In this study, we synthesized a library of eNTRy-rule-compliant compounds by introducing ionizable nitrogen atoms to an antimalarial compound. These positively charged derivatives gained activity against both Gram-negative and -positive bacteria, Mycobacterium tuberculosis, and boosted Plasmodium falciparum inhibition to the double-digit nanomolar range. Overcoming and remaining inside the cell envelope of Gram-negative bacteria (GNB) is one of the major difficulties in antibacterial drug discovery and development. The eNTRy rules (N = ionizable nitrogen, T = low three-dimensionality, R = rigidity) can be a useful structural guideline to improve accumulation of small molecules in GNB. With the aim of unlocking Gram-negative activity, we added amines and (cyclic) N-alkyl guanidines to an already flat and rigid pyrazole-amide class as a representative example for our investigation. To test their performance, we compared these eNTRy-rule-compliant compounds to closely related noncompliant ones through phenotypic screening of various pathogens (P. falciparum, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, and M. tuberculosis), obtaining a handful of broad-spectrum hits. The results support the working hypothesis and even extend its applicability. The studied pyrazole-amide class adheres to the eNTRy rules; noncompliant compounds do not kill any of the bacteria tested, while compliant compounds largely showed growth inhibition of Gram-negative, -positive, and M. tuberculosis bacteria in the single-digit micromolar range.

In the search for new antitubercular compounds, we leveraged target-directed dynamic combinatorial chemistry (tdDCC) as an efficient hit-identification method. In tdDCC, the target selects its own binders from a dynamic library generated in situ, reducing the number of compounds that require synthesis and evaluation. We combined a total of 12 hydrazides and six aldehydes to generate 72 structurally diverse N-acylhydrazones. To amplify the best binders, we employed anti-infective target 4-diphosphocytidyl-2C-methyl-d-erythritol kinase (IspE) from Mycobacterium tuberculosis (Mtb). We successfully validated the use of tdDCC as hit-identification method for IspE and optimized the analysis of tdDCC hit determination. From the 72 possible N-acylhydrazones, we synthesized 12 of them, revealing several new starting points for the development of IspE inhibitors as antibacterial agents.

Aspartate transcarbamoylase (ATCase) plays a key role in the second step of de novo pyrimidine biosynthesis in eukaryotes and has been proposed to be a target to suppress cell proliferation in E. coli, human cells and the malarial parasite. We hypothesized that a library of ATCase inhibitors developed for malarial ATCase (PfATCase) may also contain inhibitors of the tubercular ATCase and provide a similar inhibition of cellular proliferation. Of the 70 compounds screened, 10 showed single-digit micromolar inhibition in an in vitro activity assay and were tested for their effect on M. tuberculosis cell growth in culture. The most promising compound demonstrated a MIC90 of 4 & mu;M. A model of MtbATCase was generated using the experimental coordinates of PfATCase. In silico docking experiments showed this compound can occupy a similar allosteric pocket on MtbATCase to that seen on PfATCase, explaining the observed species selectivity seen for this compound series.