Bridging Gaps in Drug Discovery: Allosteric Atlases Illuminate Therapeutic Avenues in KRAS Oncogene Inhibition

Many proteins are regarded as “undruggable” because of their activity through protein-protein interactions, despite the fact that they have been discovered as therapeutic targets in human diseases. A thorough allosteric map for proteins like KRAS, which is mutated in one out of every ten human malignancies, is required to address this. The goal of this map is to illustrate KRAS inhibitory allosteric communication. The researchers from the Centre for Genomic Regulation conducted a study on this. The effects of more than 26,000 mutations on KRAS folding and binding to six interaction partners were examined in this work. From genetic interactions in double mutants, over 22,000 causal free energy changes could be inferred from large-scale biophysical studies. These energy landscapes map inhibitory allosteric sites for a therapeutic target and quantify how mutations affect the binding selectivity of a signaling protein. Although allosteric mutations generally prevent binding to all effectors that have been studied, they can also alter the binding specificity, which may have implications for regulation, evolution, and therapy.

Journey of KRAS Research

Approximately 10% of all malignancies, including pancreatic, colorectal, lung, and multiple myeloma, have somatically altered protein GTPase KRas (KRAS) versions. KRAS cycles between the inactive GDP-bound and the active GTP-bound states, acting as the prototypical molecular switch. Allostery, or the long-range transfer of information in a protein, is exemplified by the changed shape and activity of KRAS upon GTP binding. The KRAS effectors examples are RALGDS, PIK3CG, and RAF1. Mutations in cancer driver proteins disrupt this cycle, leading to an increase in the number of active GTP-bound effector-binding states.

Out of thousands of scholarly articles, over three hundred published structures, and 40 years of discovery as an oncoprotein, only one KRAS inhibitor (sotorasib), a covalent binder of one driver mutation, KRAS(G12C), has been authorized for clinical usage. In order to lock KRAS(G12C) in inactive GDP-bound states and reduce effector binding, sotorasib, an allosteric inhibitor, binds outside of the nucleotide and effector binding sites. This reduces effector binding and clinically validates the effectiveness of allosteric KRAS inhibition.

Allosteric site atlases hold significant promise for expediting drug discovery, particularly for the numerous human proteins deemed “undruggable” due to their inability to bind an adequate active site or their ability to function through interfaces between proteins that are difficult to inhibit. Among their many advantages, allosteric medications frequently target conserved active sites with a better specificity than orthosteric medications.

Key Findings

1. KRAS contains several allosteric inhibitory sites. 
2. The majority of allosteric mutations show the potential to significantly reduce KRAS activity by blocking binding to all three KRAS effectors. 
3. The enrichment of allosteric mutations near binding sites implies that the primary allosteric mechanism is local energetic propagation. 
4. Allosteric communication is anisotropic, with a high degree of effectiveness in communicating across KRAS’s core beta-sheet. 
5. There is significant potential for regulatory, evolutionary, and therapeutic regulation of signaling bias due to the ability of mutations to also allosterically influence binding selectivity. 
6. KRAS exhibits allosteric activity in all four of its surface pockets, with the impact of mutations in the distant and unexplored pockets being more noticeable. 

Understanding KRAS

KRAS inhibitors have been studied for their potential to inhibit RAF1 binding. The binding site of KRAS is divided into four pockets, including the nucleotide-binding pocket. Pocket 1 is the binding site for multiple inhibitors, while Pocket 2 is the binding site of sotorasib, a clinically approved allosteric inhibitor. Mutations in these pockets can allosterically inhibit KRAS activity. Pocket 3 is the most distant pocket from the RAF1 binding interface and has received little attention for therapeutic development. However, pocket 3 is allosterically active, with 20 mutations in 6 residues inhibiting binding to RAF1. Pocket 4 is located behind the flexible effector binding loop and contains 105 allosteric mutations in 9 residues that do not contact RAF1. These findings validate 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 Energetic and Allosteric Landscape for KRAS Inhibition

KRAS, an oncoprotein, binds various proteins for physiological and disease-relevant functions. Because these interaction partners connect to KRAS’s effector-binding interface, a common surface, the idea of multi-specificity in molecular recognition becomes intriguing. No protein’s impact of mutations on binding energies for various interaction partners has been thoroughly examined. Measurement of KRAS binding to several interaction partners offers a way to measure the specificity and conservation of allosteric effects within a signaling hub. More than 26,000 KRAS variants were measured to bind to six different interaction partners, including the GEF SOS1, two DARPins, K27 and K55, and the three KRAS effector proteins, RAF1, PIK3CG, and RALGDS. The two known interfaces for SOS1 and other known binding surfaces on KRAS were identified using the binding energies. These seven free energy landscapes represent more than 22,000 thermodynamic measurements, which is comparable to the total amount of measurements found in the scientific literature for proteins.

The main allosteric sites for each interaction were identified, and the investigation concentrated on the specificity of mutational effects outside of binding interfaces. With a median of nine major allosteric sites in the nucleotide-binding pocket and five extra sites for each interaction, nine major allosteric sites were found for each of the six binding partners. For every mutation at these sites, binding free energy changes were evaluated amongst the six interaction partners. All six interactions were suppressed by substitutions at G10, G15, S17, D57, F78, P110, and V112. At least five interactions were prevented by substituting non-aromatic amino acids (F28), charged amino acids (I55), and hydrophobic amino acids (A18 and A83). For 4/6 partners, allostery mutations were enriched at residues G, P, F, and T, whereas for 6/6 partners, they were deficient at charged residues.

Conclusion

The first comprehensive comparison map of mutations on the free energy of binding a protein to many interaction partners and the first global map of inhibitory allosteric sites for any protein have been reported. With more than 22,000 free energy measurements, the dataset is an important tool for computational biology and protein biophysics. Trade-offs between fitness and recognition of structural diversity govern the evolution of the KRAS effector interface. Nonetheless, KRAS’s binding selectivity is very malleable, changing in a variety of ways upon a single amino acid replacement. These modified binding profiles can serve as valuable experimental instruments to examine the roles of individual molecules and their combinations. 

The information showed that allosteric sites can be used to detect regulatory sites in a wide variety of proteins and that they are more common than is often believed. Drug research is expected to benefit greatly from the mapping of allosteric sites, which will provide the groundwork for therapeutically targeting proteins that were previously thought to be “undruggable.”

Article Source: Reference Paper | Reference Article

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