Protein structural biomarkers to guide targeted chemotherapies

Abstract

A rapid, infrared spectroscopic method has been developed to assess the efficacy of targeted chemotherapeutics against the structure of the polypeptide target, based on the effect of natural polymorphic sequence variation on the conformation of the protein. This method has an advantage over the current genomics-based screening, as the new method provides a direct readout of the structural, and hence functional, outcome of polymorphisms to the protein region targeted by drugs. It allows rapid measurement of a protein's susceptibility to therapeutic targeted agents, prior to using the drug as treatment in the patient. This method can be used to identify biomarkers for a response for a protein to a drug which can be readily tested, interpreted, and used in a clinical setting.

1 .- 24 . (canceled) 
     
     
         25 . An in vitro method for identifying protein structural biomarkers useful for determining drug sensitivity and/or drug efficacy in a subject in need thereof, the method comprising:
 (a) measuring the conformation of each polymorphic variant x of a protein from a patient in the absence of drug compound, of a polymorphic variant y in the absence of drug compound, and of a polymorphic variant y when bound to the drug compound by vibrational spectroscopy using a spectrometer;   (b) determining whether the vibrational spectrum in an amide I region of each polymorphic variant x directly overlays onto the amide I band frequency when superimposed to: (i) the vibrational spectrum of polymorphic variant y, in the absence of the drug compound, or (ii) the vibrational spectrum of polymorphic variant y when bound to the drug compound;   (c) resolving whether each of one or more polymorphic variants x: (i) will not be resistant to the therapeutic effect of the drug compound if the vibrational spectrum in the amide I region directly overlays onto the amide I band frequency when superimposed to the vibrational spectrum of polymorphic variant y, in the absence of the drug compound; or (ii) will be resistant to the therapeutic effect of the drug compound if the vibrational spectrum in the amide I region directly overlays onto the amide I band frequency when superimposed to the vibrational spectrum of polymorphic variant y, when bound to the drug compound; and   (d) administering to the subject an effective amount of the drug compound identified in step (c)(i),   wherein the vibrational spectroscopic spectrum in the amide I region is determined as absorption or transmittance at one or more wavelengths.   
     
     
         26 . The method of  claim 25 , wherein step (a) is conducted by infrared or Raman spectroscopic measurement. 
     
     
         27 . The method of  claim 25 , wherein step (a) is conducted by Fourier-transform infrared spectroscopy. 
     
     
         28 . The method of  claim 25 , wherein the drug compound is a chemotherapeutic agent. 
     
     
         29 . The method of  claim 28 , wherein the drug compound is a chemotherapeutic agent that acts as an allosteric inhibitor of the protein. 
     
     
         30 . The method of  claim 28 , wherein the drug compound inhibits mitosis. 
     
     
         31 . The method of  claim 25 , wherein the protein is an ATPase. 
     
     
         32 . The method of  claim 31 , wherein the ATPase is selected from the group consisting of muscle myosin II, Na + ,K + -ATPase, P glycoprotein, H + ,K + -pump, vacuolar H + -ATPase, and DNA topoisomerase II. 
     
     
         33 . The method of  claim 25 , wherein the protein is a kinesin. 
     
     
         34 . The method of  claim 33 , wherein the protein is a kinesin selected from the group consisting of Eg5, Kif5A, Kif21A, Kif1B, and Kif6. 
     
     
         35 . The method of  claim 25 , wherein the protein is kinesin Eg5, and wherein the drug compound inhibits kinesin Eg5. 
     
     
         36 . The method of  claim 28 , wherein the drug is selected from the group consisting of monastrol, S-trityl-L-cysteine and its derivatives, quinazolines (e.g., ispinesib), adociasulfates, thetrahydroishquinolines, dihydropyrazoles, thiophenes, pyrrolotriazines, thiazoles, gossypol, indoles, and biphenyls. 
     
     
         37 . The method of  claim 28 , wherein the drug is monastrol. 
     
     
         38 . The method of  claim 28 , wherein the drug is S-trityl-L-cysteine. 
     
     
         39 . The method of  claim 25 , wherein the protein is a tyrosine kinase. 
     
     
         40 . The method of  claim 39 , wherein the tyrosine kinase is BCR-ABL. 
     
     
         41 . The method of  claim 28 , wherein the drug is Gleevec. 
     
     
         42 . The method of  claim 25 , wherein one or more polymorphic variants x have an amino acid sequence or a DNA sequence different than polymorphic variant y. 
     
     
         43 . The method of  claim 25 , wherein the polymorphic variants of the protein are found in a human population. 
     
     
         44 . The method of  claim 25 , wherein at least one polymorphic variant x of the protein or the DNA sequence encoding the polymorphic variant x of the protein is isolated from a human patient sample and wherein the responsiveness of polymorphic variant x to the drug compound is used to predict the responsiveness of the human patient to the drug compound. 
     
     
         45 . The method of  claim 25 , wherein the protein is a drug target for a disease. 
     
     
         46 . The method of  claim 25 , wherein the protein is the drug target for the drug compound. 
     
     
         47 . An in vitro analytical method for determining drug potency and/or efficacy of a cytotoxic drug targeted to a specific protein in a patient in need thereof, the method comprising:
 (a) measuring the conformation of a polymorphic variant of the protein isolated from the patient (variant x) in the absence of the cytotoxic drug, of a polymorphic variant of the protein that is known to be responsive to the cytotoxic drug (variant y) in the absence of the cytotoxic drug, and of polymorphic variant y when bound to the cytotoxic drug by vibrational spectroscopy using a spectrometer;   (b) determining whether the vibrational spectrum in an amide I region of polymorphic variant x directly overlays onto the amide I band frequency when superimposed to: (i) the vibrational spectrum of polymorphic variant y in the absence of the cytotoxic drug, or (ii) the vibrational spectrum of polymorphic variant y when bound to the cytotoxic drug;   (c) resolving whether the patient (i) will not be resistant to the efficacy or potency of the cytotoxic drug if the vibrational spectrum in the amide I region of polymorphic variant x directly overlays onto the amide I band frequency when superimposed to the vibrational spectrum of polymorphic variant y, in the absence of the cytotoxic drug; or (ii) will be resistant to the efficacy or potency of the cytotoxic drug if the vibrational spectrum in the amide I region of polymorphic variant x directly overlays onto the amide I band frequency when superimposed to the vibrational spectrum of polymorphic variant y, when bound to the cytotoxic drug; and   (d) administering to the patient an effective amount of the cytotoxic drug identified in step (c)(i),   wherein the vibrational spectroscopic spectrum in the amide I region is determined as absorption or transmittance at one or more wavelengths.   
     
     
         48 . The method of  claim 47 , wherein the cytotoxic drug is a chemotherapeutic drug. 
     
     
         49 . The method of  claim 47 , wherein the cytotoxic drug is an allosteric inhibitor. 
     
     
         50 . A method of treating and/or ameliorating cancer, a neurodegenerative disease, or a heart disease in a subject in need thereof, the method comprising:
 (a) measuring the conformation of a polymorphic variant of the protein isolated from the subject (variant x) in the absence of the cytotoxic drug, of a polymorphic variant of the protein that is known to be responsive to the cytotoxic drug (variant y) in the absence of the cytotoxic drug, and of polymorphic variant y when bound to the cytotoxic drug by vibrational spectroscopy using a spectrometer;   (b) determining whether the vibrational spectrum in an amide I region of polymorphic variant x directly overlays onto the amide I band frequency when superimposed to: (i) the vibrational spectrum of polymorphic variant y in the absence of the cytotoxic drug, or (ii) the vibrational spectrum of polymorphic variant y when bound to the cytotoxic drug;   (c) resolving whether the subject (i) will not be resistant to a therapeutic effect of the cytotoxic drug if the vibrational spectrum in the amide I region of polymorphic variant x directly overlays onto the amide I band frequency when superimposed to the vibrational spectrum of polymorphic variant y, in the absence of the cytotoxic drug; or (ii) will be resistant to the therapeutic effect of the cytotoxic drug if the vibrational spectrum in the amide I region of polymorphic variant x directly overlays onto the amide I band frequency when superimposed to the vibrational spectrum of polymorphic variant y, when bound to the cytotoxic drug; and   (d) administering to the subject an effective amount of the cytotoxic drug identified in step (c)(i),   wherein the vibrational spectroscopic spectrum in the amide I region is determined as absorption or transmittance at one or more wavelengths.   
     
     
         51 . The method of  claim 50 , wherein the cytotoxic drug is a chemotherapeutic drug. 
     
     
         52 . The method of  claim 50 , wherein the cytotoxic drug is an allosteric inhibitor.