Design, synthesis and antimicrobial studies of some polymyxin analogues.

To find novel polymycin analogues with high antimicrobial activities and low toxicity, 36 novel polymyxin analogues were synthesized, and in which TZ40-J and TZ40-K were evaluated for their antimicrobial activities using broth microdilution method and for their haemolytic toxicity with sterile sheep blood. Preliminary safety assessments of those two compounds were carried out via the MTT cell viability assay in vitro and acute toxicity assay in vivo. Experimental data demonstrated that TZ40-J and TZ40-K were less toxic and indicate higher activities against Pseudomonas aeruginosa than polymyxin B.

Polymyxin derivatives as broad-spectrum antibiotic agents.

We designed a few polymyxin derivatives which exhibit broad-spectrum antimicrobial activity. Lead compound P1 could disrupt bacterial membranes rapidly without developing resistance, inhibit biofilms formed by E. coli, and exhibit excellent in vivo activity in an MRSA-infected thigh burden mouse model.

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.

Novel synthetic polymyxins kill Gram-positive bacteria.

Background: Staphylococcus aureus, including 'superbug' MRSA, is a major cause of nosocomial infections. In the European Union, up to 171 200 new nosocomial MRSA infections are acquired annually, and in the USA S. aureus causes more deaths than HIV/AIDS and tuberculosis combined. MRSA is also the first group of pathogens that infect the pulmonary tract in young patients with cystic fibrosis. Objectives: We describe two newly developed and synthesized colistin (polymyxin E)-inspired molecules. Methods: A collection of several isolates of S. aureus [including MRSA and vancomycin-resistant S. aureus (VRSA)] was tested. To check the antimicrobial activity, we performed time-kill curves, growth curves, biofilm eradication, toxicity and isothermal titration calorimetry. Results: Both peptides showed high antimicrobial activities (MIC 4 mg/L) and low relative toxicities (selectivity index close to 23). Conclusions: Successful production of polymyxin-scaffold molecules active against S. aureus, both MRSA and VRSA, opens up new approaches to the treatment of these complicated infections.

Development of dilipid polymyxins: Investigation on the effect of hydrophobicity through its fatty acyl component.

Continuous development of new antibacterial agents is necessary to counter the problem of antimicrobial resistance. Polymyxins are considered as drugs of last resort to combat multidrug-resistant Gram-negative pathogens. Structural optimization of polymyxins requires an in-depth understanding of its structure and how it relates to its antibacterial activity. Herein, the effect of hydrophobicity was explored by adding a secondary fatty acyl component of varying length onto the polymyxin structure at the amine side-chain of l-diaminobutyric acid at position 1, resulting to the development of dilipid polymyxins. The incorporation of an additional lipid was found to confer polymyxin activity against Gram-positive bacteria, to which polymyxins are inherently inactive against. The dilipid polymyxins showed selective antibacterial activity against Pseudomonas aeruginosa. Moreover, dilipid polymyxin 1 that consists of four carbon-long aliphatic lipids displayed the ability to enhance the antibacterial potency of other antibiotics in combination against P. aeruginosa, resembling the adjuvant activity of the well-known outer membrane permeabilizer polymyxin B nonapeptide (PMBN). Interestingly, our data revealed that dilipid polymyxin 1 and PMBN are substrates for the MexAB-OprM efflux system, and therefore are affected by efflux. In contrast, dilipid polymyxin analogs that consist of longer lipids and colistin were not affected by efflux, suggesting that the lipid component of polymyxin plays an important role in resisting active efflux. Our work described herein provides an understanding to the polymyxin structure that may be used to usher the development of enhanced polymyxin analogs.

Novobiocin Enhances Polymyxin Activity by Stimulating Lipopolysaccharide Transport.

Gram-negative bacteria are challenging to kill with antibiotics due to their impenetrable outer membrane containing lipopolysaccharide (LPS). The polymyxins, including colistin, are the drugs of last resort for treating Gram-negative infections. These drugs bind LPS and disrupt the outer membrane; however, their toxicity limits their usefulness. Polymyxin has been shown to synergize with many antibiotics including novobiocin, which inhibits DNA gyrase, by facilitating transport of these antibiotics across the outer membrane. Recently, we have shown that novobiocin not only inhibits DNA gyrase but also binds and stimulates LptB, the ATPase that powers LPS transport. Here, we report the synthesis of novobiocin derivatives that separate these two activities. One analog retains LptB-stimulatory activity but is unable to inhibit DNA gyrase. This analog, which is not toxic on its own, nevertheless enhances the lethality of polymyxin by binding LptB and stimulating LPS transport. Therefore, LPS transport agonism contributes substantially to novobiocin-polymyxin synergy. We also report other novobiocin analogs that inhibit DNA gyrase better than or equal to novobiocin, but bind better to LptB and therefore have even greater LptB stimulatory activity. These compounds are more potent than novobiocin when used in combination with polymyxin. Novobiocin analogs optimized for both gyrase inhibition and LPS transport agonism may allow the use of lower doses of polymyxin, increasing its efficacy and safety.

The Road from Host-Defense Peptides to a New Generation of Antimicrobial Drugs.

Host-defense peptides, also called antimicrobial peptides (AMPs), whose protective action has been used by animals for millions of years, fulfill many requirements of the pharmaceutical industry, such as: (1) broad spectrum of activity; (2) unlike classic antibiotics, they induce very little resistance; (3) they act synergically with conventional antibiotics; (4) they neutralize endotoxins and are active in animal models. However, it is considered that many natural peptides are not suitable for drug development due to stability and biodisponibility problems, or high production costs. This review describes the efforts to overcome these problems and develop new antimicrobial drugs from these peptides or inspired by them. The discovery process of natural AMPs is discussed, as well as the development of synthetic analogs with improved pharmacological properties. The production of these compounds at acceptable costs, using different chemical and biotechnological methods, is also commented. Once these challenges are overcome, a new generation of versatile, potent and long-lasting antimicrobial drugs is expected.

Synthesis and Bioactivity Investigation of the Individual Components of Cyclic Lipopeptide Antibiotics.

In this paper, 26 natural polymyxin components and a new derivative S2 were synthesized, and their differences in efficacy and toxicity have been investigated. Almost all of the synthesized components showed strong activity against both susceptible and resistant strains of E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii. The toxicities were obviously different between the components. Only some of the components were tested for toxicity in vivo. Compounds E2, E2-Val, A2, M2, D2, and S2 showed obviously lower renal cytotoxicity and acute toxicity than polymyxins B and E. The in vivo nephrotoxicity of E2, M2, and S2 was similar to that of polymyxin E. Compound S2, with four positive charges, was especially interesting as it possessed both increased efficacy and decreased toxicity. The SAR and toxicity studies indicated that further structural modification could concentrate on polymyxin S. The results also indicated that S2 could be a new drug candidate.

The first total synthesis and solution structure of a polypeptin, PE2, a cyclic lipopeptide with broad spectrum antibiotic activity.

The first total synthesis of a polypeptin, PE2, as well as its solution structure is reported. Synthesis in optically pure form confirms the proposed stereochemistry of the polypeptins at the 3-position on the 3-hydroxy depsipeptide moiety. We have also determined the NMR structure of PE2 in aqueous solution, showing it to form a stable ring conformation. The synthetic peptide shows anti-bacterial activity consistent with reports for naturally derived counterparts.

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.

A Novel Chemical Biology Approach for Mapping of Polymyxin Lipopeptide Antibody Binding Epitopes.

Polymyxins B and E (i.e., colistin) are a family of naturally occurring lipopeptide antibiotics that are our last line of defense against multidrug resistant (MDR) Gram-negative pathogens. Unfortunately, nephrotoxicity is a dose-limiting factor for polymyxins that limits their clinical utility. Our recent studies demonstrate that polymyxin-induced nephrotoxicity is a result of their extensive accumulation in renal tubular cells. The design and development of safer, novel polymyxin lipopeptides is hampered by our limited understanding of their complex structure-nephrotoxicity relationships. This is the first study to employ a novel targeted chemical biology approach to map the polymyxin recognition epitope of a commercially available polymyxin mAb and demonstrate its utility for mapping the kidney distribution of a novel, less nephrotoxic polymyxin lipopeptide. Eighteen novel polymyxin lipopeptide analogues were synthesized with modifications in the polymyxin core domains, namely, the N-terminal fatty acyl region, tripeptide linear segment, and cyclic heptapeptide. Surface plasmon resonance epitope mapping revealed that the monoclonal antibody (mAb) recognition epitope consisted of the hydrophobic domain (N-terminal fatty acyl and position 6/7) and diaminobutyric acid (Dab) residues at positions 3, 5, 8, and 9 of the polymyxin molecule. Structural diversity within the hydrophobic domains and Dab 3 position are tolerated. Enlightened with an understating of the structure-binding relationships between the polymyxin mAb and the core polymyxin scaffold, we can now rationally employ the mAb to probe the kidney distribution of novel polymyxin lipopeptides. This information will be vital in the design of novel, safer polymyxins through chemical tailoring of the core scaffold and exploration of the elusive/complex polymyxin structure-nephrotoxicity relationships.

Design, synthesis, and evaluation of a new fluorescent probe for measuring polymyxin-lipopolysaccharide binding interactions.

Fluorescence assays employing semisynthetic or commercial dansyl-polymyxin B have been widely employed to assess the affinity of polycations, including polymyxins, for bacterial cells and lipopolysaccharide (LPS). The five primary γ-amines on diaminobutyric acid residues of polymyxin B are potentially derivatized with dansyl-chloride. Mass spectrometric analysis of the commercial product revealed a complex mixture of di- or tetra-dansyl-substituted polymyxin B. We synthesized a mono-substituted fluorescent derivative, dansyl[Lys]¹polymyxin B3. The affinity of polymyxin for purified gram-negative LPS and whole bacterial cells was investigated. The affinity of dansyl[Lys]¹polymyxin B3 for LPS was comparable to polymyxin B and colistin, and considerably greater (K(d)<1 µM) than for whole cells (K(d)∼6-12µM). Isothermal titration calorimetric studies demonstrated exothermic enthalpically driven binding between both polymyxin B and dansyl[Lys]¹polymyxin B3 to LPS, attributed to electrostatic interactions. The hydrophobic dansyl moiety imparted a greater entropic contribution to the dansyl[Lys]¹polymyxin B3-LPS reaction. Molecular modeling revealed a loss of electrostatic contact within the dansyl[Lys]¹polymyxin B3-LPS complex due to steric hindrance from the dansyl[Lys]¹ fluorophore; this corresponded with diminished antibacterial activity (MIC≥16µg/mL). Dansyl[Lys]¹polymyxin B3 may prove useful as a screening tool for drug development.

Solid-phase synthesis of polymyxin B1 and analogues via a safety-catch approach.

As part of a program towards the development of novel antibiotics, a convenient method for solid-phase synthesis of the cyclic cationic peptide polymyxin B1 and analogues thereof is described. The methodology, based on cleavage-by-cyclization using Kenner's safety-catch linker, yields crude products with purities ranging from 37-67%. Antibacterial assays revealed that analogues 23-26, in which the (S)-6-methyloctanoic acid moiety is replaced with shorter acyl chains, exhibit distinct antimicrobial activity. The results suggest that the length of the acyl chain is rather critical for antimicrobial activity. On the other hand, substitution of the hydrophobic ring-segment D-Phe-6/Leu-7 in polymyxin B1 with dipeptide mimics (i.e. analogues 27-33) resulted in almost complete loss of antimicrobial activity.

The outer membrane permeability-increasing action of deacylpolymyxins.

The outer membrane permeability-increasing action of deacylpolymyxins was compared to the well-known potent action of polymyxin B nonapeptide (PMBN). Deacylpolymyxin B (DAPB), prepared by treating polymyxin B with polymyxin acylase, was found to be a slightly more effective permeabilizer than PMBN. As low a DAPB concentration as 1 microgram/ml sensitized Escherichia coli to the probe antibiotics (rifampin, fusidic acid, erythromycin, clindamycin, novobiocin) by factors 30-100 and Salmonella typhimurium by factors 10-100. A higher concentration (3 micrograms/ml) of DAPB elicited further sensitization. Also deacylcolistin (DAC) was found to be an effective permeabilizer.

Solid-phase synthesis of a cyclic decapeptide, analog of the antibiotic polymyxin M.

Pelargonoyl-cyclo[Dab-Thr-Dab-Dab(Pel)-Dab-D-Leu-Thr-Dab-Dab-Thr]. 5 HCl (5), analog of the polymyxin M, has been synthesized by the solid phase method. The intermediate linear Boc-protected decapeptide-resin (1) was assembled on the solid support by the stepwise addition of Boc-amino acids in the presence of DCC. For side-chain protection of diaminobutyric acid, Z-group was used. The peptide was cleaved from the resin in the form of methyl ester by triethylamine-catalyzed transesterification with methanol. Alkaline hydrolysis of the peptide ester with aqueous KOH (1 mol/l) afforded the Bocpenta-benzyloxycarbonyl-decapeptide acid (2) in 60% yield (based on Thr/resin). After removal of the Boc-group, the linear peptide 3 was cyclized with DCC in high dilution in DMF (10(-3) mol/l) to give 4 in 51% yield. Hydrogenolysis of 4 in 80% formic acid afforded the pelargonyl cyclic decapeptide 5 in 74% yield. The synthetic peptide 5 retained the same activity of the natural polymyxin M against Bacillus subtilis and Staphylococcus aureus, whereas it showed only 3% of the activity of Escherichia coli.

Synthesis of pelargonoyl-cyclic decapeptide analog of the antibiotic polymyxin B.1.

The synthesis of pelargonoyl-cyclic decapeptide, analog of polymycin B1, is described. The open-chain protected decapeptide was synthesized on a polymer support, starting with threonine as the C-terminal amino acid residue, and was cleaved satisfactorily by hydrazinolysis. The alpha-amino protecting Boc-group was removed with HCl (1 mol/l) acetic acid and the resulting decapeptide hydrazide was converted to the azide with nitrous acid. Cyclization of the azide in pyridine at peptide concentration 2.5.10(-5) mol/l gave the penta-benzyloxycarbonyl-substituted decapeptide in 65% yield. Catalytic hydrogenation afforded the pelargonoyl cyclic decapeptide pentahydrochloride (6) in 75% yield. The synthetic product (6) exhibited an activity comparable to that of the natural polymyxin B1 against Bacillus subtilis and Staphylococcus aureus, whereas it showed only 2% of the activity to Escherichia coli2.