KRAS Inhibitors and Target Engagement Technology: From Undruggable to Druggable Targets in Cancer Therapeutics

Important Compound Classes

Titles

Vinyl Imidazole Compounds as Inhibitors of KRAS; and Target Engagement Assay for RAS Proteins

Patent Publication Numbers

WO 2022/047093 A1 (URL: https://patents.google.com/patent/WO2022047093A1/en?oq=WO+2022%2f047093+A1); and WO 2022/047186 A1 (URL: https://patents.google.com/patent/WO2022047186A1/en?oq=WO+2022%2f047186+A1)

Publication Date

March 3, 2022

Priority Applications

US63/071,875; US63/071,694; US63/117,080; and US63/160,120

Priority Dates

August 28, 2020; November 23, 2020; and March 12, 2021

Inventors

Carlsen, P.; Wang, X.; Sokolsky, A.; Gan, P.; Policarpo, R.; Yao, W. (WO 2022/047093 A1); and Robers, M. B.; Walker, J. R.; Vasta, J.; Corona, C. (WO 2022/047186 A1)

Assignee Company

Incyte Corporation [US/US], 1801 Augustine Cut-off, Wilmington, DE 19803, USA; and Promega Corporation [US/US], 2800 Woods Hollow Rd., Madison, WI 53711-5399, USA

Disease Area

Cancer

Biological Target

RAS proteins (KRAS^G12C mutant)

Summary

Cancer is the second leading cause of death worldwide, accounting for nearly 10 million deaths in 2020, or nearly one in six deaths [“Cancer”, who.int fact sheet), assessed 3/20/2022]. Cancer is simply an abnormal and rapid growth of cells that permeate beyond regular cell dimensions and can develop in any part of the body. Furthermore, cancer-causing infections, such as hepatitis and human papillomavirus (HPV), are the cause of approximately 30% of cancer cases in low- and lower-income countries. However, many cancers can be cured if detected early and treated effectively.

Protein–protein interactions (PPIs) are important for basically all biological processes and constitute more than 0.5 million estimated interactions in humans. The rat sarcoma viral oncogene homologue (Ras) proteins are part of the family of small GTPases that are activated by growth factors and various extracellular stimuli. In addition, the Ras family regulates intracellular signaling pathways responsible for migration, growth, differentiation, and survival of a cell.

Activation of Ras proteins at the cell membrane results in the binding of key effectors and initiates a cascade of intracellular signaling pathways within the cell, including the PI3K and RAF kinase pathways. RAS is encoded by KRAS, NRAS, and HRAS, functioning as cellular signal transducers, which cycle between an active GTP-bound state and an inactive GDP-bound state. While the identification and subsequent clinical success of the irreversible KRAS^G12C mutants, which occupy the induced switch II pocket, are pivotal milestones in drug discovery, their inhibitory activity relies on a reactive warhead in order to form a stable covalent bond with the mutant Cys12. In contrast, the most prevalent mutants (KRAS^G12D, found in 33% of KRAS mutant tumors) do not presently have clinical agents, due in part to the lack of a reactive residue adjacent to the switch II pocket.

Oncogenic mutation of KRAS is closely linked to tumorigenesis, which is found in 22% of cancer patients [pancreatic cancer (61%), colon cancer (33%), and lung cancer (17%)]. In addition, oncogenic KRAS induces several inflammatory cytokines, chemokines, and signaling pathways, which may promote carcinogenesis or have an influence on endothelial cells, fibroblast, extracellular matrix, and intrusiveness in these cancers.

Novel approaches are required for the identification of a new class of selective KRAS inhibitors that enhances binding to the shallow pocket between the switch I and II pockets. KRAS cycles between the GDP-bound inactive state and the GTP-bound active state. It is regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins, which promote the exchange of GDP with GTP and the hydrolysis of GTP, respectively. The Son of Sevenless Homologue 1 (SOS1) is the primary and most well-studied GEF for regulating the GDP–GTP cycle of KRAS. SOS1 interfaces with KRAS at the catalytic site, catalyzes the exchange of GDP to GTP, and then activates the MAPK pathway. The modulation in the regulation of the GDP–GTP cycle of KRAS by the SOS1 modulators leads to changes of downstream signals, such as the phosphorylated ERK (pERK) in the RAS-RAF-MEK-ERK pathway.

Although the development of irreversible KRAS^G12C inhibitors is an active research area, limited cellular activity has been observed for most of the analogs. For example, recently reported cyclic peptide KRAS^G12D inhibitors demonstrated better biochemical activity compared to the indole analogs; however, their physicochemical properties limited their cellular potency. Nonetheless, several tool compounds targeting other KRAS mutation variants are at different stages in various pipelines and have yet to enter into clinical trials.

Research technology such as a target engagement strategy in the exploration of new drug design strategies is currently highly needed. Patent application WO 2022047186 A1 discloses tracers for identifying RAS binding compounds by using a RAS binding agent which comprises a RAS binding moiety and a functional element. For example, the KRAS binding moiety binds to one site on the KRAS protein and can successfully interrogate engagement at another KRAS binding site, which enables a broad use of live cell target engagement assays for identifying KRAS binding compounds such as KRAS inhibitors. The method was used to measure KRAS target engagement in cells. Furthermore, luminescence was produced from either NanoLuc (Nluc) tagging of KRAS as the bioluminescence resonance energy transfer (BRET) donor or NanoBiT tagging of KRAS, a technology in which the BRET donor signal originates from a KRAS multimeric species in cells. Also, KRAS^G12C homomultimeric complexes were measured in cells using an enzyme complementation–NanoBiT oligomer configuration with a HiBiT tag.

Key Structures

Biological Assay

GDP–GTP exchange assay, luminescent viability assay, whole blood pERK1/2 HTRF assay, and RAS activation Elisa assay.

Biological Data

The table below show a KRAS_G12C exchange assay and a KRAS_G12C pERK assay, where the symbols indicate IC₅₀ for † ≤ 100 nM, †† > 100 nM but ≤ 1 μM, ††† > 1 μM but ≤ 5 μM, and †††† > 5 μM but ≤ 10 μM.

The author declares no competing financial interest.