Synthesis of Small Molecules That Break Colistin Resistance

Addressing the escalating threat of antibiotic-resistant bacteria is imperative for the survival of modern healthcare systems. Among all bacterial pathogens, the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) are responsible for many of the most difficult to treat infections as a result of high levels of multidrug resistance. Given the lack of investment from big pharmaceutical companies in discovering new classes of antibiotics against Gram-negative bacteria in recent decades, physicians have turned to the use of polymyxins, which are typically viewed as the antibiotics of last resort for the treatment of multidrug-resistant (MDR) Gram-negative infections. However, the nephrotoxic side effects that once caused the suspension of colistin should not be ignored. In parallel with the development of new conventional antibiotics is the use of non-microbicidal small molecule adjuvants that disrupt bacterial resistance mechanisms. In the context of colistin resistance, we and others have identified a number of adjuvants that suppress resistance in bacterial strains that harbor either chromosomally or plasmid encoded resistance determinants. A series of screening efforts using eukaryotic kinase and phosphatase inhibitors yielded several compounds as highly active colistin adjuvants.

The following research describes the synthesis of derivatives of three scaffolds identified from these screens, and evaluation of their biological activity. For benzimidazole derivatives, structure-activity relationship (SAR) studies were conducted, leading to the identification of four derivatives with distinct activities, which were further investigated to understand their mechanism of action and potential pharmaceutical concerns. Unexpectedly, MALDI mass spectrometry detected no changes in the lipid A composition of strains with a colistin resistant phenotype, while quantitative RT-PCR (qRT-PCR) disclosed insignificant changes in the expression of genes involved in resistance pathways. Therefore, the mechanism of action of how these compounds potentiate colistin still remains unclear. By measuring interaction of the active compounds with IKKß via a luciferase assay, we proved it is possible to decouple colistin adjuvant activity from eukaryotic kinase activity for the benzimidazole scaffold. Similar research was also conducted with 6-bromoindirubin-3’-oxime derivatives. We have synthesized and tested a library of indirubin analogs for restoration of colistin susceptibility in colistin resistant bacteria. The most active compound lowered the colistin MIC in KPB9 below the breakpoint. No hemolysis was noted for the most active compound up to 400 μM (80× its active concentration against all strains tested), while mechanism of action studies indicate that colistin potentiation is not dependent upon full reversal of lipid A modification.

We also recently discovered the indolic marine natural product meridianin D, which is also a kinase inhibitor, as a highly active colistin potentiator against representative colistin resistant K. pneumoniae and A. baumannii strains. This discovery inspired us to find potential antibiotic potentiators from other natural products. The National Cancer institute (NCI) Developmental Therapeutics Program is a library that includes more than 230,000 natural products purified from various plant, marine, and microbial samples. From a subset of this library, another natural product variabiline (4.1) was found to be active against colistin susceptible K. pneumoniae 2146 and A. baumannii 5075 strains. We performed the total synthesis and SAR study of this natural product. Chiral resolution followed by X-ray analysis was also conducted to resolve a ¹H NMR discrepancy between our data and published data. Both enantiomers of variabiline were determined to have same level of activity. This observation reinforces our expectation that variabiline increasescolistin activity by increasing membrane permeability. Mechanism of action studies suggest that variabiline increases the permeability of Gram-negative outer membrane (OM), but not through direct interaction with lipopolysaccharide (LPS).