The Pharmaceutical Journal
Vol 277 No 7483 p725-727
22/29 December 2007

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Frogs and snails and invertebrate tales

Before recombinant DNA technology, animals were the only viable source of many hormones. Insulin and growth hormone were obtained from pigs and cows and conjugated oestrogens were collected from the urine of pregnant mares (hence the name Premarin). More recently, however, attention has turned to what venomous creatures might offer, with several new drugs being developed. Christopher Shaw takes a look

Christmas miscellany 2007 index

The publication of the first draft of the human genome in 2001 was generally agreed by scientists from many disciplines as a milestone in human endeavour of a magnitude worthy of marking the threshold of a new millennium.

In terms of pharmaceutical development, this was meant to provide a host of new drug targets and provide a base for rational drug design that would usher in a golden age of profitability for the industry and serve to generate a plethora of new therapies for the major intractable diseases of humankind.

Alas to date, this has not been realised. In fact, the pipelines of many if not most major pharmaceutical companies have been reduced to trickles if they have not effectively ceased to flow.

The mainstay of drug discovery for the industry during most of the past century was natural product chemistry, which involved large scale panning of molecular libraries contained in crude extracts from plants and micro-organisms. Although this was not directed by logic in most instances, it produced morphine, aspirin, quinine, many antibiotics, artemisinin and some front-line anticancer drugs, such as paclitaxel. This non-rational approach to drug discovery made the pharmaceutical industry one of the most profitable sectors in the world economy.

Serendipity, essentially the paradigm in operation for drug discovery for almost a century, weighed heavily within the breasts of industry chief executives and accountants who would have much preferred a more rational and predicable approach to filling product pipelines, as exemplified by James Black in the discovery of two fundamental drug classes, the beta-blockers and the H₂-receptor antagonists.

Both were “designed” to interact with highly specific and defined molecular targets. In addition, “designer drugs” — essentially new chemical entities that may not exist in the universe outside the medicinal chemist’s test-tube — were easily patentable and protectable, in contrast to “natural products”.

However, massive investment in this approach has failed to provide the array of templates required to fill drug pipelines leading to the dearth of new drugs that we have today.

Food for thought

Scientists like myself now believe that we need to evaluate all of these parameters critically in the light of the “new biology” and to return essentially to unravelling the accumulated molecular wisdom of nature, arrived at after aeons of natural selection and resulting in fit-for-purpose molecular design.

Peptides and proteins are no strangers to living things — both have coevolved for, perhaps, 3.5 billion years and although many proteins perform structural or catalytic functions within living systems, most peptides are messenger molecules conveying information between cells. It is these attributes that render many proteins drug targets and most peptides as their cognate ligands.

Although many peptides have expanded the coffers of the pharmaceutical industry while alleviating much human mortality and morbidity (insulin being the archetype of this class), the pinnacle of peptide signalling and targeting evolution resides in the arsenals of smart weaponry found in animal venoms.

Venoms are produced by many animals as an effective means of subduing prey or as agents of defence or both. Almost every unfortunate incident involving human envenomations are either accidental (eg, jellyfish stings) or as a result of the venomous animal attempting to defend itself or its kin (eg, snake bites and bee stings).

Nevertheless, anyone who has ever suffered such envenomation and survived, will testify to the profound biological effects that follow, ranging from intense pain and inflammation to clinical emergency.

All venoms are complex mixtures and often the spectrum of biological effects results from interaction and co-operation of components. Dissection of these components by physicochemical separation of the mixture, can often identify individual molecules with potential therapeutic uses either directly or as exquisitely targeted carriers for chosen toxic payloads.

A classic example of the former are the bradykinin-potentiating peptides originally found in the venom of a South American viper (Bothrops jararaca) and, subsequently, in the venoms of many viperine snakes. These were found to be responsible for the profound hypotension experienced by humans following envenomation and, as this appeared to occur due to inhibition of angiotensin-converting enzyme (ACE), became the lead compounds for ACE inhibitors that have become the mainstay of long-term hypertension management in the developed world.

The strategy adopted by vipers in general is one of interference with cardiovascular regulation and haemostasis. In the latter instance, components with profound anticoagulant and clot-busting effects have found their way into clinical practice.

Ancrod, an enzyme from the venom of the Malayan pit viper is a powerful defibrinogenating agent and has been used to dissolve clots following both cardiac and cerebral infarcts. Likewise, tirofiban (Aggrastat) developed from the venom of the African saw-scale viper, has been used to prevent myocardial infarction.

The disintegrins found in many snake venoms are powerful platelet anti-adherence agents and have been used to unravel the molecular mechanisms of platelet aggregation. Drugs such as tirofiban and eptifibatide (Integrilin), were produced following acquisition of this knowledge and the anticell adhesion function of disintegrins has found possible fundamental applications in antiangiogenesis and cancer cell biology.

Other protein factors from snake venom are powerful procoagulants and these have found efficacy in application to bleeding wounds in accident victims (for example, those trapped in car wreckage) whose delivery to hospital is delayed by such a degree that death would be probable.

Although effective therapeutic leads have been developed for some time now from snake venoms (partially as a result of focused research attention on their uses in many traditional medicines and the primitive behavioural aversion of humans to these reptiles), the most recent and dramatic appearances on the therapeutic stage have been from venom components from the most unexpected sources.

Another source of agents affecting haemostasis has been provided by observational study of haematophagous invertebrates, such as leeches and ticks, followed by biochemical analyses on their saliva. Hirudin (eg, lepirudin [Refludan]) from leech saliva is a potent and highly specific inhibitor of thrombin with applications in thrombolysis.

However, keeping to a reptilian theme, the most exciting development in recent times for the treatment of type 2 diabetes, which has become of epidemic status in developed countries and is set to reach pandemic status in rapidly developing countries, resides in a venom polypeptide from the Gila monster (Heloderma suspectum), a rotund lizard from the deserts of the south western US, bordering Mexico.

The story of its discovery is an epic in drug development, fulfilling Pasteur’s adage of “chance favouring the prepared mind”. Profound hypotension is a general effect of envenomation by this lizard, which is a rare event usually occurring in members of the local Indian tribe whose manhood rituals include placing one’s hand in the mouth of this rather bad-tempered animal.

About the time of the discovery of vasoactive intestinal peptide (VIP), a vessel-dilating and consequent hypotension-inducing peptide from mammalian intestine, scientists studying Gila monster venom decided to bioassay it for VIP using the assay available in their laboratory at the time: the stimulation of pancreatic secretion. A positive result was obtained and one of the several peptides isolated was named exendin-4.

This peptide was subsequently found not to be an analogue of VIP but of a newly discovered insulin-releasing peptide, glucagon-like peptide 1 (GLP-1). While GLP-1 was found to be relatively unstable in human plasma, exendin-4 had superior pharmacodynamics and acted as a full agonist at GLP-1 receptors on pancreatic islet beta cells effecting a glucose-dependent insulin release. An injectable exendin-4 analogue, exenatide (Byetta), is now marketed for the treatment of type 2 diabetes.

Frogs and snails

Although amphibians do not produce venoms in the strictest sense, many species do discharge a defensive skin secretion that is a complex cocktail of peptides. One of the most abundant classes of peptides in these secretions has broad-spectrum antimicrobial properties.

In the multiple drug-resistant bacterial environments of today (no, not sewage farms but hospitals) there is an ever increasing need for new antibiotics especially those with modes of action that are unlikely to induce resistance in the short term. As the frog skin peptides kill bacteria essentially by membrane lysis, it is difficult to envisage a rapid development of resistance if this will occur at all.

Thus huge efforts are under way to reduce the haemolytic effects of candidate peptides while maintaining or increasing their antibacterial activities.

In recent times, some of these peptides have been found to have antiviral effects, most notably against HIV, and this has opened up an avenue of research that may provide new therapies in a field that is sparsely populated.

In addition to the antimicrobial peptides, amphibian secretions contain protease inhibitors, mucous membrane growth factors, potent analogues of human neuropeptides involved in anxiety mediation and appetite control, and potent vasodilators. This is not an exhaustive list by any means and from personal experience over many years in this research, our group has never used any bioassay that has produced a negative result indicating the plethora of molecular targets with which individual peptides can interact.

Indeed, my interest in this field was taken after reading several reports about South American Indian “hunting magic” rituals and subsequent personal accounts of human bioassays by visiting anthropologists. Application of the “sapo” (a native name for the dried frog skin secretion), dispersed in a saliva vehicle, to self-inflicted burns, produced an immediate and massive peripheral vasodilation (the subjects turned bright red) with a profound decrease in blood pressure and increase in heart rate.

This was rapidly followed by forceful vomiting and defecation described as “total vacation of the gastrointestinal tract”. Unconsciousness followed for several hours after which the subject awakened feeling in a “god-like” state, with enhanced sensory perception, unlimited stamina and total loss of appetite for up to three days.

One can see why the natives use this before a hunt and also why a biomedicinal chemist would be intrigued. Let us just hope that there are separate agents mediating these effects. Like the natives, we can harvest the secretions in a non-invasive, non-lethal manner at monthly intervals and can gain all peptide structural and gene-coding information from even single samples of dried secretion.

The venoms from lower animals (invertebrates) have not been exempt from the searching eye of the biomedicinal chemist and, in two instances, new peptide drugs for unmet clinical needs have been developed. Chronic neuropathic pain is a most debilitating condition and rapidly becomes resistant to opioid analgesia in dosages that are below the induction of systemic sedative effects. One of the latest and effective treatments for this condition, ziconatide (Prialt), is a synthetic version of a venom peptide from a tropical marine cone shell.

Some cone shells catch and instantly stun their fish prey by means of a barbed harpoon that both penetrates and injects a complex peptide-based venom. Many of the peptides are potent blockers of ion channels — multi-subunit proteins in cell membranes that are highly conserved between species. For this reason, accidental cone shell envenomation of humans can have a fatal outcome.

However, one of the component peptides or conotoxins, is targeted to calcium channels and it is this peptide, in appropriate dosage and delivered locally, that can ameliorate chronic neuropathic pain via an opiate-independent mechanism and without the tolerance induction of these drugs.

Cancer therapeutics remain one of the major foci of interest to the biomedicinal chemist and it is here that venom-derived peptides are expected to have a major impact. A recent advance in the treatment of what was once-considered to be an untreatable tumour (the glioma) has been found within the venom of a denizen of the deserts of Israel: the giant yellow scorpion (Leiurus quinquestriatus).

Scorpion venoms are complex mixtures of peptides, many of which are precision-targeted like the cone shell toxins, to ion channels on the surfaces of cells. One such toxin named chlorotoxin was found to specifically block chloride channels that happen to be almost specific for glial cells and to be greatly over-expressed in malignant gliomas. Using this precision weapon on this discrete target, it was found that chlorotoxin could effectively deliver a cytotoxic payload and in fact, was capable of inhibiting tumour growth in its underivatised state.

The results are promising and what is even more interesting is that chlorotoxin binding has been found to be high in malignant melanoma and in small cell lung tumours; both are highly invasive tumours with few effective therapeutic options. The effectiveness of this toxin in these conditions is now being assessed by Transmolecular Inc, of Birmingham, Alabama, which discovered and developed this arachnid anticancer drug.

What the future could hold

I have described some of the major examples of venom-derived peptides that have progressed to clinical use but many are in the pipeline as research and development scientists become convinced that they may hold the key to successful drug development programmes in many areas of unmet clinical need.

Venom peptides for the most part have cruise missile precision in seeking their target proteins that are nearly exclusively located on the outer surface of cell membranes. This differential target expression on cell surfaces in contrast to the need for many biochemical pathway-disrupting drugs to penetrate the cell membrane, serves to increase their likelihood of an effective target strike and to reduce that of non-specific collateral damage (after all what passes through one membrane will pass through all).

So the pharmaceutical industry has been presented with another take on natural product chemistry. Not the random extraction of organisms for their metabolic toxins and the Las Vegas-style screening of the past but rather, a sentient, informed and objective look at peptidic molecular libraries with their teams of highly specialised and purpose-designed molecular weapons, in a trek of discovery that is worthy of the new biology and the highest goal of saving human lives.

Nature could provide many of the answers — we just have to ask the right questions.