Structure of DNA-porphyrin complex.

We report the 2.4 A resolution X-ray structure of a complex in which a small molecule flips a base out of a DNA helical stack. The small molecule is a metalloporphyrin, CuTMPyP4 [copper(II) meso-tetra(N-methyl-4-pyridyl)porphyrin], and the DNA is a hexamer duplex, [d(CGATCG)]2. The porphyrin system, with the copper atom near the helical axis, is located within the helical stack. The porphyrin binds by normal intercalation between the C and G of 5' TCG 3' and by extruding the C of 5' CGA 3'. The DNA forms a distorted right-handed helix with only four normal cross-strand Watson-Crick base pairs. Two pyridyl rings are located in each groove of the DNA. The complex appears to be extensively stabilized by electrostatic interactions between positively charged nitrogen atoms of the pyridyl rings and negatively charged phosphate oxygen atoms of the DNA. Favorable electrostatic interactions appear to draw the porphyrin into the duplex interior, offsetting unfavorable steric clashes between the pyridyl rings and the DNA backbone. These pyridyl-backbone clashes extend the DNA along its axis and preclude formation of van der Waals stacking contacts in the interior of the complex. Stacking contacts are the primary contributor to stability of DNA. The unusual lack of van der Waals stacking contacts in the porphyrin complex destabilizes the DNA duplex and decreases the energetic cost of local melting. Thus extrusion of a base appears to be facilitated by pyridyl-DNA steric clashes.

Inhibition of trypsin and thrombin by amino(4-amidinophenyl)methanephosphonate diphenyl ester derivatives: X-ray structures and molecular models.

X-ray structures of trypsin from bovine pancreas inactivated by diphenyl [N-(benzyloxycarbonyl)amino](4-amidinophenyl)methanephosphonate [Z-(4-AmPhGly)P(OPh)2] were determined at 113 and 293 K to 1.8 angstrom resolution and refined to R factors of 0.211 (113 K) and 0. 178 (293 K). The structures reveal a tetrahedral phosphorus covalently bonded to the O gamma of the active site serine. Covalent bond formation is accompanied by the loss of both phenoxy groups. The D-stereoisomer of Z-(4-AmPhGly)P-(OPh)2 is not observed in the complex. The L-stereoisomer of the inhibitor forms contacts with several residues in the trypsin active site. One of the phosphonate oxygens is inserted into the oxyanion hole and forms hydrogen bonds to the amides of Gly193, Asp194, and Ser195. The second phosphonate oxygen forms hydrogen bonds to N epsilon 2 of His 57. The p-amidinophenylglycine moiety binds into the trypsin primary specificity pocket, interacting with Asp189. The amide forms a hydrogen bond to the carbonyl oxygen atom of Ser214. The inhibitor moiety, from the 113 K structure of trypsin inactivated by the reaction product of Z-(4-AmPhGly)P(OPh)2, was docked into human thrombin [Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S. R., & Hofsteenge, J. (1989) EMBO J. 8, 3467-3475] and energy minimized. The inhibitor fits well into the thrombin active site, forming favorable contacts similar to those in the trypsin complex with no bad contacts.

Structural requirements for cocaine-sensitive and -insensitive uptake of phenethylamines into the adrenal chromaffin cell.

The adrenal medullary chromaffin cell is a commonly used model for the adrenergic neuron. Although much work has been done to study the transport system in the adrenal chromaffin vesicles, relatively little is known about cellular transport, especially with regard to structural features of phenethylamines required for intracellular accumulation. We have now investigated the structural requirements of phenethylamine-related compounds for their accumulation into cultured adrenal chromaffin cells. We find that two types of cellular uptake, previously described only for dopamine, norepinephrine, and epinephrine, are also present for [3H]tyramine. Although two types of accumulation occur, tyramine accumulation occurs mainly via a cocaine-insensitive process, whereas dopamine accumulation occurs predominantly via a cocaine-sensitive process. The accumulation of [14C]-phenethylamine and p-methoxyphenethylamine is not affected by cocaine, suggesting that a ring hydroxyl substituent is necessary for cocaine-sensitive accumulation. The compounds p-hydroxyphenylpropylamine and p-hydroxyphenyl-2-aminoethyl sulfide accumulate in the cell only via a cocaine-insensitive process, indicating that lengthening of the aminoalkyl side chain prevents cocaine-sensitive accumulation. We have performed conformational analyses of this series of compounds to determine whether the conformation of these compounds can be related to the kinetic data. For dopamine, tyramine, phenethylamine, and p-methoxyphenethylamine, two groups of energy-minimized conformers were found. We find that there is an approximately linear relationship between the Km values for these phenethylamines and the differences in minimized energies between the low- and highest energy conformer groups of each compound. A similar correlation was found for p-hydroxyphenyl-2-aminoethyl sulfide. These results are consistent with the hypothesis that these compounds undergo a conformational change from the low-energy conformer to the highest energy conformer before their cocaine-insensitive accumulation.

Mechanism-based isocoumarin inhibitors for blood coagulation serine proteases. Effect of the 7-substituent in 7-amino-4-chloro-3-(isothioureidoalkoxy)isocoumarins on inhibitory and anticoagulant potency.

A series of 7-amino-4-chloro-3-(3-isothioureidopropoxy)isocoumarin (NH2-CiTPrOIC) derivatives with various substituents at the 7- and 3-positions have been synthesized as inhibitors of several blood coagulation enzymes. Isocoumarins substituted with basic groups such as guanidino or isothioureidoalkoxy groups were previously shown to be potent irreversible inhibitors of blood coagulation enzymes [Kam et al. Biochemistry 1988, 27, 2547-2557]. Substituted isocoumarins with an isothioureidoethoxy group at the 3-position and a large hydrophobic group at the 7-position are better inhibitors for thrombin, factor VIIa, factor Xa, factor XIa, factor IIa, and factor IXa than NH2-CiTPrOIC (4). PhNHCONH-CiTEtOIC (14), (S)-Ph(CH3)CHNHCONH-CiTEtOIC (25), and (R)-Ph(CH3)CHNHCONH-CiTEtOIC (26) inhibit thrombin quite potently and have kobs/[I] values of (1-4) x 10(4) M-1 s-1. Modeled structures of several isocoumarins noncovalently complexed with human alpha-thrombin suggest that H-bonding between the 7-substituent and the Lys-60F NH3+ relates to the inhibitory potency. Thrombin inhibited by 14, 25, or 26 is quite stable, and only 4-16% of enzymatic activity is regained after incubation for 20 days in 0.1 M Hepes, pH 7.5 buffer. However, 100, 67, and 65% of enzyme activity, respectively, is regained with the addition of 0.38 M hydroxylamine. With normal citrated pig or human plasma, these isocoumarin derivatives prolong the prothrombin time ca. 1.3-3.1-fold and also prolong the activated partial thromboplastin time more than 3-7-fold at 32 microM. Thus, these compounds are effective anticoagulants in vitro and may be useful in vivo.

Inhibition of porcine pancreatic elastase by 7-substituted 4-chloro-3-ethoxyisocoumarins: structural characteristics of modeled noncovalent complexes relate to the measured inhibition kinetics.

Kinetic measurements for the inhibition of porcine pancreatic elastase by 7-substituted 4-chloro-3-ethoxyisocoumarins were performed. To obtain possible explanations for the kinetic results, structures resulting from energy minimizations of inhibitor-enzyme complex structures where each inhibitor was initially positioned in 64 locations within the active site were obtained. In keeping with solution NMR studies, a positive-charged His-57 was employed. The number of low energy complex structures with Ser-195 O gamma-inhibitor benzoyl ester carbonyl carbon distances < or = 2.9 A, Ser-195 O gamma-inhibitor benzoyl ester carbonyl carbon-inhibitor benzoyl ester carbonyl oxygen angles > 91 degrees, and the inhibitor in the oxyanion hole exhibits a direct linear relationship to ln(Ki/k3). The proportion of those structures that show 7-substituent H-bonding between the inhibitor and porcine pancreatic elastase exhibits a direct relationship to k3. Assuming a direct linear relationship to ln(k3) and k3, finer differences in k3 than are experimentally observed are expected. The relationship with k3 and that with Ki/k3 are shown to be useful tools for the design of more potent 7-substituted 4-chloro-3-ethoxyisocoumarins. A novel inhibitor of this class (4-chloro-3-ethoxy-7-[(2-methyl-2- butylcarbamoyl)amino]isocoumarin) expected to be more potent is synthesized and tested. Its potency within experimental error is as predicted. Although the relationship observed with Ki/k3 involves only a twofold increase in Ki/k3 (a statistically significant increase), results with the novel inhibitor show the relationship to be valid over a four- to fivefold increase.

Michaelis complexes of porcine pancreatic elastase with 7-[(alkylcarbamoyl)amino]-4-chloro-3-ethoxyisocoumarins: translational sampling of inhibitor position and kinetic measurements.

A step leading to the formation of the covalent complexes between porcine pancreatic elastase (PPE) and 7-[(alkylcarbamoyl)amino]-4-chloro-3-ethoxyisocoumarins (alkylHNCO-EICs) is the formation of the noncovalent Michaelis complex. No average structures are available for the Michaelis complexes of PPE with alkylHNCO-EICs. We present the results of an initial step in obtaining these structures and have determined kinetic constants as well. The kinetic results indicate that formation of the Michaelis complex is what differentiates the effectiveness of these inhibitors in inactivating PPE. The structural and kinetic results together suggest that the structure of the Michaelis complex is necessary for the design of potent alkylHNCO-EIC inhibitors of PPE. Two novel alkylHNCO-EICs are predicted to be the best inhibitors of this series. An alternate mechanism for serine protease inhibition is also proposed. Evidence for, and studies that may add support to, the hypothesized mechanism are discussed.

Inverted repeat sequences can influence the melting transitions of linear DNAs.

The influence of inverted repeat sequences on the melting transitions of linear DNAs has been examined. Derivative melting curves (DMC) of a 514 base pair (bp) DNA, seven subfragments of this DNA, and four other DNAs have been compared to predictions of DNA melting theory. The 514-bp DNA contains three inverted repeat sequences that can form cruciform structures in supercoiled DNA. We refer to these sequences as c-inverted repeats. Previous work showed that the DMC of this DNA, unlike a number of other DNAs, is not accurately predicted by DNA melting theory. Since the theoretical model does not include hairpin-like structures, it was suggested that hairpin or cruciform formation in these inverted repeats may be responsible for this discrepancy. Our results support this hypothesis. Predicted DMCs are in good agreement with DNAs with no inverted repeats, or inverted repeats not evident in supercoiled DNA. Differences between the theoretical and experimental Tm's are less than or equal to 0.3 degrees C. DNA molecules that contain one or more of the three c-inverted repeats are not as accurately predicted. Experimental Tm values are lower than predicted values by 0.7-3.8 degrees C. It is concluded that some inverted repeat sequences can form hairpin-like structures during the melting of linear DNAs. These structures appear to lower overall DNA stability.

Sequence distributions associated with DNA curvature are found upstream of strong E. coli promoters.

The regions upstream from forty-three procaryotic promoters were examined for nucleotide distributions which have been associated with DNA curvature. The analysis procedure assigned a DNA curvature score based on the phasing of the 5' and 3' ends of An and Tn tracts, n greater than or equal to 3. The weighting scheme for the curvature score was based on recent studies which showed that tracts of An and Tn periodically phased with the helix repeat cause DNA curvature. Results show that promoters which have high transcription initiation rates in vivo tend to have high curvature scores in their upstream regions. Regions downstream from the transcription start-point do not have sequences correlated with DNA curvature. Four promoters which have been shown to have upstream activation regions have curvature scores above 1.5 in their -40 to -150 regions. The correlations observed lend support to the hypothesis that DNA curvature is associated with upstream activation of transcription.