Natural products serve as chemical blueprints for the majority of antibiotics in our clinical arsenal. The evolutionary process by which these molecules arise is inherently accompanied by the co-evolution of resistance mechanisms that shorten the clinical lifetime of any given class 1 . Virginiamycin acetyltransferases (Vats) are resistance proteins that provide protection against streptogramins 2 , potent Gram-positive antibiotics that inhibit the bacterial ribosome 3 . Due to the challenge of selectively modifying the chemically complex, 23-membered macrocyclic scaffold of group A streptogramins, analogs that overcome Vat resistance have not been previously accessed 2 . Here we report the design, synthesis, and antibacterial evaluation of group A streptogramin antibiotics with unprecedented structural variability. Using cryo-electron microscopy and forcefield-based refinement, we characterize the binding of eight analogs to the bacterial ribosome at high resolution, revealing new binding interactions that extend into the peptidyl tRNA binding site and towards synergistic binders that occupy the nascent peptide exit tunnel (NPET). One of these analogs has excellent activity against several streptogramin-resistant strains of S. aureus , exhibits decreased acetylation rates in vitro , and is effective at lowering bacterial load in a mouse model of infection. Our results demonstrate that the combination of rational design and modular chemical synthesis can revitalize classes of antibiotics that are limited by naturally arising resistance mechanisms.
Stochastic fluctuations at the transcriptional level contribute to isogenic cell-to-cell heterogeneity in mammalian cell populations. However, we still have no clear understanding of the repercussions of this heterogeneity, given the lack of tools to independently control mean expression and variability of a gene. Here, we engineer a synthetic circuit to modulate mean expression and heterogeneity of transgenes and endogenous human genes. The circuit, a Tunable Noise Rheostat (TuNR), consists of a transcriptional cascade of two inducible transcriptional activators, where the output mean and variance can be modulated by two orthogonal small molecule inputs. In this fashion, different combinations of the inputs can achieve the same mean but with different population variability. With TuNR, we achieve low basal expression, over 1000-fold expression of a transgene product, and up to 7-fold induction of the endogenous gene NGFR. Importantly, for the same mean expression level, we are able to establish varying degrees of heterogeneity in expression within an isogenic population, thereby decoupling gene expression noise from its mean. TuNR is therefore a modular tool that can be used in mammalian cells to enable direct interrogation of the implications of cell-to-cell variability.
Stochastic fluctuations at the transcriptional level contribute to isogenic cell-to-cell heterogeneity in mammalian cell populations. However, we still have no clear understanding of the repercussions of this heterogeneity, given the lack of tools to independently control mean expression and variability of a gene. Here, we engineered a synthetic circuit to independently modulate mean expression and heterogeneity of transgenes and endogenous human genes.1
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