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Profiling the in Vitro and in Vivo Activity of Streptothricin-F Against Carbapenem-Resistant

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Profiling the in Vitro and in Vivo Activity of Streptothricin-F Against Carbapenem-Resistant bioRxiv preprint doi: https://doi.org/10.1101/2021.06.14.448463; this version posted June 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Profiling the in vitro and in vivo activity of streptothricin-F against carbapenem-resistant 2 Enterobacterales: a historic scaffold with a novel mechanism of action 3 Kenneth P. Smith,a,b* Yoon-Suk Kang,a,b* Alex B. Greena*, Matthew G. Dowgiallo,c Brandon C. 4 Miller,c Lucius Chiaraviglio,a Katherine A. Truelsona, Katelyn E. Zulaufa,b, Shade Rodriguez,a 5 Roman Manetsch,c,d,e James E. Kirbya,# 6 7 8 9 aDepartment of Pathology, Beth Israel Deaconess Medical Center, Boston MA 10 bHarvard Medical School, Boston MA, USA 11 cDepartment of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Ave, 12 Boston, MA 02115, USA 13 dDepartment of Pharmaceutical Sciences, Northeastern University, 360 Huntington Ave, Boston, 14 MA 02115, USA 15 eCenter for Drug Discovery, Northeastern University, Boston, MA 02115, USA 16 17 Running Title: Profiling streptothricin-F activity 18 19 *equal contributions, authorship order determined by order of experiments presented in the 20 manuscript 21 22 #Address correspondence to James E. Kirby, [email protected] 23 24 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.14.448463; this version posted June 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 25 ABSTRACT 26 Streptothricins are components of the natural product, nourseothricin; each containing 27 identical streptolidine and gulosamine aminosugar moieties attached to varying numbers of linked 28 -lysines. Nourseothricin was discovered by Waksman and colleagues in the early 1940's, 29 generating intense interest because of excellent Gram-negative activity. However, the natural 30 product mixture was associated with toxicity, and subsequent exploration was limited. Here, we 31 establish the activity spectrum of nourseothricin and its main components, streptothricin-F (S-F, 32 one lysine) and streptothricin D (S-D, three lysines), purified to homogeneity, against highly drug- 33 resistant, carbapenem-resistant Enterobacterales (CRE). The MIC50 and MIC90 for S-F and S-D 34 were 2 and 4 µM, and 0.25 and 0.5 µM, respectively. S-F and nourseothricin showed rapid, 35 bactericidal activity. S-F and S-D both showed approximately 40-fold greater selectivity for 36 prokaryotic than eukaryotic ribosomes in in vitro translation assays. There was >10-fold in vitro 37 selectivity of S-F compared with S-D on LLC-PK1 and J774 cell lines. In vivo, delayed renal 38 toxicity occurred at >10-fold higher doses of S-F compared with S-D. Substantial treatment effect 39 of S-F in the murine thigh model was observed against the otherwise pandrug-resistant, NDM-1- 40 expressing Klebsiella pneumoniae Nevada strain at dosing levels without observable or minimal 41 toxicity. Resistance mutations obtained in single ribosomal operon E. coli identify novel 42 interactions with 16S rRNA helix 34, i.e., C1504A and A1196G/C conferred high level resistance 43 to nourseothricin. Based on promising, unique activity, we suggest that the streptothricin scaffold 44 deserves further pre-clinical exploration as a potential therapeutic for the treatment of CRE and 45 potentially other multidrug-resistant, gram-negative pathogens. 46 47 IMPORTANCE 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.14.448463; this version posted June 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 48 Streptothricins are a historic class of antibiotics discovered by Waksman and colleagues in the 49 1940’s. Toxicities associated with the streptothricin natural product mixture, also known as 50 nourseothricin, discouraged further development. However, we found that a component of 51 nourseothricin, streptothricin-F, retained potent activity against contemporary carbapenem- 52 resistant Enterobacterales with significant selectivity in in vitro and in vivo assays. This included 53 demonstration of rapid bactericidal activity in vitro and substantial therapeutic effect in the murine 54 thigh model against a pandrug-resistant Klebsiella pneumoniae isolate at non-toxic concentrations. 55 Through resistance mutation analysis, we identified helix 34 of 16S rRNA in the prokaryotic 56 ribosome, and specifically bases C1054 and A1196, as critical for streptothricin’s activity. The 57 mechanism of action is distinct from other known translation inhibitors. Based on promising and 58 unique activity, we believe the streptothricin scaffold deserves further pre-clinical exploration as 59 a potential therapeutic for the treatment of CRE and potentially other multidrug-resistant, Gram- 60 negative pathogens. 61 62 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.14.448463; this version posted June 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 63 INTRODUCTION 64 The rapid emergence of antimicrobial resistance presents a significant challenge for 65 treatment of bacterial infections. Carbapenem-resistant Enterobacterales (CRE) are of particular 66 concern. The gram-negative cell envelope presents a formidable barrier to potential antimicrobial 67 compounds based on a double cell membrane permeability barrier and efflux pumps. Almost all 68 approved gram-negative antimicrobials that can overcome this barrier are natural products or 69 synthetic or semi-synthetic derivatives of natural products. Unfortunately, small molecules 70 commonly available in high-throughput screening libraries rarely share similar physicochemical 71 properties associated with gram-negative penetrance and activity (1). As such, high throughput 72 screening efforts using synthetic chemical libraries with rare exceptions have been non-productive 73 (1) . As a result, there is a significant antimicrobial discovery void. 74 Moreover, there is little doubt that resistance will emerge to agents currently in the pipeline. 75 It is already known for example, that plazomicin, an aminoglycoside engineered to avoid drug- 76 modification-based resistance mechanisms, is ineffective against strains with ribosomal 77 methylase-based resistance mechanisms found in a large proportion of NDM-1 CRE strains (2, 3). 78 Concerningly, these NDM-1 strains, highly endemic in East Asia (in particular India) (4-8), are 79 emerging in the US, exemplified by the report of a lethal NDM-1 CRE infection in Nevada that 80 was resistant to all available agents (4). We are therefore clearly in need of several new gram- 81 negative agents that are unique in terms of antimicrobial class and potential vulnerabilities, and 82 which can diversify our antimicrobial therapeutic portfolio (9). 83 Here, we further consider the properties of a historic antibiotic scaffold. Streptothricin was 84 originally isolated by Waksman and Woodruff in 1942 from a soil Actinomyces (10). It generated 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.14.448463; this version posted June 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 85 initial excitement based on activity against gram-negative organisms and Mycobacterium 86 tuberculosis (11). It was also noted to completely cure Brucella abortus infection in guinea pigs 87 (12) and otherwise lethal Salmonella paratyphi B infection in mice (13). Based on these attributes, 88 it went into fermentative production at Merck. However, in a limited human trial (details not 89 published) (14), the natural product streptothricin induced reversible kidney toxicity, and, 90 therefore, was not further pursued as a therapeutic. 91 Studies forty years ago using in vitro translation systems determined that the natural 92 product inhibited prokaryotic translation and induced substantial ribosomal miscoding activity, 93 similar to aminoglycosides (15). Notably, however, it did not inhibit translation in rat liver extracts, 94 suggesting prokaryotic selectivity. 95 Streptothricin is now known to be a natural product mixture, often referred to as 96 nourseothricin. For clarity, we will henceforth refer to the natural product mixture as 97 nourseothricin and individual components by their specific streptothricin designation. Each 98 streptothricin in the nourseothricin natural product mixture consists of three linked components, a 99 streptolidine, a carbamoylated gulosamine moiety, and a β-lysine homopolymer of varying length. 100 The individual streptothricins are designated by letter suffixes (A-F and X) based on the number 101 of lysine residues with streptothricin F (S-F) and streptothricin D (S-D) having one and three lysine 102 moieties, respectively (see Fig. 1). 103 Initial studies on toxicity in animals from the 1940's used only semi-quantitative activity 104 measures and impure compound with large doses presumably exceeding 100 mg/kg (16) and are 105 therefore problematic to interpret. More recent studies in mice with fractionated streptothricins 106 showed that toxicity varies with the length of the poly--lysine chain: specifically, a murine LD50 5 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.14.448463; this version posted June 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
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