Molecular Glue Promotes Glucose Uptake Independent of Insulin
On July 24, 2025, a team led by Frank McCormick and Dhirendra K. Simanshu from the Frederick National Laboratory for Cancer Research, and Jun Tanaka and Kazuishi Kubota from Daiichi Sankyo Co., Ltd., published a study in Science titled "Molecular glues that facilitate RAS binding to PI3Kα to promote glucose uptake without insulin."
Phosphoinositide 3-Kinase α (PI3Kα) is a key molecule in cell signaling and the regulation of insulin homeostasis. In skeletal muscle, heart, and adipose tissue, PI3Kα signals through the insulin receptor, activating downstream kinases 3-Phosphoinositide-dependent Kinase 1 (PDK1) and AKT, which in turn promote the translocation of Glucose Transporter Type 4 (GLUT4) to the cell membrane, thereby facilitating glucose uptake. Defects in this process can lead to insulin resistance, type 2 diabetes, and metabolic syndrome. Loss of the PI3Kα-encoding gene, PIK3CA, also causes glucose intolerance and hyperinsulinemia, highlighting its central role in glucose homeostasis.
Diabetes treatment has long relied on insulin supplementation or enhancing insulin sensitivity, but existing therapies remain limited for patients with severe insulin resistance or deficiency. Therefore, identifying novel mechanisms that activate glucose uptake independently of insulin is a crucial approach to addressing this bottleneck in diabetes treatment.
In this study, researchers screened and identified two molecular glues, D223 and D927, that significantly enhance RAS binding to the PI3Kα catalytic subunit, p110α, thereby promoting glucose uptake in the absence of insulin. Using chemical proteomics, structural biology, and animal model experiments, the research team revealed the mechanism of action of these molecular glues and validated their potential application in improving diabetic symptoms, providing new insights into diabetes treatment.
To identify small molecules that activate glucose uptake in the absence of Insulin, the research team screened compounds in differentiated rat L6 myocytes expressing myc-tagged GLUT4 (L6-GLUT4 myc). They ultimately identified two active compounds, D223 and D927. These two compounds share the same backbone, differing only in substituents.
Both D223 and D927 stimulate GLUT4 translocation to the cell membrane in a concentration-dependent manner, similar to insulin. Insulin recruits insulin receptor substrate 1 (IRS-1) through the RAS protein, thereby activating the RAF-ERK1/2 and PI3Kα-AKT pathways. However, D927 specifically activates the PI3Kα-AKT pathway, has no significant effect on the RAF-ERK1/2 Pathway, and does not alter the expression of the PI3Kα catalytic subunit p110α. Furthermore, co-treatment with D927 and insulin synergistically enhances AKT activity.
Fig. 1 Effects of D223 and D927 on GLUT4 translocation and the PI3Kα signaling pathway. (Terayama, et al. 2025)
To elucidate the molecular mechanism by which D223 and D927 activate the PI3Kα pathway independently of the insulin receptor, the research team used immunoprecipitation combined with mass spectrometry and found that D223 significantly enhances the interaction between the RAS-associated protein RRAS2 and PI3Kα. Further experiments revealed that in HEK293T cells, D927 promoted the binding of RAS Family proteins (including KRAS, MRAS, and RRAS2) to the PI3Kα catalytic subunit p110α, with higher binding affinity for the KRAS G12D mutant. This mechanism may be related to the mutant's primary existence in the GTP-bound active form.
Isothermal titration calorimetry (ITC) analysis revealed that both D223 and D927 directly bound to p110α. Under physiological conditions, active KRAS has a low affinity for p110α, but the presence of D223 and D927 significantly enhanced their binding. Notably, even in the presence of D223 or D927, GDP-bound KRAS failed to form a stable association with p110α. These results suggest that D223 and D927 function as a molecular glue that directly binds to PI3Kα, recruiting active RAS proteins to form a complex with them, thereby promoting PI3Kα activation.
The research team used X-ray crystallography to determine the crystal structures of D927 and D223 in complex with the RAS-binding domain (RBD) of p110α. The results revealed that the two molecules bind to a pocket region between the β-sheet and α-helix of the RBD.
To elucidate the molecular mechanism by which molecular glues significantly enhance the affinity of RAS for p110α, the research team further determined the crystal structures of p110α and KRAS complexes in the presence of D223 or D927. Structural Analysis reveals that at the RAS-p110α interface, the amide groups of D223 and D927 form hydrogen bonds with the R41 backbone atom of KRAS and interact with the Y40 side chain of KRAS via van der Waals forces. Upon binding to p110α, the molecular glue stabilizes the first two α-helices and the intervening loop in the RBD, enabling the interface residues to form an optimal conformation for RAS binding. Furthermore, compared to D927, the N-tert-butyloxycarbonyl group of D223 forms additional interactions with p110α, while the methoxyl group of D927 faces the solvent phase, resulting in weaker interactions with the p110α-RBD.
Notably, the interface residues in KRAS that interact with p110α are highly conserved across RAS family members, including HRAS, NRAS, KRAS, RRAS, RRAS2, and MRAS, consistent with the interactions of D927 and D223 with these RAS family members.
Fig. 2 Structural Basis for D223 and D927 Enhanced RAS Binding to p110α. (Terayama,et al. 2025)
To investigate the mechanism of action of wild-type KRAS in D927-induced PI3Kα activation, the research team used mouse embryonic fibroblasts (MEFs) with HRAS and NRAS knockouts and induced KRAS knockout with 4-hydroxytamoxifen. When KRAS was GDP-bound, D927 only slightly increased AKT phosphorylation. However, when KRAS was GTP-bound, AKT phosphorylation increased significantly, and D927 further enhanced this effect, confirming that D927's action depended on active GTP-bound RAS. Notably, KRAS knockout in serum-starved cells actually increased D927-induced AKT phosphorylation. Because these cells lack HRAS and NRAS, it is hypothesized that other RAS family proteins (RRAS, RRAS2, and MRAS) may compensate for KRAS deficiency. Experiments have confirmed that the GTP-bound forms of these proteins significantly accumulate after KRAS knockout. Further CRISPR Knockout Experiments revealed that RRAS2 is the primary contributor to the D927 effect. These results suggest that D927 activates the PI3Kα signaling pathway by enhancing the interaction between RAS family proteins (particularly RRAS2) and p110α, and that this process is independent of KRAS.
To investigate the pharmacological effects of D927 on glucose homeostasis in vivo, the research team observed in insulin-resistant Zucker obese rats that a single oral administration of D927 rapidly lowered blood glucose levels, significantly increased glucose infusion rate and glucose turnover, and reduced plasma insulin levels. In obese and insulin-resistant type 2 diabetic db/db mice, D927 acutely improves hyperglycemia independently of insulin secretion, and its effect on lowering glycated hemoglobin (HbA1c), a marker of chronic hyperglycemia, is comparable to that of insulin. In insulin-deficient mice treated with streptozotocin (STZ), D927 significantly reduces HbA1c levels. In normal Sprague-Dawley rats, D927 lowers blood glucose in a time- and dose-dependent manner. In normal C57BL/6j mice, D927, like insulin, promotes AKT phosphorylation in soleus and gastrocnemius muscles, liver, heart, and white adipose tissue. These results demonstrate that D927 can improve glucose homeostasis in Diabetic Animal Models independently of insulin.
In summary, this study discovered a novel class of molecular glues, D223 and D927, that significantly enhance the binding affinity of RAS proteins to PI3Kα, thereby promoting glucose uptake in the absence of insulin. These molecular glues bind to the RAS-binding domain (RBD) of PI3Kα, stabilizing its secondary structure and maintaining the RAS-bound conformation. They also directly interact with specific residues of the RAS protein. In animal models, D927 rapidly lowered blood glucose levels, enhanced glucose metabolism efficiency, and ameliorated the hyperglycemic phenotype in both type 1 and type 2 diabetes models, with significant effects even in insulin-deficient diabetic models. These findings not only provide potential drug candidates for diabetes treatment but also provide key insights into the structural basis of the PI3Kα-RAS interaction.
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