Single-molecule platform for drug discovery: methods and apparatuses for drug discovery, including discovery of anticancer and antiviral agents
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Publication Number
US-9862984-B2
Publication Date
2018-01-09
Expiration Date
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.
Core Innovation
The invention provides a single-molecule drug screening and validation platform (NANO-VALID™) that uses optical or magnetic tweezers to measure real-time mechanical signatures of target enzyme-substrate interactions. In particular, it measures how nucleic acid polymerases affect mechanical behavior of an immobilized substrate under constant tension, with baseline mechanical signatures recorded without drug candidates and candidate drugs then added to detect deviations in mechanical traces.
The approach correlates mechanical response signals with polymerase velocity and uses those measured mechanical signatures to infer interference mechanisms. The documented examples identify effects such as initiation inhibition, premature termination, and exonuclease/proof-reading mode, based on changes in the mechanical traces.
The platform includes constant-tension optical trapping with feedback control, including force-feedback to maintain a set force. Instrumentation and software/GUI-controlled acquisition are described as enabling near real-time measurement using microplate wells, with readouts including template-length measurement and polymerase position/termination detection tied to processed mechanical signals for drug candidate validation.
Claims Coverage
The independent claim covers identifying nucleic acid polymerization modifying drug candidates by measuring a constant-tension feedback response correlated with nucleic acid polymerase velocity, both without and with the drug candidates. The claim set includes multiple inventive feature refinements across dependent claims specifying the polymerase type, substrate/template context, and constant-tension implementations.
Constant-tension immobilized substrate maintained by feedback control
Contacting a target nucleic acid polymerase with an immobilized substrate of the target polymerase in the absence of one or more nucleic acid polymerization modifying drug candidates, wherein the immobilized substrate is maintained under constant tension by at least partial stretching of the immobilized substrate using feedback control.
Baseline force-feedback response correlated with velocity without drugs
Detecting and recording a baseline feedback response signal of the applied force necessary to maintain the tension on the immobilized substrate in the presence of the target polymerase, wherein the baseline feedback response signal is correlated with the velocity of the target polymerase along the bound substrate of the target polymerase, but in the absence of the one or more nucleic acid polymerization modifying drug candidates.
Force-feedback response correlated with velocity in the presence of drug candidates
Contacting the target polymerase and the substrate of the target polymerase with a nucleic acid polymerization modifying drug candidate and detecting and recording, in the presence of the one or more nucleic acid polymerization modifying drug candidates, a feedback response signal correlated with the velocity of the target polymerase along the bound substrate of the target polymerase.
Overall, the claim coverage centers on maintaining constant tension on an immobilized nucleic acid polymerase substrate using feedback control, recording a baseline force-feedback response correlated with polymerase velocity without drugs, and recording a corresponding force-feedback response correlated with velocity after contacting with nucleic acid polymerization modifying drug candidates.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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