Development of a sensitive high-throughput enzymatic assay capable of measuring sub-nanomolar inhibitors of SARS-CoV2 Mpro Kovar, Peter In: 2024. @article{noKey,
title = {Development of a sensitive high-throughput enzymatic assay capable of measuring sub-nanomolar inhibitors of SARS-CoV2 Mpro},
author = {Kovar, Peter},
url = {https://www.sciencedirect.com/science/article/pii/S2472555224000418},
doi = {https://doi.org/10.1016/j.slasd.2024.100179},
year = {2024},
date = {2024-09-01},
abstract = {The SARS-CoV-2 main protease (Mpro) is essential for viral replication because it is responsible for the processing of most of the non-structural proteins encoded by the virus. Inhibition of Mpro prevents viral replication and therefore constitutes an attractive antiviral strategy. We set out to develop a high-throughput Mpro enzymatic activity assay using fluorescently labeled peptide substrates. A library of fluorogenic substrates of various lengths, sequences and dye/quencher positions was prepared and tested against full length SARS-CoV-2 Mpro enzyme for optimal activity. The addition of buffers containing strongly hydrated kosmotropic anion salts, such as citrate, from the Hofmeister series significantly boosted the enzyme activity and enhanced the assay detection limit, enabling the ranking of sub-nanomolar inhibitors without relying on the low-throughput Morrison equation method. By comparing cooperativity in citrate or non-citrate buffer while titrating the Mpro enzyme concentration, we found full positive cooperativity of Mpro with citrate buffer at less than one nanomolar (nM), but at a much higher enzyme concentration (∼320 nM) with non-citrate buffer. In addition, using a tight binding Mpro inhibitor, we confirmed there was only one active catalytical site in each Mpro monomer. Since cooperativity requires at least two binding sites, we hypothesized that citrate facilitates dimerization of Mpro at sub-nanomolar concentration as one of the mechanisms enhances Mpro catalytic efficiency. This assay has been used in high-throughput screening and structure activity relationship (SAR) studies to support medicinal chemistry efforts. IC50 values determined in this assay correlates well with EC50 values generated by a SARS-CoV-2 antiviral assay after adjusted for cell penetration.},
keywords = {TEMPEST},
pubstate = {published},
tppubtype = {article}
}
The SARS-CoV-2 main protease (Mpro) is essential for viral replication because it is responsible for the processing of most of the non-structural proteins encoded by the virus. Inhibition of Mpro prevents viral replication and therefore constitutes an attractive antiviral strategy. We set out to develop a high-throughput Mpro enzymatic activity assay using fluorescently labeled peptide substrates. A library of fluorogenic substrates of various lengths, sequences and dye/quencher positions was prepared and tested against full length SARS-CoV-2 Mpro enzyme for optimal activity. The addition of buffers containing strongly hydrated kosmotropic anion salts, such as citrate, from the Hofmeister series significantly boosted the enzyme activity and enhanced the assay detection limit, enabling the ranking of sub-nanomolar inhibitors without relying on the low-throughput Morrison equation method. By comparing cooperativity in citrate or non-citrate buffer while titrating the Mpro enzyme concentration, we found full positive cooperativity of Mpro with citrate buffer at less than one nanomolar (nM), but at a much higher enzyme concentration (∼320 nM) with non-citrate buffer. In addition, using a tight binding Mpro inhibitor, we confirmed there was only one active catalytical site in each Mpro monomer. Since cooperativity requires at least two binding sites, we hypothesized that citrate facilitates dimerization of Mpro at sub-nanomolar concentration as one of the mechanisms enhances Mpro catalytic efficiency. This assay has been used in high-throughput screening and structure activity relationship (SAR) studies to support medicinal chemistry efforts. IC50 values determined in this assay correlates well with EC50 values generated by a SARS-CoV-2 antiviral assay after adjusted for cell penetration. |
Discovery of an antivirulence compound that targets the Staphylococcus aureus SaeRS two-component system to inhibit toxic shock syndrome toxin-1 production Dufresne, Karine In: 2024. @article{noKey,
title = {Discovery of an antivirulence compound that targets the Staphylococcus aureus SaeRS two-component system to inhibit toxic shock syndrome toxin-1 production},
author = {Dufresne, Karine},
url = {https://www.jbc.org/article/S0021-9258(24)01956-2/fulltext},
doi = {https://doi.org/10.1016/j.jbc.2024.107455},
year = {2024},
date = {2024-07-01},
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 the 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 the 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 the virulence of S. aureus, and 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 the 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 the 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 the virulence of S. aureus, and 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. |
Discovery of JNJ-74856665: A Novel Isoquinolinone DHODH Inhibitor for the Treatment of AML DeRatt, Lindsey G. In: 2024. @article{noKey,
title = {Discovery of JNJ-74856665: A Novel Isoquinolinone DHODH Inhibitor for the Treatment of AML},
author = {DeRatt, Lindsey G.},
url = {https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c00809},
doi = {https://doi.org/10.1021/acs.jmedchem.4c00809},
year = {2024},
date = {2024-06-18},
abstract = {Acute myelogenous leukemia (AML), a heterogeneous 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 and preclinical findings demonstrated that DHODH is a metabolic vulnerability in AML as inhibitors can induce differentiation across multiple AML subtypes. As a result of virtual screening and structure-based drug design approaches, a novel series of isoquinolinone DHODH inhibitors was identified. Further lead optimization afforded JNJ-74856665 as an orally bioavailable, potent, and selective DHODH inhibitor with favorable physicochemical properties selected for clinical development in patients with AML and myelodysplastic syndromes (MDS).},
keywords = {TEMPEST},
pubstate = {published},
tppubtype = {article}
}
Acute myelogenous leukemia (AML), a heterogeneous 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 and preclinical findings demonstrated that DHODH is a metabolic vulnerability in AML as inhibitors can induce differentiation across multiple AML subtypes. As a result of virtual screening and structure-based drug design approaches, a novel series of isoquinolinone DHODH inhibitors was identified. Further lead optimization afforded JNJ-74856665 as an orally bioavailable, potent, and selective DHODH inhibitor with favorable physicochemical properties selected for clinical development in patients with AML and myelodysplastic syndromes (MDS). |
Cell analysis Lamond, Angus Iain In: 2024. @article{noKey,
title = {Cell analysis},
author = {Lamond, Angus Iain},
url = {https://patents.google.com/patent/US20240125770A1/en},
doi = {US20240125770A1},
year = {2024},
date = {2024-04-18},
abstract = {Methods of studying eukaryotic cell responses to a perturbation, or of stratifying eukaryotic cells or cell lines into one or more subgroups are described. The methods involve perturbing a library of cells or cell lines in the same manner, and observing how the cells respond to the same perturbation. The observation may be via a high throughput screening method, for example, cell painting; and the perturbation may be, for example, exposure to a therapeutic agent.The methods may be used for grouping cells or cell lines that respond similarly to a given therapeutic agent, which may be useful for identifying patient groups and selecting appropriate treatments.},
keywords = {TEMPEST},
pubstate = {published},
tppubtype = {article}
}
Methods of studying eukaryotic cell responses to a perturbation, or of stratifying eukaryotic cells or cell lines into one or more subgroups are described. The methods involve perturbing a library of cells or cell lines in the same manner, and observing how the cells respond to the same perturbation. The observation may be via a high throughput screening method, for example, cell painting; and the perturbation may be, for example, exposure to a therapeutic agent.The methods may be used for grouping cells or cell lines that respond similarly to a given therapeutic agent, which may be useful for identifying patient groups and selecting appropriate treatments. |
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 = {2024-02-27},
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. |
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 = {2024-02-07},
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 = {2024-01-04},
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. |
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 = {2023-12-06},
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. |
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 = {2023-12-05},
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. |
Mechanistic plasticity in ApmA enables aminoglycoside promiscuity for resistance Bordeleau, Emily In: 2023. @article{noKey,
title = {Mechanistic plasticity in ApmA enables aminoglycoside promiscuity for resistance},
author = {Bordeleau, Emily},
url = {https://www.nature.com/articles/s41589-023-01483-3},
doi = {https://doi.org/10.1038/s41589-023-01483-3},
year = {2023},
date = {2023-11-16},
abstract = {The efficacy of aminoglycoside antibiotics is waning due to the acquisition of diverse resistance mechanisms by bacteria. Among the most prevalent are aminoglycoside acetyltransferases (AACs) that inactivate the antibiotics through acetyl coenzyme A-mediated modification. Most AACs are members of the GCN5 superfamily of acyltransferases which lack conserved active site residues that participate in catalysis. ApmA is the first reported AAC belonging to the left-handed β-helix superfamily. These enzymes are characterized by an essential active site histidine that acts as an active site base. Here we show that ApmA confers broad-spectrum aminoglycoside resistance with a molecular mechanism that diverges from other detoxifying left-handed β-helix superfamily enzymes and canonical GCN5 AACs. We find that the active site histidine plays different functions depending on the acetyl-accepting aminoglycoside substrate. This flexibility in the mechanism of a single enzyme underscores the plasticity of antibiotic resistance elements to co-opt protein catalysts in the evolution of drug detoxification.},
keywords = {TEMPEST},
pubstate = {published},
tppubtype = {article}
}
The efficacy of aminoglycoside antibiotics is waning due to the acquisition of diverse resistance mechanisms by bacteria. Among the most prevalent are aminoglycoside acetyltransferases (AACs) that inactivate the antibiotics through acetyl coenzyme A-mediated modification. Most AACs are members of the GCN5 superfamily of acyltransferases which lack conserved active site residues that participate in catalysis. ApmA is the first reported AAC belonging to the left-handed β-helix superfamily. These enzymes are characterized by an essential active site histidine that acts as an active site base. Here we show that ApmA confers broad-spectrum aminoglycoside resistance with a molecular mechanism that diverges from other detoxifying left-handed β-helix superfamily enzymes and canonical GCN5 AACs. We find that the active site histidine plays different functions depending on the acetyl-accepting aminoglycoside substrate. This flexibility in the mechanism of a single enzyme underscores the plasticity of antibiotic resistance elements to co-opt protein catalysts in the evolution of drug detoxification. |
Accelerated Hit Identification with Target Evaluation, Deep Learning and Automated Labs: Prospective Validation in IRAK1 Kamuntavicius, Gintautas In: 2023. @article{noKey,
title = {Accelerated Hit Identification with Target Evaluation, Deep Learning and Automated Labs: Prospective Validation in IRAK1},
author = {Kamuntavicius, Gintautas},
url = {https://chemrxiv.org/engage/chemrxiv/article-details/6512960eade1178b2423365f},
doi = {10.26434/chemrxiv-2023-mh22x-v3},
year = {2023},
date = {2023-09-26},
abstract = {In this study, we integrate Ro5’s target evaluation SpectraView and DL-driven virtual screening HydraScreen tools alongside Strateos' robotic cloud labs high-throughput screening platform to accelerate target and hit identification. Using SpectraView to select IRAK1 as the target, we prospectively validate HydraScreen, a structure-based deep learning model. We demonstrate that HydraScreen could identify up to 23.8% of all IRAK1 hits in the top 1% of the ranked compounds, simultaneously identifying the three most potent (nanomolar) scaffolds present in the library. The three nanomolar scaffolds identified in our project are novel for IRAK1 and lend themselves for future development. HydraScreen outperforms traditional virtual screening methods in an unbiased prospective evaluation and offers advanced features such as ligand pose confidence scoring. Thus, SpectraView and HydraScreen are innovative tools which can aid and expedite early stages of drug discovery.},
keywords = {TEMPEST},
pubstate = {published},
tppubtype = {article}
}
In this study, we integrate Ro5’s target evaluation SpectraView and DL-driven virtual screening HydraScreen tools alongside Strateos' robotic cloud labs high-throughput screening platform to accelerate target and hit identification. Using SpectraView to select IRAK1 as the target, we prospectively validate HydraScreen, a structure-based deep learning model. We demonstrate that HydraScreen could identify up to 23.8% of all IRAK1 hits in the top 1% of the ranked compounds, simultaneously identifying the three most potent (nanomolar) scaffolds present in the library. The three nanomolar scaffolds identified in our project are novel for IRAK1 and lend themselves for future development. HydraScreen outperforms traditional virtual screening methods in an unbiased prospective evaluation and offers advanced features such as ligand pose confidence scoring. Thus, SpectraView and HydraScreen are innovative tools which can aid and expedite early stages of drug discovery. |
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 = {2023-09-17},
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. |
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 = {2023-08-28},
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. |
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 = {2023-07-07},
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. |
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 = {2023-06-17},
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. |
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 = {2023-06-06},
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. |
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 = {2023-05-04},
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 = {2023-01-19},
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. |
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 = {2022-08-04},
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. |
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 = {2022-07-12},
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. |
