Supplementary Table S1: Evaluation of M3814 (MSC2490484) in kinase assays Supplementary Table S2:... more Supplementary Table S1: Evaluation of M3814 (MSC2490484) in kinase assays Supplementary Table S2: In vitro activity of M3814 in combination with a single dose of gamma radiation in a panel of 93 cell lines. Supplementary Table S3: Pharmacokinetic data for M3814 Supplementary Figure S1: 3 Gy IR - M3814 combination profiling: Bliss data Supplementary Figure S2: 70 Drug - M3814 combination profiling: Bliss data Supplementary Figure S3: Concentration of M3814 in vitro, following administration of M3814 intravenously (0.2 mg/kg) or orally (0.5 mg/kg) Supplementary Figure S4: Additional 1-week IR combination efficacy data (A549, BxPC3, Capan-1, HCT-116) Supplementary Figure S5: Scheduling of M3814 relative to IR Supplementary Figure S6: Body weight curves from efficacy studies
Supplementary Table from A New Class of Selective ATM Inhibitors as Combination Partners of DNA D... more Supplementary Table from A New Class of Selective ATM Inhibitors as Combination Partners of DNA Double-Strand Break Inducing Cancer Therapies
PDF file - 55K, Effective reduction of c-Met phosphorylation by EMD 1214063 in KP-4 pancreatic ca... more PDF file - 55K, Effective reduction of c-Met phosphorylation by EMD 1214063 in KP-4 pancreatic carcinoma tumors. To assess the pharmacodynamic effect of EMD 1214063 on pancreatic carcinoma, KP-4 tumors were obtained ex vivo at the indicated time points after treatment with 5 mg/kg EMD 1214063, lysed, and subsequently analyzed for the levels of total c-Met and c-Met phosphorylation at the Y1234/Y1235 auto-phosphorylation site and at the Y1349 adaptor binding site by a Luminex-based assay. Phosphorylation of both Y1234/Y1235 and Y1349 sites were markedly reduced between 2-6 h after treatment, in the absence of any significant changes in the levels of total c-Met. Data are representative of two separate experiments.
PDF file - 54K, Dose-dependent induction of caspase-3 cleavage by EMD 1204831. Mice bearing estab... more PDF file - 54K, Dose-dependent induction of caspase-3 cleavage by EMD 1204831. Mice bearing established (100-200mm3) Hs746T tumors received a single dose of 3, 10, 30, and 100 mg/kg of EMD 1204831. The effects on caspase-3 activation were determined by Western blot analyses, using an antibody (clone 5A1E) specifically recognizing the large fragment (17/19 kDa) of activated caspase-3 resulting from cleavage adjacent to Asp 175. This antibody does not recognize full-length caspase-3 or other cleaved caspases. Treatment with EMD 1204831 resulted in a transient increase in cleaved caspase-3 that peaked at 24 h and declined at 48 h. Data are representative of three separate experiments.
PDF file - 255K, Effective inhibition of in vitro tumor cell migration by EMD 1214063 and EMD 120... more PDF file - 255K, Effective inhibition of in vitro tumor cell migration by EMD 1214063 and EMD 1204831. A classical "wound healing" test was performed to assess the impact of EMD 1214063 (A) and EMD 1204831 (B) on tumor cell migration. In brief, H441 lung cancer cells were seeded on day 1 (3 x 105 cells/well in 24-well plate), serum-starved on day 2, and treated on day 3 with the indicated concentrations of the EMD compounds for 1 h. After "scratching" the cell layer, cells were incubated in serum-free medium containing 100 ng/mL HGF and the indicated concentrations of the compounds for 24h. Controls were untreated cells with (left) or without HGF (right). At 0 h and 24 h, cells were washed, fixed with ice-cold methanol, and stained with crystal violet (0.05% in 50% ethanol). Treatment with EMD 1214063 or EMD 1204831 resulted in a strong inhibition of tumor cell migration, preventing wound healing. Pictures were taken at 5x magnification. Data are representative o...
PDF file - 77K, In vivo tumor growth inhibition by EMD 1214063 and EMD 1204831. The effect of EMD... more PDF file - 77K, In vivo tumor growth inhibition by EMD 1214063 and EMD 1204831. The effect of EMD 1214063 and EMD 1204831 on tumor growth (T/C) was evaluated in in vivo xenograft models, using a panel of human tumor cell lines. Both inhibitors were highly active on tumor models with different types of aberrant c-Met upregulation, that (i) harbor MET gene amplification, (ii) express high levels of c-Met, or (iii) exhibit an HGF/c-Met autocrine loop, while inactive in models without signs of active c-Met signaling.
PDF file - 184K, EMD 1214063 and EMD 1204831 display anti-tumor activity in HGF-dependent and HGF... more PDF file - 184K, EMD 1214063 and EMD 1204831 display anti-tumor activity in HGF-dependent and HGF-independent xenograft tumor models. Mice bearing subcutaneous tumors derived from the human gastric Hs746T cell line (A and B) or the glioblastoma U87MG cell line (C and D) were administered daily the indicated doses of EMD 1214063 (A and C), EMD 1204831 (B and D), or vehicle for the indicated treatment duration. Each data point represents the mean � SD of the calculated tumor volumes observed in experimental groups including ten mice. Statistical significance was evaluated by one-way ANOVA (Kruskal-Wallis, Dunn's post test). Asterisks indicate statistically significant differences between treated and control experimental groups (P≤0.05).
PDF file - 74K, Effective inhibition of c-Met phosphorylation in U87MG cells. To assess the pharm... more PDF file - 74K, Effective inhibition of c-Met phosphorylation in U87MG cells. To assess the pharmacodynamic activity of EMD 1214063 and EMD 1204831 in glioblastoma, U87MG glioblastoma cells were exposed for 2 h to either EMD compound. After lysis, the phosphorylation levels of c-Met Y1234/Y1235 auto-phosphorylation site (A) and of c-Met Y1349 adaptor binding site (B) were assessed and correlated with the levels of total c-Met by a Luminex-based assay. Y1234/Y1235 EC50 values for EMD 1214063 and EMD 1204831 were 0.003 �M and 0.009 �M, respectively. For Y1349, the EC50 values were 0.011 �M and 0.005 �M for EMD 1214063 and EMD 1204831, respectively. While a dose-dependent reduction in the levels of c-Met phosphorylation could be observed upon exposure to both compounds, the levels of total c-Met remained unchanged (C). Data are cumulative of three independent experiments.
Purpose: The mesenchymal–epithelial transition factor (c-Met) receptor, also known as hepatocyte ... more Purpose: The mesenchymal–epithelial transition factor (c-Met) receptor, also known as hepatocyte growth factor receptor (HGFR), controls morphogenesis, a process that is physiologically required for embryonic development and tissue repair. Aberrant c-Met activation is associated with a variety of human malignancies including cancers of the lung, kidney, stomach, liver, and brain. In this study, we investigated the properties of two novel compounds developed to selectively inhibit the c-Met receptor in antitumor therapeutic interventions.Experimental Design: The pharmacologic properties, c-Met inhibitory activity, and antitumor effects of EMD 1214063 and EMD 1204831 were investigated in vitro and in vivo, using human cancer cell lines and mouse xenograft models.Results: EMD 1214063 and EMD 1204831 selectively suppressed the c-Met receptor tyrosine kinase activity. Their inhibitory activity was potent [inhibitory 50% concentration (IC50), 3 nmol/L and 9 nmol/L, respectively] and highl...
PDF file - 263K, Effects of EMD 1214063 on c-Met phosphorylation and downstream signaling molecul... more PDF file - 263K, Effects of EMD 1214063 on c-Met phosphorylation and downstream signaling molecules (see Figure 2C). EBC-1 cells were cultured in vitro in control medium (medium + vehicle) (lane 1), or in the presence of increasing concentrations of EMD 1214063 (A-D) or EMD 1204831(E-H) (0.01nM to 30�M, as depicted in lanes 2-15). Upon cell lysis, the levels of total c-Met, AKT, p42/44 MAPK (A, E), Gab1 (C, G), as well as phospho-c-Met, phospho-Akt, phospho-p42/44 MAPK (B, F), and phospho-Gab1 (D, H) were assessed by Western blot analyses. The levels of cofilin were used to ascertain equal loading (A, B, and F). Marked reductions in the levels of c-Met phosphorylation were observed for EMD 1214063 and EMD 1204831 at concentrations ≥0.1 �M (lanes 10-15). Data are representative of two independent experiments; cumulative results are shown in Fig. 2C.
PDF file - 53K, Characteristics of the antibodies used in the study. The specificity, isotype, cl... more PDF file - 53K, Characteristics of the antibodies used in the study. The specificity, isotype, clone number, and commercial source of the antibodies used throughout the study are indicated. All antibodies were used according to the manufacturer's instructions.
The protein kinase ataxia telangiectasia- mutated and Rad3-related (ATR) is an important componen... more The protein kinase ataxia telangiectasia- mutated and Rad3-related (ATR) is an important component of the DNA Damage Response (DDR) and a key mediator of the replication stress response (RSR). ATR is recruited to and activated by single-stranded DNA, which commonly forms as a consequence of replication stress (RS). ATR activation and signaling coordinates a multifaceted response to RS, however if left unresolved, replication forks may collapse, form double-strand breaks (DSB), lead to chromosomal rearrangements and eventually cell death. Several chemotherapeutic agents act by increasing RS in tumor cells, by directly damaging the DNA and/or depleting cellular resources required for DNA replication. Increased RS may also result from activation of oncogenes that drive dysregulated replication, a hypoxic environment, or from defects in other repair pathways even in the absence of exogenous DNA damaging insults. Inhibition of ATR activity in cancer cells disables the RSR, potentially en...
Radiotherapy and chemical DNA-damaging agents are among the most widely used classes of cancer th... more Radiotherapy and chemical DNA-damaging agents are among the most widely used classes of cancer therapeutics today. Double-strand breaks (DSB) induced by many of these treatments are lethal to cancer cells if left unrepaired. Ataxia telangiectasia-mutated (ATM) kinase plays a key role in the DNA damage response by driving DSB repair and cell-cycle checkpoints to protect cancer cells. Inhibitors of ATM catalytic activity have been shown to suppress DSB DNA repair, block checkpoint controls and enhance the therapeutic effect of radiotherapy and other DSB-inducing modalities. Here, we describe the pharmacological activities of two highly potent and selective ATM inhibitors from a new chemical class, M3541 and M4076. In biochemical assays, they inhibited ATM kinase activity with a sub-nanomolar potency and showed remarkable selectivity against other protein kinases. In cancer cells, the ATM inhibitors suppressed DSB repair, clonogenic cancer cell growth, and potentiated antitumor activit...
NIMA-related kinase 1 (Nek1) has lately garnered attention for its widespread function in cilioge... more NIMA-related kinase 1 (Nek1) has lately garnered attention for its widespread function in ciliogenesis, apoptosis, and the DNA-damage response. Despite its involvement in various diseases and its potential as a cancer drug target, no directed medicinal chemistry efforts toward inhibitors against this dark kinase are published. Here, we report the structure-guided design of a potent small-molecule Nek1 inhibitor, starting from a scaffold identified by kinase cross-screening analysis. Seven lead compounds were identified in silico and evaluated for their inhibitory activity. The top compound, 10f, was further profiled for efficacy, toxicity, and bioavailability in a zebrafish polycystic kidney disease model. Administration of 10f caused the expansion of fluorescence-labeled proximal convoluted tubules, supporting our hypothesis that Nek1-inhibition causes cystic kidneys in zebrafish embryos. Compound 10f displayed insignificant inhibition in 48 of 50 kinases in a selectivity test panel. The findings provide a powerful tool to further elucidate the function and pharmacology of this neglected kinase.
Physical and chemical DNA-damaging agents are used widely in the treatment of cancer. Double-stra... more Physical and chemical DNA-damaging agents are used widely in the treatment of cancer. Double-strand break (DSB) lesions in DNA are the most deleterious form of damage and, if left unrepaired, can effectively kill cancer cells. DNA-dependent protein kinase (DNA-PK) is a critical component of nonhomologous end joining (NHEJ), one of the two major pathways for DSB repair. Although DNA-PK has been considered an attractive target for cancer therapy, the development of pharmacologic DNA-PK inhibitors for clinical use has been lagging. Here, we report the discovery and characterization of a potent, selective, and orally bioavailable DNA-PK inhibitor, M3814 (peposertib), and provide in vivo proof of principle for DNA-PK inhibition as a novel approach to combination radiotherapy. M3814 potently inhibits DNA-PK catalytic activity and sensitizes multiple cancer cell lines to ionizing radiation (IR) and DSB-inducing agents. Inhibition of DNA-PK autophosphorylation in cancer cells or xenograft t...
Mass spectrometry (MS) is known for its label-free detection of substrates and products from a va... more Mass spectrometry (MS) is known for its label-free detection of substrates and products from a variety of enzyme reactions. Recent hardware improvements have increased interest in the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS for high-throughput drug discovery. Despite interest in this technology, several challenges remain and must be overcome before MALDI-MS can be integrated as an automated “in-line reader” for high-throughput drug discovery. Two such hurdles include in situ sample processing and deposition, as well as integration of MALDI-MS for enzymatic screening assays that usually contain high levels of MS-incompatible components. Here we adapt our c-MET kinase assay to optimize for MALDI-MS compatibility and test its feasibility for compound screening. The pros and cons of the Echo (Labcyte) as a transfer system for in situ MALDI-MS sample preparation are discussed. We demonstrate that this method generates robust data in a 1536-grid fo...
Physical or chemical agents that damage DNA such as ionizing radiation are among the most widely ... more Physical or chemical agents that damage DNA such as ionizing radiation are among the most widely used classes of cancer therapeutics today. Double strand breaks (DSB) generated in DNA by radiation induce multitude of cellular responses, including DNA repair, cell cycle arrest or cell death if the damage is left unrepaired. A complex set of molecular events are responsible for DNA repair via two major mechanisms - homologous recombination (HR) or non-homologous end joining (NHEJ). DNA-PKcs with its regulatory protein subunits, Ku70 and Ku80, is an integral component of NHEJ and considered an attractive intervention point to inhibit DNA repair. We have developed an orally bioavailable, highly potent, and selective inhibitor of DNA-PK, M3814, for cancer therapy in combination with DNA damaging modalities such as radiation, and radio-chemotherapy. Here, we present the preclinical characterization of M3814 using biochemical, cellular and human tumor xenograft models. M3814 sensitized mul...
Interleukin-6 (IL-6) is regarded as an endogenous mediator of lipopolysaccharide (LPS)-induced fe... more Interleukin-6 (IL-6) is regarded as an endogenous mediator of lipopolysaccharide (LPS)-induced fever. IL-6 is thought to act on the brain at sites that lack a blood-brain barrier, the circumventricular organs (CVOs). Cells that are activated by IL-6 respond with nuclear translocation of the signal transducer and activator of transcription 3 molecule (STAT3) and can be detected by immunohistochemistry. We investigated whether the LPS-induced release of IL-6 into the systemic circulation was accompanied by a nuclear STAT3 translocation within the sensory CVOs. Treatment with LPS (100 μg/kg) led to a slight (1 h) and then a strong increase (2–8 h) in plasma IL-6 levels, which started to decline at the end of the febrile response. Administration of both pyrogens LPS and IL-6 (45 μg/kg) induced a febrile response with IL-6, causing a rather moderate fever compared with the LPS-induced fever. Nuclear STAT3 translocation in response to LPS was observed within the vascular organ of the lami...
Journal of applied physiology (Bethesda, Md. : 1985), 2003
In guinea pigs, dose-dependent febrile responses can be induced by injection of a high (100 micro... more In guinea pigs, dose-dependent febrile responses can be induced by injection of a high (100 micro g/kg) or low (10 micro g/kg) dose of bacterial lipopolysaccharide (LPS) into artificial subcutaneously implanted Teflon chambers. In this fever model, LPS does not enter the systemic circulation from the site of localized tissue inflammation in considerable amounts but causes a local induction of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6), which can be measured in lavage fluid collected from the chamber area. Only in response to the high LPS dose, small traces of TNF are measurable in blood plasma. A moderate increase of circulating IL-6 occurs in response to administration of both LPS doses. To investigate the putative roles of TNF and prostaglandins in this fever model, a neutralizing TNF binding protein (TNF-bp) or a nonselective inhibitor of cyclooxygenases (diclofenac) was injected along with the high or low dose of LPS into the subcutaneous ...
Supplementary Table S1: Evaluation of M3814 (MSC2490484) in kinase assays Supplementary Table S2:... more Supplementary Table S1: Evaluation of M3814 (MSC2490484) in kinase assays Supplementary Table S2: In vitro activity of M3814 in combination with a single dose of gamma radiation in a panel of 93 cell lines. Supplementary Table S3: Pharmacokinetic data for M3814 Supplementary Figure S1: 3 Gy IR - M3814 combination profiling: Bliss data Supplementary Figure S2: 70 Drug - M3814 combination profiling: Bliss data Supplementary Figure S3: Concentration of M3814 in vitro, following administration of M3814 intravenously (0.2 mg/kg) or orally (0.5 mg/kg) Supplementary Figure S4: Additional 1-week IR combination efficacy data (A549, BxPC3, Capan-1, HCT-116) Supplementary Figure S5: Scheduling of M3814 relative to IR Supplementary Figure S6: Body weight curves from efficacy studies
Supplementary Table from A New Class of Selective ATM Inhibitors as Combination Partners of DNA D... more Supplementary Table from A New Class of Selective ATM Inhibitors as Combination Partners of DNA Double-Strand Break Inducing Cancer Therapies
PDF file - 55K, Effective reduction of c-Met phosphorylation by EMD 1214063 in KP-4 pancreatic ca... more PDF file - 55K, Effective reduction of c-Met phosphorylation by EMD 1214063 in KP-4 pancreatic carcinoma tumors. To assess the pharmacodynamic effect of EMD 1214063 on pancreatic carcinoma, KP-4 tumors were obtained ex vivo at the indicated time points after treatment with 5 mg/kg EMD 1214063, lysed, and subsequently analyzed for the levels of total c-Met and c-Met phosphorylation at the Y1234/Y1235 auto-phosphorylation site and at the Y1349 adaptor binding site by a Luminex-based assay. Phosphorylation of both Y1234/Y1235 and Y1349 sites were markedly reduced between 2-6 h after treatment, in the absence of any significant changes in the levels of total c-Met. Data are representative of two separate experiments.
PDF file - 54K, Dose-dependent induction of caspase-3 cleavage by EMD 1204831. Mice bearing estab... more PDF file - 54K, Dose-dependent induction of caspase-3 cleavage by EMD 1204831. Mice bearing established (100-200mm3) Hs746T tumors received a single dose of 3, 10, 30, and 100 mg/kg of EMD 1204831. The effects on caspase-3 activation were determined by Western blot analyses, using an antibody (clone 5A1E) specifically recognizing the large fragment (17/19 kDa) of activated caspase-3 resulting from cleavage adjacent to Asp 175. This antibody does not recognize full-length caspase-3 or other cleaved caspases. Treatment with EMD 1204831 resulted in a transient increase in cleaved caspase-3 that peaked at 24 h and declined at 48 h. Data are representative of three separate experiments.
PDF file - 255K, Effective inhibition of in vitro tumor cell migration by EMD 1214063 and EMD 120... more PDF file - 255K, Effective inhibition of in vitro tumor cell migration by EMD 1214063 and EMD 1204831. A classical "wound healing" test was performed to assess the impact of EMD 1214063 (A) and EMD 1204831 (B) on tumor cell migration. In brief, H441 lung cancer cells were seeded on day 1 (3 x 105 cells/well in 24-well plate), serum-starved on day 2, and treated on day 3 with the indicated concentrations of the EMD compounds for 1 h. After "scratching" the cell layer, cells were incubated in serum-free medium containing 100 ng/mL HGF and the indicated concentrations of the compounds for 24h. Controls were untreated cells with (left) or without HGF (right). At 0 h and 24 h, cells were washed, fixed with ice-cold methanol, and stained with crystal violet (0.05% in 50% ethanol). Treatment with EMD 1214063 or EMD 1204831 resulted in a strong inhibition of tumor cell migration, preventing wound healing. Pictures were taken at 5x magnification. Data are representative o...
PDF file - 77K, In vivo tumor growth inhibition by EMD 1214063 and EMD 1204831. The effect of EMD... more PDF file - 77K, In vivo tumor growth inhibition by EMD 1214063 and EMD 1204831. The effect of EMD 1214063 and EMD 1204831 on tumor growth (T/C) was evaluated in in vivo xenograft models, using a panel of human tumor cell lines. Both inhibitors were highly active on tumor models with different types of aberrant c-Met upregulation, that (i) harbor MET gene amplification, (ii) express high levels of c-Met, or (iii) exhibit an HGF/c-Met autocrine loop, while inactive in models without signs of active c-Met signaling.
PDF file - 184K, EMD 1214063 and EMD 1204831 display anti-tumor activity in HGF-dependent and HGF... more PDF file - 184K, EMD 1214063 and EMD 1204831 display anti-tumor activity in HGF-dependent and HGF-independent xenograft tumor models. Mice bearing subcutaneous tumors derived from the human gastric Hs746T cell line (A and B) or the glioblastoma U87MG cell line (C and D) were administered daily the indicated doses of EMD 1214063 (A and C), EMD 1204831 (B and D), or vehicle for the indicated treatment duration. Each data point represents the mean � SD of the calculated tumor volumes observed in experimental groups including ten mice. Statistical significance was evaluated by one-way ANOVA (Kruskal-Wallis, Dunn's post test). Asterisks indicate statistically significant differences between treated and control experimental groups (P≤0.05).
PDF file - 74K, Effective inhibition of c-Met phosphorylation in U87MG cells. To assess the pharm... more PDF file - 74K, Effective inhibition of c-Met phosphorylation in U87MG cells. To assess the pharmacodynamic activity of EMD 1214063 and EMD 1204831 in glioblastoma, U87MG glioblastoma cells were exposed for 2 h to either EMD compound. After lysis, the phosphorylation levels of c-Met Y1234/Y1235 auto-phosphorylation site (A) and of c-Met Y1349 adaptor binding site (B) were assessed and correlated with the levels of total c-Met by a Luminex-based assay. Y1234/Y1235 EC50 values for EMD 1214063 and EMD 1204831 were 0.003 �M and 0.009 �M, respectively. For Y1349, the EC50 values were 0.011 �M and 0.005 �M for EMD 1214063 and EMD 1204831, respectively. While a dose-dependent reduction in the levels of c-Met phosphorylation could be observed upon exposure to both compounds, the levels of total c-Met remained unchanged (C). Data are cumulative of three independent experiments.
Purpose: The mesenchymal–epithelial transition factor (c-Met) receptor, also known as hepatocyte ... more Purpose: The mesenchymal–epithelial transition factor (c-Met) receptor, also known as hepatocyte growth factor receptor (HGFR), controls morphogenesis, a process that is physiologically required for embryonic development and tissue repair. Aberrant c-Met activation is associated with a variety of human malignancies including cancers of the lung, kidney, stomach, liver, and brain. In this study, we investigated the properties of two novel compounds developed to selectively inhibit the c-Met receptor in antitumor therapeutic interventions.Experimental Design: The pharmacologic properties, c-Met inhibitory activity, and antitumor effects of EMD 1214063 and EMD 1204831 were investigated in vitro and in vivo, using human cancer cell lines and mouse xenograft models.Results: EMD 1214063 and EMD 1204831 selectively suppressed the c-Met receptor tyrosine kinase activity. Their inhibitory activity was potent [inhibitory 50% concentration (IC50), 3 nmol/L and 9 nmol/L, respectively] and highl...
PDF file - 263K, Effects of EMD 1214063 on c-Met phosphorylation and downstream signaling molecul... more PDF file - 263K, Effects of EMD 1214063 on c-Met phosphorylation and downstream signaling molecules (see Figure 2C). EBC-1 cells were cultured in vitro in control medium (medium + vehicle) (lane 1), or in the presence of increasing concentrations of EMD 1214063 (A-D) or EMD 1204831(E-H) (0.01nM to 30�M, as depicted in lanes 2-15). Upon cell lysis, the levels of total c-Met, AKT, p42/44 MAPK (A, E), Gab1 (C, G), as well as phospho-c-Met, phospho-Akt, phospho-p42/44 MAPK (B, F), and phospho-Gab1 (D, H) were assessed by Western blot analyses. The levels of cofilin were used to ascertain equal loading (A, B, and F). Marked reductions in the levels of c-Met phosphorylation were observed for EMD 1214063 and EMD 1204831 at concentrations ≥0.1 �M (lanes 10-15). Data are representative of two independent experiments; cumulative results are shown in Fig. 2C.
PDF file - 53K, Characteristics of the antibodies used in the study. The specificity, isotype, cl... more PDF file - 53K, Characteristics of the antibodies used in the study. The specificity, isotype, clone number, and commercial source of the antibodies used throughout the study are indicated. All antibodies were used according to the manufacturer's instructions.
The protein kinase ataxia telangiectasia- mutated and Rad3-related (ATR) is an important componen... more The protein kinase ataxia telangiectasia- mutated and Rad3-related (ATR) is an important component of the DNA Damage Response (DDR) and a key mediator of the replication stress response (RSR). ATR is recruited to and activated by single-stranded DNA, which commonly forms as a consequence of replication stress (RS). ATR activation and signaling coordinates a multifaceted response to RS, however if left unresolved, replication forks may collapse, form double-strand breaks (DSB), lead to chromosomal rearrangements and eventually cell death. Several chemotherapeutic agents act by increasing RS in tumor cells, by directly damaging the DNA and/or depleting cellular resources required for DNA replication. Increased RS may also result from activation of oncogenes that drive dysregulated replication, a hypoxic environment, or from defects in other repair pathways even in the absence of exogenous DNA damaging insults. Inhibition of ATR activity in cancer cells disables the RSR, potentially en...
Radiotherapy and chemical DNA-damaging agents are among the most widely used classes of cancer th... more Radiotherapy and chemical DNA-damaging agents are among the most widely used classes of cancer therapeutics today. Double-strand breaks (DSB) induced by many of these treatments are lethal to cancer cells if left unrepaired. Ataxia telangiectasia-mutated (ATM) kinase plays a key role in the DNA damage response by driving DSB repair and cell-cycle checkpoints to protect cancer cells. Inhibitors of ATM catalytic activity have been shown to suppress DSB DNA repair, block checkpoint controls and enhance the therapeutic effect of radiotherapy and other DSB-inducing modalities. Here, we describe the pharmacological activities of two highly potent and selective ATM inhibitors from a new chemical class, M3541 and M4076. In biochemical assays, they inhibited ATM kinase activity with a sub-nanomolar potency and showed remarkable selectivity against other protein kinases. In cancer cells, the ATM inhibitors suppressed DSB repair, clonogenic cancer cell growth, and potentiated antitumor activit...
NIMA-related kinase 1 (Nek1) has lately garnered attention for its widespread function in cilioge... more NIMA-related kinase 1 (Nek1) has lately garnered attention for its widespread function in ciliogenesis, apoptosis, and the DNA-damage response. Despite its involvement in various diseases and its potential as a cancer drug target, no directed medicinal chemistry efforts toward inhibitors against this dark kinase are published. Here, we report the structure-guided design of a potent small-molecule Nek1 inhibitor, starting from a scaffold identified by kinase cross-screening analysis. Seven lead compounds were identified in silico and evaluated for their inhibitory activity. The top compound, 10f, was further profiled for efficacy, toxicity, and bioavailability in a zebrafish polycystic kidney disease model. Administration of 10f caused the expansion of fluorescence-labeled proximal convoluted tubules, supporting our hypothesis that Nek1-inhibition causes cystic kidneys in zebrafish embryos. Compound 10f displayed insignificant inhibition in 48 of 50 kinases in a selectivity test panel. The findings provide a powerful tool to further elucidate the function and pharmacology of this neglected kinase.
Physical and chemical DNA-damaging agents are used widely in the treatment of cancer. Double-stra... more Physical and chemical DNA-damaging agents are used widely in the treatment of cancer. Double-strand break (DSB) lesions in DNA are the most deleterious form of damage and, if left unrepaired, can effectively kill cancer cells. DNA-dependent protein kinase (DNA-PK) is a critical component of nonhomologous end joining (NHEJ), one of the two major pathways for DSB repair. Although DNA-PK has been considered an attractive target for cancer therapy, the development of pharmacologic DNA-PK inhibitors for clinical use has been lagging. Here, we report the discovery and characterization of a potent, selective, and orally bioavailable DNA-PK inhibitor, M3814 (peposertib), and provide in vivo proof of principle for DNA-PK inhibition as a novel approach to combination radiotherapy. M3814 potently inhibits DNA-PK catalytic activity and sensitizes multiple cancer cell lines to ionizing radiation (IR) and DSB-inducing agents. Inhibition of DNA-PK autophosphorylation in cancer cells or xenograft t...
Mass spectrometry (MS) is known for its label-free detection of substrates and products from a va... more Mass spectrometry (MS) is known for its label-free detection of substrates and products from a variety of enzyme reactions. Recent hardware improvements have increased interest in the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS for high-throughput drug discovery. Despite interest in this technology, several challenges remain and must be overcome before MALDI-MS can be integrated as an automated “in-line reader” for high-throughput drug discovery. Two such hurdles include in situ sample processing and deposition, as well as integration of MALDI-MS for enzymatic screening assays that usually contain high levels of MS-incompatible components. Here we adapt our c-MET kinase assay to optimize for MALDI-MS compatibility and test its feasibility for compound screening. The pros and cons of the Echo (Labcyte) as a transfer system for in situ MALDI-MS sample preparation are discussed. We demonstrate that this method generates robust data in a 1536-grid fo...
Physical or chemical agents that damage DNA such as ionizing radiation are among the most widely ... more Physical or chemical agents that damage DNA such as ionizing radiation are among the most widely used classes of cancer therapeutics today. Double strand breaks (DSB) generated in DNA by radiation induce multitude of cellular responses, including DNA repair, cell cycle arrest or cell death if the damage is left unrepaired. A complex set of molecular events are responsible for DNA repair via two major mechanisms - homologous recombination (HR) or non-homologous end joining (NHEJ). DNA-PKcs with its regulatory protein subunits, Ku70 and Ku80, is an integral component of NHEJ and considered an attractive intervention point to inhibit DNA repair. We have developed an orally bioavailable, highly potent, and selective inhibitor of DNA-PK, M3814, for cancer therapy in combination with DNA damaging modalities such as radiation, and radio-chemotherapy. Here, we present the preclinical characterization of M3814 using biochemical, cellular and human tumor xenograft models. M3814 sensitized mul...
Interleukin-6 (IL-6) is regarded as an endogenous mediator of lipopolysaccharide (LPS)-induced fe... more Interleukin-6 (IL-6) is regarded as an endogenous mediator of lipopolysaccharide (LPS)-induced fever. IL-6 is thought to act on the brain at sites that lack a blood-brain barrier, the circumventricular organs (CVOs). Cells that are activated by IL-6 respond with nuclear translocation of the signal transducer and activator of transcription 3 molecule (STAT3) and can be detected by immunohistochemistry. We investigated whether the LPS-induced release of IL-6 into the systemic circulation was accompanied by a nuclear STAT3 translocation within the sensory CVOs. Treatment with LPS (100 μg/kg) led to a slight (1 h) and then a strong increase (2–8 h) in plasma IL-6 levels, which started to decline at the end of the febrile response. Administration of both pyrogens LPS and IL-6 (45 μg/kg) induced a febrile response with IL-6, causing a rather moderate fever compared with the LPS-induced fever. Nuclear STAT3 translocation in response to LPS was observed within the vascular organ of the lami...
Journal of applied physiology (Bethesda, Md. : 1985), 2003
In guinea pigs, dose-dependent febrile responses can be induced by injection of a high (100 micro... more In guinea pigs, dose-dependent febrile responses can be induced by injection of a high (100 micro g/kg) or low (10 micro g/kg) dose of bacterial lipopolysaccharide (LPS) into artificial subcutaneously implanted Teflon chambers. In this fever model, LPS does not enter the systemic circulation from the site of localized tissue inflammation in considerable amounts but causes a local induction of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6), which can be measured in lavage fluid collected from the chamber area. Only in response to the high LPS dose, small traces of TNF are measurable in blood plasma. A moderate increase of circulating IL-6 occurs in response to administration of both LPS doses. To investigate the putative roles of TNF and prostaglandins in this fever model, a neutralizing TNF binding protein (TNF-bp) or a nonselective inhibitor of cyclooxygenases (diclofenac) was injected along with the high or low dose of LPS into the subcutaneous ...
Uploads
Papers by Ulrich Pehl