01. US Patent & Trademark Office. S/N: P-154,392 License Number: 613,685.

Vlachakis D et al
Attorney Docket No. 111025-00002

02. US Patent & Trademark Office. S/N: P-154,391 License Number: 613,683.

Vlachakis D et al

Attorney Docket No. 111025-00002

03. WO/2009/125191 – 15.10.2009 (Pub) – PCT/GB2009/000936 – 09.04.2009

Brancale A, Vlachakis D, Berry C, Neyts J.

HCV ANTIVIRAL DRUG DESIGN (BENZENE DERIVATIVES): Compounds, which according to the invention can display potent inhibition against Hepatitis C Virus (HCV) helicase & potent anti-viral activity against HCV.

Hepatitis C is a liver infection caused by a flavivirus (HCV) that affects over 170 million people worldwide. In most cases the infection becomes chronic and 20% of the carriers develop cirrhosis. Currently the infection is treated with interferon, but this therapy does not completely eliminate the virus and more efficacious treatments are needed.

A new assay for the measurement of helicase enzyme activity was developed for the evaluation of the potency of potential inhibitors. This assay involves the use of a DNA or RNA duplex substrate and recombinant purified helicase. The DNA duplex consisted of a pair of oligonucleotides, one biotinylated and the other one DIG-labelled, at their respective 5' termini. This DNA duplex was immobilised, via the biotin molecule, on the surface of a neutravidin-coated 96 well plate. Helicase will initiate its unwinding activity upon activation with ATP, leading eventually in the release of the DIG labelled oligonucleotides, which translates in signal (luminescence) reduction with respect to control wells. This signal can be produced and quantified with the aid of a chemiluminescence antibody.

Other assay methods for this type of enzyme may analyse ATPase activity but assessment of the effects on helicase unwinding activity has been identified as the best way to evaluate inhibitors of this class of enzyme (1). Helicase activity assays rely on the ability of the enzyme to displace one strand (release strand) of DNA or RNA from another (template) strand. Many reports use displacement of radio-labelled release strands, detected either after gel electrophoresis (eg (2)), thin layer chromatography (eg (3)) or scintillation counting (eg (4)). The latter method may be amenable to high-throughput screening but carries the practical disadvantages of the use of radioactive materials. Further methods have incorporated DIG labelled release strands to allow detection by ELISA (5). We propose a new combined method that uses chemiluminescent antibody detection of residual release strand to give a robust helicase assay, that does not employ radiolabeled compounds and gives a stable read-out that is highly suited to high-throughput screening.

As proof of concept, the HCV helicase was expressed and isolated using recombinant protein techniques and then used in our assay. We found this assay to be highly reproducible since only slight variation was observed when a total of 96 helicase reactions (including controls) were performed on one plate. Therefore, our suggested rapid helicase assay is fast, convenient and highly reproducible while obviating the need to employ radiochemicals. These criteria make it suitable for high throughput screening of potential helicase inhibitors.

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04. GR/2012/0100266 – 21.05.2012 (Pub) – 201201605 – 21.05.2012

Kossida S, Vlachakis D, Tsiliki G, Pavlopoulou A.

3D PHARMACOPHORE FOR THE DESIGN OF HUMAN POLYADENOSINE RIBONUCLEASE(PARN) INHIBITORS. This invention is about a highly specific, custom-made three dimensional Pharmacophore model for the catalytic site of the human poly-adenosine ribonuclease (PARN).

The present invention relates to means and methods for the design of highly specific anti-PARN agent using our 3D pharmacophore model. Using a library of previously published modified nucleoside analog substrates acting in the proximity of the scissile bond of PARN, we conducted structure – activity relationship (SAR) analysis, structural characterization and complex (receptor-ligand) based molecular dynamics simulations that led to the design of a PARN-specific pharmacophore model. Our full pharmacophore model consists of five pharmacophoric annotation points, which include two hydrogen donating PAPs, one hydrophobic PAP, one aromatic PAP and a hydrogen accepting PAP, in the vicinity of human poly-Adenosine ribonuclease catalytic site.

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Dimitrios P. Vlachakis