Deoxyadenosine-based DNA polymerase photoprobes: design, synthesis, and characterization as inhibitors of the Escherichia coli DNA polymerase I Klenow fragment.

DNA polymerase photoprobes 2-[(4-azidophenacyl)thio]-2'-deoxyadenosine 5'-triphosphate (1), 2-[(4-azidophenylsulfenyl)thio]-2'-deoxyadenosine 5'-triphosphate (2), and 2-[(4-azido-2-nitrophenyl)-thio]-2'-deoxyadenosine 5'-triphosphate (3) were designed from a thermodynamic model of DNA polymerase 1-substrate interactions such that the triphosphate would anchor the inhibitor and allow the phenyl azide to interact with the complementary template binding site. Photoprobes 1-3 were synthesized by condensation of 2-thio-2'-deoxyadenosine or its phosphate with p-azidophenacyl bromide, N-(4-azidophenylsulfenyl)phthalimide, and 4-azido-1-fluoro-2-nitrobenzene, respectively, and characterized as reversible and photoinduced irreversible inhibitors of the DNA polymerase I Klenow fragment and HIV I reverse transcriptase. The aryl azides decomposed with irradiation at 300 and 350 nm with half-lives ranging from 0.98 to 2.33 min and 2.15 to 5.38 min, respectively, with quantum efficiencies ranging from 0.29 to 0.55 and no apparent photodecomposition of the 2-thio-2'-deoxyadenosine nucleotide. Photoprobes 1-3 showed mixed noncompetitive inhibition of the Klenow fragment polymerase activity versus poly(dA).(T)10 as variable substrate with apparent competitive inhibition constants of 2.1, 36, and 29 microM, respectively, evidence suggesting that these photoprobes bind to both the free enzyme form and the enzyme-template-primer binary complex. Of the three photoprobes, only nucleotide 1 photoinactivates the Klenow fragment; in the presence of a 200-fold excess of nitrene scavenger, photoprobe 1 inactivates 92% of the Klenow fragment polymerase activity with saturation observed at 9.7 microM and an IC50 of about 2 microM. This evidence demonstrates that photoprobe 1 does bind to the Klenow fragment in the absence of template-primer and that it is an efficient photoprobe.

DNA polymerase photoprobe 2-[(4-azidophenacyl)thio]-2'-deoxyadenosine 5'-triphosphate labels an Escherichia coli DNA polymerase I Klenow fragment substrate binding site.

The nucleotide photoprobe 2-[(4-azidophenacyl)thio]-2'-deoxyadenosine 5'-triphosphate (1) was evaluated as a photoaffinity label of the DNA polymerase I Klenow fragment. Photolabel [3H]-1 covalently labeled the Klenow fragment with photolysis at 300 nm, reaching saturation at an approximate 1:1 mole ratio at 5.7 microM and with an EC50 (the effective concentration at 50% maximum photoincorporation) of about 0.74 microM. Saturating concentrations of poly(dA).(T)10 protect the Klenow fragment from [3H]-1 photoincorporation, and TTP at a concentration approximately equal to its KD for the free enzyme form shifts the dose-response curve for photoincorporation of [3H]-1 into the Klenow fragment by a factor of 2, indicating a competitive relationship between TTP and 1. Additionally, the photoincorporation of [3H]-1 into the Klenow fragment has an absolute requirement for magnesium, with no significant photoincorporation observed at concentrations of 1 up to 10 microM in the absence of magnesium. These results demonstrate that, as designed, photoprobe 1 binds to both the dNTP and a portion of the template-primer binding sites on the Klenow fragment. Photoaffinity labeling of the Klenow fragment by 1 yielded a single radiolabeled tryptic fragment which was isolated by HPLC; sequence analysis identified Asp732 in the peptide fragment Asp732-Ile733-His734-Arg735 as the site of covalent modification. Molecular modeling and complementary NMR analysis of the conformation of 1 indicated preferred C3'-exo and C2'-exo-C3'-endo symmetrical twist furanose ring puckers, with a high antibase conformation and a +sc C-5 torsional angle. Docking studies using Asp732 as an anchor point for the azide alpha-nitrogen on the photolabel indicate that the dNTP binding site is at the edge of the DNA binding cleft opposite the exonuclease site and that the template binding site includes helix O in the finger motif of the Klenow fragment.

Physical and conformational properties of synthetic idealized signal sequences parallel their biological function.

Transported proteins often contain an extension sequence called the signal peptide. The alkaline phosphatase (PhoA) signal sequence represents a typical signal peptide for comparison to idealized sequences both in vivo and in vitro. We have designed a series of idealized signal sequences which vary in amino terminal charge and core region hydrophobicity with minimal variation in amino acid composition. The idealized core regions contain different proportions of leucine and alanine residues, effectively producing hydrophobicities above and below the threshold level required for efficient secretion. The flanking amino and carboxyl termini were designed to maintain the general features and relative hydrophobicity of their counterparts in the wild-type PhoA signal sequence. Using the phoA gene, the signal peptide region was modified to generate mutants corresponding to the model sequences. Transport studies in Escherichia coli confirmed that completely idealized signal sequences, which lack a helix-breaking proline or glycine residue, can be functional if the core region is sufficiently hydrophobic and that one positively charged residue in the amino terminus is adequate for efficient transport. The corresponding peptides were chemically synthesized and exhibited HPLC retention times that reflect the relative hydrophobicities of the sequences. Structural analyses of the isolated peptides by circular dichroism demonstrate solvent dependence and exceptionally stable alpha-helix formation by the functional signal peptides in trifluoroethanol. Although leucine and alanine residues are often predicted to have similar propensities for forming an alpha-helix, considerably higher alpha-helical content is observed in the signal peptides which contain predominantly polyleucine core regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Conformational properties of the proline region of porcine neuropeptide Y by CD and 1H-nmr spectroscopy.

We synthesized porcine neuropeptide Y (pNPY) N-terminal fragments by solid-phase synthesis techniques and analyzed them for solution conformational properties by CD and 1H-nmr spectroscopy. The analogues pNPY1-9 and pNPY1-14 displayed CD spectra indicative of random structures and showed no evidence for induced alpha-helical structures in trifluoroethanol (TFE) up to 50%. However, the CD spectra of pNPY1-9 suggested a conformational shift in tetrahydrofuran. Although in aqueous solution the CD spectra of pNPY1-21 indicated random structures with induction of only a small percentage of alpha-helix in aqueous TFE, pNPY1-25 displayed 13% alpha-helical structure in aqueous solution that increased to 40 and 41% by the addition of TFE and methanol, respectively. The nmr spectra of pNPY1-9 and the proline region of pNPY1-25 indicated extended structures with all-trans conformers at Pro5 and Pro8 for pNPY1-9 and at Pro5, Pro8, and Pro13 for pNPY1-25; in each case the Tyr1-Pro2 amide bond was in both cis and trans conformations. However, observed nuclear Overhauser effect correlations and HN exchange experiments indicated an alpha-helical segment in pNPY1-25 initiated by Pro13 and extending from residues 14 to 25. Thus, the N-terminal polyproline region of NPY has no propensity to fold into a regular secondary structure, although Pro13 is a helix initiator, a result consistent with the proposed role of this amino acid in the NPY structural model.

Neuropeptide Y N-terminal deletion fragments: correlation between solution structure and receptor binding activity at Y1 receptors in rat brain cortex.

N alpha-Acetyl (Ac), N-terminal deletion fragments of porcine neuropeptide Y (NPY) have been synthesized and characterized for solution conformation properties by circular dichroism and for receptor binding activity at benextramine-sensitive Y1 binding sites in rat brain cortex. Sequential deletion of Tyr1, Pro2, and Ser3 had no effect on the structural (alpha-helical content of 32.5, 30.6, and 30.7%, respectively, at 1 x 10(-5) M) or aggregation (monomer to dimer transition for N alpha-Ac-NPY3-36 and N alpha-Ac-NPY4-36) properties of NPY. In contrast, deletion of Tyr1 decreased receptor binding activity in rat brain cortex by 4-fold (IC50 = 13.0 nM versus 3.75 nM for NPY), but further deletion of Pro2-Ser3 had no additional detrimental effect on receptor binding activity relative to the desTyr1 analog. Thus, Pro2 and Ser3 do not contribute either to the stability of the NPY tertiary structure nor directly to the receptor-ligand interactions. Additional removal of N-terminal amino acids Lys4-Pro5 decreased the helical content and abolished aggregation to a dimeric form of the resultant analog, results suggesting that the residues around Pro5 are important for formation of NPY's compact, pancreatic polypeptide (PP)-fold structure. This loss in structure also correlated with a further 2-3 fold drop in receptor binding activity. These structure-activity correlations provide evidence for the importance of the PP-fold structure in the activity of NPY at Y1 receptors in rat brain cortex.

Conformation of a peptide corresponding to T4 lysozyme residues 59-81 by NMR and CD spectroscopy.

The conformation, in solution, of a peptide corresponding to residues 59-81 from T4 lysozyme [LYS(59-81)] has been determined by 1H NMR and CD spectroscopy. This peptide spans the region corresponding to helix C in the crystal structure of T4 lysozyme. Secondary structure predictions indicated that the peptide would possibly be helical in an aqueous environment, but in a more hydrophobic environment the peptide would certainly adopt a helical conformation. This prediction was confirmed by the far-UV CD and NMR studies, which showed the peptide to be relatively unstructured in aqueous solution and significantly helical in the presence of either TFE or SDS micelles, although the 1H NMR results did give some indication of the presence of nascent helix in aqueous solution. For LYS(59-81), in TFE, the three-dimensional structure derived from the NMR data showed that the helix had a more pronounced curvature than the gradual bend observed in the crystal structure.

Nonpeptide peptidomimetic antagonists of the neuropeptide Y receptor: benextramine analogs with selectivity for the peripheral Y2 receptor.

We synthesized a new series of benextramine analogs as neuropeptide Y (NPY) functional group mimetics and tested them for N-[propionyl-3H]NPY ([3]NPY) displacement activity in rat brain membrane homogenates and for NPY receptor antagonist activity in the rat femoral artery. The tetraamine, carbon analog N,N'-bis[6-[N-(2-naphthylmethyl)amino]hexyl]-1,6-hexanediamine (15) was equipotent with benextramine (based on comparison of the relevant IC50's) in a rat brain [3H]NPY displacement assay, suggesting that the disulfide is not a necessary feature of benextramine's [3H]NPY displacement activity, although this analog maintained selectivity for the benextramine-sensitive binding site population. The bis(N,N-dialkylguanyl) disulfide and carbon analogs 14a-c were 3-4 times more potent than their respective controls in displacing [3H]NPY from rat brain membrane homogenates with IC50's ranging from 15 to 18 microM and maintained selectivity for the benextramine-sensitive, Y1 binding site population. However, the activity of the carbon analog N,N'-bis[6-[N-(2-naphthylmethyl)amino]hexyl]-N,N'-(1,6- hexanediyl)diguanidine tetrahydrochloride (14b) showed a different profile in a femoral artery vasoconstriction assay; at 1.0 nM, this analog shifted the concentration-effect curve of the Y2-selective agonist NPY13-36 to the right (pA2 = 9.2; Kd = 0.63 nM) without a significant change in the maximum effect, while even at 1.0 mM it had no effect on the vasoconstrictive activity of the Y1-selective agonist [Leu31,Pro34]NPY. Thus, the bis(N,N-dialkylguanidine) analogs of benextramine are selective, competitive antagonists of the postsynaptic NPY receptor in the femoral artery.

Neuropeptide Y acylation chemistry in aqueous solution: significance to synthesis of a peptide-based photoaffinity label.

Treatment of neuropeptide Y (NPY, 1) for 20 hr with a 20 equivalent excess of N-propionyl succinimide (2) in 10 mM phosphate buffer, pH 6.0, yields NPY and N alpha-propionyl-NPY (3) as major products, and at pH 7.5 the major product is N alpha, N epsilon-dipropionyl-NPY. However, acylation of NPY with one equivalent of N-(5-azido-2-nitrobenzolyloxy)-succinimide (5) is more rapid, yielding N alpha-(5-azido-2-nitrobenzoyl)-NPY (6) in 70% conversion yield after only 5 min. Thus, in spite of its increased reactivity, the N-hydroxysuccinimide active ester shows enhanced alpha- vs. epsilon-NH2 selectivity relative to 2. The activities of 3, 4, and 6 as reversible, competitive ligands at rat brain NPY binding sites and of 6 as an irreversible photoaffinity label are reported.

Characterization of vascular postsynaptic NPY receptor function and regulation and differential sensitivity of Y1 and Y2 receptor function to changes in extracellular calcium availability and prior in vitro peptide exposure.

Effects of calcium-free buffer, nifedipine, or prior cumulative neuropeptide Y (NPY) receptor agonist concentration exposure on vasoconstrictive responsiveness to the agonists were assessed in norepinephrine (NE)-conditioned isolated rat femoral artery rings. Calcium-free buffer and nifedipine partially inhibited responsiveness to initial NPY exposure; residual responsiveness to NPY re-exposure was unaffected. In contrast, these treatments markedly inhibited responsiveness to the Y2 agonist NPY13-36, the calcium channel agonist BAY K 8644 (BAY) and the partial alpha 1 adrenoceptor agonist indanidine but did not alter to the Y1 agonist [Leu31,Pro34]NPY. Responsiveness to NPY and NPY13-36 but not to BAY or indanidine was markedly reduced 120 min following conditioning regardless of prior ring exposure to the same peptide; only prior exposure reduced responsiveness to [Leu31,Pro34]NPY. Responsiveness changes to NPY at various times or after various numbers of NE and/or NPY exposures indicated that pre-exposure and time-related responsiveness reductions were discriminable and temporally unrelated to conditioning. Postsynaptic vascular Y2 receptor activation therefore accounts for the known sensitivity of NPY-induced pressor and vasoconstrictive actions to nifedipine. The 'time-dependent' loss of Y2 receptor function may also explain prior failures to observe postsynaptic arterial Y2 receptors in vitro.

The contribution of helical potential to the in vitro receptor binding activity of a neuropeptide Y N-terminal deletion fragment.

In its dimeric form neuropeptide Y (NPY) folds into a compact structure in which the antiparallel oriented proline and alpha-helices apparently associate to form a primitive hydrophobic core. To investigate the contribution of helical stability to the receptor binding activity of NPY and its N-terminal deletion fragments, we synthesized and studied the solution conformational properties and in vitro activities of NPY, N alpha-acetyl-NPY2-36, NPY15-36, N alpha-propionyl-NPY15-36, and N alpha-succinyl-NPY15-36. NPY15-36 is significantly less helical than both NPY and N alpha-acetyl-NPY2-36, and this decreased helical potential is attributed to the absence of the intramolecular stabilizing interaction afforded by the proline helix in the latter analogues. However, in accord with the helix dipole model, the helical potential of NPY15-36 is significantly increased by N-terminal succinylation, whereas propionylation has no effect. In addition to an increase in helical potential, N alpha-succinyl-NPY15-36 is 2.5 and 4.6 times more active than NPY15-36 and N alpha-propionyl-NPY15-36, respectively, and is equipotent with N alpha-acetyl-NPY2-36 in displacing 1 nM [3H]-NPY from specific binding sites in rat brain membranes. The demonstration of a positive correlation between % alpha-helix content and in vitro binding activity suggests that the helical potential of N-terminal NPY deletion fragments contributes to their in vitro activity in the rat brain, and that a second role of the proline helix might be to stabilize the receptor-active conformation of the NPY alpha-helix.

Characterization of vascular postsynaptic neuropeptide Y receptor function and regulation. 1. NPY-induced constriction in isolated rat femoral artery rings is mediated by both Y1 and Y2 receptors: evidence from benextramine protection studies.

Neuropeptide Y (NPY), a potent pressor agent and vasoconstrictor, is thought to contribute to the sympathetically mediated postsynaptic regulation of blood pressure primarily through activation of vascular Y1 rather than Y2 NPY receptors. However, data are available that conflict with this conclusion. In this article, the relative roles of postsynaptic Y1 and Y2 receptors as mediators of direct NPY-induced isolated rat femoral artery ring vasoconstriction were evaluated through use of selective Y1 and Y2 agonists, [Leu31, Pro34]NPY ([Leu, Pro]NPY) and NPY13-36 [NPY(13-36)], respectively, and the NPY receptor antagonist benextramine (BXT). NPY, [Leu, Pro]NPY and NPY(13-36) were equipotent as vasoconstrictors, and constriction induced by each peptide, but not by the calcium channel agonist BAY K 8644 (BAY), was almost completely blocked by 10 microM BXT. Each of the three peptides also induced self- and cross-desensitization and protection from BXT blockade, except that [Leu, Pro]NPY neither desensitized nor protected NPY(13-36)-associated responses. NPY also failed to protect [Leu, Pro]NPY- and NPY(13-36)-elicited constriction, and NPY(13-36) failed to provide self-protection, from BXT blockade. However, in these instances, as opposed to the [Leu, Pro]NPY-NPY(13-36) cross-protection experiments, the occurrence of protection was probably masked by the relatively large magnitude of desensitization concurrently induced by the protecting peptide. Taken together, the present findings suggest that NPY-induced rat femoral artery vasoconstriction is mediated by separate, BXT-sensitive, postsynaptic Y1 ([Leu, Pro]NPY-activated) and Y2 [NPY(13-36)-activated] receptors.

Benextramine-neuropeptide Y receptor interactions: contribution of the benzylic moieties to [3H]neuropeptide Y displacement activity.

Analogs of N,N'-bis[6-[(2-methoxybenzyl)amino]hex-1-yl]cystamine (benextramine, BXT, 2) were synthesized using solution-phase peptide synthesis methodology and analyzed for activity in displacing specifically bound 1 nM N-[propionyl-3H]neuropeptide Y([3H]NPY) from benextramine-sensitive neuropeptide Y (NPY) binding sites in rat brain. Our new synthetic approach to these analogs began with the acylation of cystamine with the N-hydroxysuccinimide ester of tert-butyloxycarbonyl (t-Boc) protected 6-aminohexanoic acid, followed by deprotection of the t-Boc groups with 4 N HCl in dioxane. Acylation of this symmetric diamine with N-hydroxysuccinimide esters of appropriately substituted benzoic acids, followed by reduction of the resultant tetraamides with diborane in refluxing THF, afforded the target compounds. The BXT analog lacking the benzylic group (i.e., compound 11) had no [3H]NPY displacement activity at concentrations up to 1.4 x 10(-3) M. The 9-fold range in activities observed for the ortho, meta, and para regioisomers of the methoxy, chloro, and hydroxy benextramine analogs at benextramine-sensitive NPY rat brain binding sites does not differ from the range of potencies observed at alpha-adrenoceptors. However, the order of potencies at [3H]NPY sites differs from the order of potencies at alpha-adrenoceptors, with the m-methoxyphenyl (9a), m-hydroxyphenyl (10b), and 2-naphthyl (9f) analogs being the most active at [3H]NPY binding sites. The present results demonstrate the importance of the benzylic moiety for BXT's NPY antagonist activity, and suggest that the BXT binding site on the NPY receptor is significantly distinct from that on the alpha-adrenoceptor.

Benextramine-neuropeptide Y (NPY) binding site interactions: characterization of 3H-NPY binding site heterogeneity in rat brain.

Pre-incubation of rat brain membranes with 200 microM benextramine followed by extensive dilution and washing to remove unbound ligand reduced Bmax for N-[propionyl-3H]-NPY (3H-NPY) specific binding by 61% relative to control membranes treated identically but in the absence of benextramine. When rat brain membranes were co-incubated with 3H-NPY and 57 microM benextramine, there was a significant shift to the right; the apparent Kd for 3H-NPY binding increased two-fold relative to control membranes. These data are consistent with the hypothesis that benextramine is a competitive and irreversible ligand for a population (60-65%) of rat brain NPY binding sites. 'Paired tube' assays were then used to determine the selectivity of these benextramine-sensitive and insensitive 3H-NPY binding site populations. PYY, NPY and NPY13-36 each displaced 100% of 3H-NPY from rat brain membrane binding sites both in the absence and presence of 1 mM benextramine. In contrast, [Leu31,Pro34]NPY displayed the same binding site selectivity as benextramine in displacing 65% of 3H-NPY from specific binding sites on untreated rat brain membranes, and it failed to displace 3H-NPY from membranes treated with 1 mM benextramine. Thus the selectivity of the benextramine-insensitive 3H-NPY binding site population--PYY > = NPY > NPY13-36 >> [Leu31,Pro34]NPY--is characteristic of a Y2-like NPY binding site population, while the benextramine-sensitive 3H-NPY binding sites appear to be a Y1-like binding site population.