It's all about Zanamivir (Relenza)

Introduction

 

 

Figure 1: Example of Zanamivir (Relenza) 

 

Relenza is a medication that contains zanamivir as an active ingredient. Zanamivir is an antiviral drug which is being used in the treatment and prevention of influenza A and B (Figure 1). It is a selective inhibitor of viral neuraminidase enzyme which is necessary for the release of newly formed viruses from the surface of infected host cells. [1]

Zanamivir (a white to off-white powder) is an orally inhaled drug. It is administered through oral inhalation by using Diskhaler as it will provide direct access to the respiratory system where the influenza virus usually resides and replicates. [2]

 

Details of Zanamivir [2]

 

Chemical Name	5-(acetylamino)-4-[(aminoiminomethyl)-amino]-2,6-anhydro-3,4,5-trideoxy-D-glycero-D-galacto-non-2-en onic acid.
Molecular Formula	C12H20N4O7
Structural Formula
Molecular Weight	332.31 g/mol
Melting Point	>2450° C (lit.)
Solubility	Soluble in water

 

Mechanism of Action

The proposed mechanism of action of zanamivir is via inhibition of influenza virus neuraminidase with the possibility of alteration of virus particle aggregation and release. By binding and inhibiting the neuraminidase protein, the drug renders the influenza virus unable to escape its host cell and infect others. [1]

 

Figure 2: Mechanism of Action of Zanamivir

Dosage Details [3]

Treatment of Influenza A and B

Adult: 10 mg (2 inhalations) twice a day for 5 days, administered as soon as possible (within 48 hour) after the onset of symptoms.

 

Child: >7 yr: 10 mg (2 inhalations) twice a day for 5 days, administered as soon as possible (within 48 hr) after the onset of symptoms.

Prophylaxis Influenza A and B

Adult:

For community outbreak: 10 mg (2 inhalations) once daily for 28 days, to be started within 5 days of outbreak.

For prophylaxis in household setting: 10 mg (2 inhalations) once daily for 10 days, to be started within 36 hours of exposure.

Child:

For community outbreak: >12 yr: 10 mg (2 inhalations) once daily for 28 days, started within 5 days of outbreak. 

For prophylaxis in household setting: ≥5 yr: 10 mg (2 inhalations) once daily for 10 days, to be started within 36 hours of exposure.

History [4]

Figure 3: Picture of Mark von Itzstein [4]

Zanamivir was discovered in 1989 by scientists led by Mark von Itzstein at the Victorian College of Pharmacy, Monash University in collaboration with the Commonwealth Scientific and Industrial Research Organisation, CSIRO. [5] Far back then, influenza virus's enzyme was first discovered by George Hirst that they can destroy receptors on red blood cells in humans. Later, Robin Valentine, W. Graeme Laver, Norbert Bischofberger and Robert G. Webster interpreted the result from discovery that, there were hemagglutinin and neuraminidase, activities of the virus resided in two different spikes on the surface of the influenza virus.

Each type of flu neuraminidase contained an amino acid sequence that differed from each other apart from one small sequence. When the neuraminidase polypeptide folded up to form the active enzyme, the conserved amino acids will form an active catalytic site of the neuraminidase enzyme. Thus, 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) which fit into the active site and inhibit the neuraminidase activity, but it was not enough to treat flu in humans.  Thus, Mark von Itzstein and colleagues improvise by replacing the OH at the 4 position of sialic acid with a guanidino group leading to strong binding of the drug to the neuraminidase enzyme. They named it as GG167 or Zanamivir and until now. [6]

Figure 4: OH in position 4 is replaced by Guanidino group in Relenza [7]

Figure 5: Chart above shows the flow of the discovery of Zanamivir

 

Introduction to GRID

Figure 6: GRID Energy Function 

GRID is an infrastructure for people to share computing and storage resources. This GRID system provided computing capability beyond clusters by combining clusters that are geographically dispersed into a collective environment. This GRID force field has been tuned to fit experimental observations in crystal structures. [8]

One of the applications of GRID is firstly, GRID software is able to detect the binding sites on macromolecules as well as estimate the selectivity between the macromolecules and ligands with disparate functional groups. In addition, drug design GRID software is capable of identifying the hydration sites and designing to either include or exclude the interfacial water molecules. Next, GRID software is used to dock the molecules into targeted macromolecules binding sites. Furthermore, GRID software is used to compute the molecular interaction in order to study the Quantitative structure activity relationship (QSAR) and predict the ADME in vivo as well [9].

            In drug discovery, GRID offers high-speed computing and is able to manage a huge database of chemical compounds. Moreover, it studies the possible variant tangents, in turn helps the medicinal chemists to respond to major instant threats in a short time.

 

 

 Figure 7: Interaction of α-N-acetylneuraminic acid with influenza virus sialidase [10]

 

Figure 8: The use of GREATER to run GRID [11]

Figure 9: The use of GREATER to run GRID [11]

Figure 10: The use of GREATER to run GRID [11]

 

Discovery of Zanamivir using GRID 

New inhibitors were discovered by utilising GRID molecular modelling software to evaluate likely binding regions within a model with active site. GRID software was used to design the analogue DANA, that is able to bind with the amino acid residues of the catalytic site, causing inhibition effect of enzyme. Basically GRID set-up of a series of grid points within the active site. The probe will place at each point to measure interaction s between the probe and amino acid residues. Different atomic probes were used to represent various functional groups. Probes include the oxygen of carboxylate group, the carbon of a methyl group as well as multi-atom probes. Each atom of the probe was placed at each grid point and in turn was marked by the probes in the grid. Thus, energy will be calculated for all the atoms within the probe in the energy at each grid point. The probe also keeps rotated and turning to search for the orientation for the perfect fit of hydrogen-bonding interaction. [12]

Figure 11: Binding interaction of aminium and guanidium moieties at C-4 with active site of neuraminidase [12]

The main objective the discovery that the binding region normally occupied by the 4-OH of sialic acid could also interact with an aminium or guanidinium ion resulting sialic acid analogues having an amino or guanidyl group at C-4, instead of a hydroxyl group, were modelled in the active site to study the binding interactions and to check whether there was room for the groups to fit. [8] 4-Amino-Neu5Ac2en contains the aminium group and was found to be more potent than Neu5Ac2en. Thus, confirming the binding pattern predicted by the molecular modelling due to a crystal structure of the inhibitor bound to the enzyme.

Figure 12: X-ray crystal structure of neuraminidase tetramer [13]

Molecular modelling suggested that the larger guanidinium group is capable of even greater bonding interactions, and favorable van der Waals interaction. The relevant Zanamivir structure was found to be more potent inhibitor having a 100-fold increase in activity. X-ray crystallographic studies of the enzyme-inhibitor complex proved the expected binding interactions. Furthermore, the larger guanidinium group was found to expel a water molecule from this binding pocket which is thought to contribute a beneficial entropic effect. Zanamivir is a slow-binding inhibitor with a high binding affinity to influenza A NA. Unfortunately, the polar nature of the molecule means it has poor oral bioavailability (<5%), and it is administered via inhalation. [12]

 

a.  Neu5Ac2en (PDB code 1F8B)  b. Relenza (PDB code 1NNC)

Figure 13(a) and 13(b): chemical structures of Neu5Ac2en and Relenza respectively [13]

Figure 14: Hydrogen bonding interaction between sialic acid and active site of neuraminidase [12]

Relenza was developed through the use of the program GRID which shows a potentially strong binding site for an NH₃⁺ group with a calculated binding energy of −16 kcal/mol in the vicinity of the position that normally occupied by the O4 hydroxyl of sialic acid. Using Neu5Ac2en as the scaffold, substitution of O4 with an amino group gained two orders of binding over Neu5Ac2en, meanwhile substitution by a guanidino group gained five orders of binding over Neu5Ac2en. [13]

In complexes of Relenza with both influenza A and influenza B virus NA, the guanidino group interacts most efficiently with Asp151 and Glu227. Glu119 is also close enough to make a charge-to-charge interaction, although Glu119 may be neutral in the case of 4-amino-Neu5Ac2en binding. Relenza is a successful inhibitor of influenza A and B NA, but due to its highly polar nature has necessitated administration as a powder, it requires an inhaler with all the inherent problems of use. Replacement of the glycerol group of Relenza by a series of hydrophobic dihydropyrancaboxamides has provided inhibitors with similar binding affinity to Relenza for influenza A NA, but with only micromolar inhibition of influenza B NA. [13]

 

Conclusion

 

Zanamivir can be discovered by using GRID program. This program is a computational procedure for determining energetically favorable binding sites on molecules of known structure. It is used to study individual molecules such as drugs and macromolecules. This technique is also used to probe the active site of enzymes and used to attempt to design derivatives of DANA that would bind tightly to the amino acid residues of the catalytic site. As a result, it would be potent and specific inhibitors of the enzyme. [14]

This antiviral drug is discovered using the GRID program by using Neu5Ac2en as the scaffold. The oxygen at carbon-4 is substituted with an amino group and produces two orders of binding over Neu5Ac2en. The substitution by the guanidino group with oxygen at carbon-4 gained five orders of binding over Neu5Ac2en. The complexes between Zanamivir and both influenza A and B of virus NA, the guanidino group will interact almost ideally with Asp151 and Glu227. [15]

 

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