Rimantadine: A Patient Case Related to Medicinal Chemistry and Drug Design
This Jmol Exploration was created using the Jmol Exploration Webpage Creator from the MSOE Center for BioMolecular Modeling.
The influenza virus affects thousands of people in the United States annually. In the 2012-2013 flu season there were 73,130 reported cases of influenza. The influenza is an acute viral infection that is primarily located in the human respiratory tract. Common symptoms are inflammation of the nasal mucosa, the pharynx, and conjunctiva; also a severe headache and generalized myalgia are associated(1). There are 4 FDA approved antiviral medications for the treatment of influenza virus in the United States: amantadine, rimantadine, zanamivir, and oseltamivir. Rimantadine is a member of the Adamantane class, and is an antiviral used to treat influenza A strains(2).
A 46 year old male presents to his regular Costco pharmacy with a new prescription for rimantadine 100mg daily for the treatment of influenza stain A. The pharmacist noticed that the patient received his annual flu vaccination yesterday from the pharmacy. The patient received the live intranasal FLuMist(R) upon request. He is under the age of 49, thus was able to get the FLuMist(R). The pharmacist then discussed with the patient how it is not recommended to use rimantadine since he received the live flu vaccination this year. There is an interaction between the rimantadine and FLuMist(R) that would interfere with how the FLuMist (R) would replicate. Also there is a growing resistance the amantadine class drugs and the influenza strain A. The pharmacist then proceeded to call the prescribing physician to change the medication to treat the patient's flu. (**Note This is a fictional case for educational purposes and did NOT actually take place in a pharmacy.)
Influenza A is one of the infective strains of the common flu virus. Vaccinations have been the most effective form of preventing influenza infection(7). However, various patient specific characteristics can influence how effective the vaccine will be for the patient. The age of the patient as well as immune status can influence a vaccine's efficacy, as well as which strain of influenza the patient was infected with compared to the influenza strain the patient was vaccinated with. This led researchers to develop a drug that worked on the virus itself, indifferent to age or immune status of the patient.
Rimantadine, brand name Flumadine, was approved by the FDA on September 17th of 1993. Flumadine is available by prescription only, in 100mg tablet strength(6).
A patient should avoid rimantadine 48 hours prior to and 2 weeks after vaccination with the live attenuated intranasal spray influenza vaccine(5). If administered during this time period, rimantadine may inactivate the live vaccine, decreasing the effectiveness of the vaccine.
The influenza virus contains a pH-gated proton channel in the viral envelope formed by the M2 membrane protein. The proton channel lowers the pH of the viral interior, which is required for unpacking of the genome, and therefore needed for replication. Rimantadine inhibits the movement of protons through the M2 channel, and thereby inhibits viral replication(6). The M2 protein is also required to transport protons from the transgolgi membrane to the host cell cytoplasm to stabilize the hemagglutinin during its transport to the host cell membrane(11). Life cycle of the virus is seen in figure 2.
Rimantadine binds to the exterior of the M2 receptor(9). The nitrogen of rimantadine is in contact with the polar side chain of Asp44 and Arg45, and in contact with the indole amine of Trp41. Ile42 contributes to the hydrophobic walls of the binding pocket that interact with the adamantane group, which consists of four connected cyclohexane rings(6).
Rimantadine is no longer recommended for use. Recommendations from the CDC in January of 2013 state that no circulating viruses existed that are susceptible to rimantadine during 2012-2013, and is therefore no longer recommended for use(3). Resistance is developed to rimantadine through 5 amino acid substitutions common sites at position Leu26, Val27, Ala30, Ser31, or Gly34 of the transmembrane domain portion of the M2 receptor(10). At these positions, genetic mutations cause reduced binding of rimantadine or the pore diameter to enlarge. Both of these mechanisms do not allow rimantadine to work on the M2 pore(8).
Rimantadine is an anti-viral agent that is a derivative of its sister compound Amantadine. Both molecules contain two distinct portions of the molecule; a polar head group and a hydrophobic region (or tail). The main portion of the drug is made up by an alicyclic hydrophobic region called adamantane. The polar head is comprised of an amine group that is connected to the adamantane. Rimantadine is unique because it also contains a methyl group attached to the amine. The addition of the methyl group makes Rimantadine more effective against Influenza A, which is the most virulent human strain of the three influenza viruses, compared to its sister compound Amantadine(6).
Rimantadine Hydrogen BondingWhen rimantadine is bound it stabilizes the closed formation of the channel, thus prohibiting inward flow of protons. If protons were allowed to flow into the virion the pH would decrease. This would facilitate the unpacking of the viruses' genomic sequence and increase viral replication(6). In the closed conformation the M2 protein forms what is called a 'tryptophan gate', which is characterized by an interaction between a side chain tryptophan and an aspartic acid. Rimantadine's main mechanism of action is to inhibit the M2 proton channel (decrease proton flow); which is made up of 4 different chains. Overall, there are four individual rimantadine molecules interacting with one M2 proton channel; one individual rimantadine will interact between 2 chains (Figure 3). Both portions of the individual drug molecule will have specific interactions with amino acids from both chains. The adamantane portion of the molecule is undergoing Van der Waal interactions with Leu40 on one chain and Ile42 from another chain (Figure 4). The polar amine group is undergoing polar interactions with Asp44 from one chain and Trp41 and Arg45 from another chain (Figure 4). The methyl group and basic amine within the head group and are critical in rimantadine's mechanism of action. We can see a hydrogen bond interaction occurring between the amine from rimantadine and Asp44 (Figure 5). Rimantadine interacts with a unique polar patch within the binding pocket in an overall hydrophobic pocket. With the above interactions we can see that the binding of four rimantadine molecules causes a blockage in the M2 proton channel (Figure 6).
Rimantadine inhibits the live influenza A virus(13). When the influenza virus is taken into an endosome it is acidified by the acidic environment of the endosome(6). The acidification of the viral interior facilitates the dissociation of the matrix protein from viral nucleoproteins, a required process for unpacking the viral genome(6). Acidification of the virus is due to the transport of protons across the viral envelope. Rimantadine inhibits viral replication by attaching to the viral M2 channel and inhibiting proton transport into the virus. The lack of an acidic environment inside the virus stops it from uncoating(6). Rimantadine does not directly kill the virus but a person treated with rimantadine will have an easier time fighting off the virus.
Rimantadine is an analog of the drug amantadine. Its structure consists of an adamantane group, which is four interconnected cyclohexane rings with an alpha methylamine group(6,12). The main site of action for rimantadine is the M2 channel protein located on the viral lipid envelope6. The M2 channel is a homotetrameric protein consisting of 97 residues per subunit, with each subunit divided into extracellular N-terminal, transmembrane, and intracellular C-terminal domains(12). In an acidic environment, the M2 channel becomes active and allows the passage of protons and water into the viral interior(6,12). The M2 channel contains two very important residues in its pore, His37, and Trp41, see figure 7. His37 serves as the pH sensor, and facilitates proton conductance, while Trp41 is the 'gate'(6). Trp41 indole rings prevent the passage of water or ions through the protein due to its tight van der Waals interactions(6). Acidic environments cause His37 to become protonated. This protonation causes conformational rearrangement and destabilization of the channel. Electrostatic repulsion from the protonated His37 residues forces the four subunits apart, breaking the weak van der Waals interactions between Trp41 and Asp44(6,14), see Figure 7. The breakage allows protons and water molecules to pass through the channel(12).
There is some controversy about the exact mechanism of action of rimantadine but there are two general theories as to how it works: the blocking mechanism and the allosteric mechanism(12). The conventional theory, or blocking mechanism, is that rimantadine behaves as a blocker of the M2 channel by being placed between Val 27 and Ser31 with hydrogen bonding of the charged amine group to Ser31(12,14). The blocking of the pore by rimantadine prevents the passage of ions and water by creating a prohibitive electrostatic potential with a large amount of positive charge(14).
New evidence suggests that the allosteric mechanism is a better representation of the binding of rimantadine to the M2 channel(6,12). This mechanism states that rimantadine inhibits the M2 channel by binding directly to four sites within the pore(6). The amine head group of rimantadine forms hydrogen bonds with Asp44 and Arg45 side chains, and the indole amine of Trp41(6). The adamantane group interacts with the hydrophobic walls of the channel, specifically Ile42, Leu40, and Leu43 residues(3). Rimantadine uniquely binds to the channel by binding onto the polar patch in an otherwise hydrophobic channel(6). This binding causes conformational changes in the channel that stabilizes the closed conformation, even in an acidic environment, effectively stopping the influx of protons into the virus. This mechanism can help explain why rimantadine interferes with the live attenuated virus vaccine. More research into the crystal structure of rimantadine and the M2 protein will lead to a better understanding of the true mechanism of action and what targets can be used for drug therapy.
Rimantadine can only prevent viral replication in susceptible viral strains, however. The M2 channel can develop resistance to rimantadine and other adamantane antivirals. There are several primary mutations that confer drug resistance: L26F, V27A, A30T, S31N, G34E, and L38F(6). Mutations are named with the wild-type amino acid, the position of the mutation, and the mutant amino acid. For example, in the L26F mutant the wild-type leucine located at position 26 has been changed to a phenylalanine. The mutations at positions 27, 30, and 34 are within the pore, while those at positions 26, 31, and 38 are on the helix-helix interface. Interestingly, none of the mutations are at the external drug binding site. Schnell and Chou propose that all these mutations destabilize the helical packing of the tetramer in various ways, making the pore easier to open, even with a bound inhibitor such as rimantadine(6). The mutation at residue 27 changes valine to a smaller alanine, resulting in an enlarged pore. All other mutations involve a change from smaller amino acid to a larger amino acid which destabilizes the helical packing. This both makes the pore inherently easier to open as well as disrupts the binding site of rimantadine, which is located at the interface between helices.
However, there is another binding site within the pore composed of the amino acid residues at positions 27, 30, 31, and 34(14). Mutations at these same four residues confer resistance to rimantadine. Mutations at positions 30 and 31 prevent rimantadine binding within the pore and involve the substitution of a larger, more polar amino acid for the wild type amino acid (alanine becomes threonine at position 30, and serine becomes asparagine at position 31). The mechanism for this type of resistance seems clear; the larger amino acids block the channel so rimantadine cannot enter the binding site. Mutations at position 27 tend to retain drug binding and the amino acid is replaced by a smaller one(14). A study by Leonov, et al showed that the wild-type channel with drug bound showed a localized positive electrostatic potential, whereas the V27 mutant channel did not. The researchers concluded that the larger valine of the wild-type channel restricts the movement of the positive amine on rimantadine creating a localized positive charge that prevents the passage of protons by electrostatic repulsion. In the mutant channel, the smaller methyl group on alanine allows greater movement of the amine group. Thus there is no localized positive charge to repel the passage of protons through the channel(14). Further research needs to be done to clarify whether the main mechanism of viral resistance is through the intra-pore or allosteric binding mechanism. Knowledge of the resistance mechanism could lead to development of new, effective adamantine drugs, possibly with increased efficacy and reduced incidence of viral resistance.
As of the 2007-2008 flu season, adamantane drugs were no longer recommended for influenza A treatment or prophylaxis due to 99% resistance of the influenza A (H3N2) to adamantane drugs(15). This recommendation has continued to include the current flu season(3). As such, the patient would not have seen any benefit from taking rimantadine. For this patient, taking rimantadine would not only have been ineffective at mitigating his current flu symptoms, it would have put him at risk of additional influenza infections for the remainder of the flu season. Because the attenuated virus could be affected as well, rimantadine would bind the M2 channel of the weakened virus and prevent its replication and spread. The low viral load would induce a much smaller immune response, leading to insufficient production of protective antibodies to preclude subsequent influenza infections. Because the doctor prescribed oseltamivir as an alternative agent, the pharmacist counseled the patient to receive a repeat dose of the influenza vaccine after completing his course of antiviral therapy. All influenza antiviral medications carry the same potential interaction with the live attenuated vaccine.
1. Influenza, Human. NCBI MeSH database [database online]. Bethesda MD: National Center for Biotechnology Information, U.S. National Library of Medicine; 2013. Available at: http://www.ncbi.nlm.nih.gov/ mesh/68007251. Accessed November 27, 2013.
2. Centers for Disease Control and Prevention, National Center for Immunization and Respiratory Diseases. Antiviral Drugs for Seasonal Influenza: Additional Links and Resources. Seasonal Influenza (Flu). October 1, 2013. Available at http://www.cdc.gov/flu/professionals/antivirals/links.htm. Accessed November 27, 2013.
3. Influenza activity--United States, 2012-13 season and composition of the 2013-14 influenza vaccine. MMWR Morb Mortal Wkly Rep. 2013;62(23):473-9.
4. Flumadine drug details. Drugs@FDA [database online]. Silver Spring, MD: U.S. Food and Drug Administration; 2013. http://www.accessdata.fda.gov/scripts/cder/ drugsatfda/. Accessed November 15, 2013.
5. Rimantadine Monograph. Lexi-Comp Online, Lexi-Drugs Online, Hudson, Ohio: Wolters Kluwer Health; updated November 6, 2013. online.lexi.com/?. Accessed November 15, 2013.
6. Schnell JR, Chou JJ. Structure and mechanism of the M2 proton channel of influenza A virus. Nature. 2008;451(7178):591-5.
7. Simeonsson K, Moore Z. Prevention and control of influenza: no easy task. N C Med J. 2013;74(5):425-33.
8. Ison MG. Antivirals and resistance: influenza virus. Curr Opin Virol. 2011;1(6):563-73.
9. Chuang GY, Kozakov D, Brenke R, Beglov D, Guarnieri F, Vajda S. Binding hot spots and amantadine orientation in the influenza a virus M2 proton channel. Biophys J. 2009;97(10):2846-53.
10. Jing X, Ma C, Ohigashi Y, et al. Functional studies indicate amantadine binds to the pore of the influenza A virus M2 proton-selective ion channel. Proc Natl Acad Sci USA. 2008;105(31):10967-72.
11. Intharathep P, Rungrotmongkol T, Decha P, et al. Evaluating how rimantadines control the proton gating of the influenza A M2-proton port via allosteric binding outside of the M2-channel: MD simulations. J Enzyme Inhib Med Chem. 2011;26(2):162-8.
12. Intharathep P, Laohpongspaisan C, Rungrotmongkol T, et al. How Amantadine and Rimantadine Inhibit Proton Transport in the M2 Protein Channel. Journal of Molecular Graphics and Modeling. 2008; 27:342-348.
13. Influenza virus vaccine. Micromedex Healthcare Series. DRUGDEX System. Greenwood Village, CO: Truven Health Analytics, 2013. http://www.thomsonhc.com/. Accessed December 1, 2013.
14. Leonov H, Astrahan P, Krugliak M, Arkin IT. How do aminoadamantanes block the influenza M2 channel, and how does resistance develop?. J Am Chem Soc. 2011;133(25):9903-11.
15. Centers for Disease Control and Prevention (CDC). Update: influenza activity--United States, September 30, 2007-February 9, 2008. MMWR Morb Mortal Wkly Rep. 2008;57(7):179-83. Accessed Dec. 1, 2013.
16. CID: 5071
Bolton E, Wang Y, Thiessen PA, Bryant SH. PubChem: Rimantadine - Compound Summary: Integrated Platform of Small Molecules and Biological Activities. American Chemical Society, Washington, DC, Apr 2008.
17. Das K, Aramini JM, Ma LC, Krug RM, Arnold E. Structures of influenza A proteins and insights into antiviral drug targets. Nat Struct Mol Biol. 2010;17(5):530-8.
18. PDB ID: 2RLF
Proton Channel M2 from Influenza A in complex with inhibitor rimantadine. Nature. 2008; 451: 591-595