Discovery and innovation in natural products

Objectives: Explore approaches to discovery of natural products from nature.

Key questions: What is bioprospecting? Will natural products continue to contribute to advances in medicine?

Novel bioactive compounds from natural sources are discovered by systematic research using an array of modern scientific techniques and interdisciplinary approaches.

Bioprospecting is the systematic search for biochemical and genetic information in nature in order to develop commercially valuable products for pharmaceutical, agricultural, cosmetic and other applications. Species may be randomly selected for screening, or the biodiversity of a particular ecosystem may come under study.

Often species are selected for screening based on genomic information, traditional knowledge or historical records. Ethnobotany is the study of a region’s plants and their practical uses through the traditional knowledge of a local culture. Humanity has always engaged in bioprospecting. Such traditional knowledge informs research and exploratory efforts and can provide clues to natural sources that hold molecules with useful bioactivities.

The first stage of bioprospecting is collection of samples from the environment for study. It is important that access agreements and permissions are in place. Some endangered or at risk species are protected and collection may be prohibited. Terrestrial or marine regions may be designated protected areas of conservation. For all sample collections, sustainable harvesting practices should be followed to prevent irreparable damage to biodiversity and ecosystems.

It is important samples are catalogued and identified correctly. For plants, a voucher sample of the collection can be lodged at a herbarium that can be consulted at a later date if the species identity is in question. Evaluation of plant samples usually first involves preparation of a plant extract that can then be put forward for testing in a variety of bioassays. Bioassays are tests used to determine the potency or effect of a substance. They are set up to allow 100s or 1,000s of compounds to be rapidly tested for bioactivity, which is called high throughput screening.

High throughput screening in action

The work of the National Cancer Institute (NCI), part of the National Institutes of Health (NIH) of the US Department of Health, has had profound impact on cancer research and drug discovery. The year 1955 marked the beginning of systematic NCI screening of compounds for anticancer activity against a variety of cancer types in animal models. Libraries of compounds were screened including natural product extracts and significant discoveries were made such as the chemotherapy medications vincristine and vinblastine from the Madagascar periwinkle plant.

Advances in cell culture techniques (growing cells in a lab environment) allowed for the rapid high-throughput screening of 1,000s of compounds. Today, the NCI-60 Human Tumour Cell Lines Screen utilizes 60 different human tumour cell lines representing leukaemia, melanoma and cancers of the lung, colon, brain, ovary, breast, prostate, and kidney cancers to identify novel compounds that can kill or inhibit the growth of cancer cells. It is designed to screen up to 7,000 small molecules per year for potential activity. This screening program is unique in allowing comparison of screening results across 60 cell lines.

Pattern recognition algorithms can help predict a mechanism of action or identify when a compound is likely to have a unique mechanism. This can help pave the way for candidate drug molecules from the lab to the clinic. The NCI-60 panel has been used to comprehensively screen over 100,000 compounds, including naturally sourced compounds, and the data generated is publicly available. It continues to play a hugely significant role in advancing cancer research.

Discovery based on traditional use – the example of Artemisinin

Looking at herbal medicines and traditional medicinal practices of past generations and indeed the practices of indigenous cultures today can point to useful bioactivities of plant constituents that may have potential for development. Humanity has looked to nature for thousands of years for cures for ailments and disease and an estimated 80% of the world’s population currently still rely on herbal medicinal products and traditional medical practice as their primary healthcare source. Often traditional practice is the only healthcare available in regions of the developing world.

Artemisinin is a drug used to treat malaria that has been developed based on historical records. Artemisinin is extracted from the sweet wormwood plant (Artemisia annua), a plant that has been used in Chinese medicine for thousands of years.

The earliest record of its use is an ancient Chinese medical text in seal script on sheets of silk that was found in a tomb from the Han dynasty. Its use as an antimalarial was described in the Chinese Herbal medicine source ‘Handbook of Prescriptions for Emergencies’ from the fourth century.

Centuries later, in 1972, this text helped Tu Youyou to discover the active substance in the plant that acts as a potent antimalarial. The reference to how to extract the plant by steeping in cold water inspired her to use a low-temperature extraction method.

Youyou had been working on a plant screening research program to discover treatments for malaria under a secret military programme code-named Project 523 in which many candidates were tested from a list of thousands of traditional Chinese medicines. In 2015, Tu Youyou was awarded the Nobel Prize in Physiology and Medicine for the discovery of artemisinin. 

Discovery by serendipity – the example of Penicillin 

Penicillin was discovered by accident by Alexander Fleming in 1928 at St. Mary’s hospital in London. He was growing or culturing the bacteria Staphylococcus aureus on petri dishes in his lab. When he went on holiday, some of these dishes were left in the lab rather than disposed of. When he returned, he noticed that some were contaminated with a fungus and that the bacteria had failed to grow around this fungal colony.

The fungus was later identified as Penicillium rubrum. Fleming conducted experiments to confirm what he had observed and published his experiment in 1929 proposing the fungal extract as an antiseptic. 

‘(The discovery of penicillin) was a triumph of accident, a fortunate occurrence which happened while I was working on a purely academic bacteriological problem’ 

Alexander Fleming 

If he had cleaned up the lab before his holiday and not left the petri dishes out in the cool temperature that allowed the fungus to grow or not been observant to question what he was seeing when he returned, he would not have discovered penicillin, a drug that has saved countless lives in the last century.

It took some time however for the significance of his discovery to be truly realised. The active penicillin substance was difficult to isolate from the fungal broth but this was achieved in 1940 by a team at Oxford led by Florey and Chain. Successful treatment of patients with otherwise fatal streptococcal infections led to projects for mass production for distribution to Allied troops fighting in Europe in World War II. Penicillin greatly reduced the number of deaths and amputations caused by infected wounds.

In the following decades, modifications were made to develop new penicillins with activity against a broader range of bacterial infections. Most of us, unless we have an allergy to penicillin, have probably been treated with penicillin antibiotics at some stage in our lives.

Where should bioprospecting programs focus their efforts?

Bioprospecting programs have extensively investigated plants, fungi and microorganisms but there is still much to investigate. It is estimated that only 10 – 20% of the world’s biodiversity has been investigated for bioactive compounds.

We are still discovering species in easy to reach terrestrial areas and new technologies are allowing us to sample environments that we could never reach before such as the marine and extreme habitats.

What are extreme habitats?

Extreme habitats are environments in which most life on earth cannot thrive or survive. However, a wealth of interesting life-forms have adapted to the harsh conditions of these habitats that were once thought devoid of life. These organisms are known as extremophiles. Extremes of temperature are encountered in frozen polar regions and in hot springs and geysers. The deep ocean is cold, under high pressure and lacking solar energy. Other inhospitable environments are highly acidic or highly alkaline, conditions that would damage or kill the cells of most organisms.

Extremophiles – the new frontier?

The adaptations of extremophiles to their environments may be useful to us. Diverse metabolic systems provide a plethora of new complex compounds that we have not otherwise observed in the chemical world. These life forms have found solutions to problems that are challenges for us today. We will now look at some examples.

The polar regions are extremely low temperature environments. Scientists have discovered microorganisms, microalgae, fungi and even insects that can survive and grow well in these regions. How do they do it? They have adapted to produce antifreeze proteins that protect their DNA. The cell membrane is structurally adapted to maintain fluidity at low temperatures.

Alaska’s wood frogs (Lithobates sylvaticus) freeze in winter and become metabolically inactive but thaw in spring, waking up as if from sleep. By studying the chemicals that these frogs produce to prevent ice crystals forming in their bodies and their metabolic processes, scientists hope to discover a way to extend the amount of time that organs can be stored for transplantation. With a shortage of transplantable organs to meet patient need, the ability to store and transport organs without damaging the tissue, would drastically increase the chances of patients receiving life-saving transplants.

Thermophiles are life-forms that can survive the high temperature conditions of hot springs and geysers around the world and in deep sea hydrothermal vents. Thermophiles are sources of enzymes that can function at high temperatures and so can be useful in industrial processes.

The enzyme taq polymerase was derived from the bacterium Thermus aquaticus which was discovered in a bubbling hot spring in Yellowstone National Park in 1965. This enzyme can synthesize strands of DNA complementary to a template strand and has high thermal stability.

This enzyme revolutionized molecular biology. It is a key enzyme in genetic research used in genetic sequencing and polymerase chain reaction (PCR) techniques. PCR tests have medical applications. For example, PCR testing is the gold standard for detecting COVID-19 infections in patients.

At the bottom of the ocean, organisms must withstand high pressure as well as cold and so they can be sources of enzymes that can function optimally at low temperatures and high temperatures typical of their environment. An example of these enzymes in action can be seen in detergents that are used specifically for cold water washing.