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The highly conserved nature of protein kinase catalytic sites makes identifying selective ATP competitive inhibitors a challenging task. During the development of adhesion kinases (FAKs), a class of molecules was stumbled upon that, while inactive against FAK, showed activity and selectivity in inhibition of serine-arginine protein kinase 1-3 (SRPK1-3). The key to selectivity lies in the interaction of such molecules with the unique non-conserved hinge regions of the SRPK family. Through structure-based medicinal chemistry studies, an SRPK inhibitor was successfully obtainedmsc-1186。The molecule exhibits activity at the nanomolar level and has good selectivity. In combination with CLK inhibitors, the reduction of phosphorylation of SR proteins can be accelerated. This article is rightmsc-1186The design strategy and optimization route were sorted out in detail, which provided valuable experience for the structural optimization of similar projects.
Figure 1msc-1186optimization process.
Since 2001, the FDA approved the first kinase inhibitor, imatinib (imatinib, Figure 2), protein kinases have become major drug targets. Protein kinases regulate cellular function by transferring the -phosphate of ATP to tyrosine, serine, or threonine of the substrate protein. Research on key oncogenic factors for protein kinases, such as point mutation activation, gene fusions, and lipid kinase amplification, and develop specific kinase inhibitors based on them. More than 70 kinase drugs have been approved in clinical practice, but many of them lack selectivity, so they limit their dose and fail to achieve the desired targeted effect. The use of non-selective kinase inhibitors to elucidate the biological role of specific kinases often yields conflicting results and ambiguous conclusions. Therefore, the development of selective kinase inhibitors is a key challenge in the development of preclinical drug candidates.
Figure 2Representative kinase inhibitors.
Adhesion kinases (FAKs) belong to the non-receptor tyrosine kinase family and are closely related to the growth, invasion, and metastasis of cancer cells. At present, six FAK kinase inhibitors have entered clinical Phase I Ib clinical studies. pf-431396defactiniband its 7-azaindole derivative (Figure 2) and other FAK inhibitors, all of which have the problem of insufficient selectivity. The role of serine-arginine protein kinase (SRPK) in cancer compared to FAK has not been fully understood. Studies have shown that SRPK plays an important role in the splicing of key proteins such as MyB, BRD4, and MD24, and is also involved in the regulation of VEGF. The role of SRPKs in cancer splicing is difficult to assess due to the lack of potent and selective SRPK inhibitors. Several reversible SRPK inhibitors have been developed, such as:srpin340withsphinx31(Figure 3), but is also active against kinase families such as CDK, MAPK, GSK, and CLK. srpkin-1(Figure 3) Covalent binding to tyrosine of the SRPK protein enhances selectivity.
Figure 3Chemical structure of an SRPK inhibitor.
Recently, Merck & Co. published a study on selective SRPK inhibitors at JMC. The researchers successfully converted a class of single-toothed FAK structural fragments into a benzimidazole-pyrimidine backbone. This backbone structure contains "dual receptor hinge binding fragments", which lays the foundation for highly active, kinase-selective, and reversible SRPK inhibitors. The design and optimization process is summarized as follows:
HIT Discovery
The aminopyrimidine structure is a common kinase hinge-bound fragment capable of forming donor-acceptor-donor type interactions. Due to the presence of large hydrophobic residues at the front end of the DFG sequence, structural design for narrow posterior pocket regions enables selectivity, such as p38 inhibitorsskepinone-l(Figure 2) is the selectivity obtained by using the unique glycine flip within the hinge region. MET inhibitorsclk-t3(Fig. 2) The formation of single-tooth hinge bonding with proteins is the key to high selectivity. Reducing interactions with the hinge region while binding to other conserved regions in the non-hinge region has become an important focus in inhibitor development.
To find FAK inhibitors, the researchers screened the fragment library. Discover the fragmentsA single-toothed hinge binding is formed with proteins with a kd value of 95 M and a ligand efficiency of 046。The X-ray crystal structure of Figure 4A shows that the N atom on pyridine forms hydrogen bonds with Cys502. In addition, non-classical hydrogen bonding is formed between 2-Ch on pyridine and O atom on Cys502. By placing the fragmentwith 7-azaindolesSuperposition was performed and fragments of n transfer to tetrazolium ortho were designed(Figure 4b).
Figure 4(a) FragmentCrystal structure with fak protein;(b) FragmentsThe origin of the structure.
The activity of HEK293T transfected with FAK plasmid was selected for biological activity testing. SnippetswithEC50 of 301 m and 253 m, respectively. 2-Pyridine-benzimidazole fragmentThe EC50 is 103 m (Figure 5). The structure of defactinib that interacts with DFG-loop is introducedon, get5a, but activity is largely lost. Compound 5A was screened for activity against 100 kinases, and IC50s of 448 nM and 7 for SRPK1 and SRPK2 were found79μm。So, right5aFurther structural modifications.
Figure 5Snippetswith5achemical structure.
SAR Research
A structure-activity relationship study was performed on the substituents on the imidazole ring (Table 1), and the results showed:
1) When withdrawing electron groups, such as fluorine atoms (5c) substituted SRPK1 activity increased, cyano (5b) is also tolerated;
2) When donating an electron group, such as R6 position is a methyl group (5d), the r5 position is methoxy (5f) did not optimize the activity of SRPK1;
3) The R5 position is halogen (5g-5i), the activity is significantly increased, and5iSignificant potency is shown against both kinases (SRPK1 IC50 = 32 nM and SRPK2 IC50 = 672 nM).
4) Based on5istructural analysis (described below) to further design derivatives (5j-5m）。r4 is an F atom (5m), the most active SRPK12 inhibitor was obtained;
5) The R5 position introduces water-soluble fragments, such as morpholine (5n), N-methylpiperazine (5o) and n-methylpyrazole (5p), SRPK1 activity is tolerated, but SRPK2 activity is reduced.
5iThe crystalline structure with SRPK1 and SRPK1 is shown (Figure 6).5iThe ATP site orientation is the same as that of the classical pyrimidine N-atom acceptor and the non-classical CH donor hydrogen bonds towards the hinge region (SRPK1: LEU168 and Glu166;srpk2: leu180 and glu178);The NH atom on the glycine residue GLY169 (GLY181) in the hinge region of SRPK1 (SRPK2) is flipped to form a hydrogen bond with the N atom on imidazoleHowever, the leu168-gly169 amide bone structure is different from that of other inhibitors (Figure 7).
Figure 6(a) Crystal structure of 5i and SRPK1 (PDB:7ZKS);(b) Crystal structure of 5i and srpk1.
Cl and F atoms form van der Waals effects with his170 (his182), tyr227 (tyr239), and leu231 (met243) of srpk1 (srpk2) (Figure 6). The N atom of the pyridine ring forms hydrogen bonds with Glu217 (SRPK1) and Glu229 (SRPK2) through the mediation of water. The O atom of sulfonamide not only has intramolecular hydrogen bonds with the N atom on benzimidazole, but also forms hydrogen bonds with his171 (his183) and gly169 (gly181) of srpk1 (srpk2) through the mediation of water molecules.
Figure 7（a）5iSuperposition of LEU168 and GLY169 residues of protein crystals with other inhibitors.
Based on the above structure-activity relationship study, the most effective SRPK1 2 derivative was obtained5m。At a concentration of 1 m, it was tested on a 395 panel5mkinase selectivity (Figure 8). The results are displayed5mThe effect level for SRPK subtype and 3 was >85% with an IC50 of 27nm, 81nm and 06nm。50% < other kinase A.
Figure 85mSelectivity for 395 kinases.
5mThe crystal structure with SRPK1 indicates that its binding mode is the same as5iSame (Figure 9). The F atom at R4 is inserted into the hydrophobic region formed by Val223 and Val167, and van der Waals action is formed at Val167 and GLY168.
Figure 9（a）5mEutectic structure with SRPK1;(b) Protein-binding surface is shown.
to5hFor further modification of the starting molecule (Table 2). The results are displayed
1) Benzimidazole NH is methylated (compound), and then cannot form intramolecular hydrogen bonds with intramolecular sulfonamides, resulting in a 2-fold loss of SRPK1 activity, but SRPK2 activity is the same5hSimilar;
2) The linked NH is methylated (compound), activity is unchanged;
3) Change one n atom on pyrimidine to ch(), with5hcompared to compoundsThe potency against SRPK1 is 2 times higher and the potency against SRPK2 is higher, indicating that the hydrogen bonding of pyrimidines is not as important as expected;
5) Transform benzimidazole into an indole structure (), loses SRPKS activity. Conformational analysis reveals compoundsThe indole 3-position CH atom is located in the hinge region and loses its hydrogen bonding with LEU168.
From the structural analysis (Figures 6 and 9), it can be seen that PHE165 (PHE177), Val145 (Val157), and ALA496 (ALA540) in SRPK1 (SRPK2) form a lipophilic pocket. Based on that, right5hFurther modifications were performed (Table 3) and the results showed:
1) In pyrimidines (compounds) increases SRPK1 activity 2-fold, while SRPK2 activity is not affected;
2) Place the compound5hNH is cyclized with a pyrimidine ring (compound), reduce the degree of rotation, and increase the activity;
3) in compoundson the introduction of methyl groups (compounds), which penetrates deep into the pocket, but the activity does not increase;
4) Place the compoundThe pyrimidine ring becomes a pyridine (compound), lipophilicity is enhanced (LOGP>5), and SRPK1 activity is reduced, but SRPK2 activity is enhanced.
Physicochemical properties and cell activity
Cell viability
compounds5mThe most active and namedmsc-1186compoundsFor negative controls, namedmsc-5360。The test was performed using the SRPK live cell binding assay (Nanobret Target Engagement Assays).msc-1186For the activity of SRPK1 and SRPK3 cells, the EC50 assays are 98 nM and 40 nM, respectively (Figure 10). Since the nanobret experiment for SRPK1 was not successful, it was detected in lysed cellsmsc-1186The EC50 is 149 nm. The KD of SPRK2 was determined to be 145 nm by ITC experiment. Tested for selectivity for 135 kinases. 5 mmsc-1186Only 36% reduction in activity against MAPK14 and no significant effect on the other kinases tested.
Figure 10（a）msc-1186Activity against three SRPK isoforms;(b) Nanobret assaymsc-1186Selectivity for 135 kinases.
Physicochemical properties
msc-1186It has low solubility and is easily metabolized by liver microsomes (Table 4), thereforeIt is not suitable for animal studies, but there are currently no reports of better selective instrumental molecules
Comparison with other SRPK kinase inhibitor activity (Table 5).
srpin340It has good selectivity, but the activity is significantly weaker thanmsc-1186sphinx31It is most effective against SRPK1, has a weaker effect on SRPK2 and SRPK3, and inhibits family proteins such as CLK and DYRK. srpkin-1The activity is highly efficient, but the selectivity is low.
Characteristics of cell function
For further validationmsc-1186The researchers analyzed phosphorylated PRSF4 (PSRSF4) levels in cells (Figure 11). SRPK inhibitorsmsc-118withsrpin340The activity is very weak. However, with CLK kinase inhibitorst-025When combined, compared with monotherapyt-025, activity is significantly enhanced.
Figure 11msc-1186msc-5360withsrpin340and with CLK inhibitorst-025Effect on PSRSF4 levels when combined.
To further investigate the synergistic effects of SRPK and CLK, the researchers investigated the effects of the compound on the intracellular SR levels and location of HCT116 with high expression of pan-phosphorylated SR protein (PSR) (Figure 12). Hypophosphorylated SR proteins are mainly distributed in nuclear speckles, while phosphorylated SR proteins are more evenly distributed in the nuclei. msc-1186It was used in combination with a negative control (CLK-T3) for the Pan-CLK probe without altering PSR levels or subnuclear localization activity. Active Pan-Clk probe (CLKT3) with SRPK-negative moleculemsc-5360Reduced PSR levels in a dose-dependent manner and altered the distribution of PSR signals in the nucleus (Figure 12C). The results of the PSRSF4 experiment were similarmsc-1186Combination with the CLK inhibitor CLKT3 resulted in a decrease in PSR levels (Fig. 12D), and the combination further promoted PSR aggregation. These results suggest that the combination of SRPK CLK inhibitors not only inhibits the phosphorylation of SR4, but also affects the phosphorylation and localization of SR protein.
Figure 12msc-1186Effect of CLK inhibitors alone or in combination on PSR levels and subcellular localization.
Conclusion
When optimizing FAK activity, fragments are found incidentallyIt has SRPK activity. Pyridine isomerization is performed first), which is then converted to benzimidazole (), willCombined with the sulfonamide group of Defactinib to obtain a molecule with SRPK selectivity5aSAR optimization resulted in highly effective and highly selective SRPK inhibitorsmsc-1186。The study of the combination of selective SRPK and CLK inhibitors on the regulation of splicing has been carried out, which provides an advantageous choice of drug combination. msc-1186has been made available to the SGC consortium for use in a wider range of scientific research.
Article**
doi.org/10.1021/acs.jmedchem.2c01705
Please clickLink here**Tutorial on how to use the CyberSAR system in detail
The Cyber platform integrates drug design ideas, excavates the active structures reported in the literature and patents, and can easily and quickly obtain the target structures of interest of R&D personnel through the Cyber platform for developing ideas, such as SRPK1 inhibitors
1.Go to the CyberSAR homepage, enter "srpk" in the target drop-down field, and select "Associate srpk1 (Homo sapiens)" to search for target information about SRPK1.
2.In the target interface, select the "Cluster Space" tab under the "Chemical Space" option tab, and you can display the literature and patents on the CyberSAR platform with the activity of SRPK1-related experiments in the form of "molecular parent nucleus clustering".
3.Select the "Test Data" option in the target page to see the activity data of the SRPK1 target molecule.
4.Click on the molecular structure to see the molecule of interest to further expand the information.
5.Clicking on the "Skeleton Similarity" tab or the "Targets" tab can further provide derivative types of structures for molecular learning.
Sign-in method
Cybersar logs in on your computer's browser**:
Feel free to try it out.
Please clickLink here**Tutorial on how to use the CyberSAR system in detail
If you need further communication, please scan the QR code to add WeChat to contact Dr. Yakudo Zhao or Yaodu CyberSAR to communicate.