HBV RNAseH Purification and Inhibitor Screening

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Solution Overview

Problem

Current treatments for hepatitis B virus (HBV) infection, particularly nucleos(t)ide analogs, while effective in suppressing viral replication, often require long-term administration and can lead to antiviral resistance, with residual cccDNA persistence due to residual viral replication and low-level infection, necessitating new therapeutic targets beyond DNA polymerase inhibition.

Innovation Solution

The development of a method to isolate and purify active hepatitis B virus RNAseH, utilizing nickel-agarose affinity chromatography and imidazole elution, followed by screening for inhibitors using a recombinant RNAseH enzyme to identify compounds that can inhibit HBV RNAseH activity, potentially in combination with nucleos(t)ide analogs to achieve viral suppression below cccDNA maintenance levels.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If nucleos(t)ide analogs are used to suppress viral replication, then viral replication is inhibited, but residual cccDNA persists due to low-level replication and infection

Engineering Contradiction:

Improveviral replication suppressionVSAvoidcccDNA persistence

Core Design Contradiction:

ReliabilityVSStability of the object's composition

Solution Approach 1:

The patent extracts and isolates the RNAseH enzymatic activity from the HBV polymerase complex, enabling independent targeting and inhibition of this specific function. This extraction allows for the development of inhibitors that specifically block RNAseH without affecting other viral enzymes, thereby addressing the residual replication problem that nucleos(t)ide analogs cannot fully eliminate

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

Instead of continuing to inhibit DNA polymerase activity (the conventional approach), the patent inverts the strategy by targeting the RNAseH activity that degrades RNA templates during reverse transcription. By inhibiting RNAseH, the patent prevents the formation of cccDNA from the outset, rather than trying to eliminate persistent cccDNA later

Inventive Principle:

Principle #13The other way round (Inversion)

2Reliability

If long-term nucleos(t)ide analog administration is used, then viral replication is suppressed, but antiviral resistance develops

Engineering Contradiction:

Improveviral replication suppressionVSAvoidantiviral resistance

Core Design Contradiction:

ReliabilityVSAdaptability or versatility

Solution Approach 1:

The patent develops RNAseH inhibitors that can be used in combination with existing nucleos(t)ide analogs, creating a multi-functional therapeutic approach. This combination therapy targets two different viral enzymes (reverse transcriptase and RNAseH), providing broader coverage and reducing the likelihood of resistance development compared to single-target therapies

Inventive Principle:

Principle #6Universality (Multi-functionality)

Solution Approach 2:

The patent segments the viral replication process into distinct targetable steps by focusing on RNAseH activity as a separate target from reverse transcriptase. This segmentation allows for the development of specialized inhibitors that act at a different stage of replication, reducing the selective pressure that leads to resistance against conventional nucleos(t)ide analogs

Inventive Principle:

Principle #1Segmentation

3Ease of manufacture

If HBV RNAseH purification method is developed, then active recombinant HBV RNAseH is obtained for drug screening, but complex purification steps are required

Engineering Contradiction:

Improverecombinant enzyme productionVSAvoidpurification process complexity

Core Design Contradiction:

Ease of manufactureVSDevice complexity

Solution Approach 1:

The patent incorporates a polyhistidine tag sequence into the HBV RNAseH gene before expression in E. coli. This preliminary modification enables direct affinity purification using nickel-agarose chromatography, eliminating the need for complex multi-step purification protocols and allowing rapid isolation of active recombinant enzyme for drug screening applications

Inventive Principle:

Principle #10Preliminary action

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

This approach enables the production of enzymatically active recombinant HBV RNAseH for antiviral drug screening, guiding the identification of compounds that can inhibit HBV RNAseH, potentially enhancing treatment efficacy by targeting a novel enzymatic activity, thereby addressing the limitations of existing therapies.

Implementation Method 1

applying said lysate to a nickel-agarose affinity chromatography column

Methodology Applied

Scientific EffectAffinity chromatography: Chromatography

Implementation Method 2

nickel-agarose affinity chromatography

Methodology Applied

Scientific EffectMetal coordination: Chemical Bonding

Implementation Method 3

eluting bound material from said column with imidazole

Methodology Applied

Scientific EffectCompetitive displacement: Chemical Bonding

Data Source

PatentUS10329542B2HBV RNAse H purification and enzyme inhibitors

Publication Date: 2019.06.25 SAINT LOUIS UNIV

AI summary

Provided herein are methods for the obtention of an active HBV RNaseH preparation and its use in screening methods to identify potential inhibitors of the enzyme for possible use as therapeutic agents. Also provided are methods of treatment using agents identified according to the screen.