Induction of rapid IL-1 beta mRNA degradation in THP-1 cells mediated through the AU-rich region in the 3'UTR by a radicicol analogue.

A radicicol analogue (analogue A) was found to inhibit interleukin 1 beta (IL-1 beta) and tumour necrosis factor alpha (TNF-alpha) secretion from THP-1 cells. If added to cells activated by interferon gamma and lipopolysaccharide, radicicol analogue A not only inhibited the secretion of IL-1 beta but also induced an extremely rapid degradation of IL-1 beta, IL-6 and TNF-alpha mRNA to undetectable levels within 5-8 h. This degradation is independent of translation and of the signal inducing transcription. The common feature of these genes is the inclusion of one or more copies of the mRNA-instability sequence, AUUUA, in the 3' untranslated region. Indeed, no destabilizing effect of radicicol analogue A could be observed on mRNA derived from the expression of an IL-1 beta construct lacking the AUUUA motifs of the 3'UTR. The effect of radicicol analogue A on protein/mRNA interaction and on post-translational modifications of cytoplasmic proteins is described. This class of compound constitutes a valuable tool for the further elucidation of the mechanism of mRNA degradation of cytokines and proto-oncogenes.

Microbial biotransformation products of cyclosporin A.

In order to mimic the human metabolic pathway of cyclosporin A (CyA) a total of 28 bacterial and 72 fungal strains was screened for their ability to transform CyA. Among 3 bacteria and 11 fungi, which produced the main human metabolite OL-17 [eta HyMeBmt1]CyA, Actinoplanes sp. (ATCC 53771) achieved the best transformation rate (5.4%). Furthermore, the two N-demethylated minor products [Leu4]CyA (3.2%) and [Leu9]CyA (4.7%) were isolated, both known as minor natural metabolites and the first one also as a human biotransformation product. Microbial conversion of CyA using the actinomycete Sebekia benihana (NRRL 11111) yielded [gamma HyMeLeu4]CyA (35%), [gamma HyLeu4]CyA (4.5%) and [gamma HyMeLeu4, gamma HyMeLeu6]CyA (8.6%). The structures of these derivatives correspond with those of the human metabolic pathway. The related compounds [Nva2]CyA (CyG) and [D-MeSer3]CyA were similarly converted to the corresponding 4-gamma-hydroxylated analogues. None of the biotransformation products showed a better immunosuppressive effect than CyA, although in various cases the cyclophilin binding affinity was comparable to that of CyA.

Biosynthesis of the unusual amino acid (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine of cyclosporin A: enzymatic analysis of the reaction sequence including identification of the methylation precursor in a polyketide pathway.

3(R)-Hydroxy-4(R)-methyl-6(E)-octenoic acid, the C9-backbone of the unusual amino acid (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine (Bmt), is biosynthesized as a coenzyme A thioester from acetyl-CoA, malonyl-CoA, NADPH, and S-adenosylmethionine via a polyketide pathway. Here we present detailed enzymatic studies about the basic assembly process. After attachment of the activated building units to Bmt polyketide synthase the intermediates remained enzyme-bound throughout the cycle. Premature cutoff of biosynthesis led to the release of the intermediates from the enzyme, either as coenzyme A thioesters or, in the case of reactive C8-intermediates, as lactones. Enzyme-bound 3-oxo-4-hexenoic acid, the condensation product of the second elongation cycle, could be identified as the exclusive substrate for the introduction of the methyl group. Part of the biosynthesis including the first elongation cycle, the second condensation reaction, and the methylation step was shown to follow a processive mechanism. All activated intermediates of this processive part could be introduced into the correct pathway at the respective steps, whereas 2-methyl-3-oxo-4-hexenoyl-CoA and all following methylated intermediates were not able to enter the cycle any more. Obviously, the region of Bmt polyketide synthase responsible for this latter part of the biosynthetic pathway is inaccessible for externally supplied coenzyme A thioesters. Butyryl-CoA was recognized by Bmt polyketide synthase with an efficiency comparable to that of crotonyl-CoA and processed to 3-hydroxy-4-methyloctanoyl-CoA, the saturated analog of the natural basic assembly product, indicating a relaxed specificity of Bmt polyketide synthase with respect to the starter unit.

Reversal of multidrug resistance by novel cyclosporin A analogues and the cyclopeptolide SDZ 214-103 biosynthesized in vitro.

It was shown that cyclopeptolide SDZ 214-103 (10 microM) is more active in rhodamine-123 accumulation in actinomycin-D-resistant human lymphoma cells CCRF/ACTD400 than cyclosporin A (10 microM), but equipotent in the doxorubicin-resistant Friend erythroleukemia cell line F4-6/ADR. In F4-6/ADR cells, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay showed comparable cytotoxic effects of doxorubicin at various concentrations in the presence of SDZ 214-103 and cyclosporin A. For the other novel cyclosporin A analogues minor multidrug-resistance-modulating potency was demonstrated. At equipotent modulating doses of verapamil (10 microM) and cyclosporin A (10 microM) in the MTT assay regarding doxorubicin cytotoxicity, cyclosporin A was efficient in the rhodamine-123-uptake assay while verapamil was not active when identical incubation times were used.

In vitro biosynthesis of ring-extended cyclosporins.

Cyclosporin synthetase, a multifunctional polypeptide, catalyses the biosynthesis of the set of natural cyclosporins. We report that this enzyme is also capable of introducing a beta-alanine into position 7 or 8 of the ring instead of the alpha-alanines present at these positions in cyclosporin A. This leads to 34-membered rings in contrast to the 33-membered ring of the cyclo-undecapeptide cyclosporin A. Both [beta Ala7]CyA and [beta Ala8]CyA show immunosuppressive activity. The cyclosporin synthetase-related enzyme peptolide SDZ 214-103 synthetase, on the other hand, does not incorporate either beta-alanine into position 7 or beta-hydroxy acids into position 8, confirming the previously described higher substrate specificity of this enzyme compared with cyclosporin synthetase [Lawen and Traber (1993) J. Biol. Chem. 268, 20452-20465].

Biosynthesis of the unusual amino acid (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine of cyclosporin A. Identification of 3(R)-hydroxy-4(R)-methyl-6(E)-octenoic acid as a key intermediate by enzymatic in vitro synthesis and by in vivo labeling techniques.

The biosynthesis of (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine (abbreviation: Bmt, systematic name: 2(S)-amino-3(R)-hydroxy-4(R)-methyl-6(E)-octenoic acid) is proposed to involve two principal phases: the formation of a polyketide backbone and a subsequent transformation process to the final product. Here we report on the identification of 3(R)-hydroxy-4(R)-methyl-6(E)-octenoic acid as the end product of the first phase. The primary indication of 3(R)-hydroxy-4(R)-methyl-6(E)-octenoic acid as the key intermediate in the proposed biosynthetic route came from in vivo labeling studies with [1-13C,18O2]acetate, demonstrating retention of 18O in the 3-hydroxy group. Final identification of this intermediate in in vitro polyketide assays with enriched enzyme fractions of Tolypocladium niveum was achieved after development of highly sensitive and specific detection methods and by use of synthetic reference substances. Two additional methylated in vitro products could be detected and characterized as 4(R)-methyl-(E,E)-2,6-octadienoic acid and 4(R)-methyl-6(E)-octenoic acid by liquid chromatography-mass spectrometry analysis and comparison with synthetic reference samples. Their relevance for Bmt biosynthesis is discussed. Bmt polyketide synthase shows optimal activity at substrate concentrations of 200 microM acetyl-CoA, 150 microM malonyl-CoA, and 200 microM S-adenosylmethionine, around pH 7 and at 35 degrees C. Interestingly the Bmt backbone is released from the enzyme as a coenzyme A thioester, suggesting that subsequent transformation to Bmt takes place upon this activated intermediate.

Substrate specificities of cyclosporin synthetase and peptolide SDZ 214-103 synthetase. Comparison of the substrate specificities of the related multifunctional polypeptides.

The recently discovered multifunctional polypeptide cyclosporin synthetase is capable of synthesizing the cyclic undecapeptide cyclosporin A in a batch reaction. Substrates are the unmethylated constitutive amino acids of cyclosporin A. Exchange of one or more of these by various amino acids gives a picture of the substrate specificity of the enzyme in vitro, which is different from the known picture obtained by in vivo studies. The uncommon amino acid butenylmethylthreonine in position 1 of the cyclosporin ring can be exchanged by an unexpected large spectrum of different amino acids, showing a great flexibility of this site. Position 2, on the other hand, which shows the greatest variability in vivo, has an only slightly lower specificity in vitro. Position 3 has a very high degree of specificity; positions 4, 6, 7, 9, and 10 have marginally less. The variability of positions 5 and 11 is moderate, whereas position 8 shows only low substrate specificity in vitro. In general, most sites of SDZ 214-103 synthetase appear to be more specific than those of cyclosporin synthetase. Site 11 has nearly identical substrate specificity compared with that of cyclosporin synthetase. The D-2-hydroxy acid position (position 8) can be occupied by a large spectrum of substrates varying from D-lactic acid to D-2-hydroxyisocaproic acid. Within the limits of the present data, the addition of further functional groups to the D-2-hydroxy acid moiety are apparently not tolerated by the enzyme.

Selective 13C-labelling of cyclosporin A.

Cyclosporin A is biosynthetically labelled with 13C by growing an overproducing strain of Tolypocladium inflatum on minimal media containing either [1-13C]-, [2-13C]-, [3-13C]- or [6-13C]glucose as the only carbon source. NMR analysis of the 13C-labelled peptide showed a labelling pattern in which 13C occurs at specific sites. These can be predicted by consideration of the relevant biosynthetic pathways. Quantitation of the site-specific enrichments revealed that the 13C-label incorporation is efficient and selective. Metabolic fluxes through alternative pathways can also be estimated from these results. Isotopically labelled peptides will be a very useful tool for the study of molecular interactions with their receptors.

In vitro biosynthesis of [Thr2,Leu5,D-Hiv8,Leu10]-cyclosporin, a cyclosporin-related peptolide, with immunosuppressive activity by a multienzyme polypeptide.

A new cyclic peptolide (SDZ 214-103), which is produced by the fungus Cylindrotrichumoligospermum (Corda) BONORDEN (Dreyfuss, M. M., Schreier, M. H., Tscherter, H., and Wenger, R. (June 15, 1988) European Patent Application 0 296 123 A2) and is closely related to cyclosporin A (CyA), has as the main structural difference D-2-hydroxyisovaleric acid in ester linkage at position 8 instead of D-alanine in the cyclosporins. This peptolide exerts similar biological activities to CyA. We were able to prepare an enzyme fraction of crude extracts of the mycelium, which is capable of synthesizing the peptolide with consumption of the constitutive amino acids, D-2-hydroxyisovaleric acid, ATP, and S-adenosyl-L-methionine. The in vitro product co-chromatographs with authentic peptolide on thin layer chromatography and high performance liquid chromatography and shows similar immunosuppressive activity in vitro. The enzyme does not synthesize CyA, whereas cyclosporin synthetase does not synthesize the peptolide. Peptolide synthetase has a high molecular weight (in the same range as cyclosporin synthetase) and also does not appear to be glycosylated. The enzyme cross-reacts with antibodies directed specifically against cyclosporin synthetase.

The NMR structure of cyclosporin A bound to cyclophilin in aqueous solution.

Cyclosporin A bound to the presumed receptor protein cyclophilin was studied in aqueous solution at pH 6.0 by nuclear magnetic resonance spectroscopy using uniform 15N- or 13C-labeling of cyclosporin A and heteronuclear spectral editing techniques. Sequence-specific assignments were obtained for all but one of the cyclosporin A proton resonances. With an input of 108 intramolecular NOEs and four vicinal 3JHN alpha coupling constants, the three-dimensional structure of cyclosporin A bound to cyclophilin was calculated with the distance geometry program DISMAN, and the structures resulting from 181 converged calculations were energy refined with the program FANTOM. A group of 120 conformers was selected on the basis of the residual constraint violations and energy criteria to represent the solution structure. The average of the pairwise root-mean-square distances calculated for the backbone atoms of the 120 structures was 0.58 A. The structure represents a novel conformation of cyclosporin A, for which the backbone conformation is significantly different from the previously reported structures in single crystals and in chloroform solution. The structure has all peptide bonds in the trans form, contains no elements of regular secondary structure and no intramolecular hydrogen bonds, and exposes nearly all polar groups to its environment. The root-mean-square distance between the backbone atoms of the crystal structure of cyclosporin A and the mean of the 120 conformers representing the NMR structure of cyclosporin A bound to cyclophilin is 2.5 A.

In vitro biosynthesis of [Thr2, Leu5, D-Hiv8, Leu10]cyclosporin, a cyclosporin-related immunosuppressive peptolide.

We were able to prepare an enzyme fraction from crude extracts of the mycelium of the fungus Cylindrotrichum Bonorden, which is capable of synthesizing the new peptolide SDZ 214-103 under consumption of the constitutive amino acids, D-2-hydroxyisovaleric acid, ATP and S-adenosyl-L-methionine. The enzyme does not synthesize CyA, while cyclosporin synthetase does not synthesize the peptolide. Peptolide synthetase has a high molecular weight in the same range as cyclosporin synthetase (about 1.5 MDa).

Isolation of (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine, the characteristic structural element of cyclosporins, from a blocked mutant of Tolypocladium inflatum.

By mutagenic treatment of a strain of Tolypocladium inflatum, a cyclosporin non-producing mutant was obtained which accumulated the characteristic building unit of cyclosporins, (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine (abbreviation Bmt; systematic name: (2S,3R,4R,6E)-2-amino-3-hydroxy-4-methyl-6-octenoic acid) in free form. The isolation from a culture filtrate was performed by extraction, chromatographic separation and final crystallization from methanol - water. The structure and stereochemistry of this amino acid was determined by chemical transformation and correlation to dihydro-MeBmt, with known chirality [(2S,3R,4R)-3-hydroxy-4-methyl-2-methylamino-octanoic acid], obtained by hydrolysis of dihydrocyclosporin A.

Overcoming multidrug resistance in Chinese hamster ovary cells in vitro by cyclosporin A (Sandimmune) and non-immunosuppressive derivatives.

Cyclosporin A (Sandimmune) increased the in vitro susceptibility of 'parental' and 'multidrug-resistant' (MDR) chinese hamster ovary (CHO) cell lines to three anti-tumour drugs: colchicine, daunomycin, and vincristine. Several immunosuppressive or non-immunosuppressive derivatives of cyclosporin (Cs) were compared for their ability to sensitise both parental and MDR cells to chemotherapeutic agents. Although 5-10-fold increases of sensitivity to anti-tumour drugs could be obtained for cells of the parental line with several Cs-derivatives, the largest 'gains' of sensitivity (chemosensitisation) were obtained for the cells of the MDR line and with only some of the Cs derivatives. The MDR cells employed displayed the typical MDR phenotype. However, we found no correlation between the immunosuppressive activity of Cs derivatives and their capacity to reverse MDR and all four possible combinations of these two activities could indeed be shown among the tested Cs derivatives. This study demonstrates for the first time that some immunosuppressive Cs can be devoid of chemosensitising activity.

Cell-free biosynthesis of new cyclosporins.

An enzyme preparation, isolated from extracts of the fungus Beauveria nivea (previously designated Tolypocladium inflatum), is able to synthesize cyclosporins (Cy's) in vitro. At suboptimal temperature it was possible to yield about 50 micrograms of CyA per ml. The enzyme also produces several of the naturally occurring congeners of CyA, such as the Cy's B, C, D, G, M, O, Q, U and V and some of the analogues known to be produced by the fungus via precursor directed biosynthesis, like dihydro-CyA, [N-methyl-L-beta-cyclohexylalanine]CyA, [L-allylglycine]CyA and [D-serine]CyA. Furthermore, Cy's not obtainable by the fungus could be prepared by the enzyme system in the presence of the appropriate precursor amino acids; the synthesis of [N-methyl-(+)-2-amino-3-hydroxy-4,4-dimethyloctanoic acid]CyA, [L-norvaline, N-methyl-L-norvaline]CyA, [L-norvaline, N-methyl-L-norvaline]CyA, [L-allo-isoleucine, N-methyl-L-allo-isoleucine]CyA, [L-allo-isoleucine]CyA, [D-2-aminobutyric acid]CyA and [beta-chloro-D-alanine]CyA could be established. The immunosuppressive effects of the new derivatives are discussed.

Cyclosporins--new analogues by precursor directed biosynthesis.

Cyclosporin A (ciclosporin), a potent and clinically important immunosuppressive drug (Sandimmun), represents the main component of a group of over 25 closely related, cyclic undecapeptides produced by the fungus Tolypocladium inflatum. By feeding experiments using DL-alpha-allylglycine as precursor, specific incorporation in position 2 was attained leading to [Allylgly2]cyclosporin A. Exogenously supplied L-beta-cyclohexylalanine results in the almost exclusive production of [MeCyclohexylala1]cyclosporin A (replacement of methylbutenyl-methylthreonine-1). D-Alanine in position 8 can be successfully substituted by D-serine. The new [D-Ser8] analogues of the cyclosporins A, C, D and G as well as [Allylgly2]cyclosporin A exhibit high immunosuppressive effects.