Modulation of Cellular Signaling by Protein-Protein Interaction Inhibitor



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Serotonin GPCR subtype 2C (5-HT2CR) plays an important role in reducing the rate of relapse in drug addiction by regulating neuronal firing in dopaminergic reward pathway. When PTEN (protein phosphatase and tensin homologue) interacts with 5-HT2CR, intracellular signaling of 5-HT2CR ceases. An eight-residue peptide (3L4F-F1) of 5-HT2CR disrupted this interaction to restore intracellular signaling of 5-HT2CR. Arginine (R)-alanine (A)-asparagine (N) sequence in the 3L4F-F1 peptide was modified into RGD- or isoDGR- sequence, which interacts with integrin in the cellular membrane for endocytosis for cellular uptake. Using an alanine scan, arginine, asparagine, and aspartic acid were determined to be important for binding with PTEN. α-helix mimic peptides containing said amino acids were synthesized and evaluated by calcium assay. Those α-helix mimic peptides showed promising inhibition activity in positive allosteric modulation (PAM) assay. PTEN is comprised of catalytic domain, C2 domain, and PTD in C-terminal. To find the binding site in the interface between PTEN and 3L4F, photoaffinity probe of diazirine was synthesized and tagged to 3L4F-F1 peptide, which will be bound to PTEN. Autophagy is a cellular degradative pathway that plays diverse roles such as homeostasis, and immune defense system. Beclin-1 in PtIns3K complex is the key protein inducing autophagy. GAPR-1 protein as negative regulator of autophagy interacts with ECD 265-284 (evolutionary conserved domain) of Beclin-1 to inhibit autophagy. The synthetic peptide of ECD 265-284 blocks the interface of the GAPR-1 and Beclin-1 to restore autophagy induction. Based on the result of the alanine scan, the L-17 peptide was composed of the key residues for inducing autophagy. We synthesized α-helix mimic cyclic L-17 peptide for better binding efficiency and α-helix mimetic small organic compound based on the aryl template. 5-HT2CR plays an important role to suppress the neuronal firing in response to drug intake. 5-HT2CR/5-HT2AR GPCR dimer is formed between TM4 and TM5. We synthesized truncated TM4 of 5-HT2CR coupled to PEG (MW=5,000) by disulfide bridge to modulate the crosstalk between 5-HT2CR and 5-HT2AR by inhibiting the dimerization of the two GPCRs. In luciferase complementary assay (LCA), the PEG-SS-TM4 showed promising inhibitory effect against the dimerization of 5-HT2CR and 5-HT2AR.



Protein-protein interaction inhibitor, PTEN, 5-HT2CR, Autophagy, Crosstalk