Lilibeth A. Salvador

Anticolon Cancer Activity of Largazole, a Marine-Derived Tunable Histone Deacetylase Inhibitor

Histone deacetylases (HDACs) are validated targets for anticancer therapy as attested by the approval of suberoylanilide hydroxamic acid (SAHA) and romidepsin (FK228) for treating cutaneous T cell lymphoma. We recently described the bioassay-guided isolation, structure determination, synthesis, and target identification of largazole, a marine-derived antiproliferative natural product that is a prodrug that releases a potent HDAC inhibitor, largazole thiol. Here, we characterize the anticancer activity of largazole by using in vitro and in vivo cancer models. Screening against the National Cancer Institutes 60 cell lines revealed that largazole is particularly active against several colon cancer cell types. Consequently, we tested largazole, along with several synthetic analogs, for HDAC inhibition in human HCT116 colon cancer cells. Enzyme inhibition strongly correlated with the growth inhibitory effects, and differential activity of largazole analogs was rationalized by molecular docking to an HDAC1 homology model. Comparative genomewide transcript profiling revealed a close overlap of genes that are regulated by largazole, FK228, and SAHA. Several of these genes can be related to largazoles ability to induce cell cycle arrest and apoptosis. Stability studies suggested reasonable bioavailability of the active species, largazole thiol. We established that largazole inhibits HDACs in tumor tissue in vivo by using a human HCT116 xenograft mouse model. Largazole strongly stimulated histone hyperacetylation in the tumor, showed efficacy in inhibiting tumor growth, and induced apoptosis in the tumor. This effect probably is mediated by the modulation of levels of cell cycle regulators, antagonism of the AKT pathway through insulin receptor substrate 1 down-regulation, and reduction of epidermal growth factor receptor levels.

Cytotoxic Halogenated Macrolides and Modified Peptides from the Apratoxin-Producing Marine Cyanobacterium Lyngbya bouillonii from Guam

Collections of the marine cyanobacterium Lyngbya bouillonii from shallow patch reefs in Apra Harbor, Guam, afforded three hitherto undescribed analogues of the glycosidic macrolide lyngbyaloside, namely, 2-epi-lyngbyaloside (1) and the regioisomeric 18E- and 18Z-lyngbyalosides C (2 and 3). Concurrently we discovered two new analogues of the cytoskeletal actin-disrupting lyngbyabellins, 27-deoxylyngbyabellin A (4) and lyngbyabellin J (5), a novel macrolide of the laingolide family, laingolide B (6), and a linear modified peptide, lyngbyapeptin D (7), along with known lyngbyabellins A and B, lyngbyapeptin A, and lyngbyaloside. The structures of 1-7 were elucidated by a combination of NMR spectroscopic and mass spectrometric analysis. Compounds 1-6 were either brominated (1-3) or chlorinated (4-6), consistent with halogenation being a hallmark of many marine natural products. All extracts derived from these L. bouillonii collections were highly cytotoxic due to the presence of apratoxin A or apratoxin C. Compounds 1-5 showed weak to moderate cytotoxicity to HT29 colorectal adenocarcinoma and HeLa cervical carcinoma cells.

Veraguamides A–G, Cyclic Hexadepsipeptides from a Dolastatin 16-Producing Cyanobacterium Symploca cf. hydnoides from Guam

Cytotoxicity-directed purification of a Symploca cf. hydnoides sample from Cetti Bay, Guam, afforded seven new cyclic depsipeptides, veraguamides A-G (1-7), together with the known compound dolastatin 16. The planar structures of 1-7 were elucidated using NMR and MS experiments, while enantioselective HPLC and Moshers analysis of acid and base hydrolysates, respectively, were utilized to assign the absolute configurations of the stereocenters. Veraguamides A-G (1-7) are characterized by the presence of an invariant proline residue, multiple N-methylated amino acids, an α-hydroxy acid, and a C8-polyketide-derived β-hydroxy acid moiety with a characteristic terminus as either an alkynyl bromide, alkyne, or vinyl group. These compounds and a semisynthetic analogue (8) showed moderate to weak cytotoxic activity against HT29 colorectal adenocarcinoma and HeLa cervical carcinoma cell lines. Preliminary structure-activity relationship analysis identified several sensitive positions in the veraguamide scaffold that affect the cytotoxic activity of this compound class. Dolastatin 16 showed only weak cytotoxic activity on both cell lines tested. The complete stereostructure of dolastatin 16 was proposed for the first time through degradation followed by a combination of advanced Marfeys analysis and modified Moshers analysis using phenylglycine methyl ester as a chiral anisotropic reagent.

Potent elastase inhibitors from cyanobacteria: structural basis and mechanisms mediating cytoprotective and anti-inflammatory effects in bronchial epithelial cells.

We discovered new structural diversity to a prevalent, yet medicinally underappreciated, cyanobacterial protease inhibitor scaffold and undertook comprehensive protease profiling to reveal potent and selective elastase inhibition. Structure-activity relationship (SAR) studies and X-ray cocrystal structure analysis allowed a detailed assessment of critical and tunable structural elements. To realize the therapeutic potential of these cyclodepsipeptides, we probed the cellular effects of a novel and representative family member, symplostatin 5 (1), which attenuated the downstream cellular effects of elastase in an epithelial lung airway model system, alleviating clinical hallmarks of chronic pulmonary diseases such as cell death, cell detachment, and inflammation. This compound attenuated the effects of elastase on receptor activation, proteolytic processing of the adhesion protein ICAM-1, NF-κB activation, and transcriptomic changes, including the expression of pro-inflammatory cytokines IL1A, IL1B, and IL8. Compound 1 exhibited activity comparable to the clinically approved elastase inhibitor sivelestat in short-term assays and demonstrated superior sustained activity in longer-term assays.

Caylobolide B, a Macrolactone from Symplostatin 1-Producing Marine Cyanobacteria Phormidium spp. from Florida

A Phormidium spp. collection from Key West, Florida, afforded caylobolide B (1), an analogue of the known macrolactone caylobolide A, previously isolated from a Lyngbya majuscula collection from the Bahamas. The planar structure of 1 was determined using NMR and MS experiments. The relative configuration for subunits C7-C9 and C25-C29 was assigned using Kishis Universal NMR Database. Caylobolide B (1) displayed cytotoxic activity against HT29 colorectal adenocarcinoma and HeLa cervical carcinoma cells with IC(50) values of 4.5 and 12.2 μM, respectively.

Cyanobacterial Peptides as a Prototype for the Design of Potent beta-Secretase Inhibitors and the Development of Selective Chemical Probes for Other Aspartic Proteases

Inspired by marine cyanobacterial natural products, we synthesized modified peptides with a central statine-core unit, characteristic for aspartic protease inhibition. A series of tasiamide B analogues inhibited BACE1, a therapeutic target in Alzheimers disease. We probed the stereospecificity of target engagement and determined additional structure-activity relationships with respect to BACE1 and related aspartic proteases, cathepsins D and E. We cocrystallized selected inhibitors with BACE1 to reveal the structural basis for the activity. Hybrid molecules that combine features of tasiamide B and an isophthalic acid moiety-containing sulfonamide showed nanomolar cellular activity. Compounds were screened in a series of rigorous complementary cell-based assays. We measured secreted APP ectodomain (sAPPβ), membrane bound carboxyl terminal fragment (CTF), levels of β-amyloid (Aβ) peptides and selectivity for β-secretase (BACE1) over γ-secretase. Prioritized compounds showed reasonable stability in vitro and in vivo, and our most potent inhibitor showed efficacy in reducing Aβ levels in the rodent brain.

Evaluation of class I HDAC isoform selectivity of largazole analogues.

Largazole is a potent class I selective histone deacetylase (HDAC) inhibitor. The majority of largazole analogues to date have modified the thiazole-thiazoline and the warhead moiety. In order to elucidate class I-specific structure-activity relationships, a series of analogues with modifications in the valine or the linker region were prepared and evaluated for their class I isoform selectivity. The inhibition profile showed that the C2 position of largazole has an optimal steric requirement for efficient HDAC inhibition and that substitution of the trans-alkene in the linker with an aromatic group results in complete loss of activity. This data will aid the design of class I isoform selective HDAC inhibitors.

Discovery and Mechanism of Natural Products as Modulators of Histone Acetylation

Small molecules that modulate histone acetylation by targeting key enzymes mediating this posttranslational modification - histone acetyltransferases and histone deacetylases - are validated chemotherapeutic agents for the treatment of cancer. This area of research has seen a rapid increase in interest in the past decade, with the structurally diverse natural products-derived compounds at its forefront. These secondary metabolites from various biological sources target this epigenetic modification through distinct mechanisms of enzyme regulation by utilizing a diverse array of pharmacophores. We review the discovery of these compounds and discuss their modes of inhibition together with their downstream biological effects.

Largazole Pharmacokinetics in Rats by LC-MS/MS

A highly sensitive and specific LC-MS/MS method for the quantitation of largazole thiol, the active species of the marine-derived preclinical histone deacetylase inhibitor, largazole (prodrug), was developed and validated. Largazole thiol was extracted with ethyl acetate from human or rat plasma along with the internal standard, harmine. Samples were separated on an Onyx Monolithic C18 column by a stepwise gradient elution with 0.1% formic acid in methanol and 0.1% aqueous formic acid employing multiple reaction monitoring (MRM) detection. Linear calibration curves were obtained in the range of 12.5–400 ng/mL with 200 µL of human plasma. The overall intra-day precision was from 3.87% to 12.6%, and the inter-day precision was from 7.12% to 9.8%. The accuracy at low, medium and high concentrations ranged from 101.55% to 105.84%. Plasma protein bindings of largazole thiol in human and rat plasma as determined by an ultrafiltration method were 90.13% and 77.14%, respectively. Plasma drug concentrations were measured by this LC-MS/MS method. The pharmacokinetics of largazole thiol in rats was studied following i.v. administration at 10 mg/kg and found to follow a two-compartment model. Largazole thiol was rapidly eliminated from systemic circulation within 2 h. The established LC-MS/MS method is suitable for the analysis of largazole thiol in human plasma, as well.

HDAC Inhibitors and Other Histone Modifying Natural Products as Emerging Anticancer Agents

Histone deacetylase (HDAC) inhibitors represent a new class of anticancer agents that target dysregulated acetylation of histone lysines, an epigenetic rather than a genetic event. Certain HDACs are overexpressed and hyperactive in cancer cells, and suppression of these enzymes’ activities provides superior selectivity over more traditional anticancer agents. To date, two HDAC inhibitors—vorinostat and romidepsin—have reached the market, with romidepsin being an actual natural product and vorinostat closely related to the natural product HDAC inhibitor trichostatin A. Over the past 15 years, several secondary metabolites with high structural diversity from microorganisms, marine sponges, and cyanobacteria have been discovered to possess HDAC inhibitory activity and are currently at the clinical and preclinical stages. In this chapter, we recapitulate the discovery of natural product HDAC inhibitors, enumerate the challenges in their development and provide insights in the continuing role of natural products in the discovery of HDAC inhibitors as well as new modulators for other clinically-relevant epigenetic events, including histone methylation.