Evaluation of the efficacy of marine natural products against PARP-1/2 proteins in high-grade serous ovarian cancer: insights into MD and SMD simulations.

High-grade serous ovarian cancer (HGSOC) is the most malignant and ubiquitous phenotype of epithelial ovarian cancer. Originating in the fallopian tubes and rapidly spreading to the ovaries, this highly heterogeneous disease is a result of serous tubal intraepithelial carcinoma. The proteins known as poly(ADP-ribose) polymerase (PARP) aid in the development of HGSOC by repairing the cancer cells that proliferate and spread metastatically. By using molecular docking to screen 1100 marine natural products (MNPs) from different marine environments against PARP-1/2 proteins, prominent PARP inhibitors (PARPi) were identified. Four compounds, alisiaquinone A, alisiaquinone C, ascomindone D and (+)-zampanolide referred to as MNP-1, MNP-2, MNP-3 and MNP-4, respectively, were chosen based on their binding affinity towards PARP-1/2 proteins, and their bioavailability and drug-like qualities were accessed using ADMET analysis. To investigate the structural stability and dynamics of these complexes, molecular dynamics simulations were performed for 200 ns. These results were compared with the complexes of olaparib (OLA), a PARPi that has been approved by the FDA for the treatment of advanced ovarian cancer. We determined that MNP-4 exhibited stronger binding energies with PARP-1/2 proteins than OLA by using MM/PBSA calculations. Hotspot residues from PARP-1 (E883, M890, Y896, D899 and Y907) and PARP-2 (Y449, F450, A451, S457 and Y460) showed strong interactions with the compounds. To comprehend the unbinding mechanism of MNP-4 complexed with PARP-1/2, steered molecular dynamics (SMD) simulations were performed. We concluded from the free energy landscape (FEL) map that PARP-1/2 are well-stabilised when the compound MNP-4 is bound rather than being pulled away from its binding pockets. This finding provides significant evidence regarding PARPi, which could potentially be employed in the therapeutic treatment of HGSOC.Communicated by Ramaswamy H. Sarma.

Linear (-)-Zampanolide: Flexibility in Conformation-Activity Relationships.

Through an understanding of the conformational preferences of the polyketide natural product (-)-zampanolide, and the structural motifs that control these preferences, we developed a linear zampanolide analogue that exhibits potent cytotoxicity against cancer cell lines. This discovery provides a set of three structural handles for further structure-activity relationship (SAR) studies of this potent microtubule-stabilizing agent. Moreover, it provides additional evidence of the complex relationship between ligand preorganization, conformational flexibility, and biological potency. In contrast to medicinal chemistry dogma, these results demonstrate that increased overall conformational flexibility is not necessarily detrimental to protein binding affinity and biological activity.

Synthesis and Structure-Activity Relationship Studies of C(13)-Desmethylene-(-)-Zampanolide Analogs.

We describe the synthesis and biochemical and cellular profiling of five partially reduced or demethylated analogs of the marine macrolide (-)-zampanolide (ZMP). These analogs were derived from 13-desmethylene-(-)-zampanolide (DM-ZMP), which is an equally potent cancer cell growth inhibitor as ZMP. Key steps in the synthesis of all compounds were the formation of the dioxabicyclo[15.3.1]heneicosane core by an intramolecular HWE reaction (67-95 % yield) and a stereoselective aza-aldol reaction with an (S)-BINOL-derived sorbamide transfer complex, to establish the C(20) stereocenter (24-71 % yield). As the sole exception, for the 5-desmethyl macrocycle, ring-closure relied on macrolactonization; however, elaboration of the macrocyclization product into the corresponding zampanolide analog was unsuccessful. All modifications led to reduced cellular activity and lowered microtubule-binding affinity compared to DM-ZMP, albeit to a different extent. For compounds incorporating the reactive enone moiety of ZMP, IC50 values for cancer cell growth inhibition varied between 5 and 133 nM, compared to 1-12 nM for DM-ZMP. Reduction of the enone double bond led to a several hundred-fold loss in growth inhibition. The cellular potency of 2,3-dihydro-13-desmethylene zampanolide, as the most potent analog identified, remained within a ninefold range of that of DM-ZMP.

In Vivo Evaluation of (-)-Zampanolide Demonstrates Potent and Persistent Antitumor Efficacy When Targeted to the Tumor Site.

Microtubule-stabilizing agents (MSAs) are a class of compounds used in the treatment of triple-negative breast cancer (TNBC), a subtype of breast cancer where chemotherapy remains the standard-of-care for patients. Taxanes like paclitaxel and docetaxel have demonstrated efficacy against TNBC in the clinic, however new classes of MSAs need to be identified due to the rise of taxane resistance in patients. (-)-Zampanolide is a covalent microtubule stabilizer that can circumvent taxane resistance in vitro but has not been evaluated for in vivo antitumor efficacy. Here, we determine that (-)-zampanolide has similar potency and efficacy to paclitaxel in TNBC cell lines, but is significantly more persistent due to its covalent binding. We also provide the first reported in vivo antitumor evaluation of (-)-zampanolide where we determine that it has potent and persistent antitumor efficacy when delivered intratumorally. Future work on zampanolide to further evaluate its pharmacophore and determine ways to improve its systemic therapeutic window would make this compound a potential candidate for clinical development through its ability to circumvent taxane-resistance mechanisms.

An amide mimic of desTHPdactylolide: Total synthesis and antiproliferative evaluation.

(-)-Zampanolide is a unique microtubule stabilizing agent (MSA) with covalent-binding mechanism and low nanomolar anitproliferative potency towards multi-drug resistant cancer cells. MSAs have a special connection with prostate cancer by inhibiting androgen receptor nuclear translocation. Zampanolide and the structurally related dactylolide have thus been sought after by us as lead compounds for development of anti-prostate cancer agents. DesTHPdactylolide is a simplified mimic of dactylolide and has previously been synthesized by us in both configurations, with the (17R) configuration being more potent in suppressing prostate cancer cell proliferation. The current study aims to synthesize an amide mimic of (17R) desTHPdactylolide that was anticipated to be metabolically more stable than (17R) desTHPdactylolide. To this end, the amide mimic has been successfully synthesized through a 26-step transformation from 2-butyn-1-ol. Our WST-1 cell proliferation assay in five human prostate cancer cell models indicated that the lactam moiety can serve as a bioisostere for the lactone in desTHPdactylolide.

Synthesis of Morpholine-Based Analogues of (-)-Zampanolide and Their Biological Activity.

We describe the convergent synthesis of three prototypical examples of a new class of analogues of the complex, cytotoxic marine macrolide (-)-zampanolide that incorporate an embedded N-substituted morpholine moiety in place of the natural tetrahydropyran ring. The final construction of the macrolactone core was based on a high-yielding intramolecular HWE olefination, while the hemiaminal-linked side chain was elaborated through a stereoselective, BINAL-H-mediated addition of (Z,E)-sorbamide to a macrocyclic aldehyde precursor. The synthesis of the common functionalized morpholine building block involved two consecutive epoxide openings with tosylamide and the product of the first opening reaction, respectively, as nucleophiles. Of the three morpholino-zampanolides investigated, the N-acetyl and the N-benzoyl derivatives both exhibited nanomolar antiproliferative activity, thus being essentially equipotent with the natural product. In contrast, the activity of the N-tosyl derivative was significantly reduced.

New Zampanolide Mimics: Design, Synthesis, and Antiproliferative Evaluation.

Zampanolide is a promising microtubule-stabilizing agent (MSA) with a unique chemical structure. It is superior to the current clinically used MSAs due to the covalent nature of its binding to ß-tubulin and high cytotoxic potency toward multidrug-resistant cancer cells. However, its further development as a viable drug candidate is hindered by its limited availability. More importantly, conversion of its chemically fragile side chain into a stabilized bioisostere is envisioned to enable zampanolide to possess more drug-like properties. As part of our ongoing project aiming to develop its mimics with a stable side chain using straightforward synthetic approaches, 2-fluorobenzyl alcohol was designed as a bioisosteric surrogate for the side chain based on its binding conformation as confirmed by the X-ray structure of tubulin complexed with zampanolide. Two new zampanolide mimics with the newly designed side chain have been successfully synthesized through a 25-step chemical transformation for each. Yamaguchi esterification and intramolecular Horner-Wadsworth-Emmons condensation were used as key reactions to construct the lactone core. The chiral centers at C17 and C18 were introduced by the Sharpless asymmetric dihydroxylation. Our WST-1 cell proliferation assay data in both docetaxel-resistant and docetaxel-naive prostate cancer cell lines revealed that compound 6 is the optimal mimic and the newly designed side chain can serve as a bioisostere for the chemically fragile N-acetyl hemiaminal side chain in zampanolide.

Optimized synthesis and antiproliferative activity of desTHPdactylolides.

Dactylolide and certain analogues are attractive targets for study due to their structural resemblance to zampanolide, a very promising anticancer lead compound and a unique covalent-binding microtubule stabilizing agent. The primary goal of this project is identification and synthesis of simplified analogues of dactylolide that would be easier to prepare and could be investigated for antiproliferative activity in comparison with zampanolide. Extension of Almann's concept of a simplified zampanolide analogue to dactylolide in the form of desTHPdactylolide was attractive not only for reasons of synthetic simplification but also for the prospect that analogues of dactylolide could be prepared in both (17S) and (17R) configurations. Since Altmann's overall yield for the six-step procedure leading to the C9-C18 fragment of desTHPdactylolide was only 8.7%, a study focused on optimized synthesis and antiproliferative evaluation of each enantiomer of desTHPdactylolide was initiated using Altmann's route as a framework. To this end, two optimized approaches to this fragment C9-C18 were successfully developed by us using allyl iodide or allyl tosylate as the starting material for a critical Williamson ether synthesis. Both (17S) and (17R) desTHPdactylolides were readily synthesized in our laboratory using optimized methods in yields of 37-43%. Antiproliferative activity of the pair of enantiomeric desTHPdactylolides, together with their analogues, was evaluated in three docetaxel-sensitive and two docetaxel-resistant prostate cancer cell models using a WST-1 cell proliferation assay. Surprisingly, (17R) desTHPdactylolide was identified as the eutomer in the prostate cancer cell models. It was found that (17S) and (17R) desTHPdactylolide exhibit equivalent antiproliferative potency towards both docetaxel-sensitive (PC-3 and DU145) and docetaxel-resistant prostate cancer cell lines (PC-3/DTX and DU145/DTX).

Zampanolide, a Microtubule-Stabilizing Agent, Is Active in Resistant Cancer Cells and Inhibits Cell Migration.

Zampanolide, first discovered in a sponge extract in 1996 and later identified as a microtubule-stabilizing agent in 2009, is a covalent binding secondary metabolite with potent, low nanomolar activity in mammalian cells. Zampanolide was not susceptible to single amino acid mutations at the taxoid site of ß-tubulin in human ovarian cancer 1A9 cells, despite evidence that it selectively binds to the taxoid site. As expected, it did not synergize with other taxoid site microtubule-stabilizing agents (paclitaxel, ixabepilone, discodermolide), but surprisingly also did not synergize in 1A9 cells with laulimalide/peloruside binding site agents either. Efforts to generate a zampanolide-resistant cell line were unsuccessful. Using a standard wound scratch assay in cell culture, it was an effective inhibitor of migration of human umbilical vein endothelial cells (HUVEC) and fibroblast cells (D551). These properties of covalent binding, the ability to inhibit cell growth in paclitaxel and epothilone resistant cells, and the ability to inhibit cell migration suggest that it would be of interest to investigate zampanolide in preclinical animal models to determine if it is effective in vivo at preventing tumor growth and metastasis.

Insights into the Distinct Mechanisms of Action of Taxane and Non-Taxane Microtubule Stabilizers from Cryo-EM Structures.

A number of microtubule (MT)-stabilizing agents (MSAs) have demonstrated or predicted potential as anticancer agents, but a detailed structural basis for their mechanism of action is still lacking. We have obtained high-resolution (3.9-4.2Å) cryo-electron microscopy (cryo-EM) reconstructions of MTs stabilized by the taxane-site binders Taxol and zampanolide, and by peloruside, which targets a distinct, non-taxoid pocket on ß-tubulin. We find that each molecule has unique distinct structural effects on the MT lattice structure. Peloruside acts primarily at lateral contacts and has an effect on the "seam" of heterologous interactions, enforcing a conformation more similar to that of homologous (i.e., non-seam) contacts by which it regularizes the MT lattice. In contrast, binding of either Taxol or zampanolide induces MT heterogeneity. In doubly bound MTs, peloruside overrides the heterogeneity induced by Taxol binding. Our structural analysis illustrates distinct mechanisms of these drugs for stabilizing the MT lattice and is of relevance to the possible use of combinations of MSAs to regulate MT activity and improve therapeutic potential.

Methods for studying microtubule binding site interactions: zampanolide as a covalent binding agent.

In this chapter, we describe the methods used to determine the binding site and binding profile of zampanolide, a novel microtubule-stabilizing agent (MSA) that binds covalently to tubulin. These methods can be applied to other novel MSAs in which the binding site and mechanism of binding are unknown. Using the described methods, we have shown that zampanolide binds to the taxoid site on ß-tubulin, but unlike most other MSAs is able to covalently modify this site. The purpose of this chapter is to provide a step-by-step protocol for determining the binding site of a novel MSA.

Microtubule-stabilizing drugs from marine sponges: focus on peloruside A and zampanolide.

Marine sponges are an excellent source of bioactive secondary metabolites with potential therapeutic value in the treatment of diseases. One group of compounds of particular interest is the microtubule-stabilizing agents, the most well-known compound of this group being paclitaxel (Taxol), an anti-cancer compound isolated from the bark and leaves of the Pacific yew tree. This review focuses on two of the more recent additions to this important class of drugs, peloruside A and zampanolide, both isolated from marine sponges. Peloruside A was isolated from Mycale hentscheli collected in New Zealand coastal waters, and it already shows promising anti-cancer activity. Two other potent bioactive compounds with different modes of action but isolated from the same sponge, mycalamide A and pateamine, will also be discussed. The fourth compound, zampanolide, most recently isolated from the Tongan sponge Cacospongia mycofijiensis, has only recently been added to the microtubule-stabilizing group of compounds, and further work is in progress to determine its activity profile relative to peloruside A and other drugs of this class.