Exploration of marine natural resources in Indonesia and development of efficient strategies for the production of microbial halogenated metabolites

Nature is a prolific source of organic products with diverse scaffolds and biological activities. The process of natural product discovery has gradually become more challenging, and advances in novel strategic approaches are essential to evolve natural product chemistry. Our focus has been on surveying untouched marine resources and fermentation to enhance microbial productive performance. The first topic is the screening of marine natural products isolated from Indonesian marine organisms for new types of bioactive compounds, such as antineoplastics, antimycobacterium substances, and inhibitors of protein tyrosine phosphatase 1B, sterol O-acyl-transferase, and bone morphogenetic protein-induced osteoblastic differentiation. The unique biological properties of marine organohalides are discussed herein and attempts to efficiently produce fungal halogenated metabolites are documented. This review presents an overview of our recent work accomplishments based on the MONOTORI study.


Introduction
The search for bioactive natural products from plants and microorganisms followed by marine invertebrates is called "MONOTORI", and has made a significant contribution to the discovery and development of various pharmaceutical applications for global health and care [1,2]. Important research on two natural products won the 2015 Nobel Prize in Physiology or Medicine [3]. One award winner was artemisinin, which was isolated from the Chinese folk medicinal plant Artemisia annua and is very effective against malaria [4]. Another award winner was avermectin produced by soil-derived Streptomyces avermitilis, and its dihydro-derivative, ivermectin, is clinically used to treat roundworm parasites [5]. Over the past few decades, natural product chemists have shifted their focus to bioresources with access difficulties, and marine organisms have been in the spotlight as the next suppliers of highly diverse natural products in addition to terrestrial organisms [6][7][8]. Some marine substances have been in clinical trials for the treatment of cancers, and cytarabine (a pyrimidine nucleoside), trabectedin (ET743), eribulin (a synthetic derivative of halichondrin B), brentuximab vedotin (an antibody drug conjugate of monomethylauristatin E), and plitidepsin (dehydrodidemnin B) have already been approved as anticancer agents [9]. These natural compounds introduced are "a splendid gift from the Earth" [10], and this research area will continuously provide exciting outcomes.
I and my collaborators have also been investigating MONOTORI studies aimed at marine organisms and microorganisms mainly collected in tropical and subtropical regions. We herein review the following findings of our recent studies: (i) bioactive compounds from Indonesian marine invertebrates and microorganisms; (ii) the unique biological properties of the marine organohalogen; and (iii) the efficient production of microbial halogenated metabolites.

The search for bioactive substances from Indonesian marine organisms
The Republic of Indonesia (commonly called Indonesia) is one of the Southeast Asian countries surrounding the Indian and Pacific oceans, and is the largest island country in the world, consisting of more than 10,000 islands, primarily Sumatra, Java, and Sulawesi.
Our research group has been collaborating with Sam Ratulangi University (UNSRAT: Universitas Sam Ratulangi in Indonesia) located in Manado, North Sulawesi in Indonesia. North Sulawesi is an archipelagic area on the Minahasa Peninsula of Sulawesi Island that maintains numerous natural resources (Fig. 1a, b). Abundant coral reefs are well preserved over adjacent oceans in which widely diverse native marine organisms live without invasion by foreign species (Fig. 1c). Therefore, we conducted field work in the ocean of North Sulawesi to collect marine invertebrates (ascidians and marine sponges) and marine-derived microorganisms by scuba diving (Fig. 1d), and investigate chemical constituents for their structural and biological characteristics using various bioassay screening techniques. We herein summarize the novel bioactive compounds (containing structurally known compounds) found during our search of marine bioresources in North Sulawesi that exhibit anticancer, antimycobacterial, antidiabetes, antidyslipidemia, and antiosteoblastogenic activities.

Anticancer compounds
Some marine natural products have been approved as anticancer agents. Based on this background, we initially attempted to identify cytotoxic compounds and discovered two rare types of alkaloids with interesting features, lissoclibadin 1 (1) and papuamine (2), as shown in Fig. 2 [11][12][13][14].
Papuamine (2), an unusual pentacyclic diamine alkaloid, was originally isolated as a fungicide against Trichophyton mentagrophytes from the Papua New Guinean marine sponge Haliclona sp. [21]. We also discovered the same alkaloid 2 in Indonesian Haliclona sp. (Fig. 2) and its potent cytotoxicity against the human solid cancer cell lines, MCF-7, HCT-15, Caco-2 (colon adenocarcinoma), and LNCap (prostate adenocarcinoma) [12]. Further biochemical experiments on the cytotoxic mechanism of 2 against MCF-7 cells revealed autophagosome vesicular formation by the detection of LC3, a typical marker of mammalian autophagy, and the release of cytochrome c coincided with the activation of c-Jun N-terminal kinase (JNK), indicating that compound 2 induces an earlier onset of autophagy, followed by a reduction in cell survival through mitochondrial damage and the activation of JNK in MCF-7 cells [13]. Additionally, in our examination to evaluate synergistic effects with doxorubicin (DOX), a major chemotherapeutic reagent that activates JNK, the combination of 2 and DOX exhibited stronger cytotoxicity against MCF-7 cells, which did not involve changes in the cellular accumulation of DOX and appeared to reflect the additional activation of JNK phosphorylation [14].
Mycobacterium tuberculosis causes tuberculosis (TB), which is one of the three major infectious diseases, including human immunodeficiency virus (HIV) and malaria, worldwide [33]. The treatment of TB is challenging due to the prevalence of multidrug resistance, the limited number of anti-TB agents, and long-term administration; therefore, the exploitation of new anti-TB drugs with novel modes of action globally is needed [34,35]. Experiments using M. tuberculosis are tightly restricted by the requirement of a biosafety level 3 facility and time-consuming assays because of the pathogenicity and slow growth of M. tuberculosis, respectively. Our project to search for antimycobacterium activity has applied non-pathogenic and fast-growing M. smegmatis, the susceptibility of which to anti-TB drugs is consistent with that of M. tuberculosis [36], as an alternative test strain to detect antituberculous activity [37][38][39][40][41][42].
In this screening, we found that an ethanol (EtOH) extract of the Indonesian marine sponge Haliclona sp. exhibited antimycobacterial activity against M. smegmatis [37]. ODS and HPLC separation according to bioassays gave haliclocyclamines A-C (3-5) and five known congeners, cyclostellettamines A-C, E, and F [43]. The structures of 3-5 were elucidated as new dimeric 3-alkyl pyridinium alkaloids based on their NMR spectra in combination with ESI-MS/ MS analyses (Fig. 3). The inhibitory efficacies of 3-5 against the growth of M. smegmatis were assessed using the paper disc method [44]. Compound 3 exhibited the most potent activity, in a dose-dependent manner, with an inhibition zone of 17 mm at 10 μg/disc. Since anti-M. tuberculosis activity by cyclostellettamines, compounds related to 3-5, has been demonstrated [45], compounds 3-5 are also expected to be active against M. tuberculosis.
Leucettamol A (6), a known dimeric sphingolipid (Fig. 3), was isolated as an anti-M. smegmatis component from the Indonesian marine sponge Agelas sp. [38]. The isolation of 6 from the Bermudan marine sponge Leucetta microraphis and its antimicrobial activity were initially reported by Kong and Faulkner [46], and the absolute configuration of 6 was elucidated by Dalisay et al. [47] using a deconvolution exciton coupled circular dichroism (CD) spectrum. In our study, compound 6 exhibited moderate antimycobacterial activity against M. smegmatis with an inhibition zone of 12 mm at 50 μg/disk, whereas its bis-TFA salt and N,N'diacetyl derivative showed smaller inhibition zones, suggesting that the free amino groups in 6 are a key functional group for antimycobacterial activity. Although recent studies reported inhibitory effects on the Ubc13-Uev1A interaction and modulatory effects on TRPA1 and TRPM8 channels by 6 [48,49], we were the first to demonstrate that compound 6 exhibited antimycobacterial activity.
Due to our continuous efforts, compound 2, described in the previous section, was rediscovered as an anti-M. smegmatis substance with an MIC value of 16 μg/mL from two marine sponges Halichondria panicea and Haliclona sp. collected at Iriomote Island in Okinawa, Japan [39,40]. With the isolation of 2 from Okinawan Haliclona sp., we also isolated new open-chain derivatives of 2, namely, halichondriamine C (7) and 1-epi-halichondriamine C (8), as shown in Fig. 3, and reported their antimycobacterium activities against M. smegmatis with MIC values of 8.0 and 16 μg/mL, respectively [40]. Furthermore, alkaloids 7 and 8 both inhibited the growth of M. bovis BCG as a slow growing strain similar to M. tuberculosis with the same efficacy (MIC = 0.5 μg/mL for 7 and 8), and were active against two more slowly growing mycobacterial strains, M. avium (MIC = 4.0 and 8.0 μg/mL for 7 and 8, respectively) and M. intracellulare (MIC = 0.50 μg/mL for 7 and 8), which are pathogens of M. avium complex (MAC) disease. MAC infection is an intractable pulmonary disease and its incidence has been increasing more than TB in developed countries. Anti-MAC drugs used clinically are limited and their therapeutic effects are insufficient [50,51]. In our most recent study, we established an in vivo-mimic silkworm infection assay with MAC to efficiently screen anti-MAC antibiotics candidates with in vivo therapeutic efficacy [52] and, thus, a re-evaluation using this system is currently underway.

Protein tyrosine phosphatase 1B and sterol O-acyl-transferase inhibitors
Lifestyle-related diseases, including type 2 diabetes mellitus (T2DM) and lipid metabolism disorders, are caused by unfavorable daily habits, such as a fat-rich diet, inadequate exercise, stress, and drinking/smoking, in addition to genetic factors and aging, and are now an increasing global issue [53,54]. This section describes inhibitors of protein tyrosine phosphatase 1B (PTP1B) [55][56][57][58] and sterol O-acyl-transferase (SOAT, also known as acyl-CoA: cholesterol acyltransferase) [59][60][61], which are potential molecular targets for the treatment and prevention of these diseases. PTP1B is expressed in the brain, liver, muscles, and adipose tissue and is a key negative regulator of the insulin signaling pathway [55]. Moreover, this enzyme has been shown to control the leptin signaling cascade [56], and, thus, the application of PTP1B inhibitors as anti-T2DM and obesity agents is expected [57,58]. Since their clinical use has not yet been achieved despite a number of discoveries of natural and synthetic inhibitors [62][63][64][65][66][67][68][69][70][71], structurally novel types of drug candidates are in great demand.
The fungal strain Penicillium verruculosum TPU1311 was separated from the ascidian Polycarpa aurata, and strong PTP1B inhibitory activity was observed in an extract of the culture broth. Using purification monitoring of its bioactivity, we isolated two new merosesquiterpenes, verruculides A (13) and B (14) (Fig. 4) [76], together with three known congeners, chrodrimanins A, B, and H [77][78][79]. Compound 14 had a linear sesquiterpene skeleton and may be a putative precursor before 13 is generated by the terpene cyclization reaction [80]. Although the absolute configuration of 14 at the C-10 position was not elucidated in our previous study, Gubiani and co-workers recently discovered 10S-14 assigned by the in situ dimolybdenum CD method from the culture broth of Phoma sp. nov.
LG0217 with an epigenetic modifier [81]. Compound 13 showed an IC 50 value of 8.4 μM against PTP1B activity, while compound 14 exhibited reduced activity (40% inhibition at 23.1 μM), suggesting that the linear framework of 14 is not favorable for inhibitory activity.
Insulin and leptin signaling pathways are generally suppressed by other PTPs as well as PTP1B [55]. Among this family, the catalytic domains of T-cell PTP (TCPTP) and PTP1B share high homology; however, their biological functions markedly differ [85]. Accordingly, PTP1B/TCPTP selectivity is as important property, and TCPTP inhibitory activity by 15 was examined using an in vitro enzyme assay. Compound 15 inhibited TCPTP enzyme activity with an IC 50 value of 54 μM; therefore, this compound is a dual inhibitor with equivalent potency against two PTPs, PTP1B and TCPTP. Previous studies using genetic techniques demonstrated that TCPTP knockout mice (tcptp -/-) had serious abnormalities [86,87]; however, recent studies showed that knockout mice with a one-copy deletion of PTP1B and TCPTP (ptp1B +/or tcptp +/-) remained alive without any harmful phenotypes [88]. Therefore, the simultaneous inhibition of PTP1B and TCPTP has potential as a promising therapeutic strategy for T2DM and obesity.
In addition to the novel compounds described above, our successive studies afforded known compounds from Indonesian marine organisms as new types of PTP1B inhibitors.
Melophlin C (16), a known tetramic acid derivative, was isolated as the active constituent together with a new nortriterpenoid saponin, sarasinoside S, from the Indonesian marine sponge Petrosia sp. (Fig. 4) [89]. Compound 16 was initially obtained as a mixture of four diastereomers at the C-5 and C-10 positions from the Indonesian marine sponge Melophlus sarassinorum [90], and we also purified a similar isomeric mixture of 16. However, the new saponin was inactive, whereas compound 16 inhibited PTP1B activity with an IC 50 value of 14.6 μM and an inhibition of 16-like tetramic acids was the first finding.
SOAT, an endoplasmic reticulum membrane protein, catalyzes intracellular esterification, which transfers longchain fatty acids generated by acyl-CoA to free cholesterol to biosynthesize the cholesteryl ester (CE) [59]. Therefore, this enzyme is a potential molecular target for the prevention of dyslipidemia, such as hypercholesterolemia and related diseases, caused by the excessive accumulation of CE [60,61]. Moreover, recent molecular biology studies revealed that SOAT has two SOAT isozymes, SOAT1 and SOAT2, the localization and functions of which markedly differ [59]. Since the selectivities of SOAT1 and SOAT2 are considered to be an important index [101], we have been evaluating SOAT inhibitory activity toward these two isozymes using African Green monkey-derived SOAT1 and SOAT2 geneexpressing CHO cells (SOAT1-CHO and SOAT2-CHO cells) [102][103][104][105][106][107].
The screening study on SOAT1/SOAT2 inhibitors afforded an EtOH extract of the Indonesian marine sponge Callyspongia sp., and the separation process provided two new polychlorine-containing modified dipeptides, callyspongiamides A (22) and B (23) (Fig. 5) [108], together with the known congener, dysamide A [109]. The effects of 22 and 23 on the synthesis of CE through the inhibition of SOAT1/SOAT2 isozymes were examined using SOAT1-CHO and SOAT2-CHO cell-based assays and the respective IC 50 values over SOAT1 and SOAT2 were 0.78 and 2.8 μM for 22 and 1.2 and 2.4 μM for 23, respectively. To identify the molecules of 22 and 23 inhibiting the accumulation of CE, their inhibitory activities against SOAT1/SOAT2 isozymes were also examined using an enzyme assay with microsomes prepared from SOAT1-CHO and SOAT2-CHO cells, respectively. Compounds 22 and 23 affected SOAT1/SOAT2 enzyme activities with IC 50 values of 0.23/0.86 μM for 22 and 1.0/3.2 μM for 23, respectively, which were similar to inhibitory activities in the cell-based assay. Based on these findings, compounds 22 and 23 are dual-type SOAT1 and SOAT2 inhibitors.

Inhibitors of BMP-induced osteoblastic differentiation
Bone morphogenetic protein (BMP), a member of the transforming growth factor-β superfamily, plays an important role in the formation and repair of bone [113,114]. Therefore, the disruption of BMP signaling causes several types of bone disorders.
Fibrodysplasia ossificans progressiva (FOP) is a rare congenital disorder caused by abnormal BMP signaling activated by a mutant BMP receptor [activin receptor-like kinase-2 (ALK2)], leading to progressive heterotopic ossification (HO) in soft tissues [115,116]. Since BMP signaling inhibitors are a promising strategy for the prevention of HO [117,118], we started a screening program by monitoring the BMP-induced osteoblastic differentiation of a C2C12 cell line stably expressing mutated ALK2(R206H) (C2C12(R206H) cells) [119,120].
We originally discovered compounds 26 and 27 from another Lamellodysidea sp. marine sponge collected in Indonesia; however, their biological activities were not identified in a previous study [122]. Our sustained efforts enabled the rediscovery of 26 and 27 as BMP-induced osteoblastogenesis inhibitors.

Marine-derived organohalides
We have demonstrated that marine environments offer a structurally and biologically diverse range of natural products [6,7]. Additionally, organisms living in the sea, including marine sponges, ascidians, microorganisms, cyanobacteria, algae, and mollusks, are a rich source of organohalides [6,7,[137][138][139]. Halogenated natural products have been reported to exhibit various biological activities [6,7,140], and, for example, vancomycin as a clinical antibiotic is mainly used to treat methicillin-resistant Staphylococcus aureus (MRSA) infection [141].
Agelasine G (33), which belongs to a large group of marine natural products, was originally isolated from the Okinawan marine sponge Agelas sp. by Kobayashi and coworkers in 1992 and its structure comprises bromopyrrole, N-methyladeninium, and diterpene moieties [146]. In the process of screening anti-M. smegmatis substances from the marine sponge A. nakamurai collected at Iriomote Island (Okinawa, Japan), we isolated new antimycobacterial agelasine derivatives and discovered PTP1B inhibitory activity by 33 with an IC 50 value of 15 μM for the first time, while ageline B (36) [147], a known debromo-derivative of 33 obtained from the same marine sponge, was inactive up to 19 μM. These findings indicated that a Br atom is responsible for the inhibition of PTP1B activity, which is supported by our previous findings showing that polybromodiphenyl ethers exhibited more potent PTP1B inhibitory activity than diphenyl ether derivatives without Br groups [100,122,142,148]. As described in the section on PTP1B inhibitors, PTPs are composed of 107 members, including PTP1B as non-transmembrane PTPs, and regulate various cellular functions [55]. The inhibitory effects of 33 and 36 toward three types of PTPs, TCPTP, CD4, and VHR, were evaluated using an in vitro enzyme assay. Compound 33 was only active against VHR (IC 50 = 13 μM) with a similar To demonstrate their cellular effects, the phosphorylation levels of Akt (p-Akt), a key downstream molecule of the insulin signaling pathway starting from the insulin receptor, were measured by Western blotting using human hepatoma Huh-7 cells, in which PTP1B is mainly located. In this assay, compound 33 increased insulin-stimulated p-Akt levels in Huh-7 cells in a dose-dependent manner, suggesting that the inhibition of PTP1B activity by 33 activates the insulin signaling pathway. On the other hand, compound 36, an inactive derivative, also moderately enhanced insulin-stimulated p-Akt levels in a dose-dependent manner (Fig. 9a). These findings implied that compounds 33 and 36 have additional target molecule(s) that activate the cascade besides the inhibitory effects of PTP1B activity. Therefore, the effects of 33 or 36 alone on the p-Akt level of the signaling pathway in Huh-7 cells were tested using the same experiments without the insulin stimulation. Although compounds 33 and 36 did not significantly increase p-Akt levels at 50 μM, slight dosedependent elevations in p-Akt levels were detected in Huh-7 cells (Fig. 9b). These findings suggest that compounds 33 and 36 exert insulin-like effects to activate insulin signaling at an upstream point instead of insulin.
Therefore, compounds 33 and 36 initially exert similar effects to insulin for signal transduction, and compound 33 inhibited PTP1B activity to activate downstream of the signaling pathway. Considering these compounds in terms of their chemical structures, the presence of a Br group is significant for the inhibition of PTP1B, while the terpene and/ or adenine moieties may contribute to insulin-like effects. Many types of PTP1B inhibitors have been obtained from natural and synthetic origins; however, clinically efficient drugs have not yet been developed [57,58,62]. PTP1B inhibitors with insulin-like activity are extremely rare, and, thus, we are now investigating the optimal structures for these biological properties with the aim of developing candidate agents for the treatment of T2DM and obesity.

Efficient production of halogenated metabolites by fungal strains
From the above achievements, we were further interested in halogenated natural products that exhibit significant biological activity. Therefore, we attempted fermentation study with a focus on fungal strains to efficiently produce halogencontaining metabolites [149][150][151][152][153][154][155].
Since strain TPU199 is a marine-derived fungus, the next fermentation was performed using sterilized natural seawater medium. This condition reduced the production of 37 and newly gave two peaks (40 and 41) in a seawater concentration-dependent manner. The structures of 40 and 41 isolated under the seawater condition were elucidated as the chlorinated derivatives of 38 and 39, DC1149B [161] and trichodermamide B [159], respectively, based on their spectroscopic data (Fig. 10), inferring that the Cl groups of 40 and 41 were incorporated from NaCl in the seawater medium. Subsequent conditions using 3.0% NaCl-or NaBrsupplemented medium were examined and led to the production of known halogenated gliovirin-type ETPs possessing the Cl and Br groups, DC1149B (40) and DC1149R (42), respectively ( Fig. 10) [161].
Compound 42, a brominated derivative of 38, was documented in the same patent as 40 and was semisynthetically obtained from 37 by a reaction with HBr; however, the 1 H and 13 C NMR assignments of 42 have not been reported  [161]. Therefore, we were the first to describe the isolation of 42 as a fungal fermentation product as well as the complete assignment of 1 H and 13 C NMR spectroscopic data for 42 [150].
NaI was supplemented into the culture medium in anticipation of the production of iodinated metabolites, and the HPLC chromatogram of the broth with 3.0% NaI displayed a new peak, corresponding to metabolite 43, with similar UV spectrum to those of 37, 38, 40, and 42. Newly emerging 43 was purified by an ODS column and HPLC from the broth extract and 1D and 2D NMR analyses revealed the structure of 43 to be a new iodinated derivative of 38, named iododithiobrevamide ( Fig. 10) [151]. Various bromine-added metabolites were previously reported to be generated by fermentation with inorganic bromides [162][163][164]; however, obtaining the I derivative using the fermentation method with NaI is a rare and interesting finding.
Our precise purification of strain TPU199 on the NaIcontaining culture broth more recently resulted in the isolation of two new gliovirin-type ETPs 44 and 45 [151], which were elucidated as 5-epi-and 5-epi-trithio-38, respectively (Fig. 10). An N-methyl derivative of 44, designed as pretrichodermamide F, was initially reported as a gliovirintype ETP with a 5α-oriented substituent [165], and, thus, our findings are the second documented report of these ETPs. Pretrichodermamide derivatives with the 5α-oriented substituent, 44 and 45, may be generated via nucleophilic substitution from iodinated gliovirin-type ETP, such as 43. NaI-supplemented cultivations represent a versatile method to yield structurally diversified metabolites, not only the production of iodinated metabolites, but also stereoisomers.
Furthermore, in the course of investigations on culture conditions, a seawater culture of the TPU199 strain with 1.0% dimethyl sulfoxide (DMSO) provided a new gliovirintype ETP named chlorotrithiobrevamide (46), the structure of which was confirmed to be a trithio-derivative of 40 (Fig. 10). In contrast to this condition, the production of 46 was not detected by the addition of DMSO to a freshwater medium [152]. Compound 46 was the first example of a trithio-derivative in the gliovirin-type ETP, and recent studies added outovirin C and penicisulfuranol C produced by P. raciborskii TRT59 and P. janthinellum HDN13-309, respectively, as the second and third examples followed by 45 in this series of ETPs [150,166,167].
The fungal strain Cladosporium sp. TMPU1621 isolated from the leaves of Okinawan Achyranthes aspera var. rubrofusca was identified as the second producer with the productivity of organohalides [155]. The TMPU1621 strain produced a series of cladosporol derivatives [168][169][170], including a chlorinated congener under freshwater medium conditions, and the supplementation of 3.0% NaCl into the medium increased the production of chlorinated cladosporol. Therefore, we examined 3.0% NaBr-supplemented medium to induce the production of a new brominated derivative, and, as expected, obtained 2-bromo-cladosporol D (47) (Fig. 11). Compound 47 exhibited modest anti-MRSA activity with an MIC value of 25 μM, whereas the chlorinated congener was inactive by 50 μM. However, iodinated cladosporols have not yet been isolated from the culture broth of the strain with NaI-containing medium. Since its HPLC chromatogram differs from those obtained under other culture conditions, further studies are warranted (unpublished data).
Although these culture methods are very simple and easy, the probability of discovering objective strains with the desired characteristics is still low due to the extremely poor growth of microorganisms in medium containing halide salts or DMSO. Therefore, our aim is to develop novel strategic approaches to produce halogenated microbial metabolites, and the data obtained will be published in the near future.

Conclusion
Countless bioactive products have historically been reported from plants and microbes, and notable examples, such as paclitaxel (antineoplastic), artemisinin (antimalarial drug), penicillin (antibiotic), lovastatin (antihyperlipidemic agent), tacrolimus (immune suppressant), and ivermectin (antiparasitic), have contributed to breakthroughs in modern medicine. However, it has become increasingly difficult to identify novel natural products with excellent biological activities, and, thus, unutilized natural resources (ocean or extreme environments) and innovative searching strategies are required to expand this research field.
We herein described various Indonesian marine biological substances obtained by a collaborative screening program with UNSRAT. Marine natural product chemistry has rapidly advanced in a short period of time and has already provided several clinical agents. Therefore, we expect our findings to serve as drug seed/lead compounds for clinical applications to cancer, TB, T2DM, dyslipidemia, and FOP. This review also described the induced production of fungal organohalogens on which we started to work with inspiration based on the unique biological activities of marine halogenated products. The present methods may not be straightforward strategies, but simple techniques would enable researchers in chemical laboratories to access new compounds. These findings will facilitate and accelerate drug discovery and development in natural product chemistry. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.