Discovery of Alternative Binding Poses through Fragment-Based Identification of DHODH Inhibitors DeRatt, Lindsey G. In: 2024. @article{noKey,
title = {Discovery of Alternative Binding Poses through Fragment-Based Identification of DHODH Inhibitors},
author = {DeRatt, Lindsey G.},
url = {https://pubs.acs.org/doi/abs/10.1021/acsmedchemlett.3c00543},
doi = {https://doi.org/10.1021/acsmedchemlett.3c00543},
year = {2024},
date = {2024-01-01},
abstract = {Dihydroorotate dehydrogenase (DHODH) is a mitochondrial enzyme that affects many aspects essential to cell proliferation and survival. Recently, DHODH has been identified as a potential target for acute myeloid leukemia therapy. Herein, we describe the identification of potent DHODH inhibitors through a scaffold hopping approach emanating from a fragment screen followed by structure-based drug design to further improve the overall profile and reveal an unexpected novel binding mode. Additionally, these compounds had low P-gp efflux ratios, allowing for applications where exposure to the brain would be required.},
keywords = {TEMPEST},
pubstate = {published},
tppubtype = {article}
}
Dihydroorotate dehydrogenase (DHODH) is a mitochondrial enzyme that affects many aspects essential to cell proliferation and survival. Recently, DHODH has been identified as a potential target for acute myeloid leukemia therapy. Herein, we describe the identification of potent DHODH inhibitors through a scaffold hopping approach emanating from a fragment screen followed by structure-based drug design to further improve the overall profile and reveal an unexpected novel binding mode. Additionally, these compounds had low P-gp efflux ratios, allowing for applications where exposure to the brain would be required. |
Unique and Common Agonists Activate the Insect Juvenile Hormone Receptor and the Human AHR Sedlak, David In: 2024. @article{noKey,
title = {Unique and Common Agonists Activate the Insect Juvenile Hormone Receptor and the Human AHR},
author = {Sedlak, David},
url = {https://www.biorxiv.org/content/10.1101/2024.01.03.574093v1},
doi = {https://doi.org/10.1101/2024.01.03.574093},
year = {2024},
date = {2024-01-01},
abstract = {Transcription factors of the bHLH-PAS family play vital roles in animal development, physiology, and disease. Two members of the family require binding of low-molecular weight ligands for their activity: the vertebrate aryl hydrocarbon receptor (AHR) and the insect juvenile hormone receptor (JHR). In the fly Drosophila melanogaster, the paralogous proteins GCE and MET constitute the ligand-binding component of JHR complexes. Whilst GCE/MET and AHR are phylogenetically heterologous, their mode of action is similar. JHR is targeted by several synthetic agonists that serve as insecticides disrupting the insect endocrine system. AHR is an important regulator of human endocrine homeostasis and it responds to environmental pollutants and endocrine disruptors. Whether AHR signaling is affected by compounds that can activate JHR has not been reported. To address this question, we screened a chemical library of 50,000 compounds to identify 93 novel JHR agonists in a reporter system based on Drosophila cells. Of these compounds, 26% modulated AHR signaling in an analogous reporter assay in a human cell line, indicating a significant overlap in the agonist repertoires of the two receptors. To explore the structural features of agonist-dependent activation of JHR and AHR, we compared the ligand-binding cavities and their interactions with selective and common ligands of AHR and GCE. Molecular dynamics modeling revealed ligand-specific as well as conserved side chains within the respective cavities. Significance of predicted interactions was supported through site-directed mutagenesis. The results have indicated that synthetic insect juvenile hormone agonists might interfere with AHR signaling in human cells.},
keywords = {TEMPEST},
pubstate = {published},
tppubtype = {article}
}
Transcription factors of the bHLH-PAS family play vital roles in animal development, physiology, and disease. Two members of the family require binding of low-molecular weight ligands for their activity: the vertebrate aryl hydrocarbon receptor (AHR) and the insect juvenile hormone receptor (JHR). In the fly Drosophila melanogaster, the paralogous proteins GCE and MET constitute the ligand-binding component of JHR complexes. Whilst GCE/MET and AHR are phylogenetically heterologous, their mode of action is similar. JHR is targeted by several synthetic agonists that serve as insecticides disrupting the insect endocrine system. AHR is an important regulator of human endocrine homeostasis and it responds to environmental pollutants and endocrine disruptors. Whether AHR signaling is affected by compounds that can activate JHR has not been reported. To address this question, we screened a chemical library of 50,000 compounds to identify 93 novel JHR agonists in a reporter system based on Drosophila cells. Of these compounds, 26% modulated AHR signaling in an analogous reporter assay in a human cell line, indicating a significant overlap in the agonist repertoires of the two receptors. To explore the structural features of agonist-dependent activation of JHR and AHR, we compared the ligand-binding cavities and their interactions with selective and common ligands of AHR and GCE. Molecular dynamics modeling revealed ligand-specific as well as conserved side chains within the respective cavities. Significance of predicted interactions was supported through site-directed mutagenesis. The results have indicated that synthetic insect juvenile hormone agonists might interfere with AHR signaling in human cells. |
Discovery of an anti-virulence compound that targets the Staphylococcus aureus SaeRS two-component system to inhibit toxic shock syndrome toxin 1 (TSST-1) production Dufresne, Karine In: 2024. @article{noKey,
title = {Discovery of an anti-virulence compound that targets the Staphylococcus aureus SaeRS two-component system to inhibit toxic shock syndrome toxin 1 (TSST-1) production},
author = {Dufresne, Karine},
url = {https://www.biorxiv.org/content/10.1101/2024.02.27.582338v1},
doi = {https://doi.org/10.1101/2024.02.27.582338},
year = {2024},
date = {2024-01-01},
abstract = {Menstrual toxic shock syndrome (mTSS) is a rare but severe disorder associated with the use of menstrual products such as high-absorbency tampons and is caused by Staphylococcus aureus strains that produce the toxic shock syndrome toxin-1 (TSST-1) superantigen. Herein, we screened a library of 3920 small bioactive molecules for the ability to inhibit transcription of the TSST-1 gene without inhibiting growth of S. aureus. The dominant positive regulator of TSST-1 is the SaeRS two-component system (TCS), and we identified phenazopyridine hydrochloride (PP-HCl) that repressed production of TSST-1 by inhibiting the kinase function of SaeS. PP-HCl competed with ATP for binding of the kinase SaeS leading to decreased phosphorylation of SaeR and reduced expression of TSST-1 as well as several other secreted virulence factors known to be regulated by SaeRS. PP-HCl targets virulence of S. aureus, but it also decreases the impact of TSST-1 on human lymphocytes without affecting the healthy vaginal microbiota. Our findings demonstrate the promising potential of PP-HCl as a therapeutic strategy against mTSS.},
keywords = {TEMPEST},
pubstate = {published},
tppubtype = {article}
}
Menstrual toxic shock syndrome (mTSS) is a rare but severe disorder associated with the use of menstrual products such as high-absorbency tampons and is caused by Staphylococcus aureus strains that produce the toxic shock syndrome toxin-1 (TSST-1) superantigen. Herein, we screened a library of 3920 small bioactive molecules for the ability to inhibit transcription of the TSST-1 gene without inhibiting growth of S. aureus. The dominant positive regulator of TSST-1 is the SaeRS two-component system (TCS), and we identified phenazopyridine hydrochloride (PP-HCl) that repressed production of TSST-1 by inhibiting the kinase function of SaeS. PP-HCl competed with ATP for binding of the kinase SaeS leading to decreased phosphorylation of SaeR and reduced expression of TSST-1 as well as several other secreted virulence factors known to be regulated by SaeRS. PP-HCl targets virulence of S. aureus, but it also decreases the impact of TSST-1 on human lymphocytes without affecting the healthy vaginal microbiota. Our findings demonstrate the promising potential of PP-HCl as a therapeutic strategy against mTSS. |
BacterAI maps microbial metabolism without prior knowledge Dama, Adam C. In: 2023. @article{noKey,
title = {BacterAI maps microbial metabolism without prior knowledge},
author = {Dama, Adam C.},
url = {https://www.nature.com/articles/s41564-023-01376-0},
doi = {https://doi.org/10.1038/s41564-023-01376-0},
year = {2023},
date = {2023-01-01},
abstract = {Training artificial intelligence (AI) systems to perform autonomous experiments would vastly increase the throughput of microbiology; however, few microbes have large enough datasets for training such a system. In the present study, we introduce BacterAI, an automated science platform that maps microbial metabolism but requires no prior knowledge. BacterAI learns by converting scientific questions into simple games that it plays with laboratory robots. The agent then distils its findings into logical rules that can be interpreted by human scientists. We use BacterAI to learn the amino acid requirements for two oral streptococci: Streptococcus gordonii and Streptococcus sanguinis. We then show how transfer learning can accelerate BacterAI when investigating new environments or larger media with up to 39 ingredients. Scientific gameplay and BacterAI enable the unbiased, autonomous study of organisms for which no training data exist.},
keywords = {TEMPEST},
pubstate = {published},
tppubtype = {article}
}
Training artificial intelligence (AI) systems to perform autonomous experiments would vastly increase the throughput of microbiology; however, few microbes have large enough datasets for training such a system. In the present study, we introduce BacterAI, an automated science platform that maps microbial metabolism but requires no prior knowledge. BacterAI learns by converting scientific questions into simple games that it plays with laboratory robots. The agent then distils its findings into logical rules that can be interpreted by human scientists. We use BacterAI to learn the amino acid requirements for two oral streptococci: Streptococcus gordonii and Streptococcus sanguinis. We then show how transfer learning can accelerate BacterAI when investigating new environments or larger media with up to 39 ingredients. Scientific gameplay and BacterAI enable the unbiased, autonomous study of organisms for which no training data exist. |
An ependymal cell census identifies heterogeneous and ongoing cell maturation in the adult mouse spinal cord that changes dynamically on injury Albors, Aida Rodrigo, et al. In: 2023. @article{noKey,
title = {An ependymal cell census identifies heterogeneous and ongoing cell maturation in the adult mouse spinal cord that changes dynamically on injury},
author = {Albors, Aida Rodrigo, et al.},
url = {https://pubmed.ncbi.nlm.nih.gov/36706756/},
doi = {https://doi.org/10.1016/j.devcel.2023.01.003},
year = {2023},
date = {2023-01-01},
abstract = {The adult spinal cord stem cell potential resides within the ependymal cell population and declines with age. Ependymal cells are, however, heterogeneous, and the biological diversity this represents and how it changes with age remain unknown. Here, we present a single-cell transcriptomic census of spinal cord ependymal cells from adult and aged mice, identifying not only all known ependymal cell subtypes but also immature as well as mature cell states. By comparing transcriptomes of spinal cord and brain ependymal cells, which lack stem cell abilities, we identify immature cells as potential spinal cord stem cells. Following spinal cord injury, these cells re-enter the cell cycle, which is accompanied by a short-lived reversal of ependymal cell maturation. We further analyze ependymal cells in the human spinal cord and identify widespread cell maturation and altered cell identities. This in-depth characterization of spinal cord ependymal cells provides insight into their biology and informs strategies for spinal cord repair.},
keywords = {TEMPEST},
pubstate = {published},
tppubtype = {article}
}
The adult spinal cord stem cell potential resides within the ependymal cell population and declines with age. Ependymal cells are, however, heterogeneous, and the biological diversity this represents and how it changes with age remain unknown. Here, we present a single-cell transcriptomic census of spinal cord ependymal cells from adult and aged mice, identifying not only all known ependymal cell subtypes but also immature as well as mature cell states. By comparing transcriptomes of spinal cord and brain ependymal cells, which lack stem cell abilities, we identify immature cells as potential spinal cord stem cells. Following spinal cord injury, these cells re-enter the cell cycle, which is accompanied by a short-lived reversal of ependymal cell maturation. We further analyze ependymal cells in the human spinal cord and identify widespread cell maturation and altered cell identities. This in-depth characterization of spinal cord ependymal cells provides insight into their biology and informs strategies for spinal cord repair. |
iTAG an optimized IMiD-induced degron for targeted protein degradation in human and murine cells Bouguenina, Habib In: 2023. @article{noKey,
title = {iTAG an optimized IMiD-induced degron for targeted protein degradation in human and murine cells},
author = {Bouguenina, Habib},
url = {https://www.cell.com/iscience/fulltext/S2589-0042(23)01136-7?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS2589004223011367%3Fshowall%3Dtrue},
doi = {https://doi.org/10.1016/j.isci.2023.107059},
year = {2023},
date = {2023-01-01},
abstract = {To address the limitation associated with degron based systems, we have developed iTAG, a synthetic tag based on IMiDs/CELMoDs mechanism of action that improves and addresses the limitations of both PROTAC and previous IMiDs/CeLMoDs based tags. Using structural and sequence analysis, we systematically explored native and chimeric degron containing domains (DCDs) and evaluated their ability to induce degradation. We identified the optimal chimeric iTAG(DCD23 60aa) that elicits robust degradation of targets across cell types and subcellular localizations without exhibiting the well documented “hook effect” of PROTAC-based systems. We showed that iTAG can also induce target degradation by murine CRBN and enabled the exploration of natural neo-substrates that can be degraded by murine CRBN. Hence, the iTAG system constitutes a versatile tool to degrade targets across the human and murine proteome.},
keywords = {TEMPEST},
pubstate = {published},
tppubtype = {article}
}
To address the limitation associated with degron based systems, we have developed iTAG, a synthetic tag based on IMiDs/CELMoDs mechanism of action that improves and addresses the limitations of both PROTAC and previous IMiDs/CeLMoDs based tags. Using structural and sequence analysis, we systematically explored native and chimeric degron containing domains (DCDs) and evaluated their ability to induce degradation. We identified the optimal chimeric iTAG(DCD23 60aa) that elicits robust degradation of targets across cell types and subcellular localizations without exhibiting the well documented “hook effect” of PROTAC-based systems. We showed that iTAG can also induce target degradation by murine CRBN and enabled the exploration of natural neo-substrates that can be degraded by murine CRBN. Hence, the iTAG system constitutes a versatile tool to degrade targets across the human and murine proteome. |
Screening for variable drug responses using human iPSC cohorts Platani, Melpomeni In: 2023. @article{noKey,
title = {Screening for variable drug responses using human iPSC cohorts},
author = {Platani, Melpomeni},
url = {https://www.biorxiv.org/content/10.1101/2023.06.16.545161v1.full},
doi = {https://doi.org/10.1101/2023.06.16.545161},
year = {2023},
date = {2023-01-01},
abstract = {We have used a cohort of human induced pluripotent stem cell (hiPSC) lines to develop a laboratory-based drug screening platform to predict variable drug responses of potential clinical relevance. Our approach is based on the findings that hiPSC lines reflect the genetic identity of the donor and that pluripotent hiPSC lines express a broad repertoire of gene transcripts and proteins. We demonstrate that a cohort of hiPSC lines from different donors can be screened efficiently in their pluripotent state using high-throughput cell painting assays, allowing detection of variable phenotypic responses to a wide range of clinically approved drugs, across multiple disease areas. Furthermore, we provide information on mechanisms of drug-cell interactions underlying the observed variable responses by using quantitative proteomic analysis to compare sets of hiPSC lines that had been stratified objectively using cell painting data. We propose that information derived from comparative drug screening using curated libraries of hiPSC lines can help to increase the success rate of drug development pipelines and improve the delivery of safe new drugs suitable for a broader ethnic and gender diversity within human populations.},
keywords = {TEMPEST},
pubstate = {published},
tppubtype = {article}
}
We have used a cohort of human induced pluripotent stem cell (hiPSC) lines to develop a laboratory-based drug screening platform to predict variable drug responses of potential clinical relevance. Our approach is based on the findings that hiPSC lines reflect the genetic identity of the donor and that pluripotent hiPSC lines express a broad repertoire of gene transcripts and proteins. We demonstrate that a cohort of hiPSC lines from different donors can be screened efficiently in their pluripotent state using high-throughput cell painting assays, allowing detection of variable phenotypic responses to a wide range of clinically approved drugs, across multiple disease areas. Furthermore, we provide information on mechanisms of drug-cell interactions underlying the observed variable responses by using quantitative proteomic analysis to compare sets of hiPSC lines that had been stratified objectively using cell painting data. We propose that information derived from comparative drug screening using curated libraries of hiPSC lines can help to increase the success rate of drug development pipelines and improve the delivery of safe new drugs suitable for a broader ethnic and gender diversity within human populations. |
A Degron Blocking Strategy Towards Improved CRL4CRBN Recruiting PROTAC Selectivity Bouguenina, Habib In: 2023. @article{noKey,
title = {A Degron Blocking Strategy Towards Improved CRL4CRBN Recruiting PROTAC Selectivity},
author = {Bouguenina, Habib},
url = {https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202300351},
doi = {https://doi.org/10.1002/cbic.202300351},
year = {2023},
date = {2023-01-01},
abstract = {Small molecules inducing protein degradation are important pharmacological tools to interrogate complex biology and are rapidly translating into clinical agents. However, to fully realise the potential of these molecules, selectivity remains a limiting challenge. Herein, we addressed the issue of selectivity in the design of CRL4CRBN recruiting PROteolysis TArgeting Chimeras (PROTACs). Thalidomide derivatives used to generate CRL4CRBN recruiting PROTACs have well described intrinsic monovalent degradation profiles by inducing the recruitment of neo-substrates, such as GSPT1, Ikaros and Aiolos. We leveraged structural insights from known CRL4CRBN neo-substrates to attenuate and indeed remove this monovalent degradation function in well-known CRL4CRBN molecular glues degraders, namely CC-885 and Pomalidomide. We then applied these design principles on a previously published BRD9 PROTAC (dBRD9-A) and generated an analogue with improved selectivity profile. Finally, we implemented a computational modelling pipeline to show that our degron blocking design does not impact PROTAC-induced ternary complex formation. We believe that the tools and principles presented in this work will be valuable to support the development of targeted protein degradation.},
keywords = {TEMPEST},
pubstate = {published},
tppubtype = {article}
}
Small molecules inducing protein degradation are important pharmacological tools to interrogate complex biology and are rapidly translating into clinical agents. However, to fully realise the potential of these molecules, selectivity remains a limiting challenge. Herein, we addressed the issue of selectivity in the design of CRL4CRBN recruiting PROteolysis TArgeting Chimeras (PROTACs). Thalidomide derivatives used to generate CRL4CRBN recruiting PROTACs have well described intrinsic monovalent degradation profiles by inducing the recruitment of neo-substrates, such as GSPT1, Ikaros and Aiolos. We leveraged structural insights from known CRL4CRBN neo-substrates to attenuate and indeed remove this monovalent degradation function in well-known CRL4CRBN molecular glues degraders, namely CC-885 and Pomalidomide. We then applied these design principles on a previously published BRD9 PROTAC (dBRD9-A) and generated an analogue with improved selectivity profile. Finally, we implemented a computational modelling pipeline to show that our degron blocking design does not impact PROTAC-induced ternary complex formation. We believe that the tools and principles presented in this work will be valuable to support the development of targeted protein degradation. |
Protomer selectivity of type II RAF inhibitors within the RAS/RAF complex Vasta, James D. In: 2023. @article{noKey,
title = {Protomer selectivity of type II RAF inhibitors within the RAS/RAF complex},
author = {Vasta, James D.},
url = {https://www.cell.com/cell-chemical-biology/fulltext/S2451-9456(23)00247-7?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS2451945623002477%3Fshowall%3Dtrue},
doi = {https://doi.org/10.1016/j.chembiol.2023.07.019},
year = {2023},
date = {2023-01-01},
abstract = {RAF dimer inhibitors offer therapeutic potential in RAF- and RAS-driven cancers. The utility of such drugs is predicated on their capacity to occupy both RAF protomers in the RAS-RAF signaling complex. Here we describe a method to conditionally quantify drug-target occupancy at selected RAF protomers within an active RAS-RAF complex in cells. RAF target engagement can be measured in the presence or absence of any mutant KRAS allele, enabling the high-affinity state of RAF dimer inhibitors to be quantified in the cellular milieu. The intracellular protomer selectivity of clinical-stage type II RAF inhibitors revealed that ARAF protomer engagement, but not engagement of BRAF or CRAF, is commensurate with inhibition of MAPK signaling in various mutant RAS cell lines. Our results support a fundamental role for ARAF in mutant RAS signaling and reveal poor ARAF protomer vulnerability for a cohort of RAF inhibitors undergoing clinical evaluation.},
keywords = {TEMPEST},
pubstate = {published},
tppubtype = {article}
}
RAF dimer inhibitors offer therapeutic potential in RAF- and RAS-driven cancers. The utility of such drugs is predicated on their capacity to occupy both RAF protomers in the RAS-RAF signaling complex. Here we describe a method to conditionally quantify drug-target occupancy at selected RAF protomers within an active RAS-RAF complex in cells. RAF target engagement can be measured in the presence or absence of any mutant KRAS allele, enabling the high-affinity state of RAF dimer inhibitors to be quantified in the cellular milieu. The intracellular protomer selectivity of clinical-stage type II RAF inhibitors revealed that ARAF protomer engagement, but not engagement of BRAF or CRAF, is commensurate with inhibition of MAPK signaling in various mutant RAS cell lines. Our results support a fundamental role for ARAF in mutant RAS signaling and reveal poor ARAF protomer vulnerability for a cohort of RAF inhibitors undergoing clinical evaluation. |
Sperm Cell Painting: A Mechanism Driven Approach for Drug Discovery in Human Spermatozoa Johnston, Zoe C In: 2023. @article{noKey,
title = {Sperm Cell Painting: A Mechanism Driven Approach for Drug Discovery in Human Spermatozoa},
author = {Johnston, Zoe C},
url = {https://www.biorxiv.org/content/10.1101/2023.09.15.557919v2},
doi = {https://doi.org/10.1101/2023.09.15.557919},
year = {2023},
date = {2023-01-01},
abstract = {We have adapted the cell painting assay developed by Carpenter and colleagues on cultured U2OS cells to human spermatozoa. In Sperm Cell Painting (SCP) we assemble an image-based quantitative fingerprint of the functional state of sperm. We use this assay to gain insight into the mechanism of action of compounds that modify sperm function and as a platform for contraceptive discovery.},
keywords = {TEMPEST},
pubstate = {published},
tppubtype = {article}
}
We have adapted the cell painting assay developed by Carpenter and colleagues on cultured U2OS cells to human spermatozoa. In Sperm Cell Painting (SCP) we assemble an image-based quantitative fingerprint of the functional state of sperm. We use this assay to gain insight into the mechanism of action of compounds that modify sperm function and as a platform for contraceptive discovery. |
Lessons from assembling a microbial natural product and pre-fractionated extract library in an academic laboratory Cook, Michael A In: 2023. @article{noKey,
title = {Lessons from assembling a microbial natural product and pre-fractionated extract library in an academic laboratory},
author = {Cook, Michael A},
url = {https://academic.oup.com/jimb/advance-article/doi/10.1093/jimb/kuad042/7459345},
doi = {https://doi.org/10.1093/jimb/kuad042},
year = {2023},
date = {2023-01-01},
abstract = {Microbial natural products are specialized metabolites that are sources of many bioactive
compounds including antibiotics, antifungals, antiparasitics, anticancer agents, and probes of
biology. The assembly of libraries of producers of natural products has traditionally been the
province of the pharmaceutical industry. This sector has gathered significant historical
collections of bacteria and fungi to identify new drug leads with outstanding outcomes - upwards of 60% of drug scaffolds originate from such libraries. Despite this success, the repeated rediscovery of known compounds and the resultant diminishing chemical novelty contributed to a pivot from this source of bioactive compounds toward more tractable synthetic compounds in the drug industry. The advent of advanced mass spectrometry tools, along with rapid whole genome sequencing and in silico identification of biosynthetic gene clusters that encode the machinery necessary for the synthesis of specialized metabolites, offers the opportunity to revisit microbial natural product libraries with renewed vigor. Assembling a suitable library of microbes and extracts for screening requires the investment of resources and the development of methods that have customarily been the proprietary purview of large pharmaceutical companies. Here, we
report a perspective on our efforts to assemble a library of natural product-producing microbes
and the establishment of methods to extract and fractionate bioactive compounds using resources available to most academic labs. We validate the library and approach through a series of screens for antimicrobial and cytotoxic agents. This work serves as a blueprint for establishing libraries of microbial natural product producers and bioactive extract fractions suitable for screens of bioactive compounds.},
keywords = {TEMPEST},
pubstate = {published},
tppubtype = {article}
}
Microbial natural products are specialized metabolites that are sources of many bioactive
compounds including antibiotics, antifungals, antiparasitics, anticancer agents, and probes of
biology. The assembly of libraries of producers of natural products has traditionally been the
province of the pharmaceutical industry. This sector has gathered significant historical
collections of bacteria and fungi to identify new drug leads with outstanding outcomes - upwards of 60% of drug scaffolds originate from such libraries. Despite this success, the repeated rediscovery of known compounds and the resultant diminishing chemical novelty contributed to a pivot from this source of bioactive compounds toward more tractable synthetic compounds in the drug industry. The advent of advanced mass spectrometry tools, along with rapid whole genome sequencing and in silico identification of biosynthetic gene clusters that encode the machinery necessary for the synthesis of specialized metabolites, offers the opportunity to revisit microbial natural product libraries with renewed vigor. Assembling a suitable library of microbes and extracts for screening requires the investment of resources and the development of methods that have customarily been the proprietary purview of large pharmaceutical companies. Here, we
report a perspective on our efforts to assemble a library of natural product-producing microbes
and the establishment of methods to extract and fractionate bioactive compounds using resources available to most academic labs. We validate the library and approach through a series of screens for antimicrobial and cytotoxic agents. This work serves as a blueprint for establishing libraries of microbial natural product producers and bioactive extract fractions suitable for screens of bioactive compounds. |
Overcome the challenge for intratumoral injection of STING agonist for pancreatic cancer by systemic administration Li, Keyu In: 2023. @article{noKey,
title = {Overcome the challenge for intratumoral injection of STING agonist for pancreatic cancer by systemic administration},
author = {Li, Keyu},
url = {https://hal.science/hal-04326175/},
doi = {hal-04326175},
year = {2023},
date = {2023-01-01},
abstract = {Objective: Due to the challenge for intratumoral administration, innate agonists have not made it beyond preclinical studies for efficacy testing in most of tumor types. Pancreatic ductal adenocarcinoma (PDAC) has a T-cell excluded or deserted tumor microenvironment. Innate agonist treatments may serve as a T cell priming mechanism to sensitize PDACs to anti-PD-1 antibody (a-PD-1) treatment. Design: Using a transplant murine model with spontaneously formed liver metastasis and also the genetically engineered KPC mouse model that spontaneously develops PDAC, we compared the antitumor efficacy between intrahepatic/intratumoral and intramuscular systemic administration of BMS-986301, a next-generation STING agonist. Flow cytometry, Nanostring, and cytokine assays were used to evaluate local and systemic immune responses. Results: The study demonstrated that administration of STING agonist systemically via intramuscular injection is equivalent or potentially superior to its intratumoral injection in inducing both effector T cell response and antitumor efficacy. Compared to intratumoral administration, T cell exhaustion and immunosuppressive signals induced by systemic administration were attenuated. Nonetheless, either local or systemic treatment of STING agonist was associated with increased expression of CTLA-4 in the tumors. However, the combination of a-PD-1 and anti-CTLA-4 antibody with systemic STING agonist demonstrated the antitumor efficacy in the KPC mouse spontaneous PDAC model. Our study also demonstrated the feasibility and antitumor efficacy of systemic administration of BMS-986299, a new NLRP3 agonist. Conclusion: For the first time, our study supports the clinical development of innate agonists via systemic administration, instead of local administration, for treating PDAC.},
keywords = {TEMPEST},
pubstate = {published},
tppubtype = {article}
}
Objective: Due to the challenge for intratumoral administration, innate agonists have not made it beyond preclinical studies for efficacy testing in most of tumor types. Pancreatic ductal adenocarcinoma (PDAC) has a T-cell excluded or deserted tumor microenvironment. Innate agonist treatments may serve as a T cell priming mechanism to sensitize PDACs to anti-PD-1 antibody (a-PD-1) treatment. Design: Using a transplant murine model with spontaneously formed liver metastasis and also the genetically engineered KPC mouse model that spontaneously develops PDAC, we compared the antitumor efficacy between intrahepatic/intratumoral and intramuscular systemic administration of BMS-986301, a next-generation STING agonist. Flow cytometry, Nanostring, and cytokine assays were used to evaluate local and systemic immune responses. Results: The study demonstrated that administration of STING agonist systemically via intramuscular injection is equivalent or potentially superior to its intratumoral injection in inducing both effector T cell response and antitumor efficacy. Compared to intratumoral administration, T cell exhaustion and immunosuppressive signals induced by systemic administration were attenuated. Nonetheless, either local or systemic treatment of STING agonist was associated with increased expression of CTLA-4 in the tumors. However, the combination of a-PD-1 and anti-CTLA-4 antibody with systemic STING agonist demonstrated the antitumor efficacy in the KPC mouse spontaneous PDAC model. Our study also demonstrated the feasibility and antitumor efficacy of systemic administration of BMS-986299, a new NLRP3 agonist. Conclusion: For the first time, our study supports the clinical development of innate agonists via systemic administration, instead of local administration, for treating PDAC. |
N-Heterocyclic 3-Pyridyl Carboxamide Inhibitors of DHODH for the Treatment of Acute Myelogenous Leukemia (Ed.) In: 2022. @article{noKey,
title = {N-Heterocyclic 3-Pyridyl Carboxamide Inhibitors of DHODH for the Treatment of Acute Myelogenous Leukemia},
url = {https://pubs.acs.org/doi/full/10.1021/acs.jmedchem.2c00788?casa_token=jh1g8UmTdE4AAAAA%3A0i9zZt2kybEwFqfPFfJ0HVuPzoQRpcP1Ajo89655vV3P_0k9ARe-ucUvvGRiOe0pgDfdY4Cw78q4ze1z},
doi = {https://doi.org/10.1021/acs.jmedchem.2c00788},
year = {2022},
date = {2022-01-01},
abstract = {Acute myelogenous leukemia (AML), a disease of the blood and bone marrow, is characterized by the inability of myeloblasts to differentiate into mature cell types. Dihydroorotate dehydrogenase (DHODH) is an enzyme well-known in the pyrimidine biosynthesis pathway; however, small molecule DHODH inhibitors were recently shown to induce differentiation in multiple AML subtypes. Using virtual screening and structure-based drug design approaches, a new series of N-heterocyclic 3-pyridyl carboxamide DHODH inhibitors were discovered. Two lead compounds, 19 and 29, have potent biochemical and cellular DHODH activity, favorable physicochemical properties, and efficacy in a preclinical model of AML.},
keywords = {TEMPEST},
pubstate = {published},
tppubtype = {article}
}
Acute myelogenous leukemia (AML), a disease of the blood and bone marrow, is characterized by the inability of myeloblasts to differentiate into mature cell types. Dihydroorotate dehydrogenase (DHODH) is an enzyme well-known in the pyrimidine biosynthesis pathway; however, small molecule DHODH inhibitors were recently shown to induce differentiation in multiple AML subtypes. Using virtual screening and structure-based drug design approaches, a new series of N-heterocyclic 3-pyridyl carboxamide DHODH inhibitors were discovered. Two lead compounds, 19 and 29, have potent biochemical and cellular DHODH activity, favorable physicochemical properties, and efficacy in a preclinical model of AML. |
Structural and molecular rationale for the diversification of resistance mediated by the Antibiotic_NAT family Stogios, Peter J., Bordeleau, Emily In: 2022. @article{noKey,
title = {Structural and molecular rationale for the diversification of resistance mediated by the Antibiotic_NAT family},
author = {Stogios, Peter J., Bordeleau, Emily},
url = {https://www.nature.com/articles/s42003-022-03219-w},
doi = {https://doi.org/10.1038/s42003-022-03219-w},
year = {2022},
date = {2022-01-01},
abstract = {The environmental microbiome harbors a vast repertoire of antibiotic resistance genes (ARGs) which can serve as evolutionary predecessors for ARGs found in pathogenic bacteria, or can be directly mobilized to pathogens in the presence of selection pressures. Thus, ARGs from benign environmental bacteria are an important resource for understanding clinically relevant resistance. Here, we conduct a comprehensive functional analysis of the Antibiotic_NAT family of aminoglycoside acetyltransferases. We determined a pan-family antibiogram of 21 Antibiotic_NAT enzymes, including 8 derived from clinical isolates and 13 from environmental metagenomic samples. We find that environment-derived representatives confer high-level, broad-spectrum resistance, including against the atypical aminoglycoside apramycin, and that a metagenome-derived gene likely is ancestral to an aac(3) gene found in clinical isolates. Through crystallographic analysis, we rationalize the molecular basis for diversification of substrate specificity across the family. This work provides critical data on the molecular mechanism underpinning resistance to established and emergent aminoglycoside antibiotics and broadens our understanding of ARGs in the environment.},
keywords = {TEMPEST},
pubstate = {published},
tppubtype = {article}
}
The environmental microbiome harbors a vast repertoire of antibiotic resistance genes (ARGs) which can serve as evolutionary predecessors for ARGs found in pathogenic bacteria, or can be directly mobilized to pathogens in the presence of selection pressures. Thus, ARGs from benign environmental bacteria are an important resource for understanding clinically relevant resistance. Here, we conduct a comprehensive functional analysis of the Antibiotic_NAT family of aminoglycoside acetyltransferases. We determined a pan-family antibiogram of 21 Antibiotic_NAT enzymes, including 8 derived from clinical isolates and 13 from environmental metagenomic samples. We find that environment-derived representatives confer high-level, broad-spectrum resistance, including against the atypical aminoglycoside apramycin, and that a metagenome-derived gene likely is ancestral to an aac(3) gene found in clinical isolates. Through crystallographic analysis, we rationalize the molecular basis for diversification of substrate specificity across the family. This work provides critical data on the molecular mechanism underpinning resistance to established and emergent aminoglycoside antibiotics and broadens our understanding of ARGs in the environment. |
Bioluminescent Zebrafish Transplantation Model for Drug Discovery Hason, Martina, Jovicic, Jovana In: 2022. @article{noKey,
title = {Bioluminescent Zebrafish Transplantation Model for Drug Discovery},
author = {Hason, Martina, Jovicic, Jovana},
url = {https://www.frontiersin.org/articles/10.3389/fphar.2022.893655/full},
doi = {https://doi.org/10.3389/fphar.2022.893655},
year = {2022},
date = {2022-01-01},
abstract = {In the last decade, zebrafish have accompanied the mouse as a robust animal model for cancer research. The possibility of screening small-molecule inhibitors in a large number of zebrafish embryos makes this model particularly valuable. However, the dynamic visualization of fluorescently labeled tumor cells needs to be complemented by a more sensitive, easy, and rapid mode for evaluating tumor growth in vivo to enable high-throughput screening of clinically relevant drugs. In this study we proposed and validated a pre-clinical screening model for drug discovery by utilizing bioluminescence as our readout for the determination of transplanted cancer cell growth and inhibition in zebrafish embryos. For this purpose, we used NanoLuc luciferase, which ensured rapid cancer cell growth quantification in vivo with high sensitivity and low background when compared to conventional fluorescence measurements. This allowed us large-scale evaluation of in vivo drug responses of 180 kinase inhibitors in zebrafish. Our bioluminescent screening platform could facilitate identification of new small-molecules for targeted cancer therapy as well as for drug repurposing.},
keywords = {TEMPEST},
pubstate = {published},
tppubtype = {article}
}
In the last decade, zebrafish have accompanied the mouse as a robust animal model for cancer research. The possibility of screening small-molecule inhibitors in a large number of zebrafish embryos makes this model particularly valuable. However, the dynamic visualization of fluorescently labeled tumor cells needs to be complemented by a more sensitive, easy, and rapid mode for evaluating tumor growth in vivo to enable high-throughput screening of clinically relevant drugs. In this study we proposed and validated a pre-clinical screening model for drug discovery by utilizing bioluminescence as our readout for the determination of transplanted cancer cell growth and inhibition in zebrafish embryos. For this purpose, we used NanoLuc luciferase, which ensured rapid cancer cell growth quantification in vivo with high sensitivity and low background when compared to conventional fluorescence measurements. This allowed us large-scale evaluation of in vivo drug responses of 180 kinase inhibitors in zebrafish. Our bioluminescent screening platform could facilitate identification of new small-molecules for targeted cancer therapy as well as for drug repurposing. |
Anti-membrane Antibodies Persist at Least One Year and Discriminate Between Past Coronavirus Disease 2019 Infection and Vaccination Amjadi, Maya F. In: 2022. @article{noKey,
title = {Anti-membrane Antibodies Persist at Least One Year and Discriminate Between Past Coronavirus Disease 2019 Infection and Vaccination},
author = {Amjadi, Maya F.},
url = {https://academic.oup.com/jid/advance-article/doi/10.1093/infdis/jiac263/6618636?login=true},
doi = {https://doi.org/10.1093/infdis/jiac263},
year = {2022},
date = {2022-01-01},
abstract = {Background The consequences of past coronavirus disease 2019 (COVID-19) infection for personal and population health are emerging, but accurately identifying distant infection is a challenge. Anti-spike antibodies rise after both vaccination and infection and anti-nucleocapsid antibodies rapidly decline. Methods We evaluated anti-membrane antibodies in COVID-19 naive, vaccinated, and convalescent subjects to determine if they persist and accurately detect distant infection. Results We found that anti-membrane antibodies persist for at least 1 year and are a sensitive and specific marker of past COVID-19 infection. Conclusions Thus, anti-membrane and anti-spike antibodies together can differentiate between COVID-19 convalescent, vaccinated, and naive states to advance public health and research.},
keywords = {TEMPEST},
pubstate = {published},
tppubtype = {article}
}
Background The consequences of past coronavirus disease 2019 (COVID-19) infection for personal and population health are emerging, but accurately identifying distant infection is a challenge. Anti-spike antibodies rise after both vaccination and infection and anti-nucleocapsid antibodies rapidly decline. Methods We evaluated anti-membrane antibodies in COVID-19 naive, vaccinated, and convalescent subjects to determine if they persist and accurately detect distant infection. Results We found that anti-membrane antibodies persist for at least 1 year and are a sensitive and specific marker of past COVID-19 infection. Conclusions Thus, anti-membrane and anti-spike antibodies together can differentiate between COVID-19 convalescent, vaccinated, and naive states to advance public health and research. |
Identification of Two Non-Peptidergic Small Molecule Inhibitors of CBX2 Binding to K27 Trimethylated Oligonucleosomes Lercher, Lukas, Simon, Nina In: 2022. @article{noKey,
title = {Identification of Two Non-Peptidergic Small Molecule Inhibitors of CBX2 Binding to K27 Trimethylated Oligonucleosomes},
author = {Lercher, Lukas, Simon, Nina},
url = {https://www.sciencedirect.com/science/article/pii/S2472555222136713},
doi = {https://doi.org/10.1016/j.slasd.2022.04.003},
year = {2022},
date = {2022-01-01},
abstract = {The dysregulation of the PRC1/2 complex plays a key role in lineage plasticity in prostate cancer and may be required to maintain neuroendocrine phenotype. [1] CBX2, a key component of the canonical PRC1 complex, is an epigenetic reader, recognizing trimethylated lysine on histone 3 (H3K27me3) [2] and is overexpressed in metastatic neuroendocrine prostate cancer. [3,4] We implemented a screening strategy using nucleosome substrates to identify inhibitors of CBX2 binding to chromatin. Construct design and phosphorylation state of CBX2 were critical for successful implementation and execution of an HTS library screen. A rigorous screening funnel including counter and selectivity assays allowed us to quickly focus on true positive hit matter. Two distinct non-peptide-like chemotypes were identified and confirmed in orthogonal biochemical and biophysical assays demonstrating disruption of CBX2 binding to nucleosomes and direct binding to purified CBX2, respectively.},
keywords = {TEMPEST},
pubstate = {published},
tppubtype = {article}
}
The dysregulation of the PRC1/2 complex plays a key role in lineage plasticity in prostate cancer and may be required to maintain neuroendocrine phenotype. [1] CBX2, a key component of the canonical PRC1 complex, is an epigenetic reader, recognizing trimethylated lysine on histone 3 (H3K27me3) [2] and is overexpressed in metastatic neuroendocrine prostate cancer. [3,4] We implemented a screening strategy using nucleosome substrates to identify inhibitors of CBX2 binding to chromatin. Construct design and phosphorylation state of CBX2 were critical for successful implementation and execution of an HTS library screen. A rigorous screening funnel including counter and selectivity assays allowed us to quickly focus on true positive hit matter. Two distinct non-peptide-like chemotypes were identified and confirmed in orthogonal biochemical and biophysical assays demonstrating disruption of CBX2 binding to nucleosomes and direct binding to purified CBX2, respectively. |
A high throughput screen with a clonogenic endpoint to identify radiation modulators of cancer Gomes, Nathan P. In: 2022. @article{noKey,
title = {A high throughput screen with a clonogenic endpoint to identify radiation modulators of cancer},
author = {Gomes, Nathan P.},
url = {https://www.biorxiv.org/content/10.1101/2022.05.24.493331v2.full},
doi = {https://doi.org/10.1667/RADE-22-00086.1},
year = {2022},
date = {2022-01-01},
abstract = {Clonogenic assays evaluate the ability of single cells to proliferate and form colonies. This process approximates the regrowth and recurrence of tumors after treatment with radiation or chemotherapy, and thereby provides a drug discovery platform for compounds that block this process. However, because of their labor-intensive and cumbersome nature, adapting canonical clonogenic assays for high throughput screening (HTS) has been challenging. We overcame these barriers by developing an integrated system that automates cell- and liquid-handling, irradiation, dosimetry, drug administration, and incubation. Further, we developed a fluorescent live-cell based automated colony scoring methodology that identifies and counts colonies precisely based upon actual nuclei number rather than colony area, thereby eliminating errors in colony counts caused by radiation induced changes in colony morphology. We identified 13 cell lines from 7 cancer types, where radiation is a standard treatment module, that exhibit identical radiation and chemoradiation response regardless of well format and are amenable to miniaturization into small-well HTS formats. We performed pilot screens through a 1584 compound NCI Diversity Set library using two cell lines representing different cancer indications. Radiation modulators identified in the pilot screens were validated in traditional clonogenic assays, providing proof-of-concept for the screen. The integrated methodology, hereafter ‘clonogenic HTS’, exhibits excellent robustness (Z’ values >0.5) and shows high reproducibility (>95%). We propose that clonogenic HTS we developed can function as a drug discovery platform to identify compounds that inhibit tumor regrowth following radiation therapy, to identify new efficacious pair-wise combinations of known oncologic therapies, or to identify novel modulators of approved therapies.},
keywords = {TEMPEST},
pubstate = {published},
tppubtype = {article}
}
Clonogenic assays evaluate the ability of single cells to proliferate and form colonies. This process approximates the regrowth and recurrence of tumors after treatment with radiation or chemotherapy, and thereby provides a drug discovery platform for compounds that block this process. However, because of their labor-intensive and cumbersome nature, adapting canonical clonogenic assays for high throughput screening (HTS) has been challenging. We overcame these barriers by developing an integrated system that automates cell- and liquid-handling, irradiation, dosimetry, drug administration, and incubation. Further, we developed a fluorescent live-cell based automated colony scoring methodology that identifies and counts colonies precisely based upon actual nuclei number rather than colony area, thereby eliminating errors in colony counts caused by radiation induced changes in colony morphology. We identified 13 cell lines from 7 cancer types, where radiation is a standard treatment module, that exhibit identical radiation and chemoradiation response regardless of well format and are amenable to miniaturization into small-well HTS formats. We performed pilot screens through a 1584 compound NCI Diversity Set library using two cell lines representing different cancer indications. Radiation modulators identified in the pilot screens were validated in traditional clonogenic assays, providing proof-of-concept for the screen. The integrated methodology, hereafter ‘clonogenic HTS’, exhibits excellent robustness (Z’ values >0.5) and shows high reproducibility (>95%). We propose that clonogenic HTS we developed can function as a drug discovery platform to identify compounds that inhibit tumor regrowth following radiation therapy, to identify new efficacious pair-wise combinations of known oncologic therapies, or to identify novel modulators of approved therapies. |
Structure–Activity Relationship of para-Carborane Selective Estrogen Receptor β Agonists Sedlak, David In: 2021. @article{noKey,
title = {Structure–Activity Relationship of para-Carborane Selective Estrogen Receptor β Agonists},
author = {Sedlak, David},
url = {https://pubs.acs.org/doi/full/10.1021/acs.jmedchem.1c00555},
doi = {https://doi.org/10.1021/acs.jmedchem.1c00555},
year = {2021},
date = {2021-01-01},
abstract = {Selective agonism of the estrogen receptor (ER) subtypes, ERα and ERβ, has historically been difficult to achieve due to the high degree of ligand-binding domain structural similarity. Multiple efforts have focused on the use of classical organic scaffolds to model 17β-estradiol geometry in the design of ERβ selective agonists, with several proceeding to various stages of clinical development. Carborane scaffolds offer many unique advantages including the potential for novel ligand/receptor interactions but remain relatively unexplored. We synthesized a series of para-carborane estrogen receptor agonists revealing an ERβ selective structure–activity relationship. We report ERβ agonists with low nanomolar potency, greater than 200-fold selectivity for ERβ over ERα, limited off-target activity against other nuclear receptors, and only sparse CYP450 inhibition at very high micromolar concentrations. The pharmacological properties of our para-carborane ERβ selective agonists measure favorably against clinically developed ERβ agonists and support further evaluation of carborane-based selective estrogen receptor modulators.},
keywords = {TEMPEST},
pubstate = {published},
tppubtype = {article}
}
Selective agonism of the estrogen receptor (ER) subtypes, ERα and ERβ, has historically been difficult to achieve due to the high degree of ligand-binding domain structural similarity. Multiple efforts have focused on the use of classical organic scaffolds to model 17β-estradiol geometry in the design of ERβ selective agonists, with several proceeding to various stages of clinical development. Carborane scaffolds offer many unique advantages including the potential for novel ligand/receptor interactions but remain relatively unexplored. We synthesized a series of para-carborane estrogen receptor agonists revealing an ERβ selective structure–activity relationship. We report ERβ agonists with low nanomolar potency, greater than 200-fold selectivity for ERβ over ERα, limited off-target activity against other nuclear receptors, and only sparse CYP450 inhibition at very high micromolar concentrations. The pharmacological properties of our para-carborane ERβ selective agonists measure favorably against clinically developed ERβ agonists and support further evaluation of carborane-based selective estrogen receptor modulators. |
Development and Evaluation of a Microbial Natural Product Prefractionation Library Pallant, Daniel In: 2021. @article{noKey,
title = {Development and Evaluation of a Microbial Natural Product Prefractionation Library},
author = {Pallant, Daniel},
url = {https://macsphere.mcmaster.ca/handle/11375/27282},
doi = {http://hdl.handle.net/11375/27282},
year = {2021},
date = {2021-01-01},
abstract = {Ongoing antibiotic drug discovery is vital as antimicrobial resistance continues to be a significant issue faced in the clinic. Natural products have long been a highly productive source to mine for new antimicrobials. While it has been challenging to discover new and unique antimicrobial natural products, numerous drugs have been derived from studying how natural products function as secondary metabolites. Previous studies suggested that screening natural product extract fraction libraries for antimicrobials can be more productive than screening crude extracts alone. These studies from large industrial enterprises are generally not directly portable to an academic setting due to significant infrastructure costs. We developed a screening platform consisting of low pressure reversed-phase chromatographic separation of methanolic extracts of bacteria and fungi to generate a prefractionated natural product library. This platform is suitable for academic labs to screen for antimicrobial compounds. A large growth inhibitor screen against multiple pathogens and lab strains of microbes was conducted to assess the validity of the advantages of screening fraction libraries versus crude extract libraries and to search for potential new drug-like compounds. Hits were investigated for reproducibility, isolated, and purified. One compound was discovered in an antifungal screen which may be a novel lipopeptide.},
keywords = {TEMPEST},
pubstate = {published},
tppubtype = {article}
}
Ongoing antibiotic drug discovery is vital as antimicrobial resistance continues to be a significant issue faced in the clinic. Natural products have long been a highly productive source to mine for new antimicrobials. While it has been challenging to discover new and unique antimicrobial natural products, numerous drugs have been derived from studying how natural products function as secondary metabolites. Previous studies suggested that screening natural product extract fraction libraries for antimicrobials can be more productive than screening crude extracts alone. These studies from large industrial enterprises are generally not directly portable to an academic setting due to significant infrastructure costs. We developed a screening platform consisting of low pressure reversed-phase chromatographic separation of methanolic extracts of bacteria and fungi to generate a prefractionated natural product library. This platform is suitable for academic labs to screen for antimicrobial compounds. A large growth inhibitor screen against multiple pathogens and lab strains of microbes was conducted to assess the validity of the advantages of screening fraction libraries versus crude extract libraries and to search for potential new drug-like compounds. Hits were investigated for reproducibility, isolated, and purified. One compound was discovered in an antifungal screen which may be a novel lipopeptide. |
