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The spontaneous deamination of cytosine is a major source of transitions from C•G to T•A base pairs, which account for half of known pathogenic point mutations in humans. The ability to efficiently convert targeted A•T base pairs to G•C could therefore advance the study and treatment of genetic diseases. The deamination of adenine yields inosine, which is treated as guanine by polymerases, but no enzymes are known to deaminate adenine in DNA. Here we describe adenine base editors (ABEs) that mediate the conversion of A•T to G•C in genomic DNA. We evolved a transfer RNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPR-Cas9 mutant. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs that convert targeted A•T base pairs efficiently to G•C (approximately 50% efficiency in human cells) with high product purity (typically at least 99.9%) and low rates of indels (typically no more than 0.1%). ABEs introduce point mutations more efficiently and cleanly, and with less off-target genome modification, than a current Cas9 nuclease-based method, and can install disease-correcting or disease-suppressing mutations in human cells. Together with previous base editors, ABEs enable the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage.
Thalidomide [α-(N-phthalimido)glutarimide] (1) is a sedative and antiemetic drug originally introduced into the clinic in the 1950s for the treatment of morning sickness. Although marketed as entirely safe, more than 10 000 babies were born with severe birth defects. Thalidomide was banned and subsequently approved for the treatment of multiple myeloma and complications associated with leprosy. Although known for more than 5 decades, the mechanism of teratogenicity remains to be conclusively understood. Various theories have been proposed in the literature including DNA damage and ROS and inhibition of angiogenesis and cereblon. All of the theories have their merits and limitations. Although the recently proposed cereblon theory has gained wide acceptance, it fails to explain the metabolism and low-dose requirement reported by a number of groups. Recently, we have provided convincing structural evidence in support of the presence of arene oxide and the quinone-reactive intermediates. However, the ability of these reactive intermediates to impart toxicity/teratogenicity needs investigation. Herein we report that the oxidative metabolite of thalidomide, dihydroxythalidomide, is responsible for generating ROS and causing DNA damage. We show, using cell lines, the formation of comet (DNA damage) and ROS. Using DNA-cleavage assays, we also show that catalase, radical scavengers, and desferal are capable of inhibiting DNA damage. A mechanism of teratogenicity is proposed that not only explains the DNA-damaging property but also the metabolism, low concentration, and species-specificity requirements of thalidomide.
Extracts from the rhizome of the turmeric plant are widely consumed as anti-inflammatory dietary supplements. Turmeric extract contains the three curcuminoids, curcumin (≈80% relative abundance), demethoxycurcumin (DMC; ≈15%), and bisdemethoxycurcumin (BDMC; ≈5%). A distinct feature of pure curcumin is its instability at physiological pH, resulting in rapid autoxidation to a bicyclopentadione within 10-15 min. Here, we describe oxidative transformation of turmeric extract, DMC, and BDMC and the identification of their oxidation products using LC-MS and NMR analyses. DMC autoxidized over the course of 24 h to the expected bicyclopentadione diastereomers. BDMC was resistant to autoxidation, and oxidative transformation required catalysis by horseradish peroxidase and H2O2 or potassium ferricyanide. The product of BDMC oxidation was a stable spiroepoxide that was equivalent to a reaction intermediate in the autoxidation of curcumin. The ability of DMC and BDMC to poison recombinant human topoisomerase IIα was significantly increased in the presence of potassium ferricyanide, indicating that oxidative transformation was required to achieve full DNA cleavage activity. DMC and BDMC are less prone to autoxidation than curcumin and contribute to the enhanced stability of turmeric extract at physiological pH. Their oxidative metabolites may contribute to the biological effects of turmeric extract.
Quinolones, which target gyrase and topoisomerase IV, are the most widely prescribed antibacterials worldwide. Unfortunately, their use is threatened by the increasing prevalence of target-mediated drug resistance. Greater than 90% of mutations that confer quinolone resistance act by disrupting enzyme-drug interactions coordinated by a critical water-metal ion bridge. Quinazolinediones are quinolone-like drugs but lack the skeletal features necessary to support the bridge interaction. These compounds are of clinical interest, however, because they retain activity against the most common quinolone resistance mutations. We utilized a chemical biology approach to determine how quinazolinediones overcome quinolone resistance in Bacillus anthracis topoisomerase IV. Quinazolinediones that retain activity against quinolone-resistant topoisomerase IV do so primarily by establishing novel interactions through the C7 substituent, rather than the drug skeleton. Because some quinolones are highly active against human topoisomerase IIα, we also determined how clinically relevant quinolones discriminate between the bacterial and human enzymes. Clinically relevant quinolones display poor activity against topoisomerase IIα because the human enzyme cannot support drug interactions mediated by the water-metal ion bridge. However, the inclusion of substituents that allow quinazolinediones to overcome topoisomerase IV-mediated quinolone resistance can cause cross-reactivity against topoisomerase IIα. Therefore, a major challenge in designing drugs that overcome quinolone resistance lies in the ability to identify substituents that mediate strong interactions with the bacterial, but not the human, enzymes. On the basis of our understanding of quinolone-enzyme interactions, we have identified three compounds that display high activity against quinolone-resistant B. anthracis topoisomerase IV but low activity against human topoisomerase IIα.
Analogues of the tripyrrolic natural product prodigiosin bearing an additional methyl and a carbonyl group at the C-ring were synthesised and evaluated. In vitro anticancer activity screening (NCI) and the study of modes of action (copper-mediated cleavage of double-stranded DNA and transmembrane transport of chloride anions) showed that the presence of the methyl group is not detrimental to activity. Furthermore, although the presence of an ester conjugated to the prodigiosene C-ring seems to decrease both pK(a) and chloride transport efficiency compared to the natural product, these analogues still exhibit a high rate of chloride transport. All analogues exhibit good in vitro anticancer activity and reduced toxicity compared to the natural product: compare an acute systemic toxicity of 100 mg kg(-1) in mice vs. 4 mg kg(-1) for prodigiosin, pointing towards a larger therapeutic window than for the natural product.
(-)-Epigallocatechin gallate (EGCG) is the most abundant and biologically active polyphenol in green tea (Camellia sinensis) leaves, and many of its cellular effects are consistent with its actions as a topoisomerase II poison. In contrast to genistein and several related bioflavonoids that act as interfacial poisons, EGCG was the first bioflavonoid shown to act as a covalent topoisomerase II poison. Although studies routinely examine the effects of dietary phytochemicals on enzyme and cellular systems, they often fail to consider that many compounds are altered during cooking or cellular metabolism. To this point, the majority of EGCG and related catechins in green tea leaves are epimerized during the brewing process. Epimerization inverts the stereochemistry of the bond that bridges the B- and C-rings and converts EGCG to (-)-gallocatechin gallate (GCG). Consequently, a significant proportion of EGCG that is ingested during the consumption of green tea is actually GCG. Therefore, the effects of GCG and related epimerized green tea catechins on human topoisomerase IIα and IIβ were characterized. GCG increased levels of DNA cleavage mediated by both enzyme isoforms with an activity that was similar to that of EGCG. GCG acted primarily by inhibiting the ability of topoisomerase IIα and IIβ to ligate cleaved DNA. Several lines of evidence indicate that GCG functions as a covalent topoisomerase II poison that adducts the enzyme. Finally, epimerization did not affect the reactivity of the chemical substituents (the three hydroxyl groups on the B-ring) that were required for enzyme poisoning. Thus, the activity of covalent topoisomerase II poisons appears to be less sensitive to stereochemical changes than interfacial poisons.
Although quinolones are the most commonly prescribed antibacterials, their use is threatened by an increasing prevalence of resistance. The most common causes of quinolone resistance are mutations of a specific serine or acidic residue in the A subunit of gyrase or topoisomerase IV. These amino acids are proposed to serve as a critical enzyme-quinolone interaction site by anchoring a water-metal ion bridge that coordinates drug binding. To probe the role of the proposed water-metal ion bridge, we characterized wild-type, GrlA(E85K), GrlA(S81F/E85K), GrlA(E85A), GrlA(S81F/E85A) and GrlA(S81F) Bacillus anthracis topoisomerase IV, their sensitivity to quinolones and related drugs and their use of metal ions. Mutations increased the Mg(2+) concentration required to produce maximal quinolone-induced DNA cleavage and restricted the divalent metal ions that could support quinolone activity. Individual mutation of Ser81 or Glu85 partially disrupted bridge function, whereas simultaneous mutation of both residues abrogated protein-quinolone interactions. Results provide functional evidence for the existence of the water-metal ion bridge, confirm that the serine and glutamic acid residues anchor the bridge, demonstrate that the bridge is the primary conduit for interactions between clinically relevant quinolones and topoisomerase IV and provide a likely mechanism for the most common causes of quinolone resistance.
Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
The polyphenol curcumin is the principal flavor and color component of the spice turmeric. Beyond its culinary uses, curcumin is believed to positively impact human health and displays antioxidant, anti-inflammatory, antibacterial, and chemopreventive properties. It also is in clinical trials as an anticancer agent. In aqueous solution at physiological pH, curcumin undergoes spontaneous autoxidation that is enhanced by oxidizing agents. The reaction proceeds through a series of quinone methide and other reactive intermediates to form a final dioxygenated bicyclopentadione product. Several naturally occurring polyphenols that can form quinones have been shown to act as topoisomerase II poisons (i.e., they increase levels of topoisomerase II-mediated DNA cleavage). Because several of these compounds have chemopreventive properties, we determined the effects of curcumin, its oxidative metabolites, and structurally related degradation products (vanillin, ferulic acid, and feruloylmethane) on the DNA cleavage activities of human topoisomerase IIα and IIβ. Intermediates in the curcumin oxidation pathway increased the level of DNA scission mediated by both enzymes ~4-5-fold. In contrast, curcumin and the bicyclopentadione, as well as vanillin, ferulic acid, and feruloylmethane, had no effect on DNA cleavage. As found for other quinone-based compounds, curcumin oxidation intermediates acted as redox-dependent (as opposed to interfacial) topoisomerase II poisons. Finally, under conditions that promote oxidation, the dietary spice turmeric enhanced topoisomerase II-mediated DNA cleavage. Thus, even within the more complex spice formulation, oxidized curcumin intermediates appear to function as topoisomerase II poisons.
Topoisomerase II resolves intrinsic topological problems of double-stranded DNA. As part of its essential cellular functions, the enzyme generates DNA breaks, but the regulation of this potentially dangerous process is not well understood. Here we report single-molecule fluorescence experiments that reveal a previously uncharacterized sequence of events during DNA cleavage by topoisomerase II: nonspecific DNA binding, sequence-specific DNA bending, and stochastic cleavage of DNA. We have identified unexpected structural roles of Mg(2+) ions coordinated in the TOPRIM (topoisomerase-primase) domain in inducing cleavage-competent DNA bending. A break at one scissile bond dramatically stabilized DNA bending, explaining how two scission events in opposing strands can be coordinated to achieve a high probability of double-stranded cleavage. Clamping of the protein N-gate greatly enhanced the rate and degree of DNA bending, resulting in a significant stimulation of the DNA cleavage and opening reactions. Our data strongly suggest that the accurate cleavage of DNA by topoisomerase II is regulated through a tight coordination with DNA bending.