Wychimicins, a new class of spirotetronate polyketides from Actinocrispum wychmicini MI503-A4

In the course of our screening program for new anti-methicillin-resistant Staphylococcus aureus antibiotics, four novel antibiotics, termed wychimicins A–D, were isolated from the culture broth of the rare actinomycete Actinocrispum wychmicini strain MI503-AF4. Wychimicins are spirotetronates possessing a macrocyclic 13-membered ring containing trans-decalin and β-d-xylo-hexopyranose moieties connected to C-17 by an O-glycosidic linkage according to MS, NMR and X-ray analyses. In X-ray crystal structure analysis, the Flack constant was 0.10 (11). The stereochemistry of the spirocarbon C-25 was R. Wychimicins had a minimum inhibitory concentration of 0.125–2 µg ml−1 against methicillin-resistant Staphylococcus aureus.


Introduction
Natural products are considered to have pharmaceutical potential because of their pharmacophore-like structures and unique chemical spaces. According to Newman et al., natural products accounted for~33% of all new drugs launched between 1980 and 2014, and~32% of new drugs were developed with natural products as the lead compounds. Therefore, it is considered that 65% of drugs are derived from natural products [1]. In the field of infectious diseases in particular, natural products account for a large proportion of pharmaceuticals, and thus, it is expected that natural products can play an active role in development of treatments to overcome antimicrobial resistance (AMR).
Staphylococcus aureus is one of the most widespread bacterial pathogenic causes of nosocomial infections, causing both serious invasive infections and simple skin infections [2,3]. It is a major cause of pneumonia and other respiratory tract infections, surgical-site infections, septic arthritis, cardiovascular infections, skin and soft tissue infections, and bacteremia [4]. S. aureus infections are particularly problematic because of the frequent development of AMR, with methicillin-resistant Staphylococcus aureus (MRSA) being the most clinically important antibiotic-resistant isolate [5]. MRSA has the highest mortality rate among all antibiotic-resistant isolates, being responsible for~20,000 deaths in 2018 according to the CDC [6]. MRSA, a nosocomial drug-resistant S. aureus isolate, was first reported in 1961, the year after methicillin was launched [7]. Nosocomial MRSA has since spread rapidly and become a major cause of infection in immunocompromised inpatients, affecting medical facilities globally. During the recent SARS-CoV-2 pandemic, many patients requiring invasive mechanical ventilation were admitted to the intensive care unit (ICU) because of severe SARS-CoV-2-related respiratory failure. Increased entry into the ICU and the increased use of invasive mechanical ventilation have been reported to increase the risk of mechanical ventilation-related pneumonia (VAP) and bloodstream infection (BSI). In addition, treatment with tocilizumab and dexamethasone poses a potential risk of secondary or co-bacterial infections. This is associated with high mortality rates and medical costs and long ICU stays. S. aureus, particularly MRSA, is frequently involved in the etiology of VAP and BSI, thereby making treatment more difficult [8][9][10][11].
Vancomycin has been considered the antibiotic of last resort for MRSA infections. However, MRSA that has acquired resistance to vancomycin has been detected. In particular, vancomycin-intermediate-resistant S. aureus (VISA) strains are important because of their spread in many medical facilities around the world [12]. MRSA is also problematic because of the frequent acquisition of antibiotic resistance, and the strain has already acquired resistance to effective antibacterial agents launched in the 21st century. Therefore, the development of new antibacterial agents effective against multidrug-resistant MRSA strains such as VISA/vancomycin-resistant S. aureus (VRSA) is desired.
The majority of the scaffolding structures that comprise antibiotics were obtained from microbial metabolites. It is believed that 99.9% of naturally occurring microorganisms have not yet been discovered, and many naturally occurring microorganisms provide scaffolding structures for new antibiotics. Even microorganisms in soil, which are often studied as sources of antibiotic isolation, represent vast reserves of chemical diversity [13][14][15][16].
We consider newly discovered microorganisms to be important sources of bioactive natural products in the search for interesting molecules and new therapeutic agents. As part of our efforts to overcome AMR, we conducted exploratory research on antibiotics with a novel skeleton and efficacy against MRSA. As a screening source, actinomycetes isolated from soil were used for molecular phylogenetic analyses of bacteria using a phenotype and 16 S rDNA to eliminate similar microorganisms and collect more novel microorganisms. As a result, Actinocrispum, a novel genus containing a rare actinomycete, was reported [17]. In this report, we discovered new 13-membered macrocyclic spirotetronates with a trans-decalin glycoside moiety termed wychimicins A-D ( Fig. 1) exhibiting strong antibacterial activity against MRSA strains including VRSA in the culture broth of A. wychmicini strain MI503-A4 collected from soil in Kuroishi-shi, Aomori prefecture, Japan [17]. This paper describes the isolation, structure determination, and biological properties of these compounds.

Results and discussion
Fermentation and isolation of wychimicins A, B, C and D A slant culture of MI503-A4 was inoculated into a 500 ml baffled Erlenmeyer flask containing 110 ml of medium consisting of galactose, dextrin, Bacto Soytone, corn steep liquor, glycerol, (NH 4 ) 2 SO 4 , and CaCO 3 . The culture (5 l) was incubated on a rotary shaker (200 rpm) at 30°C for 7 days. Active compounds were isolated from the extracts of mycelium cakes with MeOH and ethyl acetate followed by fermentation broth with ethyl acetate. The organic layer was purified by silica gel column chromatography and eluted with ethyl acetate:methanol:formic acid (100:0:0, 40:1:0.1, 36:4:0.1, and 40:10:0.125) via reversed-phase octadecylsilyl silica gel column chromatography using a Sephadex LH20 column. The active fractions were further purified via reversed-phase octadecylsilyl silica gel HPLC to obtain four novel compounds, which were termed wychimicins A (82.8 mg), B (47.0 mg), C (62.8 mg), and D (35.5 mg).

Structure elucidation of wychimicins A-D
The physicochemical properties of these compounds are summarized in the supplementary information (S-1). Wychimicins were brown solid compounds. The UV spectra of wychimicins revealed absorption maxima at 281-283 nm in acidic methanol and bathochromic shifts to 317-327 nm in alkaline conditions, as presented in Table S1. The molecular formulae of wychimicins A (1), B (2), C (3), and D (4) were C 47 H 60 ClNO 11 , C 47 H 61 ClNO 11 , C 46 H 58 ClNO 11 , and C 46 H 59 NO 11 , respectively, as determined by HRESI-MS and NMR spectra. 1 H and 13 C NMR data are summarized in Tables S2-5.

Absolute structure of wychimicins
Because the complete structure of wychimicins including their stereochemistry could not be determined by NMR, X-ray crystallographic analysis was attempted. Crystallization of wychimicins A-D was performed in various solvents. Only 4 was able to obtain a single crystal in ethyl acetate/methanol, which was used to perform a single-crystal X-ray diffraction study. The Flack parameter of 0. 10 (11) permitted definition of the configuration as 4S, 5R, 6S, 8S, 9R, 10S, 13R, 17R, 20S, 23S, 25R, 1'R, 3'S, 4'S, 5'R ( Fig. 3a, b).
According to physicochemical analyses, including CD spectra (Figs. S29-33), and NMR studies, wychimicins have the same aglycone and sugar moiety. Only the phenyl moieties differ in structure. These results allowed the absolute configuration of wychimicins A-D to be determined in Fig. 1.

Biological activities
The minimum inhibitory concentrations of wychimicins were determined by the agar dilution method. All compounds exhibited excellent antimicrobial activities against gram-positive bacteria such as S. aureus (including MRSA) and Enterococcus faecalis/faecium (including VRE). However, only 1 and 3 exhibited strong antibacterial activity against S. aureus and E. faecalis/faecium, and lower activity were noted for 2 and 4. This result indicated that the presence of Cl at the 4″ position in the benzoic acid moiety enhances antibacterial activity.

General
The optical rotations of the purified compounds were measured using a P-1030 polarimeter (JASCO, Tokyo, Japan). The UV spectra were recorded using a U-2800 UV-Vis spectrophotometer (Hitachi High-Technologies, Tokyo, Japan). The IR spectra were recorded using an FT/IR-4100 Fourier transform infrared spectrometer (JASCO). CD spectra were obtained using a J-1500 Circular Dichroism spectrometer (JASCO). The 1 H and 13 C NMR spectra were measured by an ECZ600R spectrometer (JEOL RESO-NANCE, Tokyo, Japan) at 25°C using tetramethylsilane as an internal reference. The mass spectra were recorded using an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) or an ACQUITY QDa mass detector (Waters, Milford, MA, USA).