Genus Nocardiopsis: A Prolific Producer of Natural Products

Actinomycetes are currently one of the major sources of bioactive secondary metabolites used for medicine development. Accumulating evidence has shown that Nocardiopsis, a key class of actinomycetes, has the ability to produce novel bioactive natural products. This review covers the sources, distribution, bioactivities, biosynthesis, and structural characteristics of compounds isolated from Nocardiopsis in the period between March 2018 and 2021. Our results reveal that 67% of Nocardiopsis-derived natural products are reported for the first time, and 73% of them are isolated from marine Nocardiopsis. The chemical structures of the Nocardiopsis-derived compounds have diverse skeletons, concentrating on the categories of polyketides, peptides, terphenyls, and alkaloids. Almost 50% of the natural products isolated from Nocardiopsis have been discovered to display various bioactivities. These results fully demonstrate the great potential of the genus Nocardiopsis to produce novel bioactive secondary metabolites that may serve as a structural foundation for the development of novel drugs.


Introduction
Actinomycetes belong to Gram-positive bacteria and are one of the biggest bacterial phyla [1,2]. The high G+C DNA content of actinomycetes implied their enormous biosynthetic potential to produce various natural products with diverse structures and important commercial applications [1,2]. Two-thirds of all naturally derived antibiotics have been discovered from actinobacteria [3,4]. Approximately 70% of the pharmaceutically active natural products which are currently used in clinics are isolated from actinobacteria [5][6][7][8], including a series of anticancer, antifungal, antibacterial, antihelminthic, and immunosuppressive drugs [3].
Bennur et al. reviewed the bioactive natural products derived from Nocardiopsis prior to 2015 [12], while Ibrahim et al. conducted a literature review regarding all of the secondary metabolites discovered from Nocardiopsis from 2016 to February 2018 [5]. In this review, the sources, distribution, bioactivities, biosynthesis, and structural characteristics of the compounds isolated from Nocardiopsis between March 2018 and 2021 are comprehensively summarized.

Polyketides
Three new angucyclines, nocardiopsistins A-C (1-3), were obtained from the deep-sea sponge-derived Nocardiopsis sp. strain HB-J378 ( Figure 1) [48]. The antibacterial activities of compounds 1-3 were tested against MRSA (methicillin-resistant Staphylococcus aureus) and 1-3 exhibited antibacterial activity with MIC values of 3.12-12.5 µg/mL. Three core genes were identified through bioinformatic analysis of the sketch genome of the strain HB-J378 in a biosynthetic gene cluster encoding a typical aromatic or type II polyketide synthase (PKS) system, including acyl carrier protein (ACP), ketoacyl: ACP synthase α-subunit (KS α ) and β-subunit (KS β ). The brief biosynthetic route for 1-3 was proposed according to the discovered oviedomycin pathway [49]. Compounds 1-3 were supposed to be biosynthesized by taking advantage of one molecule of isobutyral-CoA and nine molecules of malonyl-CoA, through complex enzymatic reactions to obtain the key angucycline biosynthetic intermediate UWM6, an analog of 1-3 [50], to acquire 1-3 (see Figure 1) [48]. The new strain Nocardiopsis sp. CG3 (DSM 106572), collected from the saltpan of Kenadsa (22 km west of Bechar, located in southwest Algeria), was discovered to have the ability to produce new bioactive natural products in a screening program [51]. Chemical investigation of the strain led to the isolation of five new polyene macrolactams, kenalactams A−E (4−8) (Figure 2) [51]. The biosynthetic pathway of polyketide kenalactam A (4) was studied by feeding experiments and was found to used L-alanine as the nitrogen-bearing starter unit. Compounds 6−8 exhibited cytotoxic activity against HeLa (cervical cancer cells KB3.1) and PC-3 (human prostate cancer) cell lines, with IC 50 values ranging from 2.1 to 6.8 µM [51]. Eight new α-pyrone derivatives, nocahypyrones A-H (9-16), along with one known analog, germicidin G (17) (Figure 3) were isolated from the strains Nocardiopsis sp. HDN154-146 and HDN154-168, which were collected from soil samples derived from the Takla Makan desert area in Xinjiang Province, China. This was the first time it was reported that compounds 13 and 16 showed cytoprotective activity by inducing the expression of phase II detoxifying enzymes [52]. Aldo-keto reductase family1 member C1 (AKR1C1), human NAD(P)H: quinone oxidoreductase 1 (NQO1), superoxide dismutase 2 (SOD2), and heme oxygenase 1(HO-1), belonging to phase II detoxifying enzymes, have been illustrated to possess significant roles in defending mammalian cells against oxidative damage and excessive inflammatory reaction [53,54]. Compounds 13 and 16 displayed cytoprotective activity by inducing the expression level of SOD2 and HO-1 in HaCaT cells [52].
Three new macrolides, borrelidins C−E, along with one known analog, borrelidin, which showed antibacterial and anticancer activities, were isolated from a saltern-derived halophilic Nocardiopsis sp. in 2017 [43]. These borrelidins were found to have the ability to relieve the amyloid-β induced toxicity in the HT22 cell line in 2021, indicating the potential of borrelidins to be developed as Alzheimer's disease drugs [57].

Alkaloids
The approach of an optimized nitroso-based probe (dienophile probe), promoting the detection of compounds containing conjugated alkenes in crude broth extracts, was employed in the chemical investigation of a marine-derived Nocardiopsis sp. CNY-503 and gained the isolation of one new polyketide alkaloid, named nocarditriene (24), which contained an unprecedented epoxy-2,3,4,5-tetrahydropyridine structure ( Figure 4) [58]. The structure of nocarbenzoxazole G (25), isolated from the marine-derived actinomycete N. lucentensis DSM 44048 in 2015 [60], was revised into the nocarbenzoxazole G (26) molecule by total synthesis in 2019. The benzoxazole skeleton was constructed with microwave assistance and continued by carbon-carbon bond formation with relevant aryl bromides [59]. Compound 26 was found to display moderate cytotoxicity against HepG2 and HeLa cell lines with IC 50 values of 16 and 14 µM, respectively [60].  Compound 29 was produced successfully by the allogenetic expression of the complete loo gene cluster in a vicarious host, Streptomyces lividans K4−114. The indolocarbazole skeleton of 27−29, belonging to the family of indolocarbazole alkaloids, is structurally similar to that of rebeccamycin and staurosporine with an additional rare modified tryptophan ring. The molecular bases of these modifications were investigated by carefully analyzing loo BGC for further genome mining and combinational biosynthetic research. The loo gene cluster sustains a ∼36 kb continuous DNA sequence, including 25 open reading frames which take charge of biosynthesis, regulation, and resistance ( Figure 6A). A possible biosynthetic pathway for loonamycin was detected based on this bioinformatic analysis ( Figure 6B). In particular, compound 27 showed potent cytotoxic activities toward eight cancer cell lines, including Sum1315 (breast cancer), SH-SY5Y (neuroblastoma), HCT116 (colorectal cancer), HT29 (colorectal cancer), HCC78 (lung cancer), HeLa (cervical cancer), SW620 (colorectal cancer), and SW872 (liposarcoma), with IC 50 values of 41−283 nM [61].
The broth culture crude extracts of actinobacterium Nocardiopsis sp. SCA30, derived from marine sediments collected from Havelock Island, Andaman, and the Nicobar Islands, India (11.96 • N, 93.00 • E), displayed cytotoxic activities against a series of cell lines, including HT 29, HCT 15, MDA-MB 468, and MCF 7 at concentrations ranging from 62.5 to 1000 µg/mL. The strain extracts also showed antibacterial activities against MRSA ATCC NR-46171 and NR-46071 with MIC values of 7.81 and 15.62 µg/mL, respectively. Compound 1-acetyl-4-4(hydroxyphenyl)piperazine (30) ( Figure 5) was isolated from the crude extracts of Nocardiopsis sp. SCA30 through LC-MS analysis and NMR chemical structural identification approved to be an antibacterial and cytotoxic compound [22]. A marine sediment-derived actinobacterium N. dassonvillei JS106 showed potent antiquorum sensing activities against S. aureus and Pseudomonas aeruginosa [63]. The marine sediment sample was gathered from Lianyungang, China. Secondary metabolites research of the strain JS106 led to the isolation of one new compound, 2-hydroxyacetate-3hydroxyacetamido-phenoxazine (HHP, 33), and one known analog questiomycin A (34) ( Figure 5). Both of these two compounds (33 and 34) exhibited antibiofilm activity against Chromobacterium violaceum 12472 with IC 50 values of 23.59 and 6.82 µg/mL, respectively [63].

Peptides
The secondary metabolites research of the broth culture of Nocardiopsis sp. UR67A, derived from the marine sponge Callyspongia sp. collected from the Red Sea in 2018, resulted in the purification of one new cyclic hexapeptide, nocardiotide A (35) (Figure 8) [64]. Compound 35 was synthesized by combining solid-and solution-phase synthesis methods in 2021 [65]. Compound 35 exhibited obvious cytotoxic activities against HeLa, CT26 (murine colon carcinoma), and MM.1S (human multiple myeloma) cell lines with IC 50 values ranging from 8 to 12 µM [64].
One new diketopiperazine, 1-demethylnocazine A (36) (Figure 8), was separated from the broth culture of the actinomycete Nocardiopsis sp. TRM20105, which was derived from a local cotton field in Tarim Basin, through the approach of antifungal activity tracking purification against Candida albicans. Compound 36 displayed weak antifungal activity against C. albicans, with an MIC value of 3.16 mM [66].
Compound 36 is belonging to the family of nocazine and is proposed to be synthesized through a CDPS-dependent pathway ( Figure 11) [76]. Compounds 38-45 are modified CDP and are also deduced to be synthesized by the CDPS assembly line.
A rare actinomycete Nocardiopsis sp. SCA21, deriving from a marine sediment sample gathered from Havelock Island, Andaman, and the Nicobar Islands, India, exhibited the potential ability to produce bioactive secondary metabolites. Chemical investigation of the broth culture of the strain resulted in the purification of two known bioactive compoundsa bromophenol derivative, 4-bromophenol (63), and a phthalate ester, Bis (2-ethylhexyl) phthalate (64) (Figure 13). Compounds 63 and 64 displayed notable enzyme-inhibitory activities against α-glucosidase. However, there was no inhibited activity against α-amylase of compound 64. Both compounds 63 and 64 demonstrated potent free radical scavenging activities against DPPH and ABTS radicals. Additionally, 63 and 64 also revealed broadspectrum inhibitory activities against MRSA ATCC-46071, MRSA ATCC NR-46171, S. aureus ATCC 12600, B. subtilis ATCC 6633, and Klebsiella pneumonia ATCC 13883 [80].
A novel sterol (65) with an unidentified substitution was isolated from marine actinomycete N. alba MCCB 110. Compound 65 was found to display antibacterial activity against the aquaculture pathogen V. harveyi, and did not exhibit toxicities to the VERO cell line and shrimp hemocytes, up to 1000 ppm [81].

Conclusions
From March 2018 to 2021, a total of 63 natural products have been isolated from the genus Nocardiopsis, and 67% of them are first-discovered compounds. These findings sufficiently demonstrate that actinomycetes Nocardiopsis have great potential to produce compounds with novel structures. The structures of the isolated compounds with diverse skeletons are mainly concentrated on the classes of polyketides, peptides, terphenyls, and alkaloids (Table 1 and Figure 14).  The sources of the genus Nocardiopsis are distributed throughout diverse ecological systems, including desert, marine, mangrove, and saltpan areas ( Figure 15). More than two out of three natural products were isolated from marine-derived Nocardiopsis, demonstrating that the ocean is a vast treasure house with abundant microbial sources to produce various novel natural products. From March 2018 to 2021, the percentage of the marinederived compounds increased by 10%, compared with the period 2015−February 2018 [5], which further demonstrates that more and more attention has been concentrated on the development of the marine resources. Among the marine-derived natural products, the sources of Nocardiopsis strains are mainly collected from marine sediment and sponges ( Figure 15). The genus Nocardiopsis has the potential ability to produce a great diversity of bioactive secondary metabolites, including antibacterial, cytotoxic, cytoprotective, enzyme inhibitory, free radical scavenging, antiquorum sensing, colorectal cancer motility inhibitory, and antifungal compounds (Figure 16). Almost 50% of the Nocardiopsis-derived natural products have been discovered to exhibit various bioactivities. The activities of the bioactive compounds are mainly focused on the categories of antibacterial and cytotoxic activities (Figure 16), which serves as a reminder that there is huge potential for new antibiotics and anticancer compounds to be developed from Nocardiopsis. There were 13 antibacterial natural products isolated from Nocardiopsis in the period March 2018 to 2021 (Table 2). Among them, compounds 2, 30, and 53 exhibit strong antibacterial activity against MRSA, and compounds 20 and 21 display significant antibacterial activity against MRCNS (Table 2). These five compounds might be developed into new antibiotics to respond to the challenge of increasing antibacterial resistance. Compound 52 shows significant antibacterial activities against a series of strains, which has the potential to be exploited into novel antibiotics with a broad spectrum of antibacterial activity ( Table 2). Compound 65 with antibacterial activity displays no toxic against VERO cell line and shrimp hemocytes up to 1000 ppm, could be developed as an environmentally friendly antibiotic ( Table 2). All of the isolated antibacterial secondary metabolites provide the structural basis for novel antibiotic research.
There were 13 cytotoxic natural products isolated from Nocardiopsis in the period March 2018 to 2021 (Table 3). Compound 27 shows extremely significant and broadspectrum cytotoxicity against a series of cell lines with the IC 50 values at the nM level, and compounds 46 and 49−51 display potent and broad-spectrum cytotoxicity ( Table 3). All four compounds have the potential to be developed as new anticancer drugs. Compounds 8, 31, 32, 35, and 37 with moderate cytotoxicity provide structural inspiration for the research and development of new anticancer drugs (Table 3).     Eight secondary metabolites isolated from Nocardiopsis have various bioactivities, including cytoprotective, antiquorum sensing, colorectal cancer motility and enzyme inhibitory, and free radical scavenging activities (Table 3).
Although the Nocardiopsis-derived natural products exhibit excellent diverse bioactivities, further research on their bioactive mechanisms is deficient. Only three compounds, 13, 16, and 37, have been studied for their active mechanisms between March 2018 and 2021. To the best of our knowledge, all the activity tests were evaluated in vitro, and bioactive evaluations in vivo are also lacking between March 2018 and 2021. In terms of the current situation, it is still a long way from the drugs being approved for clinical use.
In this review, we summarized the secondary metabolites isolated from Nocardiopsis in the period between March 2018 and 2021. The literature survey comprehensively indicates that actinomycetes Nocardiopsis have great potential as producers to generate abundant and diverse novel bioactive secondary metabolites. Some potent antibacterial and cytotoxic compounds isolated from Nocardiopsis have the potential to be developed into new drugs. Additionally, all the natural products isolated from Nocardiopsis provide a structural foundation for drug design.

Conflicts of Interest:
The authors declare no conflict of interest.