Sample paper for Drug Design Project using Accelrys Discovery Studio for preparing the protein & applying docking

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Abstract:
HCV polymerase NS5B is the key enzyme in viral replication by catalyzing synthesis of
complementary negative strand RNA using positive strand RNA genome as template. That's
why many of NS5B inhibitors are used for HCV treatment including Indole C2 Acyl
Sulfonamides. With our knowledge of the SAR we developed new ligands and tested their
receptor-ligand interactions by docking the ligands in Accelrys Discovery Studio (ADS) and
comparing their scores to the lead compound, then discussing the modifications applied to
ligand with highest score and its interaction with the binding site.
Introduction:
According to the World Health Organization (WHO), about 3% of the world’s population
has been infected with the hepatitis C virus (HCV). Of those, approximately 170 million are
chronic HCV carriers at risk of developing cirrhosis and hepatocellular carcinoma (HCC)
contributing to a large percentage of liver transplantations in Europe and the United States.
Egypt is known to be the largest epidemic in the world (estimated among adults at 10 and 20%
in urban and rural areas, respectively).The origin of the epidemic has been attributed to mass
campaigns of parenteral anti-schistosomiasis treatment in rural areas in the 1960s–70s.
Since then, the virus has continued to spread, mainly through intravenous injections and
other medical procedures and the incidence of new infections remains the highest,
worldwide.
Two classes of NS5B inhibitors have been developed: nucleoside/nucleotide and non-
nucleoside polymerase inhibitors. Nucleoside/nucleotide analogues act as natural
polymerase substrates leading to termination of RNA chain elongation by inhibition of the
active site of the HCV RdRp. They are Mostly synthetic prodrugs of nucleotides are
administered to facilitate re-absorption, and additional steps of intracellular
phosphorylation are required to gain full functional activity as a nucleoside triphosphate.
Non-nucleoside inhibitors on the other hand bind outside the active site and target allosteric
sites on the surface of the enzyme, down-regulating the RdRp activity through induction of
conformational changes.

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The current standard of care is treatment with a combination of subcutaneous pegylated
interferon administration with oral dosing of the cytotoxic nucleoside drug ribavirin. The
response rate is >75% for HCV patients with genotypes 2 and 3 after a 24 week treatment
regimen, while genotype 1 patients have a response rate of less than 50% after 48 weeks of
treatment. Recent FDA approval of Victrelis (boceprevir) has invigorated interest in small
molecule inhibitors of HCV. With a clear opportunity to improve clinical outcomes, and given
the side effects associated with the current standard of care, it is valuable to discover potent
inhibitors of HCV replication that will improve outcomes and shorten treatment duration.
Several crystal structures of NS5B polymerase in several crystalline forms have been
determined. Its structure can be represented by a right hand shape with fingers, palm, and
thumb.
Fig 1: A Structural representation of the HCV NS5B polymerase. Palm, thumb and fingers sub-domains of NS5B are color
coded red, green, and blue, respectively.
Sequence variation analysis suggests that residues lining the active site cavity (‘palm site’) are more
conserved than in other regions, making the palm site an attractive target for inhibition of the viral
polymerase. Clinical efficacy has been demonstrated with non-nucleoside inhibitors binding at the
palm, thumb, and finger-loop sub-domains. Indole C2 Acyl Sulfonmides act as non-nucleoside palm
site inhibitors of HCV NS5B polymerase. The unique feature of this class of inhibitors was the bi-
dentate hydrogen bonding interactions with the polypeptide backbone of Tyr-448 and Gln-446.
Objective and Molecular modeling docking study:
Our aim was to find a ligand with better binding to the receptor than the lead:
1-[(2-aminopyridin-4-yl) methyl]-5-chloro- N-({3-[(methylsulfonyl) amino] phenyl}
sulfonyl)-3-(2-oxo-1, 2-dihydropyridin-3-yl)-1H-indole- 2-sulfonamide.
We started by adding more H-bond donors, H-bond acceptors and we also tried
improving the hydrophobic interaction with the binding site without affecting the essential
pharmacophore which is the indole acyl sulfonamide nucleus. Eight molecules came up
from those lead modifications from which we wanted to find out which had the best
binding.

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Fig 2: The lead (upper left) and other suggested modifications.
From Protein Data Bank we managed to obtain the lead binding to HCV Polymerase, then
we opened it through Accelrys Discovery Studio (ADS), removed water and other
unwanted files we didn't want in the interaction. To add hydrogens and correct any
missing parts we used Clean Protein tool, we also ran the Prepare Protein Protocol, and
then we defined the binding site and the sphere corresponding to the active site from
Define and Edit Binding Site tool.

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After applying final adjustments to the receptor, it was ligands preparation time. Before
running Prepare Ligands Protocol, we adjusted the parameters so that Change Ionization,
Generate Tautomers, Generate Isomers and Parallel Processing are set "False" while both
Lipinski's Filter and Generate 3D are set "True".
As both protein and ligands became prepared the only remaining thing was to Dock
Ligands. The Parameters were adjusted so that; Hot Spot Number "200", Docking
Preferences: User Specified & Max Hit To Save "1", Conformation Generation "Best". Finally
we pressed run the LibDock Protocol.
Results and Discussion:
In the following table the ligands are classified from the highest to the lowest
LibDockScore.

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From the results Molecule-8 had the highest LibDockScore, even higher than the lead followed by
Molecule-7 while Molecule-9 came last. After checking Molecule-8 2D interaction diagram with the
binding site, we noticed that the higher score that was obtained by Molecule-8 is mainly due to the
hydrophobic interaction between the newly introduced thiophene ring and Tyr415 even though the
bi-dentate hydrogen bonding interactions with the Tyr-448 and Gln-446 that was a unique feature of
the lead wasn't preserved. We also noticed that the addition of small hydrophobic substituents to the
acyl sulfonamide moiety instead of the phenyl sulfonamide resulted in lower LibDockScores as with
ethyl in Molecule-9, methyl in Molecule-2 and cyclopropyl in Molecule-3.
Fig 3: 2D interaction diagram between Molecule-8 and active site.

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Fig 4: 3D interaction between Molecule-8 and active site.
Conclusion:
As already mentioned the current HCV standard of care is associated with many side effects, that's
why the discovery of small potent inhibitors to HCV replication is a valuable topic for research. HCV
NS5B Polymerase has variety of inhibitors and which opens doors for interested researchers,
especially with the use of computer-aided drug design (CADD) that helped our team test the results
of the modifications applied to the lead and find a new indole acyl sulfonamide derivative with
higher LibDockScore than the lead (Molecule-8).
:References
1. New therapeuticstrategies in HCV: polymerase inhibitors - Liver InternationalISSN 1478-3223.
By: Ludmila Gerber, Tania M. Welzel and Stefan Zeuzem.
2. Novel HCV NS5B polymerase inhibitors: Discovery of indole C2 acyl.
Sulfonamides - Bioorganic & MedicinalChemistry Letters 22 (2012) 713–717.
By: Gopinadhan N. Anilkumar.
http://www.rcsb.org:3. Protein Data Bank (PDB)
http://www.who.int/csr/disease/hepatitis/whocdscsrlyo2003/en/index3.html4. WHO. Hepatitis C: Available at
5. Expert opinion on the treatment of patients with chronic hepatitis C.J Viral Hepatitis 2009; 16: 75–90.
By: Zeuzem S, Berg T, Moeller B, et al.
6. Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus Proc Nat Acad Sci 1996; 96: 13034–9.
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