Oxandrastins: Antibacterial Meroterpenes from an Australian Mud Dauber Wasp Nest-Associated Fungus, Penicillium sp. CMB-MD14

The ethyl acetate extract of an ISP-2 agar cultivation of the wasp nest-associated fungus Penicillium sp. CMB-MD14 exhibited promising antibacterial activity against vancomycin-resistant enterococci (VRE), with a bioassay guided chemical investigation yielding the new meroterpene, oxandrastin A (1), the first andrastin-like metabolite with an extra oxygenation at C-2. A culture media optimisation strategy informed a scaled-up rice cultivation that yielded 1, together with three new oxandrastins B–D (2–4), two known andrastins C (5) and F (6), and a new meroterpene of the austalide family, isoaustalide F (7). Structures of 1–7 were assigned based on detailed spectroscopic analysis and chemical interconversion. A GNPS molecular networking analysis of the rice cultivation extract detected the known austalides B (8), H (9), and H acid (10), tentatively identified based on molecular formulae and co-clustering with 7. That the anti-VRE properties of the CMB-MD14 extract were exclusively attributed to 1 (IC50 6.0 µM, MIC99 13.9 µM), highlights the importance of the 2-OAc and 3-OAc moieties to the oxandrastin anti-VRE pharmacophore.


Introduction
The widespread emergence of antibiotic resistance is seriously undermining the capacity of modern antibiotics to protect against infectious disease. Antibiotic resistance is not just a problem for patients presenting with a primary infection, as secondary infections also drive high levels of morbidity and mortality. For example, patients with genetic, cardiovascular, and/or respiratory diseases, or with compromised immune systems, or even post-operative patients, are at risk from secondary infection. The magnitude of the antibiotic crisis is further exacerbated by the fact that most pharmaceutical companies have exited the antibiotic space, leaving an under resourced and depleted drug development pipeline desperately in need of innovation and renewal. As a result, current infection control is hostage to the residual efficacy of a handful of resistance compromised antibiotics.
For the latter half of the last century, science and industry successfully exploited microbial chemical defences, fuelling a knowledge revolution that inspired new pharmaceuticals and agrochemicals, driving global commerce, and improving quality of life. This led to some of the most widely recognised and successful antibiotic classes, including the penicillins, erythromycins, cephalosporins, tetracyclines, streptomycins, vancomycins, and rifampicins. Notwithstanding this success, when confronted by lower returns on investment and the perception of a near exhausted microbial resource, late last century industry turned elsewhere for inspiration. Two decades on, and confronted by surging antibiotic resistance, and a community demand for better, safer, and more effective antibiotics, the need for inspiration is more urgent than ever.
As part of our ongoing investigations into the chemistry of Australian fungi, we assembled a library of fungi co-isolated from a mud dauber wasp (×15), and a mud dauber wasp nest (×21), both sourced from an urban location in Pullenvale, Queensland, Australia. Collectively, this wasp-derived fungal library proved to be an excellent source of novel natural products with Aspergillus sp. CMB-W031, yielding an unprecedented nitro depsipeptide diketopiperazine (waspergillamide A) [1]. Talaromyces sp. CMB-W045 yielding new non-cytotoxic siderophores (talarazines A-E) [2], a new a p-terphenyl (talarophenol sulfate), and polyketides (talarophilones) [3]. Penicillium sp. CMB-MD22 yielded an extensive family of stereo-complex and selectively antifungal neobulgarone bianthrones [4]. Turning our attention to the need for new antibiotic classes, and prompted by promising antibacterial activity against vancomycin-resistant enterococci (VRE), this current report describes an investigation into the antibacterial natural products of the mud dauber wasp nest-derived Penicillium sp. CMB-MD14. Bioassay guided fractionation of a scaled-up ISP-2 agar cultivation yielded the new anti-VRE meroterpene 1, closely related to the known antibacterial inactive fungal meroterpene andrastins [5,6] as well as the atlantinones [7,8], citreohybridones, isocitreohybridones and citreohybriddiones [9,10]. The first andrastin-like metabolite to be reported with C-2 oxygenation, 1 was assigned the trivial name oxandrastin A. To better explore the anti-VRE oxandrastin pharmacophore a media optimisation strategy was employed, with a scaled-up rice cultivation of CMB-MD14 yielding oxandrastin A (1), as well as three new oxandrastins B-D (2-4), two known andrastins C (5), F (6) [5,6] and a new meroterpene of the austalide family, isoaustalide F (7) (Figure 1). A GNPS molecular networking analysis of the unfractionated CMB-MD14 rice cultivation extract confirmed separate clusters for oxandrastins/andrastins and isoaustalides/austalides ( Figure 2), with the latter including nodes tentatively attributed to the known austalides B (8) [11,12], H (9) [13] and H acid (10) [14]. Subsequent UPLC-MS analysis with single ion extraction (SIE) detected 1-10 in the fresh extract, which together with GNPS analyses confirmed their natural product status. Structures were assigned 1-7 based on detailed spectroscopic analysis and chemical interconversion, and tentatively to 8-10 on the basis of molecular formulae and GNPS co-clustering with 7. An account of these structure determinations together with commentary on anti-VRE structure activity and biosynthetic relationships is outlined below.

General Experimental Procedures
Chiroptical measurements ([α] D ) were obtained on a JASCO P-1010 polarimeter (JASCO International Co. Ltd., Tokyo, Japan) in a 100 × 2 mm cell at specified temperatures. Electronic Circular Dichroism (ECD) measurement were obtained on a JASCO J-810 spectropolarimeter (JASCO International Co. Ltd., Tokyo, Japan) in a 0.1 cm pathlength cell. Nuclear magnetic resonance (NMR) spectra were acquired on a Bruker Avance 600 MHz spectrometer (Bruker Pty. Ltd., Alexandria, NSW, Australia) with either a 5 mm PASEL 1 H/D-13 C Z-Gradient probe or 5 mm CPTCI 1 H/ 19 F-13 C/ 15 N/DZ-Gradient cryoprobe. In all cases, spectra were acquired at 25 • C deuterated solvents as indicated, with referencing to residual 1 H or 13 C signals. High-resolution ESIMS spectra were obtained on a Bruker micrOTOF mass spectrometer (Bruker Daltonik Pty. Ltd., Preston, VIC, Australia) by direct injection in MeOH at 3 µL/min using sodium formate clusters as an internal calibrant. Liquid chromatography-diode array-mass spectrometry (HPLC-DAD-MS) data were acquired either on an Agilent 1260 series separation module equipped with an Ag-ilent G6125B series LC/MSD mass detector (Agilent Technologies Inc., Mulgrave, VIC, Australia) and diode array detector or on Shimadzu LCMS-2020 LCMS. Semi-preparative HPLCs were performed using Agilent 1100 series HPLC instruments (Agilent Technologies Inc., Mulgrave, VIC, Australia) with corresponding detectors, fraction collectors, and software inclusively. UPLC chromatograms were obtained on Agilent 1290 infinity UPLC system equipped with diode array multiple wavelength detector (Zorbax C 8 RRHD 1.8 µm, 50 × 2.1 mm column, 0.417 mL/min with a 2.50 min gradient from 90% H 2 O/MeCN to MeCN with a constant 0.01% TFA modifier). UPLC-QTOF analysis was performed on UPLC-QTOF instrument comprising an Agilent 1290 Infinity II UPLC (Zorbax C 8 RRHD 1.8 µm, 50 × 2.1 mm column, eluting at 0.417 mL/min with a 2.50 min gradient elution from 90% H 2 O/MeCN to 100% MeCN with a constant 0.1% formic acid modifier) coupled to an Agilent 6545 Q-TOF. MS/MS analysis was performed on the same instrument for ions detected in the full scan, at an intensity above 1000 counts at 10 scans/s, with an isolation width of 4~m/z using a fixed collision energy and a maximum of 3 selected precursors per cycle. Chemicals were purchased from Sigma-Aldrich or Merck unless otherwise specified. Analytical-grade solvents were used for solvent extractions. Chromatography solvents were of HPLC grade supplied by Labscan or Sigma-Aldrich and filtered/degassed through 0.45 µm polytetrafluoroethylene (PTFE) membrane prior to use. Deuterated solvents were purchased from Cambridge Isotopes. Microorganisms were manipulated under sterile conditions using a Laftech class II biological safety cabinet and incubated in either MMM Friocell incubators (Lomb Scientific, NSW, Australia) or an Innova 42R incubator shaker (John Morris, NSW, Australia).

Fungal Isolation
The fungus Penicillium sp. CMB-MD14 was isolated from a specimen of mud dauber wasp nests collected from an urban location in Pullenvale, Queensland, Australia, by cultivation on ISP-2 agar plate at 26.5 • C for 8 days.

Global Natural Product Social (GNPS) Molecular Networking
Aliquots (1 µL) of dried fraction (100 µg/mL in MeOH) were analysed on an Agilent 6545 Q-TOF LC/MS equipped with an Agilent 1290 Infinity II UPLC system, utilising an Agilent SB-C8 1.8 µm, 2.1 × 50 mm column, eluting with 90% H 2 O/MeCN to MeCN at a 0.417 mL/min over 2.5 min with an isocratic 0.1% formic acid modifier. UPLC-QTOF-(+)MS/MS data acquired for all samples at collision energy of 10, 20, and 40 eV were converted from Agilent MassHunter data files (.d) to mzXML file format using MSConvert software and transferred to the GNPS server (gnps.ucsd.edu). Molecular networking was performed using the GNPS data analysis workflow [22] using the spectral clustering algorithm with a cosine score of 0.6 and a minimum of 5 matched peaks. The resulting spectral network was imported into Cytoscape software (version 3.7.1) [23] and visualized using a ball-stick layout where nodes represent parent masses and the cosine score was reflected by edge thickness. Furthermore, group abundances were set as pie charts, which reflected the intensity of MS signals.

Extraction and Fractionation of a Rice Cultivation of CMB-MD14
A culture flask (2 L) containing medium grain rice (200 g) inoculated with the fungus CMB-MD14 was incubated at room temperature for 6 weeks, after which it was extracted with EtOAc (2 × 400 mL), and the combined organic phase filtered and concentrated in vacuo at 40 • C to yield the EtOAc extract (1.6 g). The EtOAc extract was subjected to solvent trituration (2 × 50 mL) to afford after concentration in vacuo n-hexane (850 mg) and MeOH (750 mg) solubles. A portion of the MeOH solubles (600 mg) was fractionated by preparative reversed phase HPLC (Phenomenex Luna-C 8

Antibiotic Assays
Antibacterial and antifungal assays were performed using prior published methods [24][25][26] as documented in the Supporting Information ( Figure S37).

Supplementary Materials:
The following are available online, Fungal taxonomy, NMR spectra and tabulated data for 1-7, bioassay methods and results, and GNPS analyses.