Shigeko Harada

Tripropeptin C Blocks the Lipid Cycle of Cell Wall Biosynthesis by Complex Formation with Undecaprenyl Pyrophosphate

ABSTRACT Tripropeptin C (TPPC) is a naturally occurring cyclic lipodepsipeptide antibiotic produced by a Lysobacter sp. TPPC exhibits potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and penicillin-resistant Streptococcus pneumoniae. This antibiotic also inhibits the incorporation of N-acetylglucosamine into the peptidoglycan of S. aureus at a 50% inhibitory concentration (IC50) of 0.7 μM, which is proportional to its MIC (0.87 μM; equivalent to 1.0 μg/ml). Treatment of exponential-phase S. aureus cells with TPPC resulted in accumulation of UDP-MurNAc-pentapeptide in the cytoplasm. The antimicrobial activity of TPPC was weakened by the addition of prenyl pyrophosphates but not by prenyl phosphates, UDP-linked sugars, or the pentapeptide of peptidoglycan. The direct interaction between TPPC and undecaprenyl pyrophosphate (C55-PP) was observed by mass spectrometry and thin-layer chromatography analysis, indicating that TPPC can potentially inhibit C55-PP phosphatase activity, which plays a crucial role in the lipid cycle of peptidoglycan synthesis. As expected, TPPC inhibits this enzymatic reaction at an IC50 of 0.03 to 0.1 μM in vitro, as does bacitracin. From the analysis of accumulation of lipid carrier-related compounds, TPPC was found to cause the accumulation of C55-PP in situ, leading to the accumulation of a glycine-containing lipid intermediate. This suggested that the TPPC/C55-PP complex also inhibits the transglycosylation step or flippase activity, adding to the inhibition of C55-PP dephosphorylation. This mode of action is different from that of currently available drugs such as vancomycin, daptomycin, and bacitracin.

Natural lipopeptide antibiotic tripropeptin C revitalizes and synergistically potentiates the activity of beta-lactams against methicillin-resistant Staphylococcus aureus

Tripropeptin C (TPPC) is a natural calcium-ion-dependent lipopeptide antibiotic that inhibits peptidoglycan biosynthesis by binding to prenyl pyrophosphate. It displays very potent antimicrobial activity both in vitro and in a mouse model of methicillin-resistant Staphylococcus aureus (MRSA) septicemia. The combination of TPPC with all classes of beta-lactams tested (including penam, carbapenem, cephem and oxacephem) showed highly synergistic (SYN) effects against MRSA strains, but not against methicillin-sensitive S. aureus strains. These SYN effects were observed with both a checkerboard methodology and a time-kill analysis. The TPPC analog, bis-methyl ester-TPPC, which has neither antimicrobial activity nor the ability to bind prenyl pyrophosphate, also potentiated the activity of beta-lactams. This result indicates that the mechanism of the SYN activity of TPPC is independent of its binding to prenyl pyrophosphate. Therefore, synergistically enhancing the anti-MRSA activities of TPPC and beta-lactams by combining them is a novel and potentially powerful therapeutic strategy for MRSA infections.

PIPERASTATIN A, A NEW SELECTIVE SERINE CARBOXYPEPTIDASE INHIBITOR PRODUCED BY ACTINOMYCETE. I. TAXONOMY, PRODUCTION, ISOLATION AND BIOLOGICAL ACTIVITIES

Piperastatin A (structure, N-formyl-allo Ile-Thr-Leu-Val-Pip-Leu-Pip, Pip = hexahydropyridadine-3-carboxylic acid; molecular weight, 809), a new inhibitor of serine carboxypeptidase was discovered in the fermentation broth of Streptomyces lavendofoliae MJ908-WF13. It was purified by activated charcoal chromatography, YMC gel ODS-A chromatography and centrifugal partition chromatography (CPC) by monitoring its inhibitory activity against carboxypeptidase Y (CP-Y), and finally obtained as colourless needles. Piperastatin A is a competitive inhibitor of the enzyme with Ki = 52 +/- 6.2 nM. Piperastatin A is a highly specific inhibitor of the serine carboxypeptidases, CP-Y and platelet deamidase with little effect on related enzymes, has no antimicrobial activity and has low toxicity.

BELACTINS A AND B, NEW SERINE CARBOXYPEPTIDASE INHIBITORS PRODUCED BY ACTINOMYCETE. I: TAXONOMY, PRODUCTION, ISOLATION AND BIOLOGICAL ACTIVITIES

Belactins A and B, new inhibitors of serine carboxypeptidase were discovered in the fermentation broth of Saccharopolyspora sp. MK19-42F6. They were purified by ethyl acetate extraction, silica gel chromatography, Sephadex LH20 chromatography, Capcellpak C18 SG120 reversed phase HPLC and centrifugal partition chromatography (CPC) following their inhibitory activity against carboxypeptidase Y (CP-Y). The inhibition constants (Ki) of belactins A and B against CP-Y are 0.14 and 0.27 microM respectively. Belactins A and B have highly specific inhibitory activities for CP-Y among various peptidases, have no antimicrobial activities at 100 micrograms/ml and have low toxicities.

Relative deficiency of serine proteinase activities in spleen of aged mice

We examined the relation of hydrolytic enzymes in spleen to the aging process in mice over a period of 30 months. When the enzymatic activities were expressed as activities per milligrams protein, those of serine proteinases and dipeptidyl peptidase IV (Gly-Pro-AP) significantly decreased with age, whereas that of L-leucine aminopeptidase (Leu-AP) increased significantly. However, when expressed as total activities, the enzymatic activities in spleen generally tended to increase with age, except in the case of serine proteinases, because of the age-related increase in spleen weight. The results were taken to indicate that the activities of serine proteinases become relatively more deficient in the spleen as age increases. The results of a multivariate study maintained this peculiarity of serine proteinases in comparison with other enzymes. The relative deficiency of serine proteinases in spleen may be somehow related to immunodeficiency in aged animals, as judged from similar findings in animal models of systemic erythematodes.

Piperastatin B: A New Selective Serine Carboxypeptidase Inhibitor from Streptomyces Lavendofoliae MJ908-WF13

Piperastatin B, a new inhibitor of serine carboxypeptidase was purified from a culture broth of Streptomyces lavendofoliae MJ908-WF13 as a minor component by monitoring its inhibitory activity against carboxypeptidase Y (CP-Y). Its structure was determined to be N-formyl-Val-Thr-Leu-Val-Pip-Leu-Pip (pip: piperazic acid, hexahydropyridadine-3-carboxylic acid). Piperastatin B is a highly specific competitive inhibitor of CP-Y (Ki = 55 nM) with little effect on related enzymes and resembles the major component, piperastatin A, in these respects.