Single-Molecule Platform For Drug Discovery: Methods And Apparatuses For Drug Discovery, Including Discovery Of Anticancer And Antiviral Agents

Abstract

The present application discloses methods and apparatuses for single molecule drug screening, discovery and validation. These methods and apparatuses allow a user to detect rapidly, using observation of single molecules, whether and how a drug candidate interferes with a target enzyme involved in a particular disease pathway. The methods and apparatuses described herein utilize single molecule manipulation and detection technologies (e.g., optical or magnetic tweezers) to directly detect whether the characteristic dynamics, or “mechanical signature,” of the target enzyme-substrate interaction are substantially altered or modulated by a drug candidate. Furthermore, the methods and apparatuses are useful for analyzing the modulation of the mechanical signature in order to identify potential interference mechanisms of a drug candidate. In one aspect of the invention, the methods and apparatuses disclosed herein relate to monitoring the real-time dynamic mechanical signatures of individual polymerase molecules (e.g. DNA polymerases, RNA polymerases, and reverse transcriptases) along a polynucleotide substrate in the presence of drug candidates that either inhibit or otherwise modulate the polymerization process. Identification and analysis of such drug candidates is critical for anti-viral, anti-cancer, and antibiotic drug development.

What is claimed is: 
     
         1 .- 13 . (canceled) 
     
     
         14 . A system for measuring the interaction of a polymerase and a polymerase substrate, comprising:
 (a) a sample chamber containing a bound polymerase or a bound polymerase substrate;   (b) a radiation source configured to maintain said bound polymerase or bound polymerase substrate substantially at a focus position in the sample plane of said sample chamber using radiation signal focused on said sample plane;   (c) a detector configured to detect interference patterns in radiation reflected from said bound polymerase or said bound polymerase substrate arising from spatial deviations of said bound polymerase or said bound polymerase substrate from said focus position;   (d) a computer processor configured to determine spatial deviations of said bound polymerase or said bound polymerase substrate from said focus position using said detected spatial deviations; and   (e) a force feedback system configured to apply said determined spatial deviations to correct said radiation signal to maintain said bound polymerase or bound polymerase substrate substantially at said focus position.   
     
     
         15 . The system of  claim 14 , wherein said polymerase or polymerase substrate is bound to a bead. 
     
     
         16 . The system of  claim 15 , wherein said radiation source is a laser. 
     
     
         17 . The system of  claim 16 , further comprising a two-axis acousto-optical detector, said two-axis acousto-optical detector being configured to deflect said radiation to maintain said bound polymerase or said bound polymerase substrate at said focus position. 
     
     
         18 . The system of  claim 17 , wherein said detector is a quadrupole detector configured to detect interference patterns of the signal from said laser scattering off of said bound polymerase or bound polymerase substrate. 
     
     
         19 . The system of  claim 18 , further comprising a camera, said camera being configured to determining the position of said bound polymerase or bound polymerase substrate. 
     
     
         20 . The system of  claim 14 , further comprising a two-axis acousto-optical detector, said two-axis acousto-optical detector being configured to deflect said radiation to maintain said bound polymerase or said bound polymerase substrate at said focus position. 
     
     
         21 . The system of  claim 20 , wherein said detector is a quadrupole detector configured to detect interference patterns of the IR signal from said IR laser scattering off of said bound polymerase or bound polymerase substrate. 
     
     
         22 . The system of  claim 21 , further comprising a camera, said camera being configured to determining the position of said bound polymerase or bound polymerase substrate. 
     
     
         23 . A method for measuring the interaction of a polymerase and a polymerase substrate, comprising:
 (a) providing a bound polymerase or a bound polymerase substrate in a sample chamber;   (b) irradiating said bound polymerase or bound polymerase substrate using a radiation source and maintaining said bound polymerase or bound polymerase substrate substantially at a focus position in the sample plane of said sample chamber using a radiation signal focused on said sample plane from said radiation source;   (c) detecting interference patterns in radiation reflected from said bound polymerase or said bound polymerase substrate arising from spatial deviations of said bound polymerase or said bound polymerase substrate from said focus position;   (d) determining spatial deviations of said bound polymerase or said bound polymerase substrate from said focus position using said detected spatial deviations; and   (e) applying said determined spatial deviations to correct said radiation signal to maintain said bound polymerase or bound polymerase substrate substantially at said focus position.   
     
     
         24 . The method of  claim 23 , wherein said radiation is laser radiation. 
     
     
         25 . The method of  claim 24 , wherein said detecting is performed using a two-axis acousto-optical detector configured to deflect said radiation to maintain said bound polymerase or said bound polymerase substrate at said focus position. 
     
     
         26 . The method of  claim 25 , wherein said detector is a quadrupole detector configured to detect interference patterns of the signal from said laser scattering off of said bound polymerase or bound polymerase substrate. 
     
     
         27 . The method of  claim 26 , further comprising determining the position of said bound polymerase or bound polymerase substrate using a camera. 
     
     
         28 . The method of  claim 27 , further comprising providing a drug candidate in said sample chamber. 
     
     
         29 . The method of  claim 23 , wherein said detecting is performed using a two-axis acousto-optical detector configured to deflect said radiation to maintain said bound polymerase or said bound polymerase substrate at said focus position. 
     
     
         30 . The method of  claim 29 , wherein said detector is a quadrupole detector configured to detect interference patterns of the signal from said laser scattering off of said bound polymerase or bound polymerase substrate. 
     
     
         31 . The method of  claim 30 , further comprising determining the position of said bound polymerase or bound polymerase substrate using a camera. 
     
     
         32 . The method of  claim 31 , further comprising providing a drug candidate in said sample chamber. 
     
     
         33 . The method of  claim 32 , further comprising comparing the force required to maintain the position of said bound polymerase or bound polymerase substrate without said drug candidate with the force required to maintain the position of said bound polymerase or bound polymerase substrate combined with said drug candidate.