Targeting BRD4 and PI3K signaling pathways for the treatment of medulloblastoma.

Medulloblastoma (MB) is a malignant pediatric brain tumor which shows upregulation of MYC and sonic hedgehog (SHH) signaling. SHH inhibitors face acquired resistance, which is a major cause of relapse. Further, direct MYC oncogene inhibition is challenging, inhibition of MYC upstream insulin-like growth factor/ phosphatidylinositol-4,5-bisphosphate 3-kinase (IGF/PI3K) is a promising alternative. While PI3K inhibition activates resistance mechanisms, simultaneous inhibition of bromodomain-containing protein 4 (BRD4) and PI3K can overcome resistance. We synthesized a new molecule 8-(2,3-dihydrobenzo[b] [1, 4] dioxin-6-yl)-2-morpholino-4H-chromen-4-one (MDP5) that targets both BRD4 and PI3K pathways. We used X-ray crystal structures and a molecular modeling approach to confirm the interactions between MDP5 with bromo domains (BDs) from both BRD2 and BRD4, and molecular modeling for PI3K binding. MDP5 was shown to inhibit target pathways and MB cell growth in vitro and in vivo. MDP5 showed higher potency in DAOY cells (IC50 5.5 µM) compared to SF2523 (IC50 12.6 µM), and its IC50 values in HD-MB03 cells were like SF2523. Treatment of MB cells with MDP5 significantly decreased colony formation, increased apoptosis, and halted cell cycle progression. Further, MDP5 was well tolerated in NSG mice bearing either xenograft or orthotopic MB tumors at the dose of 20 mg/kg, and significantly reduced tumor growth and prolonged animal survival.

Synthesis of C7/C8-cyclitols and C7N-aminocyclitols from maltose and X-ray crystal structure of Streptomyces coelicolor GlgEI V279S in a complex with an amylostatin GXG-like derivative.

C7/C8-cyclitols and C7N-aminocyclitols find applications in the pharmaceutical sector as α-glucosidase inhibitors and in the agricultural sector as fungicides and insecticides. In this study, we identified C7/C8-cyclitols and C7N-aminocyclitols as potential inhibitors of Streptomyces coelicolor (Sco) GlgEI-V279S based on the docking scores. The protein and the ligand (targets 11, 12, and 13) were prepared, the states were generated at pH 7.0 ± 2.0, and the ligands were docked into the active sites of the receptor via Glide™. The synthetic route to these targets was similar to our previously reported route used to obtain 4-⍺-glucoside of valienamine (AGV), except the protecting group for target 12 was a p-bromobenzyl (PBB) ether to preserve the alkene upon deprotection. While compounds 11-13 did not inhibit Sco GlgEI-V279S at the concentrations evaluated, an X-ray crystal structure of the Sco GlgE1-V279S/13 complex was solved to a resolution of 2.73 Å. This structure allowed assessment differences and commonality with our previously reported inhibitors and was useful for identifying enzyme-compound interactions that may be important for future inhibitor development. The Asp 394 nucleophile formed a bidentate hydrogen bond interaction with the exocyclic oxygen atoms (C(3)-OH and C(7)-OH) similar to the observed interactions with the Sco GlgEI-V279S in a complex with AGV (PDB:7MGY). In addition, the data suggest replacing the cyclohexyl group with more isosteric and hydrogen bond-donating groups to increase binding interactions in the + 1 binding site.

Stereoselective synthesis of a 4-⍺-glucoside of valienamine and its X-ray structure in complex with Streptomyces coelicolor GlgE1-V279S.

Glycoside hydrolases (GH) are a large family of hydrolytic enzymes found in all domains of life. As such, they control a plethora of normal and pathogenic biological functions. Thus, understanding selective inhibition of GH enzymes at the atomic level can lead to the identification of new classes of therapeutics. In these studies, we identified a 4-⍺-glucoside of valienamine (8) as an inhibitor of Streptomyces coelicolor (Sco) GlgE1-V279S which belongs to the GH13 Carbohydrate Active EnZyme family. The results obtained from the dose-response experiments show that 8 at a concentration of 1000 µM reduced the enzyme activity of Sco GlgE1-V279S by 65%. The synthetic route to 8 and a closely related 4-⍺-glucoside of validamine (7) was achieved starting from readily available D-maltose. A key step in the synthesis was a chelation-controlled addition of vinylmagnesium bromide to a maltose-derived enone intermediate. X-ray structures of both 7 and 8 in complex with Sco GlgE1-V279S were solved to resolutions of 1.75 and 1.83 Å, respectively. Structural analysis revealed the valienamine derivative 8 binds the enzyme in an E2 conformation for the cyclohexene fragment. Also, the cyclohexene fragment shows a new hydrogen-bonding contact from the pseudo-diaxial C(3)-OH to the catalytic nucleophile Asp 394 at the enzyme active site. Asp 394, in fact, forms a bidentate interaction with both the C(3)-OH and C(7)-OH of the inhibitor. In contrast, compound 7 disrupts the catalytic sidechain interaction network of Sco GlgE1-V279S via steric interactions resulting in a conformation change in Asp 394. These findings will have implications for the design other aminocarbasugar-based GH13-inhibitors and will be useful for identifying more potent and selective inhibitors.