Triazole-polyene antagonism in experimental invasive pulmonary aspergillosis: in vitro and in vivo correlation.

Combination antifungal therapy is increasingly used in the treatment of invasive aspergillosis. Whether the interaction between amphotericin B and triazoles is antagonistic against invasive aspergillosis is a controversial issue that is not likely to be resolved through a randomized clinical trial. Here, we found both in vitro and in vivo antagonism between liposomal amphotericin B and ravuconazole in simultaneous treatment of experimental invasive pulmonary aspergillosis in persistently neutropenic rabbits. Bliss independence-based drug-interaction modeling showed significant antagonism in vitro and in vivo, with the observed drug effects being 20%-69% lower than would be expected if the drugs were acting independently. These in vitro and in vivo findings of antagonism were consistent with the findings from Loewe additivity-based drug-interaction modeling. No pharmacokinetic interaction was found. The combination of a triazole and polyene may be antagonistic in the treatment of invasive pulmonary aspergillosis.

Specific triazine resistance in bacterial reaction centers induced by a single mutation in the QA protein pocket.

We report here the first example of a reaction center mutant from Rhodobacter sphaeroides, where a single mutation (M266His --> Leu) taking place in the primary quinone protein pocket confers selective resistance to triazine-type inhibitors (terbutryn, ametryn, and atrazine), which bind in the secondary quinone protein pocket, at about 13 A from the mutation site. The M266His --> Leu mutation involves one of the iron atom ligands. Interestingly, neither the secondary quinone nor the highly specific inhibitor stigmatellin binding affinities are affected by the mutation. It is noticeable that in the M266His --> Ala mutant a nativelike behavior in observed. We suggest that the long side chain of Leu in position M266 may lack space to accommodate in the Q(A) pocket therefore transferring its hindrance to the Q(B) pocket. This may occur via the structural feature formed by the Q(A)-M219His-Fe-L190His-inhibitor (or Q(B)) connection, pushing L189Leu and/or L229Ile in closer contact to the triazine molecules, therefore decreasing their bindings. This opens the possibility to finely tune, in reaction center proteins, the affinity for herbicides by designing mutations distant from their binding sites.

Inhibition of cell cycle progression by a synthetic peptide corresponding to residues 65-79 of an HLA class II sequence: functional similarities but mechanistic differences with the immunosuppressive drug rapamycin.

A synthetic peptide corresponding to a region of the alpha1 alpha-helix of DQA03011 (DQ 65-79) inhibits the proliferation of human PBL and T cells in an allele-nonspecific manner. It blocks proliferation stimulated by anti-CD3 mAb, PHA-P, and alloantigen, but not by PMA and ionomycin. Substitution of each amino acid with serine shows that residues 66, 68, 69, 71-73, and 75-79 are critical for function. Inhibition of proliferation is long lasting and is not reversible with exogenous IL-2. The peptide can be added 24 to 48 h after stimulation and still block proliferation. The DQ 65-79 peptide does not affect expression of IL-2 or IL-2R; however, IL-2-stimulated proliferation is inhibited. Cell cycle progression is blocked at the G1/S transition, and the activity of cdk2 (cyclin-dependent kinase 2) kinase is impaired by the continued presence of p27. Although these results suggest a mechanism similar to that of rapamycin, the peptide inhibition is not reversed with FK-506, which indicates a distinct mechanism.

Two distinct effects of 12S-hydroxyeicosatetraenoic acid on platelet aggregation by zooxanthellatoxin-A and ionomycin.

The marine toxin zooxanthellatoxin-A (ZT-A) and the Ca2+ ionophore ionomycin caused aggregation in rabbit platelets. While ZT-A-induced platelet aggregation was inhibited by indomethacin, ionomycin-induced aggregation was potentiated by the drug. In contrast, both ZT-A- and ionomycin-induced accumulations of thromboxane B2 (TXB2), a stable metabolite of thromboxane A2 (TXA2), were completely inhibited by indomethacin. 12S-Hydroxyeicosatetraenoic acid (12-HETE), which could be accumulated in the presence of indomethacin, inhibited ZT-A-induced aggregation, but it potentiated the ionomycin-induced one. These results suggest that the different effects of indomethacin may be attributed to the distinct effects of 12-HETE on ZT-A- and ionomycin-induced platelet aggregations.

Antascomicins A, B, C, D and E. Novel FKBP12 binding compounds from a Micromonospora strain.

5 novel ascomycin-like compounds, antascomicins A, B, C, D and E were isolated from a strain of Micromonospora. The antascomicins bind strongly to the FK506-binding protein FKBP12 and antagonize the immunosuppressive activity of FK506 and rapamycin. The strain description, fermentation, structure elucidation and biological activity of these compounds are described.

Rapamycin inhibits clonal expansion and adipogenic differentiation of 3T3-L1 cells.

Differentiating 3T3-L1 cells express an immunophilin early during the adipocyte conversion program as described in this issue [Yeh, W.-C., Li, T.-K., Bierer, B. E. & McKnight, S. L. (1995) Proc. Natl. Acad. Sci. USA 92, 11081-11085]. The temporal expression profile of this protein, designated FK506-binding protein (FKBP) 51, is concordant with the clonal-expansion period undertaken by 3T3-L1 cells after exposure to adipogenic hormones. Having observed FKBP51 synthesis early during adipogenesis, we tested the effects of three immunosuppressive drugs--cyclosporin A, FK506, and rapamycin--on the terminal-differentiation process. Adipocyte conversion was not affected by either cyclosporin A or FK506 and yet was significantly reduced by rapamycin at drug concentrations as low as 10 nM. Clonal expansion was impeded in drug-treated cultures, as was the accumulation of cytoplasmic lipid droplets normally seen late during differentiation. Rapamycin treatment likewise inhibited the expression of CCAAT/enhancer binding protein alpha, a transcription factor required for 3T3-L1 cell differentiation. All three of these effects were reversed by high FK506 concentrations, indicating that the operative inhibitory event was mediated by an immunophilin-rapamycin complex.

Rapamycin, a potent immunosuppressive drug, causes programmed cell death in B lymphoma cells.

Rapamycin, a potent immunosuppressive drug that prevents rejection of organ transplants in many animals, caused profound growth inhibition in an immature B cell lymphoma, BKS-2, at very low concentrations (2 ng/ml). Similar growth inhibition was also observed in a series of B cell lymphomas (i.e., L1.2, NFS.1.1, and WEHI-279) as well as in thymoma cells. The cell death induced by rapamycin in BKS-2 lymphoma was found to be via programmed cell death, or apoptosis. In contrast to rapamycin, neither FK506 nor CsA affected the normal growth of these cells. FK506, but not CsA antagonized the effect of rapamycin and rescued the BKS-2 cells from undergoing apoptosis. Further, suboptimal concentrations of anti-IgM antibodies and rapamycin acted synergistically in causing the growth inhibition of BKS-2 cells and this inhibitory effect was also completely reversed by FK506. Thus, rapamycin appeared to inhibit lymphoma growth by binding to FK506 binding protein. These results indicate that rapamycin should be evaluated as an effective immunosuppressive therapeutic agent to prevent the incidence of lymphoma after transplantations.

Increased LFA-1-mediated homotypic cell adhesion is associated with the G1 growth arrest induced by rapamycin in a T cell lymphoma.

The immunosuppressive macrolide, rapamycin, impedes the G1 to S cell cycle progression in cytokine-stimulated normal lymphocytes and in certain autonomously proliferating cell lines. Here, we found that the rapamycin-induced growth arrest augments homotypic aggregation in the YAC-1 T cell lymphoma. The growth arrest and increased aggregation were both blocked by the rapamycin antagonist, L-685,818, which interacts with the intracellular binding proteins mediating rapamycin's biochemical action. Moreover, rapamycin-induced aggregation was not seen in YAC-1 cells mutants selected for resistance to the drug's antiproliferative effect. Although the inhibition of G1/S progression induced by serum deprivation also resulted in increased cellular aggregation, cell cycle blockade in late G1 by mimosine, early S phase by hydroxyurea, or G2/M by nocodazole all failed to do so. Furthermore, the aggregation induced by rapamycin was blocked by antibodies to the alpha (CD11a) or beta (CD18) subunits of the integrin, LFA-1, or to its ligands, ICAM-1 and ICAM-2, and did not occur in LFA-1-deficient YAC mutants. However, the surface expression of LFA-1, ICAM-1, or ICAM-2 was not augmented in cells aggregated by rapamycin. Finally, the serine/threonine protein phosphatase inhibitor, okadaic acid, was found to abrogate rapamycin-induced aggregation. Therefore, rapamycin's impairment of YAC-1 cell growth in G1 is accompanied by enhanced LFA-1-mediated homotypic cell adhesion that may reflect an increase of the integrin's avidity for its ligands and may involve protein phosphorylation/dephosphorylation events. This suggests the existence of a link between cell cycle progression and "inside-out" LFA-1 signaling, possibly regulated by rapamycin's biochemical targets.

Induction of CD5 on B and T cells is suppressed by cyclosporin A, FK-520 and rapamycin.

The expression of CD5 can be induced on murine B-2 cells by anti-IgM, a recognized analog of thymus-independent 2 type (TI-2) antigen. Given that cyclosporin A (CsA) sensitivity is a distinguishing feature of TI-2 type B cell activation, we asked whether the in vitro induction of CD5 on B cells by anti-mu is CsA sensitive. We report that anti-mu induced CD5 expression on B-2 cells was inhibited by CsA as well as FK-520 and rapamycin. When L-685,818, a FK-520 and rapamycin antagonist, was added to anti-mu stimulated B cell cultures containing FK-520 or rapamycin, but not CsA, suppression was abrogated and complete induction of CD5 was seen. When we used either CD4+CD8+ thymocytes or peripheral T cells activated by phorbol ester and ionomycin, the cell surface induction of CD5 was also partially blocked by CsA, FK-520 and rapamycin. Moreover, in both B and T cells, the same immunosuppressive drugs did not affect constitutive CD5 expression but only blocked de novo induction. To determine the level of CD5 regulation, we activated T cells using phorbol myristate acetate (PMA)/ionomycin and report that CD5 induction was sensitive to actinomycin D (AcD). Similarly, the induction of CD5 on anti-mu activated B cells was blocked by AcD. In addition, T cells that were activated by PMA/ionomycin expressed more abundant CD5 mRNA than CsA or FK-520 treated cells. Based on the CsA-sensitive regulation of CD5 we thought that the CsA-sensitive nuclear factor of activated T cells (NFAT) might be involved in CD5 regulation. We report evidence by Western blot analysis that NFATp is expressed by both resting and TI activated B cells but apparently not CD4+CD8+CD5+ thymocytes. We conclude that in both B and T cells the induction of CD5 requires transcriptional regulation, and that the inhibition of CD5 expression by the immunosuppressive drugs CsA, FK-520 and rapamycin requires drug-immunophilin complex formation.

Effects of rapamycin on growth factor-stimulated vascular smooth muscle cell DNA synthesis. Inhibition of basic fibroblast growth factor and platelet-derived growth factor action and antagonism of rapamycin by FK506.

Rapamycin (RPM) is a potent and effective immunosuppressant which we have shown previously to inhibit intimal thickening in rat allograft and balloon-injured arteries. In this report, we have examined the effects of RPM on growth factor-induced vascular smooth muscle cell (VSMC) DNA synthesis. RPM potently inhibited platelet-derived growth factor (PDGF) (IC50 = 5 x 10(-9) M) and basic fibroblast growth factor (bFGF) (IC50 = 8 x 10(-10) M)-induced VSMC DNA synthesis. In contrast, only the highest concentrations of FK506 and CsA significantly altered PDGF- or bFGF-induced VSMC DNA synthesis. Addition of RPM (10(-9) M) at as late as 46 hr after growth factor addition still effectively suppressed bFGF- or PDGF-induced DNA synthesis by 76% and 54%, respectively. The extent of the antagonism of RPM's inhibition of bFGF-induced VSMC DNA synthesis by FK506 was inversely proportional to RPM concentration and directly proportional to FK506 concentration.

Quantitative and temporal analysis of the cellular interaction of FK-506 and rapamycin in T-lymphocytes.

The structurally related immunosuppressive macrolides FK-506 and rapamycin (RAP) exert distinct biological effects: inhibition of interleukin-2 production and inhibition of interleukin-2-induced proliferation, respectively, through binding to intracellular receptors, termed FKBPs. Although the interaction of these drugs with purified FKBPs in vitro has been well characterized, little is known about their interaction with FKBPs in living cells. Here, we used [3H]-dihydro-FK-506 as a probe to examine the binding of these macrolides in both normal mouse splenic T-cells and the human Jurkat T-cell lymphoma. These cells were found to accumulate the radioligand, predominantly in the cytosol, to a saturable level corresponding to an estimated concentration of 6 to 7 microM. Half-maximal suppression of T-cell activation was shown to require radioligand occupancy of only 3 to 5% of the pool of available intracellular binding sites (FKBPs). Moreover, the binding and immunosuppressive effect of the radioligand could not be removed by extensive washing and remained stable for at least 6 hr upon incubation of the cells at 37 degrees C. However, a molar excess of either FK-506 or RAP was found to rapidly displace [3H]-dihydro-FK-506 from its cellular binding sites. Consistently, FK-506 and RAP were able to antagonize mutually their immunosuppressive activities even when added several hr after each other to T-cell cultures. We took advantage of the reciprocal antagonism of FK-506 and RAP to define their apparent affinities for the functionally relevant cellular receptors by Schild analysis. This indicated that the drugs compete for a single cellular receptor with similar KdS and, therefore, may mediate their immunosuppressive action upon interaction with similar or highly related FKBPs.

[The effect of lovastatin on sterol synthesis and yeast resistance to polyene antibiotics]. / Vliianie lovastatina na sintez sterinov i rezistentnost' drozhzhei k polienovym antibiotikam.

Lovastatin (monocolin K) is a competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA-reductase. Its influence on the growth of a lovastatin sensitive strain of Rhodotorula rubra, the biosynthesis of ergosterol and the resistance to polyenic antibiotics was studied. It was shown that the lovastatin action on the strain depended on the inhibitor dose. In a concentration of 0.1 to 0.5 micrograms/ml lovastatin inhibited the yeast growth and ergosterol biosynthesis. Higher concentrations of the inhibitor in the medium led to the recovery of the sterol biosynthesis. It was also demonstrated that a decrease of the ergosterol level in the cells under the influence of lovastatin resulted in the development of resistance in the yeast to polyenic antibiotics such as nistatin and amphotericin B. Correlation between the ergosterol level in the yeast cells and their susceptibility to the polyenic antibiotics was observed.

The immunosuppressive and toxic effects of FK-506 are mechanistically related: pharmacology of a novel antagonist of FK-506 and rapamycin.

FK-506 inhibits Ca(2+)-dependent transcription of lymphokine genes in T cells, and thereby acts as a powerful immunosuppressant. However, its potential therapeutic applications may be seriously limited by several side effects, including nephrotoxicity and neurotoxicity. At present, it is unclear whether these immunosuppressive and toxic effects result from interference with related biochemical processes. FK-506 is known to interact with FK-binding protein-12 (FKBP-12), an abundant cytosolic protein with cis-trans peptidyl-prolyl isomerase activity (PPIase) activity. Because rapamycin (RAP) similarly binds to FKBP-12, although it acts in a manner different from FK-506, by inhibiting T cell responses to lymphokines, such an interaction with FKBP-12 is not sufficient to mediate immunosuppression. Recently, it was found that the complex of FKBP-12 with FK-506, but not with RAP, inhibits the phosphatase activity of calcineurin. Here, we used L-685,818, the C18-hydroxy, C21-ethyl derivative of FK-506, to explore further the role of FKBP-12 in the immunosuppressive and toxic actions of FK-506. Although L-685,818 bound with high affinity to FKBP-12 and inhibited its PPIase activity, it did not suppress T cell activation, and, when complexed with FKBP-12, did not affect calcineurin phosphatase activity. However, L-685,818 was a potent antagonist of the immunosuppressive activity of both FK-506 and RAP. Moreover, L-685,818 did not induce any toxicity in dogs and rats or in a mouse model of acute FK-506 nephrotoxicity, but it blocked the effect of FK-506 in this model. Therefore, FK-506 toxicity involves the disruption of biochemical mechanisms related to those implicated in T cell activation. Like immunosuppression, this toxicity is not due to the inhibition of the PPIase activity of FKBP-12, but may be linked to the inhibition of the phosphatase activity of calcineurin by the drug FKBP-12 complex.

Rapamycin and FK506 differentially inhibit mast cell cytokine production and cytokine-induced proliferation and act as reciprocal antagonists.

We have previously demonstrated that cyclosporine (CSA) and FK506 are able to selectively inhibit cytokine production by murine mast cell lines at concentrations comparable to those observed with thymus-derived lymphocytes (T cells). The selectivity of these effects were demonstrated by the failure of CSA and FK506 to inhibit cytokine-induced mast cell proliferation at equivalent or higher concentrations. In this report, we examined the ability of rapamycin (RAP) to inhibit cytokine production and cytokine-induced proliferation by a factor-dependent murine mast cell line and compared its activity to that of the structurally related macrolide FK506. The mast cell clone, MC/9, was stimulated to produce cytokines with phorbol myristate acetate plus the calcium ionophore A23187, or to proliferate in response to exogenous cytokines such as interleukin-3 and interleukin-4, produced by the helper T cell clone D10.G4. RAP did not inhibit cytokine production by MC/9, even at concentrations greater than 1000 nM. FK506 and CSA inhibited cytokine production with IC50 of 0.8 and 16.2 nM, respectively. In contrast to its lack of effect on cytokine production, RAP potently inhibited cytokine-induced proliferation of MC/9 cells with an IC50 of 1.9 nM. Because RAP and FK506 are structurally related and yet have divergent biological effects, we examined the ability of RAP to antagonize inhibitory effects of FK506 on mast cell cytokine production and the ability of FK506 to antagonize inhibitory effects of RAP on cytokine-induced mast cell proliferation. The addition of RAP in molar excess reversed inhibition of mast cell cytokine production mediated by FK506, but not that of CSA.(ABSTRACT TRUNCATED AT 250 WORDS)

[The sensitivity of yeasts from the Candida genus isolated from cancer patients to polyene antifungals]. / Sensibilidade de leveduras do gênero Candida, isoladas de pacientes com câncer, a antifúngicos poliênicos.

Candida strains susceptibility from cancer patients were compared with Candida strains susceptibility from patients, without cancer by MIC (minimal inhibitory concentration) and MFC (minimal fungicidal concentration) to Amphotericin B and Nystatin. Broth dilution method and agar dilution method were the procedure employed. The authors find no significant differences between the studied groups. The problem of Candida resistance to polyene antifungals is discussed.

Probing immunosuppressant action with a nonnatural immunophilin ligand.

The immunosuppressants FK506 and rapamycin bind to the same immunophilin, FK506 binding protein (FKBP), and inhibit distinct signal transduction pathways in T lymphocytes. A nonnatural immunophilin ligand, 506BD, which contains only the common structural elements of FK506 and rapamycin, was synthesized and found to be a high-affinity ligand of FKBP and a potent inhibitor of FKBP rotamase activity. Whereas 506BD does not interfere with T cell activation, it does block the immunosuppressive effects of both FK506 and rapamycin. Thus, the common immunophilin binding element of these immunosuppressants, which is responsible for rotamase inhibition, is fused to different effector elements, resulting in the inhibition of different signaling pathways. Inhibition of rotamase activity is an insufficient requirement for mediating these effects.

The immunosuppressive macrolides FK-506 and rapamycin act as reciprocal antagonists in murine T cells.

The structurally related immunosuppressive macrolides FK-506 and rapamycin (RAP) were previously shown to inhibit T cell stimulation through different mechanisms. FK-506 acts similarly to cyclosporin A (CsA) and prevents IL-2 production and IL-2R expression. RAP has little or no effect on these events but markedly impedes the response to IL-2. The present study was initiated to examine the possibility of a complementation between the immunosuppressive actions of RAP and FK-506 or CsA on various murine T cell responses. RAP potentiated the effect of CsA on proliferation and IL-2R expression in T cells stimulated with ionomycin + PMA. However, in the same system, RAP acted as a potent antagonist of FK-506 suppression. RAP also blocked FK-506- but not CsA-mediated inhibition of IL-2 mRNA induction. By using model systems sensitive to inhibition by RAP but not FK-506 we further demonstrated that FK-506 reciprocally behaves as an antagonist of RAP. In one such model, the stimulation of splenic T cells with IL-2 + PMA, FK-506, but not CsA, reversed the suppressive effect of RAP on proliferation. FK-506 also antagonized RAP-mediated inhibition with respect to the induction of Ly-6E Ag expression by IFN in YAC cells. To explore further the competition between the two macrolides at the cellular level, we performed binding experiments with a radiolabeled derivative of FK-506. Both FK-506 and RAP, but not CsA, inhibited the binding of this probe in YAC cells. Taken together, these data demonstrate that FK-506 and RAP antagonize each other's biologic activity and physically interact with a common receptor site(s) in T cells. Moreover, CsA acts at a site distinct from the cellular target(s) of FK-506 or RAP.

[Yeast resistance to polyene antibiotics. VII. The interaction of the mutant alleles of the nystatin resistance genes in Saccharomyces cerevisiae]. / Rezistentnost' drozhzhei k polienovym antibiotikam. Soobshchenie VII. Vzaimodeistvie mutantnykh allelei genov nistatinustoichivosti u Saccharomyces cerevisiae.

The data on allele interactions of nystatin resistance genes are presented. It has been shown that the mutant phenotype of heteroallelic hybrids NYS1, NYS4 and, probably, NYS3 is strengthened. The intragenic complementation has been found in NYS2 gene, allowing to imply the multimeric structure of delta 8----delta 7 isomerase which is controlled by this gene.