiu, X., Stockman, B., Thanabal, V., Varghese, A., Ward, J., Withka, J., and Ahn, K. (2010) J. Biol. Chem. 285, 8340-8351). Rather, the data suggest that STACs interact directly with SIRT1 and activate SIRT1-catalyzed deacetylation through an allosteric mechanism.