Jens Sydor - Academia.edu (original) (raw)
Papers by Jens Sydor
Drug metabolism and disposition: the biological fate of chemicals, Mar 19, 2016
Venetoclax (also known as ABT-199) is a B-cell lymphoma-2 (Bcl-2) family protein inhibitor and is... more Venetoclax (also known as ABT-199) is a B-cell lymphoma-2 (Bcl-2) family protein inhibitor and is currently in clinical development for the treatment of chronic lymphocytic leukaemia (CLL) and other hematological malignancies. The objective of this study was to characterize the absorption, metabolism, and excretion of venetoclax in humans. Following a single oral dose of 200 mg (100 μ - Ci) of [(14)C]venetoclax to four healthy volunteers, recovery of total radioactive dose was 100% (± 5%), with feces being the major route of elimination of the administered dose, whereas urinary excretion was minimal (<0.1%). The extent of absorption was estimated to be at least 65%. Venetoclax was primarily cleared by hepatic metabolism (~66% of the administered dose). ~33% of the administered dose was recovered as parent drug and its nitro reduction metabolite M30 in feces, which is likely due to unabsorbed parent drug. Biotransformation of venetoclax in human primarily involves enzymatic oxidat...
Biochemistry Usa, 1998
Transient kinetic methods have been used to analyze the interaction between the Ras-binding domai... more Transient kinetic methods have been used to analyze the interaction between the Ras-binding domain (RBD) of c-Raf-1 and a complex of H-Ras and a GTP analogue. The results obtained show that the binding is a two-step process, with an initial rapid equilibrium step being followed by an isomerization reaction occurring at several hundred per second. The reversal of this step determines the rate constant for dissociation, which is on the order of 10 s-1. The lifetime of the complex is therefore on the order of 50-100 ms, which is much shorter than the lifetime of GTP at the active site of H-Ras as determined by the intrinsic GTPase reaction. This suggests that multiple interactions of a single activated Ras molecule and Raf can occur, the number being limited by the competing interaction with GAP. The GDP complex of H-Ras binds more than 2 orders of magnitude more weakly than the GTP-analogue complex, mainly due to a significant weakening of the initial binding equilibrium reaction in the GDP state, thereby avoiding even short-lived recruitment of Raf to the plasma membrane by the inactive Ras form.
Bioconjugate Chemistry, Jul 1, 2002
Intein-mediated protein ligation is a recently developed method that enables the C-terminal label... more Intein-mediated protein ligation is a recently developed method that enables the C-terminal labeling of proteins. This technique requires a correctly folded intein mutant that is fused to the C-terminus of a target protein to create a thioester, which allows the ligation of a peptide with an N-terminal cysteine (1, 2). Here we describe the establishment of this method for the labeling, under denaturing conditions, of target proteins that are expressed insolubly as intein fusion proteins. A GFPuv fusion protein with the Mycobacterium xenopi gyrA intein was expressed in inclusion bodies in Escherichia coli and initially used as a model protein to verify intein cleavage activity under different refolding conditions. The intein showed activity after refolding in nondenaturing and slightly denaturing conditions. A construct of the same intein with an anti-neutravidin single-chain antibody was also expressed in an insoluble form. The intein-mediated ligation was established for this single chain antibody-intein fusion protein under denaturing conditions in 4 M urea to prevent significant precipitation of the fusion protein during the first refolding step. Under optimized conditions, the single-chain antibody was labeled with a fluorescent peptide and used for antigen screening on a biochip after final refolding. This screening procedure allowed the determination of binding characteristics of the scFv for avidin proteins in a miniaturized format.
Analytical Chemistry, 2003
A new chip-based method to identify protein-protein interactions was developed using the guanine ... more A new chip-based method to identify protein-protein interactions was developed using the guanine nucleotide exchange factor GRF2 and two interacting proteins, Ras and calmodulin, as model proteins. A generic immobilization strategy for FLAG-tagged bait proteins on a protein-repellent streptavidin chip surface was implemented by presentation of an oriented anti-FLAG antibody. A flow cell device, integrating different chip surfaces, was developed, and the interaction of immobilized GRF2 with the two analytes was verified by fluorescence assays. On-chip tryptic digest assays were then performed on the capture surface and analyzed by microLC-MS/MS. The interaction of GRF2 with calmodulin and Ras was demonstrated, and the lower limit of detection was determined. We also implemented an on-chip immunoprecipitation assay to identify GRF2-binding partners from complex protein mixtures. Cells overexpressing FLAG-GRF2 were lysed and then incubated with the anti-FLAG chip. In addition to detecting GRF2, we also identified calmodulin, demonstrating that this technique can successfully identify endogenous levels of proteins, bound to recombinant bait proteins. This chip-based method has the advantage that no subsequent gel separations of protein complexes prior to LC-MS analysis are required and is therefore amenable to miniaturized high-throughput determination of protein-protein interactions.
Proteome science, Jan 10, 2003
The completion of the human genome sequence has led to a rapid increase in genetic information. T... more The completion of the human genome sequence has led to a rapid increase in genetic information. The invention of DNA microarrays, which allow for the parallel measurement of thousands of genes on the level of mRNA, has enabled scientists to take a more global view of biological systems. Protein microarrays have a big potential to increase the throughput of proteomic research. Microarrays of antibodies can simultaneously measure the concentration of a multitude of target proteins in a very short period of time. The ability of protein microarrays to increase the quantity of data points in small biological samples on the protein level will have a major impact on basic biological research as well as on the discovery of new drug targets and diagnostic markers. This review highlights the current status of protein expression profiling arrays, their development, applications and limitations.
Recent evidence suggests that blocking aberrant Hedgehog pathway signaling may be a potential the... more Recent evidence suggests that blocking aberrant Hedgehog pathway signaling may be a potential therapeutic strategy for the treatment of several types of cancer. Cyclopamine, a plant Veratrum alkaloid natural product, is an antagonist of the Hedgehog pathway and was used as a starting point for the development of new Hedgehog pathway antagonists. Herein, we describe the synthesis of D-homo cyclopamine analogs by a sequence of chemoselective cyclopropanation followed by stereoselective acid-catalyzed rearrangement. These D-homo analogs are more stable to acid-catalyzed degradation than the natural product. Further modifications of the A-ring generated three new series of analogs, which were characterized in vitro and evaluated in vivo for biological activity and pharmacokinetic properties. These studies led to the discovery of IPI-926, a novel semi-synthetic cyclopamine analog with improved pharmaceutical properties and potency, and a superior plasma pharmacokinetic profile relative t...
Encyclopedia of Molecular Cell Biology and Molecular Medicine, 2006
ABSTRACT Protein microarrays allow for the parallel analysis of a large number of proteins with r... more ABSTRACT Protein microarrays allow for the parallel analysis of a large number of proteins with respect to their abundance or activity and, therefore, are playing an emerging role in proteome research.Protein expression profiling, detection of enzymatic activity and protein interaction mapping are the most important applications of protein microarrays. The biological content, as well as the detection strategies, vary significantly between these applications, making the protein microarray technologies much more varied than those used with DNA microarrays.In most cases, the biological content on the microarray, as well as the analytes, are proteins, which, due to their delicate nature and the heterogeneity of their properties, complicate the manufacturing and assay design. The potential advantages of protein microarrays over nonparallel, macroscopic technologies cannot, however, be overestimated.Keywords:Capture Agents;Enzyme Activity Profiling;Multiplexed Protein Assay;Protein Expression Profiling;Protein Interaction Profiling;Protein Microarray
European Journal of Cancer Supplements, 2008
Xenobiotica, 2013
1. IPI-926 is a novel semisynthetic cyclopamine derivative that is a potent and selective Smoothe... more 1. IPI-926 is a novel semisynthetic cyclopamine derivative that is a potent and selective Smoothened inhibitor that blocks the hedgehog signal transduction pathway. 2. The in vivo clearance of IPI-926 is low in mouse and dog and moderate in monkey. The volume of distribution is high across species. Oral bioavailability ranges from moderate in monkey to high in mouse and dog. Predicted human clearance using simple allometry is low (24 L h(-1)), predicted volume of distribution is high (469 L) and predicted half-life is long (20 h). 3. IPI-926 is highly bound to plasma proteins and has minimal interaction with human α-1-acid glycoprotein. 4. In vitro metabolic stability ranges from stable to moderately stable. Twelve oxidative metabolites were detected in mouse, rat, dog, monkey and human liver microsome incubations and none were unique to human. 5. IPI-926 is not a potent reversible inhibitor of CYP1A2, 2C8, 2C9 or 3A4 (testosterone). IPI-926 is a moderate inhibitor of CYP2C19, 2D6 and 3A4 (midazolam) with KI values of 19, 16 and 4.5 µM, respectively. IPI-926 is both a substrate and inhibitor (IC50 = 1.9 µM) of P-glycoprotein. 6. In summary, IPI-926 has desirable pre-clinical absorption, distribution, metabolism and excretion properties.
![Research paper thumbnail of Design, total chemical synthesis, and binding properties of a [Leu-91-N1-methyl-7-azaTrp]Ras-binding domain of c-Raf-1](https://attachments.academia-assets.com/46492544/thumbnails/1.jpg)
](Proceedings of the National Academy of Sciences, 1999
The Ras-binding domain (RBD) of c-Raf-1 has been synthesized chemically, taking advantage of the ... more The Ras-binding domain (RBD) of c-Raf-1 has been synthesized chemically, taking advantage of the chemical ligation of two peptide fragments of the protein. This procedure allowed incorporation of an unnatural amino acid (N 1 -methyl-7-azatryptophan) at position 91 of RBD, producing a protein with f luorescent properties distinct from and distinguishable from those of proteins containing the natural f luorophore tryptophan. The resulting protein was shown to interact with Ras in a manner that was almost indistinguishable from that of unmodified RBD based on transient kinetic monitoring of the binding event. Modified RBD containing the L-isomer of the unnatural amino acid or its racemic D,L mixture appeared to interact identically with Ras. The approach demonstrates a general procedure for the introduction of unnatural amino acids that can be used for monitoring protein-protein interactions and for the introduction of an unnatural backbone structure at strategic positions.
Proceedings of the National Academy of Sciences, 2006
analyzed data; and J.R.S. wrote the paper. The authors declare no conflict of interest. Freely av... more analyzed data; and J.R.S. wrote the paper. The authors declare no conflict of interest. Freely available online through the PNAS open access option. Abbreviations: Hsp90, heat shock protein 90; MM, multiple myeloma; IPI-504, 17allylamino-17-demethoxygeldanamycin hydroquinone hydrochloride; NQO1, NADPHquinone oxidoreductase 1; 17-AGG, 17-allylamino-17-demethoxygeldanamycin; 17-AG, 17-(amino)-17-demethoxygeldanamycin; LC-MS͞MS, liquid chromatography-tandem mass spectrometry.
Journal of Medicinal Chemistry, 2008
Herein is reported the synthesis of a novel class of hedgehog antagonists derived from cyclopamin... more Herein is reported the synthesis of a novel class of hedgehog antagonists derived from cyclopamine. The acid sensitive D-ring of cyclopamine was homologated utilizing a sequence of chemoselective cyclopropanation and stereoselective acid-catalyzed rearrangement. Further modification of the A/B-ring homoallylic alcohol to the conjugated ketone led to the discovery of new cyclopamine analogues with improved pharmaceutical properties and in vitro potency (EC 50) ranging from 10 to 1000 nM.
Journal of Medicinal Chemistry, 2009
Recent evidence suggests that blocking aberrant hedgehog pathway signaling may be a promising the... more Recent evidence suggests that blocking aberrant hedgehog pathway signaling may be a promising therapeutic strategy for the treatment of several types of cancer. Cyclopamine, a plant Veratrum alkaloid, is a natural product antagonist of the hedgehog pathway. In a previous report, a seven-membered D-ring semisynthetic analogue of cyclopamine, IPI-269609 (2), was shown to have greater acid stability and better aqueous solubility compared to cyclopamine. Further modifications of the A-ring system generated three series of analogues with improved potency and/or solubility. Lead compounds from each series were characterized in vitro and evaluated in vivo for biological activity and pharmacokinetic properties. These studies led to the discovery of IPI-926 (compound 28), a novel semisynthetic cyclopamine analogue with substantially improved pharmaceutical properties and potency and a favorable pharmacokinetic profile relative to cyclopamine and compound 2. As a result, complete tumor regression was observed in a Hh-dependent medulloblastoma allograft model after daily oral administration of 40 mg/kg of compound 28.
FEBS Letters, 1999
It has previously been shown that the transient kinetics of the interaction between the Ras-bindi... more It has previously been shown that the transient kinetics of the interaction between the Ras-binding domain of c-Raf-1 and the proto-oncoprotein Ras can be followed by stopped-flow measurements using the 2',3'-(N-methylanthraniloyl) fluorescence of 2',3'-(N-methylanthraniloyl) guanyl-5'-yl-imidodiphosphate-labelled Ras. In continuation of this work, we demonstrate that the His-tagged Ras-binding domain of c-Raf-1 can also be synthesized in a cell-free expression system. After purification by Ni2+ affinity chromatography, His-tagged Ras-binding domain of c-Raf-1 could be isolated in sufficient amounts for biochemical and biophysical investigations. The results obtained describe the first example of a cell-free synthesized protein which has been used for stopped-flow measurements to determine the transient kinetics of protein-protein interactions with an effector.
FEBS Letters, 2000
Sensory rhodopsin II (pSRII), the photophobic receptor from Natronobacterium pharaonis, has been ... more Sensory rhodopsin II (pSRII), the photophobic receptor from Natronobacterium pharaonis, has been studied by time-resolved resonance Raman (RR) spectroscopy using the rotating cell technique. Upon excitation with low laser power, the RR spectra largely reflect the parent state pSRII(500) whereas an increase of the laser power leads to a substantial accumulation of long-lived intermediates contributing to the RR spectra. All RR spectra could consistently be analysed in terms of four component spectra which were assigned to the parent state pSRII(500) and the long-lived intermediates M(400), N(485) and O(535) based on the correlation between the C = C stretching frequency and the absorption maximum. The parent state and the intermediates N(485) and O(535) exhibit a protonated Schiff base. The C = N stretching frequencies and the H/D isotopic shifts indicate strong hydrogen bonding interactions of the Schiff base in pSRII(500) and O(535) whereas these interactions are most likely very weak in N(485).
Biophysical Journal, 1998
The photocycle of the photophobic receptor sensory rhodopsin II from N. pharaonis was analyzed by... more The photocycle of the photophobic receptor sensory rhodopsin II from N. pharaonis was analyzed by varying measuring wavelengths, temperature, and pH, and by exchanging H 2 O with D 2 O. The data can be satisfactorily modeled by eight exponents over the whole range of modified parameters. The kinetic data support a model similar to that of bacteriorhodopsin (BR) if a scheme of irreversible first-order reactions is assumed. Eight kinetically distinct protein states can then be identified. These states are formed from five spectrally distinct species. The chromophore states S i correspond in their spectral properties to those of the BR photocycle, namely pSRII 510 (K), pSRII 495 (L), pSRII 400 (M), pSRII 485 (N), and pSRII 535 (O). In comparison to BR, pSRII 400 is formed ϳ10 times faster than the M state; however, the back-reaction is almost 100 times slower. Comparison of the temperature dependence of the rate constants with those from the BR photocycle suggests that the differences are caused by changes of ⌬S. The rate constants of the pSRII photocycle are almost insensitive to the pH variation from 9.0 to 5.5, and show only a small H 2 O/D 2 O effect. This analysis supports the idea that the conformational dynamics of pSRII controls the kinetics of the photocycle of pSRII.
Bioconjugate Chemistry, 2002
Intein-mediated protein ligation is a recently developed method that enables the C-terminal label... more Intein-mediated protein ligation is a recently developed method that enables the C-terminal labeling of proteins. This technique requires a correctly folded intein mutant that is fused to the C-terminus of a target protein to create a thioester, which allows the ligation of a peptide with an N-terminal cysteine (1, 2). Here we describe the establishment of this method for the labeling, under denaturing conditions, of target proteins that are expressed insolubly as intein fusion proteins. A GFPuv fusion protein with the Mycobacterium xenopi gyrA intein was expressed in inclusion bodies in Escherichia coli and initially used as a model protein to verify intein cleavage activity under different refolding conditions. The intein showed activity after refolding in nondenaturing and slightly denaturing conditions. A construct of the same intein with an anti-neutravidin single-chain antibody was also expressed in an insoluble form. The intein-mediated ligation was established for this single chain antibody-intein fusion protein under denaturing conditions in 4 M urea to prevent significant precipitation of the fusion protein during the first refolding step. Under optimized conditions, the single-chain antibody was labeled with a fluorescent peptide and used for antigen screening on a biochip after final refolding. This screening procedure allowed the determination of binding characteristics of the scFv for avidin proteins in a miniaturized format.
Biochemistry, 1998
Transient kinetic methods have been used to analyze the interaction between the Ras-binding domai... more Transient kinetic methods have been used to analyze the interaction between the Ras-binding domain (RBD) of c-Raf-1 and a complex of H-Ras and a GTP analogue. The results obtained show that the binding is a two-step process, with an initial rapid equilibrium step being followed by an isomerization reaction occurring at several hundred per second. The reversal of this step determines the rate constant for dissociation, which is on the order of 10 s-1. The lifetime of the complex is therefore on the order of 50-100 ms, which is much shorter than the lifetime of GTP at the active site of H-Ras as determined by the intrinsic GTPase reaction. This suggests that multiple interactions of a single activated Ras molecule and Raf can occur, the number being limited by the competing interaction with GAP. The GDP complex of H-Ras binds more than 2 orders of magnitude more weakly than the GTP-analogue complex, mainly due to a significant weakening of the initial binding equilibrium reaction in the GDP state, thereby avoiding even short-lived recruitment of Raf to the plasma membrane by the inactive Ras form.
Drug metabolism and disposition: the biological fate of chemicals, Mar 19, 2016
Venetoclax (also known as ABT-199) is a B-cell lymphoma-2 (Bcl-2) family protein inhibitor and is... more Venetoclax (also known as ABT-199) is a B-cell lymphoma-2 (Bcl-2) family protein inhibitor and is currently in clinical development for the treatment of chronic lymphocytic leukaemia (CLL) and other hematological malignancies. The objective of this study was to characterize the absorption, metabolism, and excretion of venetoclax in humans. Following a single oral dose of 200 mg (100 μ - Ci) of [(14)C]venetoclax to four healthy volunteers, recovery of total radioactive dose was 100% (± 5%), with feces being the major route of elimination of the administered dose, whereas urinary excretion was minimal (<0.1%). The extent of absorption was estimated to be at least 65%. Venetoclax was primarily cleared by hepatic metabolism (~66% of the administered dose). ~33% of the administered dose was recovered as parent drug and its nitro reduction metabolite M30 in feces, which is likely due to unabsorbed parent drug. Biotransformation of venetoclax in human primarily involves enzymatic oxidat...
Biochemistry Usa, 1998
Transient kinetic methods have been used to analyze the interaction between the Ras-binding domai... more Transient kinetic methods have been used to analyze the interaction between the Ras-binding domain (RBD) of c-Raf-1 and a complex of H-Ras and a GTP analogue. The results obtained show that the binding is a two-step process, with an initial rapid equilibrium step being followed by an isomerization reaction occurring at several hundred per second. The reversal of this step determines the rate constant for dissociation, which is on the order of 10 s-1. The lifetime of the complex is therefore on the order of 50-100 ms, which is much shorter than the lifetime of GTP at the active site of H-Ras as determined by the intrinsic GTPase reaction. This suggests that multiple interactions of a single activated Ras molecule and Raf can occur, the number being limited by the competing interaction with GAP. The GDP complex of H-Ras binds more than 2 orders of magnitude more weakly than the GTP-analogue complex, mainly due to a significant weakening of the initial binding equilibrium reaction in the GDP state, thereby avoiding even short-lived recruitment of Raf to the plasma membrane by the inactive Ras form.
Bioconjugate Chemistry, Jul 1, 2002
Intein-mediated protein ligation is a recently developed method that enables the C-terminal label... more Intein-mediated protein ligation is a recently developed method that enables the C-terminal labeling of proteins. This technique requires a correctly folded intein mutant that is fused to the C-terminus of a target protein to create a thioester, which allows the ligation of a peptide with an N-terminal cysteine (1, 2). Here we describe the establishment of this method for the labeling, under denaturing conditions, of target proteins that are expressed insolubly as intein fusion proteins. A GFPuv fusion protein with the Mycobacterium xenopi gyrA intein was expressed in inclusion bodies in Escherichia coli and initially used as a model protein to verify intein cleavage activity under different refolding conditions. The intein showed activity after refolding in nondenaturing and slightly denaturing conditions. A construct of the same intein with an anti-neutravidin single-chain antibody was also expressed in an insoluble form. The intein-mediated ligation was established for this single chain antibody-intein fusion protein under denaturing conditions in 4 M urea to prevent significant precipitation of the fusion protein during the first refolding step. Under optimized conditions, the single-chain antibody was labeled with a fluorescent peptide and used for antigen screening on a biochip after final refolding. This screening procedure allowed the determination of binding characteristics of the scFv for avidin proteins in a miniaturized format.
Analytical Chemistry, 2003
A new chip-based method to identify protein-protein interactions was developed using the guanine ... more A new chip-based method to identify protein-protein interactions was developed using the guanine nucleotide exchange factor GRF2 and two interacting proteins, Ras and calmodulin, as model proteins. A generic immobilization strategy for FLAG-tagged bait proteins on a protein-repellent streptavidin chip surface was implemented by presentation of an oriented anti-FLAG antibody. A flow cell device, integrating different chip surfaces, was developed, and the interaction of immobilized GRF2 with the two analytes was verified by fluorescence assays. On-chip tryptic digest assays were then performed on the capture surface and analyzed by microLC-MS/MS. The interaction of GRF2 with calmodulin and Ras was demonstrated, and the lower limit of detection was determined. We also implemented an on-chip immunoprecipitation assay to identify GRF2-binding partners from complex protein mixtures. Cells overexpressing FLAG-GRF2 were lysed and then incubated with the anti-FLAG chip. In addition to detecting GRF2, we also identified calmodulin, demonstrating that this technique can successfully identify endogenous levels of proteins, bound to recombinant bait proteins. This chip-based method has the advantage that no subsequent gel separations of protein complexes prior to LC-MS analysis are required and is therefore amenable to miniaturized high-throughput determination of protein-protein interactions.
Proteome science, Jan 10, 2003
The completion of the human genome sequence has led to a rapid increase in genetic information. T... more The completion of the human genome sequence has led to a rapid increase in genetic information. The invention of DNA microarrays, which allow for the parallel measurement of thousands of genes on the level of mRNA, has enabled scientists to take a more global view of biological systems. Protein microarrays have a big potential to increase the throughput of proteomic research. Microarrays of antibodies can simultaneously measure the concentration of a multitude of target proteins in a very short period of time. The ability of protein microarrays to increase the quantity of data points in small biological samples on the protein level will have a major impact on basic biological research as well as on the discovery of new drug targets and diagnostic markers. This review highlights the current status of protein expression profiling arrays, their development, applications and limitations.
Recent evidence suggests that blocking aberrant Hedgehog pathway signaling may be a potential the... more Recent evidence suggests that blocking aberrant Hedgehog pathway signaling may be a potential therapeutic strategy for the treatment of several types of cancer. Cyclopamine, a plant Veratrum alkaloid natural product, is an antagonist of the Hedgehog pathway and was used as a starting point for the development of new Hedgehog pathway antagonists. Herein, we describe the synthesis of D-homo cyclopamine analogs by a sequence of chemoselective cyclopropanation followed by stereoselective acid-catalyzed rearrangement. These D-homo analogs are more stable to acid-catalyzed degradation than the natural product. Further modifications of the A-ring generated three new series of analogs, which were characterized in vitro and evaluated in vivo for biological activity and pharmacokinetic properties. These studies led to the discovery of IPI-926, a novel semi-synthetic cyclopamine analog with improved pharmaceutical properties and potency, and a superior plasma pharmacokinetic profile relative t...
Encyclopedia of Molecular Cell Biology and Molecular Medicine, 2006
ABSTRACT Protein microarrays allow for the parallel analysis of a large number of proteins with r... more ABSTRACT Protein microarrays allow for the parallel analysis of a large number of proteins with respect to their abundance or activity and, therefore, are playing an emerging role in proteome research.Protein expression profiling, detection of enzymatic activity and protein interaction mapping are the most important applications of protein microarrays. The biological content, as well as the detection strategies, vary significantly between these applications, making the protein microarray technologies much more varied than those used with DNA microarrays.In most cases, the biological content on the microarray, as well as the analytes, are proteins, which, due to their delicate nature and the heterogeneity of their properties, complicate the manufacturing and assay design. The potential advantages of protein microarrays over nonparallel, macroscopic technologies cannot, however, be overestimated.Keywords:Capture Agents;Enzyme Activity Profiling;Multiplexed Protein Assay;Protein Expression Profiling;Protein Interaction Profiling;Protein Microarray
European Journal of Cancer Supplements, 2008
Xenobiotica, 2013
1. IPI-926 is a novel semisynthetic cyclopamine derivative that is a potent and selective Smoothe... more 1. IPI-926 is a novel semisynthetic cyclopamine derivative that is a potent and selective Smoothened inhibitor that blocks the hedgehog signal transduction pathway. 2. The in vivo clearance of IPI-926 is low in mouse and dog and moderate in monkey. The volume of distribution is high across species. Oral bioavailability ranges from moderate in monkey to high in mouse and dog. Predicted human clearance using simple allometry is low (24 L h(-1)), predicted volume of distribution is high (469 L) and predicted half-life is long (20 h). 3. IPI-926 is highly bound to plasma proteins and has minimal interaction with human α-1-acid glycoprotein. 4. In vitro metabolic stability ranges from stable to moderately stable. Twelve oxidative metabolites were detected in mouse, rat, dog, monkey and human liver microsome incubations and none were unique to human. 5. IPI-926 is not a potent reversible inhibitor of CYP1A2, 2C8, 2C9 or 3A4 (testosterone). IPI-926 is a moderate inhibitor of CYP2C19, 2D6 and 3A4 (midazolam) with KI values of 19, 16 and 4.5 µM, respectively. IPI-926 is both a substrate and inhibitor (IC50 = 1.9 µM) of P-glycoprotein. 6. In summary, IPI-926 has desirable pre-clinical absorption, distribution, metabolism and excretion properties.
Proceedings of the National Academy of Sciences, 1999
The Ras-binding domain (RBD) of c-Raf-1 has been synthesized chemically, taking advantage of the ... more The Ras-binding domain (RBD) of c-Raf-1 has been synthesized chemically, taking advantage of the chemical ligation of two peptide fragments of the protein. This procedure allowed incorporation of an unnatural amino acid (N 1 -methyl-7-azatryptophan) at position 91 of RBD, producing a protein with f luorescent properties distinct from and distinguishable from those of proteins containing the natural f luorophore tryptophan. The resulting protein was shown to interact with Ras in a manner that was almost indistinguishable from that of unmodified RBD based on transient kinetic monitoring of the binding event. Modified RBD containing the L-isomer of the unnatural amino acid or its racemic D,L mixture appeared to interact identically with Ras. The approach demonstrates a general procedure for the introduction of unnatural amino acids that can be used for monitoring protein-protein interactions and for the introduction of an unnatural backbone structure at strategic positions.
Proceedings of the National Academy of Sciences, 2006
analyzed data; and J.R.S. wrote the paper. The authors declare no conflict of interest. Freely av... more analyzed data; and J.R.S. wrote the paper. The authors declare no conflict of interest. Freely available online through the PNAS open access option. Abbreviations: Hsp90, heat shock protein 90; MM, multiple myeloma; IPI-504, 17allylamino-17-demethoxygeldanamycin hydroquinone hydrochloride; NQO1, NADPHquinone oxidoreductase 1; 17-AGG, 17-allylamino-17-demethoxygeldanamycin; 17-AG, 17-(amino)-17-demethoxygeldanamycin; LC-MS͞MS, liquid chromatography-tandem mass spectrometry.
Journal of Medicinal Chemistry, 2008
Herein is reported the synthesis of a novel class of hedgehog antagonists derived from cyclopamin... more Herein is reported the synthesis of a novel class of hedgehog antagonists derived from cyclopamine. The acid sensitive D-ring of cyclopamine was homologated utilizing a sequence of chemoselective cyclopropanation and stereoselective acid-catalyzed rearrangement. Further modification of the A/B-ring homoallylic alcohol to the conjugated ketone led to the discovery of new cyclopamine analogues with improved pharmaceutical properties and in vitro potency (EC 50) ranging from 10 to 1000 nM.
Journal of Medicinal Chemistry, 2009
Recent evidence suggests that blocking aberrant hedgehog pathway signaling may be a promising the... more Recent evidence suggests that blocking aberrant hedgehog pathway signaling may be a promising therapeutic strategy for the treatment of several types of cancer. Cyclopamine, a plant Veratrum alkaloid, is a natural product antagonist of the hedgehog pathway. In a previous report, a seven-membered D-ring semisynthetic analogue of cyclopamine, IPI-269609 (2), was shown to have greater acid stability and better aqueous solubility compared to cyclopamine. Further modifications of the A-ring system generated three series of analogues with improved potency and/or solubility. Lead compounds from each series were characterized in vitro and evaluated in vivo for biological activity and pharmacokinetic properties. These studies led to the discovery of IPI-926 (compound 28), a novel semisynthetic cyclopamine analogue with substantially improved pharmaceutical properties and potency and a favorable pharmacokinetic profile relative to cyclopamine and compound 2. As a result, complete tumor regression was observed in a Hh-dependent medulloblastoma allograft model after daily oral administration of 40 mg/kg of compound 28.
FEBS Letters, 1999
It has previously been shown that the transient kinetics of the interaction between the Ras-bindi... more It has previously been shown that the transient kinetics of the interaction between the Ras-binding domain of c-Raf-1 and the proto-oncoprotein Ras can be followed by stopped-flow measurements using the 2',3'-(N-methylanthraniloyl) fluorescence of 2',3'-(N-methylanthraniloyl) guanyl-5'-yl-imidodiphosphate-labelled Ras. In continuation of this work, we demonstrate that the His-tagged Ras-binding domain of c-Raf-1 can also be synthesized in a cell-free expression system. After purification by Ni2+ affinity chromatography, His-tagged Ras-binding domain of c-Raf-1 could be isolated in sufficient amounts for biochemical and biophysical investigations. The results obtained describe the first example of a cell-free synthesized protein which has been used for stopped-flow measurements to determine the transient kinetics of protein-protein interactions with an effector.
FEBS Letters, 2000
Sensory rhodopsin II (pSRII), the photophobic receptor from Natronobacterium pharaonis, has been ... more Sensory rhodopsin II (pSRII), the photophobic receptor from Natronobacterium pharaonis, has been studied by time-resolved resonance Raman (RR) spectroscopy using the rotating cell technique. Upon excitation with low laser power, the RR spectra largely reflect the parent state pSRII(500) whereas an increase of the laser power leads to a substantial accumulation of long-lived intermediates contributing to the RR spectra. All RR spectra could consistently be analysed in terms of four component spectra which were assigned to the parent state pSRII(500) and the long-lived intermediates M(400), N(485) and O(535) based on the correlation between the C = C stretching frequency and the absorption maximum. The parent state and the intermediates N(485) and O(535) exhibit a protonated Schiff base. The C = N stretching frequencies and the H/D isotopic shifts indicate strong hydrogen bonding interactions of the Schiff base in pSRII(500) and O(535) whereas these interactions are most likely very weak in N(485).
Biophysical Journal, 1998
The photocycle of the photophobic receptor sensory rhodopsin II from N. pharaonis was analyzed by... more The photocycle of the photophobic receptor sensory rhodopsin II from N. pharaonis was analyzed by varying measuring wavelengths, temperature, and pH, and by exchanging H 2 O with D 2 O. The data can be satisfactorily modeled by eight exponents over the whole range of modified parameters. The kinetic data support a model similar to that of bacteriorhodopsin (BR) if a scheme of irreversible first-order reactions is assumed. Eight kinetically distinct protein states can then be identified. These states are formed from five spectrally distinct species. The chromophore states S i correspond in their spectral properties to those of the BR photocycle, namely pSRII 510 (K), pSRII 495 (L), pSRII 400 (M), pSRII 485 (N), and pSRII 535 (O). In comparison to BR, pSRII 400 is formed ϳ10 times faster than the M state; however, the back-reaction is almost 100 times slower. Comparison of the temperature dependence of the rate constants with those from the BR photocycle suggests that the differences are caused by changes of ⌬S. The rate constants of the pSRII photocycle are almost insensitive to the pH variation from 9.0 to 5.5, and show only a small H 2 O/D 2 O effect. This analysis supports the idea that the conformational dynamics of pSRII controls the kinetics of the photocycle of pSRII.
Bioconjugate Chemistry, 2002
Intein-mediated protein ligation is a recently developed method that enables the C-terminal label... more Intein-mediated protein ligation is a recently developed method that enables the C-terminal labeling of proteins. This technique requires a correctly folded intein mutant that is fused to the C-terminus of a target protein to create a thioester, which allows the ligation of a peptide with an N-terminal cysteine (1, 2). Here we describe the establishment of this method for the labeling, under denaturing conditions, of target proteins that are expressed insolubly as intein fusion proteins. A GFPuv fusion protein with the Mycobacterium xenopi gyrA intein was expressed in inclusion bodies in Escherichia coli and initially used as a model protein to verify intein cleavage activity under different refolding conditions. The intein showed activity after refolding in nondenaturing and slightly denaturing conditions. A construct of the same intein with an anti-neutravidin single-chain antibody was also expressed in an insoluble form. The intein-mediated ligation was established for this single chain antibody-intein fusion protein under denaturing conditions in 4 M urea to prevent significant precipitation of the fusion protein during the first refolding step. Under optimized conditions, the single-chain antibody was labeled with a fluorescent peptide and used for antigen screening on a biochip after final refolding. This screening procedure allowed the determination of binding characteristics of the scFv for avidin proteins in a miniaturized format.
Biochemistry, 1998
Transient kinetic methods have been used to analyze the interaction between the Ras-binding domai... more Transient kinetic methods have been used to analyze the interaction between the Ras-binding domain (RBD) of c-Raf-1 and a complex of H-Ras and a GTP analogue. The results obtained show that the binding is a two-step process, with an initial rapid equilibrium step being followed by an isomerization reaction occurring at several hundred per second. The reversal of this step determines the rate constant for dissociation, which is on the order of 10 s-1. The lifetime of the complex is therefore on the order of 50-100 ms, which is much shorter than the lifetime of GTP at the active site of H-Ras as determined by the intrinsic GTPase reaction. This suggests that multiple interactions of a single activated Ras molecule and Raf can occur, the number being limited by the competing interaction with GAP. The GDP complex of H-Ras binds more than 2 orders of magnitude more weakly than the GTP-analogue complex, mainly due to a significant weakening of the initial binding equilibrium reaction in the GDP state, thereby avoiding even short-lived recruitment of Raf to the plasma membrane by the inactive Ras form.