Dawn is dawning—the way to attack KRAS G12C star inhibitor (part 1)

Ras oncogene mutation is the most common activation mutation in human cancer, which occurs in 30% of human tumorsAlthough it is one of the earliest oncogenes, three decades of efforts have failed to identify clinically feasible KRAS protein inhibitorsThere are two main reasons: (1) KRAS binds to GDP and GTP with picomolar affinity, which seriously hinders the efforts to develop nucleotide competitive inhibitors; (2) KRAS protein lacks other deep surface hydrophobic pockets, It hinders the search for high affinity allosteric inhibitors [1] [2] [3]< br / > there was a major breakthrough in 2013Shokat et al [4] reported a new strategy to overcome these challenges, which uses covalent inhibitors to target the active cysteine of krasg12cThe crystal structure of the combination of wild KRAS and GDP is shown in the left figure of Figure 3 [5]Crystallographic studies show that a new pocket is formed under the switch-ii region where the effector bindsIt is worth noting that the mutant cys12 is located near the nucleotide pocket and switching region related to the interaction of the effector (as shown in the right figure of Figure 1 below), The fragment screening method based on disulfide was used to screen the library with 480 chain compounds under the condition of GDP bindingThe fragments 6h05 and 2e07 were obtained, and the two fragments did not react with wild-type K-ras containing 3 natural cysteine residues< br / > Figure 1Schematic diagram of the combination of wild-type KRAS (left) and krasg12c (right) GDP < br / > select the optimal fragment 6h05 to study the structure-activity relationship, and obtain the highest active chemical (6)The binding mode is shown in the right figure (as shown in the left figure of Figure 2 below)Compound 6 does not bind in the nucleotide mouth bag, but extends from cys12 to the adjacent bag mainly composed of switch-iiThis completely formed pocket is not obvious in other published structures of RAS, although grooves can be seen in some casesPrevious studies have shown that there are allosteric sites in this regionThe composite binding area is called switch-ii pocket (s-iip) [4]< br / > Fig 2 Structure activity relationship study diagram (left); schematic diagram of eutectic structure of compound 6 (cyan) combined with GDP (gray) and Ca2 + (green) (right) < br / > s-iip is located between β - fold and α 2 - (switch II) and α 3-helix in the center of RAS, clear electronic density shows the position of compound 6 in the interior of s-iip, and confirms the disulfide bond between 6 and cys12 (as shown in the left of Figure 3 below) The hydrophobic dichlorophenyl of 6 forms several hydrophobic contacts Glu99 and gly60 form direct hydrogen bonds to 6 (as shown on the right of Figure 3 below) However, switch-ii has an obvious rearrangement effect on the formation of s-iip, and the configuration of switch-i has not changed from GDP binding state [4] < br / > Fig 3 Electron cloud density of compound 6 (left); the surface of s-iip is around the compound, showing hydrogen bond (yellow line) Instead of using disulfide based compounds, the indicator residue has hydrophobic contact with 6 (right) < br / > shokat and so on Instead of using disulfide based compounds, they have turned to carbon based electrophilic agents, acrylamide and vinyl sulfonamide to obtain efficient acrylamide compounds, of which compound 12 has good activity, as shown in Figure 6 below It is also proved that compound 12 does not react with the cysteine of wild type KRAS Covering multiple eutectic structures shows that the compound follows a similar trajectory through the pocket and projects the functional group into the (o -) and (P -) subpackages Although there are considerable differences in terminal benzene rings, these compounds satisfy similar hydrophobic interactions, supporting the key role of this region of s-iip (as shown in Figure 4 below) [4] < br / > Figure 4 Adduct formation of 10 mm compound after 24 hours (left); eutectic structure covering 8, 9 and 11 with GDP bound K-ras (G12C) (right) < br / > EDTA's catalytic effect on nucleotide exchange shows that although the compound may subtly affect metal binding, leading to changes in nucleoside acid affinity, Mg2 + cannot be excluded even when s-iip is occupied Structural analysis also predicts that the function of the exchange factor SOS may be affected by its combination with s-iip Using compounds 8 or 12 to block the SOS catalyzed nucleotide exchange, K-ras (G12C) is treated As mentioned above, these compounds will not damage the EDTA catalyzed GDP exchange The specific results are shown in Figure 5 [4] < br / > Fig 5 SOS catalyzed nucleotide exchange of full-length K-ras (G12C) alone (a), or in the form of 8 (b) or 12 (c) labeled krasg12c; (a) a-c.e; (E) half-life of a-C diagram < br / > next, immunocoprecipitation showed that compound 12 would destroy the conformation of GTP state of Ras and damage the interaction with effectors (such as RAF) The role of compound 12 was studied in a small number of lung cancer cell lines with gene phenotypes As expected, cell lines with G12C mutations (h1792, H358, H23, and Calu-1) showed reduced survival and increased apoptosis after treatment compared to groups without G12C mutations (h1437, H1299, and A549), as shown in Figure 6 (left) below Highly sensitive h1792 cells showed low levels of k-rasgtp, consistent with the preferred binding of inhibitors to K-ras GDP, as shown in Figure 6 (middle) They are highly K-ras dependent as shown in Figure 6 (right) below It is worth noting that K-Ras-dependent (A549) and non-dependent (H1299) cell lines lacking G12C are not sensitive to compound 12 The EC50 of compound 12 in h1792 cells was 0.32 ± 0.01 μ m [4] < br / > Figure 6 Sensitivity of lung cancer cells to inhibitors, K-ras GTP level and dependence on K-ras Compared with the percentage of survival rate (left) after 72 hours of DMSO treatment, glutathione S-transferase (GST) - labeled RBD (RAS binding domain of c-raf) was incubated with lysate to determine the level of K-ras GTP (medium), and the cell survival rate (right) < br / > was evaluated after 72 hours of KRAS siRNA transfection In general, the cell data provided a conceptual validation for the genotype specific use of s-iip binding complex in k-rasg12c driven cancer < br / > Matthew P Patricelli et al [6] continued to explore on the basis of compound 12, and found that compound 12 did not show substantial krasg12c CO binding in nci-h358 (H358) cells containing krasg12c mutation, even after treatment with 100 μ mol / L compound for 6 hours In order to solve the possible reason of lacking cell activity of compound 12, we need more effective switch II pocket inhibitors We designed a covalent krasg12c targeting reagent based on iterative structure, and tested their activity to purified recombinant krasg12c and their ability to bind to KRAS G12C in cells Ars-853, which gives great hope, is shown in Figure 7 below < br / > Figure 7 Structure activity improvement of compound 12 < br / > in biochemical determination, the binding rate constant of ars-853 and krasg12c is 76m-1s-1, which is more than 600 times higher than compound 12, and the cell binding IC50 is 1.6 μ mol / L in 6 hours In the presence of GDP, the high-resolution crystal structure of the ligand binding krasg12c determines the binding site of ars-853 as the switch II pocket described above In this structure, ars853 is covalently connected to C12 and extends to the switch II pocket area between the central β - fold and α 2 and α 3 helices of KRAS Compared with the structure of other published switch II binding compounds, ars853 induced the rotation of α 2-helix, accompanied by the displacement of M72, to adapt to the ligands in different hydrophobic pockets The hydrophobic pocket is occupied by the aromatic ring of ars-853 Chlorine and methylcyclopropyl substituent provide close van der Waals contact, while the phenolic hydroxyl forms hydrogen bond with D69 The carbonyl group of ars-853 acrylamide forms hydrogen bond with the conserved K16 and a water molecule coordinated with the usual magnesium ion, and occupies a similar position with the terminal phosphoric acid of GTP binding form of KRAS In general, several features of the structure indicate that krasg12c combined with ars-853 represents the inactive state of KRAS See Figure 8 [6] for details < br / > Fig 8 Signal intensity and LC / MS-MS analysis of krasg12c digested by trypsin in H358 cells after 6h treatment (left); the crystal structure of ars-853 combined with krasg12c highlights the key hydrogen bond and hydrophobic interaction in switch II pocket (right) < br / > Matthew R Janes et al [6] through further experiments, ars-853 inhibits the function of krasg12c oncoprotein in cells, Ars-853 cells are involved in the rapid cycle of nucleotides on the required KRAS G12C oncoprotein, and the level of KRAS G12C GTP is regulated by upstream signaling factors < br / > despite the above breakthroughs, revealing previously unknown Ras binding pockets, researchers have also carried out a large number of screening tests, but they only lead to limited proof, and at the same time, there is a serious lack of convincing evidence of in vivo response to targeted mechanism of action [7] [8] Matthew R Janes et al [9] Based on the structure of drug design, continue to improve the efficacy of mutation targeting and KRAS specific inhibitors and drug-like properties It was found that the main disadvantages of ars-853 series compounds were short plasma metabolic stability (T1 / 2 < 20min) and poor oral bioavailability (f < 2%), which made them unable to be used for further in vivo study The limited structure activity relationship also limits the further improvement of ars-853 series The emphasis is shifted to different scaffolds of different design structures, which properly locate the conformation and trace of acrylamide, and allow the best hydrophobic binding part to be properly placed in s-iip We assume that the flexible 2-amino-1 - (π - piperazine-1-yl) ethane-1-one linker of ars-853 series can be shortened and replaced by a more rigid double ring scaffold, and is suitable for the free region between the S-II and A3 helices repackaged by the previously reported ars-853 eutectic structure [6] After extensive stent optimization, we determined that the core of quinazoline is a multifunctional stent, which can overcome the SAR limitation of ars-853 and has better drug like properties See Figure 9 below < br / > Figure 9 Ars-853 research on scaffold structure optimization < br / > this progress has led to a series based on quinazoline, and after the systematic optimization of substituents around the scaffold, it has resulted in a significant improvement in ADME / PK and KRAS covalent binding activity, as shown in Figure 10 [9] < br / > Fig 10 The structure-activity study of the biochemical rate constants of g12c-te related to the 7-and 8-substituents of quinazoline nucleus, The reaction of s-atropine to the isomer of kras-12 cysteine is shown in Figure 11 [9] < br / > Fig 11 Chemical structure of ars-1620 < br / > the eutectic structure of ars-1620 and krasg12c shows the unique binding mode and binding locus from the initial s-iip krasg12c inhibitor to cys-12, and reveals the acquisition of additional key interactions with His-95, thus obtaining a more rigid and favorable covalent reaction conformation than that of ars-853 series compounds (as shown in FIG 12 and 13 below) [9] < br / > Figure 12 Krasg12c eutectic structure of ars-1620 (gold) combined with GDP, located in switch 1 (blue), switch 2 (pink) and induced switch 2 Pocket (s-iip); from the perspective of eutectic structure, the combination mode of ars-1620 and krasg12c (right); < br / > figure 13 Comparison of combination mode and key interaction between ars-1620 (gold) and ars-853 (green) < br / > in biochemical experiments, The observation rate of ars-1620 covalently modified krasg12c is 10 times higher than that of ars-853, which proves that the modification effect of ars-1620 on KRAS is significantly higher than that of the previous compounds Ars-1620 preferentially interacts with the GDP binding state of krasg12c, lacking detectable reactivity to any residue of wt-kras protein In addition, KRAS G12C covalently bound to ars-1620 lacks the ability of nucleotide exchange mediated by SOS and EDTA At the same time, the inhibitory activity of ars-1620 and the selectivity of Pan RAS to krasg12c were confirmed at the cell level [9] < br / > a major risk of covalent inhibitors is the possibility of non-specific reactions with non target proteins There are 200 kinases with reactive cysteine near the ATP pocket [10], which also extend to the non kinase protein with reactive cysteine [11], if targeted, it may produce no