Researchers at the Centre for Genomic Regulation and the Wellcome Sanger Institute have comprehensively identified the allosteric control sites found in the protein KRAS, which is one of the most frequently mutated genes in many types of cancer. The scientists used a technology known as deep mutational scanning to quantify the impact of more than 26,000 mutations on the folding of KRAS and its binding to six interaction partners.

The KRAS allosteric control sites represent potentially key targets for drug development, and the new findings could point to vulnerabilities that may be exploited to control the effects of this important cause of cancer. The researchers said their study also presents the first complete control map for any protein.

“The big challenge in medicine isn’t knowing which proteins are causing diseases but not knowing how to control them,” said ICREA research professor Ben Lehner, PhD, at the Centre for Genomic Regulation and the Wellcome Sanger Institute. “Our study represents a new strategy to target these proteins and speed up the development of drugs to control their activity. The nature of targeting allosteric sites means that the resulting drugs are likely to be safer, more effective treatments than the ones we have right now.”

Lehner is senior author of the team’s published paper in Nature titled, “The energetic and allosteric landscape for KRAS inhibition.” In their paper the team stated, “We have presented here the first global map of inhibitory allosteric sites for any protein and the first comprehensive comparative map of the effects of mutations on the free energies of binding of a protein to multiple interaction partners. The dataset constitutes >22,000 free energy measurements, which is a rich resource for protein biophysics and computational biology.”

The GTPase KRas (KRAS) is mutated in 1 in 10 human cancers, including about 90% of pancreatic adenocarcinomas, about 40% of all colorectal adenocarcinomas, and 35% of lung adenocarcinomas, the authors explained. Discovered in 1982, KRAS has also been called the “Death Star” protein because of its spherical shape and lack of a good site to target with drugs. For this reason, the protein has been historically considered undruggable.

The only effective strategy for controlling KRAS has been by targeting its allostery communication system. “KRAS functions as an archetypal molecular switch, cycling between inactive GDP-bound and active GTP-bound states,” the authors further explained. This altered conformation and activity of KRAS on GTP binding is an example of allostery, which is, effectively, “the long-range transmission of information from one site to another in a protein.”

The KRAS allostery system functions as molecular signals that work through a remote-control lock and key mechanism. To control a protein, you need a key (a chemical compound or drug) that can open a lock (active site). Proteins can also be influenced by a secondary lock (allosteric site) which lies elsewhere on its surface. When a molecule binds to an allosteric site, it causes a change in the protein’s shape, which can alter the protein’s activity or its ability to bind to other molecules, for example, by changing the internal structure of its main lock.

Allosteric sites are often preferred for drug development as they offer greater specificity, reducing the likelihood of side effects. They can also change a protein’s activity more subtly, offering potential for fine-tuning its function. Drugs that target allosteric sites are generally safer and more effective compared to drugs targeting active sites.

However, allosteric sites are highly elusive. Despite four decades of research, tens of thousands of scientific publications, and more than three hundred published structures of KRAS, only two drugs have been approved for clinical use—sotorasib and adagrasib. The drugs work by attaching to a pocket adjacent to the active site, inducing an allosteric conformational change in the protein that prevents it from being activated. As the authors commented, “As for many medically important proteins, the development of therapeutics against KRAS is limited by the lack of information about inhibitory allosteric sites to target.”

The ability to generate atlases of allosteric sites could greatly accelerate drug development, especially for the many human proteins considered undruggable because of the lack of an appropriate active site or because they function via difficult to inhibit protein-protein interaction interfaces. However, as the investigators further pointed out, a comprehensive map of allosteric sites has not been generated for any oncoprotein, disease target, or complete protein in any species.

For their newly reported study the team mapped the KRAS allosteric sites by using a technique called deep mutational scanning. It involved creating over 26,000 variations of the KRAS protein, changing only one or two amino acids (aa) at a time. “In total, the library consists of >26,500 variants of KRAS, including >3,200 single aa substitutions and >23,300 double aa substitutions,” they wrote. The team checked how these different KRAS variations bind to six other proteins, including those critical for KRAS to cause cancer. “We quantified the binding of the >26,000 KRAS variants to six interaction partners: the three KRAS effector proteins RAF1, PIK3CG, and RALGDS, the GEF SOS1, and two DARPins, K27 and K55, synthetic antibody-like molecules selected to bind GDP- and GTP-bound KRAS, respectively.” They first focused on quantifying the binding of the KRAS variants to the RAS-binding domain (RBD) of the oncoprotein effector RAF1,” they noted. The researchers then used AI software to analyse the data, detect allostery and identify the location of known and new therapeutic target sites.

A three-dimensional image showing the human protein KRAS (blue) interacting with RAF1 (yellow), one of its main partners. The blue-to-red colour gradient indicates increasing potential for allosteric effects.
A three-dimensional image showing the human protein KRAS (blue) interacting with RAF1 (yellow), one of its main partners. The blue-to-red color gradient indicates increasing potential for allosteric effects. [Weng, Faure and Escobedo/Centro de Regulación Genómica]
The technique revealed that KRAS has many more strong allosteric sites than expected. Mutations in these sites inhibited the protein’s binding to all three of its main partners, suggesting that broadly inhibiting the activity of KRAS is possible. A subset of these sites are particularly interesting as they are located in four different structural pockets easily accessible on the surface of the protein, and represent promising targets for future drugs.

The findings, the scientists noted, validated all four surface pockets of KRAS as allosterically active, with perturbations in all pockets having large inhibitory effects on RAF1 binding. “This strongly argues for the development of molecules targeting all four pockets as potential KRAS inhibitors.” The authors highlighted one of these pockets, “pocket 3” as particularly interesting. This pocket is located far away from the active site of KRAS and so has previously received very little attention for therapeutic development, they acknowledged. “However, our data reveal that pocket 3 is allosterically active, with 20 mutations in 6 residues in pocket 3 inhibiting binding to RAF1.”

The researchers also found that small alterations in KRAS can drastically change its behavior with its partners, making the protein prefer one over another. This has important implications because it could lead to new strategies which control the aberrant activity of KRAS without hampering its normal function in non-cancerous tissues. Sparing normal versions of KRAS means fewer side effects, and safer, more effective treatments. Researchers could also use this knowledge to dig further into the biology of KRAS and explain how the protein behaves in various scenarios, which could be key to determining its role in different cancer types.

Study co-author André Faure, PhD, staff scientist at the Centre for Genomic Regulation, further pointed out, “It took decades to produce a working drug against KRAS partly because we lacked tools to identify allosteric sites at scale, meaning we were looking for therapeutic target sites in the dark.” In this study we demonstrate a new approach that can map allosteric sites systematically for entire proteins. For the purposes of drug discovery, it’s like turning the lights on and laying bare the many ways we can control a protein.”

The study provides the first ever complete map of allosteric sites for any complete protein in any species. The study also showed that with the right tools and techniques it may be possible to uncover new vulnerabilities for many different medically important proteins that have historically been considered “undruggable.”

“The data presented here and in additional recent studies have revealed that allosteric sites are much more prevalent than is widely appreciated,” the team stated. “Moreover, the approach that we have applied here to KRAS is quite general and can be used to identify allosteric sites in many different proteins.” The investigators believe that using their general strategy will make it possible to systematically map the regulatory sites to target in many important proteins. “Mapping allosteric sites is likely to play an increasingly important role in drug development, laying the foundations for therapeutically targeting proteins previously considered to be ‘undruggable’,” they concluded.

“The unique selling point of our method is its scalability,” explained first author Chenchun Weng, PhD, a postdoctoral researcher at the Centre for Genomic Regulation. “In this work alone we made more than 22,000 biophysical measurements, a similar number as the total ever made for all proteins before we started harnessing the remarkable strides in DNA sequencing and synthesis methodologies. This is an enormous acceleration and demonstrates the power and potential of the approach.”

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