Peter Karuso

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.

A gloverin-like antibacterial protein is synthesized in Helicoverpa armigera following bacterial challenge.

A bacteria inducible antibacterial protein, P2, was isolated from the old world bollworm Helicoverpa armigera. Fifth-instar larvae were injected with live Escherichia coli NCTC 8196. P2 was isolated by HPLC using reversed-phase and size-exclusion columns. In addition, P2 was isolated by an alternative method of sequential cation-exchange and reversed-phase HPLC. The structure of P2 was determined by N-terminal Edman degradation and mass spectrometry. P2 had similar mass (14.1 kDa) structure and activity to gloverin, an inducible glycine-rich antibacterial protein isolated from Hyalophora gloveri [Axén, A.; Carlsson, A.; Engström, A.; Bennich, H. Eur. J. Biochem. 247:614-619; 1997]. At the N-terminus P2 had approximately 60% identity with gloverin. P2 is basic, heat stable, and displayed rapid antibacterial action. P2 was active against the Gram-negative bacteria tested and was inactive against the Gram-positive bacteria, Candida albicans, a bovine turbinate cell line, and pestivirus.

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.

Fluorescence anisotropy assay for the traceless kinetic analysis of protein digestion.

A novel fluorescence polarization assay based on the natural fluorophore epicocconone has been developed. This assay allows the rapid and accurate determination of enzyme kinetic parameters as well as inhibition constants through the measurement of fluorescence anisotropy on the actual substrate of the protease. It takes advantage of epicocconones ability to reversibly react with proteins to form an internal charge-transfer complex that is highly fluorescent. The protein-substrate is labeled in situ without the need for prior incubation and/or derivatization steps, which saves time and effort compared to methods employing specifically labeled protein-substrates. The assay can be carried out in 96- or 384-well plates, making it suitable for high-throughput applications in drug development and biotechnology.

Natural Products From Sponges of the Genus Agelas - on the Trail of a [2+2]-Photoaddition Enzyme

By specifically targeting sponges likely to contain oroidin derivatives, we have, for the first time, identified Australian sponges that contain sceptrin (2) and related compounds. Using a simple extraction technique and HPLC (with a photodiode array detector) in combination with LC-MS and MS-MS we have been able to quickly identify known compounds and flag the presence of some new compounds in the extracts. Further work will entail isolation and structure elucidation of the new compounds and collection of fresh Agelas sp.1 with the aim of isolating the enzyme that catalyses the [2 + 2] dimerisation or oroidin to sceptrin

Design and Synthesis of Epicocconone Analogues with Improved Fluorescence Properties

Epicocconone is a natural latent fluorophore that is widely used in biotechnology because of its large Stokes shift and lack of fluorescence in its unconjugated state. However, the low photostability and quantum yields of epicocconone have limited its wider use, and in the absence of a total synthesis, this limitation has been a long-standing problem. Here we report a general strategy for the synthesis of epicocconone analogues that relies on a 2-iodoxybenzoic acid-mediated dearomatization and on the replacement of the triene tail of the natural product by an aromatic ring. This design element is general and the synthesis is straightforward, providing ready access to libraries of polyfunctional fluorophores with long Stokes shifts based on the epicocconone core. Our structural modifications resulted in analogues with increased photostability and quantum yields compared with the natural product. Staining proteomic gels with these new analogues showed significant lowering of the detection limit and a 30% increase in the number of low-abundance proteins detected. These epiccoconone analogues will substantially improve the discovery rate of biomarker needles in the proteomic haystack.

Ultrafast dynamics of epicocconone, a second generation fluorescent protein stain

Femtosecond upconversion experiment has been carried out for epicocconone and its butylamine adduct in acetonitrile and tert-butanol. An ultrafast component is found to dominate the decay of fluorescence of epicocconone in acetonitrile solution. Upon reacting with butylamine, a model for the epicocconone-protein adduct, this ultrafast component remains almost unaffected but an additional rise time occurs, indicating the formation of a highly emissive species from the locally excited state. This phenomenon is central to the extraordinary applications of epicocconone in biotechnology. The magnitude of the rise time of the butylamine adduct is similar to that of the longer component of the decay of epicocconone in acetonitrile, suggesting that the dynamics of epicocconone and its butylamine adduct are similar. The ultrafast component is slowed upon increasing the viscosity of the solvent. This results in a marked increase in quantum yield and suggests that it corresponds to rapid bond isomerization, leading to a nonradiative decay. Surprisingly, in water/sucrose mixtures, the ultrafast component remains unaffected but there is still an increase in quantum yield, suggesting that there are at least two nonradiative pathways, one involving bond isomerization and another involving proton transfer. The correct interpretation of these data will allow the design of second generation protein stains based on the epicocconone scaffold with increased quantum yields and photostability.

A one-pot synthesis and biological activity of ageladine A and analogues

A one-pot synthesis of ageladine A and analogues is reported. The key Pictet-Spengler reaction between 2-aminohistamine and aryl aldehydes has been successfully utilized for the synthesis of the natural product and 14 analogues. These compounds were screened for their matrix metalloprotease (MMP) and kinase inhibition to develop the first structure-activity relationship of ageladine A analogues. One compound, which showed significant kinase activity but little MMP inhibitory activity, was found to be highly active in an antiangiogenic screen, suggesting that the angiogenic activity of ageladine A is not associated with MMP inhibition but rather kinase inhibitory activity. Cytotoxicity was excluded as a mode of action by the assay of ageladine A and an analogue against 60 human cell lines.

Short Communication: Secondary Metabolites from Basotho Medicinal Plants. II. Bulbine capitata

The medicinal plant Bulbine capitatais used by the Basotho of southern Africa as a mild purgative and to cure gonorrhea infections. Organic extracts of the powdered root were found to be rich in anthraquinones, which may be responsible for the reported laxative effects. Examination of B. capitata led to the isolation of five known anthraquinones: chrysophanol, knipholone, isoknipholone, 10,7´-bichrysophanol and chrysalodin, and one new anthraquinone glycoside, M-4´-demethylknipholone-2´-O-l-D-glucopyranoside.

Mass spectrometric compatibility of Deep Purple and SYPRO Ruby total protein stains for high-throughput proteomics using large-format two-dimensional gel electrophoresis†

In order to identify putative biomarkers from two-dimensional (2D) gel electrophoresis it is necessary to use a visualization technique that is sensitive, has a large dynamic range and does not interfere with the identification of the protein. As mass spectrometry increases in sensitivity more pressure is placed on visualization techniques that facilitate proteomic workflows but do not interfere with downstream processing. Two stains reported to meet these requirements are SYPRO Ruby (Invitrogen) and Deep Purple (GE Healthcare). This study examined the compatibility of these stains with protein identification by selecting spots from replicate 2D gels of human plasma and subjecting these to protein identification using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Using a test of two populations of proportions it was found that proteins were statistically more likely to be identified from gels stained with Deep Purple. Additionally, the identifications from Deep Purple stained gels are of higher quality because they are based on multiple peptides.