89 research outputs found
AI is a viable alternative to high throughput screening: a 318-target study
: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNetÂź convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNetÂź model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery
Screening for effective cell-penetrating peptides with minimal impact on epithelial cells and gut commensals <i>in vitro</i>
One of the biggest challenges for oral drug absorption is the epithelial barrier of the gastrointestinal tract. The use of cell-penetrating peptides (CPPs) to modulate the epithelial barrier function is known to be an effective strategy to improve drug absorption and bioavailability. In this study we compare side-by-side, 9 most promising CPPs to study their cytotoxicity (Cytotox Red dye staining) and cell viability (AlamarBlue staining) on epithelial cells and their effects on paracellular permeability of the intestinal barrier in vitro in a differentiated Caco-2 epithelial monolayer model. The data revealed that 4 out of 9 well-studied CPPs significantly improved Caco-2 paracellular permeability without compromising on cellular health. To assess the impact of CPPs on the human microbiota we studied the antimicrobial effects of the 4 effective CPPs from our permeation studies against 10 representative strains of the gut microbiota in vitro using microbroth dilution. Our data revealed that these 4 CPPs affected the growth of almost all tested commensal strains. Interestingly, we found that two synthetic CPPs (Shuffle and Penetramax) outperformed all the other CPPs in their ability to increase intestinal paracellular permeability at 50 ”M and had only a small to moderate effect on the tested gut commensal strains. Based on these data Shuffle and Penetramax represent relevant CPPs to be further characterized in vivo for safe delivery of poorly absorbed therapeutics while minimizing negative impacts on the gut microbiota
<i>Saccharomyces boulardii</i> promoters for control of gene expression in vivo
Background: Interest in the use of engineered microbes to deliver therapeutic activities has increased in recent years. The probiotic yeast Saccharomyces boulardii has been investigated for production of therapeutics in the gastrointestinal tract. Well-characterised promoters are a prerequisite for robust therapeutic expression in the gut; however, S. boulardii promoters have not yet been thoroughly characterised in vitro and in vivo. Results: We present a thorough characterisation of the expression activities of 12 S. boulardii promoters in vitro in glucose, fructose, sucrose, inulin and acetate, under both aerobic and anaerobic conditions, as well as in the murine gastrointestinal tract. Green fluorescent protein was used to report on promoter activity. Promoter expression was found to be carbon-source dependent, with inulin emerging as a favourable carbon source. Furthermore, relative promoter expression in vivo was highly correlated with expression in sucrose (R = 0.99). Conclusions: These findings provide insights into S. boulardii promoter activity and aid in promoter selection in future studies utilising S. boulardii to produce therapeutics in the gut.</p
Characterization of Eight Bacterial Biosensors for Microbial Diagnostic and Therapeutic Applications
The engineering of microbial cells to produce and secrete
therapeutics
directly in the human body, known as advanced microbial therapeutics,
is an exciting alternative to current drug delivery routes. These
living therapeutics can be engineered to sense disease biomarkers
and, in response, deliver a therapeutic activity. This strategy allows
for precise and self-regulating delivery of a therapeutic that adapts
to the disease state of the individual patient. Numerous sensing systems
have been characterized for use in prokaryotes, but a very limited
number of advanced microbial therapeutics have incorporated such sensors.
We characterized eight different sensors that respond to physiologically
relevant conditions and molecules found in the human body in the probiotic
strain Escherichia coli Nissle 1917.
The resulting sensors were characterized under aerobic and anaerobic
conditions and were demonstrated to be functional under gut-like conditions
using the nematode Caenorhabditis elegans as an in vivo model. We show for the first time how a biosensor
is able to detect in vivo the bile acid-like molecule Î4-dafachronic acid, a small molecule in C. elegans that regulates lifespan. Furthermore,
we exemplify how bacterial sensors can be used to dynamically report
on changes in the intestinal environment of C. elegans, by demonstrating the use of a biosensor able to detect changes
in lactate concentrations in the gut lumen of individual C. elegans. The biosensors presented in this study
allow for dynamic control of expression in vivo and represent a valuable
tool in further developing advanced microbiome therapeutics
Effects of broad-spectrum antibiotics on the colonisation of probiotic yeast Saccharomyces boulardii in the murine gastrointestinal tract
Mouse models are commonly used to study the colonisation profiles of microorganisms introduced to the gastrointestinal tract. Three commonly used mouse models include conventional, germ-free, and antibiotic-treated mice. However, colonisation resistance in conventional mice and specialised equipment for germ-free mice are usually limiting factors in their applications. In this study, we sought to establish a robust colonisation model for Saccharomyces boulardii, a probiotic yeast that has caught attention in the field of probiotics and advanced microbiome therapeutics. We characterised the colonisation of S. boulardii in conventional mice and mice treated with a cocktail of broad-spectrum antibiotics, including ampicillin, kanamycin, metronidazole and vancomycin. We found colonisation levels increased up to 10,000-fold in the antibiotic-treated mice compared to nonantibiotic-treated mice. Furthermore, S. boulardii was detected continuously in more than 75% of mice for 10Â days after the last administration in antibiotic-treated mice, in contrast to in nonantibiotic-treated mice where S. boulardii was undetectable in less than 2Â days. Finally, we demonstrated that this antibiotic cocktail can be used in two commonly used mouse strains, C57BL/6 and ob/ob mice, both achievingâ~â10(8)Â CFU/g of S. boulardii in faeces. These findings highlight that the antibiotic cocktail used in this study is an advantageous tool to study S. boulardii based probiotic and advanced microbiome therapeutics
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