Andreas Fredenhagen

Mode of action of the lanthionine-containing peptide antibiotics duramycin, duramycin B and C, and cinnamycin as indirect inhibitors of phospholipase A2

Effects of the lanthionine-containing peptide antibiotics duramycin, duramycin B, duramycin C and cinnamycin on the activity of phospholipase A2 from six different sources were studied, and their mode of action was investigated. The four antibiotics inhibited potently all tested phospholipases A2, with IC50 values of around 1 microM, using phosphatidylethanolamine or [1-14C]oleate-labelled Escherichia coli, whose phospholipids are rich in phosphatidylethanolamine, as substrates. No inhibition was observed when the substrate was phosphatidylcholine. Binding of the antibiotics to the lipid fraction of E. coli could be demonstrated by co-sedimentation with whole, but not with lipid-depleted E. coli. In addition, preincubation of duramycin B with vesicles of phosphatidylethanolamine, but not those of phosphatidylcholine, prevented the inhibition of phospholipase A2 activity. The interaction of duramycin B and C, but not that of the biologically inactive compounds actagardine and the duramycin B trisulphoxide, with phosphatidylethanolamine was demonstrated using circular dichroism studies. On the other hand, no interaction of duramycin B with phosphatidylcholine could be demonstrated. A strict correlation between the physico-chemical interaction of the studied lantibiotics, demonstrated by circular dichroism spectroscopy, and their inhibition of phospholipase A2 was observed. These results suggest that lanthionine-containing peptide antibiotics inhibit phospholipase A2 indirectly by specifically sequestering the substrate phosphatidylethanolamine. This mode of action is analogous to the one described for the protein lipocortin.

Inhibitory activity and selectivity of staurosporine derivatives towards protein kinase C

Abstract The synthesis and in vitro protein kinase C (PKC) inhibition of a series of staurosporine derivatives is described. Essential for activity is a free NH of the lactam portion of the molecule. A large variety of substituents is tolerated at the secondary amine, although in most cases these modifications lead to a decrease in activity. Acylation of the methylamino group leads generally to the most selective derivatives with respect to other serine/threonine and tyrosine kinases.

Structure Determination of Neoefrapeptins A to N: Peptides with Insecticidal Activity Produced by the Fungus Geotrichum candidum

The structures of neoefrapeptins A to N, peptides with insecticidal activity, were elucidated. They showed a close similarity to efrapeptin. However, all neoefrapeptins contained the very rare amino acid 1-amino-cyclopropane-carboxylic acid and some of them also contained (2S,3S)-3-methylproline. The neoefrapeptins are the first case, in which these amino acids are found as building blocks for linear peptides. They were identified by comparison of the silylated hydrolyzate to reference material by GC/MS (EI-mode). The sequence was elucidated using mass spectrometry (ESI+ mode). Full scan spectra showed two fragments in high yield, even under mild ionization conditions. MS/MS spectra of these two fragments yielded fragment rich spectra from which the sequence of the compounds was determined almost completely. The proteolytic cleavage with the proteinase papain yielded products that allowed to prove the rest of the sequence and the identity of the C-terminus to efrapeptin. The proteolytic cleavage products allowed furthermore to determine the position of the isobaric amino acids, pipecolic acid and 3-methylproline in neoefrapeptin F, as well as the location of R-isovaline and S-isovaline. Papain digestion was such established as a tool for structure elucidation of peptides rich in α,α-dialkylated amino acids. CD spectra suggested a 310 helical structure for neoefrapeptins A and F.

Biocatalytic Synthesis and Structure Elucidation of Cyclized Metabolites of the Deacetylase Inhibitor Panobinostat (LBH589)

Panobinostat (LBH589) is a novel pan-deacetylase inhibitor that is currently being evaluated in phase III clinical trials for treatment of Hodgkins lymphoma and multiple myeloma. Under catalysis of recombinant human CYP3A4 and CYP2D6 coexpressed with human cytochrome P450 reductase in Escherichia coli JM109, five metabolites of panobinostat were produced via whole-cell biotransformation. The structures of the metabolites were elucidated with the spectroscopic methods mass spectrometry (MS) and NMR and revealed an oxidative cyclization of the ethyl-amino group to the methylindole moiety. The MS2 spectrum of the cyclized metabolite showed a base peak, where the closed ring is reopened and that, taken as sole base for structure proposals, would have lead to wrong conclusions. The metabolites were substantially less potent deacetylase inhibitors than the parent compound.

New stereoselective beckmann-type rearrangement leading to ring contraction

Abstract In a new stereospecific reaction the known oxime TAN-1030A gave rise to a ring contraction to yield compound 2 closely related to the metabolite K-252a. The structure was elucidated by spectroscopic comparison with K-252a. The compound strongly inhibited protein kinase C with IC50 values of 0.18 μM. This reaction suggests that TAN-1030A is a biosynthetic precursor of K-252. The absolute stereochemistry of K-252a was assigned by comparison of CD spectra.

KAE609 (Cipargamin), a new spiroindolone agent for the treatment of malaria: Evaluation of the Absorption, Distribution, Metabolism and Excretion of a single oral 300 mg dose of [14C]KAE609 in healthy male subjects

KAE609 [(1′R,3′S)-5,7′-dichloro-6′-fluoro-3′-methyl-2′,3′,4′,9′-tetrahydrospiro[indoline-3,1′-pyridol[3,4-b]indol]-2-one] is a potent, fast-acting, schizonticidal agent in clinical development for the treatment of malaria. This study investigated the absorption, distribution, metabolism, and excretion of KAE609 after oral administration of [14C]KAE609 in healthy subjects. After oral administration to human subjects, KAE609 was the major radioactive component (approximately 76% of the total radioactivity in plasma); M23 was the major circulating oxidative metabolite (approximately 12% of the total radioactivity in plasma). Several minor oxidative metabolites (M14, M16, M18, and M23.5B) were also identified, each accounting for approximately 3%–8% of the total radioactivity in plasma. KAE609 was well absorbed and extensively metabolized, such that KAE609 accounted for approximately 32% of the dose in feces. The elimination of KAE609 and metabolites was primarily mediated via biliary pathways. M23 was the major metabolite in feces. Subjects reported semen discoloration after dosing in prior studies; therefore, semen samples were collected once from each subject to further evaluate this clinical observation. Radioactivity excreted in semen was negligible, but the major component in semen was M23, supporting the rationale that this yellow-colored metabolite was the main source of semen discoloration. In this study, a new metabolite, M16, was identified in all biologic matrices albeit at low levels. All 19 recombinant human cytochrome P450 enzymes were capable of catalyzing the hydroxylation of M23 to form M16 even though the extent of turnover was very low. Thus, electrochemistry was used to generate a sufficient quantity of M16 for structural elucidation. Metabolic pathways of KAE609 in humans are summarized herein and M23 is the major metabolite in plasma and excreta.

Evaluation of the optimization space for atmospheric pressure photoionization (APPI) in comparison with APCI

The usefulness of atmospheric pressure photoionization (APPI) is difficult to evaluate for unknowns due to the fragmented literature. Specifically, the variation of dopants with a wide set of compounds or the use of APPI in the negative mode have rarely been explored. Thirty compounds were selected that were not suitable for ESI with a wide variety of functional groups and investigated with atmospheric pressure chemical ionization (APCI) and APPI in the positive and negative ion modes. The influence of the mobile phase (eluents containing acetonitrile or methanol) and--for APPI--four different dopants (acetone, chlorobenzene, toluene, and toluene/anisole) were explored. Stepwise variation of the organic mobile phase allowed to elucidate the ionization mechanism. Atmospheric pressure photoionization was especially useful for compounds, where the M(●+) and not the [M + H](+) was formed. The dopants chlorobenzene and anisole promoted the formation of molecular ions M(●+) for about half of the compounds, and its formation was also positively influenced by the use of mobile phases containing methanol. In the negative ion mode, APPI offered no advantage toward APCI. Best results were generally achieved with the dopant chlorobenzene, establishing that this dopant is suitable for a wide set of compounds. For one quarter of the compounds, significantly better results were achieved with mobile phases containing methanol for both APPI and APCI than those with acetonitrile, but only in the positive mode. With either of the methods--APPI or APCI--about 10% of the compounds were not detected. Strategies to get results quickly with difficult unknowns will be discussed.

Cladosporin Derivatives Obtained by Biotransformation Provide Guidance for the Focused Derivatization of this Antimalarial Lead Compound

Cladosporin, a natural product known for decades, has recently been discovered to display potent and selective antiplasmodial activity by inhibition of lysyl‐tRNA synthetase. It was subjected to a panel of oxidative biotransformations with one fungal and two actinomycetes strains, as well as a triple mutant bacterial CYP102A1, yielding eight, mostly hydroxylated, derivatives. These new compounds covered a wide chemical space and contained two pairs of epimers in the tetrahydropyran ring. Although less potent than the parent compound, all analogues showed activity in a cell‐based synthetase assay, thus demonstrating uptake and on‐target activity in living cells with varying degrees of selectivity for the enzyme lysyl‐tRNA synthetase from Plasmodium falciparum and highlighting sites suitable for synthesis of future cladosporin analogues. Compounds with adjacent hydroxy functions showed different MS/MS fragmentation that can be explained in terms of an, in some cases, regioselective loss of water followed by a retro‐Diels–Alder reaction.