Aptamers as reagents for high-throughput screening.

The identification of new drug candidates from chemical libraries is a major component of discovery research in many pharmaceutical companies. Given the large size of many conventional and combinatorial libraries and the rapid increase in the number of possible therapeutic targets, the speed with which efficient high-throughput screening (HTS) assays can be developed can be a rate-limiting step in the discovery process. We show here that aptamers, nucleic acids that bind other molecules with high affinity, can be used as versatile reagents in competition binding HTS assays to identify and optimize small-molecule ligands to protein targets. To illustrate this application, we have used labeled aptamers to platelet-derived growth factor B-chain and wheat germ agglutinin to screen two sets of potential small-molecule ligands. In both cases, binding affinities of all ligands tested (small molecules and aptamers) were strongly correlated with their inhibitory potencies in functional assays. The major advantages of using aptamers in HTS assays are speed of aptamer identification, high affinity of aptamers for protein targets, relatively large aptamer-protein interaction surfaces, and compatibility with various labeling/detection strategies. Aptamers may be particularly useful in HTS assays with protein targets that have no known binding partners such as orphan receptors. Since aptamers that bind to proteins are often specific and potent antagonists of protein function, the use of aptamers for target validation can be coupled with their subsequent use in HTS.

Intimal hyperplasia recurs after removal of PDGF-AB and -BB inhibition in the rat carotid artery injury model.

Several antagonists specific for platelet-derived growth factor (PDGF) or its receptors have recently been developed and shown to inhibit intimal hyperplasia formation in various animal models, but data investigating the durability of this intervention is limited. The present study was designed to investigate the potency of PDGF B-chain aptamer, a novel type of PDGF-AB and -BB antagonist, in the rat carotid model and to characterize intermediate-term effects on lesion formation. One hundred thirty-four animals were randomized to aptamer treatment or placebo. Daily treatment with the antagonist resulted in a 50% reduction in lesion size at 2 weeks (P<0.001). The beneficial effect involved increased apoptosis and possibly an interference with smooth muscle cell migration. Discontinuing administration 1 week earlier did not give any significant benefit compared with phosphate-buffered saline-treated controls. When the antagonist was administered for 2 weeks and the vessels analyzed 6 weeks later, the beneficial effect was lost and the treated lesions had a higher intima-media and area-cell ratio compared with the treated lesions in the 2-week-endpoint study. Our findings confirm a role of PDGF B-chain in intimal hyperplasia, but the successful use of PDGF antagonists may require either prolonged treatment or combination therapy with other agents.

Estimates of repolarization dispersion from electrocardiographic measurements.

BACKGROUND: Repolarization dispersion (Rd) is frequently mentioned as a predictor of cardiac abnormalities. We present a new measure of Rd based on the root-mean-square (RMS) curve of an ECG lead set and compare its performance with that of the commonly used QT dispersion (QTd) measure with the use of recovery times measured from directly recorded canine electrograms. METHODS AND RESULTS: Using isolated, perfused canine hearts suspended in a torso-shaped electrolytic tank, we simultaneously recorded electrograms from 64 epicardial sites and ECGs from 192 "body surface" sites. RMS curves were derived from 4 lead sets: epicardial, body surface, precordial, and a 6-lead optimal set. Repolarization was altered by changing cycle length, temperature, and activation sequence. Rd, calculated directly from recovery times of the 64 epicardial potentials, was then compared with the width of the T wave of the RMS curve and with QTd for each of these 4 lead sets. The correlation between T-wave width and Rd for each lead set, respectively, was epicardium, 0.91; body surface, 0.84; precordial, 0.72; and optimal leads, 0.81. The correlation between QTd and Rd for each lead set was epicardium, 0.46; body surface, 0.47; precordial, 0.17; and optimal leads, 0.11. CONCLUSIONS: RMS curve analysis provides an accurate method of estimating Rd from the body surface. In contrast, QTd analysis provides a poor estimate of Rd.

Estimates of repolarization and its dispersion from electrocardiographic measurements: direct epicardial assessment in the canine heart.

This study investigates a technique to estimate dispersion based on the root mean square (RMS) signal of multiple electrocardiographic leads. Activation and recovery times were measured from 64 sites on the epicardium of canine hearts using acute in situ or Langendorff perfused isolated heart preparations. Repolarization and its dispersion were altered by varying cycle length, myocardial temperature, or ventricular pacing site. Mean and dispersion of activation and recovery times, and activation-recovery interval (ARI) were calculated for each beat. The waveform was then calculated from all leads. Estimates of mean and dispersion of activation and recovery times and mean ARI were derived using only inflection points from the RMS waveform. QT intervals were also measured and QT dispersion was determined. Estimates determined from the RMS waveform provided accurate estimates of repolarization and were, in particular, a better measure of repolarization dispersion than QT dispersion.

Catabolism of alpha-ketoglutarate by a sucA mutant of Bradyrhizobium japonicum: evidence for an alternative tricarboxylic acid cycle.

A complete tricarboxylic acid (TCA) cycle is generally considered necessary for energy production from the dicarboxylic acid substrates malate, succinate, and fumarate. However, a Bradyrhizobium japonicum sucA mutant that is missing alpha-ketoglutarate dehydrogenase is able to grow on malate as its sole source of carbon. This mutant also fixes nitrogen in symbiosis with soybean, where dicarboxylic acids are its principal carbon substrate. Using a flow chamber system to make direct measurements of oxygen consumption and ammonium excretion, we confirmed that bacteroids formed by the sucA mutant displayed wild-type rates of respiration and nitrogen fixation. Despite the absence of alpha-ketoglutarate dehydrogenase activity, whole cells of the mutant were able to decarboxylate alpha-[U-(14)C]ketoglutarate and [U-(14)C]glutamate at rates similar to those of wild-type B. japonicum, indicating that there was an alternative route for alpha-ketoglutarate catabolism. Because cell extracts from B. japonicum decarboxylated [U-(14)C]glutamate very slowly, the gamma-aminobutyrate shunt is unlikely to be the pathway responsible for alpha-ketoglutarate catabolism in the mutant. In contrast, cell extracts from both the wild type and mutant showed a coenzyme A (CoA)-independent alpha-ketoglutarate decarboxylation activity. This activity was independent of pyridine nucleotides and was stimulated by thiamine PP(i). Thin-layer chromatography showed that the product of alpha-ketoglutarate decarboxylation was succinic semialdehyde. The CoA-independent alpha-ketoglutarate decarboxylase, along with succinate semialdehyde dehydrogenase, may form an alternative pathway for alpha-ketoglutarate catabolism, and this pathway may enhance TCA cycle function during symbiotic nitrogen fixation.

Novel approach to specific growth factor inhibition in vivo: antagonism of platelet-derived growth factor in glomerulonephritis by aptamers.

Mesangial cell proliferation and matrix accumulation, driven by platelet-derived growth factor (PDGF), contribute to many progressive renal diseases. In a novel approach to antagonize PDGF, we investigated the effects of a nuclease-resistant high-affinity oligonucleotide aptamer in vitro and in vivo. In cultured mesangial cells, the aptamer markedly suppressed PDGF-BB but not epidermal- or fibroblast-growth-factor-2-induced proliferation. In vivo effects of the aptamer were evaluated in a rat mesangioproliferative glomerulonephritis model. Twice-daily intravenous (i.v.) injections from days 3 to 8 after disease induction of 2.2 mg/kg PDGF-B aptamer, coupled to 40-kd polyethylene glycol (PEG), led to 1) a reduction of glomerular mitoses by 64% on day 6 and by 78% on day 9, 2) a reduction of proliferating mesangial cells by 95% on day 9, 3) markedly reduced glomerular expression of endogenous PDGF B-chain, 4) reduced glomerular monocyte/macrophage influx on day 6 after disease induction, and 5) a marked reduction of glomerular extracellular matrix overproduction (as assessed by analysis of fibronectin and type IV collagen) both on the protein and mRNA level. The administration of equivalent amounts of a PEG-coupled aptamer with a scrambled sequence or PEG alone had no beneficial effect on the natural course of the disease. These data show that specific inhibition of growth factors using custom-designed, high-affinity aptamers is feasible and effective.

Liposome-anchored vascular endothelial growth factor aptamers.

Nuclease-resistant aptamers identified from randomized nucleic acid libraries represent a novel class of drug candidates. Aptamers are synthesized chemically and therefore can be readily modified with functional groups that modulate their properties. We report here on the preparation, initial characterization, and functional properties of a nuclease-resistant vascular endothelial growth factor (VEGF) aptamer anchored in liposome bilayers through a lipid group on the aptamer. While the high-affinity binding to VEGF is maintained, the plasma residence time of the liposome-anchored aptamer is considerably improved compared with that of the free aptamer. The lipid group attachment and/or liposome anchoring leads to a dramatic improvement in inhibitory activity of the aptamer toward VEGF-induced endothelial cell proliferation in vitro and vascular permeability increase and angiogenesis in vivo.

2'-Fluoropyrimidine RNA-based aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF165). Inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain.

Vascular endothelial growth factor (VEGF) has been implicated in the pathological induction of new blood vessel growth in a variety of proliferative disorders. Using the SELEX process (systematic evolution of ligands by exponential enrichment), we have isolated 2'-F-pyrimidine RNA oligonucleotide ligands (aptamers) to human VEGF165. Representative aptamers from three distinct sequence families were truncated to the minimal sequence capable of high affinity binding to VEGF (23-29 nucleotides) and were further modified by replacement of 2'-O-methyl for 2'-OH at all ribopurine positions where the substitution was tolerated. Equilibrium dissociation constants for the interaction of VEGF with the truncated, 2'-O-methyl-modified aptamers range between 49 and 130 pM. These aptamers bind equally well to murine VEGF164, do not bind to VEGF121 or the smaller isoform of placenta growth factor (PlGF129), and show reduced, but significant affinity for the VEGF165/PlGF129 heterodimer. Cysteine 137 in the exon 7-encoded domain of VEGF165 forms a photo-inducible cross-link to a single uridine residue in each of the three aptamers. The aptamers potently inhibit the binding of VEGF to the human VEGF receptors, KDR and Flt-1, expressed by transfected porcine aortic endothelial cells. Furthermore, one of the aptamers is able to significantly reduce intradermal VEGF-induced vascular permeability in vivo.

Isocitrate dehydrogenase and glyoxylate cycle enzyme activities in Bradyrhizobium japonicum under various growth conditions.

Bradyrhizobium japonicum, the nitrogen-fixing symbiotic partner of soybean, was grown on various carbon substrates and assayed for the presence of the glyoxylate cycle enzymes, isocitrate lyase and malate synthase. The highest levels of isocitrate lyase [165-170 nmol min-1 (mg protein)-1] were found in cells grown on acetate or beta-hydroxybutyrate, intermediate activity was found after growth on pyruvate or galactose, and very little activity was found in cells grown on arabinose, malate, or glycerol. Malate synthase activity was present in arabinose- and malate-grown cultures and increased by only 50-80% when cells were grown on acetate. B. japonicum bacteroids, harvested at four different nodule ages, showed very little isocitrate lyase activity, implying that a complete glyoxylate cycle is not functional during symbiosis. The apparent Km of isocitrate lyase for D,L-isocitrate was fourfold higher than that of isocitrate dehydrogenase (61.5 and 15.5 microM, respectively) in desalted crude extracts from acetate-grown B. japonicum. When isocitrate lyase was induced, neither the Vmax nor the D,L-isocitrate Km of isocitrate dehydrogenase changed, implying that isocitrate dehydrogenase is not inhibited by covalent modification to facilitate operation of the glyoxylate cycle in B. japonicum.

QT interval dispersion: dispersion of ventricular repolarization or dispersion of QT interval?

The QT interval (QTI) has long been useful as a clinical index of the duration of ventricular repolarization, particularly as a marker of prolonged repolarization and its well-established association with arrhythmogenic cardiac states. Likewise, inhomogeneity (dispersion) of repolarization has been linked definitively to increased susceptibility to reentrant arrhythmias. Recent studies have reported the use of QTI dispersion as a meaningful clinical index to identify patients at risk, but the interpretation of the measurement has been controversial. A Langendorff-perfused, isolated canine heart suspended in a torso-shaped, electrolytic tank filled with NaCl-sucrose solution was used to investigate the relationship between body surface QTIs and ventricular repolarization measured directly from the cardiac surface by using activation-recovery intervals, which have been documented to reflect the duration of local action potentials as well as local refractory periods. The data showed poor correlation between cardiac surface activation-recovery intervals and QTIs, as well as the insensitivity of QTIs to regional repolarization shortening in the presence of prolonged repolarization elsewhere. Furthermore, the data confirmed that torso tank QTI dispersion does not reflect directly the full range of measured ventricular repolarization inhomogeneity. It is concluded that body surface QTI dispersion is not a reliable index of repolarization dispersion.

The Formation of Nitrogen-Fixing Bacteroids Is Delayed but Not Abolished in Soybean Infected by an [alpha]-Ketoglutarate Dehydrogenase-Deficient Mutant of Bradyrhizobium japonicum.

A mutant strain of Bradyrhizobium japonicum USDA 110 devoid of [alpha]-ketoglutarate dehydrogenase activity (LSG184) was used to test whether this tricarboxylic acid cycle enzyme is necessary to support nitrogen fixation during symbiosis with soybean (Glycine max). LSG184 formed nodules about 5 d later than the wild-type strain, and the nodules, although otherwise normal in structure, contained many fewer infected host cells than is typical. At 19 d after inoculation cells infected with the mutant strain were only partially filled with bacteroids and showed large accumulations of starch, but by 32 d after inoculation the host cells infected with the mutant appeared normal. The onset of nitrogen fixation was delayed about 15 d for plants inoculated with LSG184, and the rate, on a per nodule fresh weight basis, reached only about 20% of normal. However, because nodules formed by LSG184 contained only about 20% of the normal number of bacteroids, it could be inferred that the mutant, on an individual bacteroid basis, was fixing nitrogen at near wild-type rates. Therefore, the loss of [alpha]-ketoglutarate dehydrogenase in B. japonicum does not prevent the formation or the functioning of nitrogen-fixing bacteroids in soybean.

Grain Development Mutants of Barley ([alpha]-Amylase Production during Grain Maturation and Its Relation to Endogenous Gibberellic Acid Content).

Barley (Hordeum vulgare L. Himalaya) mutants with altered grain morphology were isolated to investigate whether defects in grain development, possibly involving gibberellins (GAs) and abscisic acid, would lead to altered patterns of [alpha]-amylase gene expression. Following treatment with sodium azide, 75 mutants, typically showing grain shriveling, were identified. At grain maturity 15 of the 75 mutants had higher [alpha]-amylase activities in shriveled grains compared with either phenotypically normal grains that developed on the same heterozygous plant or with grains of cv Himalaya. Studies of four of these mutants demonstrated increased levels of both high- and low-isoelectric point [alpha]-amylase isozymes midway through grain development. This category of mutant has been designated pga, for premature grain [alpha]-amylase. One such mutant (M326) showed an endosperm-determined inheritance pattern. When crossed into a (GA-deficient) dwarfing background there was a 10- to 20-fold reduction in [alpha]-amylase activity, suggesting a requirement for GA biosynthesis. Endogenous GAs and abscisic acid were quantified by combined gas chromatography-specific ion monitoring in normal and mutant grains of heterozygous M326 plants during the period of [alpha]-amylase accumulation. Mutant grains had significantly higher (5.8-fold) levels of the bioactive GA1 compared with normal grains but much lower (approximately 10-fold) levels of the 2[beta]-hydroxylated ("inactive") GAs, typical of developing barley grains (e.g. GA8, GA34, GA48). We propose that a reduced extent of 2[beta]-hydroxylation in the mutant grains results in an increased level of GA1, which is responsible for premature [alpha]-amylase gene expression.

Bradyrhizobium japonicum does not require alpha-ketoglutarate dehydrogenase for growth on succinate or malate.

The sucA gene, encoding the E1 component of alpha-ketoglutarate dehydrogenase, was cloned from Bradyrhizobium japonicum USDA110, and its nucleotide sequence was determined. The gene shows a codon usage bias typical of non-nif and non-fix genes from this bacterium, with 89.1% of the codons being G or C in the third position. A mutant strain of B. japonicum, LSG184, was constructed with the sucA gene interrupted by a kanamycin resistance marker. LSG184 is devoid of alpha-ketoglutarate dehydrogenase activity, indicating that there is only one copy of sucA in B. japonicum and that it is completely inactivated in the mutant. Batch culture experiments on minimal medium revealed that LSG184 grows well on a variety of carbon substrates, including arabinose, malate, succinate, beta-hydroxybutyrate, glycerol, formate, and galactose. The sucA mutant is not a succinate auxotroph but has a reduced ability to use glutamate as a carbon or nitrogen source and an increased sensitivity to growth inhibition by acetate, relative to the parental strain. Because LSG184 grows well on malate or succinate as its sole carbon source, we conclude that B. japonicum, unlike most other bacteria, does not require an intact tricarboxylic acid (TCA) cycle to meet its energy needs when growing on the four-carbon TCA cycle intermediates. Our data support the idea that B. japonicum has alternate energy-yielding pathways that could potentially compensate for inhibition of alpha-ketoglutarate dehydrogenase during symbiotic nitrogen fixation under oxygen-limiting conditions.

Three-dimensional distribution of ST-T wave alternans during acute ischemia.

INTRODUCTION: A canine model of reversible ischemia was used to measure the magnitude and transmural distribution of repolarization alternans. METHODS AND RESULTS: Twenty-four multielectrode needles were inserted into a reversibly ischemic region created by 8 minutes of coronary occlusion. One hundred ninety-two unipolar electrograms were simultaneously recorded at 1-minute intervals for 8 minutes of ischemia and 3 minutes of reflow recovery. Beat-to-beat repolarization alternans was quantified for all electrograms using the standard deviation of QRST integrals. When alternans from animals that fibrillated was compared with alternans from animals that did not, the magnitude of alternans in the fibrillation group was an average standard deviation of 1125 +/- 99.7 mV-msec at the time of fibrillation and 409 +/- 183 mV-msec at 8 minutes of ischemia in the animals that did not fibrillate. The increase in alternans occurred mainly in the mid-myocardial and epicardial regions in the animals that fibrillated. QRS morphology of sequential electrograms did not differ in beat-to-beat comparison, suggesting that repolarization alternans measured was not due to alternating conduction block in the region of reversible ischemia. CONCLUSION: During acute ischemia, the magnitude and distribution of repolarization alternans are greater and differ in hearts that experience ventricular fibrillation. This observation may have clinical utility in arrhythmia prediction. It also is consistent with the possibility there may be multiple mechanisms for repolarization alternans.

Inhibitory DNA ligands to platelet-derived growth factor B-chain.

We have identified a group of DNA molecules that bind to platelet-derived growth factor (PDGF)-AB with subnanomolar affinity from a randomized DNA library using in vitro selection. Individual ligands cloned from the affinity-enriched pool bind to PDGF-AB and PDGF-BB with comparably high affinity (Kd approximately 10(-10) M) and to PDGF-AA with lower affinity (> 10(-8) M), indicating specific recognition of the PDGF B-chain in the context of the hetero- or homodimer. The consensus secondary structure motif for most of the high-affinity ligands is a three-way helix junction with a three-nucleotide loop at the branch point. Photo-cross-linking experiments with 5-iodo-2'-deoxyuridine-substituted ligands establish a point contact between a thymidine nucleotide in the helix junction loop region and phenylalanine 84 of the PDGF-B chain. Representative minimal DNA ligands inhibit the binding of 125I-PDGF-BB but not of 125I-PDGF-AA to PDGF alpha- or beta-receptors expressed in porcine aortic endothelial (PAE) cells in a concentration-dependent manner with half-maximal effects of approximately 1 nM. The same ligands also exhibit a similar inhibitory effect on PDGF-BB-dependent [3H]thymidine incorporation in PAE cells expressing the PDGF beta-receptors. These DNA ligands represent a novel class of specific and potent antagonists of PDGF-BB and, by inference, PDGF-AB.

Genetically defined therapy of inherited long-QT syndrome. Correction of abnormal repolarization by potassium.

BACKGROUND: Many members of families with inherited long-QT (LQT) syndrome have mutations in HERG, a gene encoding a cardiac potassium channel that is modulated by extracellular potassium. We hypothesized that an increase in serum potassium would normalize repolarization in these patients. METHODS AND RESULTS: We studied seven subjects with chromosome 7-linked LQT syndrome and five normal control subjects. Repolarization was measured by ECG and body surface potential mapping during sinus rhythm, exercise, and atrial pacing, before and after serum potassium increase. Potassium administration improved repolarization in the LQT syndrome. At baseline, LQT subjects differed from control subjects: resting corrected QT interval (QTc, 627 +/- 90 versus 425 +/- 25 ms, P = .0007), QTc dispersion (133 +/- 62 versus 36 +/- 9 ms, P = .009), QT/RR slope (0.35 +/- 0.08 versus 0.24 +/- 0.07, P = .04), and global root-mean-square QT interval (RMS-QTc; 525 +/- 68 versus 393 +/- 22, P = .002). All LQT subjects had biphasic or notched T waves. After administration of potassium, the LQT group had a 24% reduction in resting QTc interval (from 617 +/- 92 to 469 +/- 23 ms, P = .004) compared with a 4% reduction among control subjects (from 425 +/- 25 to 410 +/- 45 ms, P > .05). The reduction was significantly greater in LQT subjects (P = .018). QT dispersion became normal in LQT subjects and did not change in control subjects. The slope of the relation between QT interval and cycle length (QT/RR slope) decreased toward normal. T-wave morphology improved in six of seven LQT subjects. The LQT group had a greater reduction in RMS-QTc than control subjects (P = .04). CONCLUSIONS: An increase in serum potassium corrects abnormalities of repolarization duration, T-wave morphology, QT/ RR slope, and QT dispersion in patients with chromosome 7-linked LQT.

Optimal lead selection for detection of ST segment shifts.

A comparison was made to determine the ability of optimal sets of 2-6 unipolar leads and a normal Holter lead set to estimate ST potential distributions changes induced by balloon inflation during angioplasty. The performance of these lead sets was compared to measurements observed in recorded 32-lead body surface maps. Unipolar lead potentials were estimated using a linear, least mean squared error estimator of the total body surface map. The correlation between maximum ST potential change in the body surface map and that predicted by the unipolar lead sets ranged from 0.84-0.93. The correlation between maximum ST segment change measured from the body surface map and measured from the Holter leads was 0.29. Therefore, shifts in ST segment potentials can accurately be estimated from a small number of unipolar leads. In contrast, current bipolar ambulatory recording techniques may introduce significant bias to such estimates.

Nuclease-resistant nucleic acid ligands to vascular permeability factor/vascular endothelial growth factor.

BACKGROUND: Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a potent inducer of new blood vessel growth (angiogenesis) that contributes to the pathology of many angiogenesis-associated disease states such as psoriasis, rheumatoid arthritis and cancer. Few molecular entities capable of binding to VPF/VEGF with high affinity and specificity have been described to date. RESULTS: Nuclease-resistant 2'-amino-2'-deoxypyrimidine nucleotide RNA (2'-aminopyrimidine RNA) ligands that bind to VPF/VEGF with high affinity have been identified by iterative rounds of affinity-selection/amplification from two independent random libraries. The sequence information that confers high affinity binding to VPF/VEGF is contained in a contiguous stretch of 24 nucleotides, 5'-CCCUGAUGGUAGACGCCGGGGUG-3' (2'-aminopyrimidine nucleotides are designated with italic letters). Of the 14 ribopurines in this minimal ligand, 10 can be substituted with the corresponding 2'-O-methylpurine nucleotides without a reduction in binding affinity to VPF/VEGF. In fact, the 2'-O-methyl substitution at permissive positions leads to a approximately 17-fold improvement in the binding affinity to VPF/VEGF. The higher affinity results from the reduction in the dissociation rate constant of the 2'-O-methyl-substituted RNA ligand from the protein compared to the unsubstituted ligand. The 2'-O-methyl-substituted minimal ligand, which folds into a bulged hairpin motif, is also more thermally stable than the unsubstituted ligand. Nuclease resistance of the ligand is further improved by the 2'-O-methyl substitutions and the addition of short phosphorothioate caps to the 3'- and 5'-ends. CONCLUSIONS: We have used the SELEX (systematic evolution of ligands by exponential enrichment) process in conjunction with post-SELEX modifications to define a highly nuclease-resistant oligonucleotide that binds to VPF/VEGF with high affinity and specificity.