Pain management is currently one of the most challenging therapeutic goals. Chronic pain is pain that persists beyond the usual time of healing and is associated with numerous conditions, including cancer, diabetes, multiple sclerosis and HIV/AIDS. According to the World Health Organization chronic pain will affect 20% of the population in developed countries. Current therapeutics provide relief for most patients but are ineffective in many cases, decline in efficacy over time due to the development of tolerance, or produce severe adverse drug reactions. Thus there is a rising need to develop new analgesic agents that can improve the quality of life for millions of people.
Classical analgesic drugs, notably opiates and non-steroidal anti-inflammatory drugs (NSAIDs) originate from natural products. In fact, natural products from terrestrial plants and microbes have been employed in disease therapy for hundreds of years, whereas marine natural products (MNPs) are a relatively new source of drug molecules that hold huge potential for further pharmaceutical development. This thesis focuses on MNPs, more specifically on disulfide-rich peptides from cone snails and marine sponges and the beginning of their journey towards the pharmaceutical market.
The venom produced by marine cone snails comprises a group of small bioactive peptides, conotoxins, which target a variety of membrane receptors and ion channels with high selectivity and potency. Many of these pharmacological targets are involved in nociceptive pathways and consequently conotoxins, in particular α-conotoxins, are very interesting leads for drug development, or as drug themselves. One of their recently discovered targets, the γ-aminobutyric acid type B receptor (GABABR) is a key receptor in neuronal signaling and its malfunction has been implicated in a range of neurological and psychiatric disorders, including analgesia, depression and drug addiction.
This thesis examines peptides from marine sponges and cone snail venom as potential sources of novel pharmaceuticals, in particular for the treatment of chronic pain. Chapter 1 provides a background of these natural marine wonders and their rich arsenal of disulfide-rich peptides. Chapter 2 focuses on conotoxins as drugs for the treatment of pain, and in particular on the development of approaches to overcome some of the disadvantages of peptide-based drugs, with an emphasis on engineering orally active compounds. In Chapters 3−6, efforts are made to develop α-conotoxins into drug-like compounds and to explore their interaction with their newly discovered target for analgesia, the GABABR.
Chapter 3 describes the design and synthesis of a selective and potent GABABR cyclic analog of the α-conotoxin Vc1.1. In Chapter 4, seven α-conotoxins were chosen based on sequence similarity to previously discovered conotoxins that target the GABABR and subsequently synthesized and structurally characterized by NMR and CD spectroscopy and further tested for their effects at this target, including selected nicotine acetylcholine receptors (nAChRs). Two novel α-conotoxins, Pn1.2 and Pu1.2, were found to be potent inhibitors of calcium channel currents (ICa) via GABABR activation.
For any compound to progress to the clinic, the issues of bioavailability and membrane permeability must be addressed. In Chapter 5 the cyclization linker sequence of α-conotoxin cVc1.1 was modified to improve its stability and pharmacokinetic properties. Three new linker analogs were synthesized, characterized and analyzed for their biological activity. Surprisingly, none of the analogs were found to modulate ICa despite the structural conservation of the analogs to cVc1.1, as shown by NMR. Caco-2 cells and parallel artificial membrane permeability assays (PAMPA) indicated that none of the analogs were taken up by passive transport across the intestinal mucosa.
The interaction between α-conotoxins and the GABABR was analyzed in Chapter 6 by performing a variety of radioligand binding assays using tritiated and iodinated derivatives of Vc1.1 and different cell lines, including dorsal root ganglion (DRG) neurons. Binding experiments revealed that Vc1.1 did not bind to the same binding site as the selective GABABR antagonist, CGP54626. Further studies suggested that Vc1.1 do not bind the GABABR, as experiments that did show binding were inconsistent and not reproducible.
Chapter 7 discusses the extraction, isolation and characterization of two novel peptides from the marine cold-water sponge Geodia barretti. Their sequences were elucidated by tandem MS sequencing supported by amino acid analysis and found to only differ at one position. The three-dimensional structure for one of the peptides was determined by NMR spectroscopy and showed to comprise a β-hairpin followed by a tight turn starting at Pro20. Interestingly, Pro20 is in the cis configuration.
The continual exploration of new drug leads from diverse pharmaceutical sources is crucial to help doctors give the best medical treatment to patients. This thesis investigated and characterized peptides from cone snail venom and marine sponges, contributing several potential novel drug leads for the treatment of a range of neurological conditions, including pain.