Single molecule drug discovery

• US 20070254279A1
• Filed: 04/23/2007
• Published: 11/01/2007
• Est. Priority Date: 04/21/2006
• Status: Active Grant

• Current Assignee: Nanobiosym, Inc.
• Original Assignee: Nanobiosym, Inc.
• Inventors: Goel, Anita
• Granted Patent: US 9,862,984 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/00 Measuring or testing processes involving enzymes, nucleic acids or microorganisms measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34; Compositions therefor; Processes of preparing such compositions
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: G01N 2333/16 HIV-1, HIV-2
• CPC Class Codes: G01N 2333/91245 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: G01N 2500/02 Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

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.

Claims

1. A method for screening drug candidates, wherein the method comprises:

(a) contacting a target enzyme with a substrate of the target enzyme;

(b) determining a baseline mechanical signature of the target enzyme in the presence of the substrate of the target enzyme by using a single-molecule detection apparatus to make a mechanical measurement;

(c) contacting the target enzyme and the substrate of the target enzyme with one or more drug candidates;

(d) determining a mechanical signature of the target enzyme in the presence of the substrate of the target enzyme and one or more drug candidates by using a single-molecule detection apparatus to make a mechanical measurement; and

(e) comparing the baseline mechanical signature of step (b) with the mechanical signature of step (d);

wherein;

the baseline mechanical signature of step (b) and the mechanical signature of step (d) are determined using the same single-molecule detection apparatus and the same mechanical measurement.

2. The method of claim 1, wherein the mechanical measurement is selected from the group consisting of:

a measurement of the time-dependent velocity of the target enzyme along the substrate of the target enzyme, a measurement of the time-dependent change in the length of the substrate of the target enzyme, a measurement of a change in the elasticity of the substrate of the target enzyme, and a measurement of the efficiency or the accuracy of substrate binding and processing by the target enzyme.

3. The method of claim 1, wherein the single-molecule detection apparatus is selected from the group consisting of:

an apparatus that utilizes magnetic or optical trapping, an apparatus that utilizes high-resolution fluorescent imaging coupled with quantum-dot labeling, and an apparatus that utilizes atomic force microscopy.

4. The method of claim 1, wherein the target enzyme is a polymerase.

5. The method of claim 4, wherein the polymerase is selected from the group consisting of:

a DNA polymerase, an RNA polymerase, and a reverse transcriptase.

6. The method of claim 4, wherein the polymerase is a human polymerase.

7. The method of claim 4, wherein the polymerase is a viral polymerase.

8. The method of claim 7, wherein the viral polymerase is a viral reverse transcriptase.

9. The method of claim 8, wherein the viral reverse transcriptase is an HIV-1 reverse transcriptase.

10. The method of claim 4, wherein the polymerase is a bacterial polymerase.

11. The method of claim 1, wherein the target enzyme is a polymerase, the substrate of the target enzyme is a polynucleotide, and the molecular measurement is a measurement of the movement of the polymerase along the polynucleotide substrate or a measurement of the time-dependent change in the length of the polynucleotide substrate during polymerization.

12. The method of claim 11, wherein the molecular measurement is made using a single-molecule detection apparatus that utilizes magnetic or optical trapping.

13. The method of claim 1, wherein the method provides detailed enzymatic dynamics data which precisely characterizes the inhibition and/or interference mechanism of the drug candidate tested.