Study On The Structure And Function Of Kv1.3 Specific Inhibitor RTX-? And Its Application

Potassium channel is a transmembrane protein which mediated K⁺efflux.It is widely distributed in various cells or tissues,such as skeletal muscle,heart,nervous system,gastrointestinal tract system and lymphocytes.And it is a big family contained the most number of ion channel subtypes and the most complex function.Kv1.3 is one of the members of voltage-gated potassium channels,and is highly expressed in lymphocytes.Recently,several studies reported that Kv1.3 is largely associated with the processes of generation and development of human autoimmune diseases.Compared to the healthy person,there is higher expression of Kv1.3 in peripheral blood T_EMM cells and lesions of autoimmune diseases patients.And the symptoms of model animals with autoimmune diseases,such as Psoriasis,Rheumatoid Arthritis and Multiple Sclerosis,apparently attenuated with treatment of Kv1.3 inhibitors.Moreover,there has a peptide toxin inhibitor of Kv1.3 that entered clinical phase II experiments for the treatment of psoriasis.Multiple sclerosis,psoriasis,rheumatoid arthritis,type-1 diabetes,and Crohn's disease,etc.are all autoimmune diseases and currently the main treatments are dependentonbroad-spectrumimmunosuppressiveagentsand anti-inflammatory drugs.These drugs alleviated the syndromes of autoimmune diseases at a certain extent but with obvious side effects.That is because the drugs act on the unclear targets and may damage other normal tissues or organs of the body.Therefore,there is an urgent requirement for efficient drugs with specific target applied in the field of autoimmune diseases treatment.Although many drugs targeting Kv1.3 have been discovered and used in the clinical trials,we still need to screen leading molecules with more affinity and selectivity for Kv1.3.And at the same time,the leading molecules could serve as a probe to further elaborate the relationships between Kv1.3 and more autoimmune diseases.Based on the previously research,we discovered a peptide toxin inhibitor of Kv1.3 from Macrothele raveni venom,named RTX-? and develop a study related to autoimmune diseases.RTX-? consists of 35amino acid residues and contains six cysteines forming 3 pairs of disulfide bond.It inhibited the Kv1.3 current with an IC50 of 36.3 nM.For more sample access,we primarily synthesized RTX-? by chemical synthesis,and then this method was given up because of the low reductive yield of linear peptide,about 15%.Subsequently,RTX-? was obtained by recombinant expression with Shuffle T7 E.coli strain.The activity of recombinant peptide is similar to that of natural RTX-?,and the expression level of this peptide amounted to 15 mg/L.Sufficient sample was finally used to treat the animal models with autoimmune diseases.RTX-? inhibited the current of Kv1.3 heterogenously expressed in Hek293t cell lines,whether it could be an effective preparation for the treatment of autoimmune disease models remains to be further verified.Thus,we performed a multiple sclerosis rat model?experimental encephalomyelitis,EAE?and a K14-VEGF psoriasis mouse model,and evaluated the therapeutic effect of RTX-? on those two types of model animals.The results showed that 1 mg/kg RTX-? obviously attenuated the symptoms of EAE model rats and reduced the secretion of inflammatory factors in the treatment group.And RTX-? also showed a good therapeutic effect on K14-VEGF psoriasis mouse.In pharmacokinetics experiments and toxicity experiments,we found that RTX-? reached the maximum value at 30 min after administration,and completely degraded at 1 h.It is mainly metabolism by the liver,and a small part is metabolism by the kidney.Interestingly,the usage of RTX-? didn't cause toxic side effects of liver and kidney and didn't affect the behavior of animals at a 100 times effective doses.Although the relationship between Kv1.3 and multiple sclerosis and psoriasis is clearly interpreted in previous researches,the relationship between Kv1.3 and Crohn's disease remains to be explored.We firstly studied their relationships,1.At the clinical level:T_EMM cells were isolated from peripheral blood of Crohn's patients and normal subjects and were recorded the current of Kv1.3 by patch clamp technology.We found that the expression level of Kv1.3 in patients T_EMM cells is three times higher than that in normal subjects.The mRNA levels of the former were about 5times higher than that of the latter by RT-qPCR.Similarly,the results of immune-histochemistry experiments showed that the Kv1.3 expression level of Crohn's patients is higher than that of the normal group.Thus,we concluded that Crohn's patients are characterized with high mRNA and protein expression.2.At the model level:we performed a Crohn's disease model with rats and appropriate dose of RTX-? was used treat these model rats.The usage of 1 mg/kg and 2 mg/kg RTX-? can effectively alleviate the symptoms of Crohn's rat at a certain degree and reduced the expression of Kv1.3 at mRNA and protein levels in vivo.Moreover,the secretion of inflammatory factors was apparently reduced in the experimental group compared to the control group,such as IL-2,TNF-a,IFN-gand IL-17A,and its efficacy is basically consistent with the efficacy of the clinical drug infliximab.In summary,the therapeutic effect of RTX-? on Crohn's disease could achieve the same therapeutic effects as infliximab.A large number of peptide inhibitors of Kv1.3 have been identified in scorpion and sea anemone venom and a few of them resourced from spider venom.Almost of these peptide toxins inhibit the current of Kv1.3 with low IC50 value,usually at picomolar levels.Compared to the existing peptide toxins,RTX-? has several obvious advantages in the following aspects:1.ShK-186 is a modified peptide based on the anemone peptide toxin ShK,which is now used in clinical experiments.It potently inhibits Kv1.3 current with IC50 value of 71±4 pM,and its selectivity is more than97 time for Kv1.1?7±0.5 nM?.While RTX-? inhibits Kv1.3 current with IC50 value of 36 nM,and the selectivity is greater than 3000 times for Kv1.1?100mM?.That is to say,the selectivity of RTX-? is more predominant than that of ShK-186.2.The acquisition processes of ShK-186 is relatively complicated,specifically mentioned in following three steps:replacing the twenty-third amino acid to a methionine based on ShK,coupling the amide compound at the C-terminus,and adding a phosphorylated tyrosine at the N-terminus.Therefore,the acquisition of ShK-186 is must dependent on chemical synthesis technology,and the active peptide is subjected to refolding in redox system.The acquisition of RTX-? is easier by genetic engineering.Thus,RTX-? also has an advantage in sample preparation.In summary,we successfully addressed the acquisition problem of Kv1.3 inhibitor RTX-? by recombinant expression,and proved that RTX-? showed beneficial therapeutic effects in EAE and psoriasis animal model.Moreover,we firstly clarified the relationships between Kv1.3 and Crohn's disease and demonstrated that RTX-? also presented a potential application effects in rat Crohn's disease model,which enriched our understanding of the relationships between Crohn's disease and Kv1.3.