molecules

Review Marine Cyanobacteria: A Source of Lead Compounds and their Clinically-Relevant Molecular Targets

Lik Tong Tan * and Ma Yadanar Phyo Natural Sciences and Science Education, National Institute of Education, Nanyang Technological University, Singapore 637616, Singapore; [email protected] * Correspondence: [email protected]; Tel.: +65-6790-3842



Academic Editor: Tatsufumi Okino
 Received: 25 March 2020; Accepted: 5 May 2020; Published: 8 May 2020 Abstract: The prokaryotic filamentous marine cyanobacteria are photosynthetic microbes that are found in diverse marine habitats, ranging from epiphytic to endolithic communities. Their successful colonization in nature is largely attributed to genetic diversity as well as the production of ecologically important natural products. These cyanobacterial natural products are also a source of potential drug leads for the development of therapeutic agents used in the treatment of diseases, such as cancer, parasitic infections and inflammation. Major sources of these biomedically important natural compounds are found predominately from marine cyanobacterial orders Oscillatoriales, Nostocales, Chroococcales and Synechococcales. Moreover, technological advances in genomic and metabolomics approaches, such as mass spectrometry and NMR spectroscopy, revealed that marine cyanobacteria are a treasure trove of structurally unique natural products. The high potency of a number of natural products are due to their specific interference with validated drug targets, such as proteasomes, proteases, histone deacetylases, microtubules, actin filaments and membrane receptors/channels. In this review, the chemistry and biology of selected potent cyanobacterial compounds as well as their synthetic analogues are presented based on their molecular targets. These molecules are discussed to reflect current research trends in drug discovery from marine cyanobacterial natural products.

Keywords: marine cyanobacteria; natural products; drug discovery; molecular targets

1. Introduction The photosynthetic filamentous marine cyanobacteria are prokaryotic microorganisms found in diverse habitats, including epiphytic, epilithic, endolithic and microbial mats communities, in coral reef ecosystems [1]. Natural products research on marine cyanobacteria has revealed its impressive biosynthetic capacity in producing structurally novel bioactive secondary metabolites [2]. The high success rate of marine cyanobacteria in colonizing different aquatic habitats could be attributed to the ecological roles of these compounds, such as UV-radiation protection, feeding deterrence, allelopathy and signaling [3]. To date, more than 550 secondary metabolites have been reported from diverse marine cyanobacterial genera, including Lyngbya, Moorea, Symploca and Oscillatoria [4]. A majority of these biomolecules are nitrogen-containing and are products of the modular biosynthetic enzymes, such as the non-ribosomal peptide synthetases (NRPS), polyketide synthases (PKS) and hybrid NRPS-PKS [5]. Moreover, research on the biosynthetic machinery of these microbial systems revealed unusual mechanistic and enzymatic features, resulting in the production of diverse chemical structures [5]. Several pharmacological trends have been observed amongst the various marine cyanobacterial secondary metabolites. A significant number of molecules have been reported to possess either potent cytotoxic (e.g., largazole, coibamide A and curacin A), neuromodulating (e.g., antillatoxin, kalkitoxin and jamaicamides) or antiinfective (e.g., almiramides and gallinamide A) properties [6–9]. The high

Molecules 2020, 25, 2197; doi:10.3390/molecules25092197 www.mdpi.com/journal/molecules Molecules 2020, 25, 2197 2 of 30 potency of these compounds is due to their specific disruption/interference with validated drug targets implicated in various human diseases, including cancer, inflammation and neurodegenerative disorders. These drug targets include enzymes, e.g., proteasomes, proteases, and histone deacetylases, cellular cytoskeletal structures, e.g., microtubules and actin filaments, as well as membrane channels, e.g., voltage-gated sodium channels and Sec61 protein translocation channels. As such, these natural products make excellent lead compounds for drug discovery and development. For instance, a number of marine cyanobacterial compounds and their synthetic analogues formulated as Antibody-Drug Conjugates (ADCs), including dolastatin 10, auristatin E and OKI-179, have undergone/undergoing clinical trials for the treatment of cancer diseases [10,11]. Integrated genomic and metabolomics approaches have been used to mine marine cyanobacteria for structurally unique natural products. In particular, the MS/MS-based metabolomics platform, Global Natural Product Social Molecular Networking (GNPS), is a powerful tool for rapid chemical profiling of natural products mixtures [12]. The use of GNPS, in combination with genomic or biological assays, has extended the accessible chemical space for the discovery of novel bioactive cyanobacterial compounds. For instance, a new class of acyl amides, columbamides, with cannabinomimetic activity was uncovered based on genomic and mass spectrometric profiling of three marine cyanobacterial strains of the genus Moorea [13]. In addition, the use of bioassay-guided fractionation and MS-based molecular networking resulted in the isolation of a cytotoxic cyclic octapeptide, samoamide A [14]. Moreover, recent advances in NMR spectroscopy have enabled new strategies for detection of new natural products from cyanobacterial extracts. One such tool is the recently developed Small Molecule Accurate Recognition Technology (SMART), which is based on a convolutional neural network to classify 2D NMR spectra, such as HSQC spectra, of natural products using pattern recognition principles [15]. Such innovative NMR-based method has led to the detection of several new cyclic depsipeptides belonging to the viequeamide class of molecules as well as new chimeric swinholide-like macrolide, symplocolide A [16,17]. Due to the sheer number of reported bioactive marine cyanobacterial compounds, it is probably impossible to provide a comprehensive coverage of all potent compounds from marine cyanobacteria in this review. There are a number of reviews on the diversity of marine cyanobacterial compounds as well as their pharmaceutical importance that readers can refer to [2,18–25]. Instead, this mini review will feature selected potent natural products and their clinically relevant molecular targets, including both enzyme and non-enzyme-based targets/pathways. The selection includes cyanobacterial molecules that have been identified as drug leads for further structural optimization as well as SAR studies. These natural products and their synthetic analogues are discussed based on their interference with molecular targets/pathways, such as histone deacetylases, proteasomes, proteases, actin and microtubule filaments, and membrane receptors/channels. Furthermore, this review serves to complement and provide an update on information of two reviews previously published by Tan as well as Salvador-Reyes and Luesch [21,22].

2. Marine Cyanobacterial Drug Leads and their Molecular Targets

2.1. Histone Deacetylases

2.1.1. Largazole Largazole (1) (Figure1), a cyclic depsipeptide, is a highly potent class I histone deacetylase (HDAC) inhibitor originally discovered from the marine cyanobacterium, Symploca sp., collected from Key Largo (FL, USA) [26]. Largazole consists of a number of unusual structural features, including a 3-hydroxy-7-mercaptohept-4-enoic acid unit and the linkage of a 4-methylthiazoline unit to a thiazole. The compound is found to be a potent inhibitor on the growth of transformed human mammary epithelial cells (MDA-MB-231) with GI50 of 7.7 nM. In addition, compound 1 showed exquisite antiproliferative activity against transformed fibroblastic osteosarcoma U2OS cells (GI50 55 nM) over Molecules 2020, 25, 2197 3 of 30

Molecules 2019, 24, x FOR PEER REVIEW 3 of 30 non-transformed fibroblasts NIH3T3 (GI50 480 nM) when compared to paclitaxel, actinomycin D, and nM) over non-transformed fibroblasts NIH3T3 (GI50 480 nM) when compared to paclitaxel, doxorubicin [26]. actinomycin D, and doxorubicin [26].

O O S O S S N NH N NH N NH N N S N S O O O O O O O O O O 19 HS N S N HS N H H H

ar azo e o Largazole (1) L g l thi l (2) 3

O O O S S N N N NH NH NH N N S N S O O O O O O O O O O O HS HS - N N n C7H15 S N S N H H H H 4 5 6 Figure 1. Largazole and related compounds. Figure 1. Largazole and related compounds. Largazole is a prodrug and upon hydrolysis of the thioester provides the highly active largazole Largazole is a prodrug and upon hydrolysis of the thioester provides the highly active largazole thiol (2) (Figure1)[ 27]. Due to the impressive anticancer property and unique structure of largazole, it thiol (2) (Figure 1) [27]. Due to the impressive anticancer property and unique structure of largazole, has attracted interest from synthetic chemists on its total synthesis and generation of synthetic analogues. it has attracted interest from synthetic chemists on its total synthesis and generation of synthetic To date, more than a dozen reports on its total synthesis have been successfully accomplished [28]. analogues. To date, more than a dozen reports on its total synthesis have been successfully A scale-up synthesis of largazole was also recently reported by Chen et al., where the syntheses of accomplished [28]. A scale-up synthesis of largazole was also recently reported by Chen et al., where each fragment and final product were optimized to achieve an overall yield of 21% in eight steps [29]. the syntheses of each fragment and final product were optimized to achieve an overall yield of 21% In addition, various in vivo and in vitro biological as well as epigenetic studies performed on largazole in eight steps [29]. In addition, various in vivo and in vitro biological as well as epigenetic studies revealed its potential use in broad spectrum therapy (Table1)[30–41]. performed on largazole revealed its potential use in broad spectrum therapy (Table 1) [30–41].

Table 1. Summary of pharmacological studies on largazole (1). Table 1. Summary of pharmacological studies on largazole (1). Disease/Target Significant Biological Activity Reference Disease/Target Significant Biological Activity Reference Angiogenesis- associated Topical application of the compound attenuated alkali-induced [31] • Angiogenesisdiseases - • Topicalcorneal application neovascularization of the compound in mouse attenuated model. alkali-induced [31] Down-regulated the expression of the pro-angiogenic factors and associated diseases •corneal neovascularization in mouse model. up-regulated the expression of the anti-angiogenic factors in vivo. • Down-regulated the expression of the pro-angiogenic factors and up- Lung cancer Potently inhibits the proliferation and clonogenic activity in lung [32] • regulatedcancer the cells. expression of the anti-angiogenic factors in vivo. Arrests cell cycle at G1 phase and up-regulates the expression of Lung cancer • •Potently inhibits the proliferation and clonogenic activity in lung [32] cyclin-dependent kinase inhibitor p21 in lung cancer cells. cancerAn cells. E2F1-targeting cell cycle inhibitor, which is overexpressed in lung • cancer tumor. • Arrests cell cycle at G1 phase and up-regulates the expression of Rheumatoid arthritis Activates p38 and Akt pathways and increase expression of HDAC6 [33] • cyclinby-dependent more than 200% kinase in rheumatoidinhibitor p21 arthritis in lung synovial cancer fibroblasts. cells. Increases HDAC6 expression, which led to enhancement of the • •An E2F1-targeting cell cycle inhibitor, which is overexpressed in lung detrimental effects of TNF-α in RA synovial fibroblasts. cancer tumor. Liver fibrosis Reduces liver fibrosis and angiogenesis by inhibition of transforming [34] • Rheumatoid arthritis • Activatesgrowth p38 factor-b and Akt as well pathways as vascular and endothelial increase expression growth of HDAC6 [33] factor signalling. by more than 200% in rheumatoid arthritis synovial fibroblasts. Breast cancer Cooperates with dexamethasone to induce localization of E-cadherin [35] • • Increasesto the HDAC6 plasma membrane expression, in breast which cancers led to as enhancement well as to suppress of the detrimentalin vitro cellular effects invasionof TNF- ofα in cancer RA cells.synovial fibroblasts. Liver fibrosis • Reduces liver fibrosis and angiogenesis by inhibition of transforming [34] growth factor-b as well as vascular endothelial growth factor signalling.

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Table 1. Summary of pharmacological studies on largazole (1).

Disease/Target Significant Biological Activity Reference

Protein ubiquitination Largazole and its ester and ketone analogs selectively inhibit ubiquitin [36] • conjugation to p27Kip1 and TRF1 in vitro.

Epstein-Barr virus Induces expression of EBV lytic-phase gene and sensitize lymphoma [37] • (EBV)-associated cells to nucleoside antiviral drugs. lymphomas

Bone-related disorders Exhibits in vitro and in vivo osteogenic activity via increased [38] • expression of Runx2 (runt-related transcription factor 2) and BMPs (bone morphogenetic proteins). Induces expression of alkaline phosphatase (ALP) and osteopontin • (OPN).

Colon cancer Strongly stimulated histone hyperacetylation in tumor in vivo by [39] • using a human HCT116 xenograft mouse model. Efficacy in inhibiting tumor growth and induced apoptosis in • the tumor. Regulates transcription of genes involved in the induction of cell cycle • arrest and apoptosis.

Oncogenic Decreases RNA polymerase II accumulation at super-enhancers (SEs) [40] • super-enhancers and preferentially suppresses SE-driven transcripts associated with oncogenic activities in transformed cells.

Antiretroviral therapy Combination of largazole and bryostatin analogues are potent [41] • activators of latent HIV without global T-cell activation within resting CD4+ T-cells.

Extensive SAR studies on synthetic largazole-based analogues were carried out and the results of these studies were summarized recently by Poli et al. [42]. In general, the thiazole-thiazoline unit of largazole is the recommended moiety where structural modifications should be carried out to generate new analogues with selective inhibition on HDAC as well as anticancer activity. For instance, pyridine analogues (e.g., 3) and bipyridine analogue, 4, provided potent anticancer and HDAC inhibitory activities (Figure1, Table2)[ 43,44]. Modification to the L-valine unit in largazole is well tolerated and in some cases able to confer higher selectivity for cancer cells over normal cells. In addition, changes to the conformation and the flexibility of the macrocyclic core can impact HDAC activity as well as on the selectivity for HDACs 1-3 over HDAC6 [45]. Furthermore, largazole analogues with modified warheads generally showed decreased in activity. However, a number of analogues containing zinc binding groups, including α-thioacetamide (e.g., 5) or mercaptosulfide (e.g., 6), have altered selectivity profile, such as conferring selectivity for HDAC6 or HDAC10 (Figure1, Table2)[ 46,47]. Largazole analogues with selectivity for HDAC6 or HDAC10 have possible usage for the treatment of CNS disorders or ovarian cancer, respectively [48,49]. Recent study on the incorporation of monofluoro and gem-difluoro substitution at the Zn2+-binding thiolate side chain of largazole thiol 2 revealed that C19-position fluoro-substituted largazole analogue exhibited high selectivity against HDAC1 over HDAC6 [50]. Due to the exceptional activity of largazole, new studies on the design of synthetic analogues with enhanced potency and selectivity are expected to be pursued to expand the chemical space of this class of compounds. Molecules 2019, 24, x FOR PEER REVIEW 5 of 30

Molecules 2020, 25, 2197 5 of 30 Table 2. HDAC inhibitory activities of largazole-based synthetic analogues 3 to 6 (IC50 nM).

Compound Table 2. HDAC inhibitory activitiesHDAC of largazole-based synthetic analogues 3 to 6 (IC50 nM). 3 4 5 6 Compound HDAC 1 2.20 21.0 1.0 40.0 3 4 5 6 2 4.42 28.0 1.9 90.0 1 2.20 21.0 1.0 40.0 23 4.422.31 28.027.0 1.5 1.943.0 90.0 35 2.31 27.0 1.5>10000 43.0 5 >10,000 66 35.16 35.16 13,00013000 0.24 0.242800 2800 88 101.8 101.8 9600 9600 10 3.6 10 3.6

AmongstAmongst thethe variousvarious largazolelargazole analogues, analogues, aa synthetic synthetic compound, compound, OKI-179,OKI-179, hashas recentlyrecently beenbeen identifiedidentified as as a a drug drug candidate candidate and and subsequently, subsequently, proceeded proceeded to to Phase Phase I I clinical clinical trial trial for for the the treatment treatment ofof advanced advanced solid solid tumors tumors [ 51[51,52,52].]. OKI-179 OKI-179 waswas developeddeveloped basedbased onon several several chemical chemical optimization optimization stepssteps involving involving largazole largazole thiol thiol and and OKI-006. OKI-006. OKI-179 OKI-179 showed showed potent potent inhibition inhibition of of the the ClassClass 11 HDACs,HDACs, namelynamely HDAC HDAC 1, 1, 2 2 and and 3, 3, with with IC IC5050s’s’ valuesvalues ofof 1.2,1.2, 2.42.4 andand 2.02.0 nM,nM, respectively.respectively.Moreover, Moreover, it it shows shows impressiveimpressive antiproliferative antiproliferative and and apoptotic apoptotic properties properties against against a a wide wide range range of of cancer cancer cell cell lines, lines, activity activity inin xenograft xenograft cancer cancer models models as as well well as as good good oral oral pharmacokinetic pharmacokinetic properties properties in in mouse, mouse, rat rat and and dog dog models.models.Results Results fromfrom the the firstfirst dosingdosing cohorts cohorts revealed revealed that that the the drug drug was was well-tolerated well-tolerated and and achieved achieved goodgood exposure exposure following following oral oral administration administration [ 52[52].].

2.1.2. Santacruzamate A 2.1.2. Santacruzamate A A picomolar-range histone deacetylase inhibitor, santacruzamate A (7) (Figure2), was obtained A picomolar-range histone deacetylase inhibitor, santacruzamate A (7) (Figure 2), was obtained from the organic extracts of a tuft-forming marine cyanobacterium collected from Coiba National from the organic extracts of a tuft-forming marine cyanobacterium collected from Coiba National Park, Panama [53]. Due to the structural similarities of santacruzamate A with the clinically approved Park, Panama [53]. Due to the structural similarities of santacruzamate A with the clinically approved HDAC inhibitor, suberoylanilide hydroxamic acid (= SAHA, 8) (Figure2), the natural product was HDAC inhibitor, suberoylanilide hydroxamic acid (= SAHA, 8) (Figure 2), the natural product was further evaluated in a series of anti-HDAC assays. This led to the discovery of santacruzamate A as further evaluated in a series of anti-HDAC assays. This led to the discovery of santacruzamate A as a potent inhibitor of HDAC2, a Class I HDAC, with an IC50 of 119 pM. Compound 7 also showed a potent inhibitor of HDAC2, a Class I HDAC, with an IC50 of 119 pM. Compound 7 also showed cytotoxicity against HCT116 and Hut-78 cancer cell lines with GI50 of 28.3 µM and 1.4 µM, respectively. cytotoxicity against HCT116 and Hut-78 cancer cell lines with GI50 of 28.3 µM and 1.4 µM, Total synthesis, achieved in two steps, of santacruzamate A as well as a synthetic hybrid compound, respectively. Total synthesis, achieved in two steps, of santacruzamate A as well as a synthetic hybrid 9 (Figure2), was reported [54]. compound, 9 (Figure 2), was reported [54].

O O O O H H O N N HO N HO N N N H O H H H O Santacruzamate A (7) SAHA (8) SAHA hybrid (9)

O H O O R N H N O N H N OH O H O

10 R = OH = 12 11 R F FigureFigure 2. 2.Santacruzamate Santacruzamate A, A, SAHA SAHA and and related related compounds. compounds.

SyntheticSynthetic compoundcompound 9 waswas found found to to be be 30 30time timess less less potent potent compared compared to santacruzamate to santacruzamate A when A whentested tested against against HDAC2. HDAC2. In another In another SAR SAR study study by by Randino Randino et et al. al.,, two two classes ofof santacruzamatesantacruzamate derivativesderivatives havinghaving severalseveral aromaticaromatic aminesamines containingcontaining eithereither thethe ethylethyl carbamatecarbamate unitunit (class(class I)I) oror oxamicoxamic acid acid moiety moiety (class (class II) II) as as zinc-binding zinc-binding groups groups were were synthesized synthesized [ 55[55].]. The The study study identified identified three three syntheticsynthetic molecules,molecules, includingincluding 1010,, 1111and and12 12(Figure (Figure2 ),2), having having potent potent antiproliferative antiproliferative activity activity in in

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HCT116 cancer cells, with IC50s’ of 0.476 µM, 0.825 µM and 0.814 µM, respectively [55]. However, HDAC inhibitory activity was not detected in these synthetic derivatives, which underscore the HCT116 cancer cells, with IC50s’ of 0.476 µM, 0.825 µM and 0.814 µM, respectively [55]. However, HDACimportance inhibitory of the activityzinc binding was notgroup detected positioning in these into synthetic the HDAC derivatives, catalytic site which as a crucial underscore step in the the importanceinhibition process. of the zinc binding group positioning into the HDAC catalytic site as a crucial step in the inhibitionRecent process. pharmacological study using santacruzamate A, in combination with other HDAC inhibitors,Recent such pharmacological as HDAC1 inhibitor, study using tacedinaline, santacruzamate HDAC1/2 A, common in combination inhibitor, withromidepsin other HDAC(FK228) inhibitors,and global such HDAC as HDAC1 inhibitor, inhibitor, vorinostat tacedinaline, (SAHA), HDAC1 to /treat2 common hepatocellular inhibitor, romidepsincarcinoma (FK228)(HCC) andwas globalreported HDAC [56]. inhibitor, The combined vorinostat chemotherapy (SAHA), to treatled to hepatocellular the inhibition carcinoma of HDAC1/2 (HCC) as waswell reportedas changes [56 ].in TheHCC combined cell morphology, chemotherapy growth led inhibition, to the inhibition cell cycle of blockage HDAC1 /2and as apoptosis well as changes in vitro in and HCC growth cell morphology,suppression growthof subcutaneous inhibition, HCC cell cycle xenograft blockage tumors and apoptosisin vivo [56in]. vitroSantacruzamateand growth A suppression has also been of subcutaneousexplored as HCCpotential xenograft candidate tumors leadin vivocompound[56]. Santacruzamate for the treatment A has of also endoplasmic been explored reticulum as potential (ER) candidatestress- and lead compoundunfolded protein for the treatment response of endoplasmic(UPR)-related reticulum neurodegenerative (ER) stress- and disorders, unfolded such protein as responseAlzheimer (UPR)-related’s disease [57 neurodegenerative]. disorders, such as Alzheimer’s disease [57].

2.2.2.2. Proteasome Proteasome

CarmaphycinsCarmaphycins CarmaphycinsCarmaphycins A A (13 (13) and) and B (B14 ()14 (Figure) (Figure3) are 3) potentare potent novel novel proteasome proteasome inhibitors inhibitors isolated isolated in low in yieldlow fromyield organic from organic extracts ofextractsSymploca of sp.Symploca obtained sp. fromobtained CARMABI from beach,CARMABI Curacao beach, [50]. Curacao Structurally, [50]. theStructurally, carmaphycins the consistcarmaphycins of a leucine-derived consist of aα ,leucineβ-epoxyketone-derived warheadα,β-epoxyketone directly attachedwarhead to directly either aattached methionine to either sulfoxide a methionine in 13 or a methioninesulfoxide in sulfone13 or a inmethionine14, which sulfone is linked in to 14 a, valinewhich andis linked an alkyl to a chainvaline terminal and an tail.alkyl Their chain total terminal synthesis tail. wasTheir accomplished total synthesis using was anaccomplished efficient and using scalable an effi convergentcient and methodscalable [58 convergent]. The proteasome method inhibitory[58]. The propertiesproteasome of inhibitory carmaphycins properties A and of B werecarmaphycins evaluated A against and B Saccharomyceswere evaluated cerevisiae against20S Saccharomyces proteasome cerevisiae and found 20S to proteasome have comparable and found IC50 tovalues have comparable of 2.5 nM and IC50 2.6values nM, respectively.of 2.5 nM and Such 2.6 inhibitory nM, respectively. activities are Such comparable inhibitory with activities those reported are comparable for epoxomicin with those (15) andreported the marine-derived for epoxomicin salinosporamide (15) and the marine A (16-derived), with IC salinosporamide50 values of 2.7 nMA (16 and), with 1.4 nM, IC50 respectively values of 2.7 (FigurenM and3). 1.4 In addition,nM, respectively cytotoxic (Figure assay showed 3). In addition, carmaphycins cytotoxic to be assay particularly showed active carmaphycins against solid to be tumorparticul cellarly lines, active including against humansolid tumor lung cell adenocarcinoma lines, including and human colon lung cancer adenocarcinoma cell lines. Preliminary and colon structuralcancer cell biology lines. Preliminary investigation structural of the carmaphycins biology investigation suggested of distinct the carmaphycins binding site suggested as compared distinct to epoxomicin,binding site salinosporamide as compared to epoxomicin, A and bortezomib. salinosporamide A and bortezomib.

R

O O HO H H O O O O N N H H N N N O H N N Cl NH O O O H O O O O OH S O Carmaphycin A 1 R = ( 3) CH3 Epoxomicin (15) Salinosporamide A (16) O O

Carmaphycin B 14 R = S ( ) CH3

NH O O H N O O O N N O O H H H H H H N N O O N N N O N O H O H O O S O O O NH NH 2 Analog 18 17 ( ) 19 - Carmaphycin 17 (18)

FigureFigure 3. 3.Carmaphycins, Carmaphycins, epoxomicin, epoxomicin, salinosporamide salinosporamide A A and and related related compounds. compounds.

TheTheα α,β,β-epoxyketone-epoxyketone warheadwarhead inin carmaphycinscarmaphycins isis also a structural feature in in epoxomicin epoxomicin ( (1212),), a aknown known proteasome proteasome inhibitor, as wellwell asas thethe FDA-approvedFDA-approved anticancer anticancer drug, drug carfilzomib., carfilzomib. Their Their

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irreversible inhibition of proteasome is due to the formation of the morpholino adduct between the irreversibleepoxyketone inhibition warheads of with proteasome the Thr1 is residues due to the in the formation catalytic of sites the morpholino of the 20S proteasome adduct between core. The the epoxyketonemechanism involves warheads warhead with the carbonyl Thr1 residues and epoxide in the undergoing catalytic sites two of successive the 20S proteasome nucleophilic core. attacks The mechanismby Thr1 Oγ involvesand Thr1 warhead N, respectively carbonyl (Scheme and epoxide 1) [59 undergoing]. A novel mechanism two successive of proteasome nucleophilic inhibition attacks byvia Thr1 hydroamination Oγ and Thr1 using N, respectively alkene derivatives (Scheme1)[ of59 the]. Acarmaphycin novel mechanism was recently of proteasome reported inhibition by Trivella via hydroaminationand co-workers using[60]. The alkene action derivatives of the carmaphycin of the carmaphycin enone electrophile was recently was reported found by to Trivellabe partially and co-workersreversible and [60]. this The could action provide of the carmaphycin further insights enone on electrophilethe design of was proteasome found to be inhibitors partially for reversible cancer andtreatment. this could provide further insights on the design of proteasome inhibitors for cancer treatment.

_ O O OH O O O H O H O + H N H N NH2 H H O ox e one H OH Ep yk t H war ea o or o no a uc h d f M ph li dd t natural product - rm n r N T e i al Th on ro easome p t α β SchemeScheme 1.1. MechanismMechanism ofof actionaction ofof carmaphycinscarmaphycins withwith α,β, -epoxyketone-epoxyketone warhead.warhead. A number of synthetic analogues of carmaphycin B have been explored as lead molecules for A number of synthetic analogues of carmaphycin B have been explored as lead molecules for the treatment of protozoan and metazoan parasitic infection as well as cancer. One such synthetic the treatment of protozoan and metazoan parasitic infection as well as cancer. One such synthetic compound is analog 18 (17) (Figure3), which was synthesized based on the structure of carmaphycin compound is analog 18 (17) (Figure 3), which was synthesized based on the structure of carmaphycin B[61]. Analog 18 was shown to possess potent in vitro antimalarial activity against asexual blood B [61]. Analog 18 was shown to possess potent in vitro antimalarial activity against asexual blood stages and gametocytes as well as strongly inhibits the activity of Plasmodium proteasome. Its potent stages and gametocytes as well as strongly inhibits the activity of Plasmodium proteasome. Its potent activity was due to specific inhibition of the β5 subunit of the proteasome. Moreover, the study showed activity was due to specific inhibition of the β5 subunit of the proteasome. Moreover, the study that incorporating a d-amino acid at the P3 position can significantly modulate host cytotoxicity without showed that incorporating a D-amino acid at the P3 position can significantly modulate host interfering significantly with anti-plasmodial activity [61]. In another study, a carmaphycin-related cytotoxicity without interfering significantly with anti-plasmodial activity [61]. In another study, a synthetic analogue, carmaphycin-17 (18, Figure3), was found to have greater inhibitory activity against carmaphycin-related synthetic analogue, carmaphycin-17 (18, Figure 3), was found to have greater Trichomonas vaginalis than the reference drug metronidazole [62]. Trichomonas vaginalis is the causative inhibitory activity against Trichomonas vaginalis than the reference drug metronidazole [62]. agent of the sexually transmitted disease, trichomoniasis. In addition, 18 was found to be selective Trichomonas vaginalis is the causative agent of the sexually transmitted disease, trichomoniasis. In for T. vaginalis due to its increased potency against the β1 and β5 catalytic subunits of the T. vaginalis addition, 18 was found to be selective for T. vaginalis due to its increased potency against the β1 and proteasome as compared to the human proteasome subunits [62]. Compound 18 is also shown to β5 catalytic subunits of the T. vaginalis proteasome as compared to the human proteasome subunits be selective for the Schistosoma mansoni proteasome (about 13.2-fold more potent) over the human [62]. Compound 18 is also shown to be selective for the Schistosoma mansoni proteasome (about 13.2- proteasome [63]. Treatment of adult S. mansoni with 18 resulted in the same phenotypic changes as the fold more potent) over the human proteasome [63]. Treatment of adult S. mansoni with 18 resulted in FDA-approved drugs, bortezomib and carfilzomib. the same phenotypic changes as the FDA-approved drugs, bortezomib and carfilzomib. In another development, a library of carmaphycin B analogues containing amine handles were In another development, a library of carmaphycin B analogues containing amine handles were synthesized and screened for their cytotoxic properties as Antibody-Drug Conjugates [64]. The synthesized and screened for their cytotoxic properties as Antibody-Drug Conjugates [64]. The screening effort resulted in identifying a highly potent analog of carmaphycin B, 19 (Figure3), screening effort resulted in identifying a highly potent analog of carmaphycin B, 19 (Figure 3), containing a 4-sulfonylaniline handle as an attachment point for the linker antibody. However, the containing a 4-sulfonylaniline handle as an attachment point for the linker antibody. However, the study found that the free analogues, such as 19, gave better cytotoxic activity at sub-nanomolar levels study found that the free analogues, such as 19, gave better cytotoxic activity at sub-nanomolar levels as compared to their ADCs when tested against the SKBR3 and MDA-MB-231 cancer cell lines. In as compared to their ADCs when tested against the SKBR3 and MDA-MB-231 cancer cell lines. In addition, it was revealed that the cytotoxicity of free analogues with linear amines was greatly reduced addition, it was revealed that the cytotoxicity of free analogues with linear amines was greatly as compared to free analogues with aromatic amines. The reduced potency of the ADCs could be due reduced as compared to free analogues with aromatic amines. The reduced potency of the ADCs to protein-mediated degradation of the conjugated carmaphycin backbone, lysosomal degradation of could be due to protein-mediated degradation of the conjugated carmaphycin backbone, lysosomal the analogues or inappropriate ADC trafficking [64]. degradation of the analogues or inappropriate ADC trafficking [64]. 2.3. Protease Enzymes 2.3. Protease Enzymes Proteases are ubiquitous proteolytic enzymes found in eukaryotic and prokaryotic cells. The Proteases are ubiquitous proteolytic enzymes found in eukaryotic and prokaryotic cells. The classification of these enzymes is based on key catalytic group present in the active site, such as serine, classification of these enzymes is based on key catalytic group present in the active site, such as serine, threonine, cysteine, aspartate, glutamate or zinc in metalloproteases. They account for about 2% of the threonine, cysteine, aspartate, glutamate or zinc in metalloproteases. They account for about 2% of genes in human cells and are involved primarily in proteins activation, synthesis and turnover [65]. the genes in human cells and are involved primarily in proteins activation, synthesis and turnover

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Due to their involvement in many signaling pathways, they represent potential drug targets in human diseases, including cardiovascular disorders, inflammation, cancers as well as parasitic and viral infections [66]. It has been reported that filamentous marine cyanobacteria are sources of potent protease inhibitors, particular molecules targeting serine and cysteine proteases.

2.3.1. Serine Protease Inhibitors Serine proteases, including elastase, chymotrypsin, and trypsin, are a large class of enzymes having different roles related to human health, such as immune response, wound healing and blood coagulation. Studies have shown that an increase or decrease of protease activity can induce pathologies, including cancer, inflammation, heart attack, stroke and pancreatitis [67]. A number of potent serine protease inhibitors have been reported from marine cyanobacteria. A majority of these serine protease inhibitors are 3-amino-6-hydroxypiperidone (Ahp)-containing cyclodepsipeptides, including lyngbyastatins 4–10 (e.g., 20) (Figure4), pompanopeptin A, symplocamide A, kempopeptins, molassamide, bouillomides, somamide B as well as recently reported molecules, loggerpeptins, kyanamide and tutuilamides (e.g., 21)[68–78]. All Ahp-cyclodepsipeptides contain an N-methyl aromatic amino acid at conserved positions in their 19-membered ring structure, while other residues are less conserved. In addition to elastase selectivity, molassamide (22) was reported to inhibit the migration of the highly invasive MDA-MB-231 breast cancer cells [76]. Other non Ahp-containing cyclodepsipeptides, such as the largamides (e.g., 23) and tiglicamides, have also been reported to possess moderate to potent serine protease inhibitory activities (Figure4)[79–81]. Molecules 2020, 25, 2197 9 of 30 Molecules 2019, 24, x FOR PEER REVIEW 9 of 30

Cl OH OH

O O O O HN O H O H O N N N N N H N H H N O H O N O O O O HN O O HN O H O H N N N N O NH2 O O HO HO n as a n Ly gby t ti 7 (20) Tutuilamide A (21)

OH O

O OH O O O HN O N H H O N O HN O N O O O H N H H H O N O N N O HN O N N N H O HO H H H N O O N O HO OH

ar am e Molassamide (22) L g id A (23)

OH Br

O O O OH O H H N N N H N N N H H O O H O HN O O O H O N N O O OH

Largamide D oxazoline (24)

OH Br

O O O OH O H H N N N H N N N H H O O H O HN O O O H O HO N N O HO OH

ar am e L g id D (25)

FigureFigure 4. 4. SerineSerine protease protease inhibitors inhibitors from from marine marine cyanobacteria. cyanobacteria.

ItIt has beenbeen proposed proposed that that these these cyanobacterial cyanobacterial serine serine protease proteas inhibitorse inhibitors may function may function as chemical as chemicaldefenses defenses against marineagainst predatorsmarine predators [82]. Ecological [82]. Ecological studies studies by Matthew by Matthew and co-workers and co-workers led to led the toisolation the isolation of an of Ahp-containing an Ahp-containing largamide largamide D derivativeD derivative24 24(Figure (Figure4), 4), formed formed via via intramolecularintramolecular condensationcondensation of of largamide largamide D D ( (2525)) (Figure (Figure 44).). This molecule, largamide D oxazolidine (24), exhibitedexhibited 1111-fold-fold and and 33 33-fold-fold reduction reduction in in activity activity against against chymotrypsin chymotrypsin and and elastase, elastase, respectively, respectively, when when comparedcompared to to largamide largamide D D [82]. [82]. The The Ahp Ahp moiety moiety is is essential essential for for serine serine protease protease inhibitio inhibitionn and and any any structuralstructural or or conformational conformational changes changes to to this this unit unit will will affect affect activity. Crystal structures of Ahp-cyclodepsipeptides in complex with serine proteases revealed that the inhibition is based on a substrate-like binding mode where distinct amino acid residues sit in the S-

Molecules 2020, 25, 2197 10 of 30

Crystal structures of Ahp-cyclodepsipeptides in complex with serine proteases revealed that the inhibition is based on a substrate-like binding mode where distinct amino acid residues sit in the S- and S0-pockets with no proteolytic cleavage. These cyanobacterial inhibitors act in a similar manner to proteinaceous canonical serine protease inhibitors, where the conserved residues stabilize the inhibitory fold, and the other less conserved residues define serine protease selectivity through optimal accommodation at the specific S and S0-sites [83]. Such finding suggests that Ahp-cyclodepsipeptides may represent a suitable scaffold for designing non-covalent S1 serine protease inhibitors. As such a number of potent Ahp-cyclodepsipeptides has been targeted for total synthesis as well as generating synthetic analogues with enhanced activity. For instance, the total synthesis of lyngbyastatin 7 (20) was carried out in 31 steps by Luo and co-workers [84]. The synthetic compound showed superior activity over the only approved elastase inhibitor drug, sivelestat. Specifically, compound 20 demonstrated strong ability in protecting bronchial epithelial cells against elastase-induced antiproliferation as well as negating the elastase-triggered induction of pro-inflammatory cytokine expression [84]. A scale-up synthesis of lyngbyastatin 7 was recently developed with improved reaction yields and simplified purification steps [85]. Based on this synthetic strategy, a pilot library of lyngbyastatin 7 analogues (e.g., 26–28) was constructed for the purpose of compound tailoring and modulation of lipophilicity/hydrophilicity via manipulation of the pendent side chain. More importantly, this study revealed the importance of the side chain for elastase inhibition in addition to the Ahp and 2-aminobutenoic acid moiety in the core cyclic structure of lyngbyastatin 7. This major development would path the way for the translation of the natural product into pharmacotherapeutics for the treatment of diseases with overactive human neutrophil elastase [85]. In another study, a practical mixed solid- and solution-phase synthesis were employed for the synthesis of tasipeptins (e.g., 29) and synthetic analogues [86]. More importantly, this synthetic strategy resulted in the generation of the human HTRA protease inhibitors (e.g., 30) with enhanced potency (Figure5). Molecules 2020, 25, 2197 11 of 30 Molecules 2019, 24, x FOR PEER REVIEW 11 of 30

OH OH

O O O O O H N O N N N H H N N O H H O O N O O HN O HN O H O H O N N N N O NH2 O O HO HO 26 27 OH

O O O H N N H N N H O N O O HN O O H O N N O NH2 O HO 28

H H N N N N O O O O O O O O N OH O O N OH O O O O H H H H N N Cbz N N N N N N N H H H H H O O O O

Tasipeptin A (29) 30

Figure 5. LyngbyastatinLyngbyastatin 7 synthetic analogues, tasipeptin A and related compound.

2.3.2. Falcipa Falcipainin Inhibitors Gallinamide A Gallinamide A Gallinamide A (= symplostatin 4) (31) (Figure6) is one of the most potent marine cyanobacterial Gallinamide A (= symplostatin 4) (31) (Figure 6) is one of the most potent marine cyanobacterial antimalarial compounds reported to date, with EC50 of 74 nM when tested against Plasmodium falciparum antimalarial compounds reported to date, with EC50 of 74 nM when tested against Plasmodium strain 3D7 [87–89]. However, gallinamide A has moderate activity against mammalian Vero cells and falciparum strain 3D7 [87–89]. However, gallinamide A has moderate activity against mammalian no detectable cytotoxicity against the NCI-H460 lung tumor and neuro-2a mouse neuroblastoma cell Vero cells and no detectable cytotoxicity against the NCI-H460 lung tumor and neuro-2a mouse lines [87]. Gallinamide A, a linear depsipeptide, has several unique structural features, including neuroblastoma cell lines [87]. Gallinamide A, a linear depsipeptide, has several unique structural a dimethylated N-terminal amino acid moiety, a 4-amino-2-pentenoic acid unit, and a C-terminal features, including a dimethylated N-terminal amino acid moiety, a 4-amino-2-pentenoic acid unit, N-acyl-pyrrolinone unit. Gallinamide A was initially reported by Linington and co-workers from an and a C-terminal N-acyl-pyrrolinone unit. Gallinamide A was initially reported by Linington and co- organic extract of a Schizothrix species collected from reef near Piedras Gallinas, Panama [87]. At the workers from an organic extract of a Schizothrix species collected from reef near Piedras Gallinas, same time, symplostatin 4, having identical planar structure with gallinamide A, was also reported Panama [87]. At the same time, symplostatin 4, having identical planar structure with gallinamide A, by Taori and co-workers from a different cyanobacterial species, Symploca sp., obtained from Florida was also reported by Taori and co-workers from a different cyanobacterial species, Symploca sp., obtained from Florida Keys [88]. Based on the detailed 1D NMR analysis of the synthetic molecules, it was concluded that the structures of gallinamide A and symplostatin 4 are identical [89,90].

Molecules 2019, 24, x FOR PEER REVIEW 12 of 30

The potent antimalarial activity of gallinamide A (31) led to investigations by Stolze and co- workers on its mode of action and SAR studies based on synthetic and biochemical methods [91]. It was observed that Plasmodium falciparum-infected red blood cells treated with gallinamide A, at Moleculesnanomolar2020, concentration,25, 2197 exhibited swollen food vacuole phenotype. Through the use of fluorescent12 of 30 probes based on rhodamine fluorophore-tagged molecules, it was revealed that the natural product is a specific inhibitor of the plasmodial cysteine proteases, falcipains 2, 2′ and 3. In addition, the Keys [88]. Based on the detailed 1D NMR analysis of the synthetic molecules, it was concluded that methoxypyrrolinone unit in gallinamide A is essential for antimalarial activity and modifications to the structures of gallinamide A and symplostatin 4 are identical [89,90]. the N-terminal end of the molecule are tolerated [91].

O O O O R H H H O N H N N N N N e N N N OMe H OM H O O CF COO O O O 3 O

- - - Gallinamide A = Symplostatin 4 31 32 R = CH 4 OH Ph ( ) ( ) = 2- - 33 R CH2 3 indole

O O NH MeN O O NH H H H H N N e N N M 2N N N OMe N N e H H OM O O O R O Me O O

34 R =

36 35 R =

O O O O H H H O N z e Cb N N M 2N N N OMe N N N e H H H OM O O O O O O

37 38

Figure 6. 6. GallinamideGallinamide A A and and related related compounds. compounds.

TheFalcipains potent antimalarialare Plasmodium activity falciparum of gallinamide cysteine proteases A (31) led involved to investigations in several by key Stolze processes and co-workers of the onerythrocytic its mode ofcycle action of the and malarial SAR studies parasite, based such onas syntheticthe hydrolysis and biochemicalof host hemoglobin, methods eryth [91rocyte]. It was observedinvasion, that andPlasmodium rupture [92]. falciparum These cysteine-infected proteases red blood therefore cells treatedconstitute with promising gallinamide molecular A, at nanomolartargets concentration,in the search for exhibited novel antimalarial swollen food agents. vacuole The proposed phenotype. mechanism Through of thegallinamide use of fluorescent A inhibition probes of falcipain 2 was deduced based on molecular dynamics simulation studies [93]. The simulation studies based on rhodamine fluorophore-tagged molecules, it was revealed that the natural product is suggested that the methyl methoxylpyrrolinone moiety in gallinamide A inserts into the active site a specific inhibitor of the plasmodial cysteine proteases, falcipains 2, 20 and 3. In addition, the of falcipain 2, facilitating Michael addition and Schiff base formation with Cys-42 and Lys-203, methoxypyrrolinone unit in gallinamide A is essential for antimalarial activity and modifications to respectively, leading to the irreversible inhibition of the enzyme (Scheme 2) [93]. the N-terminal end of the molecule are tolerated [91]. Falcipains are Plasmodium falciparum cysteine proteases involved in several key processes of the erythrocytic cycle of the malarial parasite, such as the hydrolysis of host hemoglobin, erythrocyte invasion, and rupture [92]. These cysteine proteases therefore constitute promising molecular targets in the search for novel antimalarial agents. The proposed mechanism of gallinamide A inhibition of falcipain 2 was deduced based on molecular dynamics simulation studies [93]. The simulation studies suggested that the methyl methoxylpyrrolinone moiety in gallinamide A inserts into the active site of falcipain 2, facilitating Michael addition and Schiff base formation with Cys-42 and Lys-203, respectively, leading to the irreversible inhibition of the enzyme (Scheme2)[93].

Molecules 2020, 25, 2197 13 of 30 Molecules 2019, 24, x FOR PEER REVIEW 13 of 30

N N H O S HN S N Irreversible complex - s s- -H i 174 - C y 42 (FP 2 active site) (FP 2 active site) OCH3

Schiff Base Formation - H2N - Michael S Lys 203 O A ition - dd (FP 2 active site) - O O N Cys 42 O - (FP 2 active site) S N

H3CO

a nam e H G lli id A evers e com ex OC 3 R ibl pl

SchemeScheme 2. 2Proposed. Proposed mechanism mechanism of of gallinamidegallinamide A at active site of of falcipain falcipain 2 2(FP (FP-2)-2) (a (adapteddapted from from Ref.Ref. [93 []).93]).

AA number number of of synthetic synthetic analogues, analogues, basedbased onon gallinamidegallinamide A A structural structural motif, motif, having having potent potent antiprotozoalantiprotozoal or or anticancer anticancer activity activity havehave beenbeen identified.identified. In In a a study study by by Conroy Conroy and and co co-workers,-workers, analoguesanalogues of of gallinamide gallinamide A A were were synthesized synthesized forfor SARSAR studies and and tested tested for for their their inhibitory inhibitory activit activityy againstagainst falcipains falcipains 2 and2 and 3 [394 [94].]. It It was was revealed revealed thatthat thethe αα,β,β--unsaturatedunsaturated imide imide moiety moiety of of gallinamide gallinamide A isA importantis important for for inhibitory inhibitory activity. activity. In In addition, addition, severalseveral analogues, such such as as compounds compounds 3232 andand 3333 (Figure(Figure6), 6 showed), showed potent potent inhibition inhibition of of thethe chloroquine-sensitivechloroquine-sensitive 3D7 3D7 strain strain of ofP. falciparumP. falciparum as wellas wellas as the chloroquine-resistantchloroquine-resistant Dd2 Dd2 strain strain of of P.P. falciparum falciparum (Table(Table 3)3 )[[9494]. ].A Arecent recent study study by byStoye Stoye et etal. al. created a library of gallinamide A-based analogues using a highly efficient and convergent synthetic created a library of gallinamide A-based analogues using a highly efficient and convergent synthetic route [95]. Based on this library, several synthetic analogues (e.g., 34-36) possessed potent inhibitory route [95]. Based on this library, several synthetic analogues (e.g., 34–36) possessed potent inhibitory activity against the P. falciparum falcipain 2 and falcipain 3 as well as cultured chloroquine-sensitive activity against the P. falciparum falcipain 2 and falcipain 3 as well as cultured chloroquine-sensitive (3D7) and chloroquine-resistant (W2) strains of P. falciparum (Table 3). Three synthetic lead molecules, (3D7) and chloroquine-resistant (W2) strains of P. falciparum (Table3). Three synthetic lead molecules, namely 34, 35 and 36, were subsequently evaluated for their in vivo efficacy against P. berghei 34, 35 36 P. berghei namelyinfection in andmice (Figure, were 6 subsequently). Of the three evaluated compounds, for compound their in vivo 36e curedfficacy P. against berghei-infected miceinfection in in micethe Peters (Figure 4 day6). Of-suppressive the three compounds, test when administered compound 2536 mgcured kg-P.1 intrape bergheiritoneally-infected micedaily inover the 4 Peters days 4 1 day-suppressive[95]. test when administered 25 mg kg− intraperitoneally daily over 4 days [95].

TableTable 3. Inhibition3. Inhibition of of falcipains falcipains 2, 2, 3 3 and and various various strainsstrains of P. falciparum byby gallinamide gallinamide A A analogues analogues 32–3236–36(IC (IC50 nM).50 nM).

FalcipainFalcipain P. falciparumP. falciparum CompoundCompound 22 3 33D7 3D7 Dd2 Dd2 W2 W2 32 32 5.255.25 81.4 81.4 20.0 20.0 67.067.0 33 12.0 66.7 9.7 29.0 33 12.0 66.7 9.7 29.0 34 31.0 117.0 26.0 28.0 35 34 29.031.0 117.0 79.0 26.0 42.0 28.0 49.0 36 35 33.029.0 79.0 112.0 42.0 1.0 49.0 4.0 36 33.0 112.0 1.0 4.0 Synthetic analogues of gallinamide A were recently explored as potent inhibitor of cathepsin L for the treatmentSynthetic of Chagasanalogues disease of gallinamide [96]. The study,A were conducted recently explored by Boudreau as potent and inhibitor co-workers, of cathepsin showed thatL gallinamidefor the treatment A and itsof Chagas synthetic disease analogs [96 potently]. The study, inhibit conducted cruzain, by a BoudreauTrypanosoma and cruzi co-workers,cysteine showed protease, andthat are gallinamide exquisitely toxicA and toward its syntheticT. cruzi analogsin the intracellularpotently inhibit amastigote cruzain, stage. a Trypanosoma Compound cruzi 37 cysteine (Figure6 ) wasprotease, the most and active are cruzainexquisitely inhibitor toxic toward with an T. IC cruzi50 of in 5.1 the nM. intracellular This molecule amastigote was found stage. to Compound be inactive to the3 epimastigote7 (Figure 6) was and the the most host active cell [cruzain96]. Synthetic inhibitor analogues with an IC of50 gallinamide of 5.1 nM. This A were molecule also exploredwas found for theirto anticancerbe inactive activity.to the ep Inimastigote a study by and Liu the et host al., ancell anti-cancer [96]. Synthetic stem analogues cells lead molecule,of gallinamide38, was A were found

Molecules 2020, 25, 2197 14 of 30 to significantly suppress tumor growth both in vitro and in vivo. Moreover, 38 was able to significantly reduce the number of melanoma tumor spheres and decrease the percentage of ALDH+ melanoma cells by blocking the Wnt/β-catenin signaling pathway [97].

2.3.3. Cathepsin Inhibitors Cathepsins are protease enzymes, classified by various families, including serine protease, cysteine protease and aspartyl protease. There are currently about 15 classes of cathepsins in humans. These enzymes are essential for diverse range of normal physiological functions, including digestion, blood coagulation, bone resorption, ion channel activity, innate immunity, vesicular trafficking and autophagy. However, dysregulated cathepsins have been implicated in a number of pathologies, including arthritis, periodontitis, pancreatitis, macular degeneration, muscular dystrophy, atherosclerosis, obesity, stroke, Alzheimer’s disease, schizophrenia, tuberculosis, and carcinogenesis [98]. As such, cathepsins have a diagnostic value and are promising drug targets for a variety of human diseases through the inhibition of these proteases [99]. A series of statine-containing linear depsipeptides, including grassystatins (e.g., 39), tasiamides, and symplocin A, were found to be potent inhibitors of aspartyl proteases, cathepsins D and E (Figure7). Cathepsins D and E are potential drug targets as their overexpressions have been observed in various cancer forms, such as pancreatic ductal adenoma, cervical adenocarcinoma, lung carcinoma, and gastric adenocarcinoma [100]. The linear decadepsipeptides, grassystatins A-F (e.g., 39) were isolated from Lyngbya confervoides and VPG 14-61 collected at Grassy Key, Florida and Cetti Bay, Guam, respectively [101,102]. These statine unit-containing molecules were initially isolated from a screening program by evaluating the inhibitory activities of natural products against 59 proteases. Grassystatins A (39) and B displayed potent inhibitory activity against cathepsins D and E with IC50 values averaging at 16.9 nM and 0.62 nM, respectively. In addition, grassystatin A was able to reduce antigen presentation by dendritic cells [101]. The total synthesis of grassystatin A was recently accomplished by Yang and co-workers and pharmacological studies revealed that the inhibition of cathepsin E by the molecule did not impact ovalbumin antigen processing and peptide presentation [103]. In addition, grassystatin F was shown to inhibit the cleavage of cystatin C and plasminogen activator inhibitor type-1 (PAI-1) via its inhibition on cathepsin D. Cystatin C and PAI-1 are involved in the downstream activation of cysteine cathepsins and tissue plasminogen activator (tPA), which would lead to the migration of highly aggressive triple negative breast cancer cells (MDA-MD-231) [102]. Molecules 2020, 25, 2197 15 of 30 Molecules 2019, 24, x FOR PEER REVIEW 15 of 30

H N 2 Statin O OH O O OH O O H H H O N N N N N O N N N H H O O O O O OCH O 3

Grassystatin A (39)

CONH2 CONH2 O OH O O O OH O O H H H H H H N N N N N N N N HO N N N HO N N N H H O O O O OCH O O O O OCH O 3 O 3

Tasiamide F (40) Tasiamide B (41)

O S O N CONH2 OH O O OH O O H H H H H H N N N N N N OMe N Cbz N N N H H O O O O O O

42 43

OH Me O O OH O O H H H H N N N N N Me O N N N H H O O O O O OMe

OH

Symplocin A (44) FigureFigure 7. GrassystatinGrassystatin A, A, tasiamides tasiamides B and B andF, symplocin F, symplocin A and A related and related compounds. compounds.

TasiamidesTasiamides areare grassystatin-relatedgrassystatin-related molecules molecules found found to possess to possess potent potent cathepsins cathepsins D and E D and E inhibitoryinhibitory activity.activity. In In particular, particular, tasiamides tasiamides B (41 B) ( 41and) andF (40 F) ((Figure40) (Figure 7) displayed7) displayed potent potentactivity activity against cathepsins D and E, with IC50 values of 50 nM and 9.0 nM for 41 and 57 nM and 23 nM for 40, against cathepsins D and E, with IC50 values of 50 nM and 9.0 nM for 41 and 57 nM and 23 nM for 40, respectively [104]. When tested against BACE1 (β-site Amyloid precursor protein Cleaving Enzyme respectively [104]. When tested against BACE1 (β-site Amyloid precursor protein Cleaving Enzyme type 1), an enzyme implicated in Alzheimer’s disease, compound 40 was about 12- to 30-fold less typeactive 1), as an an enzyme BACE1 inhibitor implicated compared in Alzheimer’s to cathepsins disease, D and E compound [104]. A number40 was of synthetic about 12- tasiamide to 30-fold less activeB analogues, as an BACE1 such as inhibitor compounds compared 42 and 4 to3 (Figure cathepsins 7), have D andbeen E developed [104]. A numberas selective of syntheticinhibitors of tasiamide B analogues,these aspartic such proteases as compounds [105–107]. 42Compoundand 43 (Figure 42 was 7found), have to beenbe highly developed selectivity as for selective cathepsin inhibitors D of thesewith aspartic 576-fold proteasesover catheps [105in E– 107and]. 554 Compound-fold over BACE142 was [106 found]. In another to be highly study by selectivity Li et al., synthetic for cathepsin D withcompound 576-fold 43 over (Figure cathepsin 6) displayed E and significant 554-fold overselective BACE1 inhibitory [106]. activity In another against study cathepsin by Li etD al.,with synthetic compoundIC50 of 3.2943 nM(Figure over cath6) displayedepsin E (72- significantfold) and BACE1 selective (295- inhibitoryfold) [107]. activityThese results against could cathepsin path the D with way for the development of highly selective cathepsin D inhibitors. IC50 of 3.29 nM over cathepsin E (72-fold) and BACE1 (295-fold) [107]. These results could path the Symplocin A (44) (Figure 7) is another highly potent cathepsin E inhibitor isolated from the way for the development of highly selective cathepsin D inhibitors. Bahamian cyanobacterium, Symploca sp. [108]. Symplocin A is structurally related to grassypeptins Symplocin A (44) (Figure7) is another highly potent cathepsin E inhibitor isolated from the in having a statine-unit. In addition to Marfey’s method, a new strategy using 2-naphthacyl esters of BahamianN,N-dimethylamino cyanobacterium, and 2-hydroxySymploca acidssp. were [108 employed]. Symplocin for absolute A is structurally stereochemistry related determination to grassypeptins in havingof this amolecule. statine-unit. Symplocin In addition A is a potent to Marfey’s inhibitor method,of cathepsin a new E with strategy IC50 value using of 300 2-naphthacyl pM, which is esters of N,comparableN-dimethylamino to that andof pepstatin, 2-hydroxy a acidsknown were inhibitor employed of aspartyl for absolute proteases. stereochemistry Taken together, determination the ofbiological this molecule. data of Symplocinthese series Aof isstatine a potent-containing inhibitor compounds, of cathepsin including E with grassystati IC50 valuens, tasiamides, of 300 pM, which isand comparable symplocin toA, thatshowed of pepstatin,that selectivity a known can be tuned inhibitor and these of aspartyl structural proteases. scaffolds can Taken serve together, as a the biologicalstarting point data for of development these series of selective statine-containing aspartic protease compounds, inhibitors. including grassystatins, tasiamides, and symplocin A, showed that selectivity can be tuned and these structural scaffolds can serve as a 2.3.4. β-Secretase 1 (BACE1) Inhibitors starting point for development of selective aspartic protease inhibitors.

Molecules 2020, 25, 2197 16 of 30

2.3.4. β-Secretase 1 (BACE1) Inhibitors Molecules 2019, 24, x FOR PEER REVIEW 16 of 30 Tasiamide B Tasiamide B 41 Tasiamide BB ( (4)1 (Figure) (Figure7) is7 a) statine-containingis a statine-containing linear linear depsipeptide depsipeptide shown toshown inhibit to the inhibit aspartic the protease, BACE1, with an IC50 of 0.19 µM[105,109]. BACE1, also known as β-secretase 1, is a potential aspartic protease, BACE1, with an IC50 of 0.19 µM [105,109]. BACE1, also known as β-secretase 1, is a drugpotential target drug for target the treatment for the treatment of Alzheimer’s of Alzheimer’s disease (AD) disease due (AD) to its due involvement to its involvement in the abnormal in the β productionabnormal production of -amyloid of β plaques-amyloid in plaques AD patients in AD [110 patients,111]. The[110, total111].synthesis The total ofsynthesis tasiamide of tasiamide B led to a revisionB led to a in revision stereochemistry in stereochemistry on the structure on the ofstructure the original of the reported original molecule reported [molecule112]. Several [112 synthetic]. Several tasiamidesynthetic tasiamide B-analogues B-analogues have been synthesizedhave been synthesized with superior with inhibitory superior activities inhibitory against activities BACE1. against For instance,BACE1. hybridFor instance, molecules hybrid containing molecules structural containing features ofstructural tasiamide features B and sulfonamide-containing of tasiamide B and isophthalicsulfonamide acid-containing unit, such isophthalic as compounds acid unit,45 andsuch46 as (Figurecompounds8) showed 45 and potent 46 (Figure inhibition 8) showed of BACE1 potent with IC of 128 nM and 57.2 nM, respectively [105]. Moreover, these synthetic molecules showed inhibition50 of BACE1 with IC50 of 128 nM and 57.2 nM, respectively [105]. Moreover, these synthetic γ selectivitymolecules forshowed BACE1 selectivity over -secretase for BACE1 and over compound γ-secretase38 exhibited and compoundin vivo 38activity exhibited by reducingin vivo activity levels β ofby amyloidreducing -peptidelevels of inamyloid brain of β rodent-peptide [105 in]. brain Recent of SARrodent studies [105]. based Recent on SAR 19 synthetic studies analoguesbased on 19 of tasiamidesynthetic analogues B revealed of the tasiamide importance B revealed of the hydrophobicthe importance substituents, of the hydrophobic valine, leucine, substituents, alanine, valine, and phenylalanine,leucine, alanine, for and inhibitory phenylalanine, activity for against inhibi BACE1tory activity [113]. against BACE1 [113].

O O S RN CH3 OH O O H H H N N N N N N H O O O O OCH O 3

= 45 R H 46 R = CH 3

Figure 8. Tasiamide B-relatedB-related compounds.compounds. 2.4. Interference of the Actin and Microtubule Filaments 2.4. Interference of the Actin and Microtubule Filaments Dolastatins 10/15 Dolastatins 10/15 One of the earliest examples of potent microtubule inhibitors reported from marine cyanobacteria are theOne dolastatin of the class earliest of molecules, examples including of potent dolastatins microtubule 10 (47) and inhibitors 15 (Figure reported9)[ 114]. Thefrom dolastatins marine werecyanobacteria originally are isolated the dolastatin from the Indianclass of Ocean molecules, sea hare, includingDolabella dolastatins auricularia. 10 However, (47) and these 15 (Figure cytotoxic 9) compounds[114]. The dolastatins are now known were tooriginally be produced isolated by marine from the cyanobacteria Indian Ocean due sea to the hare, isolation Dolabella of a auricularia number of. dolastatin-relatedHowever, these cytotoxic molecules compounds from these microbesare now known [115]. Dolastatins to be produced 10, 15 by and marine their synthetic cyanobacteria analogues, due includingto the isolation the auristatins, of a number have of dolastatin undergone-related a number molecules of clinical from trials these as microbes anticancer [115 agents]. Dolastatins [11]. It was 10, eventually15 and their realized synthetic that analogues, the auristatins including are the of greatauristatins, value have as payloads undergone in antibody a number drug of clinical conjugates trials (ADCs)as anticancer formulation agents [11]. [116 ].It Thiswas eventually discovery ledrealized to the that FDA-approved the auristatins ADC are of brentuximab great value vedotinas payloads (48) (Figurein antibody9) in drug 2011 forconjugat the treatmentes (ADCs) of formulation elapsed or refractory [116]. This Hodgkin’s discovery lymphomaled to the FDA as well-approved as systemic ADC anaplasticbrentuximab large vedotin cell lymphoma (48) (Figure [117 ].9) Brentuximabin 2011 for the vedotin treatment targets of CD30 elapsed of tumor or refractory cells and Hodgkin selectively’s deliverslymphoma monomethyl as well as auristatin systemic Eana (MMAE)plastic intolarge the cell cells lymphoma and induces [117 cancer]. Brentuximab cell apoptosis. vedotin Numerous targets modificationsCD30 of tumor of cells the N- and and selectively C-terminal delivers groups monomethyl in auristatins auristatin have been E synthesized. (MMAE) into A reviewthe cells on and the designinduces of cancer new auristatins cell apoptosis. and Numerous SAR analysis modifications has been published of the N- [ 118and]. C Currently,-terminal groups more than in auristatins 16 ADCs, incorporatinghave been synthesized. auristatins asA payloads,review on are the currently design inof clinicalnew auristatins trials for cancerand SAR therapy analysis [119 ].has been published [118]. Currently, more than 16 ADCs, incorporating auristatins as payloads, are currently in clinical trials for cancer therapy [119].

Molecules 2020, 25, 2197 17 of 30 Molecules 2019, 24, x FOR PEER REVIEW 17 of 30

FigureFigure 9.9. DolastatinDolastatin 10 and brentuximab vedotin.

2.5.2.5. Sec61 Sec61 Protein Protein Translocation Translocation Channels Channels

2.5.1.2.5.1. Apratoxin Apratoxin AA TheThe apratoxins apratoxins areare aa novelnovel classclass ofof potentpotent cytotoxiccytotoxic cyclodepsipeptidescyclodepsipeptides reported reported from from several several LyngbyaLyngbyasp. sp. strains strains collected collected fromfrom variousvarious locations.locations. They possess significant significant biological biological activities activities in in thethe nanomolar nanomolar rangerange whenwhen testedtested againstagainst aa panelpanel ofof cancercancer cell lines, including HT29, HT29, HeLa, HeLa, and and U2OS.U2OS. To To date, date, a a total total of of nine nine apratoxin-relatedapratoxin-related compoundscompounds have been reported with with apratoxin apratoxin A A (49(4)9) (Figure (Figure 10 10)) being being the the most most cytotoxic cytotoxic [120 [120].]. TheThe biosynthetic gene cluster cluster of of apratoxin apratoxin A A was was identifiedidentified via via a single-cella single-cell genome-amplification genome-amplification approach approach and and reveals reveals a PKS-type a PKS-type loading loading module module and nineand extension nine extension modules, modules, including including four polyketide four polyketide synthases synthases (PKS) and (PKS) five and nonribosomal five nonribosomal peptide synthetasespeptide synthetases (NRPS) [ 121(NRPS)]. It was[121 recently]. It was recently reported reported that the thattert-butyl the tert group-butyl in group apratoxin in apra Atoxin is formed A is asformed a pivaloyl as a acyl pivaloyl carrier acyl protein carrier by protein AprA, the by PKSAprA, loading the PKS module loading of the module apratoxin of the A aprat biosyntheticoxin A pathwaybiosynthetic [122 pathway]. Due to [122 the]. significantDue to the significant potency of potency apratoxins, of apratoxins, a number a number of synthetic of synthetic efforts efforts on the generationon the generation of potent synthetic of potent analogues synthetic have analogues been achieved. have been For achieved.instance, syntheticFor instance, analogues, synthetic such asanalogues, apratoxin such S4 (50 as) and apratoxin apratoxins S4 (50 S7) and (51) apratoxins to S9 (53), with S7 (51 improved) to S9 (53 antitumor), with improved activity, antitumor inhibitory secretionactivity, ofinhibitory the angiogenic secretion vascular of the endothelialangiogenic vascular growth factor endothelial –A (VEGF-A) growth andfactor tolerability –A (VEGF in-A human) and HCT116tolerability xenograft in human mouse HCT116 model xenograft at subnanomolar mouse mo ICdel50 concentrationsat subnanomolar have IC50 been concentrations reported(Table have4) (Figurebeen reported 10)[123(Table,124]. The4) (Figure structures 10) [ of123 these,124]. synthetic The structures molecules of these are basedsynthetic on themolecules structural are features based on of apratoxinsthe structural A (49 features) and E of(55 apratoxins), giving rise A to(49 the) and apratoxin E (55), Agiving/E hybrid rise structure, to the apratoxin differing A/E in various hybrid degreesstructure of, methylationdiffering in various at C34 degrees as well asof methylation epimeric configuration at C34 as well at as C30. epimeric From theconfiguration biological at data, C30. it wasFrom revealed the biological that the data, configuration it was revealed of C-34 that is the independent configuration of the of C cytotoxic-34 is independent activity (Table of the4). cytotoxic activity (Table 4).

Molecules 2020, 25, 2197 18 of 30 Molecules 2019, 24, x FOR PEER REVIEW 18 of 30

OCH3 OCH3

S S H H R1 N N R N 30 N 34 2 O OH O OH N O N O O O O O O O N N N O N O

ra ox n , = , = Apratoxin A (49) Ap t i S4 (50) 30S R1 CH 3 R2 H ra ox n , = = Ap t i S7 (51) 30S R1 R2 H ra ox n , = = Ap t i S8 (52) 30S R1 R2 CH 3 ra ox n , = , = Ap t i S9 ( 53) 3 0R R 1 C H3 R 2 H ra ox n , = = Ap t i S10 (54) 30R R1 R2 CH3

OCH3 OCH3

S O H H N N N N O O OH N O N O O O O O O O N N N O N O

ra ox n xa ratoxin A Ap t i E (55) O p (56)

FigureFigure 10.10. Apratoxin A and related compounds.compounds.

TableTable 4.4. ActivitiesActivities ofof apratoxin-relatedapratoxin-related analogues,analogues, apratoxins S4 (50),), S7S7 ((5151)–S9)–S9 ((5353)) onon viabilityviability ofof HCT116HCT116 cellcell andand secretionsecretion ofof VEGF-AVEGF-A (IC(IC5050 nM).

ApratoxinApratoxin HCT116HCT116 VEGF-A Secretion VEGF-A Secretion S4 (50)S4 (50) 1.431.43 0.32 0.32 S7 (51) 1.25 0.30 S8 (52)S7 (51) 1.991.25 0.30 0.47 S9 (53)S8 (52) 0.691.99 0.47 0.12 S9 (53) 0.69 0.12 Further studies on apratoxin S4 (50), revealed the molecule to possess potent antiangiogenic activityFurther by inhibiting studies theon activationapratoxin of S4 retinal (50), endothelialrevealed the cells molecule and pericytes to possess through potent mediating antiangiogenic multiple angiogenicactivity by pathwaysinhibiting [125the]. activation This finding of retinal could pathendothelial the way cells for the and development pericytes through of apratoxin mediating S4 as am potentialultiple angiogenic cure for prevention pathways or [125 treatment]. This offinding vision could loss. A path scalable the synthesisway for the of a development stable analogue, of apratoxinapratoxin S10S4 as (54 a) (Figurepotential 10 ),cure was for shown prevention to potently or treatment inhibit angiogenesis of vision loss.in vitro A scalableand growth synthesis of cancer of a cellsstable from analogue, vascularized apratox tumorsin S10 [(12654) ].(Figure A recent 10), study was shown revealed to another potently synthetic inhibit angiogenesis analogue, apratoxin in vitro S10,and growth as a potential of cancer anti-pancreatic cells from vascularized cancer agent tum [127ors]. [ The126]. study A recent showed study that revealed the molecule another inhibitedsynthetic analogue, apratoxin S10, as a potential anti-pancreatic cancer agent [127]. The study showed that the molecule inhibited the growth of established and patient-derived primary pancreatic cancer cells.

Molecules 2020, 25, 2197 19 of 30 the growth of established and patient-derived primary pancreatic cancer cells. The growth inhibitory activity of apratoxin S10 on pancreatic cancer cells was due to the downregulation of multiple receptor tyrosine kinases as well as inhibition of growth factor and cytokine secretion [127]. A series of mechanistic studies on apratoxin A (49) revealed its ability to interfere with specific cellular signaling pathways as well as protein interactions involved with the formation and maintenance of cancer cells. Using functional genomics approach, apratoxin A was found to exert its antiproliferative property through induction of G1 cell cycle arrest and apoptosis by antagonism of the fibroblast growth factor (FGF) signaling via Signal Transducer and Activator of Transcription 3 (STAT3) [128]. In another study based on a synthetic oxazoline analog of apratoxin A, it was found that 56 (Figure 10) stabilizes the Hsp90 (heat shock protein 90) client proteins-Hsc70 /Hsp70 interaction, thereby inhibiting the function of Hsp90 [129]. The inhibition of Hsp90 resulted in the promotion of Hsp90 client proteins degradation via chaperone-mediated autophagy. Apratoxin A was also found to inhibit the secretory pathway by preventing cotranslational translocation of newly synthesized secretory and membrane proteins into the ER [130]. The specific mode of action of apratoxin A was eventually elucidated in two studies conducted by Paatero et al. and Huang et al. [131,132]. Apratoxin A kills cancer cells by directly blocking the Sec61 protein translocation channel [131,132]. Specifically, apratoxin A prevents protein translocation into the ER by direct binding with the central subunit of the protein translocation channel, Sec61α [131]. Binding of apratoxin A on the luminal end of the Sec61 lateral gate resulted in blocking the biogenesis of a range of Sec61 clients [131]. In addition, pathologic studies revealed apratoxin A to target pancreas, resulting in severe pancreatic atrophy in apratoxin A-treated animals [132]. The importance of Sec61 as a potential drug target is a promising concept for anticancer therapy. Recently, it was reported that the function of human epidermal growth factor receptor 3 (HER3), implicated in several cancer types, can be inhibited through direct binding of Sec61 with substrate-specific Sec61 inhibitor, cotransin [133].

2.5.2. Coibamide A Coibamide A (57) (Figure 11) is a structurally novel lariat-type cyclic depsipeptide, with potent antiproliferative properties, reported from the Panamanian marine cyanobacterium, Leptolyngbya sp. [134]. The complete structure of coibamide A (57) was deduced by various 2D NMR spectroscopic experiments, including COSY, TOCSY, multiplicity-edited HSQS, HSQC-TOCSY, HMBC, H2BC, 1H-15N gHMBC, and ROESY as well as mass spectroscopic data. This molecule possesses a high degree of N-methylation with eight out of 11 residues being N-methylated. More importantly, coibamide A displayed potent cytotoxicity against NCI-H460 lung cancer cells and mouse neuro-2a cells, with LC50s reported to be less than 23 nM. The compound was evaluated in the NCI’s panel of 60 cancer cell lines and it exhibited significant activities against MDA-MB-231, LOX IMVI, HL-60(TB), and SNB-75 at IC50 values of 2.8 nM, 7.4 nM, 7.4 nM and 7.6 nM, respectively [134]. Total synthesis of coibamide A was accomplished using solid-phase peptide strategy by several synthetic groups. These synthetic efforts led to the revision on the stereochemical assignment on the original reported molecule as well as generation of potent analogues for SAR studies [135–137]. Several synthetic analogues, such as 58 and 59 (Figure 11), showed either similar inhibitory activity or increased cytotoxicity as compared to the natural product [136,137]. A number of pharmacological studies have also been conducted on coibamide A [138–140]. A recent study, using a synthetic coibamide A photoaffinity probe, showd that coibamide A directly targets the Sec61α subunit of the trimeric Sec61translocon. The binding of the molecule to Sec61 resulted in broad substrate-nonselective inhibition of ER protein import and conferred potent cytotoxicity against specific cancer cell lines [141]. Molecules 2020, 25, 2197 20 of 30 MoleculMoleculeses 2019 2019, ,24 24, ,x x FOR FOR PEER PEER REVIEW REVIEW 2020 of of 30 30

OO OO HH H N N N NN NN OO OO N OO O N N O OO O OO O O OO O OO O HN OO HHNN O O O HN OO OO H HH O O HH H HNN NN OO NN N O OO NN N O N N N N O NN NN NN O N N N O O O O OO OO O O O O O

Coibamide A 57 58 Coibamide A ((57)) 58 O HH NN N N O O N O O N O O O O O O O O HN O O O O HN O O N O N N N O N O N N N N N N N O O O O O O O O O

- - D MeAla11 epimer of coibamide A 59 [ - e a11]-e mer o co am e ( ) [D M Al ] pi f ib id A (59) Figure 11. Coibamide A and related compounds. Figure 11. Coibamide A and related compounds. 2.6.2.6. Interference Interference ofof InflammatoryInflammatory PathwaysPathways 2.6. Interference of Inflammatory Pathways Honaucins Honaucins Honaucins A (60)–C (62) (Figure 12) are potent anti-inflammatory and bacterial quorum sensing Honaucins A A (60 (60)–)–CC (62 (62) (Figure) (Figure 12 ) 12are) potent are potent anti-inflammatory anti-inflammatory and bacterial and bacterial quorum quorum sensing (QS) inhibitory molecules isolated from a collection of Leptolyngbya crossbyana found overgrowing on sensing(QS) inhibitory (QS) inhibitory molecules moleculesisolated from isolated a collection from of a Leptolyngbya collection of crossbyanaLeptolyngbya found crossbyana overgrowingfound on corals in Hawaii [142]. The structure of the major compound, honaucin A, consists of a unit each of overgrowingcorals in Hawaii on [ corals142]. The in structure Hawaii [of142 the]. major The structurecompound, of honaucin the major A, compound, consists of a honaucin unit each A,of consists(S)-3-hydroxy of a unit-γ-butyrolactone each of (S)-3-hydroxy- and 4-chlorocrotonicγ-butyrolactone acid, and connected 4-chlorocrotonic via an ester acid, linkage. connected These via (Smolecules)-3-hydroxy showed-γ-butyrolactone inhibition ofand bioluminescence 4-chlorocrotonic in Vibrioacid, connectedharveyi BB120 via andan esterlipopolysa linkage.ccharide These- anmolecules ester linkage. showed These inhibition molecules of bioluminescence showed inhibition in Vibrio of bioluminescence harveyi BB120 and in Vibriolipopolysa harveyiccharideBB120- andstimulated lipopolysaccharide-stimulated nitric oxide production in nitric the murine oxide productionmacrophage incell the line murine RAW264.7 macrophage [142]. Additional cell line stimulatedpharmacological nitric oxidestudies production performed in on the honaucin murine Ama showedcrophage that cell the line molecule’s RAW264.7 ability [142 ].to Additionalattenuate RAW264.7 [142]. Additional pharmacological studies performed on honaucin A showed that the pharmacologicalinflammation via studies activat ionperformed of the Nrf2 on- AREhonaucin pathway A showed [143]. Synthetic that the molecule’sanalogues, basedability on to honaucin attenuate molecule’s ability to attenuate inflammation via activation of the Nrf2-ARE pathway [143]. Synthetic inflammationA, revealed that via the activat halogenion of atom the atNrf2 the- ARE4-position pathway of the [1 43crotonoic]. Synthetic acid analogues, moiety was based essential on honaucin for both analogues, based on honaucin A, revealed that the halogen atom at the 4-position of the crotonoic acid A,anti revealed-inflammatory that the andhalogen QS inhibitory atom at the activities. 4-position of the crotonoic acid moiety was essential for both moietyanti-inflammatory was essential and for QS both inhibitory anti-inflammatory activities. and QS inhibitory activities. O O O OH O O Cl R O OH O O O O Cl Cl R O O O O O Cl Honaucin A (60) onauc n = H i B (61)O R CH2CH3 Honaucin A 60 onauc n = ( ) Honauci nC (62) R =CH3 H i B (61) R CH2CH3 onauc n = H i C (62) R CH3 O O O O R O O R O O 63 R = Br = 64 R = I 63 R Br 64 R = I Figure 12. Honaucins and related compounds. Figure 12. Honaucins and related compounds. Two synthetic analogues, 63 and 64 (Figure 12), were found to possess enhanced anti- inflammatoryTwo synthetic and QS analogues, inhibitory properties63 and 64 when (Figure compared 12), wereto the naturalfound toproducts possess [142]. enhanced Subsequent anti - inflammatorypharmacological and studiesQS inhibitory conducted properties on the when bromo compared-analogue to of the honaucin natural productsA, compound [142]. 63 Subsequent, showed pharmacological studies conducted on the bromo-analogue of honaucin A, compound 63, showed

Molecules 2020, 25, 2197 21 of 30

Two synthetic analogues, 63 and 64 (Figure 12), were found to possess enhanced anti-inflammatory and QS inhibitory properties when compared to the natural products [142]. Subsequent pharmacological studies conducted on the bromo-analogue of honaucin A, compound 63, showed that it inhibited osteoclastogenesis from macrophage cell line in vitro and reduced the RANKL-induced expression of osteoclast-associated genes, such as MMP9, Cath K, GAB2, C-MYC, C-SRC, MITF, PU 1 and DC-STAMP [144]. These results showed that compound 63 could have potential as a therapeutic drug for treatment of disorders associated with bone loss.

3. Conclusions This review covers a range of unique marine cyanobacterial natural products as well as their synthetic analogues having potent biological activities, including anticancer and antiinfective properties. A majority of these compounds are nitrogen-containing and products of the modular PKS-NRPS metabolic pathways. Due to their exquisite interference against clinically relevant drug targets, such as proteasomes, proteases and microtubule filaments, they represent important lead compounds for further development into therapeutic drugs. A number of noteworthy drug leads include largazole, apratoxin A, carmaphycins, gallinamide A and the dolastatin class of molecules. Drug candidates, such as the largazole-based synthetic analogue, OKI-179, and apratoxin S4 have been identified from synthetic analogues of these natural drug leads and are currently undergoing or being considered for clinical testing. In addition, a high number of auristatin-based compounds, formulated as ADCs, are undergoing clinical testing as anticancer agents. Unique structural features attributed to marine cyanobacterial compounds could account for their high potency. These include high degree of N-methylation (e.g., coibamide A), macrocycles with incorporation of polyketide moiety (e.g., apratoxins), presence of heterocycles, such as thiazole, thiazoline, oxazole oxazoline and methoxypyrrolinone (e.g., apratoxins, gallinamide A, dolastatins 10 and 15), incorporation of D- and non-proteinogenic amino acids (e.g., 2-aminobutenoic acid unit in lyngbyastatin 7) as well as peptide acylation (e.g., largazole). These structural features contribute to compound permeability and adoption of desired conformation for interaction of cellular drug targets. Moreover, combination of these structural motifs expands the chemical space of marine cyanobacterial natural products. The range of molecules presented in this review is by no means exhaustive, as there are numerous potent cyanobacterial compounds reported in the literature and are beyond the scope of this review. These compounds include the highly cytotoxic compounds, bisebromoamide (65) and aurilide-class of compounds (e.g., 66), antiinfective molecules, almiramides (e.g., 67) and janadolide (68), anti-inflammatory molecule, biseokeaniamide A (69) as well as cannabimimetics/CNS modulatory agents, such as mooreamide A and serinolamides (e.g., 70) (Figure 13)[145–153]. Further biological evaluation and synthesis of analogues have also been initiated for some of these compounds [154–159]. The search for novel bioactive cyanobacterial compounds has also been expedited with recent advances in genomic and metabolomics techniques, e.g., the integrated use of mass spectrometric based molecular networking and genomic approaches [160,161]. It is without a doubt that these prokaryotic marine cyanobacteria will continue to provide essential drug leads in drug discovery and development efforts. Molecules 2020, 25, 2197 22 of 30 Molecules 2019, 24, x FOR PEER REVIEW 22 of 30

O O N O O N O N O O N H N N N S N H N O N O N H O O HN O O OH O O

OH O Br

Bisebromoamide (65) Lagunamide A (66)

O O O H N N N N N NH2 O O O O

Almiramide A (67)

O O O N O N H N N O HN N O O O O N O S N HN N N N O O O

Janadolide (68) Biseokeaniamide A (69)

OH O O N

CH3 Serinolamide A (70)

FigureFigure 13. 13.Other Other potent potent marinemarine cyanobacteriacyanobacteria compounds.compounds.

Author Contributions: All authors discussed, commented on and wrote the manuscript. All authors have read Authorand agreed Contributions: to the publishedAll authors version discussed, of the manuscript. commented on and wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. ConflictsConflicts of of Interest: Interest:The The authors authors declare declare no no conflict conflict of of interest. interest.

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