Myt1 kinase is a member of the Wee-kinase family involved in G2/M checkpoint regulation of the ce... more Myt1 kinase is a member of the Wee-kinase family involved in G2/M checkpoint regulation of the cell cycle. So far, no peptide substrate suitable for activity-based screening has been reported, hampering systematic development of Myt1 kinase inhibitors. Myt1 inhibitors had to be identified by using either binding assays or activity assays with expensive proteinous substrates. Here, a peptide microarray approach was used to identify peptidic Myt1 substrates. Wee1 kinase was profiled for comparison using the same technology. Myt1 hits from peptide microarray experiments were verified in solution by a fluorescence polarization assay and several peptide substrates derived from cellular proteins were identified. Subsequently, phosphorylation site determination was carried out by MS fragmentation studies and identified substrates were validated by kinase inhibitor profiling.
To investigate pharmacokinetic differences between the nonhalogenated double ester prednicarbate ... more To investigate pharmacokinetic differences between the nonhalogenated double ester prednicarbate (PC) and the fluorinated monoester betamethasone 17-valerate (BM17V) their metabolism in human keratinocytes and fibroblasts as well as their permeation and biotransformation in reconstructed epidermis and excised human skin was compared. Special attention was given to the 17-monoesters because of their high receptor affinity and antiproliferative effects. Glucocorticoid penetration was determined using Franz diffusion cells, quantifying metabolite concentrations by HPLC. Chemical stability and reactivity of the monoesters was determined by molecular modeling analysis. PC accumulated in the stratum corneum. A considerable amount of penetrating PC was hydrolyzed by viable keratinocytes to prednisolone 17-ethylcarbonate (PI7EC), P17EC permeated the skin very rapidly when compared to BM17V. Overall P17EC concentrations in viable tissue were low. Inside of the acceptor fluid, but not within ...
The international journal of biochemistry & cell biology, 2009
Cancer in humans is the result of a multi-step process. This process often involves the activatio... more Cancer in humans is the result of a multi-step process. This process often involves the activation of oncogenes and/or the inactivation of tumor suppressor genes. These two steps arise not only due to mutations, but can also be the result of a translocation or an altered transcription rate. One important mechanism is the occurrence of epigenetic alterations like promotor methylation (which may lead to tumor suppressor silencing) or decreased histone acetylation (which can result in the downregulation of proteins involved in apoptosis). Today, histone acetylation and DNA methylation are epigenetic modifications which have been linked closely to the pathology of human cancers and inhibitors of both enzyme classes for clinical use are at hand. In contrast, other fields of epigenetics still lack of similarly thorough knowledge. This is especially true for the group of histone methyltransferases and their inhibitors. Since connections between histone methylation patterns and cancer progr...
One of the major challenges in computational approaches to drug design is the accurate prediction... more One of the major challenges in computational approaches to drug design is the accurate prediction of the binding affinity of novel biomolecules. In the present study an automated procedure which combines docking and 3D-QSAR methods was applied to several drug targets. The developed receptor-based 3D-QSAR methodology was tested on several sets of ligands for which the three-dimensional structure of the target protein has been solved--namely estrogen receptor, acetylcholine esterase and protein-tyrosine-phosphatase 1B. The molecular alignments of the studied ligands were determined using the docking program AutoDock and were compared with the X-ray structures of the corresponding protein-ligand complexes. The automatically generated protein-based ligand alignment obtained was subsequently taken as basis for a comparative field analysis applying the GRID/GOLPE approach. Using GRID interaction fields and applying variable selection procedures, highly predictive models were obtained. It ...
Sirtuins are a highly conserved class of NAD þ -dependent lysine deacylases. The human isotype Si... more Sirtuins are a highly conserved class of NAD þ -dependent lysine deacylases. The human isotype Sirt2 has been implicated in the pathogenesis of cancer, inflammation and neurodegeneration, which makes the modulation of Sirt2 activity a promising strategy for pharmaceutical intervention. A rational basis for the development of optimized Sirt2 inhibitors is lacking so far. Here we present high-resolution structures of human Sirt2 in complex with highly selective drug-like inhibitors that show a unique inhibitory mechanism. Potency and the unprecedented Sirt2 selectivity are based on a ligand-induced structural rearrangement of the active site unveiling a yet-unexploited binding pocket. Application of the most potent Sirtuin-rearranging ligand, termed SirReal2, leads to tubulin hyperacetylation in HeLa cells and induces destabilization of the checkpoint protein BubR1, consistent with Sirt2 inhibition in vivo. Our structural insights into this unique mechanism of selective sirtuin inhibition provide the basis for further inhibitor development and selective tools for sirtuin biology. | www.nature.com/naturecommunications Ac-Lys, acetyl-lysine; r.m.s., root mean squared. *Each data set was obtained from one single crystal. Sirt2-SirReal1-OTC and Sirt2-SirReal2-H3 were collected at 1.0 Å at the Swiss Light Source (Villigen, Switzerland), Sirt2-SirReal2-NAD þ was collected with an in-house X-ray source at 1.5418 Å. wValues in parentheses are for highest-resolution shell. | www.nature.com/naturecommunications Immunocytochemistry. HeLa cells that were incubated with SirReal2 (20 and 50 mM), SirReal6 (50 mM), AGK2 (Sigma-Aldrich, 20 mM) or DMSO as a control in Dulbecco's modified Eagle's medium supplemented with 10% FCS, antibiotics and DMSO (1% (v/v)) for 4 h, were fixed with ice-cold methanol (10 min), washed with PBS and blocked with PBS supplemented with 0.1% (v/v) Triton-X-100 and 5% (v/v) FCS (30 min). Cells were then stained with an anti-acetyl-a-tubulin antibody (Sigma-Aldrich, T6793) and then probed with a secondary Alexa 546 conjugated anti-mouse-antibody (Invitrogen). Nuclei were counterstained with NATURE COMMUNICATIONS |
Schistosomiasis, caused by the parasitic flatworm Schistosoma mansoni and related species, is a t... more Schistosomiasis, caused by the parasitic flatworm Schistosoma mansoni and related species, is a tropical disease that affects over 200 million people worldwide. A new approach for targeting eukaryotic parasites is to tackle their dynamic epigenetic machinery that is necessary for the extensive phenotypic changes during the life cycle of the parasite. Recently, we identified S. mansoni histone deacetylase 8 (smHDAC8) as a potential target for antiparasitic therapy. Here, we present results on the investigations of a focused set of HDAC (histone deacetylase) inhibitors on smHDAC8. Besides several active hydroxamates, we identified a thiol-based inhibitor that inhibited smHDAC8 activity in the micromolar range with unexpected selectivity over the human isotype, which has not been observed so far. The crystal structure of smHDAC8 complexed with the thiol derivative revealed that the inhibitor is accommodated in the catalytic pocket, where it interacts with both the catalytic zinc ion an...
Sirtuins are NAD(+)-dependent histone deacetylases (HDACs) that cleave off acetyl but also other ... more Sirtuins are NAD(+)-dependent histone deacetylases (HDACs) that cleave off acetyl but also other acyl groups from the ϵ-amino group of lysines in histones and other substrate proteins. Five sirtuin isoforms are encoded in the genome of the parasitic pathogen Schistosoma mansoni. During its life cycle, S. mansoni undergoes drastic changes in phenotype that are associated with epigenetic modifications. Previous work showed strong effects of hSirt2 inhibitors on both worm life span and reproduction. Thus, we postulate smSirt2 as a new antiparasite target. We report both the optimization of a homogeneous fluorescence-based assay and the development of a new heterogeneous fluorescence-based assay to determine smSirt2 activity. The homogeneous assay uses a coumarin-labeled acetyl lysine derivative, and the heterogeneous version is using a biotinylated and fluorescence-labeled oligopeptide. Magnetic streptavidin-coated beads allow higher substrate loading per well than streptavidin-coated ...
Purpose: Drug metabolism and pharmacokinetics (DMPK) assessment has come to occupy a place of int... more Purpose: Drug metabolism and pharmacokinetics (DMPK) assessment has come to occupy a place of interest during the early stages of drug discovery today. The use of computer modelling to predict the DMPK and toxicity properties of a natural product library derived from medicinal plants from Central Africa (named ConMedNP). Material from some of the plant sources are currently employed in African Traditional Medicine.
To provide a basis for the development of approaches to treat elastin-degrading diseases, the aim... more To provide a basis for the development of approaches to treat elastin-degrading diseases, the aim of this study was to investigate the degradation of the natural substrate tropoelastin by the elastinolytic matrix metalloproteinases MMP-7, MMP-9, and MMP-12 and to compare the cleavage site specificities of the enzymes using complementary MS techniques and molecular modeling. Furthermore, the ability of the three proteases to release bioactive peptides was studied. Tropoelastin was readily degraded by all three MMPs. Eighty-nine cleavage sites in tropoelastin were identified for MMP-12, whereas MMP-7 and MMP-9 were found to cleave at only 58 and 63 sites, respectively. Cleavages occurred predominantly in the N-terminal and C-terminal regions of tropoelastin. With respect to the cleavage site specificities, the study revealed that all three MMPs similarly tolerate hydrophobic and/or aliphatic amino acids, including Pro, Gly, Ile, and Val, at P(1)'. MMP-7 shows a strong preference for Leu at P(1)', which is also well accepted by MMP-9 and MMP-12. Of all three MMPs, MMP-12 best tolerates bulky charged and aromatic amino acids at P(1)'. All three MMPs showed a clear preference for Pro at P(3) that could be structurally explained by molecular modeling. Analysis of the generated peptides revealed that all three MMPs show a similar ability to release bioactive sequences, with MMP-12 producing the highest number of these peptides. Furthermore, the generated peptides YTTGKLPYGYGPGG, YGARPGVGVGGIP, and PGFGAVPGA, containing GxxPG motifs that have not yet been proven to be bioactive, were identified as new matrikines upon biological activity testing.
ADP-ribosyltransferases (ADP-RTs) use NAD(+) to transfer an ADP-ribosyl group to target proteins.... more ADP-ribosyltransferases (ADP-RTs) use NAD(+) to transfer an ADP-ribosyl group to target proteins. Although some ADP-RTs are bacterial toxins only few inhibitors are known. Here we present the development of fluorescence-based assays and a focussed library screening using kinase inhibitors as a new approach towards inhibitors of ADP-RTs. Different screening setups were established using surrogate small molecule substrates or the quantitation of the cofactor NAD(+). Proof-of-principle screening experiments were performed using a kinase inhibitor library in order to target the NAD(+) binding pockets. This led to the discovery of structurally different lead inhibitors for the mono-ADP-ribosyltransferases Mosquitocidal toxin (MTX) from Bacillus sphaericus SSII-1, C3bot toxin from Clostridium botulinum and CDTa from Clostridium difficile. The interaction of the inhibitors with the toxin proteins was analyzed by means of docking and binding free energy calculations. Binding at the nicotinamide subpocket, which shows a significant difference in the three enzymes, is used to explain the selectivity of the identified inhibitors and offers an opportunity for further development of potent and selective inhibitors.
The first antagonists known for the histamine H3 receptor were mono-substituted imidazole-contain... more The first antagonists known for the histamine H3 receptor were mono-substituted imidazole-containing compounds like thioperamide. Meanwhile numerous novel leads have been developed possessing improved affinities, selectivities, specificities, and pharmacokinetic properties. Scope and limitations of this promising class are discussed concerning their structure-activity relationships as well as pharmacological and potential therapeutic aspects.
The constitutive androstane receptor (CAR) belongs to the superfamily of nuclear-hormone receptor... more The constitutive androstane receptor (CAR) belongs to the superfamily of nuclear-hormone receptors that function as ligand-activated transcription factors. CAR plays an essential role in the metabolism of xenobiotics and shows--in contrast to related receptors--constitutive activity. However, the molecular basis for the constitutive activity remains unclear. In the present study, homology models of the ligand binding domain (LBD) were generated based on the crystal structures of the related pregnane X (PXR) and the vitamin D receptor (VDR). The models were used to investigate the basal activity of CAR and the effect of coactivator binding. Molecular dynamics (MD) simulations of complexed and uncomplexed receptor revealed a hypothesis for the activation mechanism. The suggested mechanism is supported by experimental results from site-directed mutagenesis. The basal activity of CAR can be explained by specific van-der-Waals interactions between amino acids on the LBD and its C-terminal activation domain (AF-2). Docking studies with the GOLD program yielded the interaction modes of structurally diverse agonists, giving insight into mechanisms by which ligands enhance CAR activity.
The constitutive androstane receptor (CAR) possesses an intrinsic basal activity whose structural... more The constitutive androstane receptor (CAR) possesses an intrinsic basal activity whose structural basis has been analysed during the last decade. Recently, we published a homology model of the CAR ligand binding domain (LBD) based on the X-ray structures of the closely related pregnane X (PXR) and vitamin D (VDR) receptor. A detailed analysis of the homology model and molecular dynamics (MD) simulations afforded us to propose a potential mechanism underlying the constitutive activity of CAR. Almost simultaneously, X-ray structures of human and mouse CAR LBD were released. In the present study, a detailed analysis and comparison of homology model and X-ray structures is carried out in order to evaluate the quality and reliability of our homology modelling procedure. The hypothesis of the constitutive activity which we proposed on the basis of our modelling results was tested for consistency with the crystal structures. In addition, the features stated to be essential for the basal activity based on the X-ray data were investigated by means of molecular dynamics simulations. Our results show that the homology modelling procedure was able to predict the CAR LBD structure with high accuracy. Structural features that have been revealed as critical for constitutive activity in the model are also observed in the X-ray structures. Furthermore, the MD simulations of the CAR X-ray structures and a detailed analysis of other NRs clarify the role of distinct structural features that have been assigned an important role for the constitutive activity. #
The dopamine D(3) receptor is recognized as a potential therapeutic target for the treatment of v... more The dopamine D(3) receptor is recognized as a potential therapeutic target for the treatment of various neurological and psychiatric disorders. Targetting high affinity and D(3) versus D(2) receptor-preferring ligands, the partial agonist BP 897 was taken as a lead structure. Variations in the spacer and the aryl moiety led to N-alkylated 1-(2-methyoxyphenyl)piperazines with markedly improved affinity and selectivity. Molecular modeling studies supported the structural development. Pharmacophore models for dopamine D(2) and D(3) receptor ligands were developed from their potentially bioactive conformation and were compared in order to get insight into molecular properties of importance for D(2)/D(3) receptor selectivity. For the 72 compounds presented here, an extended and more linear conformation in the aliphatic or aryl spacers turned out to be crucial for dopamine D(3) receptor selectivity. Structural diversity in the aryl moiety (benzamides, heteroarylamides, arylimides) had a major influence on (sub)nanomolar D(3) receptor affinity, which was optimized with more rigid aryl acrylamide derivatives. Compound 38 (ST 280, (E)-4-iodo-N-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)cinnamoylamide) displayed a most promising pharmacological profile (K(i) (hD(3)) = 0.5 nM; K(i) (hD(2L)) = 76.4 nM; selectivity ratio of 153), and above that, compound 38 offered the prospect of a novel radioligand as a pharmacological tool for various D(3) receptor-related in vitro and in vivo investigation.
NAD+-dependent histone deacetylases, sirtuins, cleave acetyl groups from lysines of histones and ... more NAD+-dependent histone deacetylases, sirtuins, cleave acetyl groups from lysines of histones and other proteins to regulate their activity. Identification of potent selective inhibitors would help to elucidate sirtuin biology and could lead to useful therapeutic agents. NAD+ has an adenosine moiety that is also present in the kinase cofactor ATP. Kinase inhibitors based upon adenosine mimesis may thus also target NAD+-dependent enzymes. We present a systematic approach using adenosine mimics from one cofactor class (kinase inhibitors) as a viable method to generate new lead structures in another cofactor class (sirtuin inhibitors). Our findings have broad implications for medicinal chemistry and specifically for sirtuin inhibitor design. Our results also raise a question as to whether selectivity profiling for kinase inhibitors should be limited to ATP-dependent targets.
Lysine and arginine methyltransferases participate in the post-translational modification of hist... more Lysine and arginine methyltransferases participate in the post-translational modification of histones and regulate key cellular functions. So far only one arginine methyltransferase inhibitor discovered by random screening was available. We present the first target-based approach to protein arginine methyltransferase (PRMT) inhibitors. Homology models of human and Aspergillus nidulans PRMT1 were generated from available X-ray structures of rat PRMTs. The NCI diversity set was filtered by a target-based virtual screening to identify PRMT inhibitors. Employing a fungal PRMT for screening and a human enzyme for validation, we have identified seven inhibitors of PRMTs in vitro. Hit validation was achieved for two new inhibitors by antibody mediated detection of histone hypomethylation as well as Western blotting in cancer cells. Functional activity was proven by an observed block of estrogen receptor activation. Thus, valuable chemical tools and potential drug candidates could be identified.
Myt1 kinase is a member of the Wee-kinase family involved in G2/M checkpoint regulation of the ce... more Myt1 kinase is a member of the Wee-kinase family involved in G2/M checkpoint regulation of the cell cycle. So far, no peptide substrate suitable for activity-based screening has been reported, hampering systematic development of Myt1 kinase inhibitors. Myt1 inhibitors had to be identified by using either binding assays or activity assays with expensive proteinous substrates. Here, a peptide microarray approach was used to identify peptidic Myt1 substrates. Wee1 kinase was profiled for comparison using the same technology. Myt1 hits from peptide microarray experiments were verified in solution by a fluorescence polarization assay and several peptide substrates derived from cellular proteins were identified. Subsequently, phosphorylation site determination was carried out by MS fragmentation studies and identified substrates were validated by kinase inhibitor profiling.
To investigate pharmacokinetic differences between the nonhalogenated double ester prednicarbate ... more To investigate pharmacokinetic differences between the nonhalogenated double ester prednicarbate (PC) and the fluorinated monoester betamethasone 17-valerate (BM17V) their metabolism in human keratinocytes and fibroblasts as well as their permeation and biotransformation in reconstructed epidermis and excised human skin was compared. Special attention was given to the 17-monoesters because of their high receptor affinity and antiproliferative effects. Glucocorticoid penetration was determined using Franz diffusion cells, quantifying metabolite concentrations by HPLC. Chemical stability and reactivity of the monoesters was determined by molecular modeling analysis. PC accumulated in the stratum corneum. A considerable amount of penetrating PC was hydrolyzed by viable keratinocytes to prednisolone 17-ethylcarbonate (PI7EC), P17EC permeated the skin very rapidly when compared to BM17V. Overall P17EC concentrations in viable tissue were low. Inside of the acceptor fluid, but not within ...
The international journal of biochemistry & cell biology, 2009
Cancer in humans is the result of a multi-step process. This process often involves the activatio... more Cancer in humans is the result of a multi-step process. This process often involves the activation of oncogenes and/or the inactivation of tumor suppressor genes. These two steps arise not only due to mutations, but can also be the result of a translocation or an altered transcription rate. One important mechanism is the occurrence of epigenetic alterations like promotor methylation (which may lead to tumor suppressor silencing) or decreased histone acetylation (which can result in the downregulation of proteins involved in apoptosis). Today, histone acetylation and DNA methylation are epigenetic modifications which have been linked closely to the pathology of human cancers and inhibitors of both enzyme classes for clinical use are at hand. In contrast, other fields of epigenetics still lack of similarly thorough knowledge. This is especially true for the group of histone methyltransferases and their inhibitors. Since connections between histone methylation patterns and cancer progr...
One of the major challenges in computational approaches to drug design is the accurate prediction... more One of the major challenges in computational approaches to drug design is the accurate prediction of the binding affinity of novel biomolecules. In the present study an automated procedure which combines docking and 3D-QSAR methods was applied to several drug targets. The developed receptor-based 3D-QSAR methodology was tested on several sets of ligands for which the three-dimensional structure of the target protein has been solved--namely estrogen receptor, acetylcholine esterase and protein-tyrosine-phosphatase 1B. The molecular alignments of the studied ligands were determined using the docking program AutoDock and were compared with the X-ray structures of the corresponding protein-ligand complexes. The automatically generated protein-based ligand alignment obtained was subsequently taken as basis for a comparative field analysis applying the GRID/GOLPE approach. Using GRID interaction fields and applying variable selection procedures, highly predictive models were obtained. It ...
Sirtuins are a highly conserved class of NAD þ -dependent lysine deacylases. The human isotype Si... more Sirtuins are a highly conserved class of NAD þ -dependent lysine deacylases. The human isotype Sirt2 has been implicated in the pathogenesis of cancer, inflammation and neurodegeneration, which makes the modulation of Sirt2 activity a promising strategy for pharmaceutical intervention. A rational basis for the development of optimized Sirt2 inhibitors is lacking so far. Here we present high-resolution structures of human Sirt2 in complex with highly selective drug-like inhibitors that show a unique inhibitory mechanism. Potency and the unprecedented Sirt2 selectivity are based on a ligand-induced structural rearrangement of the active site unveiling a yet-unexploited binding pocket. Application of the most potent Sirtuin-rearranging ligand, termed SirReal2, leads to tubulin hyperacetylation in HeLa cells and induces destabilization of the checkpoint protein BubR1, consistent with Sirt2 inhibition in vivo. Our structural insights into this unique mechanism of selective sirtuin inhibition provide the basis for further inhibitor development and selective tools for sirtuin biology. | www.nature.com/naturecommunications Ac-Lys, acetyl-lysine; r.m.s., root mean squared. *Each data set was obtained from one single crystal. Sirt2-SirReal1-OTC and Sirt2-SirReal2-H3 were collected at 1.0 Å at the Swiss Light Source (Villigen, Switzerland), Sirt2-SirReal2-NAD þ was collected with an in-house X-ray source at 1.5418 Å. wValues in parentheses are for highest-resolution shell. | www.nature.com/naturecommunications Immunocytochemistry. HeLa cells that were incubated with SirReal2 (20 and 50 mM), SirReal6 (50 mM), AGK2 (Sigma-Aldrich, 20 mM) or DMSO as a control in Dulbecco's modified Eagle's medium supplemented with 10% FCS, antibiotics and DMSO (1% (v/v)) for 4 h, were fixed with ice-cold methanol (10 min), washed with PBS and blocked with PBS supplemented with 0.1% (v/v) Triton-X-100 and 5% (v/v) FCS (30 min). Cells were then stained with an anti-acetyl-a-tubulin antibody (Sigma-Aldrich, T6793) and then probed with a secondary Alexa 546 conjugated anti-mouse-antibody (Invitrogen). Nuclei were counterstained with NATURE COMMUNICATIONS |
Schistosomiasis, caused by the parasitic flatworm Schistosoma mansoni and related species, is a t... more Schistosomiasis, caused by the parasitic flatworm Schistosoma mansoni and related species, is a tropical disease that affects over 200 million people worldwide. A new approach for targeting eukaryotic parasites is to tackle their dynamic epigenetic machinery that is necessary for the extensive phenotypic changes during the life cycle of the parasite. Recently, we identified S. mansoni histone deacetylase 8 (smHDAC8) as a potential target for antiparasitic therapy. Here, we present results on the investigations of a focused set of HDAC (histone deacetylase) inhibitors on smHDAC8. Besides several active hydroxamates, we identified a thiol-based inhibitor that inhibited smHDAC8 activity in the micromolar range with unexpected selectivity over the human isotype, which has not been observed so far. The crystal structure of smHDAC8 complexed with the thiol derivative revealed that the inhibitor is accommodated in the catalytic pocket, where it interacts with both the catalytic zinc ion an...
Sirtuins are NAD(+)-dependent histone deacetylases (HDACs) that cleave off acetyl but also other ... more Sirtuins are NAD(+)-dependent histone deacetylases (HDACs) that cleave off acetyl but also other acyl groups from the ϵ-amino group of lysines in histones and other substrate proteins. Five sirtuin isoforms are encoded in the genome of the parasitic pathogen Schistosoma mansoni. During its life cycle, S. mansoni undergoes drastic changes in phenotype that are associated with epigenetic modifications. Previous work showed strong effects of hSirt2 inhibitors on both worm life span and reproduction. Thus, we postulate smSirt2 as a new antiparasite target. We report both the optimization of a homogeneous fluorescence-based assay and the development of a new heterogeneous fluorescence-based assay to determine smSirt2 activity. The homogeneous assay uses a coumarin-labeled acetyl lysine derivative, and the heterogeneous version is using a biotinylated and fluorescence-labeled oligopeptide. Magnetic streptavidin-coated beads allow higher substrate loading per well than streptavidin-coated ...
Purpose: Drug metabolism and pharmacokinetics (DMPK) assessment has come to occupy a place of int... more Purpose: Drug metabolism and pharmacokinetics (DMPK) assessment has come to occupy a place of interest during the early stages of drug discovery today. The use of computer modelling to predict the DMPK and toxicity properties of a natural product library derived from medicinal plants from Central Africa (named ConMedNP). Material from some of the plant sources are currently employed in African Traditional Medicine.
To provide a basis for the development of approaches to treat elastin-degrading diseases, the aim... more To provide a basis for the development of approaches to treat elastin-degrading diseases, the aim of this study was to investigate the degradation of the natural substrate tropoelastin by the elastinolytic matrix metalloproteinases MMP-7, MMP-9, and MMP-12 and to compare the cleavage site specificities of the enzymes using complementary MS techniques and molecular modeling. Furthermore, the ability of the three proteases to release bioactive peptides was studied. Tropoelastin was readily degraded by all three MMPs. Eighty-nine cleavage sites in tropoelastin were identified for MMP-12, whereas MMP-7 and MMP-9 were found to cleave at only 58 and 63 sites, respectively. Cleavages occurred predominantly in the N-terminal and C-terminal regions of tropoelastin. With respect to the cleavage site specificities, the study revealed that all three MMPs similarly tolerate hydrophobic and/or aliphatic amino acids, including Pro, Gly, Ile, and Val, at P(1)'. MMP-7 shows a strong preference for Leu at P(1)', which is also well accepted by MMP-9 and MMP-12. Of all three MMPs, MMP-12 best tolerates bulky charged and aromatic amino acids at P(1)'. All three MMPs showed a clear preference for Pro at P(3) that could be structurally explained by molecular modeling. Analysis of the generated peptides revealed that all three MMPs show a similar ability to release bioactive sequences, with MMP-12 producing the highest number of these peptides. Furthermore, the generated peptides YTTGKLPYGYGPGG, YGARPGVGVGGIP, and PGFGAVPGA, containing GxxPG motifs that have not yet been proven to be bioactive, were identified as new matrikines upon biological activity testing.
ADP-ribosyltransferases (ADP-RTs) use NAD(+) to transfer an ADP-ribosyl group to target proteins.... more ADP-ribosyltransferases (ADP-RTs) use NAD(+) to transfer an ADP-ribosyl group to target proteins. Although some ADP-RTs are bacterial toxins only few inhibitors are known. Here we present the development of fluorescence-based assays and a focussed library screening using kinase inhibitors as a new approach towards inhibitors of ADP-RTs. Different screening setups were established using surrogate small molecule substrates or the quantitation of the cofactor NAD(+). Proof-of-principle screening experiments were performed using a kinase inhibitor library in order to target the NAD(+) binding pockets. This led to the discovery of structurally different lead inhibitors for the mono-ADP-ribosyltransferases Mosquitocidal toxin (MTX) from Bacillus sphaericus SSII-1, C3bot toxin from Clostridium botulinum and CDTa from Clostridium difficile. The interaction of the inhibitors with the toxin proteins was analyzed by means of docking and binding free energy calculations. Binding at the nicotinamide subpocket, which shows a significant difference in the three enzymes, is used to explain the selectivity of the identified inhibitors and offers an opportunity for further development of potent and selective inhibitors.
The first antagonists known for the histamine H3 receptor were mono-substituted imidazole-contain... more The first antagonists known for the histamine H3 receptor were mono-substituted imidazole-containing compounds like thioperamide. Meanwhile numerous novel leads have been developed possessing improved affinities, selectivities, specificities, and pharmacokinetic properties. Scope and limitations of this promising class are discussed concerning their structure-activity relationships as well as pharmacological and potential therapeutic aspects.
The constitutive androstane receptor (CAR) belongs to the superfamily of nuclear-hormone receptor... more The constitutive androstane receptor (CAR) belongs to the superfamily of nuclear-hormone receptors that function as ligand-activated transcription factors. CAR plays an essential role in the metabolism of xenobiotics and shows--in contrast to related receptors--constitutive activity. However, the molecular basis for the constitutive activity remains unclear. In the present study, homology models of the ligand binding domain (LBD) were generated based on the crystal structures of the related pregnane X (PXR) and the vitamin D receptor (VDR). The models were used to investigate the basal activity of CAR and the effect of coactivator binding. Molecular dynamics (MD) simulations of complexed and uncomplexed receptor revealed a hypothesis for the activation mechanism. The suggested mechanism is supported by experimental results from site-directed mutagenesis. The basal activity of CAR can be explained by specific van-der-Waals interactions between amino acids on the LBD and its C-terminal activation domain (AF-2). Docking studies with the GOLD program yielded the interaction modes of structurally diverse agonists, giving insight into mechanisms by which ligands enhance CAR activity.
The constitutive androstane receptor (CAR) possesses an intrinsic basal activity whose structural... more The constitutive androstane receptor (CAR) possesses an intrinsic basal activity whose structural basis has been analysed during the last decade. Recently, we published a homology model of the CAR ligand binding domain (LBD) based on the X-ray structures of the closely related pregnane X (PXR) and vitamin D (VDR) receptor. A detailed analysis of the homology model and molecular dynamics (MD) simulations afforded us to propose a potential mechanism underlying the constitutive activity of CAR. Almost simultaneously, X-ray structures of human and mouse CAR LBD were released. In the present study, a detailed analysis and comparison of homology model and X-ray structures is carried out in order to evaluate the quality and reliability of our homology modelling procedure. The hypothesis of the constitutive activity which we proposed on the basis of our modelling results was tested for consistency with the crystal structures. In addition, the features stated to be essential for the basal activity based on the X-ray data were investigated by means of molecular dynamics simulations. Our results show that the homology modelling procedure was able to predict the CAR LBD structure with high accuracy. Structural features that have been revealed as critical for constitutive activity in the model are also observed in the X-ray structures. Furthermore, the MD simulations of the CAR X-ray structures and a detailed analysis of other NRs clarify the role of distinct structural features that have been assigned an important role for the constitutive activity. #
The dopamine D(3) receptor is recognized as a potential therapeutic target for the treatment of v... more The dopamine D(3) receptor is recognized as a potential therapeutic target for the treatment of various neurological and psychiatric disorders. Targetting high affinity and D(3) versus D(2) receptor-preferring ligands, the partial agonist BP 897 was taken as a lead structure. Variations in the spacer and the aryl moiety led to N-alkylated 1-(2-methyoxyphenyl)piperazines with markedly improved affinity and selectivity. Molecular modeling studies supported the structural development. Pharmacophore models for dopamine D(2) and D(3) receptor ligands were developed from their potentially bioactive conformation and were compared in order to get insight into molecular properties of importance for D(2)/D(3) receptor selectivity. For the 72 compounds presented here, an extended and more linear conformation in the aliphatic or aryl spacers turned out to be crucial for dopamine D(3) receptor selectivity. Structural diversity in the aryl moiety (benzamides, heteroarylamides, arylimides) had a major influence on (sub)nanomolar D(3) receptor affinity, which was optimized with more rigid aryl acrylamide derivatives. Compound 38 (ST 280, (E)-4-iodo-N-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)cinnamoylamide) displayed a most promising pharmacological profile (K(i) (hD(3)) = 0.5 nM; K(i) (hD(2L)) = 76.4 nM; selectivity ratio of 153), and above that, compound 38 offered the prospect of a novel radioligand as a pharmacological tool for various D(3) receptor-related in vitro and in vivo investigation.
NAD+-dependent histone deacetylases, sirtuins, cleave acetyl groups from lysines of histones and ... more NAD+-dependent histone deacetylases, sirtuins, cleave acetyl groups from lysines of histones and other proteins to regulate their activity. Identification of potent selective inhibitors would help to elucidate sirtuin biology and could lead to useful therapeutic agents. NAD+ has an adenosine moiety that is also present in the kinase cofactor ATP. Kinase inhibitors based upon adenosine mimesis may thus also target NAD+-dependent enzymes. We present a systematic approach using adenosine mimics from one cofactor class (kinase inhibitors) as a viable method to generate new lead structures in another cofactor class (sirtuin inhibitors). Our findings have broad implications for medicinal chemistry and specifically for sirtuin inhibitor design. Our results also raise a question as to whether selectivity profiling for kinase inhibitors should be limited to ATP-dependent targets.
Lysine and arginine methyltransferases participate in the post-translational modification of hist... more Lysine and arginine methyltransferases participate in the post-translational modification of histones and regulate key cellular functions. So far only one arginine methyltransferase inhibitor discovered by random screening was available. We present the first target-based approach to protein arginine methyltransferase (PRMT) inhibitors. Homology models of human and Aspergillus nidulans PRMT1 were generated from available X-ray structures of rat PRMTs. The NCI diversity set was filtered by a target-based virtual screening to identify PRMT inhibitors. Employing a fungal PRMT for screening and a human enzyme for validation, we have identified seven inhibitors of PRMTs in vitro. Hit validation was achieved for two new inhibitors by antibody mediated detection of histone hypomethylation as well as Western blotting in cancer cells. Functional activity was proven by an observed block of estrogen receptor activation. Thus, valuable chemical tools and potential drug candidates could be identified.
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Papers by Wolfgang Sippl