Identification of novel small molecule inhibitors of 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) kinase of Gram-negative bacteria

Abstract

The biosyntheses of isoprenoids is essential for the survival in all living organisms, and requires one of the two biochemical pathways: (a) Mevalonate (MVA) Pathway or (b) Methylerythritol Phosphate (MEP) Pathway. The latter pathway, which is used by all Gram-negative bacteria, some Gram-positive bacteria and a few apicomplexan protozoa, provides an attractive target for the development of new antimicrobials because of its absence in humans. In this report, we describe two different approaches that we used to identify novel small molecule inhibitors of Escherichia coli and Yersinia pestis 4-diphosphocytidyl-2-C-methyl D-erythritol (CDP-ME) kinases, key enzymes of the MEP pathway encoded by the E. coli ispE and Y. pestisipk genes, respectively. In the first approach, we explored existing inhibitors of the GHMP kinases while in the second approach; we performed computational high-throughput screening of compound libraries by targeting the CDP-ME binding site of the two bacterial enzymes. From the first approach, we identified two compounds with 6-(benzylthio)-2-(2-hydroxyphenyl)-4-oxo-3,4-dihydro-2H-1,3-thiazine-5-carbonitrile and (Z)-3-methyl-4-((5-phenylfuran-2-yl)methylene)isoxazol-5(4H)-one scaffolds which inhibited E. coli CDP-ME kinase in vitro. We then performed substructure search and docking experiments based on these two scaffolds and identified twenty three analogs for structure-activity relationship (SAR) studies. Three new compounds from the isoxazol-5(4H)-one series have shown inhibitory activities against E. coli and Y. pestis CDP-ME kinases with the IC(50) values ranging from 7 to 13 μM. The second approach by computational high-throughput screening (HTS) of two million drug-like compounds yielded two compounds with benzenesulfonamide and acetamide moieties which, at a concentration of 20 μM, inhibited 80% and 65%, respectively, of control CDP-ME kinase activity.

Fig. 1

MEP Isoprenoid biosynthetic pathways in living cells

Fig. 2

Purification of E. coli CDP-ME kinase. Over-expression of E. coli CDP-ME kinase was induced in E. coli HMS174 cells harboring the plasmid expressing the E. coli IspE gene. The overproduced CDP-ME kinase seen in the lysate of the bacteria (marked by ⊕ in lane 2) was purified by Nickel affinity chromatography and collected in the eluate (also marked by ⊕). FT: Flow-through.

Fig. 3

Small molecule compounds with dual human GALK1 and E. coli CDP-ME kinase inhibitory properties.

Fig. 4

Predicted binding modes of compounds 1 and 5. (a) The predicted binding mode based on docking experiments of compound 1 in complex with E. coli CDP-ME kinase shown is color-by-atom structures. The active site of cytidine pocket is depicted in stick representation. The dotted line represents a hydrogen bonding interaction with thiazine-5-carbonitrile –C=O…HN- Asp141. (b) The isoxazol-5(4H)-one containing scaffold with compound 5 in complex with E. coli CDP-ME kinase.

Fig. 4

Predicted binding modes of compounds 1 and 5. (a) The predicted binding mode based on docking experiments of compound 1 in complex with E. coli CDP-ME kinase shown is color-by-atom structures. The active site of cytidine pocket is depicted in stick representation. The dotted line represents a hydrogen bonding interaction with thiazine-5-carbonitrile –C=O…HN- Asp141. (b) The isoxazol-5(4H)-one containing scaffold with compound 5 in complex with E. coli CDP-ME kinase.

Fig. 5

(a) Purification of recombinant Y. pestis CDP-ME kinase by Nickel-affinity chromatography. Comassie blue-stained SDS-PAGE showing cell lysate from E. coli bacterial cells over-expressing Y. pestis CDP-ME kinase. (b) Amino acid sequence alignment of CDP-ME kinases from E. coli, Shigella dysenteriae, Salmonella typhi, and Y. pestis. Residues highlighted in red are identical residues across all four species and residues highlighted in blue are conserved residues.

Fig. 5

(a) Purification of recombinant Y. pestis CDP-ME kinase by Nickel-affinity chromatography. Comassie blue-stained SDS-PAGE showing cell lysate from E. coli bacterial cells over-expressing Y. pestis CDP-ME kinase. (b) Amino acid sequence alignment of CDP-ME kinases from E. coli, Shigella dysenteriae, Salmonella typhi, and Y. pestis. Residues highlighted in red are identical residues across all four species and residues highlighted in blue are conserved residues.

Fig. 6

Inhibition of bacterial growth by small molecule compounds. Small molecule compounds 1, chlorohexene and a compound unrelated to compound 1 were added to growing E. coli cultures at time = +75 minutes (black arrow) at an external concentration of 50μM. The growth of bacterial cultures was followed for a period of 21 hours.