David Graves | University of Alabama at Birmingham (original) (raw)

Papers by David Graves

Biophysical Journal, 1995

A mathematical model based on receptor-ligand interactions at a cell surface has been modified an... more A mathematical model based on receptor-ligand interactions at a cell surface has been modified and further developed to represent heterogeneous DNA-DNA hybridization on a solid surface. The immobilized DNA molecules with known sequences are called probes, and the DNA molecules in solution with unknown sequences are called targets in this model. Capture of the perfectly complementary target is modeled as a combined reaction-diffusion limited irreversible reaction. In the model, there are two different mechanisms by which targets can hybridize with the complementary probes: direct hybridization from the solution and hybridization by molecules that adsorb nonspecifically and then surface diffuse to the probe. The results indicate that nonspecific adsorption of single-stranded DNA on the surface and subsequent twodimensional diffusion can significantly enhance the overall reaction rate. Heterogeneous hybridization depends strongly on the rate constants for DNA adsorption/desorption in the non-probe-covered regions of the surface, the two-dimensional (2D) diffusion coefficient, and the size of probes and targets. The model shows that the overall kinetics of DNA hybridization to DNA on a solid support may be an extremely efficient process for physically realistic 2D diffusion coefficients, target concentrations, and surface probe densities. The implication for design and operation of a DNA hybridization surface is that there is an optimal surface probe density when 2D diffusion occurs; values above that optimum do not increase the capture rate. Our model predicts capture rates in agreement with those from recent experimental literature. The results of our analysis predict that several things can be done to improve heterogeneous hybridization: 1) the solution phase target molecules should be about 100 bases or less in size to speed solution-phase and surface diffusion; 2) conditions should be created such that reversible adsorption and two-dimensional diffusion occur in the surface regions between DNA probe molecules; 3) provided that 2) is satisfied, one can achieve results with a sparse probe coverage that are equal to or better than those obtained with a surface totally covered with DNA probes.

Organic Letters, 2002

Synthesis of a spirocyclization precursor with a truncated D ring has been accomplished. Subseque... more Synthesis of a spirocyclization precursor with a truncated D ring has been accomplished. Subsequent bis-spirocyclization induced the formation of equal amounts of the natural transoidal 10R,13R bisspirocycle and its cisoidal 10R,13S epimer under an apparent thermodynamically controlled process. A new class of toxins in shellfish, the azaspiracids, has been recently observed in mussels harvested in the surrounding waters of Europe (Scheme 1). 1 Azaspiracid (1) 2 and its related structures, azaspiracids 2-5 (2-5), 3,4 have been shown to induce serious injury to the digestive tracts, liver, pancreas, thymus, and spleen in mice. In addition to their significant biological properties, the azaspiracids represent a daunting synthetic challenge as the parent structure 1 possesses 20 stereocenters and three separate spirocyclic linkages. For these reasons, the azaspiracids have garnered significant recent attention in both the biological 1-5 and synthetic communities. 6-8 This paper discloses the successful construction of the C 1-C 17 portion of azaspiracid including the crucial C 10 , C 13 transoidal bis-spirocyclic array. Strategy The major stumbling block in the synthesis of the northern portion of azaspiracid has been the effective construction of the natural transoidal bis-spirocycle at C 10 and C 13. 8 Our laboratory 6b,c as well as others 7c,g,8 have disclosed the apparent preference for the undesired cisoidal orientation of the spirocycles on systems possessing a fully functionalized surrounding architecture (Scheme 2). One possible solution for this problem would be the simplification of the surrounding functionality in order to facilitate the desired stereochemical array. Low-level molecular modeling calculations showed that truncation of the D ring allows for a significant narrowing of the energy difference between the cisoidal and transoidal stereochemical arrays. Based on these observations, we felt it was prudent to explore a strategy in which the furan D ring was included after spirocyclization. Access to the appropriate bis-spirocyclization precursor 11 should be available from our previously established Julia coupling strategy between sulfone A and aldehyde 7 (Scheme 1). 6 † This work was performed at the University of Mississippi.

Molecular Cancer Research, 2008

Toll-like receptor 9 (TLR9) belongs to the innate immune system and recognizes microbial and vert... more Toll-like receptor 9 (TLR9) belongs to the innate immune system and recognizes microbial and vertebrate DNA. We showed previously that treatment with the TLR9-agonistic ODN M362 (a CpG sequence containing oligonucleotide) induces matrix metalloproteinase-13–mediated invasion in TLR9-expressing human cancer cell lines. Here, we further characterized the role of the TLR9 pathway in this process. We show that CpG oligonucleotides induce invasion in macrophages from wild-type C57/B6 and MyD88 knockout mice and in human MDA-MB-231 breast cancer cells lacking MyD88 expression. This effect was significantly inhibited in macrophages from TLR9 knockout mice and in human MDA-MB-231 breast cancer cells stably expressing TLR9 small interfering RNA or dominant-negative tumor necrosis factor receptor-associated factor 6 (TRAF6). Sequence modifications to the CpG oligonucleotides that targeted the stem loop and other secondary structures were shown to influence the invasion-inducing effect in MDA-...

Nucleic Acids Research, 1995

The DNA photoaffinity ligands, 7-azldoactinomycin D and 8-azidoethidium, form DNA adducts that ca... more The DNA photoaffinity ligands, 7-azldoactinomycin D and 8-azidoethidium, form DNA adducts that cause chain cleavage upon treatment with piperldlne. Chemical DNA sequencing techniques were used to detect covalent binding. The relative preferences for modifications of all possible sites defined by a base pair step (e.g. GC) were determined within all quartet contexts such as (IGCJ). These preferences are described In terms of 'effective site occupations', which express the ability of a llgand to covalently modify some base in the binding site. Ideally, the effective site occupations measured for photoaffinity agents can also be related to site-specific, non-covalent association constants of the ligand. The sites most reactive with 7-azidoactlnomycin D were those preferred for non-covalent binding of unsubstituted actinomycin D. GC sites were most reactive, but next-nearest neighbors exerted significant Influences on reactivity. GC sites in 5-(pyrimidine)GC(purine)-3' contexts, particularly TGCA, were most reactive, while reactivity was strongly suppressed for GC sites with a 5'-flanking G, or a 3-flanking C. High reactivities were also observed for bases in the first (50 GG steps in TGGT, TGGG and TGGGT sequences recently shown to bind actinomycin D with high affinity. Pyrimidine-3',5'-purine steps and GG steps flanked by a T were most preferred by 8-azidoethidium, in agreement with the behavior of unsubstftuted ethidium. The good correspondence between expected and observed covalent binding preferences of these two azlde analogs demonstrates that photoaffinity labeling can identify highly preferred sites of non-covalent DNA binding by small molecules.

Polymers, 2018

Nucleic acid therapeutics have the potential to be the most effective disease treatment strategy ... more Nucleic acid therapeutics have the potential to be the most effective disease treatment strategy due to their intrinsic precision and selectivity for coding highly specific biological processes. However, freely administered nucleic acids of any type are quickly destroyed or rendered inert by a host of defense mechanisms in the body. In this work, we address the challenge of using nucleic acids as drugs by preparing stimuli responsive poly(methacrylic acid)/poly(N-vinylpyrrolidone) (PMAA/PVPON)n multilayer hydrogel capsules loaded with ~7 kDa G-quadruplex DNA. The capsules are shown to release their DNA cargo on demand in response to both enzymatic and ultrasound (US)-triggered degradation. The unique structure adopted by the G-quadruplex is essential to its biological function and we show that the controlled release from the microcapsules preserves the basket conformation of the oligonucleotide used in our studies. We also show that the (PMAA/PVPON) multilayer hydrogel capsules can ...

Biochemistry, 2009

The G-quadruplex structural motif of DNA has emerged as a novel and exciting target for anticance... more The G-quadruplex structural motif of DNA has emerged as a novel and exciting target for anticancer drug discovery. The human telomeric G-quadruplex consists of a single strand repeat of d[AGGG(TTAGGG) 3 ] that can fold into higher-order DNA structures. Small molecules that selectively target and stabilize the G-quadruplex structure(s) may serve as potential therapeutic agents and have garnered significant interest in recent years. In the work presented here, the anticancer agent, actinomycin D, is demonstrated to bind to and induce changes in both structure and stability to both the Na + and K + forms of the G-quadruplex DNA. The binding of actinomycin D to the G-quadruplex DNAs are characterized by intrinsic association constants of approximately 2 × 10 5 M −1 (strand), 2:1 molecularity, and are shown to be enthalpically driven with binding enthalpies of approximately −7 kcal/mol. The free Na + or K + forms of the quadruplex structures differ in melting temperatures by approximately 8°C (60 and 68°C, respectively), whereas both forms, when complexed with actinomycin D are stabilized with melting temperatures of approximately 79°C. The induced CD signals observed for the actinomycin D-G-quadruplex complexes may indicate that the phenoxazone ring of actinomycin D to be stacked on the G-tetrad rather than intercalated between adjacent G-tetrads. Complex formation with actinomycin D results in changes to both the Na + or K + structural isoforms to "ligand-bound" complexes having similar structural properties and stabilities. The DNA structural motif known as the G-quadruplex has recently emerged as a novel and exciting target for the discovery and design of new classes of anticancer agents (1-3). DNA sequences that can form G-quadruplex structures are found extensively throughout the genome and are located in biologically relevant regions. These sequences and their corresponding quadruplex structures were first observed to exist in telomeric regions of DNA, located at the terminal ends of chromosomes (4-6). More recently, G-quadruplex forming sequences have been mapped to the promoter regions of a number of genes and oncogenes (7-9). It is postulated that G-quadruplex structures may serve important biological functions in the regulation of gene expression (10-12). Hence, these findings have led to an increased interest in the structural and functional features of G-quadruplex structural motif and offers potentially novel targets for the development of small molecules that could selectively target and stabilize the quadruplex structure. The G-quadruplex consists of stacked G-tetrads connected by lateral, diagonal, or external loops and has been shown to possess a wide range of structural polymorphism within guanine rich sequences that exhibit the motif. Burge and coworkers and Dai and coworkers recently published extensive reviews of the topologies of quadruplex DNA structures (13,14). The observed polymorphism(s) found for G-quadruplex structures include differences in molecularity, strand orientation, loop characteristics, and structural isomers based on selected cation buffer conditions. The structural stability of quadruplex DNAs have

Oncology Letters, 2014

Toll-like receptor-9 (TLR9) is a cellular DNA sensor of the innate immune system. TLR9 is widely ... more Toll-like receptor-9 (TLR9) is a cellular DNA sensor of the innate immune system. TLR9 is widely expressed in a number of tumors, including brain cancer; however, little is known regarding its regulation and involvement in cancer pathophysiology. The present study demonstrated that hypoxia upregulates and downregulates TLR9 expression in human brain cancer cells in vitro, in a cell-specific manner. In addition, hypoxia-induced TLR9 upregulation was associated with hypoxia-induced invasion; however, such invasion was not detected in cells where hypoxia had suppressed TLR9 expression. Furthermore, suppression of TLR9 expression through TLR9 siRNA resulted in an upregulation of matrix metalloproteinase (MMP)-2,-9 and-13 and tissue inhibitor of matrix metalloproteinases-3 (TIMP-3) mRNA, and a decreased invasion of cells in normoxia, in a cell-specific manner. In cells where hypoxia induced TLR9 expression, TLR9 expression and invasion were reduced by TLR9 siRNA. The decreased invasion observed in hypoxia was associated with the decreased expression of the MMPs and a concomitant increase in TIMP-3 expression. In conclusion, hypoxia regulates the invasion of brain cancer cells in vitro in a TLR9-dependent manner, which is considered to be associated with a complex expression pattern of TLR9-regulated mediators and inhibitors of invasion.

Nucleic acids research, 1988

The equilibrium binding of the antitumor agent m-AMSA (4'-(9-acridinylamino) methanesulfon-m-ansi... more The equilibrium binding of the antitumor agent m-AMSA (4'-(9-acridinylamino) methanesulfon-m-ansidide) has been examined by optical methods. These studies which have focused on the low bound drug concentrations (r values < 0.02, base pairs) reveal m-AMSA to bind calf thymus DNA in a highly cooperative manner as indicated by the initial positive slope of the Scatchard plot. In contrast, the studies on the parent 9-aminoacridine under identical conditions demonstrate that this compound binds DNA in a noncooperative (neighbor exclusion) manner. The positive cooperative binding phenomenon of m-AMSA is probed as a function of ionic concentration and shown to exist over the range of salt concentrations examined (0.01 to 0.1 M); however, the magnitude of the cooperative binding is altered. This observation of cooperativity is consistent with earlier studies on biologically active compounds and may be related to such binding parameters as binding sequence selectivity and/or structural perturbations to the DNA structure.

$Research paper thumbnail of {"__content__"=>"Stability of the Na Form of the Human Telomeric G-Quadruplex: Role of Adenines in Stabilizing G-Quadruplex Structure.", "sup"=>{"__content__"=>"+"}}$

ACS omega, Jan 31, 2018

G-quadruplexes are higher order DNA structures that play significant roles in gene transcription ... more G-quadruplexes are higher order DNA structures that play significant roles in gene transcription and telomeric maintenance. The formation and stability of the G-quadruplex structures are under thermodynamic control and may be of biological significance for regulatory function of cellular processes. Here, we report the structural influence and energetic contributions of the adenine bases in the loop sequences that flank G-repeats in human telomeric DNA sequence. Spectroscopic and calorimetric techniques are used to measure the thermal stability and thermodynamic contributions to the stability of human telomeric G-quadruplexes that have been designed with systematic changes of A to T throughout the telomeric sequence. These studies demonstrate that the thermal stability of the G-quadruplex structure is directly related to the number and position of the adenines that are present in the telomeric sequence. The melting temperature () was reduced from 59 °C for the wild-type sequence to 4...

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Methods in Molecular Biology, Feb 1, 2001

Biochemistry Usa, 1989

Two-dimensional N M R experiments were performed on the adduct of anthramycin with d-(ATGCAT)2 to... more Two-dimensional N M R experiments were performed on the adduct of anthramycin with d-(ATGCAT)2 to obtain the assignments of the nucleotide base and sugar protons as well as the anthramycin protons. Anthramycin is covalently attached to a guanine 2-amino group, forming the d(ATa"GCAT)*d-(ATGCAT) modified duplex. The anthramycin protons in the minor groove exhibit NOES to several nucleotide protons. The network of anthramycin-nucleotide NOES and the measurement of the 10-Hz coupling constant between the anthramycin H11 and H 1 l a protons shows that anthramycin is covalently attached as the S stereoisomer at the anthramycin C11 position with the side chain of anthramycin oriented toward the 5' end of the modified strand. The N O E data show that the anthramycin-modified duplex is in a right-handed conformation with all bases in an anti conformation. Analysis of the .Ill2, coupling constants for the resolved H1' resonances shows that the S-type conformation of the sugars is highly preferred. A n t h r a m y c i n (Figure 1) is a potent antitumor antibiotic which reacts covalently with guanosine-containing duplex DNA to form an adduct that spans a four base pair region [for a review of pyrrolo[ 1,4] benzodiazepine antibiotics, see Hurley and Needham-VanDevanter (1986)l. Hurley and Petrusek (1979) and Petrusek et al. (1981) concluded that anthramycin forms a minor groove adduct with the exocyclic amino group of guanine. Carbon-13 and proton NMR experiments confirmed this conclusion and demonstrated the anthramycin C11 position as the reactive site (Graves et al., 1984). Subsequent 'H NMR studies were performed on an anthramycin-modified deoxyhexanucleotide, d(ATamG-CAT).d(ATGCAT), in which anthramycin is covalently attached to the exocyclic amino group of one of the two guanines in the duplex. The other guanine is sterically blocked from reaction with anthramycin, and thus only a 1:l anthramycin-duplex adduct is formed. These experiments showed that adduct formation had not significantly perturbed the B-DNA conformation and provided insight into solution geometry and dynamics (Graves et al., 1985). The C11 position of anthramycin could be attacked by the exocyclic amino group of guanine from either face of the molecule, leading to potential formation of R and S isomers of the adduct. Furthermore, each of these isomers has two potential orientations of the anthramycin side chain in the minor groove (Figure 1). Our previous NMR studies showed that one form of the complex predominates in solution, al-?This research was supported in part by U S. Public Health Service Research Grants CA-35251 (T.R.K.), CA-41474 (D.E.G.), and ES-03755 (M.P.S.).

J Biomol Struct Dyn, 1989

Actinomycin D (ActD) is a DNA-binding antitumor antibiotic that appears to act in vivo by inhibit... more Actinomycin D (ActD) is a DNA-binding antitumor antibiotic that appears to act in vivo by inhibiting RNA polymerase. The mechanism of DNA binding of ActD has attracted much attention because of its strong preference for 5'-dGpdC-3' sequences. Binding is thought to involve intercalation of the tricyclic aromatic phenoxazone ring into a GC step, with the two equivalent cyclic pentapeptide lactone substituents lying in the minor groove and making hydrogen bond contacts with the 2-amino groups of the nearest neighbor guanines. Recent studies have indicated, however, that binding is also influenced by next-nearest neighboring bases. We have examined this higher order specificity using 7-azido-actinomycin-D as a photoaffinity probe, and DNA sequencing techniques to quantitatively monitor sites of covalent photoaddition. We found that GC doublets were strongly preferred only if the 5'-flanking base was a pyrimidine and the 3'-flanking base was not cytosine. In addition we observed a previously unreported preference for binding at a GG doublet in the sequence 5'-TGGG-3'.

Breast cancer research and treatment, Jan 18, 2016

Toll-like receptor 9 (TLR9) is a cellular DNA-receptor widely expressed in cancers. We previously... more Toll-like receptor 9 (TLR9) is a cellular DNA-receptor widely expressed in cancers. We previously showed that synthetic and self-derived DNA fragments induce TLR9-mediated breast cancer cell invasion in vitro. We investigated here the invasive effects of two nuclease-resistant DNA fragments, a 9-mer hairpin, and a G-quadruplex DNA based on the human telomere sequence, both having native phosphodiester backbone. Cellular uptake of DNAs was investigated with immunofluorescence, invasion was studied with Matrigel-assays, and mRNA and protein expression were studied with qPCR and Western blotting and protease activity with zymograms. TLR9 expression was suppressed through siRNA. Although both DNAs induced TLR9-mediated changes in pro-invasive mRNA expression, only the telomeric G-quadruplex DNA significantly increased cellular invasion. This was inhibited with GM6001 and aprotinin, suggesting MMP- and serine protease mediation. Furthermore, complexing with LL-37, a cathelicidin-peptide ...

... Specific Agents; Hurley, LH, Ed.; JAI Press: Greenwich, CT, 1992; pp. 25-50. 36. Rill, RL; Ma... more ... Specific Agents; Hurley, LH, Ed.; JAI Press: Greenwich, CT, 1992; pp. 25-50. 36. Rill, RL; Marsch, GA; Graves, DE Nucleic Acids Res. 1995 23, 1252-1259. 37. Reinhardt, CG; Krugh, TR Biochemistry 1978, 17, 4845-4854. 186 DAVID E. GRAVES 38. Kastrup, RV; Young, MA ...

Journal of analytical toxicology, Jan 20, 2015

Current methods of methadone analysis in untreated urine are traditionally limited to enzyme immu... more Current methods of methadone analysis in untreated urine are traditionally limited to enzyme immunoassays (EIA) while confirmation techniques require specimen processing (i.e., sample clean-up) before analyzing by gas or liquid chromatography coupled with mass spectrometry (GC-MS or LC-MS-MS). EIA and traditional confirmation techniques can be costly and, at times inefficient. As an alternative approach, we present Direct Analysis in Real Time (DART™) coupled with both time-of-flight and triple quadrupole linear ion trap (Q-TRAP™) mass spectrometers for screening and confirming methadone in untreated urine specimens. These approaches require neither expensive kits nor sample clean-up for analysis. More importantly, the total combined analysis time for both screening and confirmation methods was <5 min per sample; in contrast to the 3-5 day process required by traditional EIA, GC-MS and LC-MS-MS techniques. To examine the fundamental protocol and its applicability for routine drug...