Christian Kersten - Academia.edu (original) (raw)
Papers by Christian Kersten
ABSTRACTWNT/β-catenin signaling endows cancer cells with proliferative capacity and immune-evasiv... more ABSTRACTWNT/β-catenin signaling endows cancer cells with proliferative capacity and immune-evasive functions that impair anti-cancer immunosurveillance by conventional, cytoxtoic T cells. However, the impact of dysregulated WNT signalling on unconventional, tissue-resident T cells, specifically in colon cancer is unknown. Here, we show that cancer cells in Apc-mutant mouse models escape immunosurveillance from gut-resident intraepithelial lymphocytes (IELs) expressing γδ T cell receptors (γδTCRs). Analysis of late-stage tumors from mice and humans revealed that γδIELs are largely absent from the tumor microenvironment, and that butyrophilin-like (BTNL) molecules, which can critically regulate γδIEL through direct γδTCR-interactions, are also downregulated. We could attribute this to β-catenin stabilization, which rapidly decreased expression of the transcription factors, HNF4A and HNF4G, that we found to bind promoter regions of Btnl genes, thereby driving their expression in normal...
Clinical and Translational Oncology, 2021
Purpose A significant percentage of colorectal cancer patients proceeds to metastatic disease. We... more Purpose A significant percentage of colorectal cancer patients proceeds to metastatic disease. We hypothesised that mitochondrial DNA (mtDNA) polymorphisms, generated by the high mtDNA mutation rate of energy-demanding clonal immune cell expansions and assessable in peripheral blood, reflect how efficiently systemic immunity impedes metastasis. Patients and methods We studied 44 rectal cancer patients from a population-based prospective biomarker study, given curative-intent neoadjuvant radiation and radical surgery for high-risk tumour stage and followed for metastatic failure. Blood specimens were sampled at the time of diagnosis and analysed for the full-length mtDNA sequence, composition of immune cell subpopulations and damaged serum mtDNA. Results Whole blood total mtDNA variant number above the median value for the study cohort, coexisting with an mtDNA non-H haplogroup, was representative for the mtDNA of circulating immune cells and associated with low risk of a metastatic ...
Annals of Surgical Oncology, 2020
Background Systemic inflammatory response (SIR) is an adverse prognostic marker in colorectal can... more Background Systemic inflammatory response (SIR) is an adverse prognostic marker in colorectal cancer (CRC) patients. The ScotScan Colorectal Cancer Group was established to examine how markers of the SIR differ between populations and may be utilised to guide prognosis. Patients and Methods Patients undergoing resection of stage I–III CRC from two prospective datasets in Scotland and Norway were included. The relationship between the modified Glasgow Prognostic Score (mGPS; combination of C-reactive protein and albumin) and overall survival (OS) was examined. The relationship between OS, adjuvant chemotherapy regime and mGPS was examined in patients with stage III colon cancer. Results A total of 2295 patients were included. Patients from Scotland were more inflamed despite controlling for associated characteristics using multivariate logistic regression or propensity score matching (OR 2.82, 95% CI 1.98–4.01, p < 0.001). mGPS had similar independent prognostic value in both coho...
British journal of anaesthesia, 2015
Neurobiological work has demonstrated that expression of mitogen-activated protein kinases (MAPK)... more Neurobiological work has demonstrated that expression of mitogen-activated protein kinases (MAPK) is upregulated on neurones and glial cells after nerve damage. Furthermore, the epidermal growth factor receptor (EGFR) has been identified as having a key role in this process and subsequent interruption of this using EGFR-Inhibitors (EGFR-I), may improve neuropathic pain. The aim of this report was to explore if EGFR-I attenuated neuropathic pain in humans. A selection of patients with neuropathic pain were treated off-label with one of four EGFR-Is, approved for the treatment of cancer. All patients had chronic and severe neuropathic pain (as defined by diagnostic criteria). Pain intensity, interference with function, and adverse events were prospectively registered. Twenty patients were treated. Eighteen patients experienced clinically significant pain relief after treatment with EGFR-I. Median observed pain reduction for all patients was 8.5 (IQR=5-9.5) points on a 0-10 numeric rat...
Morphological and clinical heterogeneity of advanced colorectal cancer is probably caused by gene... more Morphological and clinical heterogeneity of advanced colorectal cancer is probably caused by genetic vari- ability in putative cancer stem cell genes, including Lgr5. Here, we investigated 23 variants of the Lgr5 gene in nor- mal tissue, primary tumors, lymph node metastases and distant metastases of stage III and stage IV colorectal can- cer patients. These data were compared to results of immunohistochemical Lgr5 expression analysis and to prognos- tic clinical parameters. No differences were found comparing germline and somatic Lgr5 genotype in primary tumors, but additional Lgr5 gene alterations could be demonstrated in lymph node and distant metastases. Significant nega- tive correlation was seen between Lgr5 allelic variation and Lgr5 protein expression (p=0.0394), which mainly can be attributed to the negative influence of non-coding Lgr5 gene variations on Lgr5 protein expression (p=0.0166). Lgr5 gene variants could be found more frequently in primary tumors of stage III pat...
Annals of Oncology, 2021
Background: Neoadjuvant chemoradiotherapy (NCRT) has been consolidated as the main strategy for t... more Background: Neoadjuvant chemoradiotherapy (NCRT) has been consolidated as the main strategy for the treatment of locally advanced rectal cancer (LARC).However, heterogeneous responses are observed, with only about 15-20% of patients presenting a complete pathological response (pCR).Thus, countless studies evaluate possible biomarkers capable of predicting pCR and more intense neoadjuvant treatments to increase these rates. The objective of this prospective study was to analyze whether the protein expression of RAD23 homolog B (RAD23B) in the circulating tumor cells (CTCs) (at baseline-C1) could correlate with the response to NCRT. Methods: Between 2016 and 2020, 63 patients (pts) with LARC who underwent NCRT followed by radical surgery, were included in the study. Blood samples were collected before the beginning of NCRT (C1) and the evaluation of RAD23B protein expression in CTCs was correlated with the anatomopathological examination of response of patients undergoing surgery (n:56). CTCs were isolated and quantified by ISETÒ. RAD23B protein was analyzed by immunocytochemistry and visualized by bright field microscopy. Results: The mean age was 56 years old (34-92). Among the pts analyzed, 34 (54%) carried tumors at the distal rectum, 57 (90%) had clinical tumor stage (cT) T3/T4, 58 (92%) clinical nodal (cN) positive and 32 (52%) had preoperative carcinoembryonic antigen (CEA) >3 ng/mL Thirteen (23,2%) patients had pCR with NCRT. RAD23B expression in CTCs was present in 54% of non-responders, while in pCR group, it was absent in the majority of pts (91.7%; p¼0.019). In multivariate logistic regression models for pCR, including CEA, gender, cT and RAD23B expression in CTCs, the latter was an important independent prognostic factor [Odds Ratio (OR) 0.064; 95% confidence interval (CI): 0.006 e 0.751; p¼0.029]. Conclusions: This prospective study demonstrated the correlation between the absence of expression of RAD23B in CTCs (C1) and pCR, being an important result for future clinical studies. This analysis may identify NCRT responders candidates, helping to choose the best therapeutic approach for each individual.
Neurobiology of Disease, 2020
• ErbB1 inhibitors reverse hypersensitivity in neuropathic pain models, as clinically • Nocicepto... more • ErbB1 inhibitors reverse hypersensitivity in neuropathic pain models, as clinically • Nociceptors in DRG selectively express ErbB1 and ErbB2 but not ErbB3 or ErbB4 • ErbB1 drives Akt-/AS160-dependent trafficking of nociceptor Na + and Ca 2+ channels • ErbB1-and Akt-inhibitors prevent channel trafficking and hypersensitivity • Dual blockade of the ErbB1/2 signalling hub produces synergistic analgesia
Nucleic Acids Research
RNA methyltransferases (MTases) are ubiquitous enzymes whose hitherto low profile in medicinal ch... more RNA methyltransferases (MTases) are ubiquitous enzymes whose hitherto low profile in medicinal chemistry, contrasts with the surging interest in RNA methylation, the arguably most important aspect of the new field of epitranscriptomics. As MTases become validated as drug targets in all major fields of biomedicine, the development of small molecule compounds as tools and inhibitors is picking up considerable momentum, in academia as well as in biotech. Here we discuss the development of small molecules for two related aspects of chemical biology. Firstly, derivates of the ubiquitous cofactor S-adenosyl-l-methionine (SAM) are being developed as bioconjugation tools for targeted transfer of functional groups and labels to increasingly visible targets. Secondly, SAM-derived compounds are being investigated for their ability to act as inhibitors of RNA MTases. Drug development is moving from derivatives of cosubstrates towards higher generation compounds that may address allosteric sites...
<b>Copyright information:</b>Taken from "BMP-6 inhibits growth of mature human B... more <b>Copyright information:</b>Taken from "BMP-6 inhibits growth of mature human B cells; induction of Smad phosphorylation and upregulation of Id1"BMC Immunology 2005;6():9-9.Published online 9 May 2005PMCID:PMC1134658.Copyright © 2005 Kersten et al; licensee BioMed Central Ltd. CD19B cells were cultured in X-vivo 15 over night before treatment with BMP-6 for the indicated time points and cell lysates were prepared. One representative experiment of three is shown. HeLa cells were used as a positive control for Id1 protein detection.
<b>Copyright information:</b>Taken from "BMP-6 inhibits growth of mature human B... more <b>Copyright information:</b>Taken from "BMP-6 inhibits growth of mature human B cells; induction of Smad phosphorylation and upregulation of Id1"BMC Immunology 2005;6():9-9.Published online 9 May 2005PMCID:PMC1134658.Copyright © 2005 Kersten et al; licensee BioMed Central Ltd. Quantifications of Id1, Id2 and Id3 protein levels were performed using β-actin as normalization and expressed as mean ± SEM (Id1: n = 4, *p ≤ 0.020; Id2 and Id3: n = 3).
HardwareX, 2022
Differential scanning fluorimetry (DSF) is a widely used biophysical technique with applications ... more Differential scanning fluorimetry (DSF) is a widely used biophysical technique with applications to drug discovery and protein biochemistry. DSF experiments are commonly performed in commercial real-time polymerase chain reaction (qPCR) thermal cyclers or nanoDSF instruments. Here, we report the construction, validation, and example applications of an open-source DSF system for 176 €, which, in addition to protein-DSF experiments, also proved to be a versatile biophysical instrument for less conventional RNA-DSF experiments. Using 3D-printed parts made of polyoxymethylene, we were able to fabricate a thermostable machine chassis for protein-melting experiments. The combination of blue high-power LEDs as the light source and stage light foil as filter components was proven to be a reliable and affordable alternative to conventional optics equipment for the detection of SYPRO Orange or Sybr Gold fluorescence. The ESP32 microcontroller is the core piece of this openDSF instrument, while the in-built I 2 S interface was found to be a powerful analog-to-digital converter for fast acquisition of fluorescence and temperature data. Airflow heating and inline temperature control by thermistors enabled highaccuracy temperature management in PCR tubes (±0.1°C) allowing us to perform highresolution thermal shift assays (TSA) from exemplary biological applications.
ACS Chemical Biology, 2021
Viral and parasitic pathogens rely critically on cysteine proteases for host invasion, replicatio... more Viral and parasitic pathogens rely critically on cysteine proteases for host invasion, replication, and infectivity. Their inhibition by synthetic inhibitors, such as vinyl sulfone compounds, has emerged as a promising treatment strategy. However, the individual reaction steps of protease inhibition are not fully understood. Using the trypanosomal cysteine protease rhodesain as a medically relevant target, we design photoinduced electron transfer (PET) fluorescence probes to detect kinetics of binding of reversible and irreversible vinyl sulfones directly in solution. Intriguingly, the irreversible inhibitor, apart from its unlimited residence time in the enzyme, reacts 5 times faster than the reversible one. Results show that the reactivity of the warhead, and not binding of the peptidic recognition unit, limits the rate constant of protease inhibition. The use of a reversible inhibitor decreases the risk of off-target side effects not only by allowing its release from an off-target but also by reducing the rate constant of binding.
Current Medicinal Chemistry, 2022
: Due to its fast international spread and substantial mortality, the coronavirus disease COVID-1... more : Due to its fast international spread and substantial mortality, the coronavirus disease COVID-19 evolved to a global threat. Since there is currently no causative drug against this viral infection available, science is striving for new drugs and other approaches to treat the new disease. Studies have shown that the cell entry of coronaviruses into host cells takes place through the binding of the viral spike (S) protein to cell receptors. Priming of the S protein occurs via hydrolysis by different host proteases. The inhibition of these proteases could impair the processing of the S protein, thereby affecting the interaction with the host-cell receptors and preventing virus cell entry. Hence, inhibition of these proteases could be a promising strategy for treatment against SARSCoV- 2. In this review, we discuss the current state of the art of developing inhibitors against the entry proteases furin, the transmembrane serine protease type-II (TMPRSS2), trypsin, and cathepsin L.
Molecular Informatics, 2020
The analysis of B-factor profiles from X-ray protein structures can be utilized for structure-bas... more The analysis of B-factor profiles from X-ray protein structures can be utilized for structure-based drug design since protein mobility changes have been associated with the quality of protein-ligand interactions. With the BANΔIT (B'-factor analysis and ΔB' interpretation toolkit), we have developed a JavaScript-based browser application that provides a graphical user interface for the normalization and analysis of B'-factor profiles. To emphasize the usability for rational drug design applications, we have analyzed a selection of crystallographic protein-ligand complexes and have given exemplary conclusions for further drug optimization including the development of a B'-factor-supported pharmacophore model for SARS CoV-2 main protease inhibitors.
Rhodesain is the lysosomal cathepsin L-like cysteine protease ofT. brucei rhodesiense, the causat... more Rhodesain is the lysosomal cathepsin L-like cysteine protease ofT. brucei rhodesiense, the causative agent of Human African Trypanosomiasis. The enzyme is essential for the proliferation and pathogenicity of the parasite as well as its ability to overcome the blood-brain barrier of the host. Lysosomal cathepsins are expressed as zymogens with an inactivating pro-domain that is cleaved under acidic conditions. A structure of the uncleaved maturation intermediate from a trypanosomal cathepsin L-like protease is currently not available. We thus established the heterologous expression ofT. brucei rhodesiensepro-rhodesain inE. coliand determined its crystal structure. The trypanosomal pro-domain differs from non-parasitic pro-cathepsins by a unique, extended α-helix that blocks the active site and whose interactions resemble that of the antiprotozoal inhibitor K11777. Interdomain dynamics between pro- and core protease domain as observed by photoinduced electron transfer fluorescence cor...
ChemMedChem, 2020
Inhibition of coronavirus (CoV)-encoded papain-like cysteine proteases (PL pro) represents an att... more Inhibition of coronavirus (CoV)-encoded papain-like cysteine proteases (PL pro) represents an attractive strategy to treat infections by these important human pathogens. Herein we report on structure-activity relationships (SAR) of the noncovalent active-site directed inhibitor (R)-5-amino-2-methyl-N-(1-(naphthalen-1-yl)ethyl) benzamide (2 b), which is known to bind into the S3 and S4 pockets of the SARS-CoV PL pro. Moreover, we report the discovery of isoindolines as a new class of potent PL pro inhibitors. The studies also provide a deeper understanding of the binding modes of this inhibitor class. Importantly, the inhibitors were also confirmed to inhibit SARS-CoV-2 replication in cell culture suggesting that, due to the high structural similarities of the target proteases, inhibitors identified against SARS-CoV PL pro are valuable starting points for the development of new pan-coronaviral inhibitors.
Journal of Medicinal Chemistry, 2019
A model system of two related enzymes with conserved binding sites, namely N-myristoyltransferase... more A model system of two related enzymes with conserved binding sites, namely N-myristoyltransferase from two different organisms, was studied to decipher the driving forces that lead to selective inhibition in such cases. Using a combination of computational and experimental tools, two different selectivity-determining features were identified. For some ligands, a change in side chain flexibility appears to be responsible for selective inhibition. Remarkably, this was observed for residues orienting their side chains away from the ligands. For other ligands, selectivity is caused by interfering with a water molecule that binds stronger to the off-target than to the target. Based on this finding, a virtual screen for selective compounds was conducted resulting in three hit compounds with the desired selectivity profile. This study delivers a guideline on how to assess selectivity-determining features in proteins with conserved binding sites and to translate this knowledge into the design of selective inhibitors.
ABSTRACTWNT/β-catenin signaling endows cancer cells with proliferative capacity and immune-evasiv... more ABSTRACTWNT/β-catenin signaling endows cancer cells with proliferative capacity and immune-evasive functions that impair anti-cancer immunosurveillance by conventional, cytoxtoic T cells. However, the impact of dysregulated WNT signalling on unconventional, tissue-resident T cells, specifically in colon cancer is unknown. Here, we show that cancer cells in Apc-mutant mouse models escape immunosurveillance from gut-resident intraepithelial lymphocytes (IELs) expressing γδ T cell receptors (γδTCRs). Analysis of late-stage tumors from mice and humans revealed that γδIELs are largely absent from the tumor microenvironment, and that butyrophilin-like (BTNL) molecules, which can critically regulate γδIEL through direct γδTCR-interactions, are also downregulated. We could attribute this to β-catenin stabilization, which rapidly decreased expression of the transcription factors, HNF4A and HNF4G, that we found to bind promoter regions of Btnl genes, thereby driving their expression in normal...
Clinical and Translational Oncology, 2021
Purpose A significant percentage of colorectal cancer patients proceeds to metastatic disease. We... more Purpose A significant percentage of colorectal cancer patients proceeds to metastatic disease. We hypothesised that mitochondrial DNA (mtDNA) polymorphisms, generated by the high mtDNA mutation rate of energy-demanding clonal immune cell expansions and assessable in peripheral blood, reflect how efficiently systemic immunity impedes metastasis. Patients and methods We studied 44 rectal cancer patients from a population-based prospective biomarker study, given curative-intent neoadjuvant radiation and radical surgery for high-risk tumour stage and followed for metastatic failure. Blood specimens were sampled at the time of diagnosis and analysed for the full-length mtDNA sequence, composition of immune cell subpopulations and damaged serum mtDNA. Results Whole blood total mtDNA variant number above the median value for the study cohort, coexisting with an mtDNA non-H haplogroup, was representative for the mtDNA of circulating immune cells and associated with low risk of a metastatic ...
Annals of Surgical Oncology, 2020
Background Systemic inflammatory response (SIR) is an adverse prognostic marker in colorectal can... more Background Systemic inflammatory response (SIR) is an adverse prognostic marker in colorectal cancer (CRC) patients. The ScotScan Colorectal Cancer Group was established to examine how markers of the SIR differ between populations and may be utilised to guide prognosis. Patients and Methods Patients undergoing resection of stage I–III CRC from two prospective datasets in Scotland and Norway were included. The relationship between the modified Glasgow Prognostic Score (mGPS; combination of C-reactive protein and albumin) and overall survival (OS) was examined. The relationship between OS, adjuvant chemotherapy regime and mGPS was examined in patients with stage III colon cancer. Results A total of 2295 patients were included. Patients from Scotland were more inflamed despite controlling for associated characteristics using multivariate logistic regression or propensity score matching (OR 2.82, 95% CI 1.98–4.01, p < 0.001). mGPS had similar independent prognostic value in both coho...
British journal of anaesthesia, 2015
Neurobiological work has demonstrated that expression of mitogen-activated protein kinases (MAPK)... more Neurobiological work has demonstrated that expression of mitogen-activated protein kinases (MAPK) is upregulated on neurones and glial cells after nerve damage. Furthermore, the epidermal growth factor receptor (EGFR) has been identified as having a key role in this process and subsequent interruption of this using EGFR-Inhibitors (EGFR-I), may improve neuropathic pain. The aim of this report was to explore if EGFR-I attenuated neuropathic pain in humans. A selection of patients with neuropathic pain were treated off-label with one of four EGFR-Is, approved for the treatment of cancer. All patients had chronic and severe neuropathic pain (as defined by diagnostic criteria). Pain intensity, interference with function, and adverse events were prospectively registered. Twenty patients were treated. Eighteen patients experienced clinically significant pain relief after treatment with EGFR-I. Median observed pain reduction for all patients was 8.5 (IQR=5-9.5) points on a 0-10 numeric rat...
Morphological and clinical heterogeneity of advanced colorectal cancer is probably caused by gene... more Morphological and clinical heterogeneity of advanced colorectal cancer is probably caused by genetic vari- ability in putative cancer stem cell genes, including Lgr5. Here, we investigated 23 variants of the Lgr5 gene in nor- mal tissue, primary tumors, lymph node metastases and distant metastases of stage III and stage IV colorectal can- cer patients. These data were compared to results of immunohistochemical Lgr5 expression analysis and to prognos- tic clinical parameters. No differences were found comparing germline and somatic Lgr5 genotype in primary tumors, but additional Lgr5 gene alterations could be demonstrated in lymph node and distant metastases. Significant nega- tive correlation was seen between Lgr5 allelic variation and Lgr5 protein expression (p=0.0394), which mainly can be attributed to the negative influence of non-coding Lgr5 gene variations on Lgr5 protein expression (p=0.0166). Lgr5 gene variants could be found more frequently in primary tumors of stage III pat...
Annals of Oncology, 2021
Background: Neoadjuvant chemoradiotherapy (NCRT) has been consolidated as the main strategy for t... more Background: Neoadjuvant chemoradiotherapy (NCRT) has been consolidated as the main strategy for the treatment of locally advanced rectal cancer (LARC).However, heterogeneous responses are observed, with only about 15-20% of patients presenting a complete pathological response (pCR).Thus, countless studies evaluate possible biomarkers capable of predicting pCR and more intense neoadjuvant treatments to increase these rates. The objective of this prospective study was to analyze whether the protein expression of RAD23 homolog B (RAD23B) in the circulating tumor cells (CTCs) (at baseline-C1) could correlate with the response to NCRT. Methods: Between 2016 and 2020, 63 patients (pts) with LARC who underwent NCRT followed by radical surgery, were included in the study. Blood samples were collected before the beginning of NCRT (C1) and the evaluation of RAD23B protein expression in CTCs was correlated with the anatomopathological examination of response of patients undergoing surgery (n:56). CTCs were isolated and quantified by ISETÒ. RAD23B protein was analyzed by immunocytochemistry and visualized by bright field microscopy. Results: The mean age was 56 years old (34-92). Among the pts analyzed, 34 (54%) carried tumors at the distal rectum, 57 (90%) had clinical tumor stage (cT) T3/T4, 58 (92%) clinical nodal (cN) positive and 32 (52%) had preoperative carcinoembryonic antigen (CEA) >3 ng/mL Thirteen (23,2%) patients had pCR with NCRT. RAD23B expression in CTCs was present in 54% of non-responders, while in pCR group, it was absent in the majority of pts (91.7%; p¼0.019). In multivariate logistic regression models for pCR, including CEA, gender, cT and RAD23B expression in CTCs, the latter was an important independent prognostic factor [Odds Ratio (OR) 0.064; 95% confidence interval (CI): 0.006 e 0.751; p¼0.029]. Conclusions: This prospective study demonstrated the correlation between the absence of expression of RAD23B in CTCs (C1) and pCR, being an important result for future clinical studies. This analysis may identify NCRT responders candidates, helping to choose the best therapeutic approach for each individual.
Neurobiology of Disease, 2020
• ErbB1 inhibitors reverse hypersensitivity in neuropathic pain models, as clinically • Nocicepto... more • ErbB1 inhibitors reverse hypersensitivity in neuropathic pain models, as clinically • Nociceptors in DRG selectively express ErbB1 and ErbB2 but not ErbB3 or ErbB4 • ErbB1 drives Akt-/AS160-dependent trafficking of nociceptor Na + and Ca 2+ channels • ErbB1-and Akt-inhibitors prevent channel trafficking and hypersensitivity • Dual blockade of the ErbB1/2 signalling hub produces synergistic analgesia
Nucleic Acids Research
RNA methyltransferases (MTases) are ubiquitous enzymes whose hitherto low profile in medicinal ch... more RNA methyltransferases (MTases) are ubiquitous enzymes whose hitherto low profile in medicinal chemistry, contrasts with the surging interest in RNA methylation, the arguably most important aspect of the new field of epitranscriptomics. As MTases become validated as drug targets in all major fields of biomedicine, the development of small molecule compounds as tools and inhibitors is picking up considerable momentum, in academia as well as in biotech. Here we discuss the development of small molecules for two related aspects of chemical biology. Firstly, derivates of the ubiquitous cofactor S-adenosyl-l-methionine (SAM) are being developed as bioconjugation tools for targeted transfer of functional groups and labels to increasingly visible targets. Secondly, SAM-derived compounds are being investigated for their ability to act as inhibitors of RNA MTases. Drug development is moving from derivatives of cosubstrates towards higher generation compounds that may address allosteric sites...
<b>Copyright information:</b>Taken from "BMP-6 inhibits growth of mature human B... more <b>Copyright information:</b>Taken from "BMP-6 inhibits growth of mature human B cells; induction of Smad phosphorylation and upregulation of Id1"BMC Immunology 2005;6():9-9.Published online 9 May 2005PMCID:PMC1134658.Copyright © 2005 Kersten et al; licensee BioMed Central Ltd. CD19B cells were cultured in X-vivo 15 over night before treatment with BMP-6 for the indicated time points and cell lysates were prepared. One representative experiment of three is shown. HeLa cells were used as a positive control for Id1 protein detection.
<b>Copyright information:</b>Taken from "BMP-6 inhibits growth of mature human B... more <b>Copyright information:</b>Taken from "BMP-6 inhibits growth of mature human B cells; induction of Smad phosphorylation and upregulation of Id1"BMC Immunology 2005;6():9-9.Published online 9 May 2005PMCID:PMC1134658.Copyright © 2005 Kersten et al; licensee BioMed Central Ltd. Quantifications of Id1, Id2 and Id3 protein levels were performed using β-actin as normalization and expressed as mean ± SEM (Id1: n = 4, *p ≤ 0.020; Id2 and Id3: n = 3).
HardwareX, 2022
Differential scanning fluorimetry (DSF) is a widely used biophysical technique with applications ... more Differential scanning fluorimetry (DSF) is a widely used biophysical technique with applications to drug discovery and protein biochemistry. DSF experiments are commonly performed in commercial real-time polymerase chain reaction (qPCR) thermal cyclers or nanoDSF instruments. Here, we report the construction, validation, and example applications of an open-source DSF system for 176 €, which, in addition to protein-DSF experiments, also proved to be a versatile biophysical instrument for less conventional RNA-DSF experiments. Using 3D-printed parts made of polyoxymethylene, we were able to fabricate a thermostable machine chassis for protein-melting experiments. The combination of blue high-power LEDs as the light source and stage light foil as filter components was proven to be a reliable and affordable alternative to conventional optics equipment for the detection of SYPRO Orange or Sybr Gold fluorescence. The ESP32 microcontroller is the core piece of this openDSF instrument, while the in-built I 2 S interface was found to be a powerful analog-to-digital converter for fast acquisition of fluorescence and temperature data. Airflow heating and inline temperature control by thermistors enabled highaccuracy temperature management in PCR tubes (±0.1°C) allowing us to perform highresolution thermal shift assays (TSA) from exemplary biological applications.
ACS Chemical Biology, 2021
Viral and parasitic pathogens rely critically on cysteine proteases for host invasion, replicatio... more Viral and parasitic pathogens rely critically on cysteine proteases for host invasion, replication, and infectivity. Their inhibition by synthetic inhibitors, such as vinyl sulfone compounds, has emerged as a promising treatment strategy. However, the individual reaction steps of protease inhibition are not fully understood. Using the trypanosomal cysteine protease rhodesain as a medically relevant target, we design photoinduced electron transfer (PET) fluorescence probes to detect kinetics of binding of reversible and irreversible vinyl sulfones directly in solution. Intriguingly, the irreversible inhibitor, apart from its unlimited residence time in the enzyme, reacts 5 times faster than the reversible one. Results show that the reactivity of the warhead, and not binding of the peptidic recognition unit, limits the rate constant of protease inhibition. The use of a reversible inhibitor decreases the risk of off-target side effects not only by allowing its release from an off-target but also by reducing the rate constant of binding.
Current Medicinal Chemistry, 2022
: Due to its fast international spread and substantial mortality, the coronavirus disease COVID-1... more : Due to its fast international spread and substantial mortality, the coronavirus disease COVID-19 evolved to a global threat. Since there is currently no causative drug against this viral infection available, science is striving for new drugs and other approaches to treat the new disease. Studies have shown that the cell entry of coronaviruses into host cells takes place through the binding of the viral spike (S) protein to cell receptors. Priming of the S protein occurs via hydrolysis by different host proteases. The inhibition of these proteases could impair the processing of the S protein, thereby affecting the interaction with the host-cell receptors and preventing virus cell entry. Hence, inhibition of these proteases could be a promising strategy for treatment against SARSCoV- 2. In this review, we discuss the current state of the art of developing inhibitors against the entry proteases furin, the transmembrane serine protease type-II (TMPRSS2), trypsin, and cathepsin L.
Molecular Informatics, 2020
The analysis of B-factor profiles from X-ray protein structures can be utilized for structure-bas... more The analysis of B-factor profiles from X-ray protein structures can be utilized for structure-based drug design since protein mobility changes have been associated with the quality of protein-ligand interactions. With the BANΔIT (B'-factor analysis and ΔB' interpretation toolkit), we have developed a JavaScript-based browser application that provides a graphical user interface for the normalization and analysis of B'-factor profiles. To emphasize the usability for rational drug design applications, we have analyzed a selection of crystallographic protein-ligand complexes and have given exemplary conclusions for further drug optimization including the development of a B'-factor-supported pharmacophore model for SARS CoV-2 main protease inhibitors.
Rhodesain is the lysosomal cathepsin L-like cysteine protease ofT. brucei rhodesiense, the causat... more Rhodesain is the lysosomal cathepsin L-like cysteine protease ofT. brucei rhodesiense, the causative agent of Human African Trypanosomiasis. The enzyme is essential for the proliferation and pathogenicity of the parasite as well as its ability to overcome the blood-brain barrier of the host. Lysosomal cathepsins are expressed as zymogens with an inactivating pro-domain that is cleaved under acidic conditions. A structure of the uncleaved maturation intermediate from a trypanosomal cathepsin L-like protease is currently not available. We thus established the heterologous expression ofT. brucei rhodesiensepro-rhodesain inE. coliand determined its crystal structure. The trypanosomal pro-domain differs from non-parasitic pro-cathepsins by a unique, extended α-helix that blocks the active site and whose interactions resemble that of the antiprotozoal inhibitor K11777. Interdomain dynamics between pro- and core protease domain as observed by photoinduced electron transfer fluorescence cor...
ChemMedChem, 2020
Inhibition of coronavirus (CoV)-encoded papain-like cysteine proteases (PL pro) represents an att... more Inhibition of coronavirus (CoV)-encoded papain-like cysteine proteases (PL pro) represents an attractive strategy to treat infections by these important human pathogens. Herein we report on structure-activity relationships (SAR) of the noncovalent active-site directed inhibitor (R)-5-amino-2-methyl-N-(1-(naphthalen-1-yl)ethyl) benzamide (2 b), which is known to bind into the S3 and S4 pockets of the SARS-CoV PL pro. Moreover, we report the discovery of isoindolines as a new class of potent PL pro inhibitors. The studies also provide a deeper understanding of the binding modes of this inhibitor class. Importantly, the inhibitors were also confirmed to inhibit SARS-CoV-2 replication in cell culture suggesting that, due to the high structural similarities of the target proteases, inhibitors identified against SARS-CoV PL pro are valuable starting points for the development of new pan-coronaviral inhibitors.
Journal of Medicinal Chemistry, 2019
A model system of two related enzymes with conserved binding sites, namely N-myristoyltransferase... more A model system of two related enzymes with conserved binding sites, namely N-myristoyltransferase from two different organisms, was studied to decipher the driving forces that lead to selective inhibition in such cases. Using a combination of computational and experimental tools, two different selectivity-determining features were identified. For some ligands, a change in side chain flexibility appears to be responsible for selective inhibition. Remarkably, this was observed for residues orienting their side chains away from the ligands. For other ligands, selectivity is caused by interfering with a water molecule that binds stronger to the off-target than to the target. Based on this finding, a virtual screen for selective compounds was conducted resulting in three hit compounds with the desired selectivity profile. This study delivers a guideline on how to assess selectivity-determining features in proteins with conserved binding sites and to translate this knowledge into the design of selective inhibitors.