Profiling thein vitroandin vivoactivity of streptothricin-F against carbapenem-resistant Enterobacterales: a historic scaffold with a novel mechanism of action
Authors: Smith Kenneth P. , Kang Yoon-Suk , Green Alex B. , Dowgiallo Matthew G. , Miller Brandon C. , Chiaraviglio Lucius , Truelson Katherine A. , Zulauf Katelyn E. , Rodriguez Shade , Manetsch Roman , Kirby James E. 
Published: June 2021

ABSTRACTStreptothricins are components of the natural product, nourseothricin; each containing identical streptolidine and gulosamine aminosugar moieties attached to varying numbers of linked β-lysines. Nourseothricin was discovered by Waksman and colleagues in the early 1940’s, generating intense interest because of excellent Gram-negative activity. However, the natural product mixture was associated with toxicity, and subsequent exploration was limited. Here, we establish the activity spectrum of nourseothricin and its main components, streptothricin-F (S-F, one lysine) and streptothricin D (S-D, three lysines), purified to homogeneity, against highly drug-resistant, carbapenem-resistant Enterobacterales (CRE). The MIC50and MIC90for S-F and S-D were 2 and 4 µM, and 0.25 and 0.5 µM, respectively. S-F and nourseothricin showed rapid, bactericidal activity. S-F and S-D both showed approximately 40-fold greater selectivity for prokaryotic than eukaryotic ribosomes inin vitrotranslation assays. There was >10-foldin vitroselectivity of S-F compared with S-D on LLC-PK1 and J774 cell lines.In vivo, delayed renal toxicity occurred at >10-fold higher doses of S-F compared with S-D. Substantial treatment effect of S-F in the murine thigh model was observed against the otherwise pandrug-resistant, NDM-1-expressingKlebsiella pneumoniaeNevada strain at dosing levels without observable or minimal toxicity. Resistance mutations obtained in single ribosomal operonE. coliidentify novel interactions with 16S rRNA helix 34, i.e., C1504A and A1196G/C conferred high level resistance to nourseothricin. Based on promising, unique activity, we suggest that the streptothricin scaffold deserves further pre-clinical exploration as a potential therapeutic for the treatment of CRE and potentially other multidrug-resistant, gram-negative pathogens.IMPORTANCEStreptothricins are a historic class of antibiotics discovered by Waksman and colleagues in the 1940’s. Toxicities associated with the streptothricin natural product mixture, also known as nourseothricin, discouraged further development. However, we found that a component of nourseothricin, streptothricin-F, retained potent activity against contemporary carbapenem-resistant Enterobacterales with significant selectivity inin vitroandin vivoassays. This included demonstration of rapid bactericidal activityin vitroand substantial therapeutic effect in the murine thigh model against a pandrug-resistantKlebsiella pneumoniaeisolate at non-toxic concentrations. Through resistance mutation analysis, we identified helix 34 of 16S rRNA in the prokaryotic ribosome, and specifically bases C1054 and A1196, as critical for streptothricin’s activity. The mechanism of action is distinct from other known translation inhibitors. Based on promising and unique activity, we believe the streptothricin scaffold deserves further pre-clinical exploration as a potential therapeutic for the treatment of CRE and potentially other multidrug-resistant, Gram-negative pathogens.