| It is well known that Kv1.3 channel has been a clinical target for numerous autoimmune diseases,such as multiple sclerosis,rheumatoid arthritis,and psoriasis.For a long time,the Kv1.3 channel-acting small chemical molecule drug candidates have difficulty entering clinical trials,due to their low selectivity and serious potential toxic side effects.The Kv1.3 channle-blocking peptides have 3 or more intramolecular disulfide bonds,which display higher selectivity against other potassium channels.The sea anemone toxin peptide derived analog Sh K-186 has completed the clinical phase I test,which provides a promising direction for the development of potassium channel Kv1.3 immunosuppressive peptide drug.Based on the long-term work of our group on the interaction bettween scorpion toxin polypeptides and potassium channels,we will further conduct the research studies on discovery and design novel potassium channel Kv1.3 immunosuppressive peptides in this thesis.In the first part of this thesis,We carried out the research study mainly about the potassium channel Kv1.3 immunosuppressive peptide drugs screening.Based on the accumulation of previous work of our research group,we focused on screening the potential immunosuppressive peptides with novel structural features,such as Kunitz-type protease inhibitor peptide and defensin peptide.We first constructed a novel Kunitz-type protease derivative H2 based on the template of Hg1 by truncating the C-terminal region.The truncated peptide H2 showed potent blocking affinity for Kv1.3channel with an IC50 value of approximately 8.5 n M.The electrophysiological experiment revealed that h BD4 displayed weak affinity for Kv1.3 channel,and 1μM h BD4 could inhibit 34.0±0.2%Kv1.3 channel current.Channel selectivity data indicated that h BD4 had no significant activity against Kv1.1,Kv1.2,SKCa3 and IKCa channels at 1μM concentration.Moreover,we found that a fungal defensin Micasin could inhibit Kv1.3 channel,with 34.7±3.5%current blocked by 1μM concentration.Our work demonstrated that more efforts and costs should paid to find t Kv1.3 channel-specific peptide drug candidates,and the large scale peptides drug screening was accompanided with unpredictable risks.Due to the high cost of peptide preparation,developing the novel strategies to design potassium channel Kv1.3 immunosuppressive peptide drugs has become an important task in this thesis.Secondly,we applied the regulation function of the acidic amino acid residues in the binding interface of peptide blockers to start the research study on the design of Kv1.3 channel immunosuppressive peptide drugs.In the previous work,our group has previously proposed a novel strategy for the modification of the potassium channel-peptide binding mode,by regulating the distribution of acidic amino acid residues in peptides.Here,we found that scorpion toxin Bm KTX,with unusual binding mode,was found to contact both differential turret and filter region of extracellular pore region of h Kv1.3 and other potassium channels,while scorpion toxin Bm KTX-D33H,with the classical binding mode,could mainly contact differential turret of h Kv1.3 and other potassium channels.Using Bm KTX as the template,a highly h Kv1.3 channel-selective ADIP-6 blocker was designing through maintaining Bm KTX-like binding mode by slightly changing the distribution of the toxin negatively charged residues.The ADIP-6 blocked h Kv1.3,h Kv1.1,h Kv1.2,h Kv1.6 channels with IC50 values of 0.8±0.1 n M,408.0±74.9 n M,184.5±75.0 n M and 1489.3±102.5 n M,respectively.In addition,1μM ADIP-6 showed much less effect on h Kv1.5,h Kv3.2,h Kv4.1,h Kv4.2,h Kv4.3,h Kv7.1and h Kv11.1 channels.As expected,ADIP-6 could effective inhibit the currents of Kv1.3 channel and IL-2 production in Jurkat cells,and significantly ameliorate the delayed-type hypersensitivity in rats.Together,a highly h Kv1.3 channel-selective ADIP-6 immunosuppressant was successfully designed based on the specific toxin binding mode.These findings provided a strategy of designing selective h Kv1.3channel blocker through effectively contacting both the differential turret and filter region of the extracellular pore region between h Kv1.3 and other potassium channel in the future.We further conducted the structure optimization of ADIP-6 in this part of work.Based on sequence alignment and structural analysis ofα-KTX.3x family,we speculated that substitution of Asp33 residues in Bm KTX may affect the selectivity of Kv1.3 channel–acting peptides.In order to improve the selectivity of ADIP-6,we constructed a series of ADIP-6-derived peptides（AIDP-6-K33A,AIDP-6-K33H,AIDP-6-K33R,AIDP-6-K33T and AIDP-6-K33Y）to explore the optimal 33 amino acid residues.Electrophysiological experiments showed that the five newly constructed derivative peptides of ADIP-6（AIDP-6-K33A,AIDP-6-K33H,AIDP-6-K33R,AIDP-6-K33T and AIDP-6-K33Y）all have high Kv1.3 channel inhibitory activity,with an IC50 value of 1.0±0.1 n M,0.5±0.1 n M,0.7±0.2 n M,6.5±0.8 n M and 4.5±1.1 n M,respectively.Compared with the wild-type polypeptide AIDP-6,the amino acid change at position 33 did not affect the inhibitory activity of the Kv1.3 channel,but was related to the selectivity of the channel.The phamarcological profile experiments demonstrated that AIDP-6-K33H showed the best the best activity and selectivity against Kv1.3channel.The ADIP-6-K33H blocked h Kv1.2,h Kv1.6 channels with IC50 values of322.2±50.8 n M and 1217.2±90.3 n M,respectively.In addition,1μM ADIP-6-K33H could block 54.1±1.8%h Kv1.1 channel currents,and showed much less effect on h Kv1.5,h Kv3.2,h Kv4.1,h Kv4.2,h Kv4.3,h Kv7.1 and h Kv11.1 channels.Compared with the wild-type polypeptide ADIP-6,ADIP-6-K33H showed a better selectivity.In this work,we improve the selectivity and affinity of ADIP-6,through the optimization of functional residues in the binding interface,which provides a reference for the future development of Bm KTX-like immunosuppressive polypeptides.In the last part of this thesis,we used the regulation function of the acidic amino acid residues in the binding interface of peptide blockers,to carry out research study on the fine regulation of the interaction mode between Bm KTX and Kv1.3 channel.We used Bm KTX as a template to construct a series of derived peptides by slightly changing the position of acidic amino acids.By using acidic amino acid scanning,we constructed 14 Bm KTX-like polypeptides,in which the positions of acidic amino acids are distributed in various regions of the polypeptide’s space.Through recombinant expression technology,we obtained these high-purity derived peptides,and electrophysiological experiments showed that most of these Bm KTX-like peptides have high inhibitory activity against Kv1.3 channels,and the IC50 value is approximate5 n M.In order to study the interface between these derived peptides and the Kv1.3channel,we selected two key basic amino acids（arginine 23 and 26 lysine）to investigate the effects on the Kv1.3 inhinitory activity of different derivatives.Alanine substitutions were made to determine the effect of these two amino acids in the above-described derivative polypeptides by using site-directed mutagenesis techniques for these two positions.Whole-cell patch clamp results showed that the effects of these two amino acids varied with the position of the acidic amino acid,indicating that acidic amino acids play a key role in the interaction of the polypeptide with the potassium channel.By regulating the distribution of acidic amino acids,we have successfully obtained a series of peptides with high inhibitory activity on Kv1.3 channels,which brings new hope for the discovery of highly selective peptides in the future.In summary,we conducted the research studies on discovery and design novel potassium channel Kv1.3 immunosuppressive peptides in this thesis.In the case of the“uncertainty”of peptide drug screening,we mainly adopted the unique role of the acidic amino acid in the modulating the binding interface of the neuropeptides,and obtained a highly selective Kv1.3 channel-acting peptide AIDP-6.Moreover,we further improve the selectivity and affinity of ADIP-6,through the optimization of functional residues in the binding interface.These work have further promoted the design and development of Kv1.3 channel immunsuppressive peptides,and also provided theoretical and practical basis for the future research of Kv1.3-specific peptide drugs. |
