The cDNA for PRMT7, a recently discovered human protein-arginine methyltransferase (PRMT), was cloned and expressed in Escherichia coli and mammalian cells. Immunopurified PRMT7 actively methylated histones, myelin basic protein, a fragment of human fibrillarin (GAR) and spliceosomal protein SmB. Amino acid analysis showed that the modifications produced were predominantly monomethylarginine and symmetric dimethylarginine (SDMA). Examination of PRMT7 expressed in E. coli demonstrated that peptides corresponding to sequences contained in histone H4, myelin basic protein, and SmD3 were methylated. Furthermore, analysis of the methylated proteins showed that symmetric dimethylarginine and relatively small amounts of monomethylarginine and asymmetric dimethylarginine were produced. SDMA was also formed when a GRG tripeptide was methylated by PRMT7, indicating that a GRG motif is by itself sufficient for symmetric dimethylation to occur. Symmetric dimethylation is reduced dramatically compared with monomethylation as the concentration of the substrate is increased. The data demonstrate that PRMT7 (like PRMT5) is a Type II methyltransferase capable of producing SDMA modifications in proteins.

The emergence of multiple-drug-resistant (MDR) bacterial pathogens in hospitals (nosocomial infections) presents a global threat of growing importance, especially for Gram-negative bacteria with extended spectrum β-lactamase (ESBL) or the novel New Delhi metallo-β-lactamase 1 (NDM-1) resistance. Starting from the antibacterial peptide apidaecin 1b, we have optimized the sequence to treat systemic infections with the most threatening human pathogens, such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. The lead compound Api88 enters bacteria without lytic effects at the membrane and inhibits chaperone DnaK at the substrate binding domain with a K(D) of 5 μmol/L. The Api88-DnaK crystal structure revealed that Api88 binds with a seven residue long sequence (PVYIPRP), in two different modes. Mice did not show any sign of toxicity when Api88 was injected four times intraperitoneally at a dose of 40 mg/kg body weight (BW) within 24 h, whereas three injections of 1.25 mg/kg BW and 5 mg/kg BW were sufficient to rescue all animals in lethal sepsis models using pathogenic E. coli strains ATCC 25922 and Neumann, respectively. Radioactive labeling showed that Api88 enters all organs investigated including the brain and is cleared through both the liver and kidneys at similar rates. In conclusion, Api88 is a novel, highly promising, 18-residue peptide lead compound with favorable in vitro and in vivo properties including a promising safety margin.

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The APO-type proline-arginine-rich host defense peptides exhibit potent in vitro killing parameters against Enterobacteriaceae but not to other bacteria. Because of the excellent in vivo properties against systemic and local infections, attempts are regularly made to further improve the activity spectrum. A C-terminal hydrazide analog of the Chex1-Arg20 amide (ARV-1502) shows somewhat improved minimal inhibitory concentration against Moraxellaceae. Here we compared the activity of the two peptides as well as an inactive dimeric reverse amide analog in a systemic Acinetobacter baumannii infection. Only the narrow spectrum amide derivative reduced the 6-h blood bacterial burden by >2 log10 units reaching statistical significance (p = 0.03 at 5 mg/kg and 0.031 at 2 mg/kg administered intramuscularly). The hydrazide derivative, probably due to stronger activity on cell membranes, lysed erythrocytes at lower concentrations, and caused toxic effects at lower doses (10 mg/kg vs. 25 mg/kg). In a limited study, the amide induced a >5-fold production of the anti-inflammatory cytokine IL-10 over untreated naïve mice and minor increases in the anti-inflammatory IL-4 and pro-inflammatory cytokines TNF-α and IL-6, in blood. The blood of hydrazide-treated mice exhibited significantly lowered levels of IL-10 and slightly decreased IL-4 and TNF-α. These results suggest that the improved efficacy of the narrow-spectrum amide analog is likely associated with increased anti-inflammatory cytokine production and better stimulation of the immune system. Although blood IL-6 and TNF-α levels are frequently used as markers of potential toxicity in drug development, we did not observe any notable increase in mice receiving the toxic polyamide antibiotic colistin.

Both PrAMPs were not immunogenic and Onc72 concentrations in blood were well above the minimal inhibitory concentrations for Enterobacteriaceae further confirming their potential as novel antibiotics.