Heptapeptide Core Research Articles

Altering glycopeptide antibiotic biosynthesis through mutasynthesis allows incorporation of fluorinated phenylglycine residues.

Glycopeptide antibiotics (GPAs) are peptide natural products used as last resort treatments for antibiotic resistant bacterial infections. They are produced by the sequential activities of a linear nonribosomal peptide synthetase (NRPS), which assembles the heptapeptide core of GPAs, and cytochrome P450 (Oxy) enzymes, which perform a cascade of cyclisation reactions. The GPAs contain proteinogenic and nonproteinogenic amino acids, including phenylglycine residues such as 4-hydroxyphenylglycine (Hpg). The ability to incorporate non-proteinogenic amino acids in such peptides is a distinctive feature of the modular architecture of NRPSs, with each module selecting and incorporating a desired amino acid. Here, we have exploited this ability to produce and characterise GPA derivatives containing fluorinated phenylglycine (F-Phg) residues through a combination of mutasynthesis, biochemical, structural and bioactivity assays. Our data indicate that the incorporation of F-Phg residues is limited by poor acceptance by the NRPS machinery, and that the phenol moiety normally present on Hpg residues is essential to ensure both acceptance by the NRPS and the sequential cyclisation activity of Oxy enzymes. The principles learnt here may prove useful for the future production of GPA derivatives with more favourable properties through mixed feeding mutasynthesis approaches.

Synthesis of the cyclic heptapeptide core of callipeltin A

Macrolactonisation of a suitable heptapeptide precursor provides the cyclic depsipeptide core of callipeltin A in excellent yield.

1056. Safety and Tolerability of Dalbavancin in Vancomycin Allergic Patients – A Case Series

BackgroundVVancomycin and dalbavancin, both in the glycopeptide class of antibiotics, are used in the treatment of Gram-positive infections, including methicillin-resistant Staphylococcus aureus. Antibiotics in this class contain a heptapeptide core that has potential for cross-sensitivity. Due to this risk, dalbavancin carries a warning in the package insert for use in patients with a glycopeptide allergy. Dalbavancin, a semi-synthetic derivative of vancomycin, has lipophilic side chains which reduce the risk of cross-sensitivity to vancomycin. This case series evaluated patients with a listed vancomycin allergy in their electronic health record who received dalbavancin as an outpatient infusion.MethodsThis study was a non-randomized, retrospective chart review of adult patients who had a documented vancomycin allergy and received dalbavancin between February 2016 and February 2021 for any indication in the outpatient setting. The primary objective was to evaluate dalbavancin tolerability in patients allergic to vancomycin. Patient characteristics and the specifics of dalbavancin infusion – dose, volume, infusion rate, intravenous line access, and receipt of premedication before infusion – were collected on each patient.Results559 unique patients received dalbavancin over the time frame. Of these, ten had a documented, subjective vancomycin allergy. Patient-reported allergic reactions were rash (4), hives (3), anaphylaxis (2), red man syndrome (2), renal failure (2), and general malaise (1). Six patients had at least 1 additional subjective drug allergy. The various infections treated included cellulitis/abscess (8), osteomyelitis (1), and bacteremia (2). Most patients received 1500mg (2 received 1125mg) of dalbavancin in 300-500mL of dextrose 5% in water infused at either 600 or 1000mL/hr via a peripheral (6) or central (4) intravenous line. All patients tolerated the infusion with no adverse events reported and no receipt of premedication before administration. ConclusionDalbavancin may be a reasonable treatment option in vancomycin allergic patients, despite possible cross-sensitivity. Further investigation into cross-sensitivity between vancomycin, dalbavancin, and other glycopeptide class agents is warranted.Disclosures Kerry O. Cleveland, M.D., AbbVie (Speaker’s Bureau)Merck (Speaker’s Bureau)Pfizer (Speaker’s Bureau) Bruce M. Jones, PharmD, BCPS, Abbvie (Consultant, Advisor or Review Panel member, Speaker’s Bureau)La Jolla (Speaker’s Bureau)Melinta (Consultant)Merck (Consultant)Paratek (Consultant, Speaker’s Bureau)

A few atoms make the difference: Synthetic, CD, NMR and computational studies on antiviral and antibacterial activities of glycopeptide antibiotic aglycon derivatives

Despite the close structural similarity between the heptapeptide cores of the glycopeptide antibiotics teicoplanin and ristocetin, synthetically modified derivatives of their aglycons show significantly different antibacterial and antiviral properties. The teicoplanin aglycon derivatives with one exception proved to be potent antibacterials but they did not exhibit anti-influenza virus activity. In contrast, the aglycoristocetin derivatives generally showed high anti-influenza virus activity and possessed moderate antibacterial activity. A systematic structure-activity relationship study has been carried out on ristocetin and teicoplanin aglycon derivatives, to explore which structural differences are responsible for these markedly different biological activities. According to electronic circular dichroism and in silico conformational studies, it was found that the differences in anti-influenza virus activity are mainly determined by the conformation of the heptapeptide core of the antibiotics controlled by the presence or absence of chloro substituents. Knowledge of the bioactive conformation will help to design new analogs with improved anti-influenza virus activity. For the teicoplanin derivatives, it was shown that derivatization to improve the antiviral efficacy was accompanied by a significant decrease in antibacterial activity.

Glycopeptide Sulfation Evades Resistance

The incidence of antibiotic resistance among pathogenic microorganisms is increasing at an alarming rate. Resistance against front-line therapeutics such as the glycopeptide antibiotic vancomycin has emerged and has spread to highly virulent pathogens, including Staphylococcus aureus. Glycopeptide antibiotics are natural products from the Actinomycetes that have a characteristic heptapeptide core. The chemical diversity of the class is achieved through glycosylation, halogenation, methylation, and acylation of the core, modifications that are implicated in improved solubility, stability, or activity of the molecule. Sulfation is yet another modification observed infrequently in glycopeptides, but its role is not known. Although glycopeptide sulfotransferases are found in the environmental metagenome and must therefore serve an evolutionary purpose, all previous studies have reported decreased antibiotic activity with sulfation. We report that sulfation of glycopeptides has little effect on the compound's ability to bind its target, the d-Ala-d-Ala peptidoglycan precursors of the bacterial cell wall. However, sulfation does impact glycopeptide dimerization, and importantly, sulfated glycopeptides are significantly less potent inducers of the resistance gene cluster vanHAX in actinomycetes. Our results begin to unravel the mystery of the biological role of glycopeptide sulfation and offer a potential new strategy for the development of new antibiotics that avoid resistance.

Oritavancin: Mechanism of Action

Oritavancin is a semisynthetic lipoglycopeptide analogue of vancomycin that contains the heptapeptide core common to all glycopeptides. It differs from vancomycin by the presence of a hydrophobic N-4-(4-chlorophenyl)benzyl (also referred to as 4'-chlorobiphenylmethyl) substituent on the disaccharide sugar, the addition of a 4-epi-vancosamine monosaccharide to the amino acid residue in ring 6, and the replacement of the vancosamine moiety by 4-epi-vancosamine. One mechanism of action of oritavancin is inhibition of transglycosylation (important in peptidoglycan synthesis) by binding to D-alanyl-D-alanine stem termini in Gram-positive bacteria. The inhibition of peptidoglycan synthesis via inhibition of transglycosylation is common to all glycopeptides (vancomycin) and lipoglycopeptides. Secondary binding of oritavancin to the pentaglycyl (Asp/Asn) bridging segment in peptidoglycan also occurs, which distinguishes it from vancomycin and contributes to oritavancin's activity versus vancomycin-resistant organisms. The presence of the hydrophobic 4'-chlorobiphenylmethyl group allows for interaction and disruption of the cell membrane, resulting in depolarization, permeabilization, and concentration-dependent, rapid cell death. This mechanism is shared with telavancin but not vancomycin and results in activity against daptomycin-nonsusceptible organisms. In conclusion, oritavancin's mechanism of action involves at least 3 known mechanisms: inhibition of transglycosylation, inhibition of transpeptidation, and cell membrane interaction/disruption. Oritavancin's multiple mechanisms of action confer activity against vancomycin-susceptible and -resistant organisms, as well as rapid, concentration-dependent killing versus actively growing, stationary phase, and biofilm-producing Gram-positive bacteria.

New Lipoglycopeptides

Dalbavancin, oritavancin and telavancin are semisynthetic lipoglycopeptides that demonstrate promise for the treatment of patients with infections caused by multi-drug-resistant Gram-positive pathogens. Each of these agents contains a heptapeptide core, common to all glycopeptides, which enables them to inhibit transglycosylation and transpeptidation (cell wall synthesis). Modifications to the heptapeptide core result in different in vitro activities for the three semisynthetic lipoglycopeptides. All three lipoglycopeptides contain lipophilic side chains, which prolong their half-life, help to anchor the agents to the cell membrane and increase their activity against Gram-positive cocci. In addition to inhibiting cell wall synthesis, telavancin and oritavancin are also able to disrupt bacterial membrane integrity and increase membrane permeability; oritavancin also inhibits RNA synthesis. Enterococci exhibiting the VanA phenotype (resistance to both vancomycin and teicoplanin) are resistant to both dalbavancin and telavancin, while oritavancin retains activity. Dalbavancin, oritavancin and telavancin exhibit activity against VanB vancomycin-resistant enterococci. All three lipoglycopeptides demonstrate potent in vitro activity against Staphylococcus aureus and Staphylococcus epidermidis regardless of their susceptibility to meticillin, as well as Streptococcus spp. Both dalbavancin and telavancin are active against vancomycin-intermediate S. aureus (VISA), but display poor activity versus vancomycin-resistant S. aureus (VRSA). Oritavancin is active against both VISA and VRSA. Telavancin displays greater activity against Clostridium spp. than dalbavancin, oritavancin or vancomycin. The half-life of dalbavancin ranges from 147 to 258 hours, which allows for once-weekly dosing, the half-life of oritavancin of 393 hours may allow for one dose per treatment course, while telavancin requires daily administration. Dalbavancin and telavancin exhibit concentration-dependent activity and AUC/MIC (area under the concentration-time curve to minimum inhibitory concentration ratio) is the pharmacodynamic parameter that best describes their activities. Oritavancin's activity is also considered concentration-dependent in vitro, while in vivo its activity has been described by both concentration and time-dependent models; however, AUC/MIC is the pharmacodynamic parameter that best describes its activity. Clinical trials involving patients with complicated skin and skin structure infections (cSSSIs) have demonstrated that all three agents are as efficacious as comparators. The most common adverse effects reported with dalbavancin use included nausea, diarrhoea and constipation, while injection site reactions, fever and diarrhoea were commonly observed with oritavancin therapy. Patients administered telavancin frequently reported nausea, taste disturbance and insomnia. To date, no drug-drug interactions have been identified for dalbavancin, oritavancin or telavancin. All three of these agents are promising alternatives for the treatment of cSSSIs in cases where more economical options such as vancomycin have been ineffective, in cases of reduced vancomycin susceptibility or resistance, or where vancomycin use has been associated with adverse events.

Biosynthesis of Sulfated Glycopeptide Antibiotics by Using the Sulfotransferase StaL

The unique glycopeptide antibiotic A47934, produced by Streptomyces toyocaensis, possesses a nonglycosylated heptapeptide core that is sulfated on the phenolic hydroxyl of the N-terminal 4-hydroxy-L-phenylglycine residue. Genetic and biochemical experiments confirmed that StaL is a sulfotransferase capable of sulfating the predicted crosslinked heptapeptide substrate to produce A47934 both in vivo and in vitro. Incubation of purified His(6)-StaL with various substrates in vitro revealed substrate specificity and yielded two sulfo-glycopeptide antibiotics: sulfo-teicoplanin aglycone and sulfo-teicoplanin. Quantification of the antibacterial activity of desulfo-A47934, A47934, teicoplanin, and sulfo-teicoplanin demonstrated that sulfation slightly increased the minimum inhibitory concentration. This unique modification by sulfation expands glycopeptide diversity with potential application for the development of new antibiotics.

Glycopeptide derivatives.

Resistance to glycopeptides in enterococci, which first emerged in the late 1980's and is now widespread mainly in the United States, is posing a serious clinical problem due to the lack of alternative and efficacious therapeutic options, particularly against infections caused by VanA strains that are highly resistant to glycopeptides and almost all other antibiotics. In addition, isolates of Staphylococcus aureus, known as GISA, that are poorly susceptible to vancomycin and teicoplanin have been identified. Thus, there is an urgent need to develop new and more potent glycopeptides that are active against these problematic organisms. The following review will focus on the development of second-generation glycopeptides, namely LY333328 (Eli Lilly) and BI 397 (Biosearch Italia, in license to Versicor for North America), which are currently undergoing clinical trials in humans for their promising activity against VanA enterococci (LY333328), staphylococci (BI 397), and penicillin-resistant pneumococci. Both compounds were identified as the result of chemical programs that were aimed at pursuing activity of vancomycin-like or teicoplanin-like natural glycopeptides against VanA enterococci and multidrug-resistant staphylococci. More recent approaches toward glycopeptides modified in their heptapeptide core are also described. These include compounds in which amino acids 1 and 3 are replaced with other amino acid moieties such as in the modification of the asparagine side chain on residue 3 as well as attempts to change the structure of the heptapeptide backbone in positions that are critical for the molecular interaction with susceptible D-Ala-D-Ala and resistant D-Ala-D-Lactate targets. Covalently linked glycopeptide dimers and vancomycin derivatives in which vancosamine is suitably replaced with other sugar moieties will also be covered.

Molecular cloning of a novel melanocortin receptor.

Using the technique of the polymerase chain reaction primed with oligonucleotides based on the homologous transmembrane regions of seven transmembrane G protein-linked receptors, we isolated three full-length human genes that encode a novel subgroup of this receptor family. Recently, two of these receptors were identified as specific for alpha-melanocyte-stimulating hormone (alpha-MSH) and adrenocorticotropic hormone. We report the molecular cloning and pharmacologic characterization of a third member of this subgroup. The gene for this receptor encodes a protein of 361 amino acids in length. Its pharmacology characterizes it as an MSH receptor specific to the heptapeptide core common to adrenocorticotropic hormone and alpha-, beta-, and gamma-MSH. By Northern blot hybridization and polymerase chain reaction, it is expressed in brain, placental, and gut tissues but not in melanoma cells or in the adrenal gland. These findings may yield insight into the physiology of peptides derived from pro-opiomelanocortin post-translational processing.

Actinoidins A and A2: structure determination using 2D NMR methods.

The structural analysis of the intact glycopeptide antibiotics, actinoidins A (1a) and A2 (1b), by two-dimensional 1H NMR is described. The location of the single chlorine at the A3 position and the sites of attachment of the four carbohydrate substituents in actinoidin A are elucidated based on correlation spectroscopy (COSY), double quantum coherence experiments (DQCE), homonuclear Hartmann-Hahn experiments (HOHAHA) and nuclear Overhauser spectroscopy (NOESY). Similar 2D correlation and NOE NMR experiments are then performed on the novel analog, actinoidin A2, to determine its structure. The structural difference between actinoidins A and A2 is shown to reside in the presence of L-rhamnose in actinoidin A2 in place of L-acosamine in actinoidin A. All questions concerning the stereo-chemistry of the chiral centers in both the heptapeptide core and the carbohydrate moieties in each of these antibiotics could be successfully addressed with the exception of Gl', the alpha-carbon on the N-terminal amino acid which is known to have the R-configuration from previous studies.

A neuroheptapeptide influence on cognitive functioning in the elderly

The heptapeptide core common to melanocyte-stimulating hormone (MSH) and adrenocorticotropin (ACTH) was administered as a single subcutaneous dose of 30 mg to 13 elderly human subjects (9 men, 4 women) in a double-blind, cross-over design. Significant improvement was found in the Benton Visual Retention Test after MSH/ACTH 4–10 as compared with administration of saline. This appeared to be greater in men than women. No side effects or laboratory abnormalities were observed. The behavioral results are consistent with our earlier findings in men and rats of improved visual attention after administration of MSH and extend them to the elderly population.

A β-MSH in the pituitary gland of the spotted dogfish ( Scyliorhinus canicula): Isolation and structure

A β-type MSH has been isolated from the pituitary of the common dogfish ( Scyliorhinus canicula). It differs from all MSH peptides so far isolated in that glycine replaces glutamic acid in the so-called heptapeptide core. By conventional methods the sequence was deduced to be Asx.Glx.Ile.Asx.Tyr.Lys.Met.Gly.His.Phe.Arg.Trp.Gly.Ala.Pro.Met.Asp.Lys.

Structural studies of alpha-melanocyte-stimulating hormone and a novel beta-melanocyte-stimulating hormone from the neurointermediate lobe of the pituitary of the dogfish Squalus acanthias.

A melanocyte-stimulating hormone (MSH) has been isolated from extracts of the neurointermediate lobe of the pituitary of the dogfish Squalus acanthias by gel-filtration and ion-exchange chromatography. It had approximately 1% of the potency of mammalian alpha-MSH on bioassays in vitro on frog skin and dogfish skin. Sequence analysis revealed it to be a hexadecapeptide with the following primary structure: Asp-Gly-Asp-Asp-Tyr-Lys-Phe-Gly-His-Phe-Arg-Trp-Ser-Val-Pro-Leu. It appears to be related to the beta-MSH species of mammalian species but has only the sequence -His-Phe-Arg-Trp- in common with the heptapeptide core -Met-Glu-His-Phe-Arg-Trp-Gly- which is characteristic not only of the MSH peptides but also of the adrenocorticotrophins and lipotrophins studied so far. An alpha-MSH was also isolated, 50% of which was amidated at the C-terminus group. Sequence data from this study taken in conjunction with those from a previous study (Lowry & Chadwick, 1970b) revealed it to be a tridecapeptide which is identical with the N-terminal sequence of dogfish adrenocorticotrophin.