Thursday, June 12, 2014

Tigecycline - A New Weapon Against Antibacterial Resistance

As soon as Alexander Fleming accidentally discovered the medicinal benefit of antibiotics, bacteria began fighting back.  Penicillin appeared as the kind of “magic bullet” Paul Ehrlich envisioned, but the ability of microbes to evolve resistance mechanisms has led to an evolutionary and pharmacological arms race over the last century.  Antibiotics attack susceptible bacteria through specific mechanisms based on the cellular and metabolic differences between prokaryotes and eukaryotes.  One class of antibiotics, the tetracyclines, prevents the synthesis of new proteins by binding to the bacterial ribosomes.  Unfortunately, the bacteria responded by modifying their ribosomes and pumping the antibiotic out of the cell.1  Tigecycline, a glycycline built of the tetracycline scaffold, possesses a unique functional group that allows it to block the bacterial ribosome and simultaneously evade bacterial resistance mechanisms.2  Developed under the name GAR-936 by Wyeth Pharmaceuticals in Philadelphia, tigecycline (brand name: Tygacil) provides a new weapon in the rapidly changing battle of antibacterial resistance.
As a new tetracycline, tigecycline uses the same mechanism of action of its tetracycline ancestors – preventing protein synthesis.  Invading bacteria require new proteins to survive, which are made by ribosomes.  A copy of the genetic information is transcribed into an RNA message that can be translated by a ribosome.  During the translational process, the mRNA feeds between the two subunits presenting the codon that determines the appropriate amino acid to be added to the growing peptide chain.  The transfer RNA (tRNA) with the correct anticodon enters into the A-site with the specified amino acid.  From here, the amino-acyl-tRNA pivots to the P-site for the peptidyl-transfer reaction.2  After attaching, the tRNA slides to the E-site and ejects from the ribosome as a new aminoacyl-tRNA enters to continue the elongation of the peptide chain.
Tetracyclines and tigecycline specifically target the 30S subunit of the bacterial ribosome by binding reversibly to the A-site, and preventing the accommodation of the tRNA.3  Within the 30S subunit, tetracylines interfere with the H34 helical region of the 16S rRNA through hydrogen bonds between the hydrophilic parts of the drug and the phosphate backbone.  The resulting steric hindrance prevents aminoacyl-tRNA from pivoting to the P-site and blocks the elongation of the peptide chain.  While tetracyclines bind to the subunit in one direction, tigecycline blocks the A-site although through a different orientation. This unique binding increases tigecycline’s affinity almost 5-fold giving the antibiotic its bacteriostatic properties and a method of circumventing the resistance proteins in some strains of bacteria.2 
All antibiotic products face the challenge of bacterial resistance.  Formerly susceptible bacterial strains employ two major resistance mechanisms against tetracycline antibiotics.  First, bacteria up-regulate efflux pumps to expel the harmful antibiotics from the cell before it can perform its inhibitory activity.  When Gram-negative bacteria encounter a tetracycline product, efflux genes known in sequence as TetA-E turn on to a decrease the concentration of drug within the cell.  Gram-positive bacteria remove tetracyclines through the same efflux process, but with the genes TetG-L.  These gram-positive bacteria also produce extra small proteins that block the location of tetracycline binding through the genes TetM or TetO.1,4  The protective proteins compete with the tetracycline and reduce the effectiveness of the drug.  Researchers designed tigecycline with the intention of avoiding the Tet efflux pumps by including a bulky side chain that would stay in the cell to continue working, but they also found that it continued to work in the presence TetM proteins
During development in the early 1990’s, tigecycline became the main focus of a new antibiotic class of glycyclines that retained the ribosome blocking mechanisms of tetracycline but avoided the evolved Tet resistance.4  Starting with tetracycline’s four-ring structure, researchers developed new generations of antibiotics by attaching various functional groups to evade efflux and avoid TetM proteins.  For example, doxycycline moves a hydroxyl group from the C-ring to the B-Ring, while minocycline includes a dimethylamine to the 7-position on the D-ring.

Figure 1 Structures of Tetracycline, Doxycycline, Minocycline and Tigecycline Tetracycline, the original member of the family, provides a four-ring backbone which interacts with the 30S subunit of bacterial ribosomes.  The rings are identified A-D beginning with the amide-containing ring on the right side as A.  Notice that tetracycline contains a methyl and hydroxyl group attached to the C-ring.  Doxycycline moves the hydroxyl group from the C-ring to the B-ring, as a second-generation tetracycline.  Minocycline, also a second-generation tetracycline, removes the methyl and hydroxyl from the C-ring and includes a second dimethylamin in the C-7 position of the D-ring.  Tigecycline, a third-generation tetracycline or glycycline, builds on the minocycline backbone and includes the tert-butyl-glycylamido group to the C-9 position of the D-ring.  The long, bulky chain in the 9th position produces the anti-resistance properties of tigecycline.
(Taken from Garrison M, Neumiller J, Setter S. Tigecycline: An investigational glycycline antimicrobial with activity against resistant gram-positive organisms. Clin Thera. 2005;27(1):12-22.)
Minocycline proved to be a strong antibiotic, but eventually succumbed to similar mechanisms of resistance.  Researchers attempted to improve minocycline by including a new functional group to C-9 position of the D-ring.  An early glycycline, dimethylglycylamido-minocycline (DMG-MINO) provided a hopeful avenue, but tigecycline showed the most promise against the more clinically relevant, multidrug resistant pathogens.5,6  Structurally, tigecycline, a chemical descendant of minocycline, adds a tert-butyl-glycylamido group to the D-Ring, which provides the anti-resistance benefits while maintaining antibacterial properties with three main improvements.  First, the molecule became more lipophilic allowing for easier transport into the cell.  Because tetracyclines act on ribosomes, they must reach the cytosol to perform their inhibitory mechanisms.  The additional side chain increases tigecycline’s volume of distribution to 7-9 L/kg, showing that more of the drug resides tissues than in plasma.3  Secondly, the extra-long alkyl group creates enough steric hindrance to prevent efflux from the cell.  Where tetracyclines would be removed, the bulky tigecycline continues to work.  Thirdly, tigecycline shows five times more affinity to the 30S ribosome compared to tetracycline.2  The 9-tert-butyl-glycylamido group shifts the orientation of tigecycline within the 30S ribosome and generates more stable hydrogen bond stacking, shielding the molecule from TetM protective proteins.7,8  The important glycylamido addition to the minocycline backbone provides the biochemical opportunity for the pharmacological improvements of tigecycline over minocycline and other tetracyclines.  Unfortunately, some anaerobic bacteria have displayed resistance to tigecycline through the upregulation of a different set of efflux pumps.  Enterobacter and Acinetobacter increase the number of RND pumps on their membranes, which are not blocked by the bulky side chain and remove tigecycline from the cell.2 
Interest in tigecycline led to worldwide testing process as many countries sponsored research into the effectiveness of tigecycline through the TEST Program (Tigecycline Evaluation and Surveillance Trial).  Tigecycline showed similar or improved results to treatment by minocycline (the closest relative), to a vancomycin-azetrenam combination, and to an imipenem-cilastatin combination.  Specifically, tigecycline provides hope to treat the various drug-resistant strains like vancomycin-resistant Enterococci (VRE), methicillin-resistant Staphalococcus aureus (MRSA), penicillin-resistant Streptococcus pneumonia, and beta-lactamase producing E. coli.  However, the TEST program has shown regional variations in the activities of tigecycline against resistant bacteria and a growing resistance to tigecycline itself.  The FDA approved tigecycline for the treatment of complicated intra-abdominal infections, community-acquired bacterial pneumonia, and complicated skin infections against a wide range of species.3,4,9 The FDA Black Box Warning suggests a 0.6% increase in morality risk and advises the use of tigecycline only when alternatives are not available.9
Unsusceptible to the normal methods of tetracycline resistance, tigecycline continues to inhibit the 30S ribosomal subunit despite Tet efflux transporters or TetM proteins that protect the accommodation site of the ribosome. Tigecycline demonstrates the creative ability biochemistry provides to overcome bacterial resistance by building on the structure of minocycline. Tigecycline’s bulky side chain generates the improved characteristics of tigecycline.  At this point, tigecycline remains the only FDA approved glycycline and offers a potent weapon against antibacterial resistance.

References
1. Projan S. Preclinical pharmacology of GAR-936, a novel glycycline antibacterial agent. Pharmacotherapy. 2000;20(9):219-223.
2.  Seputiene V, Povilonis J, Armalyte J, Suziedelis K, Pavilonis A, Suziedeliene E. Tigecycline - how powerful is it in the fight against antibiotic-resistant bacteria? Medicina (Kaunas). 2010;46(4):240-248.
3.     Doan T, Fung H, Mehta D, Riska P. Tigecycline: A glycylcyline antimicrobial agent. Clin Thera. 2006;28(8):1079-1106.
4.     Peterson L. A review of tigecycline - the first glycycline. International Journal of Antimicrobial Agents. 2008;32(S4):S215-S222.
5.     Garrison M, Neumiller J, Setter S. Tigecycline: An investigational glycycline antimicrobial with activity against resistant gram-positive organisms. Clin Thera. 2005;27(1):12-22.
6.     Loh E, Ellis-Grosse E, Petersen P, Sum P, Projan S. Tigecycline: A case study. Expert Opin Drug Discov. 2007;2(3):403-418. 
7.     Olson M, Ruzin A, Feyfant E, Rush T, O'Connell J, Bradford P. Functional, biophysical, and structural bases for antibacterial activity of tigecycline. Antimicrob Agents Chemother. 2006;50(6):2156-2166.
8.     Jenner L, Starosta A, Terry D, et al. Sturctural basis for potent inhibitory activity of the antibiotic tigecycline during protein synthesis. PNAS. 2013;110(10):3812-3816.

9.     Tygacil  [package insert]. Philadelphia, PA: Wyeth Pharmaceuticals Inc; September 2013.

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