Clinically used RAF inhibitors are ineffective in RAS mutant tumors because they enhance homo- and heterodimerization of RAF kinases, leading to paradoxical activation of ERK signaling. Overcoming enhanced RAF dimerization and the resulting resistance is a challenge for drug design. Combining multiple inhibitors could be more effective, but it is unclear how the best combinations can be chosen. We built a next-generation mechanistic dynamic model to analyze combinations of structurally different RAF inhibitors, which can efficiently suppress MEK/ERK signaling. This rule-based model of the RAS/ERK pathway integrates thermodynamics and kinetics of drug-protein interactions, structural elements, posttranslational modifications, and cell mutational status as model rules to predict RAF inhibitor combinations for inhibiting ERK activity in oncogenic RAS and/or BRAFV600E backgrounds. Predicted synergistic inhibition of ERK signaling was corroborated by experiments in mutant NRAS, HRAS, and BRAFV600E cells, and inhibition of oncogenic RAS signaling was associated with reduced cell proliferation and colony formation.

Bacteria grown in space experiments under microgravity conditions have been found to undergo unique physiological responses, ranging from modified cell morphology and growth dynamics to a putative increased tolerance to antibiotics. A common theory for this behavior is the loss of gravity-driven convection processes in the orbital environment, resulting in both reduction of extracellular nutrient availability and the accumulation of bacterial byproducts near the cell. To further characterize the responses, this study investigated the transcriptomic response of Escherichia coli to both microgravity and antibiotic concentration. E. coli was grown aboard International Space Station in the presence of increasing concentrations of the antibiotic gentamicin with identical ground controls conducted on Earth. Here we show that within 49 h of being cultured, E. coli adapted to grow at higher antibiotic concentrations in space compared to Earth, and demonstrated consistent changes in expression of 63 genes in response to an increase in drug concentration in both environments, including specific responses related to oxidative stress and starvation response. Additionally, we find 50 stress-response genes upregulated in response to the microgravity when compared directly to the equivalent concentration in the ground control. We conclude that the increased antibiotic tolerance in microgravity may be attributed not only to diminished transport processes, but also to a resultant antibiotic cross-resistance response conferred by an overlapping effect of stress response genes. Our data suggest that direct stresses of nutrient starvation and acid-shock conveyed by the microgravity environment can incidentally upregulate stress response pathways related to antibiotic stress and in doing so contribute to the increased antibiotic stress tolerance observed for bacteria in space experiments. These results provide insights into the ability of bacteria to adapt under extreme stress conditions and potential strategies to prevent antimicrobial-resistance in space and on Earth.

Even initially sensitive bacteria can rapidly thwart antibiotic treatment through stress response processes known as adaptive resistance. Adaptive resistance fosters transient tolerance increases and the emergence of mutations conferring heritable drug resistance. In order to extend the applicable lifetime of new antibiotics, we must seek to hinder the occurrence of bacterial adaptive resistance; however, the regulation of adaptation is difficult to identify due to immense heterogeneity emerging during evolution. This study specifically seeks to generate heterogeneity by adapting bacteria to different stresses and then examines gene expression trends across the disparate populations in order to pinpoint key genes and pathways associated with adaptive resistance. The targets identified here may eventually inform strategies for impeding adaptive resistance and prolonging the effectiveness of antibiotic treatment.

Multidrug-resistant (MDR) bacteria pose a grave concern to global health, which is perpetuated by a lack of new treatments and countermeasure platforms to combat outbreaks or antibiotic resistance. To address this, we have developed a Facile Accelerated Specific Therapeutic (FAST) platform that can develop effective peptide nucleic acid (PNA) therapies against MDR bacteria within a week. Our FAST platform uses a bioinformatics toolbox to design sequence-specific PNAs targeting non-traditional pathways/genes of bacteria, then performs in-situ synthesis, validation, and efficacy testing of selected PNAs. As a proof of concept, these PNAs were tested against five MDR clinical isolates: carbapenem-resistant Escherichia coli, extended-spectrum beta-lactamase Klebsiella pneumoniae, New Delhi Metallo-beta-lactamase-1 carrying Klebsiella pneumoniae, and MDR Salmonella enterica. PNAs showed significant growth inhibition for 82% of treatments, with nearly 18% of treatments leading to greater than 97% decrease. Further, these PNAs are capable of potentiating antibiotic activity in the clinical isolates despite presence of cognate resistance genes. Finally, the FAST platform offers a novel delivery approach to overcome limited transport of PNAs into mammalian cells by repurposing the bacterial Type III secretion system in conjunction with a kill switch that is effective at eliminating 99.6% of an intracellular Salmonella infection in human epithelial cells.

The evolution of antibiotic resistance has engendered an impending global health crisis that necessitates a greater understanding of how resistance emerges. The impact of nongenetic factors and how they influence the evolution of resistance is a largely unexplored area of research. Here we present a novel application of CRISPR-Cas9 technology for investigating how gene expression governs the adaptive pathways available to bacteria during the evolution of resistance. We examine the impact of gene expression changes on bacterial adaptation by constructing a library of deactivated CRISPR-Cas9 synthetic devices to tune the expression of a set of stress-response genes in Escherichia coli. We show that artificially inducing perturbations in gene expression imparts significant synthetic control over fitness and growth during stress exposure. We present evidence that these impacts are reversible; strains with synthetically perturbed gene expression regained wild-type growth phenotypes upon stress removal, while maintaining divergent growth characteristics under stress. Furthermore, we demonstrate a prevailing trend toward negative epistatic interactions when multiple gene perturbations are combined simultaneously, thereby posing an intrinsic constraint on gene expression underlying adaptive trajectories. Together, these results emphasize how CRISPR-Cas9 can be employed to engineer gene expression changes that shape bacterial adaptation, and present a novel approach to synthetically control the evolution of antimicrobial resistance.