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. 2024 Apr 19;15(5):696–705. doi: 10.1021/acsmedchemlett.4c00094

Structure–Activity Relationship Study of Biselyngbyolide B Reveals Mitochondrial Fission-Induced Cytotoxicity in Cancer

Pratiti Mandal §,#, Debobrata Paul ∥, Himangshu Sharma ∥, Sanu Saha ∥, Partha Chakrabarti §,#,*, Rajib Kumar Goswami ∥,*
PMCID: PMC11089557  PMID: 38746877

Abstract

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A systematic structure–activity relationship study of the potent anticancer marine macrolide biselyngbyolide B has been accomplished. A total of 11 structural variants of the parent natural product, of which 2 are natural analogues, have been studied against a human colorectal carcinoma cell line. The requisite functional units of the parent molecule responsible for the cytotoxic activities have been disclosed. Biselyngbyolide C, one of the natural analogues of biselyngbyolide B, has been studied in depth to explore its molecular mechanism. Interestingly, the in vitro data demonstrated an induction of dynamin-related protein 1-mediated mitochondrial fission and reactive oxygen species production which led to activation of ASK1/P38/JNK-mediated apoptosis in colon cancer cells as an important pathway for biselyngbyolide B-mediated cytotoxicity. Notably, this study revealed that a macrolide participated in mitochondrial fission to promote apoptosis of cancer cells, providing new insight.

Keywords: Marine macrolide, Biselyngbyolide B, Structure−activity relationship study, Mitochondrial fission, Reactive oxygen species, Apoptosis

Marine natural products are regarded as treasure troves of architecturally challenging and bioactive secondary metabolites, many of which contributed quite remarkably to the therapy of tumor-related diseases.1−5 Finding and synthesizing new natural scaffolds and derivatives of them are thus important for the development of novel anticancer therapeutics. Mitochondria, the powerhouse of cells, regulate many key processes, including cell growth and cell cycle, and mitochondrial apoptosis is considered as one of the important targets for cancer chemotherapy.6−10 There are a few recent reports where the natural products are shown to serve such activities, but modulation of mitochondrial-dependent pathways is diverse.11−14 Thus, uncovering new chemotherapeutics affecting the mitochondrial dynamics could be quite impactful in cancer research.15−17 The use of macrolides in drug development is increasing steadily due to their rigid architectures and requisite structural complexity.18−21 Suenaga and co-workers discovered a series of 18-membered marine macrolides—biselyngbyolide B (1), biselyngbyaside (2), biselyngbyolide A (3), biselyngbyaside B (4), biselyngbyolide C (5), biselyngbyaside E (6), biselyngbyaside D (7), and biselyngbyaside C (8) (see Figure 1)—from cyanobacterium Lyngbya sp.22−26 Many of them exhibited growth inhibitory activities against a number of human carcinoma cell lines in submicromolar concentrations. Some of these members impeded osteoclastogenesis and controlled intracellular Ca2+ concentrations for instigating endoplasmic reticulum stress.27 The C3-glycosylated versions of these macrolides were found to have less apoptosis-inducing ability compared to their aglycon variations. Notably, biselyngbyolides A and C could be considered as the natural analogues of biselyngbyolide B, as hydroxylation and saturation, respectively, at the C4–C5 olefin of the parent molecule could generate them. The interesting structural features as well as promising bioactivities of these macrolides attracted attention from the scientific community which has culminated in the partial28−30 and total31−34 syntheses of many of them. Suenaga and co-workers disclosed elegant synthetic routes for the total synthesis of biselyngbyolide A (3),31 biselyngbyolide B (1),32 and biselyngbyaside (2)33 using the Stille macrocyclization strategy. We have also developed synthetic routes for the total synthesis of biselyngbyolide B (1),35 biselyngbyolides A (3) and C (5)36 adopting the Heck macrocyclization approach, whereas Maier et al.34 reported a successful total synthesis of biselyngbyolide B (1) following the same macrocyclization strategy.

Figure 1.

Figure 1

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Structure of the biselyngbyaside family of macrolides.

Furthermore, Suenaga and co-workers reported the structure–activity relationship (SAR) study of biselyngbyolide B against malarial parasites using three variants prepared by introducing hydrophilic functionalities (alcohol, amide) on the C20–C23 side chain.37 However, there is no such study on systematic modification of biselyngbyolide B nor on the mechanistic basis for any of its congeners known for cytotoxic activity to date, which prompted us to dig into the molecular basis of biselyngbyolide B and its analogues more in-depth. Herein, we report a SAR study of biselyngbyolide B using 11 of its analogues, of which 2 are natural variants, namely biselyngbyolides A (3) and C (5). This study discloses that macrolides promote mitochondrial dysfunction via fission in cancer cells and subsequent cellular apoptosis.

The systematic SAR study of the marine macrolide biselyngbyolide B or any of the members of this family remained largely unexplored. Building on the chemistry that we had developed for the total synthesis of biselyngbyolide B and its natural variants biselyngbyolides A and C,35,36 we planned to evaluate the necessity of different structural units of the parent natural product for its cytotoxicity. These SAR inquiries involved the investigation of the effect of modification of upper and lower halves of the molecule. During this study, the conjugated olefin (C12–C15) was not considered for modifications, as it was found to be an essential unit for the activity conceived from the data reported for biselyngbyaside C (7),23,24 one of the members of this family of natural products. We initially became interested to understand the role of the stereochemistry of the C17-hydroxy center as well as the unsaturation in the C18–C23 segment of the parent molecule and planned to synthesize compounds 9 (configuration of C17-OH inverted), 10 (C18–C23 segment replaced with n-pentyl group), and 11 (C18–C23 segment replaced with n-pentyl group along with inversion of C17-OH stereochemistry) (Figure 2). Notably, the design of compound 11 was conceived to ascertain the configurational effect of the C17-OH center further, in addition to compound 9, which could be insightful in the subsequent design of other structural variants.

Figure 2.

Figure 2

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Initially designed unnatural analogues of biselyngbyolide B (9–11).

The synthesis of the initially planned analogues of biselyngbyolide B by modification of the upper segment is depicted in Scheme 1. The known alcohol 12(35) was oxidized to the corresponding aldehyde using DMP and treated further with (−)-Ipc2B(allyl)borane following the Brown allylation protocol to provide alcohol 13 in 77% yield with good enantioselectivity.35,36 Next, the known compound 14(35) mixed with its minor inseparable Z-vinyl iodide (E/Z ∼4:1) was esterified with alcohol 13 mixed with its inseparable minor enantiomer. The esterified counterpart generated from the minor enantiomer of 13 was discarded during the purification process. The corresponding esterified major isomer bearing the minor Z-vinyl counterpart was subjected to intramolecular Heck coupling to get the corresponding cyclized product in pure form, as the minor Z-vinyl corresponding ester did not respond to the macrocyclization reaction.35,36 The requisite cyclized product was further reacted with TBAF to access compound 9 in a good yield. On the other hand, commercially available hexanal (15) was converted to alcohols 16 and ent-16 in a following Brown allylation sequence which were subsequently transmuted to compounds 10 and 11 separately following the same chemistry as for compound 9. Notably, the minor enantiomer produced during allylation in both cases was purified in the esterification step.

Scheme 1. Completion of Synthesis of Analogues 9–11.

Scheme 1

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Having the synthesized analogues (9–11) in hand, we then evaluated the in vitro cytotoxic effect in human colorectal carcinoma (HCT116) and in noncancerous human embryonic kidney (HEK293T) cell lines using the MTT assay (Table 1). It was revealed that the inversion of stereochemistry of C17-OH (9) as well as introduction of an n-pentyl group (10) by replacing the C18–C23 segment of the parent molecule reduced their cytotoxic effect around 2.5 times compared to biselyngbyolide B (1). Inversion of the C17-OH center of analogue 10, i.e., giving analogue 11, showed further reduction of activity in HCT116 cells and reached a similar effect as observed for noncancerous HEK293T. These initial findings confirmed unambiguously that the stereochemistry of the C17-OH center played a significant role. Thus, the synthesis of other variants of the parent natural product has been planned for a subsequent study, keeping the stereochemistry of the C17-OH center unaltered.

Table 1. Cellular IC50 Values for Biselyngbyolide B (1) and Its Analogues 9–11.

  IC50 Value (μM)
Compound HEK293T HCT116
1 20.6 3.2
9 18.1 8.9
10 14.3 8.1
11 20.2 23.0
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Other unnatural analogues (17–22) of biselyngbyolide B are shown in Figure 3. The removal of C21-olefin (17) and replacement of the hydrophobic C18–C23 part with the tolyl counterpart (18) in the upper half as well as the removal of C10-methyl (19) or C8-methyl (20), change of C7-OMe to C7-OH (21), and removal of C3-hydroxy (22) in the lower half of biselyngbyolide B were taken into account in the next course of our study.

Figure 3.

Figure 3

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Other unnatural analogues of biselyngbyolide B (17–22).

The synthesis of analogues 17 and 18 is described in Scheme 2. Following the identical chemistry as for analogue 9, analogue 17 was prepared from the known alcohol 23(38) via intermediate 24, whereas analogue 18 was constructed from commercially available aldehyde 25 similarly via intermediate 26 in good overall yield.

Scheme 2. Completion of Synthesis of Analogues 17 and 18.

Scheme 2

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The synthesis of analogues 19 and 20 is described in Scheme 3. The known compounds 27(35) and 28(39) were oxidized separately using Swern conditions and subjected to Wittig olefination using triphenylcarbethoxyethylidinephosphorane and triphenylcarbethoxymethylenephosphorane, respectively, to obtain compounds 29 and 30, respectively, in good yield and diastereoselectivity. The purified major isomers were then treated separately with DIBAL-H cautiously to get the corresponding aldehydes, which were reacted further with the known thiothiazolidine 31 following the Crimmins acetate aldol protocol to access compounds 32 and 33, respectively, as the major isomers.35,36 The corresponding minor aldol products (epi-32 and epi-33) were discarded during the chromatographic purification. Next, compounds 32 and 33 were treated separately with NaBH4 followed by TrCl/Et3N/DMAP to provide compounds 34 and 35, respectively, which were further methylated and subjected to TBDPS ether deprotection to furnish compounds 36 and 37, respectively, in good overall yield. The free primary alcohols of compounds 36 and 37 were oxidized using IBX separately, and the resultant aldehydes were subjected to Takai olefination followed by in situ trityl deprotection to access compounds 38 and 39, respectively.35,36 The minor Z-isomers produced in the olefination reaction remained inseparable at this stage and were removed in a later step of the synthesis. Next, compounds 38 and 39 were transmuted separately to sulfones 41 and 42, respectively, using 1-phenyl-1H-tetrazole-5-thiol (40) following the Mitsunobu protocol and subsequent oxidation by (NH4)6Mo7O24·4H2O/H2O2. Sulfones 41 and 42 were then subjected to Julia–Kocienski olefination35,36 separately with the known aldehyde 43(35) to access compounds 44 and 45, respectively. The minor Z-isomers generated during Julia–Kocienski olefination were separated by silica gel column chromatographic purification. The primary TBS ether forms of compounds 44 and 45 were then deprotected separately, and the resulting alcohols were oxidized to acids 46 and 47, respectively, following two-step oxidation protocols. Both acids 46 and 47 were esterified separately with the known alcohol ent-13(35) mixed with its inseparable minor enantiomer (13). The requisite purified esters were then subjected to Heck macrocyclization35,36 to furnish pure compounds 48 and 49, respectively. The esterified products bearing inseparable Z-vinyl iodide did not participate in Heck cyclization, similar to our earlier observation. Finally, the TBS ether forms of both compounds 48 and 49 were deprotected to obtain the required analogues 19 and 20, respectively, in good overall yield.

Scheme 3. Synthesis of Analogues 19 and 20.

Scheme 3

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The synthesis of analogues 21 and 22 is shown in Scheme 4. The known compound 50(35) was treated with NaBH4 to get the corresponding alcohol, which was subjected to acetonide protection followed by TBDPS ether deprotection to produce compound 51. Next, it was oxidized and subsequently exposed to Takai olefination to access compound 52 (E/Z = 10:1). Compound 52 mixed with its inseparable Z-isomer was then transmuted in four steps to alcohol 53, which was subjected further to Mitsunobu reaction using thiol 40 followed by oxidation using (NH4)6Mo7O24·4H2O/H2O2 to access the corresponding sulfone. It was then subjected to Julia–Kocienski olefination with the known aldehyde 43(35) to get compound 54 (E/Z = 10:1). Next, compound 54 mixed with its inseparable minor Z-vinyl iodide was treated with CSA, and the resulting alcohol was oxidized to acid 55 following a two-step oxidation protocol. Acid 55 was esterified to the known alcohol ent-13(35) mixed with its inseparable minor enantiomer (13). The corresponding minor esterified product was removed during the purification. The required major esterified product was subjected further to Heck macrocyclization to access the requisite macrolide having no counterpart corresponding to the Z-vinyl iodide. Finally, all silyl groups were deprotected using TBAF to produce compound 21. On the other hand, the known sulfone 56(35) mixed with its minor Z-counterpart was subjected to Julia–Kocienski olefination with the above aldehyde prepared from alcohol 27 to produce the corresponding coupled products mixed with its corresponding minor Z-vinyl iodide similarly as observed earlier.

Scheme 4. Synthesis of Analogues 21 and 22.

Scheme 4

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The corresponding minor Julia–Kocienski olefination product was removed easily during the purification. Next, the respective major Julia–Kocienski olefination product was treated with TBAF to yield compound 57 mixed with its inseparable minor Z-vinyl counterpart, which was converted to acid 58 in three steps and subjected further for esterification with the known alcohol ent-13. The corresponding pure esterified product was then cyclized using Heck macrocyclization and finally treated with TBAF to obtain pure compound 22. No cyclized product from the minor Z-vinyl counterpart was formed in this case, too.

The cytotoxic effects of the synthesized analogues (17–22) have been evaluated (Table 2) against HCT116 and HEK293T cell lines. Our previously synthesized natural analogues of biselyngbyolide B, i.e., biselyngbyolides A (3) and C (5),36 were also included in this MTT assay (Table 2). The study revealed clearly that any modifications except in the C4–C5 region of biselyngbyolide B reduced the cytotoxicity of the parent natural product considerably. Interestingly, biselyngbyolides A (3) and C (5) exhibit superior and selective cytotoxic effects on cancerous cell. We had observed earlier that biselyngbyolides A (3) and C (5) triggered antitumorigenic effects and induced cell death via an apoptotic pathway36 against different cancer cell lines. However, their mode of action was not known.

Table 2. Cellular IC50 value of Analogues (3, 5, and 17–22) of Biselyngbyolide B.

  IC50 Value (μM)
Compound HEK293T HCT116
17 11.3 8.1
18 16.5 7.8
19 12.5 6.8
20 11.8 7.1
21 21.6 6.9
22 15.2 15.6
3 20.8 0.1
5 23.7 0.1
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The most active natural analogue biselyngbyolide C (5) and an inactive unnatural analogue (11) were chosen for elucidating the molecular basis of cytotoxicity of the biselyngbyolide class of macrolides. HCT116 cells were first stained with mitotracker red, which accumulated in the mitochondria upon proton gradient, and CellROX, which exhibited fluorescence in the presence of reactive oxygen species (ROS). As shown in Figure 4A, biselyngbyolide C (5) caused a dose-dependent decrease in mitochondrial membrane potential with a corresponding increase in ROS production, while unnatural analogue 11 had no significant impact. Quenching of ROS by N-acetyl cysteine (NAC) suppressed cleavage of two apoptosis markers, caspase 3 and PARP, indicating that biselyngbyolide C-induced cytotoxicity is ROS dependent (Figure 4B). To further elucidate whether ROS was sourced from the mitochondria, we transfected the cells with mito-GFP and stained them with CellROX. Treatment of biselyngbyolide C (5) led to significant colocalization of green and red fluorescence, suggesting a mitochondrial origin of the ROS (Figure 4C). Treating the cells with biselyngbyolide C (5) and scavenging mitochondrial ROS by MitoTEMPO consistently revealed a drop in MitoSOX fluorescence, an indicator of mitochondrial superoxide and the number of dead cells by live–dead assay (Figure 4D).

Figure 4.

Figure 4

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Biselyngbyolide C (5) induces mitochondrial ROS and apoptosis. (A) HCT116 cells were treated with or without biselyngbyolide C (5) (0.5,1 μM) and unnatural analogue 11 for 16 h. Mitochondria were stained with MitoTracker Deep Red FM (upper panel) or CellROX Red FM (lower panel) probes for 30 min and observed with a confocal microscope. Scale bar, 10 μm. Data were quantified with the mean fluorescence intensity from five different fields (right panels). (B) Immunoblotting analysis of CC3 and PARP1 expression in HCT116 cells pretreated (1 h) with 5 mM N-acetyl cysteine (NAC) and treated with or without biselyngbyolide C (5) (1 μM) and unnatural analogue 11 for 16 h. β-Actin was used as the loading control. (C) HCT116 cells were transfected with CellLight Mitochondria-GFP, treated with or without biselyngbyolide C (5) (1 μM) and unnatural analogue 11 for 16 h, and stained with CellROX Red FM for 30 min. Colocalization of intensities of Mitochondria-GFP and CellROX Red was quantified (right panel). Scale bar, 10 μm. (D) Mitochondrial ROS were scavenged by 1 h pretreatment of 10 μM MitoTEMPO in HCT116 cells and were treated with or without biselyngbyolide C (5) (1 μM) and unnatural analogue 11 for 16 h. Mitochondria were stained with MitoSOX Red FM, and mitochondrial ROS accumulation was analyzed by confocal microscopy (upper panel). Cytotoxity was analyzed by using a LIVE/DEAD Viability/Cytotoxicity kit. Representative images of mitochondrial ROS and live cells stained with Calcein-AM (green) and dead cells stained with EthD-1 (red) are shown (lower panel). The intensities of MitoSOX Red and percentage of dead cells were quantified. Scale bar, 10 μm. NS, not significant, *p < 0.05, **p < 0.001.

Mitochondrial ROS production has an intricate relationship with mitochondrial fission and fusion dynamics.40 Hence, we looked into the status of fission and fusion proteins following treatment with biselyngbyolide C (5) and unnatural analogue 11. Biselyngbyolide C (5) induced activatory phosphorylation at S616 and conversely downregulated the inhibitory phosphorylation (S637) of mitochondrial fission protein dynamin-related protein (DRP)1 in a time- and dose-dependent manner, while treatment of unnatural analogue 11 induced DRP1 S616 phosphorylation but had no impact on S637 (Figure 5A,B). Levels of fusion protein mitofusin and other structural proteins such as OPA1 and MFF remained unaltered, suggesting biselyngbyolide C (5) might cause mitochondrial fission in colon cancer cells. For direct visualization of mitochondrial morphology, we determined the mitochondrial length and area, two parameters for mitochondrial architecture. We found that biselyngbyolide C (5) and not the unnatural analogue 11 led to a significant deterioration in both mitochondrial length and area (Figure 5C). Time lapse microscopy with Mitotracker Red for 14 h revealed direct visualization of mitochondrial fission (Videos S1 and S2 in Supporting Information), with the emergence of mitochondrial puncta as a readout of fission (Figure 5D). Thus, both biochemical and microscopy data indicate that the treatment of biselyngbyolide C (5) caused mitochondrial fission in colon cancer cells. We next investigated whether mitochondrial ROS production is downstream of fission. To this end, cells were incubated with NAC and mitochondrial dynamics was determined. NAC treatment significantly restored the biselyngbyolide C (5)-induced altered mitochondrial architecture and partially reversed DRP1 phosphorylation (Figure 5E, F). Taken together, biselyngbyolide C (5)-dependent induction of ROS production skews the mitochondrial dynamics toward fission and cellular apoptosis. To further elucidate whether DRP1-mediated mitochondrial fission led to ROS production and cell death, we pretreated the cells with a biochemical inhibitor, mitochondrial division inhibitor-1 (Mdivi-1), which potently inhibits mitochondrial outer membrane permeabilization.41−43 Mdivi-1 potently suppressed biselyngbyolide C-dependent ROS generation and cell death (Figure 6A), with corresponding improvement in mitochondrial membrane potential, as shown by mitotracker fluorescence (Figure 6A, middle panel). Immunoblotting analysis of cleaved PARP1 and caspase 3 further supports the idea that the cytotoxicity and resultant cell death induced by biselyngbyolide C can be recovered with the effective dosage of Mdivi-1 (Figure 6B).

Figure 5.

Figure 5

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Biselyngbyolide C (5) induces cellular apoptosis through DRP1-mediated mitochondrial fission and ROS production. (A, B) Immunoblotting analysis of p-DRP1S616, p-DRP1S637, DRP1, Mitofusin1, Mitofusin2, and MFF expression in a time-dependent manner (0, 8, 16, 24 h) and p-DRP1S616, p-DRP1S637, DRP1, Mitofusin1, and OPA in a dose-dependent manner (0.5, 1 μM) in HCT116 cells treated with biselyngbyolide C (5) and unnatural analogue 11. β-Actin was used as a loading control. (C) HCT116 cells were transfected with CellLight Mitochondria-GFP and treated with or without biselyngbyolide C (5) (1 μM), and unnatural analogue 11 was added for 16 h. Representative confocal images (upper panel) of mitochondrial length and area and quantification (lower panel) were shown. Scale bar, 10 μm. (D) Size of mitochondrial puncta observed in time lapse microscopy after 14 h of biselyngbyolide C (5). (E) HCT116 cells were pretreated (1 h) with or without 5 mM NAC and treated with or without biselyngbyolide C (5) (1 μM) for 16 h. Mitochondria were stained with MitoTracker Deep Red FM probes for 30 min and observed with a confocal microscope. Representative images of mitochondrial morphology (top) with quantification (lower panel) are shown. Scale bar, 10 μm. (F) Immunoblotting analysis of p-DRP1S616, p-DRP1S637, and Mitofusin1 expression after pretreatment (1 h) with or without 5 mM NAC and treatment with or without biselyngbyolide C (5) (1 μM) for 16 h.

Figure 6.

Figure 6

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Mdivi-1 abrogates DRP1-mediated mitochondrial fission and cytotoxicity induced by biselyngbyolide C (5). (A) HCT116 cells were treated with or without biselyngbyolide C (5) (1 μM) for 16 h, and DRP1 was inhibited by treating the cells with 25 μM Mdivi1 for 8 h. Cells were stained with CellROX Red FM, MitoTracker Deep Red FM, and LIVE/DEAD Viability/Cytotoxicity reagents for 30 min and observed with a confocal microscope. Representative images of cellular ROS production (upper panel), mitochondrial morphology, and intensity of red fluorescence (middle panel) and live (green) and dead cells (red) (lower panel) are shown. Scale bar, 10 μm. Data were quantified with the mean fluorescence intensity from five different fields (right panels). (B) Immunoblotting analysis of CC3 and PARP1 expression in HCT116 cells treated with or without biselyngbyolide C (5) (1 μM) for 16 h and post-treated with 25 μM Mdivi-1 for 8 h. β-Actin was used as the loading control. *p < 0.05, **p < 0.001.

We next explored the pathway downstream of ROS responsible for biselyngbyolide C (5)-induced cell deaths. One possible pathway is headed by ASK1,42 which was established by elevated expression of the phosphorylated forms of ASK1, JNK, and p-38 following biselyngbyolide C (5) treatment (Figure 7A). Pretreatment with NAC abrogated ASK1 activation, as shown by reduced phosphorylation of JNK1 (Figure 7B), suggesting biselyngbyolide C (5)-induced ASK1 activation is ROS dependent. For determining the specificity of the ASK1 pathway in biselyngbyolide C (5)-mediated cytotoxicity, we used an ASK1 inhibitor, Selonsertib. Selonsertib not only reduced biselyngbyolide C (5)-mediated phosphorylation of JNK1 and p38 but also significantly protected cells from apoptosis (Figure 7C, D).

Figure 7.

Figure 7

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Biselyngbyolide C (5) induces cellular apoptosis mediated via activation of the ASK1/P38/JNK pathway. (A–C) Immunoblotting analysis of p-JNK, JNK, p-p38, and p-38 expressions after treatment with or without biselyngbyolide C (5) (1 μM) and unnatural analogue 11 for 16 h (A); p-JNK, JNK, p-p38, p-38 expression after pretreatment (1 h) with or without 5 mM NAC prior to biselyngbyolide C (5) treatment (B); and PARP1, CC3, p-JNK, p-p38, and p-38 expression after pretreatment (1 h) with or without 50 μM Selonsertib prior to biselyngbyolide C (5) treatment (C), in HCT116 cells. β-Actin was used as the loading control. (D) LIVE/DEAD assay in HCT116 cells was performed by confocal microscopy. Representative images of cells stained with Calcein-AM (green) and dead cells with EthD-1 (red) are shown. The percentage of dead cells was quantified (right panel). Scale bar, 10 μm.

In summary, we have demonstrated a systematic structure–activity relationship study of anticancer macrolide biselyngbyolide B. The cytotoxic activity of 11 synthesized analogues of biselyngbyolide B has been evaluated, among which natural analogues were found to be the most potent. This study has divulged the role of different functional moieties of the parent molecule toward its cytotoxicity and also disclosed the site for allowed structural modification. The molecular basis of cytotoxic activity of the biselyngbyolide class of natural products was understood using one of the most active natural analogues, biselyngbyolide C, which turned out to induce mitochondrial fission and ROS production. Furthermore, biselyngbyolide C promoted concomitant deregulation of mitochondrial morphology and metabolism along with ROS production that leads to the upregulation of pro-apoptotic ASK1/JNK/P38 pathway. Our study hereby revealed a hitherto unknown mechanism of biselyngbyolide-mediated cytotoxicity, which, however, represents one important pathway for its cytotoxicity. Since the molecular interacting partner of biselyngbyolide remains unknown, the existence of other elusive pathways leading to cytotoxicity cannot be ruled out. Since the role of the mitochondrial metabolism in tumorigenesis is known,43 this study provides insight to pave the path toward development of novel mitochondria-targeting cancer therapeutics associated with the macrolide framework.

Acknowledgments

P.M., D.P., H.S., and S.S. thank the DST-inspire, Indian Association for the Cultivation of Science, Council of Scientific and Industrial Research (CSIR), for research fellowships. Financial support from the Science and Engineering Research Board (CRG/2019/001664 and STR/2021/000002), India, to carry out this work is gratefully acknowledged. We thank Sounak Bhattacharya and Ruby Banerjee Roy for assisting with confocal microscopy and Chayan Banerjee for helping with data analysis with the mitochondrial time-lapse experiment.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.4c00094.

  • Experimental procedure for chemical synthesis of different unnatural analogues of biselyngbyolide B as well as their biological evaluations; 1H and 13C NMR and HRMS of representative compounds (PDF)

  • Video S1, time lapse microscopy with Mitotracker Red for 14 h revealed direct visualization of mitochondrial fission (AVI)

  • Video S2, time lapse microscopy with Mitotracker Red treated with compound 5 for 14 h revealed direct visualization of mitochondrial fission (AVI)

Author Contributions

† P.M., D.P., H.S., and S.S. contributed equally. R.K.G. and P.C. conceived the idea, designed the hypothesis, and managed overall manuscript preparation. P.M. was responsible for biological experiments, data analysis, and writing, and D.P., H.S., and S.S. were involved in organic synthesis and the Supporting Information preparation.

The authors declare no competing financial interest.

Supplementary Material

ml4c00094_si_001.pdf (9MB, pdf)
ml4c00094_si_002.avi (33.3MB, avi)
ml4c00094_si_003.avi (25.4MB, avi)

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