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Engineering of new pneumocandin side-chain analogues from Glarea lozoyensis by mutasynthesis and evaluation of their antifungal activity Li Chen, Yan Li, Qun Yue, Anna Loksztejn, Kenichi Yokoyama, Edd A. Felix, Xingzhong Liu, Ningyan Zhang, Zhiqiang An, and Gerald F. Bills ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00604 • Publication Date (Web): 05 Aug 2016 Downloaded from http://pubs.acs.org on August 7, 2016

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ACS Chemical Biology

Engineering of new pneumocandin side-chain analogues from Glarea lozoyensis by mutasynthesis and evaluation of their antifungal activity Li Chen,†,§ Yan Li,†,§ Qun Yue,† Anna Loksztejn,‡ Kenichi Yokoyama,‡ Edd A. Felix,& Xingzhong Liu,# Ningyan Zhang,† Zhiqiang An,*, † and Gerald F. Bills*, † †

Texas Therapeutics Institute, the Brown Foundation Institute of Molecular Medicine, the University

of Texas Health Science Center at Houston, Houston, TX 77054, USA ‡

Department of Biochemistry, Duke University School of Medicine, Nanaline H. Duke Building, Box 3711, Durham, NC 27710, USA & Phamaceutical Science Facility, Institute of Applied Cancer Science, The M. D. Anderson Cancer Center, Houston, TX 77054, USA # State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, No. 3 Park 1, Beichen West Road, Chaoyang District, Beijing 100101, China

§

These authors contributed equally to this work

*

Correspondence authors

Zhiqiang An and Gerald F. Bills Email addresses: ZA: [email protected] GB: [email protected] or [email protected]

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ABSTRACT: Pneumocandins are lipohexapeptides of the echinocandin family that inhibit fungal 1,3-β-glucan synthase. Most of the pathway steps have been identified previously. However, the lipoinitiation reaction has not yet been experimentally verified. Herein, we investigate the lipoinitiation step of pneumocandin biosynthesis in Glarea lozoyensis, and demonstrate that the gene product, GLligase, catalyzes this step. Disruption of GLHYD, a gene encoding a putative type II thioesterase and sitting upstream of the pneumocandin acyl side chain synthase gene, GLPKS4, revealed that GLHYD was necessary for optimal function of GLPKS4 and to attain normal levels of pneumocandin production. Double disruption of GLHYD and GLPKS4 did not affect residual function of the GLligase or GLNRPS4. Mutasynthesis experiments with a gene disruption mutant of GLPKS4, afforded us an opportunity to test the substrate specificity of GLligase in the absence of its native polyketide side chain to diversify pneumocandins with substituted side chains. Feeding alternative side chain precursors yielded acrophiarin and four new pneumocandin congeners with straight C14, C15, and C16 side chains. A comprehensive biological evaluation showed that one compound, pneumocandin I (5), has elevated antifungal activity and similar hemolytic activity compared to pneumocandin B0, the starting molecule for caspofungin. This study demonstrates that the lipoinitiation mechanism in pneumocandin biosynthesis involves interaction among a highly reducing PKS, a putative type II thioesterase, and an acyl AMP-ligase. A comparison of the SAR among pneumocandins with different-length acyl side chains demonstrated the potential for using GLligase for future engineering of new echinocandin analogues. Key Words: acrophiarin, acyl AMP ligase, type II thioesterase, Agrobacterium-mediated transformation, antifungal antibiotics, echinocandins, fatty acids, lipoinitiation, Penicillium arenicola


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Introduction Recently, the basic biosynthetic steps of pneumocandin B0 (1) and echinocandin B, the lipopeptide starting molecules for the first- and third-in-class antifungal drugs, caspofungin and anidulifungin, respectively, have been elucidated. To date, the side-chain synthase, GLPKS4, the core hexapeptide synthetase, GLNRPS4, and several tailoring enzymes, including GLOXY1 and GLP450-1 responsible for 3,4-hydroxyl groups on the L-homotyrosine residue respectively, GLP450-2 responsible for dihydroxylation of L-ornithine, GLOXY4 for L-leucine cyclization to form 4-methylproline, and GLOXY2 that hydroxylates L-proline to form the hydroxyl proline residue have been identified.1-6 Access to the pneumocandin biosynthetic gene cluster has provided us with opportunities to explore new SAR (structure–activity relationship) questions through genetic manipulation instead of chemical semisynthetic and total synthetic studies. For example, disruption of GLOXY2 caused exclusive production the of caspofungin precursor, pneumocandin B0,

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while gene disruption of GLOXY1,

GLP450-1, and GLP450-2 provided a unique opportunity for side-by-side exploration of how patterns of incomplete hydroxylations on the pneumocandin core affect antifungal activity.4 Although most of the biosynthetic enzymes have been identified, experimental proof of the essential step for connecting the highly reduced polyketide 10R, 12S-dimethylmyristic acyl side chain to the hexapeptide is still lacking. The addition of a polyketide side chain to the pneumocandin hexapeptide is a critical step, not only because it connects the polyketide to the non-ribosomal peptide biosynthesis, but without successful ligation of the acyl group to the first ornithine residue, peptide elongation does not proceed.2 Chemical or enzymatic removal of the side chain abolishes antifungal activity in the echinocandins.7-8 Furthermore, the diversity of acyl side chain configurations in the echinocandin family9 critically shaped the development paths to the three different approved echinocandin drugs. Palmitoyl or linoleyl linear side chains of FR901379 and of echinocandin B are significantly more hemolytic than the 10,12-dimethylmyristoyl branched side chain of pneumocandin B0 and necessitated strategies for side chain deacylation and synthetic replacement for development of anidulifungin an micafungin respectively.10-12 An additional question regarding the echinocandin lipoinitiation step that is peculiar to pneumocandin biosynthesis is the exact mechanism of the release and transfer of the GLPKS4 polyketide substructure to the NRPS’s first T (thiolation) domain that precedes the adenylation domain for ornithine activation. The terminal domain of the GLPKS4 PKS is an acyl carrier protein (ACP) domain, and not a thioesterase (TE) domain. Consequently, it is unknown whether the 10,12-methylmyristoyl product is released as a free fatty acid or if it remains tethered to the enzyme during transfer. We also questioned whether an uninvestigated putative type II TE gene (GLHYD) that lies downstream of GLPKS4 might encode a protein involved in product release. Various mechanisms are known for activation and transfer of the lipid side chains to the nonribosomal peptide synthetases (NRPSs) in fungi.2, 13-14 During the elucidation of emericellamide biosynthesis, an acyl-CoA ligase was demonstrated to be involved in the lipoinitiation step. On the



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other hand, heterologous expression and in vitro recreation of the ligase reaction in echinocandin B biosynthesis showed the pathway’s AMP-dependent-ligase mediated the activation of and shuttle of linoleic acid to the first T domain of the NRPS. In the pneumocandin biosynthetic gene cluster, a putative AMP-dependent ligase (GLligase, GLAREA_10043, EPE34349.1), and not an acyl-CoA ligase, was found. Therefore, the AMP-dependent ligase (GLligase) was proposed to be responsible for activation of the 10,12-dimethylmyristic acid and its transfer to the first T domain of GLNRPS4.5-6 The objective of this study was to investigate the functions of the putative acyl AMP-ligase gene (GLligase) that encodes the enzyme responsible for shuttling the side chain polyketide synthase (PKS) product to the core hexapeptide and a putative type II thioesterase gene (GLHYD) that sits downstream of GLPK4 by gene disruption studies. Additionally, we have exploited the mechanism of this critical step to explore the uptake of alternative acyl donors during fermentation and to evaluate how substitution of linear acyl groups for the native dimethylmyristoyl side chain affects pneumocandin antifungal activity and its hemolytic properties. Consequently, this study presents the first example of engineering new echinocandin side chain analogues by mutasynthesis including structure activity of these new analogues. RESULTS AND DISCUSSION Inactivation of GLligase abolishes production of pathway products. To test the prediction that GLligase is essential for the addition of the polyketide side chain to the hexapeptide core, gene inactivation and substrate feeding experiments were conducted. We inactivated the GLligase by Agrobacterium tumefaciens-mediated transformation and integration of a hygromycin resistance marker into the target gene. Hygromycin-resistant strains were recovered from the transformation medium. The mutants were screened for antifungal activity against Candida albicans and tested by PCR using the primers for replacement fragments adjacent to the flanking regions of the hygromycinresistance gene (Table S1, Figure S1, supporting information). HPLC analysis of the ∆GLligase strain’s fermentation extracts failed to detect pneumocandins or any new peaks that might correspond to a deacylated peptide core or free dimethylmyristic acid, and a C. albicans zone-of-inhibition assay was negative, indicating that the GLligase was essential for pneumocandin production (Figure 1AB). As a negative control, and to rule out the possibility that an alternative ligase encoded at another gene locus might be able to substitute for the function of GLligase, myristic acid (non-methylated analogue of 10,12-dimethylmyristic acid) was fed to the GLligase deletion mutant. As expected, extracts of a fermentation of the GLligase insertional mutant fed with 1.5% myristic acid did not yield any peaks related to pneumocandins and did not inhibit growth of C. albicans (Figure 1AB).

Mutasynthesis with GLPKS4 inactivation mutants. To probe the in vivo substrate specificity of GLligase, we reanalyzed a GLPKS4 deletion mutant made in a previous study.6 We hypothesized that in the absence of a functional polyketide side chain, GLligase might be able to activate and shuttle 4 ACS Paragon Plus Environment

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alternative fatty acids to complete the lipoinitiation step. In our previous study,6 we were unable to detect any pneumocandin derivatives from ∆GLPKS4, a result that might have been due to suboptimal culture and extraction conditions. In this study, we grew our strains on a medium and conditions that reliably produce pneumocandins (H medium, baffled flask, and 5-cm throw on a rotary shaker).15 Under these conditions, we detected antifungal activity from the extracts of the ∆GLPKS4 strain (Figure 1B). HPLC analysis showed that a pneumocandin A0 (2) derivative with a myristic acid side chain formed spontaneously as the main pneumocandin product. Furthermore, a tiny amount of pneumocandin A0 derivatives with a pentadecanoic or palmitic acid side chain was also observed (Figure 2A). Curiously, this experiment appeared to be the first observation of alternative side chain uptake during pneumocandin biosynthesis because previous work with pilot-plant-scale fermentations always reported minor products with a dimethylmyristoyl side chain.16 Reference to natural products databases17 also indicated that most echinocandin types, including the seven with characterized gene clusters, vary with respect to hydroxylations of core amino acids, or amino acid substitutions, but lipid side chains for each echinocandin-producing strain were generally invariable, thus seeming to indicate naturally high fidelity between each pathway ligase and its preferred acyl donor. Nonetheless, in vitro experiments have shown that acyl AMP ligase EcdI from A. pachycristatus (as A. rugulosus) likewise exhibited some degree of flexibility with regard to acyl donors.2 We asked whether the relative abundance of the myristoyl, pentadecanoyl, and palmitoyl variants might be a reflection of the relative abundance of these fatty acids in the mycelium, or whether the acyl ligase preferentially incorporated one fatty acyl group over another. Fatty acids extracted from dried wild-type mycelium of G. lozoyensis grown under pneumocandin-producing conditions in H medium were identified and quantified by GC-MS (Table S2, supporting information). As expected, the relative content of fatty acids was similar to that reported from other ascomycete fungi,18 with palmitic and linolenic acids being the by far the most abundant (Table S2, supporting information). Myristic acid and pentadeconic acids were present at very low levels in comparison (Fig. 2B, Table S2, supporting information) but presumably were sufficiently abundant for the incorporation in to pneumocandins by GLligase. The cellular localization of pneumocandin biosynthesis is yet unknown. However, assuming all fatty acids were equally available to the site of biosynthesis, then a preferential incorporation of myristic and pentadecanoic acids into pneumocandins over the much more abundant palmitic and linolenic acids was evident (Figure 2A) which would be consist with pneumocandin’s native 14C branched side chain. To increase yields of these newly observed derivatives and purify quantities sufficient for structural confirmation, we added myristic, pentadecanoic, and palmitic acids to the H medium in respective experiments to produce enough of each product to facilitate the isolation steps. As one might predict, two additional compounds, the pneumocandin B0 derivatives with myristic and palmitic acid side chains were also isolated from these fermentations along with the corresponding pneumocandin A0 derivatives (Figure 2C). We also attempted parallel experiments with lauric, 5 ACS Paragon Plus Environment


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linoleic, stearic, and aleuritic acids, but in all these cases, pneumocandin-like products could not be detected by HPLC-MS. High resolution electrospray ionization mass spectroscopy (HRESIMS) identified the new peaks as didesmethyl-pneumocandin B0, named pneumocandin H (3) (m/z calcd for [M+H]+ 1037.5401, found 1037.5413) and didesmethyl-pneumocandin A0, previously designated as acrophiarin from Penicillium arenicola S 31794/F-1 (4) (m/z calcd for [M+H]+ 1051.5558, found 1051.5557),8, 19-20 pentadecanoic acid-derived form of pneumocandin A0, pneumocandin I (5) (m/z calcd for [M+H]+ 1065.5714, found 1065.5768); palmitic acid-derived form of pneumocandin B0, pneumocandin J (6) (m/z calcd for [M+H]+ 1065.5714, found 1065.5765) and palmitic acid-derived form of pneumocandin A0, pneumocandin K (7) (m/z calcd for [M+H]+ 1079.5871, found 1079.5852) (Figure 2C , 2D and 2E). NMR characterization (Figure S2, supporting information) of the didesmethyl form and pentadecanoic and palmitic acid derived form of pneumocandins B0 and A0, confirmed that myristic, pentadecanoic and palmitic acids were substituted into the hexapeptide. By comparing the integrated peak area, we concluded that the myristic acid incorporation rate was higher than the rates for pentadecanoic and palmitic acid (the concentration of 4 was almost tenfold higher than 5 and 7). This result indicated that in the GLPKS4 deletion mutant grown in unamended H medium, GLligase activated and shuttled the myristic acid more efficiently than the other two fatty acids, while C12 and C18 fatty acids and aleurtic acid were not taken up. Therefore, we concluded that GLligase encoding an AMP-dependent ligase is essential for activating and transferring either the native polyketide, some native fatty acids from the cytoplasm, or exogenously added C14, C15, or C16 fatty acids to the first catalytic domain of the NRPS.

Disruption of GLHYD indicates a role in turnover of 10,12-dimethylmyristoyl during pneumocandin production. Like other canonical highly-reducing PKSs (HR-PKS), GKPKS4 terminates in an ACP domain and lacks a TE releasing domain that ensures correct product release in other PKSs.21 Upon reexamination of the Leotiomycete-type echinocandin gene clusters responsible for echinocandins with 10,12-dimethylmyristoyl side chains, we speculated whether a putative hydrolase gene designated GLHYD (GLAREA_10032, EPE34338.1) downstream of the PKS might bear a TE domain.22 Orthologues of GLHYD are absent in Aspergillus-type echinocandin gene clusters that encode for echinocandins with fatty-acid-derived acyl side chains, e.g., echinocandin B.9 BLAST, PFAM, Protein Data Bank, and SWISS-MODEL similarity searches with the predicted amino acid sequence of GLHYD indicated that it belongs to the alpha/beta hydrolase protein family and contains a type II TE domain. In order to test whether the protein encoded by this gene might be involved in regenerating misacylated thiol groups of 4'-phosphopantetheine cofactors of the GLPKS4 polyketide product or guiding its off-loading onto the core’s ornithine residue, we disrupted GLHYD by insertion of a hygromycin-resistance gene. Five transformants were confirmed to have the correction insertion by PCR-based assay (Table S1, Figure S3, supporting information). Inactivation of GLHYD 6 ACS Paragon Plus Environment

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significantly decreased pneumocandin production (Figure 3, Figure S3, supporting information). To compare the differences between wild-type and ∆GLHYD strains, we quantified their pneumocandin production. Pneumocandin A0 production in ∆GLHYD was 11.8 ± 1.9 µg mg-1 (production/dry mass mycelium, n=5) which was only 38% of the wild-type level (32.1 ± 2.9 µg mg-1, n=5). The pneumocandin B0 in ∆GLHYD could not be detected compared to the wild-type level of 3.05 ± 0.11 µg mg-1 (Figure 3). Previous studies on type II TEs, such as LovG from the lovastatin pathway,23 TylO from the tylosin pathway,24 RifR from rifamycin B pathway25 showed that deletion of these genes decreased the production 5%, 10%, and 40%-60%, respectively, of the levels produced by the corresponding wild-type strains. Our results were consistent with these previous studies, and therefore, we hypothesized that GLHYD exerts a similar function, either by facilitating the correct off-loading of the polyketide product and shuttling it to GLligase, or removal of aberrant residues blocking the PKS, and thereby improves the turnover of the final GLPKS4 product, 10,12-dimethylmyristoly. Previous studies also have showed that the type II TEs may also exert an editing function on NRPSs by regenerating misacylated thiol groups of 4'-phosphopantetheine cofactors attached to the peptidyl carrier proteins and thereby improving their efficiency.22, 26-27 If this was the case, we might expect to observe a reduced level of pneumocandin production from fatty acid feeding experiments in a strain with a simultaneous double disruption of GLHYD and GLPKS compared to the ∆GLPKS4 strain. To test this hypothesis, pAg1-N3 (constructed in this study) was used to construct a disruption vector for GLHYD in ∆GLPKS4. We used Agrobacterium-mediated transformation to inactivate GLHYD by insertion of a nourseothricin-resistance gene. Five transformants were confirmed to have the correction insertion by PCR-based screen (Table S1, Figure S4, supporting information). The ∆GLHYD∆GLPKS4 and ∆GLPKS4 strains were grown in medium amended with 1.5% myristic acid to produce pneumocandin derivatives for analysis. The titers of acrophiarin (4) in ∆GLHYD∆GLPKS4 and ∆GLPKS4 were 6 ± 0.34 µg mg-1and 5.4 ± 0.58 µg mg-1 respectively (production/dry mass mycelium, n=5) (Figure 4, Figure S4, supporting information). The two strains did not significantly differ in titers of acrophiarin (Student’s T test, P=0.36) (Figure 4). This result indicated that GLHYD does not interact with GLNRPS4 during pneumocandin biosynthesis. We therefore conclude that GLHYD exclusively interacts with GLPKS4 to maintain turnover of the polyketide side chain. To the best of our knowledge, this is the third report of experimental manipulation of a free-standing type II TE associated with a fungal PKS.23,

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A hypothetical lipoinitiation step for pneumocandin

biosynthesis is proposed in Figure 5. Antifungal and hemolytic activity of new pneumocandins. Biological properties are key for determining the suitability of an antifungal agent for consideration as drug a drug candidate. We conducted a serial biological evaluation (antifungal, glucan synthase [GS], and hemolytic assays) to test how the different side chain lengths affected antifungal activity in different pathogens and if any might be a superior antifungal. The in vitro antifungal and GS activities of these newly biosynthesized lipopeptides were compared. The antifungal activity of each compound was determined as the 7 ACS Paragon Plus Environment


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minimal inhibitory concentration (MIC) in seven Candida strains, including two caspofungin-resistant strains and the minimal effective concentration (MEC) in Aspergillus fumigatus compared to the reference drug, caspofungin; the GS activity was determined as the half maximal inhibitory concentration (IC50) in C. albicans ATCC 10231 (Table 1). Antifungal assays showed that all five analogues were strongly inhibitory against all the tested fungal pathogens, except for the elevated MICs for the caspofungin-resistant strains of C. albicans MDACC1 and C. glabrata MDACC1 (Table 1, Figure S5, supporting information). Assays demonstrated strong activity against C. albicans and C. glabrata with MIC values in the range of 0.1–1.6 µg mL-1. In particular, 5 and 7, two compounds substituted with pentadecanoic and palmitic acid side chains, were found to be more active than 1 and 2. Their MIC values were 0.1 µg mL-1 and 0.2 µg mL-1, respectively, which were 8-fold and 4-fold more potent compared to the corresponding wild-type fermentation products 1 and 2 against C. albicans. The MIC values of 5 and 7 against C. glabrata were 32-fold improvements compared to 1. Compounds 5 and 7 were the most effective inhibitors against caspofungin-resistant strains, albeit the MIC values were much higher than against Candida strains (Table 1, Figure S5, supporting information). Compounds 3 and 4, with a myristic acid substituted side chain, showed antifungal activities similar to 1 and 2. Interestingly, compound 6, with a palmitic-acid-substituted side chain, exhibited a higher MIC compared to 1, which suggested that same side chain combined with a different hexapeptide core within the echinocandin family will confer a different activity. As for the MEC of A. fumigatus, the most effective compounds were also 5 and 7. The β-glucan synthase inhibitory activities against C. albicans ATCC 10231 (Table 1) showed a similar relative pattern of IC50 values, but did not strictly track the MIC results. This result suggested that elevated antifungal activity may not be fully associated with the GS.29 Two distinct antifungal mechanisms have been suggested to operate in the echinocandins: a potent activity due to GS inhibition and a weaker effect due to plasma and mitochondrial membrane destabilization.30-31 In addition to the antifungal activity of pneumocandins, a critical concern to in vivo use is compound safety, including lysis of red blood cells. To evaluate the relative behaviors of these new pneumocandins, we conducted a hemolytic assay, an essential parameter for assessing the safety of any blood-contacting compound (Figure 6). At high concentrations (102.4 µg mL-1), compound 5 and 7 exhibited strong hemolytic activity, and more than 50% of human erythrocytes were lyzed. The other compounds were less hemolytic than 5 and 7. At a concentration of 56.2 µg mL-1, compound 7 also showed strong hemolytic activity (more than 40% erythrocyte lysis). However, the hemolytic activity of 5 decreased sharply, but still exhibited significant erythrocyte lysis compared to rest of the compounds which had little hemolytic effect. Compound 5 showed almost no hemolytic effect and was not significantly different compared with pneumocandin B0 at a concentration 25.6 µg mL-1 (Figure 6, Table S3, supporting information), which is almost the double the concentration of the daily administration of caspofungin for treating human fungal infections.32 Therefore, we concluded that compound 5 is comparable to pneumocandin B0 with regard to its hemolytic safety, while 5 has 8 ACS Paragon Plus Environment

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enhanced antifungal activity compared to pneumocandin B0 (Figure 6). The other derivative 7 with improved antifungal potency showed higher hemolytic activity than pneumocandin B0. Hemolysis of rest of the compounds did not significantly differ at the tested concentrations. Conclusion. Lipopeptides are a major class of fungal secondary metabolites, but only a few studies have examined their molecular-level biosynthesis. In many cases in lipopeptides with shorter chain fatty acids, the fatty acids are derived from amino acids, such as α-hydroxyisocaproic acid, which is derived from leucine. 33 Others, including cavinofungin,34 emericellamide,35 FR901469,36 apicidins,37 fusaristatin,38 W493B,38 scopularides,39 leucinostatins,28 and the echinocandins,2, 6 contain linear or branched-chain fatty acids that are derived from a fatty acid synthase or a highly reducing PKS. All these molecules are antifungal or have other significant biological activities and would be candidates for mutasynthesis experiments, such as described herein, to diversify their side chain chemistry. GLligase encodes an enzyme essential for lipoinitiation, and it can be exploited to vary the outcome of the lipoinitiation step during pneumocandin biosynthesis. Availability of disruption mutants of GLPKS4, afforded us the opportunity to investigate the specificity of GLligase in vivo for the first time. We demonstrated that GLligase can shuttle alternative C14, C15 and C16 acyl side chains to the NRPS, even without addition of exogenous fatty acids to the medium, albeit at highly reduced levels. Taking advantage of the limited substrate flexibility of GLligase, we supplemented the medium with exogenous fatty acids and purified and characterized a pneumocandin equivalent to acrophiarin plus four new derivatives of pneumocandins with alternative fatty acyl side chains. Disruption of GLHYD, a putative type II thioesterase, significantly impairs production of pneumocandins, and therefore, we infer that GLHYD cooperates with GLPKS4 and plays a critical role in maintaining efficient turnover of the polyketide product and normal levels of pneumocandin production. The biological evaluation of these new pneumocandins expanded the knowledge of the SAR between side chain length and antifungal and hemolytic activity. Finally, we found that the new congener pneumocandin I (5) possessed elevated antifungal activity and also had hemolytic properties similar to pneumocandin B0. Combining the enhanced properties of 5 with other genetic manipulations that improve antifungal potency, e.g., removal of the L-homotyrosine C4 hydroxyl group,4 has the potential to lead to the design of new starting points for the development of alternative echinocandin drug candidates. METHODS Strains and plasmid. The strains used for genetic manipulation in this study were described by Chen et al.5 Reference fungal pathogenic strains were purchased from the American Type Culture Collection and the Fungal Genetics Stock Center. Caspofungin-resistant strains of C. albicans (MDACC 1) and C. glabrata (MDACC 1) were clinical isolates provided by Prof. Dimitrios 9 ACS Paragon Plus Environment


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Kontoyiannis, M. D. Anderson Cancer Center. The plasmid pAg1-H3 for gene disruption vector construction and transformation, and genomic DNA extraction of G. lozoyensis were described by Zhang et al.40 Fungal transformation and gene disruption. The protocol for disruption vector construction and gene inactivation in G. lozoyensis was described previously.5 For double gene deletion, the plasmid pAg1-N3 was constructed as follow: the nourseothricin resistance gene, nat1, was amplified from plasmid pAg36 using the primers accompanied with ApaI and AscI restriction sites respectively (Table S1).41 The fragment was inserted into ApaI/AscI of pAg1-H3 to replace hygromycin resistance gene to generate pAg1-N3.Transformants with hygromycin and nourseothricin resistance were recovered from selection medium, and the correct gene inactivation mutants were identified by PCR (Figure S1, Figure S3, Figure S4, supporting information). PCR primers for vector construction and transformants screening are listed in Table S1, supporting information. HPLC and high resolution mass spectrometry analysis. Conidia of wild-type G. lozoyensis and mutant strains from oat bran agar (oat bran 4%, agar 2% in tap water) were inoculated into 10 ml of seed medium (KF medium). 0.4 mL of seed medium were inoculated into 10 mL of production medium (H medium) (15) in tubes at 25 oC on a rotary shaker at 220 rpm for 14 days. The cultures were extracted with equal volumes of methyl ethyl ketone (MEK), and the organic phase was evaporated to dryness and redissolved in 1 mL methanol. Ten-µl aliquots of crude extracts were injected for each run, and eluted with a solvent gradient of 10-100% B for 28 min (solvent A, 0.1% formic acid in H2O; solvent B, 0.1% formic acid in acetonitrile) on a C18 reverse phase column (Agilent Zorbax Eclipse Plus C18 4.6 × 150 mm, 5 µm). Column effluent with a flow rate of 1 mL min-1 was monitored at 210 nm. The chromatographic profiles of crude extract samples were detected with an Agilent 1260 HPLC equipped with a diode array detector (DAD), with wavelength scanning from 190 nm to 400 nm. High resolution mass spectrum analysis of the samples were analyzed on an Agilent 6538 Ultra High Definition Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) LC/MS system interfaced with an Agilent 1200 Series HPLC-Chip/MS system. The LC-Chip was used a 40-nL enrichment column 10 ACS Paragon Plus Environment

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and a 75 µm × 43 mm analytical column packed with ZORBAX 300SB-C18 5 µm. The solvents were 0.1% formic acid in water (A) and 90% acetonitrile in water with 0.1% formic acid (B). The flow rates were 3.5 nL min-1 for loading the sample on to the enrichment column, and 600 nL min-1 for the analytical column. Samples were loaded on the enrichment column using 20% B. The gradient for the analytical column was as follows: 20% B at 0 min, 90% B at 13 min, and 20% B at 15 min. The QTOF was operated in positive mode with a capillary voltage of 1800 V and drying gas flow rate of 4 L min-1 at 340 oC. Fermentation and scaled-up for compound isolation for structural elucidation. The media and procedures for growth, fermentation, and extraction of strains were the same as those described previously.4-5 For alternative substrate experiments, the different fatty acids were added to the medium (1.5 or 2% w/v) before autoclaving to help emulsify and partially solubilize them into the liquid. To scale up newly observed pneumocandins for purification, four mL of seed medium were inoculated into ten 500-mL Erlenmeyer flasks containing 100 mL of H medium (total volume was 1 L for supplementation of myristic, pentadecanoic and palmitic acids at a final concentration of 1.5%), and cultivated at 25 oC with agitation at 220 rpm for 14 days. The fermented culture were extracted with 2 L of MEK, and the organic phase was evaporated to dryness under vacuum to afford the crude extract, which was fractionated on a reversed-phase (RP) C18 column (10-100% methanol in water over 35 min; flow rate was 40 mL L-1) coupled to a Grace Reveleris X2 flash chromatography system. Fractions with desired compounds were combined , and further purified by semi-preparative RP HPLC (Agilent Zorbax SB-C18 column; 5µm; 9.4 × 250 mm; 38% acetonitrile in water with 0.1% formic acid over 20 min; 2 mL min-1) to afford compounds 3 (5.6 mg, tR 12.75 min), 4 (15.6 mg, tR 15.6 min), 5 (2.6 mg, tR 16.4 min), 6 (1.8 mg, tR 16 min) and 7 (4.2 mg, tR 17.5 min). Structures of the five new compounds were determined by MS and NMR spectroscopy (Figure 2D, Figure S2, supporting information). Antifungal assays. Procedures for antifungal assays were described previously.4 The minimal inhibitory concentration (MIC) and minimal effective concentration (MEC) were determined using serial dilution method in 96-well plates with RPMI 1640 buffered with 0.165 M MOPS as the testing medium. Test compounds and reference drug caspofungin were dissolved in DMSO and serially 11 ACS Paragon Plus Environment


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diluted in the assay medium. The assays were incubated at 35 oC. The MICs of Candida strains were determined at 24 h using PrestoBlue® resazurin dye as the viability indicator. The MIC value was defined as the lowest concentration of a testing compound that resulted in culture with no growth (100% inhibition) when compared to untreated culture (Figure S5, supporting information). The MEC values of Aspergillus fumigatus were determined by microscopic examination of the diluted wells, and the MEC was defined as the lowest concentration that caused abnormal hyphal extension resulting in short abundant branches compared to the hyphae of the strain grown in wells without drug (Figure S6, supporting information).42 Glucan synthase assay in C. albicans ATCC 10231. The IC50 was determined using membrane preparations of (1,3)-β-D-glucan synthase (GS) from C. albicans ATCC 10231. The GS-containing membrane was prepared based on the published procedure43 with modifications. Briefly, C. albicans was grown in YPD media at 30 °C and harvested at early logarithmic phase (OD600 = 0.8). Harvested cells (15 g wet weight) were re-suspended in 1 mM EDTA, and lysed by 5 cycles on a French press (20,000 psi). Subsequently, cell debris and unlysed cells were removed by centrifugation for 30 min at 1,000 × g, and the resulting supernatant was applied to ultracentrifugation for 1h at 100,000 × g. The resulting pellet, containing majority of the GS activity, was resuspended in 10 ml of storage buffer containing 50mM Tris-HCl pH 7.5, 1 mM EDTA 33% glycerol and 1 mM 2-mercaptoethanol by homogenization using a Dounce homogenizer. The resulting suspension was aliquoted and flash frozen in liquid N2 and stored in -80 oC until use. Final protein concentration was 9.7 mg mL-1 as determined by a bicinchoninic acid assay. The GS activities were assayed in 30 µL of 75 mM Tris buffer pH 7.5 containing 0.75% w/v bovine serum albumin, 25 mM KF, 0.75 mM EDTA, 20µM ɣS-GTP, 1 mM UDP-[U-14C]-Glc (specific activity, 321 cpm nmol-1) and the specified concentrations of pneumocandins. The reaction was initiated by the addition of the membrane preparation of GS (3.9 pmol min-1), and incubated at 30 °C. After 60 min, the reaction was quenched by mixing an aliquot (25 µL) of the assay solution with 200 µL of 10% (w/v) trichloroacetic acid solution. The resulting mixture was filtered using a 96well glass filter plate, and the residue containing insoluble glucan was washed three times with 200


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µL of 10%TCA followed by 200 µL ethanol. Radioactivity remaining on the filter was determined with a liquid scintillation counter (Beckman LS6500). Hemolytic assay. Human erythrocytes from healthy individuals were collected in a sterilized tube containing heparin and followed by centrifugation at 2000 rpm at 20 oC for 10 min to harvest the erythrocytes. The pellet was washed three times in 1 × phosphate-buffered saline (PBS), and the pellet was added to 1× PBS to make a 20% (v/v) erythrocyte storage solution. The 20% solution was diluted 1:10 in PBS to make a 2% working solution. Tested compounds were dissolved in 20% DMSO to yield 2.048 mg mL-1 storage solution. Compounds were serially diluted with 20% DMSO followed by the addition of 7.5 µL to the 96-well plate. The 142.5 µL of 2% erythrocytes were mixed with each compound to attain a final volume of 150 µL. 20% DMSO mixed with 2% erythrocytes was the negative control. Total hemolysis was achieved by adding 2% tween 20 to the erythrocytes. The plates were incubated for 1 h at 37 oC, followed by centrifugation for 10 min at 2000 rpm. An aliquot of 100 µL of the supernatant fluid was transferred to a new low-affinity 96-well plate, and the absorbance was measured at 450 nm. The percentage hemolysis was calculated as follows :[(A450 of compound treated erythrocytes-A450 of negative control)/ A450 of tween-20 treated erythrocytes-A450 of negative control] × 100%. Each treatment was replicated six times. Differences in treatments were statistically analyzed by pairwise T-tests between two different compounds at the same concentration. The levels of significance were set at P < 0.05. Supporting Information Tables list PCR primers, GS-MS data for mycelial fatty acids, statistical tests, and display validation of genetic constructs, LC-MS chromatograms of major products, antifungal assays, and annotated NMR spectra. This material is available free of charge via the Internet at http:_________________.

Acknowledgements This work was supported by the University of Texas Health Science Center at Houston faculty startup fund and a Kay and Ben Fortson Professorship to G.F.B., the National Natural Science Foundation of 13 ACS Paragon Plus Environment


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China (NSFC) grant 31328001 to Z.A., the Welch Foundation Grant AU00024 to Z.A., the University of Texas System Star Award to Z.A., and NIGMS R01GM115729 to K.Y. A.L. was supported by NIH T32AI052080. We thank J. Tkacz for helpful comments on the manuscript.


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References 1.

Jiang, W.; Cacho, R. A.; Chiou, G.; Garg, N. K.; Tang, Y.; Walsh, C. T., EcdGHK are three

tailoring iron oxygenases for amino acid building blocks of the echinocandin scaffold. J Am Chem Soc 2013, 135, 4457-66. 2.

Cacho, R. A.; Jiang, W.; Chooi, Y. H.; Walsh, C. T.; Tang, Y., Identification and

characterization of the echinocandin B biosynthetic gene cluster from Emericella rugulosa NRRL 11440. J. Am. Chem. Soc. 2012, 134, 16781-90. 3.

Houwaart, S.; Youssar, L.; Hüttel, W., Pneumocandin biosynthesis: Involvement of a trans-

selective proline hydroxylase. ChemBioChem 2014, 15, 2365-2369. 4.

Li, Y.; Chen, L.; Yue, Q.; Liu, X.; An, Z.; Bills, G. F., Genetic manipulation of the

pneumocandin biosynthetic pathway for generation of analogues and evaluation of their antifungal activity. ACS Chem. Biol. 2015, 10, 1702–1710. 5.

Chen, L.; Yue, Q.; Li, Y.; Niu, X.; Xiang, M.; Wang, W.; Bills, G. F.; Liu, X.; An, Z.,

Engineering of Glarea lozoyensis for the exclusive production of the pneumocandin B0 precursor of the antifungal drug caspofungin acetate. Appl. Environ. Microbiol. 2015, 81, 1550-1558. 6.

Chen, L.; Yue, Q.; Zhang, X.; Xiang, M.; Wang, C.; Li, S.; Che, Y.; Ortiz-Lopez, F. J.; Bills,

G. F.; Liu, X.; An, Z., Genomics-driven discovery of the pneumocandin biosynthetic gene cluster in the fungus Glarea lozoyensis. BMC Genom. 2013, 14, 339. 7.

Debono, M.; Turner, W. W.; LaGrandeur, L.; Burkhardt, F. J.; Nissen, J. S.; Nichols, K. K.;

Rodriguez, M. J.; Zweifel, M. J.; Zeckner, D. J., Semisynthetic chemical modification of the antifungal lipopeptide echinocandin B (ECB): structure-activity studies of the lipophilic and geometric parameters of polyarylated acyl analogs of ECB. J Med Chem 1995, 38, 3271-3281. 8.

Abbott, B. J.; Fukuda, D. S. S 31794/F-1 Nucleus. U.S. Patent 4,304,716, 1981.

9.

Yue, Q.; Chen, L.; Zhang, X.; Li, K.; Sun, J.; Liu, X.; An, Z.; Bills, G. F., Evolution of

chemical diversity in the echinocandin lipopeptide antifungal metabolites. Euk. Cell. 2015, 14, 698718. 10.

Hashimoto, S., Micafungin: A sulfated echinocandin. J. Antibiotics 2009, 62, 27-35.

11.

Balkovec, J. M.; Hughes, D. L.; Masurekar, P.; Sable, C. A.; Schwartz, R. A.; Singh, S. B.,

Discovery and development of first in class antifungal caspofungin (Cancidas). A case study. Nat. Prod. Rep. 2013, 31, 15-34. 12.

Norris, T.; VanAlsten, J.; Hubbs, S.; Ewing, M.; Cai, W.; Jorgensen, M. L.; Bordner, J.;

Jensen, G. O., Commercialization and late-stage development of a semisynthetic antifungal API: Anidulafungin/d-fructose (Eraxis). Org. Proc. Res. Develop. 2008, 12, 447-455. 13.

Chooi, Y.-H.; Tang, Y., Adding the lipo to lipopeptides: do more with less. Chem Biol 2010,

17, 791-793.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

14.

Page 16 of 27

Chiang, Y. M.; Szewczyk, E.; Nayak, T.; Davidson, A. D.; Sanchez, J. F.; Lo, H. C.; Ho, W.

Y.; Simityan, H.; Kuo, E.; Praseuth, A., Molecular Genetic Mining of the Aspergillus Secondary Metabolome: Discovery of the Emericellamide Biosynthetic Pathway. Chem Biol 2008, 15, 527-532. 15.

Tkacz, J. S.; Giacobbe, R. A.; Monaghan, R. L., Improvement in the titer of echinocandin-

type antibiotics: A magnesium-limited medium supporting the biphasic production of pneumocandins A0 and B0. J. Ind. Microbiol. 1993, 11, 95-103. 16.

Connors, N.; Pollard, D., Pneumocandin B0 production by fermentation of the fungus Glarea

lozoyensis: Physiological and engineering factors affecting titer and structual analogue formation. In Handbook of Industrial Mycology, An, Z., Ed. Marcel Dekker: New York, 2005; pp 515-538. 17.

Buckingham, J., Dictionary of Natural Products on DVD. CRC Press: Boca Raton, USA,

2011. 18.

Stahl, P. D.; Klug, M. J., Characterization and differentiation of filamentous fungi based on

fatty acid composition. Appl. Environ. Microbiol. 1996, 62, 4136-4146. 19.

Dreyfuss, M. M.; Tscherter, H. Antibiotic S 31794/F-1. U.S. Patent 4,173,629, 1979.

20.

Dreyfuss, M. M., Neue Erkenntnisse aus einem pharmakologischen Pilz-screening. Sydowia

1986, 39, 22-36. 21.

Xu, Y.; Zhou, T.; Zhang, S.; Xuan, L.-J.; Zhan, J.; Molnár, I., Thioesterase domains of fungal

nonreducing polyketide synthases act as decision gates during combinatorial biosynthesis. J. Am. Chem. Soc. 2013, 135, 10783-10791. 22.

Kotowska, M.; Pawlik, K., Roles of type II thioesterases and their application for secondary

metabolite yield improvement. Appl. Microbiol. Biotechnol. 2014, 98, 7735-7746. 23.

Xu, W.; Chooi, Y.-H.; Choi, J. W.; Li, S.; Vederas, J. C.; Da Silva, N. A.; Tang, Y., LovG:

The thioesterase required for dihydromonacolin L release and lovastatin nonaketide synthase turnover in lovastatin biosynthesis. Angewandte Chemie International Edition 2013, 52, 6472-6475. 24.

Butler, A. R.; Bate, N.; Cundliffe, E., Impact of thioesterase activity on tylosin biosynthesis in

Streptomyces fradiae. Chem. Biol. 1999, 6, 287-292. 25.

Doi-Katayama, Y.; Yoon, Y. J.; Choi, C. Y.; Yu, T. W.; Floss, H. G.; Hutchinson, C. R.,

Thioesterases and the premature termination of polyketide chain elongation in rifamycin B biosynthesis by Amycolatopsis mediterranei S699. J. Antibiotics 2000, 53, 484-495. 26.

Yeh, E.; Kohli, R. M.; Bruner, S. D.; Walsh, C. T., Type II thioesterase restores activity of a

NRPS module stalled with an aminoacyl-S-enzyme that cannot be elongated. ChemBioChem 2004, 5, 1290-1293. 27.

Schwarzer, D.; Mootz, H. D.; Linne, U.; Marahiel, M. A., Regeneration of misprimed

nonribosomal peptide synthetases by type II thioesterases. Proc. Nat. Acad. Sci. USA 2002, 99, 14083-14088.


Page 17 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


28.

Wang, G.; Liu, Z.; Lin, R.; Li, E.; Mao, Z.; Ling, J.; Yang, Y.; Yin, W.-B.; Xie, B.,

Biosynthesis of antibiotic leucinostatins in bio-control fungus Purpureocillium lilacinum and their inhibition on Phytophthora revealed by genome mining. PLoS Path. 2016, 12, e1005685. 29.

Singh, S. B.; Herath, K.; Kahn, J. N.; Mann, P.; Abruzzo, G.; Motyl, M., Synthesis and

antifungal evaluation of pentyloxyl-diphenylisoxazoloyl pneumocandins and echinocandins. Biorg. Med. Chem. Lett. 2013, 23, 3253-3256. 30.

Balkovec, J. M.; Black, R. M.; Abruzzo, G. K.; Bartizal, K.; Dreikorn, S.; Karl, N.,

Pneumocandin antifungal lipopeptides. The phenolic hydroxyl is required for 1,3-β-glucan synthesis inhibition. Biorg. Med. Chem. Lett. 1993, 3, 2039-2042. 31.

Shirey, K.; Stover, K. R.; Cleary, J.; Hoang, N.; Hosler, J., Membrane-anchored cyclic

peptides as effectors of mitochondrial oxidative phosphorylation. Biochem. 2016, 55, 2100-2111. 32.

Betts, R. F.; Nucci, M.; Talwar, D.; Gareca, M.; Queiroz-Telles, F.; Bedimo, R. J.; Herbrecht,

R.; Ruiz-Palacios, G.; Young, J.-A. H.; Baddley, J. W.; Strohmaier, K. M.; Tucker, K. A.; Taylor, A. F.; Kartsonis, N. A.; Group, f. t. C. H.-D. S., A multicenter, double-blind trial of a high-dose caspofungin treatment regimen versus a standard caspofungin treatment regimen for adult patients with invasive candidiasis. Clin. Infect. Dis. 2009, 48, 1676-1684. 33.

Wang, B.; Kang, Q.; Lu, Y.; Bai, L.; Wang, C., Unveiling the biosynthetic puzzle of

destruxins in Metarhizium species. Proc. Nat. Acad. Sci. USA 2012, 109, 1287-1292. 34.

Ortíz-López, F. J.; Monteiro, M. C.; González-Menéndez, V.; Tormo, J. R.; Genilloud, O.;

Bills, G. F.; Vicente, F.; Zhang, C.; Roemer, T.; Singh, S. B.; Reyes, F., Cyclic colisporifungin and linear cavinafungins, antifungal lipopeptides Isolated from Colispora cavincola. J. Nat. Prod. 2015, 78, 468-475. 35.

Chiang, Y. M.; Szewczyk, E.; Nayak, T.; Davidson, A. D.; Sanchez, J. F.; Lo, H. C.; Ho, W.

Y.; Simityan, H.; Kuo, E.; Praseuth, A.; Watanabe, K.; Oakley, B. R.; Wang, C. C. C., Molecular genetic mining of the Aspergillus secondary metabolome: Discovery of the emericellamide biosynthetic pathway. Chem. Biol. 2008, 15, 527-532. 36.

Fujie, A.; Iwamoto, T.; Muramatsu, H.; Okudaira, T.; Nitta, K.; Nakanishi, T.; Sakamoto, K.;

Hori, Y.; Hino, M.; Hashimoto, S.; Okuhara, M., FR901469, a novel antifungal antibiotic from an unidentified fungus No.11243. I. Taxonomy, fermentation, isolation, physico-chemical properties and biological properties. J. Antibiotics 2000, 53, 912-919. 37.

Jin, J. M.; Lee, S.; Lee, J.; Baek, S. R.; Kim, J. C.; Yun, S. H.; Park, S. Y.; Kang, S.; Lee, Y.

W., Functional characterization and manipulation of the apicidin biosynthetic pathway in Fusarium semitectum. Molec. Microbiol. 2010, 76, 456-466. 38.

Sørensen, J. L.; Sondergaard, T. E.; Covarelli, L.; Fuertes, P. R.; Hansen, F. T.; Frandsen, R. J.

N.; Saei, W.; Lukassen, M. B.; Wimmer, R.; Nielsen, K. F.; Gardiner, D. M.; Giese, H., Identification of the biosynthetic gene clusters for the lipopeptides fusaristatin A and W493 B in Fusarium graminearum and F. pseudograminearum. J. Nat. Prod. 2014, 77, 2619-2625. 17 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

39.

Page 18 of 27

Lukassen, M.; Saei, W.; Sondergaard, T.; Tamminen, A.; Kumar, A.; Kempken, F.; Wiebe,

M.; Sørensen, J., Identification of the scopularide biosynthetic gene cluster in Scopulariopsis brevicaulis. Mar. Drugs 2015, 13, 4331. 40.

Zhang, A.; Lu, P.; Dahl-Roshak, A. M.; Paress, P. S.; Kennedy, S.; Tkacz, J. S.; An, Z.,

Efficient disruption of a polyketide synthase gene (pks1) required for melanin synthesis through Agrobacterium-mediated transformation of Glarea lozoyensis. Mol. Gen. Genom. 2003, 268, 645-655. 41.

Goldstein, A. L.; McCusker, J. H., Three new dominant drug resistance cassettes for gene

disruption in Saccharomyces cerevisiae. Yeast 1999, 15, 1541-1553. 42.

Arikan, S.; Lozano-Chiu, M.; Paetznick, V.; Rex, J. H., In vitro susceptibility testing methods

for caspofungin against Aspergillus and Fusarium isolates. Antimicrob. Agents Chemother. 2001, 45, 327-330. 43.

Park, S.; Kelly, R.; Kahn, J. N.; Robles, J.; Hsu, M. J.; Register, E.; Li, W.; Vyas, V.; Fan, H.;

Abruzzo, G.; Flattery, A.; Gill, C.; Chrebet, G.; Parent, S. A.; Kurtz, M.; Teppler, H.; Douglas, C. M.; Perlin, D. S., Specific substitutions in the echinocandin target Fks1p account for reduced susceptibility of rare laboratory and clinical Candida sp. isolates. Antimicrob. Agents Chemother. 2005, 49, 3264-73.


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Table 1. In vitro antifungal and glucan synthase activity of new pneumocandin congeners (MIC, µg mL-1; IC50, µg mL-1). Glucan synthase IC50

Fungal pathogen MIC, MEC, or IC50

Compounds

Candida albicans ATCC 10231

Candida Candida albicans albicans ATCC MDACC1a 90028

Candida tropicalis ATCC 750

Candida Candida glabrata glabrata ATCC MDACC1a 2001

Candida parapsilosis ATCC 90018

Aspergillus C. albicans fumigatus ATCC 10231 FGSC A1240*

1

0.8

1.6

25.6

1.6

3.2

25.6

12.8

6.4

3.43±1.09

2

0.8

0.8

25.6

1.6

1.6

25.6

12.8

6.4

3.68±0.6

3

0.8

1.6

25.6

1.6

3.2

25.6

25.6

6.4

9.05±1.53

4

0.8

0.8

25.6

0.8

1.6

25.6

25.6

6.4

4.57±0.82

5

0.1

0.1

12.8

0.2

0.1

12.8

12.8

3.2

5.67±1.06

6

1.6

1.6

25.6

3.2

3.2

25.6

25.6

12.8

4.26±0.9

7

0.2

0.2

6.4

0.2

0.1

6.4

12.8

3.2

4.99±0.82

Caspofungin 0.2

0.2

3.2

0.1

Engineering of New Pneumocandin Side-Chain ... - ACS Publications

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