The repair of DNA damage is a complex process that relies on particular pathways to remedy specific types of damage to DNA. The range of insults to DNA includes small, modest changes in structure including mismatched bases and simple methylation events to oxidized bases, intra- and interstrand DNA crosslinks, DNA double strand breaks and protein-DNA adducts. Pathways required for the repair of these lesions include mismatch repair, base excision repair, nucleotide excision repair, and the homology directed repair/Fanconi anemia pathway. Each of these pathways contributes to genetic stability, and mutations in genes encoding proteins involved in these pathways have been demonstrated to promote genetic instability and cancer. In fact, it has been suggested all cancers display defects in DNA repair. It has also been demonstrated that the ability of cancer cells to repair therapeutically induced DNA damage impacts therapeutic efficacy. This has led to targeting DNA repair pathways and proteins to develop anti-cancer agents that will increase sensitivity to traditional chemotherapeutics. While initial studies languished and were plagued by a lack of specificity and a defined mechanism of action, more recent approaches to exploit synthetic lethal interaction and develop high affinity chemical inhibitors have proven considerably more effective. In this review we will highlight recent advances and discuss previous failures in targeting DNA repair to pave the way for future DNA repair targeted agents and their use in cancer therapy.
Programmable nucleases like the popular CRISPR/Cas9 system allow for precision genome engineering by inducing a site-specific DNA double strand break (DSB) within a genome. The DSB is repaired by endogenous DNA repair pathways, either nonhomologous end joining (NHEJ) or homology directed repair (HDR). The predominant and error-prone NHEJ pathway often results in small nucleotide insertions or deletions that can be used to construct knockout alleles. Alternatively, HDR activity can result in precise modification incorporating exogenous DNA fragments into the cut site. However, genetic recombination in mammalian systems through the HDR pathway is an inefficient process and requires cumbersome laboratory methods to identify the desired accurate insertion events. This is further compromised by the activity of the competing DNA repair pathway, NHEJ, which repairs the majority of nuclease induced DNA DSBs and also is responsible for mutagenic insertion and deletion events at off-target locations throughout the genome. Various methodologies have been developed to increase the efficiency of designer nuclease-based HDR mediated gene editing. Here, we review these advances toward modulating the activities of the two critical DNA repair pathways, HDR and NHEJ, to enhance precision genome engineering.
Design, synthesis, and in vitro bioevaluation of a new class of potential dual PPARalpha and gamma agonists discovered through a structure-driven design paradigm are described. The 7-hydroxy-benzopyran-4-one moiety (includes flavones, flavanones, and isoflavones) is the key pharmacophore of these novel molecules, exhibiting similarity to the core structure of both fibrates and thiazolidinediones. New lead PPAR ligands were identified from "natraceuticals" and synthetic analogues. In total, 77 molecules, including chalcones, flavones, flavanones, isoflavones, and pyrazole derivatives, were screened and structure-activity relationship studies of the dual agonists undertaken. Compounds 68, 70, 72, and 76 were identified as novel and potent dual PPARalpha and gamma agonists. These novel molecules may have the potential to be the future leads in PPAR-related disorders, including type II diabetes mellitus and metabolic syndrome.
The cellular entry of HIV-1 into CD4؉ T cells requires ordered interactions of HIV-1 envelope glycoprotein with C-X-C chemokine receptor type 4 (CXCR4) receptors. However, such interactions, which should be critical for rational structure-based discovery of new CXCR4 inhibitors, remain poorly understood. Here we first determined the effects of amino acid substitutions in CXCR4 on HIV-1 NL4-3 glycoprotein-elicited fusion events using site-directed mutagenesis-based fusion assays and identified 11 potentially key amino acid substitutions, including D97A and E288A, which caused >30% reductions in fusion. We subsequently carried out a computational search of a screening library containing ϳ604,000 compounds, in order to identify potential CXCR4 inhibitors. The computational search used the shape of IT1t, a known CXCR4 inhibitor, as a reference and employed various algorithms, including shape similarity, isomer generation, and docking against a CXCR4 crystal structure. Sixteen small molecules were identified for biological assays based on their high shape similarity to IT1t, and their putative binding modes formed hydrogen bond interactions with the amino acids identified above. Three compounds with piperidinylethanamine cores showed activity and were resynthesized. One molecule, designated CX6, was shown to significantly inhibit fusion elicited by X4 HIV-1 NL4-3 glycoprotein (50% inhibitory concentration [IC 50 ], 1.9 M), to inhibit Ca 2؉ flux elicited by stromal cell-derived factor 1␣ (SDF-1␣) (IC 50 , 92 nM), and to exert anti-HIV-1 activity (IC 50 , 1.5 M). Structural modeling demonstrated that CX6 bound to CXCR4 through hydrogen bond interactions with Asp97 and Glu288. Our study suggests that targeting CXCR4 residues important for fusion elicited by HIV-1 envelope glycoprotein should be a useful and feasible approach to identifying novel CXCR4 inhibitors, and it provides important insights into the mechanism by which small-molecule CXCR4 inhibitors exert their anti-HIV-1 activities.
Tonic inhibitory currents, mediated by extrasynaptic GABA receptors, are elevated at a delay following stroke. Flavonoids minimise the extent of cellular damage following stroke, but little is known about their mode of action. We demonstrate that the flavonoid, 2'-methoxy-6-methylflavone (0.1-10 µM; 2'MeO6MF), increases GABA receptor tonic currents presumably via δ-containing GABA receptors. Treatment with 2'MeO6MF 1-6 h post focal ischaemia dose dependently decreases infarct volume and improves functional recovery. The effect of 2'MeO6MF was attenuated in δ mice, indicating that the effects of the flavonoid were mediated via δ-containing GABA receptors. Further, as flavonoids have been shown to have multiple modes of action, we investigated the anti-inflammatory effects of 2'MeO6MF. Using a macrophage cell line, we show that 2'MeO6MF can dampen an LPS-induced elevation in NFkB activity. Assessment of vehicle-treated stroke animals revealed a significant increase in circulating IL1β, TNFα and IFγ levels. Treatment with 2'MeO6MF dampened the stroke-induced increase in circulating cytokines, which was blocked in the presence of the pan-AKT inhibitor, GSK690693. These studies support the hypothesis that compounds that potentiate tonic inhibition via δ-containing GABA receptors soon after stroke can afford neuroprotection.
Zingiber officinale has been used to control lipid disorders and reported to possess remarkable cholesterol-lowering activity in experimental hyperlipidaemia. In the present study, the effect of a characterized and standardized extract of Zingiber officinale on the hepatic lipid levels as well as on the hepatic mRNA and protein expression of low-density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was investigated in a high-fat diet-fed rat model. Rats were treated with an ethanol extract of Zingiber officinale (400 mg ⁄ kg) extract along with a high-fat diet for 6 weeks. The extract of Zingiber officinale significantly decreased hepatic triglyceride and tended to decrease hepatic cholesterol levels when administered over 6 weeks to the rats fed a high-fat diet. We found that in parallel, the extract up-regulated both LDL receptor mRNA and protein level and down-regulated HMG-CoA reductase protein expression in the liver of these rats. The metabolic control of body lipid homeostasis is in part due to enhanced cholesterol biosynthesis and reduced expression of LDL receptor sites following long-term consumption of high-fat diets. The present results show restoration of transcriptional and post-transcriptional changes in low-density lipoprotein and HMG CoA reductase by Zingiber officinale administration with a high-fat diet and provide a rational explanation for the effect of ginger in the treatment of hyperlipidaemia.Although cholesterol is an essential component of mammalian cell membranes, excess cellular and circulating cholesterol is detrimental and contributes to several life-threatening diseases, including atherosclerotic and coronary artery cardiovascular disease [1,2]. Cellular cholesterol homeostasis is maintained primarily through regulation of the low-density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which respectively affect the uptake and endogenous synthesis of cholesterol in the liver [3][4][5]. A large number of studies have elucidated the role of these regulatory mechanisms in vitro, in experimental animals and in humans treated with experimental diets or drugs [6][7][8]. Recent studies have also shown a regulatory effect of various phyto-ingredients on potential targets controlling in vivo cholesterol homeostasis in animal models treated with atherogenic diets [9][10][11].Zingiber officinale Roscoe (Family: Zingiberaceae) commonly known as 'ginger', is a well-known food spice which has also been used traditionally in a wide variety of ailments [12,13]. The major chemical constituents of ginger rhizome are essential volatile oils and non-volatile pungent compounds [14,15]. The volatile oil components consist mainly of various terpenoids. The non-volatile compounds include the gingerols, shogaols, paradols and zingerone. Among them, the gingerols and shogaols were identified as the major bioactive constituents of ginger, found respectively in fresh and dried ginger [16].In laboratory experiments, e...
BACKGROUND AND PURPOSEFlavonoids are known to have anxiolytic and sedative effects mediated via actions on ionotropic GABA receptors. We sought to investigate this further. EXPERIMENTAL APPROACHWe evaluated the effects of 2′-methoxy-6-methylflavone (2′MeO6MF) on native GABAA receptors in new-born rat hippocampal neurons and determined specificity from 18 human recombinant GABAA receptor subtypes expressed in Xenopus oocytes. We used ligand binding, two-electrode voltage clamp and patch clamp studies together with behavioural studies. KEY RESULTS2′MeO6MF potentiated GABA at a2b1g2L and all a1-containing GABAA receptor subtypes. At a2b2/3g2L GABAA receptors, however, 2′MeO6MF directly activated the receptors without potentiating GABA. This activation was attenuated by bicuculline and gabazine but not flumazenil indicating a novel site. Mutation studies showed position 265 in the b1/2 subunit was key to whether 2′MeO6MF was an activator or a potentiator. In hippocampal neurons, 2′MeO6MF directly activated single-channel currents that showed the hallmarks of GABAA Cl -currents. In the continued presence of 2′MeO6MF the single-channel conductance increased and these high conductance channels were disrupted by the g2(381-403) MA peptide, indicating that such currents are mediated by a2/g2-containing GABAA receptors. In mice, 2′MeO6MF (1-100 mg·kg -1 ; i.p.) displayed anxiolytic-like effects in two unconditioned models of anxiety: the elevated plus maze and light/dark tests. 2′MeO6MF induced sedative effects at higher doses in the holeboard, actimeter and barbiturate-induced sleep time tests. No myorelaxant effects were observed in the horizontal wire test. CONCLUSIONS AND IMPLICATIONS2′MeO6MF will serve as a tool to study the complex nature of the activation and modulation of GABAA receptor subtypes.
The inhibitory neurotransmitter, GABA, is a low-molecular-weight molecule that can achieve many low-energy conformations, which are recognized by GABA receptors and transporters. In this article, we assess the structure-activity relationship profiles of GABA analogs at the ionotropic ρ GABA(C) receptor. Such studies have significantly contributed to the design and development of potent and selective agonists and antagonists for this subclass of GABA receptors. With these tools in hand, the role of ρ GABA(C) receptors is slowly being realized. Of particular interest is the development of selective phosphinic acid analogs of GABA and their potential use in sleep disorders, inhibiting the development of myopia, and in improving learning and memory.
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