By Kevin She
1 Feb 2016
In the context of pharmaceuticals and natural health products, small molecules are organic compounds that may be synthetically produced, processed or isolated in some way from natural organisms, or are present in part of a natural organism. Acetylsalicylic acid in aspirin (originally discovered in willow bark as salicin and salicylic acid, but is primarily chemically synthesized now and in the acetylated form as the natural form can cause stomach and intestinal pain), nicotine in processed tobacco products or e-cigarette vape juices, or caffeine in coffee beans or tea leaves are all examples of bioactive small molecules. In this context, small molecules are the active ingredient in drugs, natural health products, and foods.
Figure 1 Acetylsalicylic acid: What’s Going On?
The figure on the left is a space-filling model for acetylsalicylic acid. The grey balls are carbon atoms, the red oxygen, and white hydrogen. The figure on the right is the skeletal formula for acetylsalicylic acid.
Most small molecule pharmaceuticals fall into three major classes; 1) synthetic copies of a small molecule found naturally in an organism, 2) naturally occurring small molecules with modifications that are chemically synthesized, or 3) synthetic small molecules inspired by naturally occurring small molecules. Small-molecule based pharmaceutical drugs that are designed completely from scratch are quite rare. Biologic drugs – those that are primarily protein or peptide based – are typically more heavily designed but selection by screening is still an important aspect of discovery and development. Part 2 of this “Small Molecules” article covers a little history of small molecule pharmaceuticals and natural health products in more detail. Part 3 is a sketch of modern small molecule drug discovery and development. This article will try to explore what small molecules are and why they naturally exist.
A conventional pharmaceutical small molecule is generally under 900 Daltons, a measure of mass for very small things. This arbitrary cutoff is mainly based on the maximum size of a molecule that can easily cross across a cell’s membrane in order to be absorbed into the bloodstream and/or to enter cells to exert their effects. However, the electrostatic charge, the hydrophobicity/hydrophilicity (something’s affinity for water), and how these properties are distributed within a small molecule all affect cellular permeability and are influenced by the shape and composition of the molecule. Many cells also have active transporters that can bring larger or otherwise low permeability molecules across the cell membrane at the expense of using up a little bit of energy.
It’s tough for most people to get a feel for how big (or how small) a small molecule is as it’s completely outside of our everyday experiences. The University of Utah’s Genetic Science Learning Center has an excellent interactive exhibit that lets you to zoom in from a representation of something the size of a coffee been down to an individual atom.
As an example, acetylsalicylic acid – the active small molecule in aspirin – weighs about 180 Daltons. Regular strength aspirin usually contains about 325 mg (milligrams) of acetylsalicylic acid which is about 1,000,000,000,000,000,000,000 (one sextillion) molecules. That’s an astronomic (or molecular) number.
Why are there small molecules?
In brief, life is fantastically very complicated and the biochemistry required for a cell to function is complex and involves a galaxy of different small molecules. Cells are constantly breaking down and building up many different molecules in order to do many things such as extracting energy, maintaining the cell’s structure, producing hormones and other signals to release out of the cell, producing the machinery to support protein synthesis and cell replication, and depending on the kind of cell potentially many more other functions. Almost all of this is done by different enzymes that break down one small molecule into two or more smaller ones, build a new molecule from smaller ones, add additional atoms or side chains to existing molecules, or modify the backbone or side chains of the molecule.
Giacoppo &al., 2014
Figure 2 The biosynthesis of the main cannabinoids.
Precursor small molecule olivetolic acid is converted to cannabigerolic acid (CBGA) which is subsequently converted into cannabichromenic acid (CBCA), cannabidiolic acid (CBDA), or tetrahydrocannabinolic acid (THCA) via the enzymes CBCA synthase, CBDA synthase, or THCA synthase. Upon drying or heating, these cannabinoid acids are decarboxylated into cannabichromene, cannabidiol, or tetrahydrocannabinol and can act on endogenous (naturally occurring, inside one’s body) cannabinoid receptors when ingested.
Bacteria and other unicellular (single-celled) organisms are relatively fairly simple as they are just single cells. Additionally, they typically grow in environments with many other different kinds of unicellular microorganisms and have to compete for locally limited nutrients. One strategy that microorganisms have adopted is very rapid generation times – they divide very quickly; E. coli, a bacteria, can divide every 17 minutes in favourable conditions. In doing so, fast growing bacteria can outnumber slower growing bacteria. Another strategy is the production of small molecules.
It is speculated that the earliest organisms did not have very efficient genomes and produced proteins that did not do anything to directly support the survival of that cell or produced enzymes (proteins that facilitate – catalyze – a chemical reaction), that produced small molecules that did not do anything (secondary metabolites). Theoretically having this non-productive genetic information is detrimental to survival; it means that more resources have to be devoted to replication compared with another organism that had a more efficient genome. However, the potential to create enzymes that produce various small molecules, even if they don’t immediately support survival of that cell, appear to be advantageous enough that persistence of silent or not-immediately-productive genetic material is tolerable.
Genes are subject to random mutations that change the shape of the protein or enzyme that they specify, much of the time this can turn a working enzyme into a non-reactive protein or one non-productive protein into another non-productive protein. Sometimes the mutation will change the shape of the enzyme so that it catalyzes a different chemical reaction giving rise to a different small molecule.
Indeed, at some point, through random mutation of an enzyme’s gene, one of these seemingly random and useless small molecules turned out to be harmful to other microorganisms (an antibiotic). This conferred increased competitive fitness (that bacteria reproduced much better than its neighbours) leading to propagation and persistence of that gene down successive generations (and many of the other genes that that particular organism possessed). The production of antibiotics (small molecules harmful to other bacteria), arose many times and continues to occur. Random mutations are not the only method microorganisms take advantage of to develop this biochemical machinery; actinomycetes, a kind of bacteria commonly found in soil and seawater, have evolved genetic structures that promote recombination of genes involved in small molecule biosynthesis (Chen &al., 2002) and are a natural source of highly varied small molecules (Doroghazi & Metcalf, 2013). The vast majority of all major antibiotics in use today originated from various bacteria (Emerson de Lima Procópio &al., 2012). The last section of Part 3 discusses actinomycetes and antibiotics in more detail. However, it is worth pointing out that the biosynthetic pathways in actinomycetes for the production of the antibiotics erythromycin and streptomycin are at least 500 million years old (Baltz 2007).
Plants, fungi, and other immobile organisms such as sponges face unique survival challenges because they can’t physically escape from predators. Among other survival strategies, the heritage of keeping seemingly useless genetic material and the production of not-immediately-useful proteins and enzymes that produce not-immediately-useful small molecules (secondary metabolites) persisted. When random mutations occur in these genes that randomly lead to the production of a small molecule that does helps the plant survive, the gene (and many of the other genes that that particular organism possessed) propagates and persists in subsequent generations. For example, the ability to produce caffeine and other methylxanthines arose independently between coffee (Coffeeae sp.), various shrubs and trees that are used to make tea, and in cocao plants (Theobroma cacao) that are used to produce chocolate (Denoeud &al., 2014). So while the gene structure of the biosynthetic machinery for producing caffeine and other methylxanthines are different between – and arose independently – in coffee, tea, and chocolate plants, the final molecule (caffeine) is identical. Caffeine and other methylxanthines, when ingested by insects, curb their feeding behaviour. At higher concentration it is directly toxic to the insect (Nathanson 1984).
Caffeine primarily exerts its effects on insects and mammals through a similar mechanism. Adenosine is a nucleoside, a small molecule that is a core component of DNA. Both insects and mammals additionally use adenosine as a signaling molecule in the nervous system and is detected by protein receptors for adenosine; caffeine has the right shape to bind to both insect and mammalian adenosine receptors and attenuates its normal inhibitory signaling (Ferré 2007) leading to easier and more frequent nerve firing. Insects, being much smaller than most mammals, are proportionally affected by caffeine and small doses are enough to induce seizures and paralysis. In mammals a sufficiently large dose can be fatal too, as it is to insects, but mammals are typically much larger and require a proportionally larger dose for deleterious effects (the LD50 – the dose lethal for half of a hypothetical population – for caffeine is around 127 mg/Kg, or about 10 grams for an average 80 Kg Canadian, or about 100x 8-ounce cups of drip coffee).
Denoeud &al., 2014
Figure 3 Relatedness of biosynthetic machinery for caffeine: What’s Going On?
At the top is a schematic for the biosynthesis of caffeine from xanthosine precursor. Below is the representation of the evolutionary relatedness of caffeine producing plants with respect to some other flowering plants. Coffee, tea, and cocao – all plants that produce caffeine – are highly unrelated, yet other similar plants – Coffea, Camellia, and Theobroma do not produce caffeine.
Likewise, nicotine is a small molecule that almost certainly arose as an insecticide to protect tobacco plants. Nicotine binds to acetylcholine receptors and activates them. Insects use acetylcholine as a neurotransmitter to control muscles and nicotine ingested by the insect first causes muscle contraction followed by paralysis. In mammals, glutamate is the primary neurotransmitter controlling muscles, however there are acetylcholine receptors in the brain. Nicotine interacts with these receptors in a very complex manner giving rise to the various effects of nicotine ingestion.
Another factor that has driven the evolution of machinery to make a vast variety of small molecules in plants is the complicated relationship between plants and its pollinators. While dioecious (two sexes) plants can usually pollinate each other via the wind, insect assisted pollination is rather more efficient and through co-evolution, can be quite precise. Plants have evolved to produce a wide variety of volatile small molecules such as terpenes that help attract insects through smell, and in many cases attract specific insects that visit only a limited number of different species of plant at specific times of the year coinciding with that plant’s maturity. While surprising, it was not completely outlandish to discover that a species of orchid that uses mosquitoes as pollinators produces volatile small molecules that are also similar to some present in human body odour (Pennisi 2016, Lahondère &al., 2016)!
Another famous example is the corpse flower or carrion flower (Amorphophallus titanum which means “giant misshapen penis” in Latin) that produces a variety of small molecules that are abundant in decomposing dead animals such as dimethyl trisulfide, dimethyl disulfide, trimethylamine, isovaleric acid, and a variety of phenols and indoles. By mimicking a dead animal, this flower attracts insects that normally feed on dead animals or lay their eggs in rotting meat to recruit them as a means of disseminating pollen (Shirasu &al., 2010).
Figure 3 Amorphophallus plant (http://www.charlottefive.com/what-does-a-corpse-flower-smell-like-people-lined-up-to-find-out-at-unc-charlotte/).
Long ago in pre-scientific times many cultures in both the West and the East believed in the “doctrine of signatures” thinking that plants that resemble parts of the body are effective in treating illnesses involving those body parts, hence the names of plants such as eyebright, liverwort, lungwort, spleenwort, and toothwort.
Figure 4 Some amusing looking organisms
From left to right, Devil’s Finger Mushroom (Clathrus archeri http://orimath.blogspot.in/2008/03/strange-mushroom-zombie-hand.html), Wood Ear Fungus (Auricularia auricular-judae https://en.wikipedia.org/wiki/Auricularia_auricula-judae#mediaviewer/File:Auricularia_auricula-judae_%28xndr%29.jpg), and Hooker’s Lips (Psychotria elata http://www.amusingplanet.com/2013/05/psychotria-elata-or-hookers-lips-most.html).
It’s well understood that this thinking is flawed, however, human inquisitiveness, curiosity, and the ability to recognize patterns have led us to discover herbs and other plant parts that really are effective in treating some illnesses and other conditions. Modern investigation has identified small molecules that are responsible for the effectiveness of herbal drugs and it is a combination of chance and a shared genetic heritage that some small molecules have biological effects, for good or ill (for example toxic tropanes such as atropine from nightshade and belladonna or ricin in castor beans). Sometimes a single kind of small molecule is sufficient to exert its effects but occasionally a combination of different small molecules is required for a polypharmaceutical or entourage effect.
One famous example is ayahuasca, a hallucinogenic beverage. Various plants including chacruna, chaliponga, and amyruca contain the hallucinogen dimetyltrptamine (DMT) that binds to serotonin and dopamine receptors, among others, in the brain. However, when consumed alone even in large quantities there are no psychedelic effects. Only when the beverage is made with ayahuasca vine tops (Banisteriopsis caapi) is there such an effect; the ayahuasca vine contains harmala alkaloids – small molecules that bind to and inhibit the enzyme monoamine oxidase A (MAO-A). Without inhibiting this enzyme, MAO-A enzymes in the stomach and small intestine inactivates the DMT before it can be absorbed into the bloodstream and then past the blood-brain-barrier to exert its effects.
Naturally occurring biosynthetic machinery evolved in response to challenges in survival and reproduction. Small molecules exert their biological activity by binding to protein enzymes or receptors and modulate the function of those enzymes or receptors. Salicylic acid is a signaling molecule in the willow tree, and other plants, that helps coordinate its growth. It is rather a coincidence that it binds to and inhibits the mammalian cyclooxygenase enzymes to exert its anti-inflammatory and anti-pain effects. Other therapeutic natural small molecules are effective as pesticides against insects and exert their effects in mammals because of conserved similarity in biochemistries between the two kingdoms. On the other hand, other small molecules, especially antibiotics, are effective in disabling various microorganisms but spare direct biological effect on mammals because of sufficient dissimilarities in biochemistry.
Baltz, RH. 2007 Antimicrobials from actinomycetes: back to the future. Microbe. 2(3):125-31
Chen, CW., Huang, CH., Lee, HH., Tsai, HH., & Kirby, R. 2002 Once the circle has been broken: dynamics and evolution of Streptomyces chromosomes. Trends. Genet. 18(10):522-9
Denoeud, F., Carretero-Paulet, L., Dereeper, A., Droc, G., Guyot, R., Pietrrella, M., Zheng, C., Alberti, A., Anthony, F., Aprea, G., Aury, JM., Bento, P., Bernard, M., Bocs, S., Campa, C., Cenci, A., Combes, MC., Crouzillat, D., Da Silva, C., Daddiego, L., De Bellis, F., Dussert, S., Garsmeur, O., Gayraud, T., Guignon, V., Jahn, K., Jamilloux, V., Joet, T., Labadie, K., Lan, T., Leclercq, J., Lepelley, M., Leroy, T., Li, LT., Librado, P., Lopez, L., Munoz, A., Noel, B., Pallavicini, A., Perrotta, G., Poncet, V., Pot, D., Priyono, Rigoreau, M., Rouard, M., Rozas, J., Tranchant-Dubreuil, C., VanBuren, R., Zhang, Q., Andrade, AC., Argout, X., Bertrand, B., de Kochko, A., Graziosi, G., Henry, RJ., Jayarama, Ming, R., Nagai, C., Rounsley, S., Sankoff, D., Giuliano, G., Albert, VA., Wincker, P., & Lashermes, P. 2014 The coffee genome provides insight into the convergent evolution of caffeine biosynthesis. Science. 345(6201): 1181-84
Doroghazi, JR., & Metcalf, WW. 2013 Comparative genomics of actinomycetes with a focus on natural product biosynthetic genes. BMC Genomics. doi: 10.1186/1471-2164-14-611
Emerson de Lima Procópio, R., Reis da Silva, I., Kassawar Martins, M., Lucio de Azevedo, J., & Magali de Araujo, J. 2012 Antibiotics produced by Streptomyces. Braz. J. Infect. Dis. 16(5): 466-71
Ferré, S. 2007 An update on the mechanism of the psychostimulant effects of caffeine. J. Neurochem. 105(4): 1067-79
Giacoppo, S., Mandolino, G., Galuppo, M., Bramanti, P., & Mazzon, E. 2014 Cannabinoids: New promising agents in the treatment of neurological diseases. Molecules. 19: 18781-816
Lahondère, C., Vinauger, C., Okubo, R. & Riffell, JA. 2016 What makes mosquitoes attracted to Platanthera orchids? Soc. Int. Comp. Biol. Annual Meeting P1-122
Nathanson, JA. 1984 Caffeine and related methylxanthines: possible naturally occurring pesticides. Science 226(4671): 184-7
Pennisi, E. 2016 Orchids give off human ‘body odor’ to attract mosquitoes. doi: 10.1126/science.aae0172
Shirasu, M., Fujioka, K., Kakishima, S., Nagai, S., Tomizawa, Y., Tsukaya, H., Murata, J., Manome, T., & Touhara, K. 2010 Chemical identity of a rotting animal-like odor emitted from the inflorescence of the titan arum (Amorphophallus titanium). Biosci. Biotechnol. Biochem. 74(12);2550-4