Andrew M. Piggott

Quality, not quantity: the role of natural products and chemical proteomics in modern drug discovery.

Chemical genetics and reverse chemical genetics parallel classical genetics but target genes at the protein level and have proven useful in recent years for screening combinatorial libraries for compounds of biological interest. However, the performance of combinatorial chemistry in filling pharmaceutical pipelines has been lower than anticipated and the tide may be turning back to Nature in the search for new drug candidates. Even though diversity oriented synthesis is now producing molecules that are natural product-like in terms of size and complexity, these molecules have not evolved to interact with biomolecules. Natural products, on the other hand, have evolved to interact with biomolecules, which is why so many can be found in pharmacopoeias. However, the cellular targets and modes of action of these fascinating compounds are seldom known, hindering the drug development process. This review focuses on the emergence of chemical proteomics and reverse chemical proteomics as tools for the discovery of cellular receptors for natural products, thereby generating protein/ligand pairs that will prove useful in identifying new drug targets and new biologically active small molecule scaffolds. Such a system-wide approach to identifying new drugable targets and their small molecule ligands will help unblock the pharmaceutical product pipelines by speeding the process of target and lead identification.

Aspergillus Sydowii Marine Fungal Bloom in Australian Coastal Waters, Its Metabolites and Potential Impact on Symbiodinium Dinoflagellates

Dust has been widely recognised as an important source of nutrients in the marine environment and as a vector for transporting pathogenic microorganisms. Disturbingly, in the wake of a dust storm event along the eastern Australian coast line in 2009, the Continuous Plankton Recorder collected masses of fungal spores and mycelia (~150,000 spores/m3) forming a floating raft that covered a coastal area equivalent to 25 times the surface of England. Cultured A. sydowii strains exhibited varying metabolite profiles, but all produced sydonic acid, a chemotaxonomic marker for A. sydowii. The Australian marine fungal strains share major metabolites and display comparable metabolic diversity to Australian terrestrial strains and to strains pathogenic to Caribbean coral. Secondary colonisation of the rafts by other fungi, including strains of Cladosporium, Penicillium and other Aspergillus species with distinct secondary metabolite profiles, was also encountered. Our bioassays revealed that the dust-derived marine fungal extracts and known A. sydowii metabolites such as sydowic acid, sydowinol and sydowinin A adversely affect photophysiological performance (Fv/Fm) of the coral reef dinoflagellate endosymbiont Symbiodinium. Different Symbiodinium clades exhibited varying sensitivities, mimicking sensitivity to coral bleaching phenomena. The detection of such large amounts of A. sydowii following this dust storm event has potential implications for the health of coral environments such as the Great Barrier Reef.

Naseseazines A and B: A New Dimeric Diketopiperazine Framework from a Marine-Derived Actinomycete, Streptomyces sp.

Chemical analysis of a Streptomyces sp. (CMB-MQ030) isolated from a Fijian marine sediment yielded two new diketopiperazines, naseseazines A and B (1, 2), featuring a new dimeric framework. Structures were determined by detailed spectroscopic analysis and C(3) Marfeys analysis.

Abyssomicins from the South China Sea deep-sea sediment Verrucosispora sp.: natural thioether michael addition adducts as antitubercular prodrugs

Tuberculosis (TB) is a leading cause of death in the world today, and is exacerbated by the prevalence of multi- (MDR-TB), extensively (XDR-TB), and totally (TDR-TB) drug resistant strains. Despite the threat to human health, existing frontline TB therapeutics remain constrained to a handful of vintage antibiotics prescribed in a combinatorial format to achieve efficacy. The current shortfall in antitubercular drugs demands urgent attention, to develop new antibiotics effective against all strains of tuberculosis.

Nocardioazines: a novel bridged diketopiperazine scaffold from a marine-derived bacterium inhibits P-glycoprotein.

An Australian marine sediment-derived isolate, Nocardiopsis sp. (CMB-M0232), yielded a new class of prenylated diketopiperazine, indicative of the action of a uniquely regioselective diketopiperazine indole prenyltransferase. The bridged scaffold of nocardioazine A proved to be a noncytotoxic inhibitor of the membrane protein efflux pump P-glycoprotein, reversing doxorubicin resistance in a multidrug resistant colon cancer cell.

Staurosporines Disrupt Phosphatidylserine Trafficking and Mislocalize Ras Proteins

Background: Ras proteins must be plasma membrane-localized for biological activity. Results: A high content screen identified staurosporines as inhibitors of Ras plasma membrane localization and K-Ras signal transmission by disrupting endosomal recycling of phosphatidylserine. Conclusion: Staurosporines are novel inhibitors of phosphatidylserine trafficking. Significance: Ras trafficking pathways and Ras spatiotemporal organization on the plasma membrane are valid targets for anti-Ras drug development. Oncogenic mutant Ras is frequently expressed in human cancers, but no anti-Ras drugs have been developed. Since membrane association is essential for Ras biological activity, we developed a high content assay for inhibitors of Ras plasma membrane localization. We discovered that staurosporine and analogs potently inhibit Ras plasma membrane binding by blocking endosomal recycling of phosphatidylserine, resulting in redistribution of phosphatidylserine from plasma membrane to endomembrane. Staurosporines are more active against K-Ras than H-Ras. K-Ras is displaced to endosomes and undergoes proteasomal-independent degradation, whereas H-Ras redistributes to the Golgi and is not degraded. K-Ras nanoclustering on the plasma membrane is also inhibited. Ras mislocalization does not correlate with protein kinase C inhibition or induction of apoptosis. Staurosporines selectively abrogate K-Ras signaling and proliferation of K-Ras-transformed cells. These results identify staurosporines as novel inhibitors of phosphatidylserine trafficking, yield new insights into the role of phosphatidylserine and electrostatics in Ras plasma membrane targeting, and validate a new target for anti-Ras therapeutics.

Heronapyrroles A−C: Farnesylated 2-Nitropyrroles from an Australian Marine-Derived Streptomyces sp.

Chemical analysis of a marine-derived Streptomyces sp. (CMB-M0423) isolated from beach sand off Heron Island, Australia, yielded three new members of the rare pyrroloterpene biosynthetic structure class. Identified by detailed spectroscopic analysis as the first reported examples of naturally occurring 2-nitropyrroles, heronapyrroles A-C (1-3) displayed promising biological activity-with low to submicromolar IC(50) activity against Gram-positive bacteria but no cytotoxicity toward mammalian cell lines.

Ircinialactams: subunit-selective glycine receptor modulators from Australian sponges of the family Irciniidae.

Screening an extract library of >2500 southern Australian and Antarctic marine invertebrates and algae for modulators of glycine receptor (GlyR) chloride channels identified three Irciniidae sponges that yielded new examples of a rare class of glycinyl lactam sesterterpene, ircinialactam A, 8-hydroxyircinialactam A, 8-hydroxyircinialactam B, ircinialactam C, ent-ircinialactam C and ircinialactam D. Structure-activity relationship (SAR) investigations revealed a new pharmacophore with potent and subunit selective modulatory properties against alpha1 and alpha3 GlyR isoforms. Such GlyR modulators have potential application as pharmacological tools, and as leads for the development of GlyR targeting therapeutics to treat chronic inflammatory pain, epilepsy, spasticity and hyperekplexia.

Rapid Identification of a Protein Binding Partner for the Marine Natural Product Kahalalide F by Using Reverse Chemical Proteomics

Kahalalide F (KF) is in phase II clinical trials as an anticancer drug against a range of difficult to treat solid tumors including prostate, breast and colon carcinomas, neuroblastomas, chondrosarcomas, and osteosarcomas and has relatively low toxicity to nontumor cells. KF was originally isolated by Hamann and Scheuer from the sacoglossan marine mollusk, Elysia rufescens, and subsequently from the sacoglossan’s food source, the green alga Bryopsis sp. In phase I clinical trials, KF had a clinical benefit for patients with advanced androgen refractory prostate cancer and other advanced tumors. KF appears to act on cell lysosomes, with treated cells swelling dramatically and forming large vacuoles. Cell death is thought to occur via oncosis, with KF inducing sub G1 cell-cycle arrest and cytotoxicity independently of MDR, HER2, p53, and blc-2. A recent study by Janmaat et al. showed that sensitivity to KF in a variety of cell lines was positively correlated to receptor protein tyrosine kinase ErbB3 (HER3) levels and that KF efficiently inhibited the phosphatidylinositol 3-kinase-Akt signaling pathway in sensitive cell lines. These findings suggest that KF is involved in a hitherto unknown oncosis signaling pathway and that disruption of lysosomes is simply the final step in a series of cascading events. Despite numerous studies into the mode of action of KF, the actual cellular receptor for the molecule remains a mystery. Chemical proteomics is a powerful tool for isolating and identifying cellular receptors for biologically active natural products, thereby facilitating subsequent rational drug design, and often providing valuable information regarding underlying biochemical and cellular processes. In chemical proteomics, a small molecule (drug) is immobilized on a solid support or is tagged with a radioactive/fluorescent label to generate an ACHTUNGTRENNUNGactivity or affinity probe, which can be used to isolate and iden ACHTUNGTRENNUNGtify a single protein or a family of proteins from an entire ACHTUNGTRENNUNGproteome. However, this technique often requires larger amounts of protein than are typically available in such experiments, particularly for low-abundance proteins, and more importantly, generally results in isolation of the most abundant binding protein, rather than the most avid binder. One solution is to provide a physical link between the protein and its corresponding gene—the so-called “genotype–phenotype link”. This construct allows affinity purification of the protein using an immobilized natural product, but also provides a means of identification through amplification (PCR) and sequencing of the attached gene. Whereas there are several methods of creating a genotype–phenotype link, such as mRNA display and ribosome display, currently the most popular is phage display, whereby the gene encoding a protein of interest is cloned into a bacteriophage (phage) in such a way that the protein is expressed on the surface of the virus. If an entire cDNA library is cloned into a phage display vector, each phage particle will contain a different gene insert and will express the protein encoded by that gene on its surface. The displayed proteins usually behave as if they were free in solution and do not suffer from many of the problems associated with protein overexpression in bacterial cells, such as toxicity or precipitation. Phage displaying the target protein can then be isolated from the library using an immobilized natural product, as per standard chemical proteomics experiments. The real power of phage display comes from the fact that phages can be amplified by transfection into E. coli and then subjected to another round of affinity selection with the immobilized small molecule. This cycle can be repeated numerous times, ACHTUNGTRENNUNGallowing the most avid binder(s) to be identified, even if they are only present in very small amounts in the original cDNA library. As the starting point is actually a transcriptome and not a proteome, we call this technique “reverse chemical proteomics”. A potential problem with all chemical proteomics and reverse chemical proteomics methods is that derivatization of the small molecule may affect its biological activity and this possibility must first be excluded. In this paper, we describe the first use of reverse chemical proteomics with T7 phage display to isolate a human protein binding partner for a marine natural product with no known receptor. A biotinylated analogue of KF 1, containing a long, hydrophilic linker, was synthesized (Scheme 1) and immobilized on a neutravidin-coated microtiter plate to generate an affinity support. In addition, a biotinylated control reagent, biotin-Bu 5, was synthesized by coupling 4 with n-butylamine to mimic only the ornithine side chain of KF. KF-NBD 6, a fluorescent analogue of KF, also derivatized through the primary amine of the ornithine side chain of KF with 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD) was similarly made for fluorescence microscopy (Figure 1). It has been shown that the biological activity of KF is lost on hydrolysis of the cyclic ester to give kahalaACHTUNGTRENNUNGlide G, suggesting that the hexadepsipeptide ring is required for activity. As the ornithine side chain of KF is some distance from the depsipeptide ring and derivatization of this group has been shown to result in retention of anticancer activity, it was reasoned that this group might not be important for [a] Dr. A. M. Piggott, Prof. P. Karuso Department of Chemistry and Biomolecular Sciences, Macquarie University Sydney, NSW, 2109 (Australia) Fax: (+61)2-9850-8313 E-mail : peter.karuso@mq.edu.au Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

Heronamides A–C, new polyketide macrolactams from an Australian marine-derived Streptomyces sp. A biosynthetic case for synchronized tandem electrocyclization

A Streptomyces sp. isolated from a shallow water sediment sample collected off Heron Island, Australia, afforded three new polyketide macrolactams, heronamides A-C (1-3). Structures were assigned to the heronamides on the basis of detailed spectroscopic analysis, chemical derivatization and biosynthetic considerations. A plausible biosynthetic pathway is proposed in which key carbocyclic ring transformations proceed via an unprecedented synchronized tandem electrocyclization. This biosynthesis provides a framework for the assignment of complete relative configurations across all heronamides, and inspires an attractive biomimetic strategy for future total syntheses. Heronamide C elicits a dramatic and reversible non-cytotoxic effect on mammalian cell morphology.