The main mechanism of carbon catabolite repression/activation in low-guanine and low-cytosine Gram-positive bacteria seems to involve phosphorylation of HPr (histidine-containing protein) at Ser-46 by the ATP-dependent HPr kinase, which in Bacillus subtilis, Lactobacillus casei, and Staphylococcus xylosus also exhibits phosphatase activity and is thus a bifunctional enzyme (HPrK/P). Since deficiency of HPrK/P in S. xylosus, L. casei, and B. subtilis mutants leads to severe growth defects, inhibitors of the enzyme could form a new family of antibiotic drugs. The aim of the study was to screen an in-house chemical library for identification of hits as inhibitors of HPrK/P in B. subtilis and to further extract additional information of structural features from hit optimization using a radioactive in vitro assay. A symmetrical bis-cationic compound LPS 02-10-L-D09 (2a) with a 12-carbon alkyl linker bridging the two 2-aminobenzimidazole moieties was identified as a non-ATP mimetic compound exhibiting an EC(50) value of 10 microM in a kinase assay with HPr as substrate. The substance also inhibited the phosphatase activity of HPrK/P triggered by the addition of inorganic phosphate. Similar results were obtained with 2a and catabolite repression HPr, which, like HPr, can be phosphorylated at Ser-46 by HPrK/P and is involved in catabolite repression. Structure-activity relationship analysis indicated the importance in its structure of a substituted 2-aminobenzimidazole. This typical heterocycle is linked through a C12 alkyl chain to a second scaffold that can bear a cationic or a noncationic moiety but in all cases should present an aromatic ring in its vicinity.