The thalidomide tragedy was the biggest “man-made disaster apart from war”.

This molecule’s structure looks simple and innocent, but a chiral carbon gives rise to two enantiomers with different pharmacological effects. While one isomer was a safe sedative, the other one led to limb malformations in thousands of babies. We will check out how this works exactly, but notably, the enantiomers interconvert in the body so there’s no way to control them.

Get this: Despite increasing doubts of its safety, the US distributor of this horror drug aggressively pushed for its approval simply to maximize sales over Christmas!

If you think the heart-breaking tragedy sealed thalidomide’s fate, you couldn’t be more wrong. Half a century after the tragedy, it generated up to $500 million sales for a pharma company! But wait, there’s more. A slightly modified, much more expensive version of thalidomide brought in eye-watering 12 billion dollars.

How is it possible that the same drug which inflicted so much damage revived and even influenced one of the biggest acquisitions in pharma history? As we will see, the answer is glue – no joke. The story of course wouldn’t be complete without classic big pharma monopoly games and price hiking. And let’s not forget everyone’s favourites – lawsuits.

In this post, we will go through history, biology, organic chemistry and pharmacology of thalidomide and its many cousins. You will also appreciate some paradoxical and morally questionable aspects of drug development.

What is Thalidomide?

Let’s get into it. Thalidomide has a very simple chemical structure. In the 1950s, the relatively small and inexperienced pharmaceutical company Chemie Grünenthal looked for new antibiotics. Instead of antibiotic activity, thalidomide seemed to be a great sedative and help with sleep or nausea. Unfortunately, it doesn’t help the story that some of Grünenthal’s leaders were ex-Nazi scientist. The research head Mückter was involved in death camp experiments and made bank during his tenure. It started all rosy. Initial safety tests in mice and rats showed good tolerability and no side effects even at remarkably high doses. Back then, you didn’t have to understand how drugs worked at all. So, thalidomide was dubbed completely safe and aggressively marketed in 1957.

It quickly became Germany’s second best-selling pharmaceutical, just behind Bayer’s Aspirin.Ironically,safety was one of its key marketing messages. Many pregnant women used thalidomide for morning sickness. The lack of toxicity was convenient as unlike barbiturates, this agent couldn’t be misused for suicide attempts. However, over the next two years, sudden increases in cases of usually rare limb defects were detected in newborns.

It’s less known that there were pretty early findings of teratogenicity and neurotoxicity from various researchers. Some of them, like these observations on tad poles, were shared with Grünenthal already in 1959 – with no response.

Thalidomide Side Effects: Beginnings

Because the incidence of deformations increased so unprecedently, German paediatricians suspected an environmental factor. In late 1961, thalidomide use by mothers during early pregnancy was the common factor. Only later did we find out that deformations were actually just the tip of the iceberg. Thalidomide induced many more miscarriages and less obvious defects like organ problems. After increasing noise on the issue, the German government pulled the drug off the market against the company’s wishes. The adverse impact of early use is so high that even a single tablet was enough to induce pregnancy loss or abnormalities – but why? To understand, we need to recap two topics.

First, we need to know about the ubiquitin proteasome system, basically, cellular garbage management. Some of you might remember the process from your biochemistry classes.

The process starts with very few, so-called E1 enzymes which are activated with ubiquitin, a small regulatory protein consisting of roughly 80 amino acids. This green ubiquitin tag is ultimately what directs the degradation of target proteins. As you can imagine, there are thousands of proteins that the cell might want to be able to degrade. However, it would be challenging to do this specifically if all you have are a few different E1 enzymes. This is ubiquitin groups are cascaded to a broader variety of E2 conjugation enzymes which finally put the tag on more than a thousand so called E3 ligases. These enzymes recognize specific substrates and upon addition of enough ubiquitin tags, the proteasome shreds up their targets into smaller peptides.

Second, we need to know about molecular glues. These work exactly like you would think. The glue molecules bind between two different proteins, aggregating or gluing them together. This can lead to several effects, but targeted protein degradation is the most important one.

How does this relate to thalidomide? Well, the molecule binds to the protein cereblon which is part of a E3 ubiquitin ligase complex. Once bound, it can act as a glue between cereblon and neo-substrates, innocent molecules. This exposes them to the E3 ligase machinery, so they are ubiquitinylated and degraded.

Remember that the adhesion arises from nuanced interactions between functional groups of the molecular glue, cereblon in purple, and its neo-substrate in green. We don’t know the natural targets of cereblon but amongst others, it’s critical for brain development, hence its name.

Thalidomide Side Effects: Cereblon

By acting via cereblon, Thalidomide-initiated protein degradation influences the body’s immune system (immunomodulatory drug). The mechanism is very complex, but one of the innocent casualties is SALL4. This is an important transcription factor that governs gene expression for normal limb development. Its absence results in deformations which is why thalidomide proved so dangerous for pregnant women. Actually, genetic deletion of SALL4 replicates a similar phenotype. But why was this missed by Grünenthal? A critical piece of information that was not known in the 1950s – rodents are resistant to thalidomide’s teratogenicity. This explains the absence of safety signals, even at high doses. Only later did people figure out that rabbits or chickens are more sensitive animal models. Why?

The susceptibility comes from a single amino acid difference in cereblon sequences. Primates and rabbits with a valine suffer thalidomide embryopathies – while rodents with an isoleucine did not show any safety signals. Interestingly, the bushbaby bears an isoleucine and is the only known resistant primate. Scientists demonstrated that mutant mice with an unnatural valine at the position become sensitive. Compared to wildtype mice, they showed statistically significant higher miscarriage rates. This is really fascinating – a slightly bulkier amino acid influences binding and degradation of substrates such as CK1 alpha but potentially also SALL4 or others.

If you had a chemistry class or two, you would know that by having one chiral center, thalidomide has two enantiomers. Much too late, it turned out that the (S) enantiomer is ten-fold more potent protein degrader. Giving only the safe isomer as a drug is not an option. The acidic proton at the chiral center triggers partial conversion to the bad enantiomer at pH levels of over 6. If you’ve watched my deuterium video, you will know that this interconversion is slower for deuterium due to its kinetic isotope effect.

How can the almost identical (R)-enantiomer be safe? Although it has the same molecular contacts with cereblon, its affinity is much lower due to an energetically unfavourable conformation that it needs to adopt upon binding. This twisting occurs because the glutarimide ring wants to minimize steric clashing with the binding pocket, particularly the highlighted tryptophan 383.

Thalidomide Synthesis & AFtermath

If we look at its synthesis, it becomes obvious why original thalidomide is a racemic mixture. The original Grünenthal synthesis starts with a condensation of L-glutamic acid and phthalic anhydride. Even though the amino acid used was chiral, the basic conditions and high temperature result in a racemic product due to the. To close the ring, the free acids linked via activation with acetic anhydride and a last treatment with urea introduced the nitrogen.

So, the aftermath entailed a large criminal trial, examining potential negligent behaviour by leading Grünenthal employees. The process was extremely drawn out, probably the dream of every lawyer. 600 thousand pages of documents without any clear verdict. Ultimately, it was said that based on the state of science at the time, the teratogenic effects of thalidomide could not have been anticipated – so the trial was terminated and settled between Grünenthal and impacted parents. The company is still providing support to affected persons through a novel foundation, with more than $100 million Euros contributed to date.

Thalidomide in the United States

The impact in the US is another ridiculous story. Grünenthal had offered the company Smith, Kline & French – today’s GSK – to market the drug in North America. SKF ran a large clinical trial which likely also resulted in several phocomelia cases, and they declined Grünenthal’s partnership offer.

However, another company Richardson-Merrell was eager to introduce it. These guys were calling up the FDA to submit a marketing authorization application in the fall of 1960. I invite you to read this nice article which contains comments of Frances Kelsey who reviewed the application at the FDA. And yes, that’s her with President John F. Kennedy, getting a medal for Distinguished Federal Civilian Service. You see, as if this story couldn’t get worse and more capitalistic, Richardson-Merrell was pushing for an early approval prior to Christmas to maximize their sales.

In a – now recognized as heroic – move Kelsey challenged the drug’s data. Something felt off about giving enormous amounts without any toxicity – so suspicions rose that other conditions could change the drug’s absorption and unveil toxic effects. Her suspicions proved to be right – but that didn’t prevent Richardson-Merrell from giving away literally millions of thalidomide tablets for “investigational use”, at the time permissible under existing regulations. The FDA cited 17 children born in America with thalidomide-associated deformities, but the true number is surely higher.

What has science learned from this tragedy? On one hand, drug controls got stricter. Prior to 1962, drug developers only had to show that new drugs were safe – and as we just saw, even that was not a given. A new pivotal amendment required strict “proof of efficacy” from well-controlled studies, and not the bro-science which Richardson-Merrell tried to pass. Drug advertising now required accurate information about side effects, and clinical trials had to include informed consent of participants prior to the study. For us in the 21^st century, this seems obvious. The FDA also launched a comprehensive assessment of drugs that were already on the market. Finally, drug testing got more robust with a requirement to use rabbits and other thalidomide-sensitive species for teratogenicity testing.

Is Thalidomide Still Used Today?

Thalidomide’s risks but also benefits continue to linger. Just shortly after its initial withdrawal, it proved efficacious in ENL, a leprosy complication. To avoid teratogenicity, access to the drug depends on so-called Risk Evaluation and Mitigation Strategies, short REMS. For instance, female patients must avoid pregnancy though regular testing and use of two or more forms of reliable contraception. In the US, thalidomide was approved for leprosy in 1998 and REMS were well regulated. However, use over decades in countries like Brazil with subpar REMS has still led to some cases of embryopathy.

Although there was research on thalidomide in cancer already in the 60s, the molecule was finally proven to have anti-cancer activity in the 90s. This time around, the company Celgene got IP rights to the drug and thoroughly interrogated its potential. A landmark trial showcased its value in multiple myeloma, a type of blood cancer. Given its unique mechanism and profile, combination with other agents was powerful. This resurrected thalidomide, turning a monster drug into a precious option for patients who relapsed or did not respond to other treatment.

Mechanistically, the anti-oncology effect arises from degradation of Ikaros and Aiolos. Unlike SALL4, these transcription factors regulate the development of B and T cells of the immune system. Ultimately, thalidomide inhibits the process of angiogenesis. As a very smart person, you will realize that lower growth of new blood vessels in turn suppresses tumor growth.

The “new early” days of thalidomide remained controversial, with a whistleblower lawsuit accusing Celgene of off-label marketing. Allegedly, they actively pushed thalidomide, which as we saw was approved for leprosy, to be off label prescribed to cancer patients prior to its approval. While these off-label prescriptions extended thousands of lives, intentional off-label marketing by companies is not compliant. The company ultimately had to settle the lawsuit for 280 million dollars.

Lenalidomide: multiple myelomA Blockbuster

Thalidomide’s legacy is even more shocking. We already know that small changes have big impacts so you shouldn’t be shocked. You see, two simple functional group modifications created lenalidomide. This is the big boy. By the way, there’s even another hybrid between the two – a bit less imaginative. Due to the new structures, these analogues enjoyed new marketing exclusivity, with great commercial success.

Lenalidomide received an orphan drug designation – a FDA incentive that gives drug developers special tax incentives and market exclusivity. With a hefty original 6-figure price tag, the drug earned Celgene double digit billions in yearly sales.

Lenalidomide didn’t sell for no reason. Molecularly, it is much more potent than thalidomide across various metrics. For instance, its IC50 inhibitory value against resistant multiple myeloma cells is orders of magnitude lower.

These molecular changes also translate into better survival outcomes for patients than thalidomide. In addition, risks of neuropathies and other adverse events were lower. This is a follow-on strategy gone well: new molecule, better efficacy and financial success.

Legal Considerations

You could also argue that given Celgene’s research is not associated with Grünenthal’s initial wrongdoings, they actually changed the world for the better. You could also call it a perverse twist – a horror drug ended up as the basis for massive profit. Some have accused Celgene of using particularly fierce ways of preventing entry of generics. Doing so, it managed to command soaring prices for the drugs, and even increase them. This includes more than a dozen of patents on their REMS system which further blocked generic competition. Remember, REMS are special activities that patients, providers and distributors need to take to prevent harm from teratogenicity.

In this case, it means that access to the drug is dependent on several criteria, such as regular pregnancy tests and surveys. This is great because it encourages safe use of the medicine, but as always, we have more potential illegal activities looming.

For many years, Celgene fought it out with the generics manufacturer Mylan. You see, to call approve a generic medicine, the FDA needs to see bioequivalence data. Essentially, we need to undoubtedly prove that the copy has the same effects. So, generic manufacturers need to buy the branded drug. Allegedly, Celgene not only refused to sell the drugs directly, but they also implemented distribution restrictions that prevented Mylan from buying thalidomide and lenalidomide. This was resolved after five years in classic fashion by paying 100 million dollars and some change to settle the claims.

As alluded to, subsequently BMS acquired Celgene and with it, the knowledge on thalidomide and friends. If you thought this was the end of the saga, think again.

Outlook: More Cereblon Modulators

The development of next-generation cereblon modulators such as iberdomide is still ongoing. At a first glance, this one might look like a simple copycat molecule.

As you can see from the crystal structure, the morpholino side chain extends into a pocket on cereblon, increasing surface interactions and binding of the molecular glue.

This enhanced affinity results in a faster protein degradation of neo-substrates, and anti-proliferative activity against multiple myeloma cell lines. In normal cells, iberdomide is much more potent – compare the red and green curves. More importantly, this analog retains activity in cells resistant to lenalidomide. Due to this higher penetration, the hope is that the drug will prove more efficacious in resistant cases. It’s currently in phase 3 trials and again, the idea is combining it with other medications to stack up the effects.

We will close with a final next-generation idea: covalent modification of cereblon. Scientists have noted that there’s a histidine residue close to the molecular glue binding site. Do you already know where this is heading? By creating analogs with electrophilic groups such as this highly reactive fluorosulfate, we can trigger a covalent bond formation with the proximal histidine.

Why is this even interesting? Well, this covalent modification triggers broader conformational changes which change cereblon’s activity. The scientists found that this covalent modulator led to the degradation of the so far elusive protein NTAQ1. Thus, such experiments might unlock even more avenues for drug discovery in different tumor types.

To not overdo it, we’ll wrap up here. As always, until next time!

Key references on thalidomide science and other information:

• Frances Oldham Kelsey. FDA medical reviewer leaves her mark on history | FDA Consum 2001, 35, 24
• The Thalidomide Syndrome | Scientific American 1962, 207, 29
• THALIDOMIDE AND CONGENITAL ABNORMALITIES | Lancet 1962, 279, 45
• The Ubiquitin Proteasome System in Neuromuscular Disorders: Moving Beyond Movement | Int J Mol Sci 2020, 21, 6429
• Molecular glues modulate protein functions by inducing protein aggregation: A promising therapeutic strategy of small molecules for disease treatment| Acta Pharmaceutica Sinica B 2022, 12, 3548
• Exploiting ubiquitin ligase cereblon as a target for small-molecule compounds in medicine and chemical biology | Cell Chem Biol 2021, 28, 987
• Crbn I391V is sufficient to confer in vivo sensitivity to thalidomide and its derivatives in mice | Blood 2018, 132, 1535
• Differentiation of antiinflammatory and antitumorigenic properties of stabilized enantiomers of thalidomide analogs | PNAS 2015, E1471
• Lawsuit Blames Thalidomide for More Birth Defects | Scientific American 2011
• Antitumor Activity of Thalidomide in Refractory Multiple Myeloma | NEJM 1999, 341, 1565
• Immunomodulatory Drugs for the Treatment of B Cell Malignancies | Int. J. Mol. Sci. 2021, 22(16), 8572
• A Cereblon Modulator (CC-220) with Improved Degradation of Ikaros and Aiolos | J Med Chem 2018, 61, 535