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This review focuses on the genetic and biotechnological aspects of the biosynthesis of ramoplanin (Rmp), enduracidin (End), and other related lipodepsipeptide antibiotics, herein named collectively ramoplanin and ramoplanin-related lipodepsipeptide (RRLDPs). These compounds exhibit a promising antimicrobial activity against Gram-positive bacterial pathogens, showing no cross-resistance with vancomycin. Rmp is in clinical development for human treatment and End has been used as animal growth promoter for decades. Other RRLDPs as ramoplanose and janiemycin had been poorly investigated in the past, whereas new molecules as chersinamycin have been recently discovered, attracting a renewed interest in this class of antibiotics. Nowadays, sequence and annotation of the biosynthetic gene clusters (BGCs) of Rmp, End, and several other RRLDPs are available, and researchers are focused on understanding the biosynthetic logic behind the production of these compounds. Interestingly, producers of Rmp and chersinamycin belong to the so-called “non-common” actinomycetes from the family Micromonosporaceae, whereas End is produced by different members of the genus Streptomyces. To the best of our knowledge, no reviews summarize and systematize the current information on the biosynthesis of RRLDPs. Therefore, in this review, we aim to fill this gap. We first describe and compare the BGCs for known RRLDPs, giving an insight on how they were discovered and developed. Next, we review the biosynthetic pathways of these antibiotics, as well as the regulation of their biosynthesis. Then, we focus on the production processes of RRLDPs, demonstrating how cultivation and nutritional factors influence their production. Finally, we provide a short outline of future directions in studying RRLDPs.

A simple, sensitive, and simultaneous method was established and validated for the active clinical components of teicoplanin and ramoplanin in environmental water by LC-MS/MS coupled with cascade elution. Moreover, a cascade elution method, which was rapid, solvent-less, and high-extraction efficient was successfully proposed to realize the extraction and purification of seven targets in one step. Under optimized conditions, the method showed excellent linearity with the correlation coefficient (R2) ≥0.998 in the range of 1.0–100.0 ng L−1. Low matrix effects and good recoveries which ranged from 86 to 114% were reached with RSDs lower than 3.0% for most targets. The limits of detection and limit of quantification were 0.1–1.3 and 0.3–4.0 ng L−1, respectively. This method was successfully applied for the determination of teicoplanin and ramoplanin in water samples from the Pearl River and the South China Sea. TA2-2,3 was quantified in only one sample with the concentration of 8.0 ng L−1.

Neisseria gonorrhoeae is a bacterial pathogen that causes gonorrhoea, a sexually transmitted infection. Increasing antimicrobial resistance in N. gonorrhoeae is providing motivation to develop new treatment options. In this study, we investigated the effectiveness of the antibiotic ramoplanin as a treatment for N. gonorrhoeae infection. We tested the effectiveness of ramoplanin in vitro against 14 World Health Organization (WHO) reference strains of N. gonorrhoeae and found that it was active against all 14 strains tested. Furthermore, in a Galleria mellonella infection model of N. gonorrhoeae WHO P, we demonstrated that ramoplanin was active in vivo without any evidence of toxicity. This suggests that ramoplanin might be a new promising antibiotic treatment for gonorrhoea.

Development of new antibiotics is of the utmost importance in the battle against antimicrobial resistance. One of the promising candidates in the fight against resistant bacteria is ramoplanin, a cyclic lipoglycodepsipeptide derived from a fermentation broth. In order to obtain effective and safe pharmaceutical product on the market, development of reliable analytical method for the evaluation of ramoplanin's purity is vital. In the present work, an LC method was developed, validated and used during stability testing of ramoplanin, in solid state and in solution. Forced degradation study was performed by subjecting the sample to different stress conditions (acid and base hydrolysis, oxidation, humidity, heat and light) and the observed degradation products were elucidated using mass spectrometry. The study revealed significant degradation of ramoplanin under alkaline conditions, leading to formation of lacton hydrolysis product. The results of the study clearly demonstrate that the hydrolysis of the lacton ring is predominant path of ramoplanin's degradation. However, other significant degradation routs were also observed. Based on the MS/MS results, degradation pathways of ramoplanin are proposed.

The threat of antimicrobial resistance to antibiotics requires a continual effort to develop alternative treatments. Arylglycines (or phenylglycines) are one of the signature amino acids found in many natural peptide antibiotics, but their propensity for epimerization in solid-phase peptide synthesis (SPPS) has prevented their use in long peptide sequences. We have now identified an optimized protocol that allows the synthesis of challenging non-ribosomal peptides including precursors of the glycopeptide antibiotics and an analogue of feglymycin (1 analogue, 20%). We have exploited this protocol to synthesize analogues of the peptide antibiotic ramoplanin using native chemical ligation/desulfurization (1 analogue, 6.5%) and head-to-tail macrocyclization in excellent yield (6 analogues, 3-9%), with these compounds extensively characterized by NMR (U-shaped structure) and antimicrobial activity assays (two clinical isolates). This method significantly reduces synthesis time (6-9 days) when compared with total syntheses (2-3 months) and enables drug discovery programs to include arylglycines in structure-activity relationship studies and drug development., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

This review focuses on the genetic and biotechnological aspects of the biosynthesis of ramoplanin (Rmp), enduracidin (End), and other related lipodepsipeptide antibiotics, herein named collectively ramoplanin and ramoplanin-related lipodepsipeptide (RRLDPs). These compounds exhibit a promising antimicrobial activity against Gram-positive bacterial pathogens, showing no cross-resistance with vancomycin. Rmp is in clinical development for human treatment and End has been used as animal growth promoter for decades. Other RRLDPs as ramoplanose and janiemycin had been poorly investigated in the past, whereas new molecules as chersinamycin have been recently discovered, attracting a renewed interest in this class of antibiotics. Nowadays, sequence and annotation of the biosynthetic gene clusters (BGCs) of Rmp, End, and several other RRLDPs are available, and researchers are focused on understanding the biosynthetic logic behind the production of these compounds. Interestingly, producers of Rmp and chersinamycin belong to the so-called "non-common" actinomycetes from the family Micromonosporaceae, whereas End is produced by different members of the genus Streptomyces. To the best of our knowledge, no reviews summarize and systematize the current information on the biosynthesis of RRLDPs. Therefore, in this review, we aim to fill this gap. We first describe and compare the BGCs for known RRLDPs, giving an insight on how they were discovered and developed. Next, we review the biosynthetic pathways of these antibiotics, as well as the regulation of their biosynthesis. Then, we focus on the production processes of RRLDPs, demonstrating how cultivation and nutritional factors influence their production. Finally, we provide a short outline of future directions in studying RRLDPs. [ABSTRACT FROM AUTHOR]

Glycopeptides such as vancomycin and telavancin are essential for treating infections caused by Gram-positive bacteria. But the dwindling availability of new antibiotics and the emergence of resistant bacteria are making effective antibiotic treatment increasingly difficult. Ramoplanin, an inhibitor of bacterial cell wall biosynthesis, is a highly effective antibiotic against a wide range of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, vancomycin-intermediate resistant Clostridium difficile and vancomycin-resistant Enterococcus sp. Here, two tailoring enzyme genes in the biosynthesis of ramoplanin were deleted by double in-frame gene knockouts to produce new ramoplanin derivatives. The deschlororamoplanin A2 aglycone was purified and its structure was identified with LC-MS/MS. Deschlororamoplanin A2 aglycone and ramoplanin aglycone showed similar activity to ramoplanin A2. The results showed that α-1,2-dimannosyl disaccharide at Hpg11 and chlorination at Chp17 in the ramoplanin structure are not essential for its antimicrobial activity. This work provides new precursor compounds for the semisynthetic modification of ramoplanin.

Glycopeptides such as vancomycin and telavancin are essential for treating infections caused by Gram-positive bacteria. But the dwindling availability of new antibiotics and the emergence of resistant bacteria are making effective antibiotic treatment increasingly difficult. Ramoplanin, an inhibitor of bacterial cell wall biosynthesis, is a highly effective antibiotic against a wide range of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, vancomycin-intermediate resistant Clostridium difficile and vancomycin-resistant Enterococcus sp. Here, two tailoring enzyme genes in the biosynthesis of ramoplanin were deleted by double in-frame gene knockouts to produce new ramoplanin derivatives. The deschlororamoplanin A2 aglycone was purified and its structure was identified with LC-MS/MS. Deschlororamoplanin A2 aglycone and ramoplanin aglycone showed similar activity to ramoplanin A2. The results showed that α-1,2-dimannosyl disaccharide at Hpg11 and chlorination at Chp17 in the ramoplanin structure are not essential for its antimicrobial activity. This work provides new precursor compounds for the semisynthetic modification of ramoplanin. [ABSTRACT FROM AUTHOR]

Adis Comments Ramoplanin [A 16686, A 16686A, MDL 62198], originated by Biosearch Italia, is a glycolipopeptide antibacterial agent derived from Actinoplanes spp. with bactericidal activity primarily against Gram-positive organisms. Ramoplanin kills microbes by blocking bacterial cell wall synthesis at a site that is different from those targeted by the β-lactams such as penicillin and the glycopeptides such as vancomycin. To date, laboratory studies have not encountered bacterial resistance with ramoplanin and no cross-resistance has been observed between ramoplanin and the β-lactams or glycopeptides. An oral and a topical formulation of ramoplanin are being investigated. Ramoplanin has potential for use in the treatment of vancomycin-resistant enterococcal (VRE) infections, for which it has been granted fast-track status by the US FDA and given an Orphan Medicinal Product designation by the European Commission. Other potential clinical uses under investigation are selective gut decontamination and eradication of Clostridium difficile from the bowels of patients with pseudomembranous colitis and eradication of nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA). Oral formulation: A placebo-controlled Phase II trial of oral ramoplanin in 150 patients with hospital-acquired vancomycin-resistant Enterococcus faecium (VREF) intestinal infections has been completed at eight centres in the USA. The study was terminated early after an independent Data Safety Monitoring Board (DSMB) determined there was sufficient evidence of effectiveness and safety to proceed with Phase III trials. Genome Therapeutics, Biosearch's North American licensing partner, is currently conducting an ongoing Phase III study in the USA of oral ramoplanin for the prevention of bloodstream infections caused by VRE infections. In March 2002, Genome Therapeutics reported that the trial was ongoing with one-third of its goal of 950 patients enrolled into the trial to date. In order to include more of the at-risk population, Genome announced an amendment to their study inclusion criteria in May 2002 to include patients with an anticipated period of chemotherapy-related neutropenia of at least 5 days (was originally at least 10 days). The company plans to file a New Drug Application with the US FDA in 2004. This study was intially started in mid-2000 by IntraBiotics. In October 2001, Genome Therapeutics licensed ramoplanin from BioSearch Italia. Topical formulation: A Phase II trial of ramoplanin ointment to prevent hospital-based infections caused by S. aureus, including MRSA, has been initiated. The goal of the phase II trial is to determine whether ramoplanin ointment can effectively eradicate nasal carriage of S. aureus in human volunteers. Nine sites across the USA will enrol a total of 250 volunteers who carry S. aureus in their nose. All volunteers will receive ramoplanin or placebo for 4 days and will be monitored for 90 days to evaluate nasal carriage of MRSA. Agreements: In October 2001, Biosearch Italia licensed exclusive rights to oral ramoplanin in the USA and Canada to Genome Therapeutics. The latter will be responsible for development of the drug, including funding of clinical trials, and will make a $US2 million initial payment, followed by up to $US8 million in milestone payments. Genome will also purchase bulk material from Biosearch and pay royalties on product sales. USA and Canadian rights to oral ramoplanin were previously licensed to IntraBiotics. As a follow-on to the delay in successfully completing the Phase III trial of oral ramoplanin in VREF infections, Biosearch Italia renegotiated this licensing agreement. IntraBiotics relinquished North American rights for ramoplanin oral powder to BioSearch, but retained the rights to topical ramoplanin for this market. However, BioSearch Italia announced in May 2002 that they had reacquired all worldwide rights for topical ramoplanin from IntraBiotics at no cost after the company failed to commence clinical trials in North America by the March 2002 deadline. During February 2001 Biosearch Italia signed an agreement with an Italian subsidiary of Aventis (Aventis Bulk SpA) for the manufacture of ramoplanin. The latter will manufacture sufficient quantities of the drug for clinical development and its eventual launch in the USA. [ABSTRACT FROM AUTHOR]

Adis Comments Ramoplanin [A 16686, A 16686A, MDL 62198], originated by Biosearch Italia, is a topical glycolipopeptide antibacterial agent derived from Actinoplanes spp. with bactericidal activity primarily against Gram-positive organisms. Ramoplanin kills microbes by blocking bacterial cell wall synthesis at a site that is different from those targeted by the β-lactams such as penicillin and the glycopeptides such as vancomycin. To date, laboratory studies have not encountered bacterial resistance with ramoplanin and no cross resistance has been observed between ramoplanin and the β-lactams or glycopeptides. Ramoplanin has potential for use in the treatment of vancomycin-resistant enterococcal infection, for which it has been granted fast-track status by the US FDA and given an Orphan Medicine designation by the European Commission. Other potential clinical uses under investigation are selective gut decontamination and eradication of Clostridium difficile from the bowels of patients with pseudomembranous colitis and eradication of nasal carriage of MRSA. A placebo-controlled phase II trial in 150 patients with hospital acquired vancomycin-resistant Enterococcus faecium(VREF) intestinal infections has been completed at eight centres in the USA. The study was terminated early after an independent Data Safety Monitoring Board (DSMB) determined there was sufficient evidence of effectiveness and safety to proceed with phase III trials. In mid-2000, IntraBiotics initiated a phase III trial of oral ramoplanin for the prevention of vancomycin-resistant enterococcal blood infection in the USA. Although the company hoped that the US phase III trials would be completed by 2001, it was announced in March 2001 that a delay of 1 year was expected because of difficulties in patient recruitment. A phase II trial of ramoplanin ointment to prevent hospital-based infections caused by Staphylococcus aureus, including MRSA, has been initiated. The goal of the phase II trial is to determine whether ramoplanin ointment can effectively eradicate nasal carriage of S. aureus in human volunteers. Nine sites across the US will enroll a total of 250 volunteers who carry S. aureus in their nose. All volunteers will receive ramoplanin or placebo for 4 days and will be monitored for 90 days to evaluate nasal carriage of MRSA. In October 2001, Biosearch Italia licensed exclusive rights to oral ramoplanin in the US and Canada to Genome Therapeutics. The latter will be responsible for development of the drug, including funding of clinical trials, and will make a $US2 million initial payment, followed by up to $US8 million in milestone payments. Genome will also purchase bulkmaterial from Biosearch and pay royalties on product sales. US and Canadian rights to oral ramoplanin were previously licensed to IntraBiotics. As a follow-on to the delay in successfully completing the phase III trial of oral ramoplanin in VREF infections, Biosearch Italia renegotiated this licensing agreement. IntraBiotics relinquished North American rights for ramoplanin oral powder to BioSearch, but retained the rights to topical ramoplanin for this market. BioSearch Italia has also imposed an ultimatum on IntraBiotics for topical ramoplanin; if IntraBiotics does not begin clinical trials by March 2002, all rights will revert to BioSearch, which would then seek another partner. During February 2001 Biosearch Italia signed an agreement with an Italian subsidiary of Aventis (Aventis Bulk SpA) for the manufacture of ramoplanin. The latter will manufacture sufficient quantities of the drug for clinical development and its eventual launch in the US. [ABSTRACT FROM AUTHOR]

An alanine scan of the ramoplanin A2 aglycon, a potent antibiotic that has inhibited bacterial cell wall biosynthesis, is described. The antimicrobial activity of the analogues has defined the importance of each residue of this complex glycopeptide antibiotic.

Ramoplanin is a novel lipoglycodepsipeptide isolated from the fermentation broths of Actinoplanes Sp. ATCC 33076. A solution phase convergent total synthesis of the ramoplanin A2 and ramoplanose aglycon is developed defining two strategically effective macrocyclization sites.

Ramoplanin is a glycolipodepsipeptide antibiotic obtained from fermentation of Actinoplanes sp. ATCC 33076 that exhibits activity against clinically important multi-drug-resistant, Gram-positive pathogens including vancomycin-resistant Enterococcus (VRE), methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-intermediate resistant Clostridium difficile. It disrupts bacterial cell wall through a unique mechanism of action by sequestering the peptidoglycan intermediate Lipid II and therefore does not show cross-resistance with other antibiotics. However, while demonstrating excellent antimicrobial activity in systemic use in animal models of infection, ramoplanin presents low local tolerability when injected intravenously. As a consequence of this limitation, new derivatives are desirable to overcome this issue. During a natural product screening program developed to discover compounds that disrupt bacterial cell wall synthesis by inhibiting peptidoglycan transglycosylation through binding to the intermediate Lipid II, 49 actinomycete strains were identified by HR-LCMS as producers of ramoplanin-related compounds. The producing strains were isolated from environmental samples collected worldwide comprising both tropical and temperate areas. To assess the diversity of this microbial population, the 49 isolates were initially identified to the genus level on the basis of their micromorphology, and 16S sequencing confirmed the initial identification of the strains. These analyses resulted in the identification of members of genus Streptomyces, as well as representatives of the families Micromonosporaceae, Nocardiaceae, Thermomonosporaceae, and Pseudonocardiaceae, suggesting that the production of ramoplanins is relatively widespread among Actinomycetes. In addition, all of these isolates were tested against a panel of Gram-positive and Gram-negative bacteria, filamentous fungi, and yeast in order to further characterize their antimicrobial properties. This work describes the diversity of actinomycete strains that produced ramoplanin-related compounds, and the analysis of the antimicrobial activity exhibited by these isolates. Our results strongly suggest the presence of new ramoplanin-analogs among these actinomycete producers. [ABSTRACT FROM AUTHOR]