Alan L. Harvey

Natural products in drug discovery

Natural products have been the single most productive source of leads for the development of drugs. Over a 100 new products are in clinical development, particularly as anti-cancer agents and anti-infectives. Application of molecular biological techniques is increasing the availability of novel compounds that can be conveniently produced in bacteria or yeasts, and combinatorial chemistry approaches are being based on natural product scaffolds to create screening libraries that closely resemble drug-like compounds. Various screening approaches are being developed to improve the ease with which natural products can be used in drug discovery campaigns, and data mining and virtual screening techniques are also being applied to databases of natural products. It is hoped that the more efficient and effective application of natural products will improve the drug discovery process.

The re-emergence of natural products for drug discovery in the genomics era

Natural products have been a rich source of compounds for drug discovery. However, their use has diminished in the past two decades, in part because of technical barriers to screening natural products in high-throughput assays against molecular targets. Here, we review strategies for natural product screening that harness the recent technical advances that have reduced these barriers. We also assess the use of genomic and metabolomic approaches to augment traditional methods of studying natural products, and highlight recent examples of natural products in antimicrobial drug discovery and as inhibitors of protein–protein interactions. The growing appreciation of functional assays and phenotypic screens may further contribute to a revival of interest in natural products for drug discovery.

Strategies for discovering drugs from previously unexplored natural products

Natural products are the most consistently successful source of drug leads. Despite this, their use in drug discovery has fallen out of favour. Natural products continue to provide greater structural diversity than standard combinatorial chemistry and so they offer major opportunities for finding novel low molecular weight lead structures that are active against a wide range of assay targets. As less than 10% of the worlds biodiversity has been tested for biological activity, many more useful natural lead compounds are awaiting discovery. The challenge is how to access this natural chemical diversity.

Characterization of a potassium channel toxin from the Caribbean sea anemone Stichodactyla helianthus

A peptide toxin, ShK, that blocks voltage-dependent potassium channels was isolated from the whole body extract of the Caribbean sea anemone Stichodactyla helianthus. It competes with dendrotoxin I and alpha-dendrotoxin for binding to synaptosomal membranes of rat brain, facilities acetylcholine release at an avian neuromuscular junction and suppresses K+ currents in rat dorsal root ganglion neurones in culture. Its amino acid sequence is R1SCIDTIPKS10RCTAFQCKHS20MKYRLSFCRK30TCGTC35. There is no homology with other K+ channel-blocking peptides, except for BgK from the sea anemone Bunodosoma granulifera. ShK and BgK appear to be in a different structural class from other toxins affecting K+ channels.

Twenty years of dendrotoxins

Dendrotoxins are small proteins that were isolated 20 years ago from mamba (Dendroaspis) snake venoms (Harvey, A.L., Karlsson, E., 1980. Dendrotoxin from the venom of the green mamba, Dendroaspis angusticeps: a neurotoxin that enhances acetylcholine release at neuromuscular junctions. Naunyn-Schmiedebergs Arch. Pharmacol. 312, 1-6.). Subsequently, a family of related proteins was found in mamba venoms and shown to be homologous to Kunitz-type serine protease inhibitors, such as aprotinin. The dendrotoxins contain 57-60 amino acid residues cross-linked by three disulphide bridges. The dendrotoxins have little or no anti-protease activity, but they were demonstrated to block particular subtypes of voltage-dependent potassium channels in neurons. Studies with cloned K(+) channels indicate that alpha-dendrotoxin from green mamba Dendroaspis angusticeps blocks Kv1.1, Kv1.2 and Kv1.6 channels in the nanomolar range, whereas toxin K from the black mamba Dendroaspis polylepis preferentially blocks Kv1.1 channels. Structural analogues of dendrotoxins have helped to define the molecular recognition properties of different types of K(+) channels, and radiolabelled dendrotoxins have also been useful in helping to discover toxins from other sources that bind to K(+) channels. Because dendrotoxins are useful markers of subtypes of K(+) channels in vivo, dendrotoxins have become widely used as probes for studying the function of K(+) channels in physiology and pathophysiology.

Medicines from nature: are natural products still relevant to drug discovery?

Historically, most drugs have been derived from natural products, but there has been a shift away from their use with the increasing predominance of molecular approaches to drug discovery. Nevertheless, their structural diversity makes them a valuable source of novel lead compounds against newly discovered therapeutic targets. Technical advances in analytical techniques mean that the use of natural products is easier than before. However, there is a widening gap between natural-product researchers in countries rich in biodiversity and drug discovery scientists immersed in proteomics and high-throughput screening.

PROTEASE INHIBITOR HOMOLOGUES FROM MAMBA VENOMS: FACILITATION OF ACETYLCHOLINE RELEASE AND INTERACTIONS WITH PREJUNCTIONAL BLOCKING TOXINS

1 Five polypeptides, which were isolated from elapid snake venoms and which are structurally related to protease inhibitors, were tested for action on isolated biventer cervicis nerve‐muscle preparations of the chick. 2 Dendrotoxin from the Eastern green mamba (Dendroaspis angusticeps) and toxins K and I from the black mamba (Dendroaspis polylepis polylepis) increased responses to indirect stimulation without affecting responses to exogenous acetylcholine, carbachol or KCl. 3 The two other protease inhibitor homologues, HHV‐II from Ringhals cobra (Hemachatus haemachatus) and NNV‐II from Cape cobra (Naja nivea) did not increase responses to nerve stimulation. Trypsin inhibitor from bovine pancreas also had no facilitatory effects on neuromuscular transmission. 4 The facilitatory toxins from mamba venoms interacted with the prejunctional blocking toxins, β‐bungarotoxin, crotoxin and notexin, but not with taipoxin. The blocking effects of β‐bungarotoxin were reduced by pretreatment with the mamba toxins, whereas the blocking actions of crotoxin and notexin were enhanced. 5 The results indicate that protease inhibitor homologues from mamba venoms form a new class of neurotoxin, which acts to increase the release of acetylcholine in response to motor nerve stimulation. 6 From the interaction studies it is concluded that the facilitatory toxins bind to motor nerve terminals at sites related to those occupied by the prejunctional blocking toxins. However, differences in interactions with individual toxins suggest that there must be several related binding sites on the nerve terminals.

Dendrotoxins: Snake toxins that block potassium channels and facilitate neurotransmitter release

This scientific review looks at dendrotoxins: snake toxins that block potassium channels and facilitate neurotransmitter release

Screening of snake venoms for neurotoxic and myotoxic effects using simple in vitro preparations from rodents and chicks.

Eight snake venoms designated by the WHO as International Reference Venoms, and one additional venom were assessed for neurotoxic and myotoxic effects in vitro using the chick biventer cervicis and the rat and mouse phrenic nerve-diaphragm preparations. The objective was to determine whether any of the preparations could be used to detect evidence of neurotoxic or myotoxic activity prior to a more detailed examination. Bungarus multicinctus venom at concentrations above 1 microgram ml-1 selectively blocked neuromuscular transmission, with no direct effect on muscle fibres. Naja naja kaouthia and Notechis scutatus venoms selectively blocked neuromuscular transmission at low concentrations, but at higher concentrations both venoms caused direct effects on skeletal muscle resulting in contractures, loss of tension following direct stimulation and a loss in sensitivity to elevated [K+]0. Vipera russelli (Thailand) venom also blocked neuromuscular transmission but it was less potent than the venoms of B. multicinctus, N. n. kaouthia and N. scutatus. It also caused contractures in the chick biventer cervicis muscle. The venoms of Echis carinatus (Iran and Mali), Crotalus atrox, Bothrops atrox asper and Trimeresurus flavoviridis had limited neuromuscular blocking activity, and most of these venoms blocked [K+]0 and cholinoceptor stimulation in the chick muscle. Although both chick and rodent muscles allowed the assessment of neurotoxic and myotoxic activity, the chick biventer cervicis was simpler and more robust in use than either of the rodent phrenic nerve-diaphragm preparations. We propose that the chick biventer cervicis muscle could be used as a standard preparation for the screening of snake venoms for neurotoxic and myotoxic effects, and that it may be possible to use this preparation as a means to check that antivenoms can neutralize neurotoxic and direct myotoxic actions of venoms.

Dendrotoxin from the venom of the green mamba, Dendroaspis angusticeps

SummaryThe venom of the green mamba, Dendroaspis angusticeps has previously been shown to produce neuromuscular facilitation by increasing acetylcholine release. After gel filtration and ion-exchange chromatography of the whole venom, a basic polypeptide with facilitatory actions was isolated. This polypeptide, named dendrotoxin, has 59 amino acid residues, probably with only 3 disulphide bonds and a blocked N-terminus.When injected into conscious mice, dendrotoxin made the mice hypersensitive to external stimuli and subsequently produced respiratory paralysis. When tested on the isolated chick biventer cervicis nerve-muscle preparation, concentrations of dendrotoxin of 0.5 μg/ml (7×10−8 M) and greater, increased responses to indirect stimulation by 200–250%, without any increase in responses to submaximal concentrations of exogeneous acetylcholine, carbachol, KCl or direct stimulation. The augmentation was slow to develop, not reversed by washing, and could last several hours before slowly waning. Dendrotoxin did not produce spontaneous twitching or contractures.It is concluded that dendrotoxin is not an anticholinesterase, does not affect receptor sensitivity or muscle contractility, but produces twitch augmentation by increasing the amount of acetylcholine released by nerve stimulation. Thus, dendrotoxin appears to represent a snake venom neurotoxin with unusual chemical and pharmacological properties.