Inhibition of Interaction of PSD93 and PSD95 with nNOS and NMDA Receptors

Abstract

PSD-95/SAP90 antisense-treated animals not only experience a significant decrease in MAC for isoflurane, but also experience an attenuation in the NMDA-induced increase in isoflurane MAC. PSD-95/SAP90 appears to mediate the role of the NMDA receptor in determining the MAC of inhalational anesthetics. Suppression of the expression of PSD-95/SAP90 in the spinal cord significantly attenuates responses to painful stimuli mediated through the N-methyl-D-aspartate receptor activation. In spinal cord neurons PSD-95/SAP90 interacts with the N-methyl-D-aspartate receptor subunits 2A/2B. Activation of the N-methyl-D-aspartate receptor in spinal hyperalgesia results in association of the N-methyl-D-aspartate receptor with PSD-95/SAP90. PSD-95/SAP90 is required for hyperalgesia triggered via the N-methyl-D-aspartate receptor at the spinal cord level.

1 . A method of screening for substances useful for relieving pain or inducing unconsciousness or sedation, comprising:
 contacting a test substance with a first protein and a second protein under conditions where the first protein and the second protein bind to each other, wherein the first protein is selected from the group consisting of PSD93, PSD95, and a combination thereof, wherein the second protein is selected from the group consisting of nNOS, NMDA receptor, NR2A subunit, NR2B subunit, and combinations thereof;   determining an amount selected from the group consisting of: free nNOS, free PSD93, free PSD95, free NMDA receptor, free NR2A subunit, free NR2B subunit, bound nNOS, bound PSD93, bound PSD95, bound NMDA receptor, bound NR2A subunit, bound NR2B subunit and combinations thereof;   identifying a test substance which increases the amount of free nNOS, free PSD93, free PSD95, free NMDA receptor, free NR2A subunit, or free NR2B subunit, or which decreases the amount of bound nNOS, bound PSD93, bound PSD95, bound NMDA receptor, bound NR2A subunit, or bound NR2B subunit as a candidate drug for relieving pain or inducing unconsciousness or sedation.   
     
     
         2 . The method of  claim 1  wherein the step of contacting is done in vitro. 
     
     
         3 . The method of  claim 1  wherein the step of contacting is done in yeast cells containing recombinant forms of the first and second proteins. 
     
     
         4 . The method of  claim 3  wherein the first and second recombinant proteins are each fused to a first and second yeast protein, wherein the first and second yeast proteins reconstitute a functional transcriptional activator when brought into physical proximity by binding of the first recombinant protein to the second recombinant protein. 
     
     
         5 . The method of  claim 1  further comprising the step of:
 testing an identified candidate drug in an animal to determine if the candidate drug relieves pain or induces unconsciousness or sedation.   
     
     
         6 . The method of  claim 1  wherein the test substance is contacted with PSD95 and nNOS. 
     
     
         7 . The method of  claim 1  wherein the test substance is contacted with PSD95 and NMDA receptor. 
     
     
         8 . The method of  claim 1  wherein the test substance is contacted with PSD95, nNOS, and NMDA receptor. 
     
     
         9 . The method of  claim 1  wherein the test substance is contacted with PSD95 and NR2A. 
     
     
         10 . The method of  claim 1  wherein the test substance is contacted with PSD95 and NR2B. 
     
     
         11 . The method of  claim 1  wherein the test substance is contacted with PSD93 and nNOS. 
     
     
         12 . The method of  claim 1  wherein the test substance is contacted with PSD93 and NMDA receptor. 
     
     
         13 . The method of  claim 1  wherein the test substance is contacted with PSD93, nNOS, and NMDA receptor. 
     
     
         14 . The method of  claim 1  wherein the test substance is contacted with PSD93 and NR2A. 
     
     
         15 . The method of  claim 1  wherein the test substance is contacted with PSD93 and NR2B. 
     
     
         16 . The method of  claim 1  wherein surface plasmon resonance is used to determine said amount. 
     
     
         17 . The method of  claim 1  wherein antibodies are used to determine said amount.