B-RAF
July 2022
Serine/threonine kinase B-Raf
B-Raf is a serine/threonine kinase involved in the regulation of the mitogen-activated protein kinase (MAPK) pathway, which controls cells proliferation. B-Raf mutation is associated with many types of cancers. The most common mutation observed is the substitution of the valine at the position 600 with a glutamic acid (V600E). It mimics the phosphorylation of the protein and induces constitutive activation of the kinase, leading to uncontrolled cell proliferation. The B-RafV600E mutation is observed in about half of all melanomas and, to varying prevalence, in many additional cancers.
Dabrafenib is an inhibitor of the mutated B-RafV600E kinase, currently approved for the treatment of advanced melanoma and metastatic non-small cell lung cancer, both alone and in combination with other drugs. However, its therapeutical application is often faced with hurdles such as rapid resistance development and fast metabolism. Moreover, severe adverse effects to the treatment have been reported, such as the appearance of secondary tumors (paradoxical effect).
This can be explained by the unwanted activation of secondary targets by the drug (off-target). One such off-target that has been recently identified is the human pregnane X receptor (PXR). PXR is a nuclear receptor whose activation causes both enhanced metabolic clearance of drugs and increased tumoral cell proliferation, in some types of cancer.
To elucidate this selectivity issue, scientists determined the crystal structure of Dabrafenib with its off-target PXR. Comparing the binding mode of the drug with the primary and the secondary target, and performing in silico screening, they were able to rationally modify the molecule and obtain inhibitors with enhanced selectivity towards B-RafV600E compared to PXR.
Image 1. Dabrafenib in complex with PXR nuclear receptor. Its tert-butyl substituent is in a hydrophobic subpocket formed by F288, W299 and Y306.
They noticed an interesting structural insight in the area around Dabrafenib’s tert-butyl group. Three residues (F288, W299 and Y306) are forming an aromatic π-trap (Image 1) that could not allocate a large and polar moiety. Therefore, scientists postulated that the replacement of the tert-butyl group with a large and polar substituent would prevent PXR binding.
On the contrary, this substitution would be well tolerated in the binding with B-RafV600E, because in this case the tert-butyl moiety points towards the solvent. In support of their hypothesis, there is a previously reported compound in the literature, which carries a large and polar cyclopropylpiperidine moiety instead of the tert-butyl group and preserves binding to B-RafV600E (CQE ligand from 4CQE structure; Image 2)
Image 2. Superposition of B-RafV600E in complex with dabrafenib (4XV2, orange) and CQE (4CQE, blue). The cyclopropylpiperidine moiety is pointing toward the solvent.
Moreover, this tert-butyl group is subjected to chemical modifications by metabolic enzymes. Its replacement by a more stable moiety would decrease the rates of metabolism and excretion of the resulting drug, prolonging its persistence in the organism and its efficacy.
With a combination of docking and machine learning, they screened many substituents to replace the tert-butyl moiety. Through experimental testing of a few selected compounds, they identified a 5-morpholine substituent as the best one. Even if the most active compound against B-RafV600E was still the original drug Dabrafenib, the newly designed inhibitor significantly decreased the affinity towards second target PXR and preserved good affinity towards the original target.
| B-RafV600E IC50 (nM) | PXR EC50 (µM) | Metabolism | |
|---|---|---|---|
| Dabrafenib | 0.5 | 0.09 | rapid |
| New inhibitor | 4 | 11.5 | slow |
With a rational drug design approach, scientists were able to address two of the major drawbacks of a clinical use cancer drug: its off-target activity towards PXR and its rapid metabolic clearance.