Tadeusz F. Molinski

Drug development from marine natural products

Drug discovery from marine natural products has enjoyed a renaissance in the past few years. Ziconotide (Prialt; Elan Pharmaceuticals), a peptide originally discovered in a tropical cone snail, was the first marine-derived compound to be approved in the United States in December 2004 for the treatment of pain. Then, in October 2007, trabectedin (Yondelis; PharmaMar) became the first marine anticancer drug to be approved in the European Union. Here, we review the history of drug discovery from marine natural products, and by describing selected examples, we examine the factors that contribute to new discoveries and the difficulties associated with translating marine-derived compounds into clinical trials. Providing an outlook into the future, we also examine the advances that may further expand the promise of drugs from the sea.

Xestospongins: Potent Membrane Permeable Blockers of the Inositol 1,4,5-Trisphosphate Receptor

Xestospongins (Xes) A, C, D, araguspongine B, and demethylxestospongin B, a group of macrocyclic bis-1-oxaquinolizidines isolated from the Australian sponge, Xestospongia species, are shown to be potent blockers of IP3-mediated Ca2+ release from endoplasmic reticulum vesicles of rabbit cerebellum. XeC blocks IP3-induced Ca2+ release (IC50 = 358 nM) without interacting with the IP3-binding site, suggesting a mechanism that is independent of the IP3 effector site. Analysis of Pheochromocytoma cells and primary astrocytes loaded with Ca2+-sensitive dye reveals that XeC selectively blocks bradykinin- and carbamylcholine-induced Ca2+ efflux from endoplasmic reticulum stores. Xes represent a new class of potent, membrane permeable IP3 receptor blockers exhibiting a high selectivity over ryanodine receptors. Xes are a valuable tool for investigating the structure and function of IP3 receptors and Ca2+ signaling in neuronal and nonneuronal cells.

NMR of natural products at the ‘nanomole-scale’

Over the last decade, dramatic improvements in mass-sensitivity of NMR through low-volume tube probes and capillary probes, coupled with cryogenically cooled radiofrequency (rf) coils and preamplifier components, have provided chemists with new capabilities for exploration of submilligram natural product samples. These innovations led to an approximate 20-fold increase in mass sensitivity compared with conventional NMR instrumentation at the same field. Now, full characterization by 1D and 2D NMR of natural products--some available only in vanishingly small amounts, down to approximately 1 nanomole--can be achieved in reasonable time-frames. In this Highlight, some recent applications of the new NMR methodology to the area of natural products discovery are discussed, along with a perspective of practical limitations and potential future applications in new areas.

Structure Elucidation at the Nanomole Scale. 1. Trisoxazole Macrolides and Thiazole-Containing Cyclic Peptides from the Nudibranch Hexabranchus sanguineus

A single specimen of Hexabranchus sanguineus, a nudibranch from the Indo-Pacific that is known to sequester kabiramides B and C and other trisoxazole macrolides, yielded new kabiramide analogues, 9-desmethylkbiramide B and 33-methyltetrahydrohalichondramide, and two new unexpected thiazole-containing cyclic peptides in submicromolar amounts. The structures of these cyclic peptides were determined by analyses of 1D and 2D NMR spectra recorded with a state-of-the-art 1 mm (1)H NMR high-temperature superconducting microcryoprobe, together with mass spectra. In addition to two proline residues, each peptide contains a thiazole- or oxazole-modified amino acid residue, together with conventional amino acid residues. All of the amino acid residues were l, as determined by Marfeys analysis of the acid hydrolysates of the peptides. This is the first report of cyclic thiazole peptides from H. sanguineus. Since thiazole-oxazole-modified peptides are typically associated with cyanobacteria and tunicates, the finding may imply a dietary component of the H. sanguineus that was previously overlooked.

Absolute configuration of phorboxazoles A and B from the marine sponge, Phorbas sp. 2. C43 and complete stereochemistry

The absolute configuration of C43 in the cytostatic macrolide phorboxazoles A was established as R by correlation with (R)-dimethyl methoxysuccinate while 43 R was also suggested for phorboxazole B by CD comparison. This completes the entire stereochemical determination of the phorboxazoles.

Sparks and Puffs in Oligodendrocyte Progenitors: Cross Talk between Ryanodine Receptors and Inositol Trisphosphate Receptors

Investigating how calcium release from the endoplasmic reticulum (ER) is triggered and coordinated is crucial to our understanding of how oligodendrocyte progenitor cells (OPs) develop into myelinating cells. Sparks and puffs represent highly localized Ca2+ release from the ER through ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs), respectively. To study whether sparks or puffs trigger Ca2+ waves in OPs, we performed rapid high-resolution line scan recordings in fluo-4-loaded OP processes. We found spontaneous and evoked sparks and puffs, and we have identified functional cross talk between IP3Rs and RyRs. Local events evoked using the IP3-linked agonist methacholine (MeCh) showed significantly different morphology compared with events evoked using the caffeine analog 3,7-dimethyl-1-propargylxanthine (DMPX). Pretreatment with MeCh potentiated DMPX-evoked events, whereas inhibition of RyRs potentiated events evoked by low concentrations of MeCh. Furthermore, activation of IP3Rs but not RyRs was critical for Ca2+ wave initiation. Using immunocytochemistry, we show OPs express the specific Ca2+ release channel subtypes RyR3 and IP3R2 in patches along OP processes. RyRs are coexpressed with IP3Rs in some patches, but IP3Rs are also found alone. This differential distribution pattern may underlie the differences in local and global Ca2+ signals mediated by these two receptors. Thus, in OPs, interactions between IP3Rs and RyRs determine the spatial and temporal characteristics of calcium signaling, from microdomains to intracellular waves.

Malignant hyperthermia susceptibility arising from altered resting coupling between the skeletal muscle L-type Ca2+ channel and the type 1 ryanodine receptor

Malignant hyperthermia (MH) susceptibility is a dominantly inherited disorder in which volatile anesthetics trigger aberrant Ca2+ release in skeletal muscle and a potentially fatal rise in perioperative body temperature. Mutations causing MH susceptibility have been identified in two proteins critical for excitation–contraction (EC) coupling, the type 1 ryanodine receptor (RyR1) and CaV1.1, the principal subunit of the L-type Ca2+ channel. All of the mutations that have been characterized previously augment EC coupling and/or increase the rate of L-type Ca2+ entry. The CaV1.1 mutation R174W associated with MH susceptibility occurs at the innermost basic residue of the IS4 voltage-sensing helix, a residue conserved among all CaV channels [Carpenter D, et al. (2009) BMC Med Genet 10:104–115.]. To define the functional consequences of this mutation, we expressed it in dysgenic (CaV1.1 null) myotubes. Unlike previously described MH-linked mutations in CaV1.1, R174W ablated the L-type current and had no effect on EC coupling. Nonetheless, R174W increased sensitivity of Ca2+ release to caffeine (used for MH diagnostic in vitro testing) and to volatile anesthetics. Moreover, in CaV1.1 R174W-expressing myotubes, resting myoplasmic Ca2+ levels were elevated, and sarcoplasmic reticulum (SR) stores were partially depleted, compared with myotubes expressing wild-type CaV1.1. Our results indicate that CaV1.1 functions not only to activate RyR1 during EC coupling, but also to suppress resting RyR1-mediated Ca2+ leak from the SR, and that perturbation of CaV1.1 negative regulation of RyR1 leak identifies a unique mechanism that can sensitize muscle cells to MH triggers.

Increased Resting Intracellular Calcium Modulates NF-κB-dependent Inducible Nitric-oxide Synthase Gene Expression in Dystrophic mdx Skeletal Myotubes

Background: The mechanisms by which NF-κB signaling is up-regulated in dystrophic muscles are unclear. Results: [Ca2+]rest is elevated in mdx myotubes as a result of both sarcolemmal Ca2+ entry and SR release, resulting in NF-κB-induced iNOS expression. Conclusion: Ca2+ alterations at rest modulate NF-κB transcriptional activity and pro-inflammatory gene expression. Significance: This allows for understanding the mechanism that relates elevated resting calcium and altered gene expression in muscular dystrophy. Duchenne muscular dystrophy (DMD) is a genetic disorder caused by dystrophin mutations, characterized by chronic inflammation and severe muscle wasting. Dystrophic muscles exhibit activated immune cell infiltrates, up-regulated inflammatory gene expression, and increased NF-κB activity, but the contribution of the skeletal muscle cell to this process has been unclear. The aim of this work was to study the pathways that contribute to the increased resting calcium ([Ca2+]rest) observed in mdx myotubes and its possible link with up-regulation of NF-κB and pro-inflammatory gene expression in dystrophic muscle cells. [Ca2+]rest was higher in mdx than in WT myotubes (308 ± 6 versus 113 ± 2 nm, p < 0.001). In mdx myotubes, both the inhibition of Ca2+ entry (low Ca2+ solution, Ca2+-free solution, and Gd3+) and blockade of either ryanodine receptors or inositol 1,4,5-trisphosphate receptors reduced [Ca2+]rest. Basal activity of NF-κB was significantly up-regulated in mdx versus WT myotubes. There was an increased transcriptional activity and p65 nuclear localization, which could be reversed when [Ca2+]rest was reduced. Levels of mRNA for TNFα, IL-1β, and IL-6 were similar in WT and mdx myotubes, whereas inducible nitric-oxide synthase (iNOS) expression was increased 5-fold. Reducing [Ca2+]rest using different strategies reduced iNOS gene expression presumably as a result of decreased activation of NF-κB. We propose that NF-κB, modulated by increased [Ca2+]rest, is constitutively active in mdx myotubes, and this mechanism can account for iNOS overexpression and the increase in reactive nitrogen species that promote damage in dystrophic skeletal muscle cells.

Haliclonadiamine, an antimicrobial alkaloid from the sponge Haliclona SP

Abstract The marine sponge Haliclona sp. from Palau contains haliclonadiamine ( 2 ) as the major antimicrobial alkaloid, together with papuamine ( 1 ). The structure of haliclonadiamine ( 2 ) was determined by X-ray analysis.

A Tetrachloro Polyketide Hexahydro-1H-isoindolone, Muironolide A, from the Marine Sponge Phorbas sp. Natural Products at the Nanomole Scale

Muironolide A, a new chemical entity with an unprecedented chlorinated hexahydro-1H-isoindolone skeleton, was isolated in only 90 microg yield from the same marine sponge, Phorbas sp. that also provided phorboxazoles A and B. The structure was solved by interpretation of NMR data obtained at 600 MHz with a 1.7 mm cryo-microprobe in combination with FTMS, exciton coupled CD, and stereochemical correlation with authentic standards prepared by Reformatsky reaction of (-)-(1R,2S)-2-chloro-1-cyclopropanecarboxaldehyde. The absolute configuration of the chlorocyclopropane ring in 1 is opposite to that of co-occurring phorbasides A-F. Muironolide A is the first described macrolide bearing an esterified trichloromethyl carbinol, and may be produced by a cyanobacterium that also makes phorbasides.