Release of the cyano moiety in the crystal structure of N-cyanomethyl-N-(2-methoxyethyl)-daunomycin complexed with d(CGATCG). (73/4054)

Doxorubicin is among the most widely used anthracycline in cancer chemotherapy. In an attempt to avoid the cardiotoxicity and drug resistance of doxorubicin therapy, several analogues were synthesized. The cyanomorpholinyl derivative is the most cytotoxic. They differ greatly from their parent compound in their biological and pharmacological properties, inducing cross-links in drug DNA complexes. The present study concerns N-cyanomethyl-N-(2-methoxyethyl)-daunomycin (CMDa), a synthetic analogue of cyanomorpholino-daunomycin. Compared to doxorubicin, CMDa displays a cytotoxic activity on L1210 leukemia cells at higher concentration but is effective on doxorubicin resistant cells. The results of fluorescence quenching experiments as well as the melting temperature (DeltaTm = 7.5 degrees C) studies are consistent with a drug molecule which intercalates between the DNA base pairs and stabilizes the DNA double helix. The crystal structure of CMDa complexed to the hexanucleotide d(CGATCG) has been determined at 1.5 A resolution. The complex crystallizes in the space group P41212 and is similar to other anthracycline-hexanucleotide complexes. In the crystal state, the observed densities indicate the formation of N-hydroxymethyl-N-(2-methoxyethyl)-daunomycin (HMDa) with the release of the cyano moiety without DNA alkylation. The formation of this degradation compound is discussed in relation with other drug modifications when binding to DNA. Comparison with two other drug-DNA crystal structures suggests a correlation between a slight change in DNA conformation and the nature of the amino sugar substituents at the N3' position located in the minor groove.  (+info)

Transport-dependent accessibility of a cytoplasmic loop cysteine in the human dopamine transporter. (74/4054)

The effect of covalent sulfhydryl modification on dopamine uptake by the human dopamine transporter was determined by rotating disc electrode voltammetry. A transporter construct, X5C, with five mutated cysteines (C90A, C135A, C306A, C319F, and C342A) and the constructs into which the wild-type cysteines were substituted back into X5C, one at a time, all showed nearly normal binding affinity for [(3)H]CFT and for cocaine, but they displayed significant reductions in K(m) and V(max) for DA uptake. Reaction of Cys-90 or Cys-306 with impermeant methanethiosulfonate derivatives enhanced dopamine uptake to a similar extent as the previously observed enhancement of [(3)H]CFT binding caused by the same reaction, suggesting that cocaine may bind preferentially to a conformation in the transport cycle. m-Tyramine increased the rate of reaction of (2-aminoethyl)methanethiosulfonate (MTSEA) with X-A342C, the construct with a cytoplasmic loop residue Cys-342 restored. This m-tyramine-induced increase in reactivity appeared to require the inward transport rather than the outward transport or external binding of m-tyramine, and it was prevented by cocaine. Thus, inward translocation of substrates may involve structural rearrangement of hDAT, which likely exposes Cys-342 to reaction with MTSEA, and Cys-342 may be located on a part of the transporter associated with cytoplasmic gating.  (+info)

Residues of the fourth transmembrane segments of the Na,K-ATPase and the gastric H,K-ATPase contribute to cation selectivity. (75/4054)

We have generated protein chimeras to investigate the role of the fourth transmembrane segments (TM4) of the Na,K- and gastric H, K-ATPases in determining the distinct cation selectivities of these two pumps. Based on a helical wheel analysis, three residues of TM4 of the Na,K-ATPase were changed to their H,K-counterparts. A construct carrying three mutations in TM4 (L319F, N326Y, and T340S) and two control constructs were heterologously expressed in Xenopus laevis oocytes and in the pig kidney epithelial cell line LLC-PK(1). Biochemical ATPase assays demonstrated a large sodium-independent ATPase activity at pH 6.0 for the pump carrying the TM4 substitutions, whereas the control constructs exhibited little or no activity in the absence of sodium. Furthermore, at pH 6.0 the K(1/2)(Na(+)) shifted to 1.5 mM for the TM4 construct compared with 9.4 and 5.9 mM for the controls. In contrast, at pH 7.5 all three constructs had characteristics similar to wild type Na,K-ATPase. Large increases in K(1/2)(K(+)) were observed for the TM4 construct compared with the control constructs both in two-electrode voltage clamp experiments in Xenopus oocytes and in ATPase assays. ATPase assays also revealed a 10-fold shift in vanadate sensitivity for the TM4 construct. Based on these findings, it appears that the three identified TM4 residues play an important role in determining both the specific cation selectivities and the E(1)/E(2) conformational equilibria of the Na,K- and H,K-ATPase.  (+info)

Characterization of TEM-56, a novel beta-lactamase produced by a Klebsiella pneumoniae clinical isolate. (76/4054)

TEM-56 produced by a Klebsiella pneumoniae clinical isolate is a novel beta-lactamase of isoelectric point 6.4 that confers a moderate resistance level to expanded-spectrum cephalosporins. The amino acid sequence deduced from the corresponding bla gene showed two amino acid replacements with respect to the TEM-2 sequence: Glu-104 to Lys and His-153 to Arg. This enzyme showed catalytic properties close to those of TEM-18. Thus, TEM-56 appears as a new TEM mutant, an intermediary between TEM-18 and the extended-spectrum beta-lactamase TEM-21.  (+info)

In vivo antitumor activity and induction of insulin-like growth factor-1-resistant apoptosis by SC-alphaalphadelta9. (77/4054)

We previously showed that SC-alphaalphadelta9 (4-(benzyl-(2-[(2, 5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)-2-decanoylami no butyric acid) is a novel antiphosphatase agent that selectively inhibits the growth of transformed cells in culture and affects elements of insulin-like growth factor-1 (IGF-1) signaling. We now show that SC-alphaalphadelta9 induces IGF-1-resistant apoptosis and kills tumor cells in vivo. In cultured murine 32D cells, SC-alphaalphadelta9 induced concentration-dependent apoptosis that was blocked by ectopic Bcl-2 expression. No apoptosis was detected in 32D cells treated with the congener SC-alpha109, which lacks the ability to disrupt IGF-1 signaling. After interleukin-3 withdrawal or etoposide treatment, exogenous IGF-1 prevented apoptosis and elevated levels of Cdc2, a biochemical indicator of a functional IGF-1 receptor pathway. In contrast, exogenous IGF-1 did not prevent apoptosis or loss of Cdc2 expression caused by SC-alphaalphadelta9. Furthermore, IGF-1 receptor overexpression failed to protect cells against SC-alphaalphadelta9-induced apoptosis. Kinetic analyses demonstrated that Cdc2 down-regulation after SC-alphaalphadelta9 treatment preceded both apoptosis and loss of the IGF-1 receptor, indicating that loss of Cdc2 was a direct effect of SC-alphaalphadelta9 treatment and not secondary to cell death. IGF-1 receptor autophosphorylation studies indicated that SC-alphaalphadelta9 did not interact directly with the IGF-1 receptor nor bind to the growth factor itself, suggesting a site of action distal to the IGF-1 receptor. In the SCCVII murine tumor model, a single i.p. injection of SC-alphaalphadelta9 caused a dose-dependent decrease in clonogenic cell survival. The IC(50) of SC-alphaalphadelta9 was 35 mg/kg, comparable to 25 mg/kg carboplatin. The ability to induce IGF-1-resistant apoptosis distinguishes SC-alphaalphadelta9 from other apoptosis-inducing agents and suggests compounds of this class deserve further study as potential anticancer agents.  (+info)

Midazolam and triazolam biotransformation in mouse and human liver microsomes: relative contribution of CYP3A and CYP2C isoforms. (78/4054)

Midazolam (MDZ) and triazolam (TRZ) hydroxylation, reactions considered to be cytochrome P-4503A (CYP3A)-mediated in humans, were examined in mouse and human liver microsomes. In both species, alpha- and 4-hydroxy metabolites were the principal products. Western blotting with anti-CYP3A1 antibody detected a single band of immunoreactive protein in both human and mouse samples: 0.45 +/- 0. 12 and 2.02 +/- 0.24 pmol/mg protein (mean +/- S.E., n = 3), respectively. Ketoconazole potently inhibited MDZ and TRZ metabolite formation in human liver microsomes (IC(50) range, 0.038-0.049 microM). Ketoconazole also inhibited the formation of both TRZ metabolites and of 4-OH-MDZ formation in mouse liver microsomes (IC(50) range, 0.0076-0.025 microM). However, ketoconazole (10 microM) did not produce 50% inhibition of alpha-OH-MDZ formation in mouse liver microsomes. Anti-CYP3A1 antibodies produced concentration-dependent inhibition of MDZ and TRZ metabolite formation in human liver microsomes and of TRZ metabolite and 4-OH-MDZ formation in mouse liver microsomes to less than 20% of control values but reduced alpha-OH-MDZ formation to only 66% of control values in mouse liver microsomes. Anti-CYP2C11 antibodies inhibited alpha-OH-MDZ metabolite formation in a concentration-dependent manner to 58% of control values in mouse liver microsomes but did not inhibit 4-OH-MDZ formation. Thus, TRZ hydroxylation appears to be CYP3A specific in mice and humans. alpha-Hydroxylation of MDZ has a major CYP2C component in addition to CYP3A in mice, demonstrating that metabolic profiles of drugs in animals cannot be assumed to reflect human metabolic patterns, even with closely related substrates.  (+info)

Leukotriene A4 hydrolase: a critical role of glutamic acid-296 for the binding of bestatin. (79/4054)

Leukotriene A(4) hydrolase is a bifunctional Zn(2+)-containing enzyme catalysing the formation of the potent chemotaxin leukotriene B(4). From an analysis of three mutants of Glu-296 we have found that this catalytic residue is critical for the binding of bestatin, a classical aminopeptidase inhibitor. For bestatin, but not for three other tight-binding inhibitors, the IC(50) values for inhibition of the epoxide hydrolase activity decreased in the mutants to 0.7-0.003% of the control. Hence Glu-296 is an important structural determinant for binding of bestatin to leukotriene A(4) hydrolase; this conclusion might also apply to other members of the M1 family of metallopeptidases.  (+info)

Structure-activity relationship study of antimicrobial dermaseptin S4 showing the consequences of peptide oligomerization on selective cytotoxicity. (80/4054)

To understand how peptide organization in aqueous solution might affect the activity of antimicrobial peptides, the potency of various dermaseptin S4 analogs was assessed against human red blood cells (RBC), protozoa, and several Gram-negative bacteria. Dermaseptin S4 had weak antibacterial activity but potent hemolytic or antiprotozoan effects. K(4)K(20)-S4 was 2-3-fold more potent against protozoa and RBC, yet K(4)K(20)-S4 was more potent by 2 orders of magnitude against bacteria. K(4)-S4 had similar behavior as K(4)K(20)-S4, but K(20)-S4 and analogous negative charge substitutions were as active as dermaseptin S4 or had reduced activity. Binding experiments suggested that potency enhancement was not the result of increased affinity to target cells. In contrast, potency correlated well with aggregation properties. Fluorescence studies indicated that K(20)-S4 and all negative charge substitutions were as aggregated as dermaseptin S4, whereas K(4)-S4 and K(4)K(20)-S4 were clearly less aggregated. Overall, the data indicated that N-terminal domain interaction between dermaseptin S4 monomers is responsible for the peptide's oligomerization in solution and, hence, for its limited spectrum of action. Moreover, bell-shaped dose-response profiles obtained with bacteria but not with protozoa or RBC implied that aggregation can have dramatic consequences on antibacterial activity. Based on these results, we tested the feasibility of selectivity reversal in the activity of dermaseptin S4. Tampering with the composition of the hydrophobic domains by reducing hydrophobicity or by increasing the net positive charge affected dramatically the peptide's activity and resulted in various analogs that displayed potent antibacterial activity but reduced hemolytic activity. Among these, maximal antibacterial activity was displayed by a 15-mer version that was more potent by 2 orders of magnitude compared with native dermaseptin S4. These results emphasize the notion that peptide-based antibiotics represent a highly modular synthetic antimicrobial system and provide indications of how the peptide's physico-chemical properties affect potency and selectivity.  (+info)