Assay for compounds that protect against sensory hair cell death and compounds identified by same

Abstract

The present invention provides methods of identifying compounds that protect against ototoxicity induced by one or more noxious stimuli, and methods of treating an individual with compounds identified using the present screening methods. Also provided are compounds demonstrated to have otoprotective effects.

1 . A method of identifying a compound that inhibits sensory hair cell death, the method comprising: 
 a) preferentially labeling the lateral line sensory hair cells of a zebrafish in comparison to other cells, wherein said labeling is substantially non-toxic to the zebrafish hair cells, wherein said label is detectably distinct in a live cell in comparison to a dying cell or a dead cell;    b) contacting the zebrafish with a test compound suspected of inhibiting sensory hair cell death;    c) contacting the zebrafish with a noxious stimulus known to cause sensory hair cell death; and    d) detecting the label, wherein a compound that inhibits sensory hair cell death is identified when the number of live lateral line hair cells is greater in zebrafish contacted with the test compound in comparison to a control zebrafish not contacted by the test compound.    
   
   
       2 . The method of  claim 1 , wherein said noxious stimulus is an ototoxic drug.  
   
   
       3 . The method of  claim 2 , wherein said ototoxic drug is selected from the group consisting of an aminoglycoside, an anticancer agent, a loop diuretic, a sesquiterpene lactone endoperoxide, a quinine, a salicylate, and an interferon polypeptide.  
   
   
       4 . The method of  claim 1 , wherein said labeling comprises contacting said zebrafish with a cytoplasmic vital dye and a nuclear vital dye.  
   
   
       5 . The method of  claim 4 , wherein said cytoplasmic vital dye preferentially labels plasma cell membranes.  
   
   
       6 . The method of  claim 4 , wherein said cytoplasmic vital dye is a styryl dye.  
   
   
       7 . The method of  claim 4 , wherein said nuclear vital dye labels dead or dying cells in the absence of cell permeabilization.  
   
   
       8 . The method of  claim 7 , wherein said nuclear vital dye is a cyanine dye.  
   
   
       9 . The method of  claim 7 , wherein said nuclear vital dye is a quinolinium dye.  
   
   
       10 . The method of  claim 8 , wherein said cyanine dye is a cyanine monomer.  
   
   
       11 . The method of  claim 1 , further comprising after step a) the step of washing away label unassociated with the lateral line hair cells of the zebrafish.  
   
   
       12 . The method of  claim 1 , wherein said sensory hair cell death is apoptosis.  
   
   
       13 . The method of  claim 1 , wherein said sensory hair cell death is necrosis.  
   
   
       14 . The method of  claim 1 , wherein said test compound comprises a thiophene-carboxamide moiety.  
   
   
       15 . The method of  claim 14 , wherein said thiophene-carboxamide moiety comprises a urea-thiophene-carboxamide moiety.  
   
   
       16 . A method of simultaneously screening a plurality of compounds that potentially inhibit sensory hair cell death, the method comprising: 
 a) preferentially labeling the lateral line hair cells of a plurality of zebrafish in comparison to other cells of the zebrafish, wherein said labeling is substantially non-toxic to the zebrafish hair cells, wherein said label is detectably distinct in a live cell in comparison to a dying cell or a dead cell;    b) contacting each member of the plurality of zebrafish with one member of a plurality of test compounds suspected of inhibiting sensory hair cell death;    c) contacting each member of the plurality of zebrafish with one or more noxious stimuli known to cause sensory hair cell death; and    d) detecting the label in each member of the plurality of zebrafish, wherein a compound that inhibits sensory hair cell death is identified when the number of live lateral line hair cells is greater in zebrafish contacted with the test compound in comparison to a control zebrafish not contacted by the test compound.    
   
   
       17 . The method of  claim 16 , further comprising after step a) the step of washing away label unassociated with the lateral line hair cells of the zebrafish.  
   
   
       18 . The method of  claim 16 , wherein said noxious stimulus is an ototoxic pharmacological agent.  
   
   
       19 . The method of  claim 16 , wherein said test compound comprises a thiophene-carboxamide moiety.  
   
   
       20 . The method of  claim 19 , wherein said test compound comprises a urea-thiophene-carboxamide moiety.  
   
   
       21 . A method of preventing a condition associated with sensory hair cell death, the method comprising administering a sufficient amount of a compound selected from the group consisting of: cepharanthine, amsacrine, drofenine, phenoxybenzamine, N,N-hexamethyleneamiloride, carvedilol and 9-amino-1,2,3,4-tetrahydroacridine.  
   
   
       22 . The method of  claim 21 , wherein said compound is administered prior to exposure to a noxious stimulus that induces ototoxicity.  
   
   
       23 . The method of  claim 21 , wherein said compound is administered at the same time as exposure to a noxious stimulus that induces ototoxicity.  
   
   
       24 . The method of  claim 21 , wherein said compound is administered after exposure to a noxious stimulus that induces ototoxicity.  
   
   
       25 . A method of preventing a condition associated with sensory hair cell death, the method comprising administering a sufficient amount of a compound comprising a thiophene-carboxamide moiety of Formula I or Formula II, wherein said compound inhibits lateral line hair cell death in a zebrafish.  
   
   
       26 . The method of  claim 25 , wherein said compound comprises a urea-thiophene-carboxamide moiety of Formula III or Formula IV.  
   
   
       27 . The method of  claim 25 , wherein said compound is selected from the group consisting of F5, H10, Compound A, Compound B, Compound C, and Compound D, and mixtures thereof.