Mometasone furoate is a less specific glucocorticoid than fluticasone propionate.

Fluticasone propionate (FP) and mometasone furoate (MF) are potent synthetic corticosteroids that are widely used as anti-inflammatory agents to treat respiratory diseases. As part of the assessment of the potential for side-effects associated with their use, their activities, not only at the glucocorticoid receptor (GR) but also at the other members of the steroid nuclear receptor family, have been compared. Cell-based functional systems were established to measure different aspects of GR function, as well as the activity at all the other steroid nuclear receptors. The effects of MF and FP on the GR were potent and indistinguishable. Neither corticosteroid showed any activity at the oestrogen receptor, while both were weak antagonists of the androgen receptor. FP was a relatively weak agonist of the progesterone receptor but MF was a very potent agonist of the progesterone receptor, giving activity at similar concentrations to those that stimulate the GR (concentration generating 50% maximal effect (EC50)=50 pM). Moreover, while FP was a weak antagonist of the mineralocorticoid receptor (concentration generating 50% maximal inhibitory effect=80 nM), MF displayed potent partial agonist activity (EC50=3 nM, 30%). Mometasone furoate is considerably less specific for the glucocorticoid receptor than fluticasone propionate, showing significant activity at other nuclear steroid receptors.

Tollip, a new component of the IL-1RI pathway, links IRAK to the IL-1 receptor.

Interleukin-1 (IL-1) is a proinflammatory cytokine that elicits its pleiotropic effects through activation of the transcription factors NF-kappaB and AP-1. Binding of IL-1 to its receptor results in rapid assembly of a membrane-proximal signalling complex that consists of two different receptor chains (IL-1Rs), IL-1RI and IL-1RAcP, the adaptor protein MyD88, the serine/threonine kinase IRAK and a new protein, which we have named Tollip. Here we show that, before IL-1beta treatment, Tollip is present in a complex with IRAK, and that recruitment of Tollip-IRAK complexes to the activated receptor complex occurs through association of Tollip with IL-1RAcP. Co-recruited MyD88 then triggers IRAK autophosphorylation, which in turn leads to rapid dissociation of IRAK from Tollip (and IL-1Rs). As overexpression of Tollip results in impaired NF-kappaB activation, we conclude that Tollip is an important constituent of the IL-1R signalling pathway.

Overexpression of an enzymically inactive interleukin-1-receptor-associated kinase activates nuclear factor-kappaB.

Upon interleukin 1 (IL-1) stimulation, the IL-1-receptor (IL-1R)-associated kinase (IRAK) is rapidly recruited to the IL-1R complex and undergoes phosphorylation. Here we demonstrate that recombinant wild-type IRAK (IRAK-WT), but not a kinase-defective mutant with Asp340 replaced by an asparagine residue (IRAK-Asp340Asn), is highly phosphorylated and is capable of auto-phosphorylation in vitro. Overexpression of both IRAK-WT and IRAK-Asp340Asn caused activation of nuclear factor kappaB, suggesting that the kinase activity of IRAK is not required outside of the IL-1R complex.

The IL1 receptor accessory protein is responsible for the recruitment of the interleukin-1 receptor associated kinase to the IL1/IL1 receptor I complex.

Following interleukin-1 (IL1) stimulation, an IL1 receptor associated kinase (IRAK) is rapidly recruited to the receptor complex. However, it is not understood if IRAK is able to interact directly with the intracellular portion of the IL1-RI or if its recruitment is mediated by a different molecule. Using the yeast two-hybrid system, we have analysed possible protein-protein interactions between IRAK, IL1-RI and IL1-RAcP. We found that IRAK is able to interact with the equivalent cytoplasmic region of the IL1-RAcP but is unable to interact with the cytoplasmic region of the IL1-RI. Immunoprecipitation of the IL1-RAcP followed by Western blot analysis using anti-IRAK antibodies revealed that IRAK co-precipitated with the IL1-RAcP. We propose that, in non-stimulated cells, IRAK is bound to the IL1-RAcP and therefore, following IL1 stimulation, both molecules are recruited simultaneously to the ILI-RI complex.

Human immunodeficiency virus. Mutations in the viral protease that confer resistance to saquinavir increase the dissociation rate constant of the protease-saquinavir complex.

Mutations in the human immunodeficiency virus (HIV) protease (L90M, G48V, and L90M/G48V) arise when HIV is passaged in the presence of the HIV protease inhibitor saquinavir. These mutations yield a virus with less sensitivity to the drug (L90M > G48V >> L90M/G48V). L90M, G48V, and L90M/G48V proteases have 1/20, 1/160, and 1/1000 the affinity for saquinavir compared to WT protease, respectively. Therefore, the affinity of mutant protease for saquinavir decreased as the sensitivity of the virus to saquinavir decreased. Association rate constants for WT and mutant proteases with saquinavir were similar, ranging from 2 to 4 x 10(7) M-1 s-1. In contrast, the dissociation rate constants for WT, L90M, G48V, and L90M/G48V proteases complexed with saquinavir were 0.0014, 0.019, 0.128, and 0. 54 s-1, respectively. This indicated that the reduced affinity for mutant proteases and saquinavir is primarily the result of larger dissociation rate constants. The increased dissociation rate constants may be the result of a decrease in the internal equilibrium between the bound inhibitor with the protease flaps up and the bound inhibitor with the flaps down. Interestingly, the affinity of these mutant proteases for VX-478, ABT-538, AG-1343, or L-735,524 was not reduced as much as that for saquinavir. Finally, the catalytic constants of WT and mutant proteases were determined for eight small peptide substrates that mimic the viral cleavage sites in vivo. WT and L90M proteases had similar catalytic constants for these substrates. In contrast, G48V and L90M/G48V proteases had catalytic efficiency (kcat/Km) values with TLNF-PISP, RKIL-FLDG, and AETF-YVDG that were 1/10 to 1/20 the value of WT protease. The decreased catalytic efficiencies were primarily the result of increased Km values. Thus, mutations in the protease decrease the affinity of the enzyme for saquinavir and the catalytic efficiency with peptide substrates.

3'-5' L-capped-antisense oligodeoxynucleotides targeted at the SV40 T-antigen gene: pharmacological and biological properties.

This paper reports on some pharmacological and biological properties of 22-mer antisense oligodeoxynucleotides which contain an L-deoxyribonucleoside at each terminus. Compared with natural compounds, of which they retain the DNA hybridizing ability and the cell uptake mechanism, the L-22-mers exhibited an increased resistance to phosphodiesterase degradation, an apparent higher intracellular concentration and a longer intracellular half life. Antiviral activity was not prominent.

In vitro selection and characterization of human immunodeficiency virus type 1 (HIV-1) isolates with reduced sensitivity to hydroxyethylamino sulfonamide inhibitors of HIV-1 aspartyl protease.

Human immunodeficiency virus type 1 (HIV-1) variants with reduced sensitivity to the hydroxyethylamino sulfonamide protease inhibitors VB-11,328 and VX-478 have been selected in vitro by two independent serial passage protocols with HIV-1 in CEM-SS and MT-4 cell lines. Virus populations with greater than 100-fold-increased resistance to both inhibitors compared with the parental virus have been obtained. DNA sequence analyses of the protease genes from VB-11,328- and VX-478-resistant variants reveal a sequential accumulation of point mutations, with similar resistance patterns occurring for the two inhibitors. The deduced amino acid substitutions in the resistant protease are Leu-10-->Phe, Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val. This is the first observation in HIV protease resistance studies of an Ile-50-->Val mutation, a mutation that appears to arise uniquely against the sulfonamide inhibitor class. When the substitutions observed were introduced as single mutations into an HIV-1 infectious clone (HXB2), only the Ile-50-->Val mutant showed reduced sensitivity (two- to threefold) to VB-11,328 and VX-478. A triple protease mutant infectious clone carrying the mutations Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val, however, showed much greater reduction in sensitivity (14- to 20-fold) to VB-11,328 and VX-478. The same mutations were studied in recombinant HIV protease. The mutant protease Ile-50-->Val displays a much lower affinity for the inhibitors than the parent enzyme (< or = 80-fold). The protease triply mutated at Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val shows an even greater decrease in inhibitor binding (< or = 270-fold). The sulfonamide-resistant HIV protease variants remain sensitive to inhibitors from other chemical classes (Ro 31-8959 and L-735,524), suggesting possibilities for clinical use of HIV protease inhibitors in combination or serially.

Analysis of resistance to human immunodeficiency virus type 1 protease inhibitors by using matched bacterial expression and proviral infection vectors.

There are already reports, from clinical trials with human immunodeficiency virus type 1 protease inhibitors, of the emergence of drug-resistant mutants which have one or more point mutations in their protease genes. To examine roles of individual and multiple amino acid substitutions in terms of altered enzyme and virus drug sensitivities, we have produced matched vectors for bacterial expression and virus production. Both vectors accept the same restriction enzyme fragment, produced by PCR or PCR-mutagenesis of the protease gene, allowing parallel expression of mutant enzymes in Escherichia coli and in recombinant viruses. The utility of this vector system was demonstrated by using protease variants glycine to valine at amino acid 48 (G48V) and leucine to methionine at amino acid 90 (L90M) identified after passage of HIV-1 in the Roche phase II clinical trial protease inhibitor Ro 31-8959 (H. Jacobsen, K. Yasargil, D. L. Winslow, J. C. Craig, A. Krohn, I. B. Duncan, and J. Mous, Virology 206:527, 1995). G48V, L90M, and G48V/L90M exhibited successively less processing in vitro than the wild-type enzyme, and the purified enzymes were 220-, 20-, and 720-fold, respectively, less sensitive to Ro 31-8959. The reduced enzyme sensitivity correlated directly with the sensitivities of the matched recombinant viruses, in that individual mutations L90M and G48V conferred 2-fold and 4- to 6-fold increases in 50% inhibitory concentration, respectively, whereas G48V/L90M was 8 to 10 times less sensitive to Ro 31-8959. A proviral vector with the entire protease gene deleted was constructed for use as an in vivo recombination target for an overlapping protease PCR fragment, generating wild-type infectious virus. Finally, direct ligation of restriction fragments, generated from random PCR mutagenesis, into the proviral vector should provide a library of protease mutations that allow extremely rapid selection of highly resistant viral variants.

Cross-resistance analysis of human immunodeficiency virus type 1 variants individually selected for resistance to five different protease inhibitors.

Human immunodeficiency virus type 1 (HIV-1) protease inhibitor-resistant variants, isolated on passage of HIV-1HXB2 in MT-4 cells with five different protease inhibitors, have been examined for cross-resistance to five inhibitors. The protease inhibitors studied were Ro 31-8959, A-77003, XM323, L-735,524, and VX-478. Resistant variants with two to four mutations within their protease sequence and 9- to 40-fold-decreased susceptibility were selected for all five inhibitors within six to eight passes in cell culture. Passage of a zidovudine-resistant mutant in Ro 31-8959 generated a dual reverse transcriptase- and protease-resistant virus. Variants were cloned directly into a modified pHXB2-D infectious clone for cross-resistance analysis. Although the resistant variants selected possessed different combinations of protease mutations for each inhibitor, many showed cross-resistance to the other inhibitors, and one showed cross-resistance to all five inhibitors. Interestingly, some mutants showed increased susceptibility to some inhibitors. Further HIV passage studies in the combined presence of two protease inhibitors demonstrated that in vitro it was possible to delay significantly selection of mutations producing resistance to one or both inhibitors. These studies indicate that there may be some rationale for combining different protease inhibitors as well as protease and reverse transcriptase inhibitors in HIV combination therapy.

Polymerase chain reaction amplification and restriction enzyme typing as an accurate and simple way to detect and identify human papillomaviruses.

A simple and economic method for the detection and identification of human papillomaviruses (HPV) is described. The method has been developed with cloned HPV DNA and DNA from clinical samples. Genomic fragments were obtained from several different HPV types, including the ones most frequently encountered in the genital tract by polymerase chain reaction (PCR) amplification directed by degenerate general primers. The amplification fragments were identified by a form of miniature fingerprinting, with a set of restriction enzymes that gave a unique digestion pattern for each HPV type. Different strategies are proposed, based on PCR and restriction analysis, and this approach to identification was compared with more classic methods such as Southern hybridisation.