A Hadal Streptomyces-Derived Echinocandin Acylase Discovered through the Prioritization of Protein Families.

Naturally occurring echinocandin B and FR901379 are potent antifungal lipopeptides featuring a cyclic hexapeptide nucleus and a fatty acid side chain. They are the parent compounds of echinocandin drugs for the treatment of severe fungal infections caused by the Candida and Aspergilla species. To minimize hemolytic toxicity, the native fatty acid side chains in these drug molecules are replaced with designer acyl side chains. The deacylation of the N-acyl side chain is, therefore, a crucial step for the development and manufacturing of echinocandin-type antibiotics. Echinocandin E (ECE) is a novel echinocandin congener with enhanced stability generated via the engineering of the biosynthetic machinery of echinocandin B (ECB). In the present study, we report the discovery of the first echinocandin E acylase (ECEA) using the enzyme similarity tool (EST) for enzymatic function mining across protein families. ECEA is derived from Streptomyces sp. SY1965 isolated from a sediment collected from the Mariana Trench. It was cloned and heterologously expressed in S. lividans TK24. The resultant TKecea66 strain showed efficient cleavage activity of the acyl side chain of ECE, showing promising applications in the development of novel echinocandin-type therapeutics. Our results also provide a showcase for harnessing the essentially untapped biodiversity from the hadal ecosystems for the discovery of functional molecules.

Unnatural tetradeoxy echinocandins produced by gene cluster design and heterologous expression.

The hydroxyl groups in the amino acid residues of echinocandin B were related to the biological activity, the instability, and the drug resistance. The modification of hydroxyl groups was expected to obtain the new lead compounds for next generation of echinocandin drug development. In this work one method for heterologous production of the tetradeoxy echinocandin was achieved. A reconstructed biosynthetic gene cluster for tetradeoxy echinocandins composed of ecdA/I/K and htyE was designed and successfully hetero-expressed in Aspergillus nidulans. The target product of echinocandin E (1) together with one unexpected derivative echinocandin F (2), were isolated from the fermentation culture of engineered strain. Both of compounds were unreported echinocandin derivatives and the structures were identified on the basis of mass and NMR spectral data analysis. Compared with echinocandin B, echinocandin E demonstrated superior stability and comparable antifungal activity.

Investigation of the enhancement for Echinocandin B fermentation with methyl oleate from transcription level.

Echinocandin B (ECB) is the key precursor compound of the antifungal drug Anidulafungin. The effects of the five precursor amino acids on ECB biosynthesis were firstly investigated. It showed that although L-threonine was a main compound of the hexapeptide scaffold of ECB, exogenous addition of L-threonine had no significant effect on the increase of ECB fermentation titer. Meanwhile, the ECB fermentation titer with methyl oleate showed two times higher than that of the other carbon sources. Transcription level analysis of the key genes for ECB biosynthesis indicated that the gene an655543 related to L-threonine biosynthesis showed higher value during the fermentation process, therefore, the exogenous addition of L-threonine had no obvious affection. Furthermore, it indicated that the transcription level of gene ecdA might be the main restriction factor for the ECB biosynthesis. The study provided the research foundation for the modification of the ECB producing strains in the following work.

Echinocandins - structure, mechanism of action and use in antifungal therapy.

With increasing number of immunocompromised patients as well as drug resistance in fungi, the risk of fatal fungal infections in humans increases as well. The action of echinocandins is based on the inhibition of ß-(1,3)-d-glucan synthesis that builds the fungal cell wall. Caspofungin, micafungin, anidulafungin and rezafungin are semi-synthetic cyclic lipopeptides. Their specific chemical structure possess a potential to obtain novel derivatives with better pharmacological properties resulting in more effective treatment, especially in infections caused by Candida and Aspergillus species. In this review we summarise information about echinocandins with closer look on their chemical structure, mechanism of action, drug resistance and usage in clinical practice. We also introduce actual trends in modification of this antifungals as well as new methods of their administration, and additional use in viral and bacterial infections.

The synthesis and activity evaluation of N-acylated analogs of echinocandin B with improved solubility and lower toxicity.

Presently, echinocandins have been recommended as the first-line drugs for the treatment of invasive candidiasis. However, low oral bioavailability and solubility limit their application. To improve this situation, this study chose amino acid and fatty acid as raw materials to modify the nucleus of echinocandin B. Six N-acylated analogs were screened from the derivatives that possessed potent antifungal activity and good water solubility. Based on antifungal susceptibility and hemolytic toxicity, compound 5 as the candidate had good antifungal activity and no hemolytic effect. Moreover, compared with anidulafungin, compound 5 showed a comparable fungicidal effect, much higher solubility, and lower toxicity. In conclusion, compound 5 has the potential for further research and development on account of reserved antifungal activity, high solubility, and low toxicity.

Effects of methyl oleate and microparticle-enhanced cultivation on echinocandin B fermentation titer.

Echinocandin B (ECB) is a key precursor of antifungal agent Anidulafungin, which has demonstrated clinical efficacy in patients with invasive candidiasis. In this study, the effects of microparticle-enhanced cultivation and methyl oleate on echinocandin B fermentation titer were investigated. The results showed that the titer was significantly influenced by the morphological type of mycelium, and mycelium pellet was beneficial to improve the titer of this secondary metabolism. First, different carbon sources were chosen for the fermentation, and methyl oleate achieved the highest echinocandin B titer of 2133 ± 50 mg/L, which was two times higher than that of the mannitol. The study further investigated the metabolic process of the fermentation, and the results showed that L-threonine concentration inside the cell could reach 275 mg/L at 168 h with methyl oleate, about 2.5 times higher than that of the mannitol. Therefore, L-threonine may be a key precursor of echinocandin B. In the end, a new method of adding microparticles for improving the mycelial morphology was used, and the addition of talcum powder (20 g/L, diameter of 45 µm) could make the maximum titer of echinocandin B reach 3148 ± 100 mg/L.

Biosynthesis of pneumocandin lipopeptides and perspectives for its production and related echinocandins.

Fungal diseases are a global public health problem. Invasive fungal infections pose a serious threat to patients with compromised immune systems, such as those undergoing organ or bone marrow transplants, cancer, or HIV/AIDS. Pneumocandins are antifungal lipohexapeptides of the echinocandin family that noncompetitively inhibit of 1,3-ß-glucan synthase of fungal cell wall and provide the precursor for the semisynthesis of caspofungin, which is widely used as first-line therapy for invasive fungal infections. Recently, the biosynthetic steps leading to formation of pneumocandin B0 and echinocandin B have been elucidated, and thus, provide a framework and attractive model for further design new antifungal therapeutics around natural variations in echinocandin structural diversities via genetic and chemical tools. In this article, we analyze the biosynthetic pathway of pneumocandins and other echinocandins, provide an update on the array of pneumocandin analogues generated by genetic manipulation, and summarize advances in the enhancement of pneumocandin B0 production by random mutagenesis and fermentation optimization. We also give offer advice on the development of improved pneumocandin drug candidates and more efficient production of pneumocandin B0.

Ex vivo Rezafungin Adsorption and Clearance During Continuous Renal Replacement Therapy.

BACKGROUND/AIMS: To determine adsorption and transmembrane clearances (CLTM) of rezafungin, a novel long-acting echinocandin, in continuous venovenous hemofiltration (CVVH). METHODS: A validated ex vivo bovine blood CVVH model using polysulfone and AN69 hemodiafilters was used to evaluate urea and rezafungin CLTM at 3 different ultrafiltrate flow rates. Rezafungin adsorption to the CRRT apparatus was determined for each hemodiafilter. RESULTS: The sieving coefficient (SC) from CVVH with 3 different ultrafiltrate flow rates was 0 for both HF1400 and Multiflow-150 hemodiafilters, while urea SC was approximately 1 at all flow rates. Hemodiafilter type and ultrafiltrate flow rate did not influence CLTM. Rezafungin adsorption to the CVVH apparatus was not observed for either hemodiafilter. CONCLUSION: Rezafungin is not removed by CVVH by membrane adsorption or via CLTM. Ultrafiltrate flow rates and hemodiafilter types are unlikely to influence rezafungin CLTM. No dosage adjustment of rezafungin is likely required for critically ill patients receiving CVVH.

Plant-derived antifungal agent poacic acid targets ß-1,3-glucan.

A rise in resistance to current antifungals necessitates strategies to identify alternative sources of effective fungicides. We report the discovery of poacic acid, a potent antifungal compound found in lignocellulosic hydrolysates of grasses. Chemical genomics using Saccharomyces cerevisiae showed that loss of cell wall synthesis and maintenance genes conferred increased sensitivity to poacic acid. Morphological analysis revealed that cells treated with poacic acid behaved similarly to cells treated with other cell wall-targeting drugs and mutants with deletions in genes involved in processes related to cell wall biogenesis. Poacic acid causes rapid cell lysis and is synergistic with caspofungin and fluconazole. The cellular target was identified; poacic acid localized to the cell wall and inhibited ß-1,3-glucan synthesis in vivo and in vitro, apparently by directly binding ß-1,3-glucan. Through its activity on the glucan layer, poacic acid inhibits growth of the fungi Sclerotinia sclerotiorum and Alternaria solani as well as the oomycete Phytophthora sojae. A single application of poacic acid to leaves infected with the broad-range fungal pathogen S. sclerotiorum substantially reduced lesion development. The discovery of poacic acid as a natural antifungal agent targeting ß-1,3-glucan highlights the potential side use of products generated in the processing of renewable biomass toward biofuels as a source of valuable bioactive compounds and further clarifies the nature and mechanism of fermentation inhibitors found in lignocellulosic hydrolysates.

Structure-Activity Relationships of a Series of Echinocandins and the Discovery of CD101, a Highly Stable and Soluble Echinocandin with Distinctive Pharmacokinetic Properties.

Echinocandins are a first-line therapy for candidemia and invasive candidiasis. They are generally safe with few drug interactions, but the stability and pharmacokinetic properties of currently approved echinocandins are such that each was developed for daily intravenous infusion. We sought to discover a novel echinocandin with properties that would enable more flexible dosing regimens, alternate routes of delivery, and expanded utility. Derivatives of known echinocandin scaffolds were generated, and an iterative process of design and screening led to the discovery of CD101, a novel echinocandin that has since demonstrated improved chemical stability and pharmacokinetics. Here, we report the structure-activity relationships (including preclinical efficacy and pharmacokinetic data) for the series of echinocandin analogs from which CD101 was selected. In a mouse model of disseminated candidiasis, the test compounds displayed clear dose responses and were generally associated with lower fungal burdens than that of anidulafungin. Single-dose pharmacokinetic studies in beagle dogs revealed a wide disparity in the half-lives and volumes of distribution, with one compound (now known as CD101) displaying a half-life that is nearly 5-fold longer than that of anidulafungin (53.1 h versus 11.6 h, respectively). In vitro activity data against panels of Candida spp. and Aspergillus spp. demonstrated that CD101 behaved similarly to approved echinocandins in terms of potency and spectrum of activity, suggesting that the improved efficacy observed in vivo for CD101 is a result of features beyond the antifungal potency inherent to the molecule. Factors that potentially contribute to the improved in vivo efficacy of CD101 are discussed.

In vitro activity of the novel echinocandin CD101 at pH 7 and 4 against Candida spp. isolates from patients with vulvovaginal candidiasis.

Background: The novel echinocandin CD101 has stability properties amenable to topical formulation for use in the treatment of acute vulvovaginal candidiasis (VVC) and recurrent VVC (RVVC). CD101 has demonstrated potent antifungal activity at pH 7, but assessment of its activity at the physiological pH of the vaginal environment is needed. Objectives: To evaluate the antifungal activity of CD101 against clinical VVC isolates of Candida spp., including azole-resistant strains, at pH 4. Methods: MIC values of CD101 and comparators (fluconazole, itraconazole, micafungin, caspofungin and anidulafungin) were assessed via broth microdilution. MIC assays were conducted at pH 7 and 4 after 24 and 48 h against a 108 VVC isolate panel of Candida spp., including Candida albicans ( n = 60), Candida glabrata ( n = 21), Candida parapsilosis ( n = 14) and Candida tropicalis ( n = 13). Results: Overall, MIC values of all drugs were slightly higher at pH 4 versus 7 and at 48 versus 24 h of incubation. CD101 MIC values typically exhibited ∼4-fold shifts at pH 4 and were not affected by azole susceptibility. C. parapsilosis susceptibility was the least affected at pH 4 and did not increase for most drugs. Conclusions: CD101 had potent activity against all Candida isolates tested, including azole-resistant strains. Although there was some reduction in activity at pH 4 versus 7, the resulting MIC values were still well below the intravaginal CD101 drug concentrations anticipated to be present following topical administration. These results support continued development of topical CD101 for the treatment of VVC/RVVC.

Cinnamic Acid Analogs as Intervention Catalysts for Overcoming Antifungal Tolerance.

Disruption of fungal cell wall should be an effective intervention strategy. However, the cell wall-disrupting echinocandin drugs, such as caspofungin (CAS), cannot exterminate filamentous fungal pathogens during treatment. For potency improvement of cell wall-disrupting agents (CAS, octyl gallate (OG)), antifungal efficacy of thirty-three cinnamic acid derivatives was investigated against Saccharomyces cerevisiaeslt2Δ, bck1Δ, mutants of the mitogen-activated protein kinase (MAPK), and MAPK kinase kinase, respectively, in cell wall integrity system, and glr1Δ, mutant of CAS-responsive glutathione reductase. Cell wall mutants were highly susceptible to four cinnamic acids (4-chloro-α-methyl-, 4-methoxy-, 4-methyl-, 3-methylcinnamic acids), where 4-chloro-α-methyl- and 4-methylcinnamic acids possessed the highest activity. Structure-activity relationship revealed that 4-methylcinnamic acid, the deoxygenated structure of 4-methoxycinnamic acid, overcame tolerance of glr1Δ to 4-methoxycinnamic acid, indicating the significance of para substitution of methyl moiety for effective fungal control. The potential of compounds as chemosensitizers (intervention catalysts) to cell wall disruptants (viz., 4-chloro-α-methyl- or 4-methylcinnamic acids + CAS or OG) was assessed according to Clinical Laboratory Standards Institute M38-A. Synergistic chemosensitization greatly lowers minimum inhibitory concentrations of the co-administered drug/agents. 4-Chloro-α-methylcinnamic acid further overcame fludioxonil tolerance of Aspergillus fumigatus antioxidant MAPK mutants (sakAΔ, mpkCΔ). Collectively, 4-chloro-α-methyl- and 4-methylcinnamic acids possess chemosensitizing capability to augment antifungal efficacy of conventional drug/agents, thus could be developed as target-based (i.e., cell wall disruption) intervention catalysts.

Caspofungin infusion solutions (50 mg/100 mL): chemical stability and antifungal activity against Candida spp.

BACKGROUND: Ready to use caspofungin infusion bags are centrally prepared in the Hospital Pharmacy, University Hospital of Heidelberg, for economic reasons and possibly occurring problems with drug shortages. The aim of this study was a quality control of the in-house preparation of caspofungin infusion bags and the preparation process. Caspofungin concentration with regard to chemical stability and antifungal activity of caspofungin preparations were defined as quality parameters. METHODS: Three caspofungin infusion bags (50 mg in 100 mL 0.9% sodium chloride) were examined every seven days for a total of four weeks. Chemical stability of caspofungin solutions was analyzed using a validated high performance liquid chromatography (HPLC) method. Antifungal activity was assessed by microdilution tests according to the EUCAST protocol. Additionally, concentration and sterility were determined in returned caspofungin infusion bags. RESULTS: The amount of caspofungin in the infusion solutions still exceeded 90% after four weeks (2-8 °C). Antifungal activity was consistent over 28 days with a MIC ≤2 mg/L for different Candida spp. In returned infusion bags, caspofungin concentration was found to be ≥90% in 12 out of 13 bags and sterility was given in all preparations. CONCLUSION: These results show that chemical stability of caspofungin infusion solutions (50 mg/100 mL) can be guaranteed for four weeks at 2-8 °C and are confirmed by corresponding results regarding sterility and antifungal activity.

Membrane-Anchored Cyclic Peptides as Effectors of Mitochondrial Oxidative Phosphorylation.

The echinocandins are membrane-anchored, cyclic lipopeptides (CLPs) with antifungal activity due to their ability to inhibit a glucan synthase located in the plasma membrane of fungi such as Candida albicans. A hydrophobic tail of an echinocandin CLP inserts into a membrane, placing a six-amino acid cyclic peptide near the membrane surface. Because processes critical for the function of the electron transfer complexes of mitochondria, such as proton uptake and release, take place near the surface of the membrane, we have tested the ability of two echinocandin CLPs, caspofungin and micafungin, to affect the activity of electron transfer complexes in isolated mammalian mitochondria. Indeed, caspofungin and micafungin both inhibit whole chain electron transfer in isolated mitochondria at low micromolar concentrations. The effects of the CLPs are fully reversible, in some cases simply via the addition of bovine serum albumin to bind the CLPs via their hydrophobic tails. Each CLP affects more than one complex, but they still exhibit specificity of action. Only caspofungin inhibits complex I, and the CLP inhibits liver but not heart complex I. Both CLPs inhibit heart and liver complex III. Caspofungin inhibits complex IV activity, while, remarkably, micafungin stimulates complex IV activity nearly 3-fold. Using a variety of assays, we have developed initial hypotheses for the mechanisms by which caspofungin and micafungin alter the activities of complexes IV and III. The dication caspofungin partially inhibits cytochrome c binding at the low-affinity binding site of complex IV, while it also appears to inhibit the release of protons from the outer surface of the complex, similar to Zn(2+). Anionic micafungin appears to stimulate complex IV activity by enhancing the transfer of protons to the O2 reduction site. For complex III, we hypothesize that each CLP binds to the cytochrome b subunit and the Fe-S subunit to inhibit the required rotational movement of the latter.

Echinocandin B biosynthesis: a biosynthetic cluster from Aspergillus nidulans NRRL 8112 and reassembly of the subclusters Ecd and Hty from Aspergillus pachycristatus NRRL 11440 reveals a single coherent gene cluster.

BACKGROUND: Echinocandins are nonribosomal lipopeptides produced by ascommycete fungi. Due to their strong inhibitory effect on fungal cell wall biosynthesis and lack of human toxicity, they have been developed to an important class of antifungal drugs. Since 2012, the biosynthetic gene clusters of most of the main echinocandin variants have been characterized. Especially the comparison of the clusters allows a deeper insight for the biosynthesis of these complex structures. RESULTS: In the genome of the echinocandin B producer Aspergillus nidulans NRRL 8112 we have identified a gene cluster (Ani) that encodes echinocandin biosynthesis. Sequence analyses showed that Ani is clearly delimited from the genomic context and forms a monophyletic lineage with the other echinocandin gene clusters. Importantly, we found that the disjunct genomic location of the echinocandin B gene cluster in A. pachycristatus NRRL 11440 on two separate subclusters, Ecd and Hty, at two loci was likely an artifact of genome misassembly in the absence of a reference sequence. We show that both sequences can be aligned resulting a single cluster with a gene arrangement collinear compared to other clusters of Aspergillus section Nidulantes. The reassembled gene cluster (Ecd/Hty) is identical to a putative gene cluster (AE) that was previously deposited at the NCBI as a sequence from A. delacroxii NRRL 3860. PCR amplification of a part of the gene cluster resulted a sequence that was very similar (97 % identity), but not identical to that of AE. CONCLUSIONS: The Echinocandin B biosynthetic cluster from A. nidulans NRRL 8112 (Ani) is particularly similar to that of A. pachycristatus NRRL 11440 (Ecd/Hty). Ecd/Hty was originally reported as two disjunct sub-clusters Ecd and Hty, but is in fact a continuous sequence with the same gene order as in Ani. According to sequences of PCR products amplified from genomic DNA, the echinocandin B producer A. delacroxii NRRL 3860 is closely related to A. pachycristatus NRRL 11440. A PCR-product from the gene cluster was very similar, but clearly distinct from the sequence published for A. delacroxii NRRL 3860 at the NCBI (No. AB720074). As the NCBI entry is virtually identical with the re-assembled Ecd/Hty cluster, it is likely that it originates from A. pachycristatus NRRL 11440 rather than A. delacroxii NRRL 3860.

Development of New Strategies for Echinocandins: Progress in Translational Research.

Echinocandins are N-acyl-substituted cyclic hexapeptides with potent in vitro and in vivo activity against Candida species that are used for primary treatment and prevention of candidemia and invasive candidiasis. Recent progress in the translational research of echinocandins has led to new approaches for treatment of central venous catheter Candida biofilms. Other studies have laid the experimental and clinical foundation for use of extended dosing intervals for administration of echinocandins in treatment and prevention of candidemia and invasive candidiasis.

Role of Echinocandins in Fungal Biofilm-Related Disease: Vascular Catheter-Related Infections, Immunomodulation, and Mucosal Surfaces.

Biofilm-related infections have become an increasingly important clinical problem. Many of these infections occur in patients with multiple comorbidities or with impaired immunity. Echinocandins (caspofungin, micafungin, and anidulafungin) exert their fungicidal activity by inhibition of the synthesis of the (1→3)-ß-d-glucan. They are active among in vitro and in vivo model systems against a number of Candida species and filamentous fungi in their planktonic and biofilm phenotype. Their superior activity against biofilms poses them in an advantageous position among the antifungal armamentarium. However, additional studies are warranted to expand our knowledge on the role of echinocandins against biofilm-related infections.

Echinocandins: The Expanding Antifungal Armamentarium.

The echinocandins are large lipopeptide molecules that, since their discovery approximately 41 years ago, have emerged as important additions to the expanding armamentarium against invasive fungal diseases. Echinocandins exert in vitro and in vivo fungicidal action against most Candida species and fungistatic action against Aspergillus species. However, the population of patients at risk for developing invasive fungal infections continues to increase. New therapeutic strategies using echinocandins are needed to improve clinical outcomes in patients with invasive fungal disease.

Antifungal-Associated Drug-Induced Cardiac Disease.

The etiology of cardiomyopathies are classified into 4 main groupings (dilated, hypertrophic, restrictive, and idiopathic) and can be mechanistically caused by myocarditis, conduction abnormalities, focal direct injury, or nutritional deficiency. Based on our review of this topic, evidence suggests that echinocandin-related cardiac dysfunction is a mitochondrial drug-induced disease caused by focal direct myocyte injury. With caspofungin or anidulafungin administration into the heart via central line, exposure is likely extreme enough to induce the acute toxicity. Chronic or low-dose exposure may lead to hypertrophic cardiomyopathy; however, only acute exposures have been explored to date.

Can physicochemical properties of antimicrobials be used to predict their pharmacokinetics during extracorporeal membrane oxygenation? Illustrative data from ovine models.

INTRODUCTION: Ex vivo experiments in extracorporeal membrane oxygenation (ECMO) circuits have identified octanol-water partition coefficient (logP, a marker of lipophilicity) and protein binding (PB) as key drug factors affecting pharmacokinetics (PK) during ECMO. Using ovine models, in this study we investigated whether these drug properties can be used to predict PK alterations of antimicrobial drugs during ECMO. METHODS: Single-dose PK sampling was performed in healthy sheep (HS, n = 7), healthy sheep on ECMO (E24H, n = 7) and sheep with smoke inhalation acute lung injury on ECMO (SE24H, n = 6). The sheep received eight study antimicrobials (ceftriaxone, gentamicin, meropenem, vancomycin, doripenem, ciprofloxacin, fluconazole, caspofungin) that exhibit varying degrees of logP and PB. Plasma drug concentrations were determined using validated chromatographic techniques. PK data obtained from a non-compartmental analysis were used in a linear regression model to predict PK parameters based on logP and PB. RESULTS: We found statistically significant differences in pH, haemodynamics, fluid balance and plasma proteins between the E24H and SE24H groups (p < 0.001). logP had a strong positive linear relationship with steady-state volume of distribution (Vss) in both the E24H and SE24H groups (p < 0.001) but not in the HS group (p = 0.9) and no relationship with clearance (CL) in all study groups. Although we observed an increase in CL for highly PB drugs in ECMO sheep, PB exhibited a weaker negative linear relationship with both CL (HS, p = 0.01; E24H, p < 0.001; SE24H, p < 0.001) and Vss (HS, p = 0.01; E24H, p = 0.004; SE24H, p = 0.05) in the final model. CONCLUSIONS: Lipophilic antimicrobials are likely to have an increased Vss and decreased CL during ECMO. Protein-bound antimicrobial agents are likely to have reductions both in CL and Vss during ECMO. The strong relationship between lipophilicity and Vss seen in both the E24H and SE24H groups indicates circuit sequestration of lipophilic drugs. These findings highlight the importance of drug factors in predicting antimicrobial drug PK during ECMO and should be a consideration when performing and interpreting population PK studies.