Ignacio Pérez-Victoria

Combined LC/UV/MS and NMR Strategies for the Dereplication of Marine Natural Products

Drug discovery from marine natural products has experienced a revival since the beginning of this century. To be successful in this field, rapid dereplication (identification of already known bioactive compounds) is essential in order to assess the chemical novelty of crude extracts and their fractions. Access to the appropriate state-of-the-art analytical instrumentation and to suitable databases is a fundamental requirement in such a task. A brief survey of the most robust LC/UV/MS- and NMR-based approaches employed for marine natural product dereplication is presented alongside a description of the procedures followed to achieve this goal in our research group.

Chemical and Physical Modulation of Antibiotic Activity in Emericella Species

The addition of epigenetic modifying agents and ion‐exchange resins to culture media and solid‐state fermentations have been promoted as ways to stimulate expression of latent biosynthetic gene clusters and to modulate secondary metabolite biosynthesis. We asked how combination of these treatments would affect a population of screening isolates and their patterns of antibiosis relative to fermentation controls. A set of 43 Emericella strains, representing 25 species and varieties, were grown on a nutrient‐rich medium comprising glucose, casein hydrolysate, urea, and mineral salts. Each strain was grown in untreated agitated liquid medium, a medium treated with suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, 5‐azacytidine, a DNA methylation inhibitor, an Amberlite non‐ionic polyacrylate resin, and the same medium incorporated into an inert static vermiculite matrix. Species‐inherent metabolic differences more strongly influenced patterns of antibiosis than medium treatments. The antibacterial siderophore, desferritriacetylfusigen (1), was detected in most species in liquid media, but not in the vermiculite medium. The predominant antifungal component detected was echinocandin B. Some species produced this antifungal regardless of treatment, although higher quantities were often produced in vermiculite. Several species are reported for the first time to produce echinocandin B (3). A new echinocandin analog, echinocandin E (2), was identified from E. quadrilineata.

New Ikarugamycin Derivatives with Antifungal and Antibacterial Properties from Streptomyces zhaozhouensis

A bioassay guided fractionation of the ethyl acetate extract from culture broths of the strain Streptomyces zhaozhouensis CA-185989 led to the isolation of three new polycyclic tetramic acid macrolactams (1–3) and four known compounds. All the new compounds were structurally related to the known Streptomyces metabolite ikarugamycin (4). Their structural elucidation was accomplished using a combination of electrospray-time of flight mass spectrometry (ESI-TOF MS) and 1D and 2D NMR analyses. Compounds 1–3 showed antifungal activity against Aspergillus fumigatus, Candida albicans and antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA).

MDN-0104, an Antiplasmodial Betaine Lipid from Heterospora chenopodii

Bioassay-guided fractionation of the crude fermentation extract of Heterospora chenopodii led to the isolation of a novel monoacylglyceryltrimethylhomoserine (1). The structure of this new betaine lipid was elucidated by detailed spectroscopic analysis using one- and two-dimensional NMR experiments and high-resolution mass spectrometry. Compound 1 displayed moderate in vitro antimalarial activity against Plasmodium falciparum, with an IC50 value of 7 μM. This betaine lipid is the first monoacylglyceryltrimethylhomoserine ever reported in the Fungi, and its acyl moiety also represents a novel natural 3-keto fatty acid. The new compound was isolated during a drug discovery program aimed at the identification of new antimalarial leads from a natural product library of microbial extracts. Interestingly, the related fungus Heterospora dimorphospora was also found to produce compound 1, suggesting that species of this genus may be a promising source of monoacylglyceryltrimethylhomoserines.

Multicomponent Analysis of the Differential Induction of Secondary Metabolite Profiles in Fungal Endophytes

Small molecule histone deacetylase (HDAC) and DNA methyltransferase (DNMT) inhibitors are commonly used to perturb the production of fungal metabolites leading to the induction of the expression of silent biosynthetic pathways. Several reports have described the variable effects observed in natural product profiles in fungi treated with HDAC and DNMT inhibitors, such as enhanced chemical diversity and/or the induction of new molecules previously unknown to be produced by the strain. Fungal endophytes are known to produce a wide variety of secondary metabolites (SMs) involved in their adaptation and survival within higher plants. The plant-microbe interaction may influence the expression of some biosynthetic pathways, otherwise cryptic in these fungi when grown in vitro. The aim of this study was to setup a systematic approach to evaluate and identify the possible effects of HDAC and DNMT inhibitors on the metabolic profiles of wild type fungal endophytes, including the chemical identification and characterization of the most significant SMs induced by these epigenetic modifiers.

Isolation and structural elucidation of cyclic tetrapeptides from Onychocola sclerotica

Three new cyclic tetrapeptides (1-3) have been isolated from the crude fermentation extract of Onychocola sclerotica. The planar structures of 1-3 were elucidated by detailed spectroscopic analyses using one- and two-dimensional NMR experiments and high-resolution mass spectrometry. The absolute configuration of the amino acid residues in each cyclotetrapeptide was established by Marfeys method. Compounds 1-3 displayed activity as cardiac calcium channel blockers (Cav1.2) but did not inhibit the hERG potassium channel and were not cytotoxic. These peptides are the first secondary metabolites ever reported from fungi of the order Arachnomycetales.

Hitting the Caspofungin Salvage Pathway of Human-Pathogenic Fungi with the Novel Lasso Peptide Humidimycin (MDN-0010)

ABSTRACT Fungal infections have increased dramatically in the last 2 decades, and fighting infectious diseases requires innovative approaches such as the combination of two drugs acting on different targets or even targeting a salvage pathway of one of the drugs. The fungal cell wall biosynthesis is inhibited by the clinically used antifungal drug caspofungin. This antifungal activity has been found to be potentiated by humidimycin, a new natural product identified from the screening of a collection of 20,000 microbial extracts, which has no major effect when used alone. An analysis of transcriptomes and selected Aspergillus fumigatus mutants indicated that humidimycin affects the high osmolarity glycerol response pathway. By combining humidimycin and caspofungin, a strong increase in caspofungin efficacy was achieved, demonstrating that targeting different signaling pathways provides an excellent basis to develop novel anti-infective strategies.

Graminin B, a furanone from the fungus Paraconiothyrium sp.

Members of the furanone structural class of natural oxygenated heterocycles have been reported from fungi of the genera Cephalosporium (for example, gregatins and graminin A), Aspergillus (huaspenone A and B or the unnamed tetronic acid derivatives from A. panamensis) and Penicillium (penicilliol).1–6 The structure initially proposed of 3-acyl-4-methoxyfuran-2(5H)-one was later revised to a structure of 4-acyl-5-methoxyfuran-3(2H)-one after the total synthesis of racemic and (þ )-gregatin B by Takaiwa and Yamashita.7,8 Very recently, a serendipitous synthesis of (þ )gregatin B9 and total syntheses of the gregatins A–D and aspertetronin A10 led to a second structural revision establishing a central core of 4-(methoxycarbonyl)furan-3(2H)-one as the right structure for the members of this family of secondary metabolites (Figure 1). As part of a continuing program to identify novel pharmaceutical lead structures from natural sources, Fundación MEDINA has been investigating new antibiotic agents from its proprietary library of over 100 000 strains of actinomycetes and fungi. The fermentation extract of a fungal strain was selected for further purification based on its antibiotic activity. A phylogenetic placement of this strain, based on its ITS/28S, identified the fungus as a species of Paraconiothyrium and closely related to Paraconiothyrium hawaiensis. Herein we report the isolation and structural elucidation of graminin B (1), a new furanone derivative obtained from fermentation broths of this species, and its activity against Escherichia coli and methicillin-resistant Staphylococcus aureus. Graminin B was isolated from acetone extracts of Paraconiothyrium sp. (CF-217411) by fractionation on SP207SS resin followed by repeated semi-preparative HPLC. The molecular formula of 1 was deduced to be C18H26O4 by accurate mass measurement (ESI-TOF, Supplementary Figure S4), requiring six degrees of unsaturation. The 13C NMR and 1H/13C HSQC spectroscopic analysis indicated the presence of 18 carbon resonances, including four resulting from methyl groups and four from sp2 methines, whereas further five signals resulting from methylene groups and five resonances were assigned to quaternary carbons (see Table 1 and Supplementary Figure S2). The 1H NMR (Supplementary Figure S1) and 1H/1H COSY spectra of 1 displayed two spin systems, one composed by aliphatic protons from H3-1 through H2-5, and another between the four olefinic protons (H-10 through H-13) and the aliphatic protons H2-14 and H3-15. The HMBC spectrum (Supplementary Figure S3) exhibited correlations from H2-5 to C-6 (dC 200.9) and C-7 (dC 107.0) (Figure 2), deduced to belong to a furanone ring. The methoxyl group H3-18, with a proton resonance in 1H NMR at d 3.81, displayed long-range heteronuclear correlations to the carbonyl C-17 (dC 164.3) of the esther group, confirming the presence of a methoxycarbonyl group in the molecule (Figure 1). The presence of a double bond between C-6 (dC 200.9) and C-7 (dC 107.0) in the furanone moiety was deduced on the basis of similar 13C NMR chemical shifts with other members of the same structural class.10 The methyl group H3-16 and the methine H-10 from the second spin system chain also showed key heteronuclear long-range correlations (Figure 2), to C-9 and to the ketone C-8 (dC 200.7), deduced to be part of the furanone moiety. An E geometry was assigned to the 10D double bond based on the existence of a coupling constant of 15.4 Hz between H-10 and H-11, whereas a coupling constant of 10.6 Hz between H-12 and H-13 secured a Z configuration for the 12D olefin. These data indicated that 1 has a structure similar to the structure of graminin A,3 the only differences between both compounds being the absence of 4D insaturation in the structure of 1 and the geometry of the 12D double bond. The trivial name graminin B is therefore proposed for compound 1. The absolute configuration at the only chiral center of the furanone ring was assumed to be R after comparison of the optical rotation value ([a]20 D 96) with those reported for the synthetic enantiomers ( )-gregatin A and (þ )aspertetronin A.10

Branimycins B and C, Antibiotics Produced by the Abyssal Actinobacterium Pseudonocardia carboxydivorans M-227

Two new antibiotics, branimycins B (2) and C (3), were produced by fermentation of the abyssal actinobacterium Pseudonocardia carboxydivorans M-227, isolated from deep seawater of the Avilés submarine Canyon. Their structures were elucidated by HRMS and NMR analyses. These compounds exhibit antibacterial activities against a panel of Gram-positive bacteria, including Corynebacterium urealyticum, Clostridium perfringens, and Micrococcus luteus, and against the Gram-negative bacterium Neisseria meningitidis. Additionally, branimycin B displayed moderate antibacterial activity against other Gram-negative bacteria such as Bacteroides fragilis, Haemophilus influenzae, and Escherichia coli, and branimycin C against the Gram-positive Enterococcus faecalis and methicillin-sensitive and methicillin-resistant Staphylococcus aureus.

Discovery of New Compounds Active against Plasmodium falciparum by High Throughput Screening of Microbial Natural Products

Due to the low structural diversity within the set of antimalarial drugs currently available in the clinic and the increasing number of cases of resistance, there is an urgent need to find new compounds with novel modes of action to treat the disease. Microbial natural products are characterized by their large diversity provided in terms of the chemical complexity of the compounds and the novelty of structures. Microbial natural products extracts have been underexplored in the search for new antiparasitic drugs and even more so in the discovery of new antimalarials. Our objective was to find new druggable natural products with antimalarial properties from the MEDINA natural products collection, one of the largest natural product libraries harboring more than 130,000 microbial extracts. In this work, we describe the optimization process and the results of a phenotypic high throughput screen (HTS) based on measurements of Plasmodium lactate dehydrogenase. A subset of more than 20,000 extracts from the MEDINA microbial products collection has been explored, leading to the discovery of 3 new compounds with antimalarial activity. In addition, we report on the novel antiplasmodial activity of 4 previously described natural products.