56 Citations
Prophages dormant bacteriophage genomes integrated within the bacterial chromosome play pivotal roles in shaping microbial communities when awakened Our current understanding of prophage activation is largely shaped by a narrow set of traditional DNA-damaging inducers such as mitomycin C and ciprofloxacin which trigger the bacterial SOS response This study employed high-throughput screening of compounds to identify novel prophage inducers using model lambdoid prophage HK We identified multiple new inducers across diverse pharmacological classes including dietary supplements and therapeutics Despite the variety in compounds all acted through SOS-dependent pathways However bleomycin an antineoplastic antibiotic demonstrated broad-spectrum and potent prophage induction exceeding ... More
Prophages, dormant bacteriophage genomes integrated within the bacterial chromosome, play pivotal roles in shaping microbial communities when awakened. Our current understanding of prophage activation is largely shaped by a narrow set of traditional DNA-damaging inducers, such as mitomycin C and ciprofloxacin, which trigger the bacterial SOS response. This study employed high-throughput screening of 3,921 compounds to identify novel prophage inducers using model lambdoid prophage HK97. We identified multiple new inducers across diverse pharmacological classes, including dietary supplements and therapeutics. Despite the variety in compounds, all acted through SOS-dependent pathways. However, bleomycin, an antineoplastic antibiotic, demonstrated broad-spectrum and potent prophage induction exceeding standard inducers, with activity validated across multiple phage-host pairings. These findings expand the repertoire of prophage inducers into commonly ingested xenobiotics and introduce bleomycin as a powerful, cost-effective tool for prophage research.
Around 75% of bacteria carry within them dormant viruses (prophages), which can awaken when the bacterium is stressed, killing the bacterium. Historically, this has been done using DNA-damaging antibiotics, but increasingly, more such signals have been discovered. Here, through a high-throughput screen, we identify phage-waking activity in several commonly consumed compounds, such as the SSRI Prozac, as well as a new DNA-damaging agent that is much more effective in waking phages than the previous gold standard. Less
Around 75% of bacteria carry within them dormant viruses (prophages), which can awaken when the bacterium is stressed, killing the bacterium. Historically, this has been done using DNA-damaging antibiotics, but increasingly, more such signals have been discovered. Here, through a high-throughput screen, we identify phage-waking activity in several commonly consumed compounds, such as the SSRI Prozac, as well as a new DNA-damaging agent that is much more effective in waking phages than the previous gold standard. Less
The increase in multidrug-resistant bacteria underscores an urgent need for additional antibiotics Here we integrate small-molecule high-throughput screening with a deep-learning-based virtual screening approach to uncover new antibacterial compounds We screen million small molecules against a sensitized Escherichia coli strain yielding thousands of hits We use these data to train a deep learning model GNEprop to predict antibacterial activity retrospectively validating robustness with respect to out-of-distribution generalization and activity cliff prediction Virtual screening of over billion synthetically accessible compounds identifies potential candidates of which exhibit antibacterial activity on the same strain illustrating a -fold improved hit rate over the high-throughput ... More
The increase in multidrug-resistant bacteria underscores an urgent need for additional antibiotics. Here, we integrate small-molecule high-throughput screening with a deep-learning-based virtual screening approach to uncover new antibacterial compounds. We screen ~2 million small molecules against a sensitized Escherichia coli strain, yielding thousands of hits. We use these data to train a deep learning model, GNEprop, to predict antibacterial activity, retrospectively validating robustness with respect to out-of-distribution generalization and activity cliff prediction. Virtual screening of over 1.4 billion synthetically accessible compounds identifies potential candidates, of which 82 exhibit antibacterial activity on the same strain, illustrating a 90-fold improved hit rate over the high-throughput screening experiment used for training. Many newly identified compounds exhibit high dissimilarity to known antibiotics, potency beyond the training bacterial strain and selectivity. Biological characterization identifies specific, validated targets, indicating promising avenues for further exploration in antibiotic discovery. Less
Discovery and artificial intelligence-guided mechanistic elucidation of a narrow-spectrum antibiotic
Current clinical antibiotics are largely broad-spectrum agents that can alter the gut microbiome and promote colonization by Enterobacteriaceae which are often drug resistant This includes adherent-invasive Escherichia coli AIEC particularly in patients with inflammatory bowel disease in which dysbiosis creates a niche for this pathogen to colonize There is an urgent and unmet need for novel narrow-spectrum and microbiome-sparing antibiotics Here we screened bioactive small molecules for antibacterial activity against AIEC and discovered enterololin an antibacterial compound with targeted activity against Enterobacteriaceae species Enterololin could overcome intrinsic and acquired resistance mechanisms in clinical isolates when combined with a subinhibitory concentration ... More
Current clinical antibiotics are largely broad-spectrum agents that can alter the gut microbiome and promote colonization by Enterobacteriaceae, which are often drug resistant. This includes adherent-invasive Escherichia coli (AIEC), particularly in patients with inflammatory bowel disease, in which dysbiosis creates a niche for this pathogen to colonize. There is an urgent and unmet need for novel narrow-spectrum and microbiome-sparing antibiotics. Here we screened 10,747 bioactive small molecules for antibacterial activity against AIEC and discovered enterololin, an antibacterial compound with targeted activity against Enterobacteriaceae species. Enterololin could overcome intrinsic and acquired resistance mechanisms in clinical isolates when combined with a subinhibitory concentration of SPR741, a polymyxin B analogue used here to increase outer membrane permeability in Gram-negative bacteria. Molecular substructure- and deep learning-guided mechanism-of-action investigations revealed that enterololin perturbs lipoprotein trafficking through a mechanism involving the LolCDE complex, laboratory-evolved resistant mutants predominantly mapped to lolC and lolE, with an in vitro frequency of resistance of ~10-8 to 10-7. Enterololin showed low mammalian cytotoxicity (HEK293 half-maximal inhibitory concentration ~100 µg ml-1) and suppressed AIEC infection in mouse models when administered in combination with SPR741, while largely preserving the overall microbiome composition. This study highlights the utility of deep learning methods for predicting molecular interactions and identifies a promising Enterobacteriaceae-specific antibacterial candidate for further development. Less
Flap endonuclease FEN is a long-standing target of interest in the DNA damage response DDR field due to its therapeutic potential in BRCA mutant cancers To-date there have only been a handful of FEN inhibitors reported in the literature most of which display modest selectivity and or weak cellular activity As such there is a need for more advanced pharmacological tools to probe the biology of FEN Here we report the discovery of MSC the first potent selective and orally bioavailable FEN inhibitor We describe our metal-chelating fragment screening approach and structure-based optimization to identify MSC using structural insights to ... More
Flap endonuclease 1 (FEN1) is a long-standing target of interest in the DNA damage response (DDR) field due to its therapeutic potential in BRCA mutant cancers. To-date there have only been a handful of FEN1 inhibitors reported in the literature, most of which display modest selectivity and/or weak cellular activity. As such, there is a need for more advanced pharmacological tools to probe the biology of FEN1. Here, we report the discovery of MSC778, the first potent, selective, and orally bioavailable FEN1 inhibitor. We describe our metal-chelating fragment screening approach and structure-based optimization to identify MSC778, using structural insights to drive design. Consistent with FEN1 inhibition, MSC778 selectively kills BRCA2-deficient cells and potentiates the activity of PARPi niraparib in vivo to induce tumor stasis in a BRCA2 KO DLD-1 mouse xenograft. Furthermore, we illustrate how development of this approach has the potential for addressing nucleases as a target class. Less
An enzyme activity-based workflow for the identification and characterization of covalent inhibitors
The field of covalent drug development has advanced rapidly offering promising therapeutic potential due to the ability of these drugs to form slowly reversible or irreversible bonds with target proteins resulting in prolonged pharmacodynamic effects This distinctive mechanism of action has sparked resurging interest in covalent inhibitors across various disease areas including oncology neurological disorders and infectious diseases However characterization of covalent inhibitors poses unique challenges highlighting the need for simplified and robust assay methods This protocol describes an enzyme activity-based workflow designed to identify and characterize covalent inhibitors efficiently By streamlining the evaluation process this approach enhances the reliability ... More
The field of covalent drug development has advanced rapidly, offering promising therapeutic potential due to the ability of these drugs to form slowly reversible or irreversible bonds with target proteins, resulting in prolonged pharmacodynamic effects. This distinctive mechanism of action has sparked resurging interest in covalent inhibitors across various disease areas, including oncology, neurological disorders, and infectious diseases. However, characterization of covalent inhibitors poses unique challenges, highlighting the need for simplified and robust assay methods. This protocol describes an enzyme activity-based workflow designed to identify and characterize covalent inhibitors efficiently. By streamlining the evaluation process, this approach enhances the reliability and reproducibility of covalent inhibitor assessment, ultimately accelerating the discovery and optimization of novel covalent therapeutics. Less
Hydrogen sulfide H S is an endogenous gasotransmitter with cardioprotective and antiviral effects In this work new cysteine-selective nucleoside-H S-donor hybrid molecules were prepared by conjugating nucleoside biomolecules with a thiol-activatable dithioacetyl group -Dithioacetate derivatives were synthesized from the canonical nucleosides uridine adenosine cytidine guanosine and thymidine and the putative -thio metabolites were also produced from uridine and adenosine According to our measurements made with an H S-specific sensor nucleoside dithioacetates are moderately fast H S donors the guanosine derivative showed the fastest kinetics and the adenosine derivative the slowest The antioxidant activity of -thionucleosides is significantly higher than that ... More
Hydrogen sulfide (H2S) is an endogenous gasotransmitter with cardioprotective and antiviral effects. In this work, new cysteine-selective nucleoside-H2S-donor hybrid molecules were prepared by conjugating nucleoside biomolecules with a thiol-activatable dithioacetyl group. 5′-Dithioacetate derivatives were synthesized from the canonical nucleosides (uridine, adenosine, cytidine, guanosine and thymidine), and the putative 5′-thio metabolites were also produced from uridine and adenosine. According to our measurements made with an H2S-specific sensor, nucleoside dithioacetates are moderately fast H2S donors, the guanosine derivative showed the fastest kinetics and the adenosine derivative the slowest. The antioxidant activity of 5′-thionucleosides is significantly higher than that of trolox, but lower than that of ascorbic acid, while intact dithioacetates have no remarkable antioxidant effect. In human Calu cells, the guanosine derivative showed a moderate anti-SARS-CoV-2 effect which was also confirmed by virus yield reduction assay. Dithioacetyl-adenosine and its metabolite showed similar acute cardiac effects as adenosine, however, it is noteworthy that both 5′-thio modified adenosines increased left ventricular ejection fraction or stroke volume, which was not observed with native adenosine. Less
The rise of drug-resistant fungal pathogens including Candida auris highlights the urgent need for novel antifungal therapies We developed a cost-effective platform combining microbial extract prefractionation with rapid MS MS-bioinformatics-based dereplication to efficiently prioritize new antifungal scaffolds Screening C auris and C albicans revealed novel lipopeptaibiotics coniotins from Coniochaeta hoffmannii WAC which were undetectable in crude extracts Coniotins exhibited potent activity against critical fungal pathogens on the WHO Fungal Priority Pathogens List including C albicans C neoformans multidrug-resistant C auris and Aspergillus fumigatus with high selectivity and low resistance potential Coniotin A targets -glucan compromising fungal cell wall integrity remodelling ... More
The rise of drug-resistant fungal pathogens, including Candida auris, highlights the urgent need for novel antifungal therapies. We developed a cost-effective platform combining microbial extract prefractionation with rapid MS/MS-bioinformatics-based dereplication to efficiently prioritize new antifungal scaffolds. Screening C. auris and C. albicans revealed novel lipopeptaibiotics, coniotins, from Coniochaeta hoffmannii WAC11161, which were undetectable in crude extracts. Coniotins exhibited potent activity against critical fungal pathogens on the WHO Fungal Priority Pathogens List, including C. albicans, C. neoformans, multidrug-resistant C. auris, and Aspergillus fumigatus, with high selectivity and low resistance potential. Coniotin A targets β-glucan, compromising fungal cell wall integrity, remodelling, and sensitizing C. auris to caspofungin. Identification of a PKS-NRPS biosynthetic gene cluster further enables the discovery of related clusters encoding potential novel lipopeptaibiotics. This study demonstrates the power of natural product prefractionation in uncovering bioactive scaffolds and introduces coniotins as promising candidates for combating multidrug-resistant fungal pathogens. Less
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 ... More
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. Less
In this study we integrate Ro 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 IRAK as the target we prospectively validate HydraScreen a structure-based deep learning model We demonstrate that HydraScreen could identify up to of all IRAK hits in the top 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 IRAK and lend themselves for future development HydraScreen outperforms traditional virtual ... More
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. Less
A series of amides of selected plant triterpenoids moronic acid and morolic acid with the tripeptides MAG and GAM was designed and synthesized Two required tripeptides and were synthesized by a step-wise chain elongation of the ethyl esters of either glycine or L-methionine at their N-terminus using Boc-protected amino acids in each step The tripeptides and were used for the synthesis of the derivatives of moronic acid and morolic acid to get a series of amide derivatives of the less frequently studied triterpenoids and The target compounds and their intermediates were subjected to an investigation of their antimicrobial antiviral and ... More
A series of amides of selected plant triterpenoids, moronic acid and morolic acid, with the tripeptides MAG and GAM, was designed and synthesized. Two required tripeptides 5 and 10 were synthesized by a step-wise chain elongation of the ethyl esters of either glycine or L-methionine at their N-terminus using Boc-protected amino acids in each step. The tripeptides 5 and 10 were used for the synthesis of 13–23, the derivatives of moronic acid (11) and morolic acid (12), to get a series of amide derivatives of the less frequently studied triterpenoids 11 and 12. The target compounds, and their intermediates, were subjected to an investigation of their antimicrobial, antiviral and cytotoxic activity. Selectivity of the pharmacological effects was found. Generally, the target compounds inhibited only the G+ microorganisms. Compound 16 inhibited Staphylococcus aureus (I = 99.6%; c = 62.5 μM) and Enterococcus faecalis (I = 85%; c = 250 μM). Several compounds showed moderate antiviral effects, both anti-HIV-1, 19 (EC50 = 57.0 ± 4.1 μM, CC50 > 100 μM), 20 (EC50 = 17.8 ± 2.1 μM, CC50 = 41.0 ± 5.2 μM) and 23 (EC50 = 12.6 ± 0.82 μM, CC50 = 38.0 ± 4.2 μM), and anti-HSV-1, 22 (EC50 = 27.7 ± 3.5 μM, CC50 > 100 μM) and 23 (EC50 = 30.9 ± 3.3 μM, CC50 > 100 μM). The target compounds showed no cytotoxicity in cancer cells, however, several of their intermediates were cytotoxic. Compound 21 showed cytotoxicity in HeLa (IC50 = 7.9 ± 2.1 μM), G-361 (IC50 = 8.0 ± 0.6 μM) and MCF7 (IC50 = 8.6 ± 0.2 μM) cancer cell lines, while being non-toxic in normal fibroblasts (BJ; IC50 > 50 μM). Less
The SARS-CoV- 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- Mpro enzyme for optimal activity The addition of buffers containing strongly hydrated kosmotropic anion salts such as citrate from the Hofmeister series significantly ... More
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. Less
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- antibody a-PD- 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- a next-generation STING agonist Flow cytometry Nanostring ... More
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. Less
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- TSST- superantigen Herein we screened a library of small bioactive molecules for the ability to inhibit transcription of the TSST- gene without inhibiting the growth of S aureus The dominant positive regulator of TSST- is the SaeRS two-component system TCS and we identified phenazopyridine hydrochloride PP-HCl that repressed the production of TSST- by inhibiting the kinase function of SaeS PP-HCl competed with ATP for ... More
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. Less
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- as an orally bioavailable potent and selective DHODH inhibitor with favorable physicochemical properties selected for clinical ... More
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). Less
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 ... More
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. Less
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
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 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 ... More
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. Less
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. Less
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 GCN superfamily of acyltransferases which lack conserved active site residues that participate in catalysis ApmA is the first reported AAC belonging to the left-handed -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 ... More
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. Less
We have adapted the cell painting assay developed by Carpenter and colleagues on cultured U OS 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
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 ... More
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. Less
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 CRL CRBN recruiting PROteolysis TArgeting Chimeras PROTACs Thalidomide derivatives used to generate CRL CRBN recruiting PROTACs have well described intrinsic monovalent degradation profiles by inducing the recruitment of neo-substrates such as GSPT Ikaros and Aiolos We leveraged structural insights from known CRL CRBN neo-substrates to attenuate and indeed remove this monovalent degradation function in well-known ... More
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. Less
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 ... More
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. Less
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 DCD aa 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 ... More
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. Less
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 ... More
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. Less
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 ... More
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. Less
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 -pyridyl carboxamide DHODH inhibitors were discovered Two lead compounds and have potent biochemical and cellular DHODH activity favorable physicochemical properties and efficacy in a preclinical model of AML
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 ... More
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. Less
Background The consequences of past coronavirus disease COVID- 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- 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 year and are a sensitive and specific marker of past COVID- infection Conclusions Thus anti-membrane and anti-spike antibodies together can differentiate between COVID- convalescent vaccinated and naive states to advance public ... More
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. Less
The dysregulation of the PRC complex plays a key role in lineage plasticity in prostate cancer and may be required to maintain neuroendocrine phenotype CBX a key component of the canonical PRC complex is an epigenetic reader recognizing trimethylated lysine on histone H K me and is overexpressed in metastatic neuroendocrine prostate cancer We implemented a screening strategy using nucleosome substrates to identify inhibitors of CBX binding to chromatin Construct design and phosphorylation state of CBX were critical for successful implementation and execution of an HTS library screen A rigorous screening funnel including counter and selectivity assays allowed us to ... More
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. Less
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 ... More
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. Less
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 Antibiotic NAT enzymes including derived from clinical isolates and from environmental metagenomic samples We find that environment-derived representatives confer high-level broad-spectrum resistance including against the atypical aminoglycoside ... More
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. Less
Identification of selective deubiquitinase DUB inhibitors is critical for probe development to further understand and explore DUB biological function Here we detail the optimization and deployment of an in vitro fluorogenic ubiquitin-rhodamine assay to conduct high-throughput screening of a small molecule library against a panel of DUBs In screening the compound library against multiple DUBs in parallel we describe an approach for identifying selective DUB inhibitors and provide a roadmap for enabling selective DUB inhibitor discovery
Deubiquitinating enzymes DUBs are a class of isopeptidases that regulate ubiquitin dynamics through catalytic cleavage of ubiquitin from protein substrates and ubiquitin precursors Despite growing interest in DUB biological function and potential as therapeutic targets few selective small-molecule inhibitors and no approved drugs currently exist To identify chemical scaffolds targeting specific DUBs and establish a broader framework for future inhibitor development across the gene family we performed high-throughput screening of a chemically diverse small-molecule library against eight different DUBs spanning three well-characterized DUB families Promising hit compounds were validated in a series of counter-screens and orthogonal assays as well as ... More
Deubiquitinating enzymes (DUBs) are a class of isopeptidases that regulate ubiquitin dynamics through catalytic cleavage of ubiquitin from protein substrates and ubiquitin precursors. Despite growing interest in DUB biological function and potential as therapeutic targets, few selective small-molecule inhibitors and no approved drugs currently exist. To identify chemical scaffolds targeting specific DUBs and establish a broader framework for future inhibitor development across the gene family, we performed high-throughput screening of a chemically diverse small-molecule library against eight different DUBs, spanning three well-characterized DUB families. Promising hit compounds were validated in a series of counter-screens and orthogonal assays, as well as further assessed for selectivity across expanded panels of DUBs. Through these efforts, we have identified multiple highly selective DUB inhibitors and developed a roadmap for rapidly identifying and validating selective inhibitors of related enzymes. Less
The cancer-associated fibroblast CAF marker podoplanin PDPN is generally correlated with poor clinical outcomes in cancer patients and thus represents a promising therapeutic target Despite its biomedical relevance basic aspects of PDPN biology such as its cellular functions and cell surface ligands remain poorly uncharacterized thus challenging drug development Here we utilize a high throughput platform to elucidate the PDPN cell surface interactome and uncover the neutrophil protein CD as a new binding partner Quantitative proteomics analysis of the CAF phosphoproteome reveals a role for PDPN in cell signaling growth and actomyosin contractility among other processes Moreover cellular assays demonstrate ... More
The cancer-associated fibroblast (CAF) marker podoplanin (PDPN) is generally correlated with poor clinical outcomes in cancer patients and thus represents a promising therapeutic target. Despite its biomedical relevance, basic aspects of PDPN biology such as its cellular functions and cell surface ligands remain poorly uncharacterized, thus challenging drug development. Here, we utilize a high throughput platform to elucidate the PDPN cell surface interactome, and uncover the neutrophil protein CD177 as a new binding partner. Quantitative proteomics analysis of the CAF phosphoproteome reveals a role for PDPN in cell signaling, growth and actomyosin contractility, among other processes. Moreover, cellular assays demonstrate that CD177 is a functional antagonist, recapitulating the phenotype observed in PDPN-deficient CAFs. In sum, starting from the unbiased elucidation of the PDPN co-receptome, our work provides insights into PDPN functions and reveals the PDPN/CD177 axis as a possible modulator of fibroblast physiology in the tumor microenvironment. Less
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 ... More
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. Less
In humans epidermal melanocytes are responsible for skin pigmentation defence against ultraviolet radiation and the deadliest common skin cancer melanoma Although there is substantial overlap in melanocyte development pathways between different model organisms species-dependent differences are frequent and the conservation of these processes in human skin remains unresolved Here we used a single-cell enrichment and RNA-sequencing pipeline to study human epidermal melanocytes directly from the skin capturing transcriptomes across different anatomical sites developmental age sexes and multiple skin tones We uncovered subpopulations of melanocytes that exhibit anatomical site-specific enrichment that occurs during gestation and persists through adulthood The transcriptional signature ... More
In humans, epidermal melanocytes are responsible for skin pigmentation, defence against ultraviolet radiation and the deadliest common skin cancer, melanoma. Although there is substantial overlap in melanocyte development pathways between different model organisms, species-dependent differences are frequent and the conservation of these processes in human skin remains unresolved. Here, we used a single-cell enrichment and RNA-sequencing pipeline to study human epidermal melanocytes directly from the skin, capturing transcriptomes across different anatomical sites, developmental age, sexes and multiple skin tones. We uncovered subpopulations of melanocytes that exhibit anatomical site-specific enrichment that occurs during gestation and persists through adulthood. The transcriptional signature of the volar-enriched subpopulation is retained in acral melanomas. Furthermore, we identified human melanocyte differentiation transcriptional programs that are distinct from gene signatures generated from model systems. Finally, we used these programs to define patterns of dedifferentiation that are predictive of melanoma prognosis and response to immune checkpoint inhibitor therapy. Less
Severe acute respiratory syndrome coronavirus SARS-CoV testing is one component of a multilayered mitigation strategy to enable safe in-person school attendance for the K school population However costs logistics and uncertainty about effectiveness are potential barriers to implementation We assessed early data from the Massachusetts K public school pooled SARS-CoV testing program which incorporates two novel design elements in-school pod pooling for assembling pools of dry anterior nasal swabs from to individuals and positive pool deconvolution using the BinaxNOW antigen rapid diagnostic test Ag RDT to assess the operational and analytical feasibility of this approach Over months individual swabs were ... More
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) testing is one component of a multilayered mitigation strategy to enable safe in-person school attendance for the K–12 school population. However, costs, logistics, and uncertainty about effectiveness are potential barriers to implementation. We assessed early data from the Massachusetts K–12 public school pooled SARS-CoV2 testing program, which incorporates two novel design elements: in-school “pod pooling” for assembling pools of dry anterior nasal swabs from 5 to 10 individuals and positive pool deconvolution using the BinaxNOW antigen rapid diagnostic test (Ag RDT), to assess the operational and analytical feasibility of this approach. Over 3 months, 187,597 individual swabs were tested across 39,297 pools from 738 schools. The pool positivity rate was 0.8%; 98.2% of pools tested negative and 0.2% inconclusive, and 0.8% of pools submitted could not be tested. Of 310 positive pools, 70.6% had an N1 or N2 probe cycle threshold (CT) value of ≤30. In reflex testing (performed on specimens newly collected from members of the positive pool), 92.5% of fully deconvoluted pools with an N1 or N2 target CT of ≤30 identified a positive individual using the BinaxNOW test performed 1 to 3 days later. However, of 124 positive pools with full reflex testing data available for analysis, 32 (25.8%) of BinaxNOW pool deconvolution testing attempts did not identify a positive individual, requiring additional reflex testing. With sufficient staffing support and low pool positivity rates, pooled sample collection and reflex testing were feasible for schools. These early program findings confirm that screening for K–12 students and staff is achievable at scale with a scheme that incorporates in-school pooling, primary testing by reverse transcription-PCR (RT-PCR), and Ag RDT reflex/deconvolution testing. Less
To identify approaches to target DNA repair vulnerabilities in cancer we discovered nanomolar potent selective low molecular weight MW allosteric inhibitors of the polymerase function of DNA polymerase Pol including ART ART inhibits the major Pol -mediated DNA repair process Theta-Mediated End Joining without targeting Non-Homologous End Joining In addition ART elicits DNA damage and synthetic lethality in BRCA - or BRCA -mutant tumour cells and enhances the effects of a PARP inhibitor Genetic perturbation screening revealed that defects in the BP Shieldin complex which cause PARP inhibitor resistance result in in vitro and in vivo sensitivity to small molecule ... More
To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance. Less
Selective agonism of the estrogen receptor ER subtypes ER and ER 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 -estradiol geometry in the design of ER 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 ER selective structure activity relationship We report ER agonists with low nanomolar potency ... More
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. Less
Rationale Clinical interest in combining targeted radionuclide therapies TRT with immunotherapies is growing External beam radiation therapy EBRT activates a type interferon IFN response mediated via stimulator of interferon genes STING and this is critical to its therapeutic interaction with immune checkpoint blockade However little is known about the time course of IFN activation after EBRT or whether this may be induced by decay of a TRT source Methods We examined the IFN response and expression of immune susceptibility markers in B and B melanomas and MOC head and neck cancer murine models using qPCR and western blot For TRT ... More
Rationale: Clinical interest in combining targeted radionuclide therapies (TRT) with immunotherapies is growing. External beam radiation therapy (EBRT) activates a type 1 interferon (IFN1) response mediated via stimulator of interferon genes (STING), and this is critical to its therapeutic interaction with immune checkpoint blockade. However, little is known about the time course of IFN1 activation after EBRT or whether this may be induced by decay of a TRT source. Methods: We examined the IFN1 response and expression of immune susceptibility markers in B78 and B16 melanomas and MOC2 head and neck cancer murine models using qPCR and western blot. For TRT, we used 90Y chelated to NM600, an alkylphosphocholine analog that exhibits selective uptake and retention in tumor cells including B78 and MOC2. Results: We observed significant IFN1 activation in all cell lines, with peak activation in B78, B16, and MOC2 cell lines occurring 7, 7, and 1 days, respectively, following RT for all doses. This effect was STING-dependent. Select IFN response genes remained upregulated at 14 days following RT. IFN1 activation following STING agonist treatment in vitro was identical to RT suggesting time course differences between cell lines were mediated by STING pathway kinetics and not DNA damage susceptibility. In vivo delivery of EBRT and TRT to B78 and MOC2 tumors resulted in a comparable time course and magnitude of IFN1 activation. In the MOC2 model, the combination of 90Y-NM600 and dual checkpoint blockade therapy reduced tumor growth and prolonged survival compared to single agent therapy and cumulative dose equivalent combination EBRT and dual checkpoint blockade therapy. Conclusions: We report the time course of the STING-dependent IFN1 response following radiation in multiple murine tumor models. We show the potential of TRT to stimulate IFN1 activation that is comparable to that observed with EBRT and this may be critical to the therapeutic integration of TRT with immunotherapies. Less
Brain metastases develop in over of advanced melanoma patients and negatively impact quality of life and prognosis In a murine melanoma model we previously showed that an in situ vaccination ISV regimen combining radiation treatment and intratumoral IT injection of immunocytokine IC anti-GD antibody fused to IL along with the immune checkpoint inhibitor anti-CTLA- robustly eliminates peripheral flank tumors but only has modest effects on co-occurring intracranial tumors In this study we investigated the ability of low-dose radiation to the brain to potentiate anti-tumor immunity against a brain tumor when combined with ISV anti-CTLA- B GD immunologically cold melanoma tumor ... More
Brain metastases develop in over 60% of advanced melanoma patients and negatively impact quality of life and prognosis. In a murine melanoma model, we previously showed that an in situ vaccination (ISV) regimen, combining radiation treatment and intratumoral (IT) injection of immunocytokine (IC: anti-GD2 antibody fused to IL2), along with the immune checkpoint inhibitor anti-CTLA-4, robustly eliminates peripheral flank tumors but only has modest effects on co-occurring intracranial tumors. In this study, we investigated the ability of low-dose radiation to the brain to potentiate anti-tumor immunity against a brain tumor when combined with ISV + anti-CTLA-4. B78 (GD2+, immunologically “cold”) melanoma tumor cells were implanted into the flank and the right striatum of the brain in C57BL/6 mice. Flank tumors (50–150 mm3) were treated following a previously optimized ISV regimen [radiation (12 Gy × 1, treatment day 1), IT-IC (50 µg daily, treatment days 6–10), and anti-CTLA-4 (100 µg, treatment days 3, 6, 9)]. Mice that additionally received whole-brain radiation treatment (WBRT, 4 Gy × 1) on day 15 demonstrated significantly increased survival compared to animals that received ISV + anti-CTLA-4 alone, WBRT alone or no treatment (control) (P < 0.001, log-rank test). Timing of WBRT was critical, as WBRT administration on day 1 did not significantly enhance survival compared to ISV + anti-CTLA-4, suggesting that the effect of WBRT on survival might be mediated through immune modulation and not just direct tumor cell cytotoxicity. Modest increases in T cells (CD8+ and CD4+) and monocytes/macrophages (F4/80+) but no changes in FOXP3+ regulatory T cells (Tregs), were observed in brain melanoma tumors with addition of WBRT (on day 15) to ISV + anti-CTLA-4. Cytokine multiplex immunoassay revealed distinct changes in both intracranial melanoma and contralateral normal brain with addition of WBRT (day 15) to ISV + anti-CTLA-4, with notable significant changes in pro-inflammatory (e.g., IFNγ, TNFα and LIX/CXCL5) and suppressive (e.g., IL10, IL13) cytokines as well as chemokines (e.g., IP-10/CXCL10 and MIG/CXCL9). We tested the ability of the alkylphosphocholine analog, NM600, to deliver immunomodulatory radiation to melanoma brain tumors as a targeted radionuclide therapy (TRT). Yttrium-86 (86Y) chelated to NM600 was delivered intravenously by tail vein to mice harboring flank and brain melanoma tumors, and PET imaging demonstrated specific accumulation up to 72 h at each tumor site (∼12:1 brain tumor/brain and ∼8:1 flank tumor/muscle). When NM600 was chelated to therapeutic β-particle-emitting 90Y and administered on treatment day 13, T-cell infiltration and cytokine profiles were altered in melanoma brain tumor, like that observed for WBRT. Overall, our results demonstrate that addition of low-dose radiation, timed appropriately with ISV administration to tumors outside the brain, significantly increases survival in animals co-harboring melanoma brain tumors. This observation has potentially important translational implications as a treatment strategy for increasing the response of tumors in the brain to systemically administered immunotherapies. Less
We previously developed a panel of one-step real-time quantitative reverse transcription PCR one-step qRT-PCR hereafter referred to as qRT-PCR assays to assess compound efficacy However these high-cost conventional qRT-PCR manual assays are not amenable to high-throughput screen HTS analysis in a time-sensitive and complex drug discovery process Here we report the establishment of an automated gene expression platform using in-house lysis conditions that allows the study of various cell lines including primary T cells This process innovation provides the opportunity to perform genotypic profiling in both immunology and oncology therapeutic areas with quantitative studies as part of routine drug discovery ... More
We previously developed a panel of one-step real-time quantitative reverse transcription PCR (one-step qRT-PCR; hereafter referred to as qRT-PCR) assays to assess compound efficacy. However, these high-cost, conventional qRT-PCR manual assays are not amenable to high-throughput screen (HTS) analysis in a time-sensitive and complex drug discovery process. Here, we report the establishment of an automated gene expression platform using in-house lysis conditions that allows the study of various cell lines, including primary T cells. This process innovation provides the opportunity to perform genotypic profiling in both immunology and oncology therapeutic areas with quantitative studies as part of routine drug discovery program support. This newly instituted platform also enables a panel screening strategy to efficiently connect HTS, lead identification, and lead optimization in parallel. Less
Bright yellow BY- tobacco cells combined with the XVE chemically inducible system are one of the most promising plant-based platforms for recombinant protein production This offers a range of benefits including the separation of the cell growth and heterologous gene expression lack of risk of infecting the end product with prions and human viruses or appropriate protein glycosylation and folding However low protein productivity remains a major obstacle that limits the extensive commercialization of bioproduction in plants A number of molecular cell culture and down processing approaches have been made to overcome this problem Media development for the specific nutritional ... More
Bright yellow (BY-2) tobacco cells combined with the XVE chemically inducible system are one of the most promising plant-based platforms for recombinant protein production. This offers a range of benefits, including the separation of the cell growth and heterologous gene expression, lack of risk of infecting the end product with prions and human viruses or appropriate protein glycosylation and folding. However, low protein productivity remains a major obstacle that limits the extensive commercialization of bioproduction in plants. A number of molecular, cell culture and down processing approaches have been made to overcome this problem. Media development for the specific nutritional and hormonal requirements of transgenic plant cells is one of the most efficient cell-culture approaches. We optimized the induction medium towards recombinant protein production in BY-2 and demonstrated the usefulness of evolutionary medium optimization for high-yield protein production in liquid plant cultures. A reliable XVE/GFP model, parallel conducting experiments in a microscale on 96-well plates, and dedicated Gene Game evolutionary optimization software allowed for an effective search of 7611 possible solutions of 11-component media. Within the 4608 formulations tested, the Induct X medium was found with a significant 107.14% increase in protein expression in relation to the standard BY-2 medium. Less
Ageing is characterised by cellular senescence leading to imbalanced tissue maintenance cell death and compromised organ function This is first observed in the thymus the primary lymphoid organ that generates and selects T cells However the molecular and cellular mechanisms underpinning these ageing processes remain unclear Here we show that mouse ageing leads to less efficient T cell selection decreased self-antigen representation and increased T cell receptor repertoire diversity Using a combination of single-cell RNA-seq and lineage-tracing we find that progenitor cells are the principal targets of ageing whereas the function of individual mature thymic epithelial cells is compromised only ... More
Ageing is characterised by cellular senescence, leading to imbalanced tissue maintenance, cell death and compromised organ function. This is first observed in the thymus, the primary lymphoid organ that generates and selects T cells. However, the molecular and cellular mechanisms underpinning these ageing processes remain unclear. Here, we show that mouse ageing leads to less efficient T cell selection, decreased self-antigen representation and increased T cell receptor repertoire diversity. Using a combination of single-cell RNA-seq and lineage-tracing, we find that progenitor cells are the principal targets of ageing, whereas the function of individual mature thymic epithelial cells is compromised only modestly. Specifically, an early-life precursor cell population, retained in the mouse cortex postnatally, is virtually extinguished at puberty. Concomitantly, a medullary precursor cell quiesces, thereby impairing maintenance of the medullary epithelium. Thus, ageing disrupts thymic progenitor differentiation and impairs the core immunological functions of the thymus. Less
Background Radiation therapy RT has the potential to enhance the efficacy of immunotherapy such as checkpoint inhibitors which has dramatically altered the landscape of treatments for many cancers but not yet for pancreatic ductal adenocarcinoma PDAC Our prior studies demonstrated that PD ligand- and indoleamine -dioxygenase IDO were induced on tumor epithelia of PDACs following neoadjuvant therapy including RT suggesting RT may prime PDAC for PD- blockade antibody PD- or IDO inhibitor IDO i treatments In this study we investigated the antitumor efficacy of the combination therapies with radiation and PD- blockade or IDO inhibition or both Methods We developed ... More
Background Radiation therapy (RT) has the potential to enhance the efficacy of immunotherapy, such as checkpoint inhibitors, which has dramatically altered the landscape of treatments for many cancers, but not yet for pancreatic ductal adenocarcinoma (PDAC). Our prior studies demonstrated that PD ligand-1 and indoleamine 2,3-dioxygenase 1 (IDO1) were induced on tumor epithelia of PDACs following neoadjuvant therapy including RT, suggesting RT may prime PDAC for PD-1 blockade antibody (αPD-1) or IDO1 inhibitor (IDO1i) treatments. In this study, we investigated the antitumor efficacy of the combination therapies with radiation and PD-1 blockade or IDO1 inhibition or both. Methods We developed and used a mouse syngeneic orthotopic model of PDAC suitable for hypofractionated RT experiments. Results The combination therapy of αPD-1 and RT improved survival. The dual combination of RT/IDO1i and triple combination of RT/αPD-1/IDO1i did not improve survival compared with RT/αPD-1, although all of these combinations offer similar local tumor control. RT/αPD-1 appeared to result in the best systemic interferon-γ response compared with other treatment groups and the highest local expression of immune-activation genes, including Cd28 and Icos. Conclusion Our RT model allows examining the immune-modulatory effects of RT alone and in combination with immune-checkpoint inhibitors in the pancreas/local microenvironment. This study highlights the importance of choosing the appropriate immune-modulatory agents to be combined with RT to tip the balance toward antitumor adaptive immune responses. Less
Interleukin- IL- is a key cytokine implicated in the pathogenesis of autoimmune disorders including psoriasis and ulcerative colitis Although targeted IL- antibody therapeutics are used clinically there are no small-molecule therapeutics that selectively inhibit IL- signaling To address this gap we developed a high-throughput screening strategy employing an IL- -responsive cell-based luciferase reporter gene assay as the primary screen with cellular cytotoxicity and off-target counter screening assays to identify IL- pathway-specific inhibitors The primary screening assay utilized avian DT cells genetically engineered to overexpress IL- R IL- R STAT and firefly luciferase in a -well format Treatment of these cells ... More
Interleukin-23 (IL-23) is a key cytokine implicated in the pathogenesis of autoimmune disorders, including psoriasis and ulcerative colitis. Although targeted IL-23 antibody therapeutics are used clinically, there are no small-molecule therapeutics that selectively inhibit IL-23 signaling. To address this gap, we developed a high-throughput screening strategy employing an IL-23-responsive cell-based luciferase reporter gene assay as the primary screen, with cellular cytotoxicity and off-target counter screening assays to identify IL-23 pathway-specific inhibitors. The primary screening assay utilized avian DT40 cells, genetically engineered to overexpress IL-23R, IL-12Rβ1, STAT5, and firefly luciferase, in a 1536-well format. Treatment of these cells with IL-23 resulted in the phosphorylation and activation of STAT5, which was completely inhibited by the pan-JAK inhibitor tofacitinib. Assay performance was robust, with signal-to-background >7-fold and Z′ > 0.5 over 40 screening plates (approximately 24,000 compounds), with a hit rate of 5% (>66.9% activity cutoff). Of these 1288 hits, 66% were identified as cytotoxic by incubating the IL-23 reporter cells with compound overnight and measuring cell viability. Further assessment of specificity via examination of impact on off-target IFN-γ signaling eliminated an additional 230 compounds, leaving 209 that were evaluated for dose–response activity. Of these compounds, 24 exhibited IC50 values of <7 µM and ≥80% inhibition of IL-23 activity, with >3-fold selectivity over IFN-γ inhibition, thus representing promising starting points for prospective IL-23 pathway small-molecule inhibitors. Less
Small-molecule drug discovery can be hindered by the formation of aggregates that act as non-selective inhibitors of drug targets Such aggregates appear as false positives in high-throughput screening campaigns and can bedevil structure-activity relationships during compound optimization Protocols are described for resonant waveguide grating RWG and dynamic light scattering DLS as microplate-based high-throughput approaches to identify compound aggregation Resonant waveguide grating and dynamic light scattering give equivalent results for the compound test set as assessed with Bland-Altman analysis
There is an urgent need to develop new methods for male contraception however a major barrier to drug discovery has been the lack of validated targets and the absence of an effective high-throughput phenotypic screening system To address this deficit we developed a fully-automated robotic screening platform that provided quantitative evaluation of compound activity against two key attributes of human sperm function motility and acrosome reaction In order to accelerate contraceptive development we screened the comprehensive collection of molecules that make up the ReFRAME repurposing library comprising nearly all the small molecules that have been approved or have undergone clinical ... More
There is an urgent need to develop new methods for male contraception, however a major barrier to drug discovery has been the lack of validated targets and the absence of an effective high-throughput phenotypic screening system. To address this deficit, we developed a fully-automated robotic screening platform that provided quantitative evaluation of compound activity against two key attributes of human sperm function: motility and acrosome reaction. In order to accelerate contraceptive development, we screened the comprehensive collection of 12,000 molecules that make up the ReFRAME repurposing library, comprising nearly all the small molecules that have been approved or have undergone clinical development, or have significant preclinical profiling. We identified several compounds that potently inhibit motility representing either novel drug candidates or routes to target identification. This platform will now allow for major drug discovery programmes that address the critical gap in the contraceptive portfolio as well as uncover novel human sperm biology. Less
In this chapter we describe Long Fragment Read LFR technology a DNA preprocessing method for genome-wide haplotyping by whole genome sequencing WGS The addition of LFR prior to WGS on any high-throughput DNA sequencer e g Complete Genomics Revolocity BGISEQ- Illumina HiSeq etc enables the assignment of single-nucleotide polymorphisms SNPs and other genomic variants onto contigs representing contiguous DNA from a single parent haplotypes with N lengths of up to Mb Importantly this is achieved independent of any parental sequencing data or knowledge of parental haplotypes Further the nature of this method allows for the correction of most amplification sequencing ... More
In this chapter, we describe Long Fragment Read (LFR) technology, a DNA preprocessing method for genome-wide haplotyping by whole genome sequencing (WGS). The addition of LFR prior to WGS on any high-throughput DNA sequencer (e.g., Complete Genomics Revolocity™, BGISEQ-500, Illumina HiSeq, etc.) enables the assignment of single-nucleotide polymorphisms (SNPs) and other genomic variants onto contigs representing contiguous DNA from a single parent (haplotypes) with N50 lengths of up to ~1 Mb. Importantly, this is achieved independent of any parental sequencing data or knowledge of parental haplotypes. Further, the nature of this method allows for the correction of most amplification, sequencing, and mapping errors, resulting in false-positive error rates as low as 10−9. This method can be employed either manually using hand-held micropipettors or in the preferred, automated manner described below, utilizing liquid-handling robots capable of pipetting in the nanoliter range. Automating the method limits the amount of hands-on time and allows significant reduction in reaction volumes. Further, the cost of LFR, as described in this chapter, is moderate, while it adds invaluable whole genome haplotype data to almost any WGS process. Less
Oral agents targeting Janus-associated kinases JAKs are promising new agents in clinical development To better understand the relationship between JAK inhibition and biological outcome compounds targeting JAKs were evaluated in peripheral human whole blood To date these analyses are low throughput and costly Here we developed a robust -well high-throughput flow-based assay approach to screen small molecules for JAK STAT signaling inhibition in human whole blood This assay platform provides a highly sensitive analysis of signaling events in blood and facilitates measurement of target engagement Further the automation technologies and process optimizations developed here overcame sample integrity handling and multiparametric ... More
Oral agents targeting Janus-associated kinases (JAKs) are promising new agents in clinical development. To better understand the relationship between JAK inhibition and biological outcome, compounds targeting JAKs were evaluated in peripheral human whole blood. To date, these analyses are low throughput and costly. Here, we developed a robust 384-well, high-throughput flow-based assay approach to screen small molecules for JAK/STAT signaling inhibition in human whole blood. This assay platform provides a highly sensitive analysis of signaling events in blood and facilitates measurement of target engagement. Further, the automation technologies and process optimizations developed here overcame sample integrity, handling, and multiparametric data analysis bottlenecks without affecting assay performance. Together these efforts dramatically increased sample throughput compared to conventional manual flow cytometric approaches and enabled development of novel JAK/STAT inhibitors. Less