The ability of metamizol to inhibit cyclooxygenase-1 and cyclooxygenase-2 activities has been evaluated using different cyclooxygenase sources. Metamizol inhibited purified cyclooxygenase-1 and cyclooxygenase-2 with an IC of about 150 mgrml. A similar IC 50 50value for cyclooxygenase-2 was obtained in lipopolysaccharide-activated broken murine macrophages. Consistent with these findings, molecular models of the complexes between cyclooxygenase-1 or cyclooxygenase-2 with 4-methylaminoantipyrine, the major active derivative of metamizol, suggested a common binding mode to both isoforms. In intact cells, however, the inhibition profiles were Ž . markedly different. The IC values of metamizol for cyclooxygenase-1 in intact bovine aortic endothelial cells BAEC cells and human 50 platelets were 1730 " 150 mgrml and 486 " 56 mgrml, respectively. Inhibition of cyclooxygenase-2 activity in murine macrophages and primary human leukocytes activated by lipopolysaccharide yielded IC values of 12 " 1.8 mgrml and 21 " 2.9 mgrml, 50 respectively. These data indicate that the IC values obtained with purified enzymes or disrupted cells cannot always be extrapolated to 50 Ž . the cyclooxygenase inhibitory activity of nonsteroidal antiinflammatory drugs NSAIDs in intact cells. The data presented here also indicate that cyclooxygenase-2 inhibition could play an important role in the pharmacological effects of metamizol. q 1999 Elsevier Science B.V. All rights reserved.Ž .

Recognition of DNA sequence information by the natural antitumor agent ecteinascidin 743 (ET743) has been proposed to operate through a direct readout mechanism involving specific hydrogen bonding interactions between the drug and the DNA minor groove prior to covalent alkylation of a guanine base. 5′-AGC and 5′-CGG are examples of high-reactivity target triplets whereas 5′-CGA is a low-reactivity sequence. Molecular dynamics computer simulations were first used to explore the stability and behavior of the precovalent complex between ET743 and a DNA nonamer containing the 5′-AGC binding site in the central region. The pre-covalent complex was stable, and some unreported distinctive features were observed that may not be amenable to direct experimental verification. A similar simulation with ET743 bound to another nonamer containing a central 5′-CGA triplet did not result in a stable association supporting the proposed role of a hydrogen bonding network in the stabilization of these complexes. The covalent complexes between ET743 and the nonamers containing the 5′-AGC and 5′-CGG target sites were then simulated. In each case the drug displayed the predicted binding mode and gave rise to a widening of the minor groove. In addition, we show that as a consequence of ET743 binding to the target sequences, positive roll is introduced that results in smooth bending of the helix toward the major groove, in agreement with results from gel electrophoresis experiments. Similar results were obtained with the synthetic compound phthalascidin, which presents a biological profile almost indistinguishable from that of ET743. The local bending elements in AGC and CGG were found to be different, and the distinct behavior of these sequences in the absence of bound drugs was in consonance with their intrinsic bending propensities.

The DNA-binding domain (DBD) of the ubiquituous transcription factor Sp1 consists of three consecutive zinc fingers that recognize a number of nucleotide sequences different from, but related to and sometimes overlapping, those recognized by the structurally better characterized early growth response protein 1 (EGR1, also known as Zif268, Krox-24, and NGFI-A). The accepted consensus binding sequence for Sp1 is usually defined by the asymmetric hexanucleotide core GGGCGG but this sequence does not include, among others, the GAG ( ¼ CTC) repeat that constitutes a high-affinity site for Sp1 binding to the wt1 promoter. Since no 3D structure of the whole DBD of Sp1 is available, either alone or in complex with DNA, a homology-based model was built and its interaction with two DNA 14-mers was studied using nanosecond molecular dynamics simulations in the presence of explicit water molecules. These oligonucleotides represent Sp1 target sites that are present in the promoters of the mdr1 and wt1 genes. For comparative purposes and validation of the protocol, the complex between the DBD of EGR1 and its DNA target site within the proximal mdr1 promoter was simulated under the same conditions. Some water molecules were seen to play an important role in recognition and stabilization of the protein-DNA complexes. Our results, which are supported by the available experimental evidence, suggest that the accuracy in the prediction of putative Sp1-binding sites can be improved by interpreting a set of rules, which are a blend of both stringency and tolerance, for the juxtaposed triplet subsites to which each zinc finger binds. Our approach can be extrapolated to WT1 and other related natural or artificial zinc-finger-containing DNA-binding proteins and may aid in the assignment of particular DNA stretches as allowed or disallowed-binding sites.

The antitumor ecteinascidin ET743 has been shown to inhibit the transcriptional activation of a number of genes at nanomolar concentrations. Cell sensitivity to subnanomolar concentrations of the drug has also been shown to specifically depend on the transcription-coupled nucleotide excision repair system. ET743 is known to bind covalently to the minor groove of a DNA double helix in regions comprising selected sets of three consecutive base pairs. Following alkylation of a central guanine, the minor groove is widened and the DNA is bent toward the major groove. We have previously shown that in the resulting adduct the DNA triplet containing the covalently modified guanine bears a strong resemblance to a DNA triplet recognized by a C(2)H(2) zinc finger. We now expand this earlier finding and use simulation methods to show that head-to-tail binding of three ET743 molecules to three adjacent optimal binding sites stabilizes a DNA structure whose conformation is intermediate between A- and B-form DNA. Furthermore, despite the increase in roll at the sites of covalent attachment, no net curvature is apparent in this complex due to cancellation of the localized bends over virtually one turn of the helix. Both observations are in good analogy to findings in zinc finger-DNA complexes. Triplets are virtually superimposable both directly and upon shifting the register one base pair. In this latter case, the central guanine in a triplet alkylated by ET743 corresponds to the third nucleic base in the triplet recognized by a zinc finger of transcription factors such as EGR1 or Sp-1. The DNA conformation found in the ET743-DNA complex is also strongly reminiscent of an RNA-DNA hybrid, as found in the RNA polymerase II elongation complex. The possible biological implications of these findings in relation to the antitumor action of ET743 are discussed.