Influence of Lipophilicity on the Antibacterial Activity of Polymyxin Derivatives and on Their Ability to Act as Potentiators of Rifampicin.

Novel polymyxin derivatives are often classified either as having direct activity against Gram-negative pathogens or as compounds inactive in their own right, which through permeabilization of the outer membrane act as potentiators of other antibiotics. Here, we report the systematic investigation of the influence of lipophilicity on microbiological activity (including against strains with reduced susceptibility to polymyxins), potentiation of rifampicin, and in vitro toxicity within a series of next-generation polymyxin nonapeptides. We demonstrate that the lipophilicity at the N-terminus and amino acids 6 and 7 in the cyclic peptide core is interchangeable and that the activity, ability to potentiate, and cytotoxicity all appear to be primarily driven by overall lipophilicity. Our work also suggests that the characterization of a polymyxin molecule as either a direct acting compound or a potentiator is more of a continuum that is strongly influenced by lipophilicity rather than as a result of fundamentally different modes-of-action.

Direct modifications of the cyclic peptide Polymyxin B leading to analogues with enhanced in vitro antibacterial activity.

Synthetic modifications have been made directly to the cyclic peptide core of polymyxin B, enabling the further understanding of structure activity relationships of this antimicrobial peptide. Such modified polymyxins are also substrates for enzymic hydrolysis, enabling the synthesis of a variety of semi-synthetic analogues, resulting in compounds with increased in vitro antibacterial activity.

Design of Next Generation Polymyxins with Lower Toxicity: The Discovery of SPR206.

Polymyxins are an important class of antibiotics for the treatment of bacterial infections due to multidrug resistant Gram-negative pathogens. However, their clinical utility is limited by nephrotoxicity. Here, we report a series of promising next generation polymyxin nonapeptides identified on the basis of our understanding of the relationship of structure with activity, cytotoxicity, and kidney compartment accumulation. We demonstrate that nonapeptides with an amine-containing N-terminal moiety of specific regio- and stereochemistry possess superior in vitro activity, together with lower cytotoxicity compared to polymyxin B. We further demonstrate that compounds with a ß-branched aminobutyrate N-terminus with an aryl substituent offer a promising combination of low cytotoxicity and kidney exposure, leading to low toxicity in the mouse. From this series, SPR206 has been selected as a development candidate.

Introducing GIST and Dieulafoy - Think of Them in GI Bleeding and Anemia.

Common presentations of gastrointestinal bleeding include hematemesis, melena, and history of dizziness, fatigue, or syncope; yet, bleeds may present in many uncommon ways. This review discusses two cases of gastrointestinal bleeds (GIBs) in middle-aged female patients who presented with anemia and recurrent unexplained GIBs. Both patients were misdiagnosed for several years before the cause of their bleeding was established. Dieulafoy lesion is said to be rare, although its precise incidence is difficult to establish due to its anatomically inaccessible location, small size, and lack of symptoms prior to presentation. This condition can result in gastrointestinal hemorrhage and quickly pose life-threatening complications. Gastrointestinal stromal tumors (GISTs) are likewise difficult to diagnose due to their location in the submucosa and lack of good diagnostic tools. Depending on tumor size, location, and spread, GISTs can have a poor prognosis even with early intervention. By presenting the two cases, this review aims to bring attention to Dieulafoy lesion and GIST as arguably not-so-rare but potentially fatal sources of GIBs. Especially troublesome is the uncommon complication of GIST to perforate and the elusive nature of Dieulafoy lesions that require diligent evaluation. For these reasons, both should be considered in the differential diagnosis in patients with recurrent unexplained GIBs and anemia.

Development of new polymyxin derivatives for multi-drug resistant Gram-negative infections.

Over the last decade, there has been a resurgence of interest in polymyxins owing to the rapid rise in multi-drug resistant Gram-negative bacteria against which polymyxins offer a last-resort treatment. Although having excellent antibacterial activity, the clinical utility of polymyxins is limited by toxicity, especially renal toxicity. There is much interest therefore in developing polymyxin analogues with an improved therapeutic index. This review describes recent work aimed at improving the activity and/or reducing the toxicity of polymyxins. Consideration to providing activity against emerging strains with reduced susceptibility to polymyxins is also made.

Antibacterial activity of the novel semisynthetic lantibiotic NVB333 in vitro and in experimental infection models.

NVB333 is a novel semisynthetic lantibiotic derived from the amide coupling of 3,5-dichlorobenzylamine to the C-terminal of deoxyactagardine B. The in vitro activity of NVB333 includes efficacy against clinically relevant pathogens including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus spp. NVB333 shows no cross-resistance with other antibiotics tested and a very low propensity for resistance development. After intravenous dosing NVB333 has high exposure in mouse plasma and shows generally improved in vivo activity compared with vancomycin in mouse infection models despite modest MIC values. In thigh infection models, promising efficacy was demonstrated against several strains of S. aureus including methicillin-resistant S. aureus (MRSA) and vancomycin-intermediate S. aureus (VISA) strains, and against Enterococcus faecalis UNT126-3. Area under the concentration curve (AUC)/MIC was shown to be the best predictor of efficacy against S. aureus UNT103-3 with an AUC/MIC of 138 (uncorrected for protein binding) achieving a static effect. NVB333 was also effective in a disseminated infection model where it conferred complete survival from the MRSA strain ATCC 33591. NVB333 showed rather modest lung penetration after intravenous dosing (AUC in lung 2-3% of plasma AUC), but because of very high plasma exposure, therapeutic levels of compound were achieved in the lung. Efficacy at least equal to vancomycin was demonstrated against an MRSA strain (UNT084-3) in a bronchoalveolar infection model. The impressive in vivo efficacy of NVB333 and strong resistance prognosis makes this compound an interesting candidate for development for treating systemic Gram-positive infections.

Rural cardiac rehabilitation: a 20-year success story.

INTRODUCTION: Data are lacking on long-term participation in a clinically supervised cardiac rehabilitation program in a rural setting. We sought to determine whether there were sustained improvements in physiologic measures and discover what restorative and deteriorative processes took place over time. METHODS: We retrospectively analyzed the records of patients who were enrolled for a least 1 year in the Healthy Hearts Cardiac Rehabilitation Program. Data from stress tests were tracked for up to 18 years to determine whether there were any sustained improvements and what factors were associated with restorative and deteriorative processes. RESULTS: We analyzed data from 85 participants. The mean age of the participants was 72 years, and the mean length of participation was 8 years. Duration of stress testing significantly (p < 0.01) increased by a mean of 15% from the first year to the second year, with a corresponding increase in estimated metabolic equivalent of task (MET) level (Cohen d = 0.82). The increase in duration was sustained into the ninth year, with an overall increase of 35% compared with the first year of testing. After the ninth year, the duration and estimated MET levels declined. CONCLUSION: Participants in the cardiac rehabilitation program demonstrated improved duration of stress testing, and stable rate-pressure product, blood pressure and resting heart rate during long-term participation in the program.

INTRODUCTION: On manque d'information sur la participation à long terme aux programmes de réadaptation cardiaque avec supervision clinique en milieu rural. Nous avons donc cherché à déterminer si ces programmes entraînent une amélioration physiologique permanente et à décrire les processus de guérison et d'aggravation qui surviennent au fil du temps. MÉTHODES: Nous avons procédé à une analyse rétrospective des dossiers de patients qui ont participé au programme Healthy Hearts pendant au moins 1 an. Des données sur leurs résultats aux épreuves d'effort sur une période maximale de 18 ans ont été recueillies pour déterminer la présence d'améliorations durables et mettre en évidence les facteurs associés à la guérison et à l'aggravation. RÉSULTATS: Nous avons étudié les dossiers de 85 patients; la durée de participation moyenne était de 8 ans, et l'âge moyen des participants, de 72 ans. La durée de l'épreuve d'effort a connu une augmentation significative de 15 % en moyenne (p < 0,01) de la première à la deuxième année, associée à une hausse correspondante de l'équivalent métabolique (MET) estimé (d de Cohen = 0,82). Cette augmentation s'est poursuivie jusqu'à la neuvième année, où la durée était supérieure de 35 % à celle de la première année. Par la suite, la durée de l'épreuve et le MET estimé ont commencé à diminuer. CONCLUSION: Au cours de leur participation prolongée au programme, les patients ont réussi à augmenter la durée de leur épreuve d'effort, et le produit de leur tension systolique par la fréquence des contractions cardiaques, leur pression artérielle et leur fréquence cardiaque au repos sont demeurés stables.

A perspective on the next generation of antibacterial agents derived by manipulation of natural products.

Natural products have been a major source of anti-infective drugs for many decades. With urgent need for new antibacterial agents to combat drug-resistant bacteria, the investigation of both new and existing classes of natural products has once again become an important focus. In this review, we highlight how a medicinal chemistry/semi-synthetic approach to natural product manipulation continues to offer a valuable strategy to overcome limitations in current therapy. Approaches to address toxicity and to improve the solubility, bioavailability and the spectrum of activity are demonstrated. Examples are drawn from aminoglycosides, glycopeptides, tetracyclines, macrolides, thiazolyl peptides, pleuromutilins and polymyxins and are taken from the current literature, patents and abstracts of symposia. In many cases, this approach has led to drug candidates currently in late stages of clinical development.

Generation of an actagardine A variant library through saturation mutagenesis.

The lantibiotic actagardine A is nineteen amino acids in length and comprises three intertwined C-terminal methyllanthionine-bridged rings and an N-terminal lanthionine-bridged ring. Produced by the actinomycete Actinoplanes garbadinensis ATCC 31049, actagardine A demonstrates antibacterial activity against important Gram-positive pathogens. This activity combined with its ribosomal synthesis makes it an attractive target for the generation of lantibiotic variants with improved biological activity. A variant generation system designed to allow the specific substitution of amino acids at targeted sites throughout the actagardine A peptide has been used to generate a comprehensive library by site-directed mutagenesis. With the exception of residues involved in bridge formation, each amino acid in the actagardine A peptide as well as the alanine (ala(0)) at position -1 relative to the mature peptide, has been systematically substituted with all remaining 19 amino acids. A total of 228 mutants have been engineered with 44 produced in good yield. The mutant V15F in particular demonstrates improved activity against a range of notable Gram-positive pathogens including Clostridium difficile, when evaluated alongside actagardine A. The scope of variants generated provides an insight into the flexibility of the actagardine A processing machinery and will undoubtedly assist in future mutational studies.

Organization of the biosynthetic genes encoding deoxyactagardine B (DAB), a new lantibiotic produced by Actinoplanes liguriae NCIMB41362.

Deoxyactagardine B (DAB) is a hitherto unknown type B lantibiotic, produced by Actinoplanes liguriae NCIMB41362. The mature peptide is 19 amino acids in length and structurally analogous to actagardine, differing by two amino acids (V15L and I16V) and the absence of a sulfoxide bond between residues 14 and 19. The biosynthetic genes encoding DAB are clustered, and in addition to the structural gene ligA include genes believed to encode for the proteins responsible for the modification, transport and regulation of DAB synthesis. Surprisingly, despite the presence of a gene that shares significant homology to the monooxygenase garO from the actagardine biosynthetic gene cluster, the oxidized form of DAB has not been detected. A lanA gene encoding the DAB peptide has been introduced into the plasmid pAGvarX and delivered into a strain of Actinoplanes garbadinensis lacking the structural gene for actagardine, garA (A. garbadinensis DeltagarA). Expression of this gene in A. garbadinensis DeltagarA resulted in the production of actagardine B, an oxidized form of DAB.

Dissecting structural and functional diversity of the lantibiotic mersacidin.

Mersacidin is a tetracyclic lantibiotic with antibacterial activity against Gram-positive pathogens. To probe the specificity of the biosynthetic pathway of mersacidin and obtain analogs with improved antibacterial activity, an efficient system for generating variants of this lantibiotic was developed. A saturation mutagenesis library of the residues of mersacidin not involved in cycle formation was constructed and used to validate this system. Mersacidin analogs were obtained in good yield in approximately 35% of the cases, producing a collection of 82 new compounds. This system was also used for the production of deletion and insertion mutants of mersacidin. The outcome of these studies suggests that this system can be extended to produce mersacidin variants with multiple changes that will allow a full investigation of the potential use of modified mersacidins as therapeutic agents.

Organization of the genes encoding the biosynthesis of actagardine and engineering of a variant generation system.

The biosynthetic pathway of the type B lantibiotic actagardine (formerly gardimycin), produced by Actinoplanes garbadinensis ATCC31049, has been cloned, sequenced and annotated. The gene cluster contains the gene garA that encodes the actagardine prepropeptide, a modification gene garM, involved in the dehydration and cyclization of the prepeptide, several putative transporter and regulatory genes as well as a novel luciferase-like monooxygenase gene designated garO. Expression of these genes in Streptomyces lividans resulted in the production of ala(0)-actagardine while deletion of the garA gene from A. garbadinensis generated a strain incapable of producing actagardine. Actagardine production was successfully restored however, by the delivery of the plasmid pAGvarX. This plasmid contains an engineered cassette of the actagardine encoding gene garA and offers an alternative route to generating extensive libraries of actagardine variants. Using this plasmid, an alanine scanning library has been constructed and the mutants analysed. Further modifications include the removal of the novel garO gene from A. garbadinensis. Deletion of this gene resulted in the production of deoxy variants of actagardine, demonstrating that the formation of the sulfoxide group is enzyme catalysed and not a spontaneous chemical modification as previously believed.

Chapter 22. Whole-cell generation of lantibiotic variants.

The generation of modified lantibiotics in whole cells has proved to be of value for the investigation of the specificity of the lantibiotic-processing enzymes and their tolerance to mutations in the primary sequence of lantibiotics. The development of methods to produce new lantibiotic variants has also enabled the investigation of the structure-activity relationships of these compounds and hence an evaluation of this hitherto underexploited class of natural products as a source of potential therapeutic drug candidates. We report the methods and strategies that have been used to engineer new lantibiotic variants and practical methods to analyze libraries of new compounds with a view toward optimizing drug properties.

Stereoinversion of beta- and gamma-substituted alpha-amino acids using a chemo-enzymatic oxidation-reduction procedure.

Both D- and L-beta- and gamma-substituted alpha-amino acids can be interconverted to their respective L- and D- diastereoisomers by treatment with an enantioselective amino acid oxidase and a chemical reducing agent.