3.3 Results
5.3.3 Pharmakokinetic studies
In the following the distribution properties of anle138b are investigated. So far it is known that anle138b is efficiently absorbed and reaches the brain after a short time. In the following study done with rats, the concentration of anle138b within liver, kidney, spleen, thymus and fat tissue at the timepoints 4, 8 and 24 hours post application was investigated. It can be seen in Fig.
5.9 that anle138b reaches very similar concentration levels in brain, liver, kidney, thymus and spleen; then the concentration declines constantly. Only in liver, where the main metabolism takes place, the maximum is reached at 8 hours before it decreases. It is a striking observation how strong anle138b accumulates in the fat tissue. The concentration in the fat is very high and increases until it reaches a putative saturation that was not further investigated here.
To test the behavior of the metabolite anle138c (M2) over time and to compare its concentration in blood plasma with anle138b, time points during 24 hours were measured from mouse blood plasma. In comparison to the other metabolites, the concentration of anle138c in the liver is not very high.
But if seen on a longer timescale of 24 hours, it becomes clear that the concentration of anle138c is constantly increasing while it is metabolized from anle138b. Anle138b is in turn decreasing in concentration. This can be seen in Fig. 5.10.
0 200 400 600 800
Brain Liver Fat Kidney Spleen Thymus
4 h 8 h 24 h
Concentration [nM/g]
Tissue homogenate
Figure 5.9: Concentration of anle138b in different rat tissue homogenates after 4, 8 and 24 hours. The compound leaves the organs after 4 hours, but accumulates in the fat tissue.
Time after application [h]
Concentration [nM/mL]
0 10 20 30 40
0 5 10 15 20 25
Anle138b Anle138c
Anle138b Anle138c
Figure 5.10: Concentration of anle138b and the metabolite anle138c in rat blood plasma at different timepoints during 24 hours. Parallel to the decrease of anle138b, the concentration of anle138c increases.
5.4. DISCUSSION & CONCLUSION 135
5.4 Discussion & Conclusion
The metabolism of the toxic protein aggregate modulator anle138b was inten-sively investigated. During this pharmacokinetic studies many informations on the ADME properties were determined. Anle138b was found to be ab-sorbed efficiently and is distributed to all organs. It passes the blood-brain-barrier and is highly concentrated in the brain. Metabolism takes place in the liver and kidney, where four metabolites were identified and their chem-ical structures were elucidated. Anle138b gets demethylenated during or hydroxylated during phase I metabolism by CYP450 enzymes. During phase II metabolism the demethylenated metabolite, anle138c, gets sulfurylated or methylated. Anle138b is metabolized and excreted from the body and no anle138b is detected in blood and organs after 24 hours. An exception is the fat tissue, were anle138b reaches high concentrations (Fig. 5.9) and a puta-tive saturation level, which was not further investigated here. A summary of the elucidated metabolism pathway in shown in Fig. 5.11.
There are several possibilities how the studies on anle138b pharmacokinet-ics could continue. First it should be investigated with CYP450 assays, which CYP450 is active in which transformation. Based on data for MDMA metabolism it is assumed that CYP2D6 is doing the demethylenation [Tucker et al., 1994, Kreth et al., 2000], but this should be further investigated.
Metabolite M3 should be synthesized to compare the NMR spectra and HPLC chromatogram of the synthetic compound with the metabolite to make sure in which position the bromine ring gets hydroxylated. If all metabolites could be synthesized, it would be advantageous to test them in animal ex-periments for their toxicity. In the end, anle138b has to be tested on humans and one would have to see, if the human body produces the same metabolites as rats and mice or different ones which could be toxic. An interesting topic would be the combination of the metabolic investigations with the NMR-based ligand binding mode determination. It could be possible to decipher the binding mode of anle138b to its demethylenation enzyme, e.g. CYP2D6.
A docking model of anle138b to the crystal structure of CYP2D6 (PDB code: 2F9Q) [Rowland et al., 2006] done with PLANTS is shown in Fig.
5.12. It proposes two distinct binding modes, that offer an explanation for the demethylenation process, as well as for the hydroxylation site.
Such a model could be refined with NMR data like STD. It would be neces-sary to investigate how the paramagnetic effects of the iron in the heme group influences the result. It should be mentioned here, that the binding mode of MPTP to its metabolizing enzyme (CYP2D6) was determined with NMR spectroscopy by using T1 relaxation measurements as distance informations of protons to the heme iron [Modi et al., 1997].
N NH Br
Phase II: Functional groups
anle138b
Figure 5.11: Metabolism and biotransformation pathway of anle138b, as revealed by the presented study. In phase I, anle138b is hydroxylized and demethylenated to anle138c by a CYP450 enzyme. Anle138c gets further sulfurylated by SULT1A3 and methylated by COMT.
5.4. DISCUSSION & CONCLUSION 137
Figure 5.12: Best scoring docking models of anle138b into the crystal structure of CYP2D6. This metabolic enzyme possesses a tunnel-like pathway to the reaction center at the heme group. On the left side the methylene group of anle138b is closest to the reaction center at the heme group. On the right side, the aromatic ring of anle138b is closest to the reaction center with proton H2. This position is thought to become hydroxylated.
Chapter 6
Structure revision of arthrofactin
6.1 Introduction
Biosurfactants are surface active substances, derived from living organisms [Cooper and Zajic, 1980]. Besides numerous industrial applications, biosur-factants are also of high relevance for medical applications. In this respect, particularly the multiple roles of biosurfactants on biofilm formation are of high interest because of the emerging problem of biofilm formation in medical devices and instruments. Knowledge about the underlying mode of action, but also the exact molecular structure of the involved molecules are essential for deeper insights and progress in this research field.
In 1993 arthrofactin was isolated from the bacteriumPseudomonassp. MIS38 (at that time identified as Arthrobacter sp.) [Morikawa et al., 1993]. This new cyclic lipopeptide (CLP) showed a capability to lower the surface ten-sion of water from 72 to 24 mNm , making it the most effective biosurfactant today, being 5-7 times more effective than surfactin. Arthrofactin belongs to the family of lipoundecapeptides, together with amphisin, lokisin, tensin and pholipeptin. The biosynthetic gene cluster of arthrofactin encodes the three nonribosomal peptide synthetase (NRPS) subunits ArfA, ArfB and ArfC, in which 11 modules and a terminal tandem thioesterase (TE) are ob-served [Roongsawang et al., 2003]. In a last step, the terminal thioesterases mediate the cyclization and release of the completed arthrofactin. Almost typical for Pseudomonas-CLP-biosynthesis gene cluster, at the C-terminal end of the NRPS a tandem of two distinct TE domains can be observed [Raai-jmakers et al., 2006, Gross and Loper, 2009].
It is crucial to know the configuration and conformation of a potential drug molecule. Unfortunately there are three different configurations and ring closures of arthrofactin in the literature. All of them lack spectroscopic
ev-139
idence and there was no crystallization of arthrofactin achieved. The first structure was presented together with the identification of arthrofactin and the ring closure was proposed to be between the 11-Asp and a CH atom of the fatty acid part, as in surfactin [Morikawa et al., 1993]. The structure of a very similar CLP, pholipeptin, was solved by NMR and the ring closure was determined to be between the residues Asp-11 and Thr-3, as a Asp-11 β peptide linkage [Ui et al., 1997]. In 2002, the crystal structure of lok-isin was solved [Sorensen et al., 2002] and confirmed a ring closure between Asp-11 and Thr-3, but via a Asp-11 α peptide linkage to the allo-isomer of threonine. A similar structure was then proposed for arthrofactin during a study of the thioesterase of arthrofactin synthetase [Roongsawang et al., 2007, Washio et al., 2010]. Nevertheless in the meantime a study on the non-ribosomal peptide synthetase (NRPS) assembly line proposed a ring closure between Asp-11α and the H3 hydroxy group of the decanoic fatty acid side chain, based on the involved enzymes [Balibar et al., 2005].
H N
Figure 6.1: Constitution and configuration of arthrofactin proposed by [Balibar et al., 2005]
A structural revision of arthofactin is done with three independent ap-proaches: i) an intensive NMR investigation, comprising the complete assign-ment and configurationa and ring closure determination, ii) a phylogenetic tree of peptide synthetase domains of bacteria was done, with a special fo-cus on the thioesterase domain. Placing the Pseudomonas strain then into this tree should reveal the nature of the ring closure mechanism and iii) the nature of the amino acids was clarified with HPLC and mass spectrometry.
6.2. MATERIALS & METHODS 141
C D-Leu-D-Asp-D-allo-Thr-D-Leu-D-Leu-D-Ser-L-Leu-D-Ser-L-Ile-L-Ile-L-Asp O
O H3C
m (m=6)
Figure 6.2: Constitution and configuration of arthrofactin proposed by [Roong-sawang et al., 2007]