Engineered protein kinases which can utilize modified nucleotide triphosphate substrates

Abstract

Engineered protein kinases which can utilize modified nucleotide triphosphate substrates that are not as readily utilized by the wild-type forms of those enzymes, and methods of making and using them. Modified nucleotide triphosphate substrates and methods of making and using them. Methods for using such engineered kinases and such modified substrates to identify which protein substrates the kinases act upon, to measure the extent of such action, and to determine if test compounds can modulate such action. Also Engineered forms of multi-substrate enzymes which covalently attach part or all of at least one (donor) substrate to at least one other (recipient) substrate, which engineered forms will accept modified substrates that are not as readily utilized by the wild-type forms of those enzymes. Methods for making and using such engineered enzymes. Modified substrates and methods of making and using them. Methods for using such engineered enzymes and such modified substrates to identify the recipient substrates the enzymes act upon, to measure the extent of such action, and to measure whether test compounds modulate such action.

1 . A mutant multi-substrate enzyme which accepts at least one orthogonal substrate analog, whereby catalytic activity results in the combination of all or part of said orthogonal substrate with at least one other substrate of said enzyme.  
     
     
         2 . The mutant enzyme of  claim 1  wherein said multi-substrate enzyme is a transferase.  
     
     
         3 . The mutant enzyme of  claim 1  wherein said multi-substrate enzyme is a signal transduction mediator.  
     
     
         4 . A mutant protein kinase which accepts an orthogonal nucleotide triphosphate analog as a phosphate donor substrate.  
     
     
         5 . The mutant protein kinase of  claim 4  wherein said mutant protein kinase binds to the orthogonal nucleotide triphosphate with an affinity which is higher than its affinity for the nucleotide triphosphate which is the primary intracellular phosphate donor substrate for the wild-type protein kinase.  
     
     
         6 . The mutant protein kinase of  claim 4  wherein said orthogonal nucleotide triphosphate analog is an orthogonal analog of ATP.  
     
     
         7 . The mutant protein kinase of  claim 4  wherein said orthogonal nucleotide triphosphate analog is a derivative of ATP having a substituent comprising at least three carbon atoms covalently attached to the N 6  position of said ATP.  
     
     
         8 . The mutant protein kinase of  claim 7  wherein said orthogonal nucleotide triphosphate analog is selected from the group consisting of N 6 -(cyclopentyl)ATP, N 6 -(cyclopentyloxy)ATP, N 6 -(cyclohexyl)ATP, N 6 -(cyclohexyloxy)ATP, N 6 -(benzyl)ATP, N 6 -(benzyloxy)ATP, N 6 -(pyrolidino)ATP, and N 6 -(pipperidino)ATP.  
     
     
         9 . The mutant protein kinase of  claim 4  wherein said orthogonal nucleotide triphosphate analog is N 6 -(cyclopentyl)ATP.  
     
     
         10 . The mutant protein kinase of  claim 4  which is a mutant protein tyrosine kinase.  
     
     
         11 . The mutant protein kinase of  claim 10  which is a mutant of a Src protein tyrosine kinase.  
     
     
         12 . The mutant protein kinase of  claim 10  which is a mutant of a Rous sarcoma virus Src protein tyrosine kinase.  
     
     
         13 . The mutant protein kinase of  claim 4  wherein the amino acid sequence differs from that of the wild type protein kinase in that at least one amino acid at a position homologous to the position selected from the group consisting of v-Src position 323 and v-Src position 338 has been replaced with an amino acid selected from the group consisting of alanine and glycine.  
     
     
         14 . The mutant protein kinase of  claim 4  wherein the amino acid at a position homologous to v-Src position 338 has been replaced with glycine.  
     
     
         15 . The mutant protein kinase of  claim 4  wherein the amino acid at a position homologous to v-Src position 323 and the amino acid at a position homologous to v-Src position 338 have been replaced with alanine.  
     
     
         16 . The mutant protein kinase of  claim 4  wherein said mutant protein kinase has been expressed as a fusion protein.  
     
     
         17 . The mutant protein kinase of  claim 16  which has been expressed as fusion protein selected from the group consisting of a glutathione-S-transferase fusion protein and a G-Histidine fusion protein.  
     
     
         18 . A nucleotide sequence which encodes a mutant multi-substrate enzyme which accepts at least one orthogonal substrate analog, whereby catalytic activity of said enzyme results in the combination of all or part of said orthogonal substrate with at least one other substrate of said enzyme.  
     
     
         19 . A nucleotide sequence which encodes a mutant protein kinase which accepts an orthogonal nucleotide triphosphate analog as a phosphate donor substrate.  
     
     
         20 . The nucleotide sequence of  claim 19  wherein said nucleotide sequence is selected from the group consisting of mRNA, cDNA, gDNA, mitochondrial DNA, chloroplast DNA, satellite DNA, plasmid DNA, viral RNA, and viral DNA.  
     
     
         21 . A method for producing a nucleic acid sequence encoding a mutant protein kinase which accepts an orthogonal nucleotide triphosphate analog as a phosphate donor substrate, comprising the steps of: 
 (a) identifying, from the crystal structure of an identical or homologous enzyme bound to its phosphate donor substrate, one or more amino acids other than glycine which are close enough to an atom of said bound phosphate donor substrate that they would sterically exclude an orthogonal substituent attached to the corresponding atom in said orthogonal nucleotide triphosphate analog; and    (b) mutating a nucleotide sequence which encodes the wild-type protein kinase such that the nucleotide triplets encoding one or more of the identified amino acids are converted to nucleotide triplets that encode amino acids having side chains that are sterically less bulky than the identified amino acids.    
     
     
         22 . The method of  claim 21  wherein said amino acids of step (a) are within about five angstroms of said atom of said bound phosphate donor substrate.  
     
     
         23 . The method of  claim 21  wherein said phosphate donor substrate is ATP.  
     
     
         24 . The method of  claim 23  wherein said atom is the N 6  amino group of ATP.  
     
     
         25 . A method for producing a mutant protein kinase which accepts an orthogonal nucleotide triphosphate analog as a phosphate donor substrate, comprising expressing the mutant sequence of  claim 21 , whereby said mutant protein kinase is produced.  
     
     
         26 . A method for producing a nucleic acid sequence encoding a mutant protein kinase which accepts an orthogonal nucleotide triphosphate analog as a phosphate donor substrate, comprising the steps of: 
 (a) identifying, from the crystal structure of an identical or homologous enzyme bound to its phosphate donor substrate, one or more amino acids other than glycine which are close enough to an atom of said bound phosphate donor substrate that they would sterically exclude the orthogonal substituent attached to the corresponding atom in said orthogonal nucleotide triphosphate analog;    (b) preparing a plurality of mutant protein kinase-encoding nucleotide sequences having one or more mutations in one or more nucleotide triplets encoding amino acids within ten amino acids of said one or more amino acids, in both the amino terminal and carboxy terminal directions;    (c) expressing said plurality of mutant kinase-encoding nucleotide sequences to produce a plurality of mutant kinases; and d. testing said plurality of mutant kinases to select one or more which have the ability to utilize said orthogonal nucleotide triphosphate analog as phosphate donor substrate.    
     
     
         27 . A method for producing a mutant protein kinase which accepts an orthogonal nucleotide triphosphate analog as a phosphate donor substrate, comprising expressing one or more mutant sequence of  claim 26  found to express such a mutant protein kinase, whereby said mutant protein kinase is produced.  
     
     
         28 . A method for producing a nucleic acid sequence encoding a mutant multi-substrate enzyme which accepts at least one orthogonal donor substrate analog, whereby catalytic activity results in the combination of all or part of said orthogonal donor substrate with at least one other, recipient substrate of said enzyme, comprising the steps of: 
 (a) identifying, from the crystal structure of an identical or homologous enzyme bound to its donor substrate, one or more amino acids other than glycine which are close enough to an atom of said bound donor substrate that they would sterically exclude an orthogonal substituent attached to the corresponding atom in said orthogonal donor substrate analog; and    (b) mutating a nucleotide sequence which encodes the wild-type form of said multi-substrate enzyme such that the nucleotide triplets encoding one or more of the identified amino acids are converted to nucleotide triplets that encode amino acids having side chains that are sterically less bulky than the identified amino acids.    
     
     
         29 . The method of  claim 28  wherein said amino acids of step (a) are within about five angstroms of said atom of said bound donor substrate.  
     
     
         30 . A method for producing a multi-substrate enzyme which accepts at least one orthogonal donor substrate analog, comprising expressing the mutant sequence of  claim 28 , whereby said mutant multi-substrate enzyme is produced.  
     
     
         31 . A method for producing a nucleic acid sequence encoding a mutant multi-substrate enzyme which accepts at least one orthogonal donor substrate analog, whereby catalytic activity results in the combination of all or part of said orthogonal donor substrate with at least one other, recipient substrate of said enzyme, comprising the steps of: 
 (a) identifying, from the crystal structure of an identical or homologous enzyme bound to its donor substrate, one or more amino acids other than glycine which are close enough to an atom of said bound phosphate donor substrate that they would sterically exclude the orthogonal substituent attached to the corresponding atom in said orthogonal donor substrate analog;    (b) preparing a plurality of mutant multi-substrate enzyme-encoding nucleotide sequences having one or more mutations in one or more nucleotide triplets encoding amino acids within ten amino acids of said one or more amino acids, in both the amino terminal and carboxy terminal directions;    (c) expressing said plurality of mutant multi-substrate enzyme-encoding nucleotide sequences to produce a plurality of mutant multi-substrate enzymes; and d. testing said plurality of mutant multi-substrate enzymes to select one or more which have the ability to utilize said orthogonal donor substrate analog as donor substrate.    
     
     
         32 . A method for producing a mutant multi-substrate enzyme which accepts at least one orthogonal donor substrate analog as a donor substrate, comprising expressing one or more mutant sequence of  claim 31  found to express such a mutant, whereby said mutant multi-substrate enzyme is produced.  
     
     
         33 . A method of detecting the one or more intracellular components that are recipient substrates for a multi-substrate enzyme that covalently transfers part or all of a donor substrate to a recipient substrate, comprising: 
 I. combining: 
 (a) cells, selected from the group consisting of permiablized cells, lysed cells, and cells which are naturally permeable to the orthogonal donor substrate analog, which cells express a mutant of said multi-substrate enzyme, which mutant accepts said orthogonal donor substrate analog as a donor substrate; and  
 (b) said orthogonal substrate analog, having a detectable moiety on the portion thereof that is catalytically transferred to a recipient substrate by said multi-substrate enzyme;  
   II. incubating said cells under conditions sufficient to allow the mutant multi-substrate enzyme to transfer part or all of the labeled orthogonal donor substrate to the recipient substrate; and    III. detecting the presence or absence of said detectable label on cellular components, where by the presence of said label on a cellular component indicates that said component is a recipient substrate for said multi-substrate enzyme, and the absence of said label on a cellular component indicates that said component is not a recipient substrate for said multi-substrate enzyme.    
     
     
         34 . A method of detecting the one or more intracellular protein substrates for a protein kinase, comprising: 
 I. combining: 
 (a) cells, selected from the group consisting of permiablised cells, lysed cells, and cells which are naturally permeable to the orthogonal nucleotide triphosphate substrate analog, which cells express a mutant of said protein kinase, which mutant accepts said orthogonal nucleotide triphosphate analog as a phosphate donor substrate; and  
 (b) said orthogonal nucleotide triphosphate analog, having a detectably labeled terminal phosphate;  
   II. incubating said cells under conditions sufficient to allow the mutant protein kinase to phosphorylate its one or more protein substrates using said orthogonal nucleotide triphosphate as phosphate donor; and    III. detecting the presence or absence of said detectably labeled phosphate on cellular proteins, whereby the presence of said label on a cellular protein indicates that said protein is a substrate for said protein kinase, and the absence of said label on a cellular protein indicates that said protein is not a substrate for said protein kinase.    
     
     
         35 . The method of  claim 34  wherein said mutant binds to said substrate with an affinity that is higher than its affinity for the primary intracellular phosphate donor substrate for the wild-type protein kinase.  
     
     
         36 . A method for determining whether a test compound modulates the activity of a multi-substrate enzyme, comprising the steps of: 
 I. combining: 
 (a) cells, selected from the group consisting of permiablized cells, lysed cells, and cells which are naturally permeable to the orthogonal donor substrate analog, which cells express a mutant of said multi-substrate enzyme, which mutant accepts said orthogonal donor substrate analog as a donor substrate; and  
 (b) said orthogonal substrate analog, having a detectable moiety on the portion thereof that is catalytically transferred to a recipient substrate by said multi-substrate enzyme; and  
 (c) said test compound;  
   II. incubating said cells under conditions sufficient to allow the mutant multi-substrate enzyme to transfer part or all of the labeled orthogonal donor substrate to the recipient substrate; and    III. detecting whether there has been an increase or decrease in the presence or absence of said detectable label on cellular components relative to that observed in one or more control experiments where said test compound was omitted, whereby a relative increase in the presence of said label on a cellular component indicates that said test compound has positively modulated the action of said multi-substrate enzyme on that component, and a relative decrease in the presence of said label on a cellular component indicates that said test compound has negatively modulated the action of said multi-substrate enzyme on that component.    
     
     
         37 . A method for determining whether a test compound modulates the activity of a protein kinase, comprising the steps of: 
 I. combining: 
 (a) cells, selected from the group consisting of permiablised cells, lysed cells, and cells which are naturally permeable to the orthogonal nucleotide triphosphate substrate analog, which cells express a mutant of said protein kinase, which mutant accepts said orthogonal nucleotide triphosphate analog as a phosphate donor substrate;  
 (b) said orthogonal nucleotide triphosphate analog, having a detectably labeled terminal phosphate; and  
 (c) said test compound;  
   II. incubating said cells under conditions sufficient to allow the mutant protein kinase to phosphorylate its one or more protein substrates using said orthogonal nucleotide triphosphate as phosphate donor; and    III. detecting whether there has been an increase or decrease in the presence or absence of said detectable label on cellular proteins relative to that observed in one or more control experiments where said test compound was omitted, whereby a relative increase in the presence of said label on a cellular protein indicates that said test compound has positively modulated the action of said protein kinase on that component, and a relative decrease in the presence of said label on a cellular protein indicates that said test compound has negatively modulated the action of said protein kinase on that component.    
     
     
         38 . An inhibitable engineered protein kinase or multi-substrate enzyme selected from kinases prepared in accordance herewith, synthetic analogs thereof, active fragments thereof, congeners thereof, and combinations thereof, for use both diagnostic and therapeutic procedures selected from drug assays, methods of treatment or intervention in disease states such as cancer, HIV or the like.  
     
     
         39 . A transgenic animal that may function as a “knock out” model for drug screening, wherein the wild-type gene corresponding to a particular kinase associated with a particular disease state is replaced with a gene encoding a mutant kinase, and said screen is used by the interaction of said model with a kinase inhibitor hereof.  
     
     
         40 . A method for the transformation of a target cell in an animal by the preparation of a vector containing DNA molecules that code on expression for a material selected from the group consisting of mutant kinases of  claim 1 , kinase inhibitors, agonists and antagonists thereto, active fragments thereof, analogs thereof, degenerate variants thereof, muteins thereof, and combinations thereof.  
     
     
         41 . A drug screen and associate screening method that utilizes an agent selected from the mutant kinase of  claim 1 , variants thereof, inhibitors thereof, active fragments thereof, analogs thereof, and combinations thereof.  
     
     
         42 . A pharmaceutical composition comprising an active agent selected from a mutant multi-substrate enzyme in accordance with  claim 1 , inhibitors thereof, agonists thereof, active fragments thereof, alleles thereof, analogs thereof, conserved variants thereof, and a pharmaceutically acceptable carrier.  
     
     
         43 . Use of the pharmaceutical composition of claim  41  for the treatment of a disease selected from cancer, HIV, Alzheimer's Disease.