Multidrug resistant (MDR) bacterial infections are on the rise and becoming a greater public health concern. Compounding this challenge is the dearth of new antibiotic classes introduced into the clinic within the last half-century, specifically those that penetrate and kill Gram-negative pathogens. The discovery of Gram-negative active antibiotics with novel targets requires new approaches to breach the outer membrane of these pathogens. This has materialized as elegant antibiotic-siderophore hybrids such as Cefiderocol (FDA approved),2 cotreatment strategies with efflux pump inhibitors such as MBX-4191 (preclinical studies),3 and outer-membrane potentiation by non-lytic polymyxins such as SPR714 (Phase I clinical trials).4,5
Alternatively, modulating the physicochemical properties of small-molecules to enable permeation and accumulation in Gram-negative bacteria represents a tractable and generalizable approach. As lack of whole-cell accumulation is frequently a point of attrition for Gram-negative antibiotics, such a strategy would greatly enable the discovery of structurally distinct Gram-negative antibiotic classes. Modulation of physicochemical properties is routinely employed in other avenues of drug-discovery such as enhancing oral bioavailability through employing Lipinksi’s rule of five,6,7 improving solubility through disrupting molecular planarity with minor structural changes such as increasing the fraction sp3 bonds, 8–11 and increasing metabolic stability through blocking oxidatively labile sites with a fluorine substituent (Fig. 1).11,12 Recent efforts in our laboratory aimed to identify factors associated with compound accumulation have unearthed a set of physicochemical features that correlate with increased accumulation in Gram-negative bacteria, including: the presence of an ionizable nitrogen (with a sterically unencumbered primary amine being most favorable), low flexibility (five or fewer rotatable bonds), and low 3-dimensionality (Globularity ≤ 0.25).13 After this initial discovery, we wanted to facilitate implementation of the eNTRy rules by making a readily accessible platform to calculate these corresponding parameters. Accordingly, we created an open-access web-based cheminformatics tool (called eNTRyway) to predict whether or not a compound has high potential for accumulation in E. coli. As a proof of concept and in order to validate this approach we sought to apply the eNTRy rules to a Gram-Positive only antibiotic.
Fig. 1: Physicochemical Modulation Strategies in Drug Discovery
Scouring the literature for such a compound revealed Debio-1452 as a prime candidate for conversion. The prodrug of Debio-1452, afabicin, is currently in Phase I clinical trials for the treatment of Staphylococcal infections.14,15 Debio-1452 acts through inhibiting the enoyl-acyl carrier protein reductase FabI, an essential enzyme involved in bacterial fatty acid biosynthesis.16 For an antibiotic in clinical studies, Debio-1452 possesses many favorable qualities including: a low frequency of resistance (S. aureus FOR = 6.6 x 1010),16 the bacterial cell target is distinct from mammalian cell fatty acid synthase17 as well as mammalian cell short-chain dehydrogenase/reductases (SDRs),18 and represents a new antibiotic drug class.19 It is important to note that the activity of a potential conversion candidate against Gram-negative pathogens should be limited by accumulation rather than the ability to inhibit the desired Gram-negative bacterial target. Although it has been reported that Debio-1452 only possesses Staphylococcus-specific activity, one report indicated that Debio-1452 was active against an efflux pump mutant strain of E. coli,20 suggesting that this compound would have activity against wild-type E. coli if it were able to accumulate in these cells. As a potential candidate for implementation of our conversion paradigm, Debio-1452 also possesses essential qualities for a conversion target such as a low number of rotatable bonds (RB = 4), low globularity (Glob = 0.093), and an x-ray crystal structure as well as structure activity relationship studies to inform strategic placement of a primary amine.
In an effort to maintain target activity, the solvent-exposed 3-position of the napthyridinone ring of Debio-1452 was selected for incorporation of a primary amine. Expansive SAR studies on the parent compound also suggested that additional functionality should be tolerated at this position. Following the synthesis of Debio-1452-NH3 and evaluation of antimicrobial activity against reference strains of WT Gram-negative bacteria, we were thrilled to observe significant bacterial burden reduction and rescue in mice with lethal infections from clinical isolates of Acinetobacter baumannii, Klebsiella pneumonia, and Escherichia coli (Fig. 2). This work demonstrates the utility and application of this conversion paradigm to aid in the discovery and development of antibiotics against Gram-negative bacteria.
Fig. 2: Brief Overview of Discovery of the Broad Spectrum Antibiotic Debio-1452-NH3
With an expanded understanding of physicochemical properties that increase the accumulation potential of small-molecules in E. coli juxtaposed with the low prevalence of primary amines in conventional chemical libraries,21 one wonders about the missed potential for small-molecule antibiotic drug discovery during the in-vitro based high-throughput screening campaigns of the genomics era. The advent of the genomics era has enabled us to uncover a new landscape of small-molecules with in-vitro activity against bacterial cell targets. If we revisit antibacterial leads generated during this era, opportunities exist to explore this conversion strategy and uncover new antibacterial leads that exhibit whole-cell activity against Gram-negative bacteria. Indeed, in our manuscript we point to (Figure 1b and Supplementary Table 1) dozens of Gram-positive-only compounds that appear to be outstanding candidates for conversion to broad-spectrum antibiotics.
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