Binary modeling

4

Following the enormous strides taken in the 19th century, interest in the alkaloids waned somewhat during the interwar period. However, isolated alkaloids continued to be identified from traditional herbal treatments. For instance, Rauwolfia serpen-tina (Indian snakeroot), a traditional treatment for insanity, yielded the antipsychotic and antihypertensive indole alkaloid reserpine.⁷⁰ The introduction of reserpine, and the synthetic chlorpromazine, both in the early 1950s, revolutionized the treatment of schizophrenia, as they represented the first mainstream pharmacological treatments of any kind for psychosis.³⁴⁶ Similarly, physostigmine, from the Calabar bean, pro-vided the first treatment for the cognitive deficits of Alzheimer’s disease in the 1970s, and its semisynthetic derivatives, such as rivastigmine, and several other plant-derived alkaloid cholinesterase inhibitors, such as galanthamine and huperzine, continue to be the only viable treatments for the disease’s symptoms to this day.¹⁹⁵ Psychotropic alkaloids also continued to provide the underlying structure for the development of semisynthetic and synthetic drugs. So, for instance, cocaine’s structure gave us a wide range of local anesthetics, including procaine and lidocaine; curare gave us a selection of muscle relaxants; hyoscyamine and atropine formed the basis for an assortment of muscarinic receptor antagonists, including the Parkinson’s disease treatment ben-ztropine; and morphine’s structure provided an extensive range of analgesics, includ-ing pethidine and tramadol.¹⁹⁵ Alkaloid drug discovery naturally encompasses many non-psychotropics as well. For instance, in the early 1960s the traditional West Indian treatment for diabetes, Catharanthus roseus, yielded the antidiabetic vinblastine and its close relative, vincristine. It transpired that both of these compounds, plus several other Catharanthus alkaloids, had potent anticancer effects, inhibiting and reversing tumor growth. They continue in widespread use in oncology to the present day.⁷⁰

From the 1950s onwards the ethnopharmacognosy approach to drug discovery gave way to programs that involved the systematic mass screening of plants for components that were active in in vitro assays. Typically these screening programs looked for bio-activity relevant to cancer and compounds that could be either used in their natural state or structurally modified for increased potency. This novel approach led to the identification of camptothecin (from Camptotheca acuminata), a quinoline alkaloid with cytotoxic properties as a consequence of its ability to inhibit topoisomerase I, an enzyme required for DNA replication.⁷⁰ On closer inspection this tree, whose name translates from Chinese as “happy tree,” happened to have had a long history of tradi-tional Chinese medicinal use as a cancer treatment.³⁴⁷

The drug discovery process rolls on, and the alkaloids synthesized by plants and fungi continue to provide a particularly rich seam of compounds. It is interesting to note that to date, in terms of both ethnopharmacognosy and mass screening, the bioactivities of the phytochemicals present in only about 15% of plants have been investigated.³⁴⁸

■ S T R U C T U R E S A N D S Y N T H E S I S

Alkaloids are a structurally diverse group of low-molecular-weight compounds that contain one or more nitrogen atoms, typically as part of an amine group. While no single classification exists, alkaloids are often grouped on the basis of the identity of their nitrogen-containing structural component. Most of the familiar psychotro-pic alkaloids belong to a handful of these alkaloid groups: tropane (e.g., atropine, scopolamine, hyoscyamine, cocaine); pyridine (e.g., nicotine, arecoline); indole

(e.g., ibogaine, psilocybin, dimethyltryptamine, bufotenin, physostigmine, ergota-mine); isoquinoline (e.g., morphine); phenethylamine (mescaline); imidazole (e.g., pi-locarpine); and purine alkaloids (e.g., caffeine, theobromine). The underlying alkaloid structure for these groups is typically derived from an amino acid, which provides the nitrogen atoms and often leaves its own carbon skeleton largely intact within the struc-ture of the alkaloid. The principal amino acid precursors here are lysine, phenylalanine/

tyrosine, tryptophan, and histidine and derivatives such as nicotinic acid and anthra-nilic acid.¹⁹⁵ A number of alkaloids are also synthesized from non-amino acid precur-sors, such as acetate and purine.¹⁹⁵

Of the 27,000-plus alkaloids identified to date in nature, more than 75% are syn-thesized by plants.¹⁹⁵ They feature as secondary metabolites in approximately 30% of higher plants and are most often found in angiosperms, although other taxa, including ferns, mosses, and gymnosperms, also express alkaloids. Naturally, plants do not have a monopoly on alkaloid synthesis, and a number of alkaloids with specific effects on brain function are synthesized by fungi, including psilocin and lysergic acid. Plants, vertebrates, and invertebrates also share the synthesis of numerous chemicals that have an “alkaloid” structure but tend to be classified as “amines.” These include several compounds that we tend to think of primarily as human neurotransmitters, includ-ing serotonin, dopamine, histamine, and noradrenaline.¹³¹ Conversely, a number of alkaloids that we think of predominantly as plant secondary metabolites are also syn-thesized endogenously by animals in small quantities and play modulatory roles in brain function. These include several β-carboline alkaloids,³⁴⁹ morphine,²⁶⁶ and sev-eral of the tryptamine “hallucinogens,” including N,N-dimethyltryptamine (DMT), 5- methoxy-DMT (5-MeO-DMT), and 5-hydroxy-dimethyltryptamine (5-HO-DMT or bufotenin).²⁶⁷ These chemicals typically play “primary” roles in animal nervous system function. However, there are also many examples of alkaloids being used by an-imals in secondary or ecological roles. For instance, a wide variety of insects and frogs sequester alkaloids from their diet either to protect themselves from predators or alter-natively to use in their chemical communications with their own and other species (see below). Occasionally these plant-derived chemicals are slightly modified by the animal prior to use, but there are also a number of examples of animals synthesizing alkaloids de novo for use in ecological roles such as defense and chemical communication. For instance, the ladybird beetle family (Coccinellidae) synthesizes up to 50 distinct alka-loids, including adaline, coccinelline, and harmonine. These compounds are used as defensive compounds that leak from joints in a process termed “reflex bleeding” when the insect is threatened.³⁵⁰ While there are few mammalian examples, it is interesting to note that the musk deer synthesizes the alkaloid muscopyridine as a component of its musk pheromone excretion,³⁵¹ whereas beavers sequester the alkaloid castoramine from the water lily and excrete it as a component of a pheromone secretion that they use to communicate with other beavers.³⁵²

Some of the alkaloids with particular relevance to brain function are shown in Figures 4.2, 4.3, 5.1, and 7.1.

■ E V O L U T I O N O F A L K A L O I D S Y N T H E S I S

As described in the previous chapter, the evolution of the plant secondary metabolite pathways took place courtesy of the expansion of plant genomes due to a number of large-scale genetic duplication events. These events generated spare genetic material

that could mutate freely without endangering the survival of the plant.¹⁰⁹ The alka-loid biosynthetic pathways then typically arose from mutations in duplicates of the genes coding for enzymes involved in primary synthetic pathways. The novel enzyme would then produce slightly modified chemical products that in turn would be modi-fied by existing, promiscuous enzymes. This process would produce novel chemicals that might, in rare cases, increase the fitness of the plant by filling an ecological need.¹¹³ The distribution of the alkaloid group would then be dictated by the point on the evo-lutionary tree at which it arose, as well as whether it arose once (monophyletically) or multiple times (polyphyletically), and whether the clades further up the evolutionary tree retained the synthetic capacity.

The benzylisoquinoline family of alkaloids, which includes morphine, codeine, and thebaine, provides a monophyletic example. This group comprises some 2,500 known structures that are synthesized by a diverse minority of clades of eudicot plants. The first committed step in the synthesis of this group involves the condensa-tion of two L- tyrosine derivatives, dopamine and 4-hydroxyphenylacetaldehyde, by (S)- norcoclaurine synthase.³⁵³ The ability to synthesize this enzyme was therefore the first key unique development that gave plants the potential to produce benzyliso-quinoline alkaloids. Molecular phylogenetic research shows that the genetic event that potentiated the synthesis of the enzyme, the mutation of one of several primary me-tabolite proteins, occurred just prior to the diversification of the eudicot and monocot clades, with a minority of groups of eudicot plants further up the ancestral tree from the group subsequently evolving elaborations of the benzylisoquinoline structure to lesser or greater extents.³⁵³ But why has synthesis of benzylisoquinoline alkaloids been restricted to only a small proportion of eudicot species? One possibility is that follow-ing the evolution of the biosynthetic pathway the capacity for synthesis remains in many plants but is switched off,⁸⁶ or alternatively, mutations in the biosynthetic genes, or other genes playing regulatory or transport protein roles, lead to inactivation of the pathway.³⁵³ In line with this a “latent molecular fingerprint” for benzylisoquinoline alkaloids can be detected in eudicot plants that do not synthesize this group of chemi-cals.³⁵³ Naturally, the factors that lead to the switching on of synthesis, or its persever-ance, and the elaboration of specific alkaloid structures must be rooted in the selective pressure of environmental factors.

In contrast, the synthesis of tropane alkaloids developed polyphyletically follow-ing independent genetic events in a number of families of plants. Polyamines, such as putrescine, spermine, and spermidine, are primary metabolites synthesized by virtu-ally all living species via conserved pathways that include the conversion of putrescine to spermidine by spermidine synthase. The resulting compounds contribute to RNA transcription and the regulation of cell growth, differentiation, and cell death.³⁵⁴ How-ever, in plants of the Solanaceae (nightshade) family, which includes genera containing plants such as tobacco, mandrake, henbane, deadly nightshade, and Datura, a genetic event following the divergence of the Solanaceae from other taxa led to the creation of a duplicate of the gene for spermidine synthase. Free from selective pressure, this gene mutated and evolved a slightly modified function as putrescine N-methyltransferase, an enzyme that catalyzes the conversion of putrescine to N-methylputrescine. This compound represents the first substrate for the synthesis of nicotine and the other tro-pane alkaloids that give the Solanaceae their distinctive psychotropic properties.¹¹⁴ In general, the Solanaceae synthesize tropane alkaloids in their roots and transport them to aerial parts. However, there are some 200 tropane alkaloids, distributed within seven

distantly related families. Recent evidence suggests that plants from the Erythroxylum genus, whose last common ancestor with the Solanaceae existed over 100 million years ago, also independently developed the synthetic pathways that lead to the synthesis of tropane alkaloids, most notably cocaine. In this genus tropane alkaloids are synthe-sized in young leaves rather than the roots.^355,356

Similarly, the pyrrolizidine alkaloids are scattered among diverse lineages, with 400 different structures identified in 600 angiosperm species. In this case, the duplication and mutation of another gene involved in polyamine biosynthesis, the gene encoding deoxyhypusine synthase, led to the novel enzyme homospermidine synthase, which participates as the first unique step in pyrrolizidine alkaloid synthesis.¹¹³ This same genetic mutation has occurred at least four times in different angiosperm families.^357,358 Across the species expressing this group of alkaloids there are wide variations within and between species in the specific compounds produced by the pathway, with indi-vidual subgroups of compounds typically restricted to one clade. The sites of alkaloid synthesis also vary between clades, with the homospermidine synthase gene expressed in diverse cell types and tissues, ranging from root tips to reproductive organs, sug-gesting individual evolution of the regulatory elements controlling the enzyme’s gene expression.¹¹³ Overall the diversity in structures and sites of synthesis suggests that the first and evolutionarily most ancient steps in pyrrolizidine alkaloid synthesis are highly conserved, whereas the subsequent diversification of their synthesis across species is exceedingly plastic.³⁵⁸

The retention of any novel chemical would require that it increases the fitness of the plant to survive. In the case of alkaloids, defense is clearly the driver underly-ing the evolution of their synthesis. Ehrlich and Raven⁹² were the first to suggest that plant chemical defenses arose as a consequence of a complex insect/plant co-evolution driven primarily by a reciprocal “arms race.” By this account evolution proceeded by a series of alternating adaptive radiations, with plants developing novel defenses, thereby taking the advantage, and insects countering with their own adaptations, taking back the advantage. The exact nature of the plant/insect relationship in evolutionary terms has since been hotly contested, with the key point of contention being whether the co-evolutionary relationship was symmetrical, as envisaged by Ehrlich and Raven, or asymmetrical. In this latter scenario plants would have evolved and exerted unidirec-tional evolutionary pressure on insects, who were then engaged in a perpetual game of catch-up, but not vice versa.^90,123 While a number of competing theories have been proposed to explain the evolution of defensive secondary metabolite synthesis, they tend to share one common feature, the notion that the key relationship is that between insects and plants, with little weight given to the possible pressures exerted by other herbivores.

■ E C O L O G I C A L R O L E S O F A L K A L O I D S

Defense Against Herbivory

Evidence suggests that the primary raison d’être of the alkaloids as a class of plant chemicals is to function as toxic defense agents against herbivorous animals.^137,359 In particular, given the close relationship between the taxa, most alkaloids act specifically as toxins or feeding deterrents to most insects.⁹⁰ Typically, alkaloid biosynthetic path-ways will involve a variety of cell types with intermediates in the pathway trafficked

from cell to cell. These processes can take place in a variety of cell types and locations within the plant,³⁶⁰ with many alkaloids being synthesized at a distance from the site of storage, for instance in the roots, and then being transported via the phloem and xylem throughout the plant, accumulating in the tissue that is most valuable to the plant in terms of survival and reproduction.^119,361 The following three chapters will describe some of the evidence confirming a defensive role against insect herbivores for a number of the key alkaloids that modulate brain function.

At this point it is worth visiting some of the phenomena thrown up by the close relationship between insects and plants that support the notion of their co-evolution.

Many of these involve alkaloid secondary metabolites. The simplest examples are the many instances of evolved tolerance to specific alkaloids exhibited by the numerous specialist insects that live via herbivory on a single species or clade of plants. A classic example is the larvae of the tobacco hornworm moth (Manduca sexta), which con-sume tobacco leaf but excrete more than 90% of the nicotine they have concon-sumed within 2 hours. In comparison, when exposed to food containing nicotine, the house-fly (Musca domestica) excretes just 10% of the nicotine it consumes within 18 hours.⁹⁰ This evolved tolerance by the specialist can make increasing the synthesis of alkaloids in the face of specialist attack futile, so plants have developed the capacity to differenti-ate the nature of the attacker, for instance through an “immune system”-like reaction to the individual species’ saliva. They can then reduce defense chemical production when faced with specialist attack and instead increase growth to compensate for lost tissue.³⁶² However, the plant must also strike a balance. So, for instance, the response to the tobacco hornworm saliva and regurgitant can still be to increase the synthesis of nicotine and reduce root growth, as the nicotine does suppress the growth of the hornworm larvae despite its evolved tolerance. Plus, any reduction in nicotine content will make the plant’s foliage more attractive to a wide range of specialist herbivores, offsetting any advantage.³⁶³ For the hornworm, despite the detrimental effect of nico-tine on its development, it still makes sense to consume tobacco leaf, as any unexcreted nicotine is sequestered and stored in its cuticle as a defense against its own preda-tors.³⁶⁴ These include a number of generalist parasitoid wasps that lay their eggs inside the hornworm’s body. However, in a further twist of the various evolved relationships, specialist parasitoids such as Cotesia congregata have themselves evolved tolerance to the nicotine sequestered by the hornworm.³⁶⁴ For the plant, the many evolved relation-ships to its alkaloid load at higher trophic levels mean that whereas specific alkaloids act as deterrents to generalist insect herbivores, they can have an opposite, attractant, effect on their own specialist herbivores.^359,365

Sequestration of alkaloids is also the driver underlying many more complex evo-lutionary and ecological relationships among diverse organisms. As an example, the pyrrolizidine alkaloids either can be synthesized de novo by the plant, or alternatively, in the case of many species of grass, can be sequestered from endophytic fungi that live symbiotically with the grass in a relationship that includes the beneficial trans-fer of the fungal alkaloids to the grass. These alkaloids then act as feeding deterrents, growth inhibitors, and toxins to the vast majority of generalist herbivore insects.

However, a variety of specialist herbivore insects, including species of moth, butter-fly, and beetle, feed on the pyrrolizidine-expressing plants and actively sequester the alkaloids, accumulating them in turn in their cuticles and wings as a defense against predation by other insects, birds, and mammals. Of course, some predators have, in turn, evolved the capacity to tolerate the pyrrolizidines. For instance, the harvestman

spider (Mitopus morio) can eat the larvae of the specialist herbivore leaf beetle cour-tesy of its own ability to metabolize or excrete the beetle’s deadly load of alkaloids.¹¹⁹ The protective effect of the alkaloids can also ascend another level of the food chain.

So, for instance, the “poison dart” frogs from the South American genus Dendrobates possess a dietary uptake system for a wide range of alkaloids, including the pyrroli-zidines, which they accumulate from their diet of specialist herbivore ants that in turn had consumed the alkaloids from plants and accumulated them in their cuticles. The frogs then express the alkaloids on their skin as a defense against predation.³⁶⁶ In a final twist, indigenous human tribespeople then use the toxic alkaloid secretions from the frogs’ skins to poison the tips of blow darts that are then used to incapacitate their own hunted prey.

To add one final level of complexity, pyrrolizidines from the diet can also function as either pheromones or their precursors for numerous specialist butterfly and moth species. The males excrete the chemicals from hair-like structures on their wings and waft them at the female during courtship. This signals to the female how well endowed the male is with pyrrolizidines, indicating its own fitness and the level of protective alkaloids that will be transferred to any eggs that result from the relationship, offering them, in turn, protection from predation.¹¹⁹

Antimicrobial Properties

Alkaloids from a wide range of structural groups have been demonstrated to possess antibacterial and antifungal properties, although it is notable that both the phenolic and terpene groups of phytochemicals have markedly more bioactive members in this respect.³⁶⁷ Most of the evidence with regard to bacteria is garnered from studies assess-ing the in vitro effects of plant chemicals on microbes that have a particular relevance as human pathogens, such as Escherichia coli and Streptococcus pneumoniae. However,

Alkaloids from a wide range of structural groups have been demonstrated to possess antibacterial and antifungal properties, although it is notable that both the phenolic and terpene groups of phytochemicals have markedly more bioactive members in this respect.³⁶⁷ Most of the evidence with regard to bacteria is garnered from studies assess-ing the in vitro effects of plant chemicals on microbes that have a particular relevance as human pathogens, such as Escherichia coli and Streptococcus pneumoniae. However,