Crystal structure of a deacetylase and inhibitors thereof

Abstract

The present invention provides three-dimensional structural information from the hyperthermophilic bacterium Aquifex aeolicus which is a histone deacetylase-like protein (HDLP). HDLP shares 35.2% amino acid sequence identity with human histone deacetylase (HDAC1). The present invention further provides three-dimensional structural information of HDLP bound by inhibitor molecules. The three-dimensional structural information of the present invention is useful to design, isolate and screen deacetylase inhibitor compounds capable of inhibiting HDLP, HDAC family members and HDLP-related molecules. The invention also relates to nucleic acids encoding a mutant HDLP which facilitates the determination of the three-dimensional structure of HDLP in the presence of a zinc atom.

1 - 19 . (canceled)  
     
     
         20 . A crystal of an enzyme comprising deacetylase activity wherein said crystal effectively diffracts X-rays for the determination of the atomic coordinates of said enzyme to a resolution of greater than 4 Å and wherein the structure of said enzyme comprises a conserved core α/β structure characteristic fold wherein said conserved α/β fold comprises an eight-stranded parallel β sheet and eight α helices and wherein four of the helices pack on either face of said parallel β sheet and wherein said structure of said enzyme comprises an rmsd of less than or equal to 1.5 Åin the positions of Cα atoms for at least ⅔ or more of the amino acids of HDLP as defined by the atomic coordinates of HDLP.  
     
     
         21 . The crystal of  claim 20 , wherein said protein structure further comprises: 
 (a) eight α helices positioned near one side of the β sheet; and    (b) at least seven large, well defined loops originating from the C-terminal ends of the β-strands of said eight-stranded parallel β sheet wherein the eight extra helices and the seven large loops are associated with a significant extension of the structure beyond the core α/β motif and wherein said extension of the structure gives rise to a deep, narrow pocket and an internal cavity adjacent to the pocket.    
     
     
         22 . The crystal of  claim 20 , wherein said enzyme comprising deacetylase activity is selected from the group consisting of HDLP, HDLP-related proteins, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC-related proteins, APAH, AcuC, and functional derivatives thereof.  
     
     
         23 . The crystal of  claim 21  further comprising a specifically bound zinc atom in the active site of said enzyme.  
     
     
         24 . The crystal of  claim 21  further comprising a specifically bound deacetylase inhibitor compound in the active site of said enzyme.  
     
     
         25 . The crystal of  claim 21  defined by the atomic coordinates according to  FIG. 16 .  
     
     
         26 . A method for solving the structure of an HDAC family member crystal comprising the steps of: 
 (a) collecting X-ray diffraction data of said crystal wherein said data diffracts to a high resolution limit of greater than 4 Å;    (b) using the atomic coordinates of HDLP according to  FIG. 16  to perform molecular replacement or refinement and difference fourier with said X-ray diffraction data of said HDAC family member crystal to determine the structure of said HDAC family member; and    (c) refining said structure of said HDAC family member.    
     
     
         27 . The method of  claim 26 , wherein said HDAC family member is HDAC1.