The art of blowing up protein

A question (of the nice variety) that students sometimes ask is about the relevance of natural products. The answer is a yes-and-no answer. I like to add to the usual arguments, an evolutionary take, which is two sided. A lot of secondary metabolites made by plants have been evolved to kill you (a mostly herbivore), but your liver has been evolved to be good at destroying them. For drug discovery, this has two opposite effects: the pro is a long list of antibacterial, antifungal and anticancer compounds to use or to adapt, the con is their ADME (absorption, distribution, metabolism, and excretion) properties have frustrated medchemists for decades and decades. Herein, the two faces of this coin are explored not by advocating for or against natural products, but exploring what they mean for medicinal chemistry.

Caveats

Herein, I am going to go over the full history,  and majorly the pros and cons of natural compounds in drug discovery. I want to instead talk about what evolution means for natural products in drug discovery. 

I am trying to not take sides, but, despite loving the biosynthesis of secondary metabolism, I am on the side of the detractors of natural products in contemporary drug design.</>

When talking about evolution the language gets stilted when trying to be fully accurate. Species, and kind of as a consequence, genomes, evolve, whereas protein and small compounds do not evolve. Protein families could be said to evolve even though a mutation happens on DNA not a protein. Therefore, when I say "a natural product evolved", what I meant to say is "the genes encoding the enzymes involved in the pathway that produces a compound', which is worse than chatGTP verbiage. The other error I make below is to make Nature or Evolution sound sentient. I therefore I should stress that all reasons for a compound being selected are casual (happen because of a cause) not teleological (happen towards a goal).

Naturalia non sunt turpia

"Quod naturalia non sunt turpia" is a common quote (actually a paraphrasing) from Virgil that you see written places trying hard to be fancy about their all natural ingredients —I last saw it on a wall in a microbrewery shop. The quote means what are natural are not foul, but in the original meaning was that nature is amoral, not that it is healthy. Do not get me wrong, I (mostly) eat vegetable over junk food and I do agree that the food industry does have a lot to be blamed for, but the remote evolutionary history of natural food did not care that you are healthy: plants do not exist to be helpful, they exist because they evolved to survive (with a recent farming twist).

Natural products are metabolic products of biochemistry. Primary metabolism products are essential for growth, while secondary metabolism increase fitness, commonly by preventing predation or knee-capping competition. Organisms such as plants, fungi or bacteria do not want to be eaten by herbivores or attacked by viruses, fungi and bacteria and often have highly evolvable secondary metabolism pathways thanks to plasticity endowed by unrestricted broad specificity / promiscuity. Plants are especially packed with phytoalexins (signals) and phytotoxins. Few people are foraging hunter-gatherers, so it is not commonly appreciated that a lot plants and fungi are toxic —did know you cannot eat daffodil bulbs? Basically, Nature does not want to increase your lifespan, but actually wants to kill you or its infection —hence the many antibiotics, narcotics, chemotherapy or immunosuppressants, but very few compounds for specific ailments.

Cancer is uncontrolled cell proliferation: curing cancer consists of killing these cells without killing all other cells. Many phytotoxins are good at the first part, not such much at the second. When talking of drug safety, the concept of therapeutic window (the gap between being effective and toxic) is used: most phytotoxins are not specific enough to be effective or safe, but some happen to be. (Strychnine, ricin and other very biologically active poisons that are synthesised at scale are not classed as pharmaceuticals)

Total synthesis

Many historical FDA drugs were derived from natural products, but had the major challenge of being hard to make by humans (i.e. total synthesis, not biosynthesis). The history of penicillin G is fascinating for example — John Sheehan in 1957 managed the total synthesis of the analogue penicillin V in 11 steps, with the ring lactam as a neat donimo reaction, whereas Penicillium rubens does it in three steps, the second closing the ring is done by Isopenicillin N synthase, a αKG-dependent hydroxylase (radical oxidation), putting the synthetic chemists to shame.

In 2014 I attended a Gordon Research conference on Biocatalysis and one panel topic was why the biocatalysis revolution had not come about (yet). Sadly, this is still true today: enzymes can win you Green Chemistry awards, but they are not the easiest to scale. Namely, biosynthetically advanced compounds suffer from poor yield hence the need for total synthesis, which in some synthetic routes would scare even Cthulhu.

Paclitaxel (taxol) is probably the archetype of natural product whose total synthesis is diabolical. The main ring system, taxadiene, is made by the usual single step terpene cyclisation by biosynthesis, whereas it takes well over two dozen steps by total synthesis.

Frequently FDA compounds are made semisynthetically, even at scale —vitamin C is one such example, which is made from glucose syrup. This applies also to some more recent, designed compounds such as Oseltamivir. Oseltamivir (Tamiflu), FDA approved in 1999 (so relatively recently), is derived from shikimate, a common metabolite, and at first required shikimate extracted from star anise which is how it started —shikimate pathway is how the aromatic amino acids. Even though the compound is near ubiquitous, these plants do make a lot of shikimate derivatives, for example anethole, the aniseed aroma compound (derived from phenylalanine via the usual cinnamic acid). In other words, not all bio-inspired 3D compounds are scary and biosynthetic building blocks are not unheard of.

Parenthetically, I should note that I am talking of trends, as there obviously there are exceptions. A counter-example is midostaurin (and lestaurtinib in clinical trials) which is a recently (2017) approved derivative of staurosporine, a fun-looking mostly planar alkaloid from a streptomycete.

Metabolism

Biosynthesis space

It is true that enzyme can achieve great feats of catalysis, but the space is actually rather limited and most compounds can be traced back to a few building blocks, such as central metabolites, polyisoprenyl-diphosphates, coumaric acid or cinnamic acid —it is fairly easy to stare at a given compound at figure out how it was biosynthesised, especially when there are many methyl groups that give terpenoids away, or hydroxyls and ketones giving polyketides away. The magic of enzymes comes from the selectivity not diversity of reactions. Pictures is nepetalactone, the active compound (for cats) in catnip: the geranyl-DP atoms can be easily spot by starting at the ester and following the scaffold around.

Pharmacokinetics

A consequence of the natural role of natural products is that our body is well evolved to not get poised easily. Pigments in food are a visual proof —you don't turn blue or pee blue if you eat blueberries (malvidin, an anthocyanin). This means that absorbed natural products are more likely to be broken down by cytochromes in the liver than more exotic compounds. Cytochromes are very proficient in the oxidation of methyl groups due to the isoprenyl building block, hence why these are commonly fluorinated in drug design. The methyl groups were evolutionary selected to be there in the first place likely because cyclisation is easier thanks to the Thrope–Ingold effect. Large high sp³ compounds are the target of cytochrome P450 3E4 for example, while aromatic compounds are generally targeted more slowly by others like 1A2 or 2C9. Molecules absorbed from the digestive tract go into a vein that passes through the liver, an safety mechanism / medchem effect known as first pass metabolism greatly diminishing oral bioavailability of compounds.

The blood brain barrier exists to stop infections spreading to the brain and to filter out toxins. Capillaries are generally 'fenestrated' with pores between the endothelial cells, except in the brain where they form tight junctions, so all metabolites need to go through the endothelial cells (also seen in lungs, skin, placenta, intestines). In order words, it is double filtered! So natural products are in part to blame for why neuronal targets are really hard in drug discovery!

Taken as intended

Antibiotics, antifungals and antivirals are small molecules that are made by the host to fight infections within the host: they have the strongest case as demonstrated by the endless list of antibiotics (penicillin, cephalosporin, vancomycin, chloramphenicol etc.), and antifungals (nystatin, fluconazole, micafungin etc). To this grouping one can easily add naturally derived insecticides, most notably pyrethrin derivatives (permethrin, deltamethrin and cypermethrin), which are used as veterinary spot-on treatment and are safe for humans to breathe.

Virus infections in plants are primarily fought by silencing RNA and viruses operate in very diverse ways. Natural neuramidase inhibitors do exist, albeit with limited efficiency. Oseltamivir as mentioned is derived from shikimate (a decorated cyclohexene), but is much larger and much much more potent. Most studies into herbal remedies against a viral infections identify a compound that is very weak.

That was the plan

The other category mentioned is to stop predation. One can conjecture a few routes for this:

1. Killing the predator by cardiac arrest: aconitine (from toadstools)
2. Killing the predator by paralysis: tetrodotoxin (puffer fish)
3. Dissuading predation: amarogentin (one of the most bitter compounds known) or capsaicin (minicks fire response, which is rather neat and I would not have thought of it)
4. Trigger a reckless behaviour in weird ways: LSD
5. Putting the predator to hypnotic sleep: morphine
6. Putting the predator in hypoglycemic coma: galegine (led to metformin)
7. Putting the predator to restorative sleep: nothing. Serotonin would seem like an easy compound to mimic, but as far as I know this is not done — 5HTP is a shared metabolite for serotonin and for whatever the Griffonia plant wants to make, while the bioactive ingredients in chamomile extract are just small greasy compounds, which happen to cross the blood brain barrier.
8. Keeping the predator youthful by activate telomerases: what, why?!

Regarding the latter, astragaloside (TA-65) activates telomerases, but it might be a promiscuous unintended effect rather than was it evolved for in the Astragalus plant. Resveratrol, a simple poly-hydroxylated stilbene from grapes, was claimed as telomerase activator, and by consequence a health benefit of drinking wine...

Don't look at hibernating Gila monsters

A parenthesis needs to made for semaglutide: this biologically inspired drug is by far and large the pharmaceutical blockbuster of the decade. This helical peptide is derived from extendin-4, which was isolated from the venom of a Gila monster, a mesoamerican lizard. Despite contaminating venom, it is actually a blood protein involved in hunger regulation for extended periods —which is what humans use it for. So not only it is a peptide and not a small molecule, it was evolved for endogenous homeostasis, therefore, whereas it does amply demonstrate that studying the natural environment can be extremely fruitful, it does not say anything on whether screening natural small compounds from different phyla of life can be.

Coincidental repurposing

So far I have been talking of natural products that activate or inhibit a target protein in the same way as it was evolved to often to different effects (e.g. mandrake evolved to produce scopolamine and to causes delirium to incapacitate not help shamans). Several pharmaceuticals have been repurposed either by leveraging protein-ligand binding with close paralogous proteins or rebalancing issues within the same pathway, however there are very few example where a drug-like compound binds an utterly different target and works wonders. Salicylic acid (ortho-hydroxylbenzoate), a signalling compound from willow trees binds to several targets in plants, mainly NPR1 to trigger stress response. In humans, it is found in topical anti-inflammatory creams (eg. Bongela) yet plants have a different immune response! The thing is, it is only 138 Da, making a fragment more than a drug. Its O-acetyl derivative, aspirin, is used as a non-steroidal anti-inflammatory as binds (both) COX enzymes stronger as it's a covalent inhibitor, but it also promiscuous as its many side-effects and off-label uses attest, plus ideally only COX2 should be inhibited. As a consequence, salicylic acid is an exception not the rule and most natural products are much larger. So what about screening natural products against targets that would not make evolutionary sense (ie. fortuitously bind)? Here lie three different but valid arguments: (a) the a priori probability of a compound binding efficiently a target diminishes the larger it and (b) the probability of a compound binding a given protein is higher if it can bind another protein, and (c) Nature is messy, who can tell why a biosynthetic pathway for a compound was selected by evolution and if even selected (secondary metabolism operates in the realm of near-neutral drift and future plasticity). The first point is extremely strong when considering that a blind docking screen is considered successful with a 1-5% validated hit rate, i.e. from compounds expected to bind. Natural product screens do give some hits, but at high concentrations and with really weak affinity, making the effort of scaling the compound down arduous. The second point overlaps with the questions of library design, which can be heavily assay dependent, and have discussed at length in the literature (example), but it is a highly valid point —even the group of Jean-Louis Reymond make a variant of GB13 (a 13 atom enumeration of chemical space) that was enriched for bioactive fingerprints. Unfortunately, the murkiness of trying to conjecture for what reason, if any, a compound exists borders on the philosophical, so it is not easy to dismiss that there is no advantage for a plant, fungus or bacterium to make a compound that affect a protein in some pathway that is involved in a specific pathology.

Conclusion

In broad terms, modern drug design has mostly moved away from natural products, to avoid the complicated total synthesis, to avoid weak binders, to avoid metabolism and to speed up drug campaigns. However, the evolutionary fight of natural products have sculpted our physiology and a lot of the inconveniences in drug discovery come from this and can be explained in an evolutionary context. Natural products were the first modern drugs and have sculpted drug development, so understanding these is essential. Natural products can be great inspirations for a mode of action, or source of building blocks. In my opinion therefore, the important thing to not be naïve by hoping that Nature has already made a compound that target an ailment that is irrelevant to other organisms.
...Now, it's time for my weekly dose of resveratrol to active my telomerases!