Krabi Krabong fighters

PROTACs, new weapons in the experimental arsenal, help flag problem proteins, marking them for destruction

Vinicius Teixeira

In Greek mythology, the chimera was a feared monster. Her powerful hybrid body sported a lion’s head, a goat’s body and a snake’s tail. Also a fire-breather, it ravaged the lands of Caria and Lycia, killing cattle and humans indiscriminately, until the hero Bellerophon mounted the winged horse Pegasus, and shot an arrow into the fire-breathing mouth, the melting of which suffocated it. You know, how gruesome mythology can get…

Modern science has come up with nicer, tamer chimeras – biological units that help fight cancer.

Chimera di Arrezo. An Etruscan ancient sculpture in the Museo Archeologico Nazionale, Florence.
Chimera di Arrezo. An ancient Etruscan sculpture in the Museo Archeologico Nazionale, Florence. CC BY 2.5

The first such chimera came from biology laboratories: CAR-Ts (Chimeric Antigen Receptor T-Cell), developed in the 1980s, has been used to get the immune system to fight cancer.

Then, in the early 2000s, the chemists’ turn to contribute with another heavyweight version: PROTACs.

The PROTAC revolution

PROteolysis TArgeting Chimera are two-headed molecules that induce protein degeneration. One head has a ligand (a part that attaches itself to a protein of interest; also comes from ‘ligatus,’ the Latin word for bind). The scientists also call such a ligand a “warhead” (see Figure 2). The other head contains a ligand that targets a E3 ubiquitin ligase.

E3 ubiquitin ligases are proteins that can call on another protein, E2 ubiquitin ligase, and form a complex with damaged or misfolded proteins. That is stage one. Then the complex E3/E2 ligase mediates the transfer of small proteins called ubiquitin to the damaged protein. These ubiquitins, when plugged to a protein signals to the cellular defense mechanisms that the protein is past its sell by date and should be destroyed. This binding of ubiquitin to the target protein is called ubiquitination.

Structure of PROTAC SJF-0628.
Structure of PROTAC SJF-0628.. Image courtesy Vinicius Teixeira

SJF-0628 (Figure 2) is an example of a PROTAC molecule. The warhead ligand carries vemurafenib, a drug that treat advanced skin cancers that were by errors in a gene called BRAF V600E. In fact, the name vermurafenib, while sounding like a spell chanted backward, comes from V600E mutated BRAF inhibition. Clever, eh? At least kind of??

Anyway, it is unfortunate that some BRAF mutations resist vemurafenib treatment, leaving some patient more vulnerable.

Yeast as intermediary

Researchers have been racing to find ways to counter this. PROTACs, of which SJF-0628 is one, is the result of that effort. Scientists at Memorial Sloan Kettering Cancer Center and Yale University have been at work on developing PROTACs. Preliminary tests suggest that SJF-0628 is it not just more effective than vemurafenib but was also able to overcome resistance to drugs, and inhibit the cancer.

One of the original PROTAC creators, Professor Raymond Deshaies, described how the researchers came up with the idea.

The modern-day Bellerophons: Raymond Deshaies and Craig Crews
Modern-day Bellerophons: Raymond Deshaies and Craig Crews bend chimeras to their purpose. Deshaies pic courtesy Amgen; Crews pic courtesy the Crews lab

“Craig Crews and I met at the Semiahmoo Resort in Blaine, Wisconsin, at a meeting for recipients of the Burroughs-Wellcome Young Investigators in the Basic Pharmacological Sciences Award [in 2000],” Deshaies says. “Craig had a poster on using the yeast two-hybrid system to identify small molecules that link two proteins together.”

At the time, Deshaies himself was working with ubiquitin ligases, the protein complex we have described as one that helps plug ubiquitin onto another protein, to stop it functioning and to prepare it for degradation.

“I … wondered whether his method could be used to identify molecules that target proteins for ubiquitination. We began to discuss this. out of the discussion emerged the idea to make PROTACs.”

Each had half the idea to make PROTACs work. Had it not been for that chance meeting, the PROTACs would not have been developed.

The mechanism of PROTAC action. BRAF contains thousands of atoms, which makes viewing complicated. The researchers use simplified models to represent these biomolecules.
The mechanism of PROTAC action. BRAF contains thousands of atoms, which makes viewing complicated. The researchers use simplified models to represent these biomolecules. Image courtesy Vinicius Teixeira

How it all works

The secret of PROTAC potency lies in the cyan ligand (E3 ligase) in figure 3. Nope, the real thing doesn’t come in color, sorry.

The traditional treatment, Vemurafenib, attaches itself on to a part of the mutant BRAF enzyme (the binding site in Figure 3) to make it dysfunctional, thus arresting the cancer. While the SJF-0628 warhead also does this. But, in addition to putting a spanner in the works of BRAF operation, it also binds to E3 ubiquitin ligase. Yes, the one Deshaies was an expert in. The resulting BRAF-PROTAC-E3 complex is bad news – for the cancer cells.

Stuck so close together, the E3 ligase tends to recognize the mutant BRAF substrate faster. Since it is always on the job, it quickly whistles up E2 ligase, which carries the ubiquitin that marks the BRAF enzyme. That’s a death sentence for the cancer cell when hulking protein complexes called proteosomes come calling. Their job is to recognize ubiquitin and destroy any non-standard molecule it is attached to – in this case the BRAF protein.

Meanwhile, since the PROTAC molecules do not carry the dreaded ubiquitin marker, are free to start a new attack against the next set of BRAFs. Since vemurafenib lacks this cyclical activity, PROTACs make for more potent cancer treatment.

The tortuous road

Though it all looks very smooth, the researchers had some problems to deal with.

“We struggled for a while on how to demonstrate degradation given the lack of cellular permeability,” says Deshaies. “We finally hit on the idea of using Xenopus extracts.”

Xenopus??

Early PROTACs were large molecules, which are hard to ram into cells. Naturally, the first PROTACs showed very low anticancer activity, but that they did was “proof of concept” that PROTACs could work. That was done with extracts from Xenopus, a clawed frog from sub-Saharan Africa.

Xenopus laevis
Xenopus laevis. Pic courtesy Emőke Dénes / Wikipedia CC SA-BY 4.0

Today, the researchers developed thousands of small molecules PROTACs, some of which, even at small doses, can destroy proteins that appeared immune to drugs. Deshaies and Crews had the former’s old colleagues at Caltech, Bill Dunphy and Akiko Kumagai, who are experts in the preparation and use of Xenopus extracts, to thank for that.

The PROTAC development, from the concept until implementation, is evidence of one of the pillars of science: collaboration.

No scientist is an island. Research is not done in obscure laboratories by an isolated genius, and may often find its spark in unlikely places – like the Semiahmoo Resort. Sometimes it materializes during idle talk over coffee at a university. And very often it comes down to luck.

Vinicius Teixeira

Vinicius Teixeira holds a Ph.D. in theoretical and computational chemistry. He has experience in research, mainly in theoretical and physical chemistry. He now uses computational methods to solve problems in general and medicinal chemistry.

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