Engineering of the start condensation domain with improved N-decanoyl catalytic activity for daptomycin biosynthesis.

Daptomycin, a lipopeptide comprising an N-decanoyl fatty acyl chain and a peptide core, is used clinically as an antimicrobial agent. The start condensation domain (dptC1) is an enzyme that catalyzes the lipoinitiation step of the daptomycin synthesis. In this study, we integrated enzymology, protein engineering, and computer simulation to study the substrate selectivity of the start condensation domain (dptC1) and to screen mutants with improved activity for decanoyl loading. Through molecular docking and computer simulation, the fatty acyl substrate channel and the protein-protein interaction interface of dptC1 are analyzed. Key residues at the protein-protein interface between dptC1 and the acyl carrier were mutated, and a single-point mutant showed more than three-folds improved catalytic efficiency of the target n-decanoyl substrate in comparing with the wild type. Moreover, molecular dynamics simulations suggested that mutants with increased catalytic activity may correlated with a more "open" and contracted substrate binding channel. Our work provides a new perspective for the elucidation of lipopeptide natural products biosynthesis, and also provides new resources to enrich its diversity and optimize the production of important components.

Dual release of daptomycin and BMP-2 from a composite of ß-TCP ceramic and ADA gelatin.

BACKGROUND: Antibiotic-containing carrier systems are one option that offers the advantage of releasing active ingredients over a longer period of time. In vitro sustained drug release from a carrier system consisting of microporous ß-TCP ceramic and alginate has been reported in previous works. Alginate dialdehyde (ADA) gelatin gel showed both better mechanical properties when loaded into a ß-TCP ceramic and higher biodegradability than pure alginate. METHODS: Dual release of daptomycin and BMP-2 was measured on days 1, 2, 3, 6, 9, 14, 21, and 28 by HPLC and ELISA. After release, the microbial efficacy of the daptomycin was verified and the biocompatibility of the composite was tested in cell culture. RESULTS: Daptomycin and the model compound FITC protein A (n = 30) were released from the composite over 28 days. A Daptomycin release above the minimum inhibitory concentration (MIC) by day 9 and a burst release of 71.7 ± 5.9% were observed in the loaded ceramics. Low concentrations of BMP-2 were released from the loaded ceramics over 28 days.

Elucidating Daptomycin's Antibacterial Efficacy: Insights into the Tripartite Complex with Lipid II and Phospholipids in Bacterial Septum Membrane.

This study elucidated the mechanism of formation of a tripartite complex containing daptomycin (Dap), lipid II, and phospholipid phosphatidylglycerol in the bacterial septum membrane, which was previously reported as the cause of the antibacterial action of Dap against gram-positive bacteria via molecular dynamics and enhanced sampling methods. Others have suggested that this transient complex ushers in the inhibition of cell wall synthesis by obstructing the downstream polymerization and cross-linking processes involving lipid II, which is absent in the presence of cardiolipin lipid in the membrane. In this work, we observed that the complex was stabilized by Ca2+-mediated electrostatic interactions between Dap and lipid head groups, hydrophobic interaction, hydrogen bonds, and salt bridges between the lipopeptide and lipids and was associated with Dap concentration-dependent membrane depolarization, thinning of the bilayer, and increased lipid tail disorder. Residues Orn6 and Kyn13, along with the DXDG motif, made simultaneous contact with constituent lipids, hence playing a crucial role in the formation of the complex. Incorporating cardiolipin into the membrane model led to its competitively displacing lipid II away from the Dap, reducing the lifetime of the complex and the nonexistence of lipid tail disorder and membrane depolarization. No evidence of water permeation inside the membrane hydrophobic interior was noted in all of the systems studied. Additionally, it was shown that using hydrophobic contacts between Dap and lipids as collective variables for enhanced sampling gave rise to a free energy barrier for the translocation of the lipopeptide. A better understanding of Dap's antibacterial mechanism, as studied through this work, will help develop lipopeptide-based antibiotics for rising Dap-resistant bacteria.

Gausemycin Antibiotic Family Acts via Ca2+-Dependent Membrane Targeting.

We report the molecular mechanism of action of gausemycins and the isolation of new members of the family, gausemycins C (1c), D (1d), E (1e), and F (1f), the minor components of the mixture. To elucidate the mechanism of action of gausemycins, we investigated the antimicrobial activity of the most active compounds, gausemycins A and B, in the presence of Ca2+, other metal ions, and phosphate. Gausemycins require a significantly higher Ca2+ concentration for maximum activity than daptomycin but lower than that required for malacidine and cadasides. Species-specific antimicrobial activity was found upon testing against a wide panel of Gram-positive bacteria. Membranoactivity of gausemycins was demonstrated upon their interactions with model lipid bilayers and micelles. The pore-forming ability was found to be dramatically dependent on the Ca2+ concentration and the membrane lipid composition. An NMR study of gausemycin B in zwitterionic and anionic micelles suggested the putative structure of the gausemycin/membrane complex and revealed the binding of Ca2+ by the macrocyclic domain of the antibiotic.

Identification of Clostridioides difficile mutants with increased daptomycin resistance.

Daptomycin is a cyclic lipopeptide antibiotic used to treat infections caused by some Gram-positive bacteria. Daptomycin disrupts synthesis of the peptidoglycan (PG) cell wall by inserting into the cytoplasmic membrane and binding multiple forms of the undecaprenyl carrier lipid required for PG synthesis. Membrane insertion requires phosphatidylglycerol, so studies of daptomycin can provide insight into assembly and maintenance of the cytoplasmic membrane. Here, we studied the effects of daptomycin on Clostridioides difficile, the leading cause of healthcare-associated diarrhea. We observed that growth of C. difficile strain R20291 in the presence of sub-MIC levels of daptomycin resulted in a chaining phenotype, minicell formation, and lysis-phenotypes broadly consistent with perturbation of membranes and PG synthesis. We also selected for and characterized eight mutants with elevated daptomycin resistance. The mutations in these mutants were mapped to four genes: cdsA (cdr20291_2041), ftsH2 (cdr20291_3396), esrR (cdr20291_1187), and draS (cdr20291_2456). Of these four genes, only draS has been characterized previously. Follow-up studies indicate these mutations confer daptomycin resistance by two general mechanisms: reducing the amount of phosphatidylglycerol in the cytoplasmic membrane (cdsA) or altering the regulation of membrane processes (ftsH2, esrR, and draS). Thus, the mutants described here provide insights into phospholipid synthesis and identify signal transduction systems involved in cell envelope biogenesis and stress response in C. difficile. IMPORTANCE: C. difficile is the leading cause of healthcare-associated diarrhea and is a threat to public health due to the risk of recurrent infections. Understanding biosynthesis of the atypical cell envelope of C. difficile may provide insight into novel drug targets to selectively inhibit C. difficile. Here, we identified mutations that increased daptomycin resistance and allowed us to better understand phospholipid synthesis, cell envelope biogenesis, and stress response in C. difficile.

Synthesis and Characterization of DOTAM-Based Sideromycins for Bacterial Imaging and Antimicrobial Therapy.

The rise of antimicrobial resistance, especially in Gram-negative bacteria, calls for novel diagnostics and antibiotics. To efficiently penetrate their double-layered cell membrane, we conjugated the potent antibiotics daptomycin, vancomycin, and sorangicin A to catechol siderophores, which are actively internalized by the bacterial iron uptake machinery. LC-MS/MS uptake measurements of sorangicin derivatives verified that the conjugation led to a 100- to 525-fold enhanced uptake into bacteria compared to the free drug. However, the transfer to the cytosol was insufficient, which explains their lack of antibiotic efficacy. Potent antimicrobial effects were observed for the daptomycin conjugate 7 (∼1 µM) against multidrug-resistant Acinetobacter baumannii. A cyanin-7 label aside the daptomycin warhead furnished the theranostic 13 that retained its antibiotic activity and was also able to label ESKAPE bacteria, as demonstrated by microscopy and fluorescence assays. 13 and the cyanin-7 imaging conjugate 14 were stable in human plasma and had low plasma protein binding and cytotoxicity.

Structural diversity, biosynthesis, and biological functions of lipopeptides from Streptomyces.

Covering: up to 2022Streptomyces are ubiquitous in terrestrial and marine environments, where they display a fascinating metabolic diversity. As a result, these bacteria are a prolific source of active natural products. One important class of these natural products is the nonribosomal lipopeptides, which have diverse biological activities and play important roles in the lifestyle of Streptomyces. The importance of this class is highlighted by the use of related antibiotics in the clinic, such as daptomycin (tradename Cubicin). By virtue of recent advances spanning chemistry and biology, significant progress has been made in biosynthetic studies on the lipopeptide antibiotics produced by Streptomyces. This review will serve as a comprehensive guide for researchers working in this multidisciplinary field, providing a summary of recent progress regarding the investigation of lipopeptides from Streptomyces. In particular, we highlight the structures, properties, biosynthetic mechanisms, chemical and chemoenzymatic synthesis, and biological functions of lipopeptides. In addition, the application of genome mining techniques to Streptomyces that have led to the discovery of many novel lipopeptides is discussed, further demonstrating the potential of lipopeptides from Streptomyces for future development in modern medicine.

A54145 Factor D Is Not Less Susceptible to Inhibition by Lung Surfactant than Daptomycin.

A54145 factor D (A5D) is a cyclic lipopeptide antibiotic that shares several structural and mechanistic features with the clinically important antibiotic daptomycin, such as their requirement for calcium and phosphatidylglycerol (PG) for activity. Studies by others have suggested that daptomycin's activity is strongly inhibited by lung surfactant while A5D's activity is not. This finding has inspired efforts, albeit unsuccessful, to develop an A5D analogue that is highly active in the presence of lung surfactant and can be used for treating community acquired pneumonia (CAP). Here we demonstrate that A5D, like daptomycin, has a strong preference for the 1,2-diacyl-sn-glycero-3-phospho-1'-sn-glycerol stereoisomer (2R,2'S configuration) of PG. This PG stereoisomer was determined to be the only stereoisomer of PG in lung surfactant. Both antibiotics are completely antagonized by approximately 1-2 mol equiv of 2R,2'S-PG. Studies performed in the presence of lung surfactant revealed that the antagonism of these peptides by surfactant is mainly due to their interaction with PG and that A5D is not significantly less susceptible to inhibition by lung surfactant than daptomycin.

Establishing the Structure-Activity Relationship between Phosphatidylglycerol and Daptomycin.

Daptomycin is a clinical antibiotic used to treat serious infections caused by Gram-positive bacteria. Although there is debate about the action mechanism of daptomycin, it is known that daptomycin requires both calcium and phosphatidylglycerol (PG) to exert its antibacterial effect. Despite the importance and uniqueness of the interaction of daptomycin with PG, very little is known about this interaction or the nascent daptomycin-PG complex. In this work, we establish a structure-activity relationship between daptomycin and PG through the synthesis of PG analogues. In total, nine PGs were synthesized using a divergent approach employing phosphoramidite chemistry. The interaction between daptomycin and these PGs was studied using fluorescence, circular dichroism, and isothermal titration calorimetry. It was determined that daptomycin is highly sensitive to the modification of the headgroup of PG and both hydroxyl groups influence membrane binding, oligomerization, and backbone structure. Methylation of each hydroxyl in the headgroup suggests that the binding pocket envelops both hydroxyl groups. A PG acyl tail chain length of at least 7-8 carbons is required for stoichiometric binding at micromolar peptide concentrations. Daptomycin binds to PG having 8-carbon, linear, unsaturated acyl groups (C8PGs) at the micromolar concentration and interacts with C8PG in essentially the same manner as when the PG is incorporated into a liposome, and thus, preassembly of individual PG moieties is not a prerequisite for binding, structural transition, and oligomerization.

Discovery of Highly Active Derivatives of Daptomycin by Assessing the Effect of Amino Acid Substitutions at Positions 8 and 11 on a Daptomycin Analogue.

Daptomycin is an important antibiotic used for treating serious infections caused by Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci. Establishing structure-activity relationships of daptomycin is important for developing new daptomycin-based antibiotics with expanded clinical applications and for tackling the ever-increasing problem of antimicrobial resistance. Toward this end, Dap-K6-E12-W13, an active analogue of daptomycin in which the uncommon amino acids in daptomycin are replaced with their common counterparts, was used as a model system for studying the effect of amino acid variation at positions 8 and 11 on in vitro biological activity against a model organism, Bacillus subtilis, and calcium-dependent insertion into model membranes. None of the new peptides were more active than Dap-K6-E12-W13; however, substitution at positions 8 and/or 11 with cationic residues resulted in little or no loss of activity, and some of these analogues were able to insert into model membranes at lower calcium ion concentrations than the parent peptide. Incorporation of these cationic residues into positions 8 and/or 11 of daptomycin itself yielded some derivatives that exhibited lower minimum inhibitory concentrations than daptomycin against B. subtilis 1046 as well as comparable and sometimes superior activity against clinical isolates of MRSA.

The Chiral Target of Daptomycin Is the 2R,2'S Stereoisomer of Phosphatidylglycerol.

Daptomycin (dap) is an important antibiotic that interacts with the bacterial membrane lipid phosphatidylglycerol (PG) in a calcium-dependent manner. The enantiomer of dap (ent-dap) was synthesized and was found to be 85-fold less active than dap against B. subtilis, indicating that dap interacts with a chiral target as part of its mechanism of action. Using liposomes containing enantiopure PG, we demonstrate that the binding of dap to PG, the structural transition that occurs upon dap binding to PG, and the subsequent oligomerization of dap, depends upon the configuration of PG, and that dap prefers the 1,2-diacyl-sn-glycero-3-phospho-1'-sn-glycerol stereoisomer (2R,2'S configuration). Ent-dap has a lower affinity for 2R,2'S liposomes than dap and cannot oligomerize to the same extent as dap, which accounts for why ent-dap is less active than dap. To our knowledge, this is the first example whereby the activity of an antibiotic depends upon the configuration of a lipid head group.

Cellulose acetate fibres loaded with daptomycin for metal implant coatings.

Multifunctional polymeric coatings containing drug delivery vehicles can play a key role in preventing/reducing biofilm formation on implant surfaces. Their requirements are biocompatibility, good adhesion, and controllable drug release. Although cellulose acetate (CA) films and membranes are widely studied for scaffolding, their applications as a protective coating and drug delivery vehicle for metal implants are scarce. The reason is that adhesion to stainless steel (SS) substrates is non-trivial. Grinding SS substrates enhances the adhesion of dip-coated CA films while the adhesion of electrospun CA membranes is improved by an electrosprayed chitosan intermediate layer. PMMA microcapsules containing daptomycin have been successfully incorporated into CA films and fibres. The released drug concentration of 3 × 10-3 mg/mL after 120 min was confirmed from the peak luminescence intensity under UV radiation of simulated body fluid (SBF) after immersion of the fibres.

Daptomycin: A Novel Macrocyclic Antibiotic as a Chiral Selector in an Organic Polymer Monolithic Capillary for the Enantioselective Analysis of a Set of Pharmaceuticals.

Daptomycin, a macrocyclic antibiotic, is here used as a new chiral selector in preparation of chiral stationary phase (CSP) in a recently prepared polymer monolithic capillary. The latter is prepared using the copolymerization of the monomers glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EGDMA) in the presence of daptomycin in water. Under reversed phase conditions (RP), the prepared capillaries were tested for the enantioselective nanoliquid chromatographic separation of fifty of the racemic drugs of different pharmacological groups, such as adrenergic blockers, H1-blockers, NSAIDs, antifungal drugs, and others. Baseline separation was attained for many drugs under RP-HPLC. Daptomycin expands the horizon of chiral selectors in HPLC.

Daptomycin suppresses tumor migration and angiogenesis via binding to ribosomal protein S19 in humans.

We have previously reported that daptomycin (DAP), a last resort antibiotic, binds to ribosomal protein S19 (RPS19) in humans and exhibits selective anti-cancer activity against MCF7 breast cancer cells. Here, we investigated the role of RPS19 in the anti-cancer effects of DAP and have found that DAP does not induce autophagy, apoptosis or cell viability but does reduce cell proliferation. Our results suggest that an extraribosomal function of RPS19 involves the regulation of vascular endothelial growth factor (VEGF) but not EGF, PDGF or FGF. Engagement of RPS19 by DAP was shown by CETSA and ITDRFCETSA assays, and knocking down of RPS19 with siRNA increased the potency of DAP in MCF7 cells. In addition, DAP suppressed the secretion of VEGF in cancer cells and thereby inhibited cell migration. Collectively, these data provide an outline of the underlying mechanism of how DAP exhibits anti-cancer activity and suggests that RPS19 could be a promising target for the development of new anticancer drugs.

Doxorubicin-loaded natural daptomycin micelles with enhanced targeting and anti-tumor effect in vivo.

Development of a simple method to enhance targeting and anti-tumor effect of the chemotherapeutic agents in vivo is a major problem. Amphipathic and natural daptomycin is biocompatible antibacterial polypeptide used in clinical practice. Herein, doxorubicin (DOX) was stabilized by zwitterionic daptomycin (Dap) micelles in aqueous solution to form a zwitterionic nanodrug (Dap-DOX micelles). The hydrodynamic size and zeta potential of Dap-DOX micelles were 85 nm and -10 mV, respectively. The study on the controlled release showed that more DOX molecules were released from Dap-DOX micelles at acidic condition of tumor tissue than that at neutral condition of normal tissue which was due to pH responsiveness of Dap-DOX micelles. Dap-DOX micelles exhibited good stability in fibrinogen solution. Moreover, MTT studies showed that Dap-DOX micelles had higher cytotoxicity than free DOX. Notably, the results of flow cytometry indicated that the average fluorescence intensity of Dap-DOX micelle-treated cells was higher than that of free DOX-treated cells, and acidic conditions were more favorable for Dap-DOX micelles than normal pH in cell uptake assay. More importantly, Dap-DOX micelles were biocompatible in vivo based on the changes of weight and blood indexes of mice. Dap-DOX micelles were selectively accumulated at tumor sites in vivo through EPR effect, which reduced the toxicity of free DOX and achieved excellent tumor inhibition effect. The tumor inhibition rate of Dap-DOX micelles reached 96%. Dap-DOX micelles also effectively inhibited the growth of bacterial. Taken together, Dap-based drug delivery systems are promising and effective in cancer therapy.

An atomic perspective on improving daptomycin's activity.

BACKGROUND: Recently, through comprehensive medicinal chemistry efforts, we have found a new daptomycin analogue, termed kynomycin, showing enhanced activity against both methicillin-resistant S. aureus and vancomycin-resistant Enterococcus in vitro and in vivo, with improved pharmacokinetics and lower cytotoxicity than daptomycin. METHODS: In this study we compared the physicochemical properties of kynomycin with those of daptomycin from an atomic perspective by using Nuclear Magnetic Resonance spectroscopy and Molecular Dynamics simulations. RESULTS AND CONCLUSION: We observed that kynurenine methylation changes daptomycin's key physicochemical properties; its calcium dependent oligomerization efficiency is improved and the modified kynurenine strengths contacts with the lipid tail and tryptophan residues. In addition, it is observed that, compared to daptomycin, kynomycin tetramer is more stable and binds stronger to calcium. The combined experiments provide key clues for the improved antibacterial activity of kynomycin. GENERAL SIGNIFICANCE: We expect that this approach will help study the calcium binding and oligomerization features of new calcium dependent peptide antibiotics.

A high-yielding solid-phase total synthesis of daptomycin using a Fmoc SPPS stable kynurenine synthon.

A high-yielding total synthesis of daptomycin, an important clinical antibiotic, is described. Key to the development of this synthesis was the elucidation of a Camps cyclization reaction that occurs in the solid-phase when conventionally used kynurenine (Kyn) synthons, such as Fmoc-l-Kyn(Boc,CHO)-OH and Fmoc-l-Kyn(CHO,CHO)-OH, are exposed to 20% 2-methylpiperidine (2MP)/DMF. During the synthesis of daptomycin, this side reaction was accompanied by intractable peptide decomposition, which resulted in a low yield of Dap and a 4-quinolone containing peptide. The Camps cyclization was found to occur in solution when Boc-l-Kyn(Boc,CHO)-Ot-Bu and Boc-l-Kyn(CHO,CHO)-OMe were exposed to 20% 2MP/DMF giving the corresponding 4-quinolone amino acid. In contrast, Boc-l-Kyn(CHO)-OMe was stable under these conditions, demonstrating that removing one of the electron withdrawing groups from the aforementioned building blocks prevents enolization in 2MP/DMF. Hence, a new synthesis of daptomycin was developed using Fmoc-l-Kyn(Boc)-OH, which is prepared in two steps from Fmoc-l-Trp(Boc)-OH, that proceeded with an unprecedented 22% overall yield. The simplicity and efficiency of this synthesis will facilitate the preparation of analogs of daptomycin. In addition, the elucidation of this side reaction will simplify preparation of other Kyn-containing natural products via Fmoc SPPS.

Structure-Activity Relationships of Daptomycin Lipopeptides.

Daptomycin is a calcium-dependent cyclic lipodepsipeptide derived from the soil saprotroph Streptomyces roseosporus, and its antibiotic properties make it a key agent for treatment of drug-resistant Gram-positive infections. It is most commonly used clinically for the treatment of Gram-positive skin and skin structure infections (SSSI), Staphylococcus aureus bacteremia, and right-sided endocarditis infections associated with S. aureus, including methicillin resistant S. aureus (MRSA). It has also been used "off-label" for Enterococcal infections. There has been a tremendous amount of research investigating its mode of action, resistance mechanisms, and biosynthesis of this clinically important antimicrobial agent. Although we cover the latter aspects in detail, the primary focus of this review is to provide the most comprehensive and up-to-date reference for the medicinal chemist on the structure-activity-toxicity of this important class of lipopeptide antibiotics.

Synthesis of gold nanoflowers stabilized with amphiphilic daptomycin for enhanced photothermal antitumor and antibacterial effects.

The development of effective agents for cancer therapy and inhibition of bacterial infection has drawn a great deal of interest. Photothermal therapy has been widely used for the thermal ablation of tumor cells. In addition, antibiotics have the ability to inhibit the growth of bacteria. Thus, the combination of photothermal therapy and antibiotics may be one of the methods to address the problem. Herein, it is the first time that daptomycin (Dap) micelles were used as the template and reducing agents to prepare stable daptomycin-gold nanoflowers (Dap-AunNFs) under mild conditions. The energy dispersive spectrometer (EDS) spectrum and X-ray diffraction (XRD) spectrum indicated that Dap-AunNFs were successfully prepared. When the molar ratio of HAuCl4 to Dap was 6, the gold nanoparticles inside of Dap-AunNFs were about 80 nm with flower-like shape. In addition, the photothermal conversion efficiency of Dap-Au6NFs was about 40%. More importantly, Dap-Au6NFs inhibited the growth of tumors and bacteria under the radiation of near-infrared light at 808 nm. The prepared Dap-Au6NFs could be used as photothermal antitumor and antibacterial agents in the future.

Decision making in the pharmaceutical industry - A tale of three antibiotics.

There is a mounting crisis in treatment of bacterial diseases. The appearance of nosocomial infections produced by multi-drug resistant bacteria is rapidly increasing and at the same time the pharmaceutical industry has been abandoning new antibiotic discovery. To help understand why, we investigated the decision-making processes behind three novel antibiotics that were initially discovered in the late 1980's and early 1990's: daptomycin, linezolid, and lysobactin. Each antibiotic was investigated by two highly qualified scientific organizations that came to opposing opinions regarding the clinical utility and commercial potential of the drug. After reviewing the literature and interviewing key scientific staff members working on each of these molecules, we have identified factors needed to generate positive development decisions. Organizational factors included decision timing, therapeutic area focus, organizational support for risk taking and the presence of a project champion. Technical factors included investment in the optimization of dosing for improved drug exposure, toxicological evaluation of the purified eutomer from a diastereomer and the failure to develop an effective research formulation.