Developing Anti-Mycobacterial Therapeutics
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Using a specific example discuss a possible target for the development of anti-mycobacterial therapeutics.
It is estimated 1.8 billion people worldwide are infected by tuberculosis (TB)- an infectious disease caused by the etiologic agent Mycobacterium tuberculosis (Mtb) (Fullam et al., 2012). This bacterium is responsible for 2 million deaths each year and remains a continuing threat (Ouellet, Johnston and Montellano, 2011). 70-90% of individuals infected carry latent TB and never develop the disease, on the other hand, 10-30% of individuals infected can develop active TB. Over the years, the threat of TB has increased alarmingly due to the rise of multi-drug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB). The rise of MDR-TB and XDR-TB threatens to overwhelm all currently available drugs (Yam et al., 2009). Hence, the need to develop new anti-mycobacterial therapeutics. Currently, there have been numerous potential targets identified for the development of novel inhibitors. This review focuses on Mtb DNA gyrase as one such possible target (Mdluli and Spigelman, 2006).
Structure and Function of DNA Gyrase
DNA gyrase is an essential tetrameric enzyme involved in DNA synthesis and is understood to be the only type II topoisomerase present in Mtb. The structure of DNA gyrase consists of two subunits called GyrA and GyrB which initially form homodimers, called A2 and B2, and then form a larger heterodimer, called A2B2. The two subunits carry out different functions with the GyrA subunit carrying out cleavage of positive supercoiled DNA, while the GyrB subunit promotes ATP hydrolysis. The GyrA and GyrB subunits are products of the gyrA gene and gyrB gene. The gyrB gene is 34 base-pairs upstream to the gyrA gene and both genes are located close to the origin of replication (Unniraman, Chatterji and Nagaraja, 2002).
Mtb reproduce by binary fission. During DNA replication, DNA helicase binds to the DNA double helix and begins to unwind the parental strands by utilising ATP to break the hydrogen bonds between the base-pairs. Single-stranded binding proteins help to stabilise the unwound DNA strands and prevent them from re-pairing. The point at which the two strands of DNA separate are known as replication fork. DNA polymerase then moves along each strand of DNA behind each replication fork synthesising new DNA nucleotides. As the replication fork expands, positive supercoils begin to accumulate ahead of the replication fork. For DNA replication to continue, the positive supercoils need to be removed. Supercoiling causes the DNA to form a more compact structure. DNA gyrase inserts negative supercoils to Mtb DNA. DNA gyrase binds to a circular, supercoiled DNA molecule and this alleviates one positive supercoils. Gyrase first introduces a double-stranded break in the DNA, then a segment of DNA passes through the break to the opposite side of the gyrase protein. This movement of the DNA requires ATP hydrolysis by gyrase, and introduces a negative supercoil into the DNA molecule. Subsequently, the break in the strands is repaired and gyrase is released from the DNA. Thus, a DNA molecule with one positive supercoil now has one negative supercoil.
The GyrA subunit consists of two domains called the GyrA N-terminal domain (GyrA-NTD) and GyrA C-terminal domain (GyrA-CTD). The GyrA-NTD whereas, the GyrA-CTD stabilises the binding of DNA gyrase to DNA. The residue Tyr-122 of GyrA is the site of covalent attachment to DNA. Similarly, the GyrB subunit consists of two domains called the GyrB N-terminal domain (GyrB-NTD) and GyrB C-terminal domain (GyrB-CTD). The GyrB-NTD contains the ATP binding sites.
DNA gyrase is absent in eukaryotic organisms even though a less homologous enzyme does exist.
Fluoroquinolones (FQs) bind to the enzyme-DNA complex.
By targeting GyrA, the duration of treatment can be shortened making it a validated target.
C-terminal Domain of GyrA
The ability of Mtb DNA gyrase to bind and insert negative supercoils into DNA is mediated by the C-terminal domain of the GyrA subunit (GyrA-CTD). Several highly-conserved residues in GyrA-CTD were selected as potentially participating in DNA binding and bending. The use of site-directed mutagenesis resulted in the identification of four key residues which were R691A, Y577A, R745A and D669A. Substitution of these four residues resulted in a total loss of DNA binding activity by GyrA. This in turn caused a loss in supercoiling activity and relaxation. The ability of Mtb DNA gyrase to carry out its function only occurs when the GyrA subunit is combined with the GyrB subunit. Mutagenesis of R691A, Y577A, R745A and D669A not only results in loss of DNA binding activity of GyrA in the absence of GyrB, but also results in a loss of DNA binding activity in the presence of GyrB. This again led to a loss in loss in supercoiling activity and relaxation. The findings of GyrA-CTD to be essential for Mtb survival strongly promotes the idea of a new potential drug target.
GyrB Subunit of Mtb DNA Gyrase
The emergence of fluoroquinolone-resistant tuberculosis has meant there is a need to develop new classes of drugs targeting Mtb DNA gyrase. A lot of emphasis is often focused on targeting the GyrA subunit and this had led to research in developing novel inhibitors targeting the GyrB subunit (Medapi et al., 2015). The GyrB subunit is an attractive target for the development of anti-mycobacterial therapeutics for several reasons. Firstly, the GyrB subunit is present in a single copy. Secondly, it is an essential gene for the survival of Mtb. Thirdly, there are no alternatives to GyrB present in Mtb which could carry out the same function if it is inhibited because it contains the ATP binding pocket. Fourthly, the various strains of Mtb have a 99.9% homology for GyrB. Fifthly, GyrB exerts the same phenotypic effects on Mtb viability as FQs. Finally, the development of inhibitors targeting GyrB can be effective in shortening the duration of TB treatment and delaying the emergence of drug resistance (Chopra et al., 2012).
The residues involved in ATP binding are found in the GyrB-NTD and are between residues 1-220. Moreover, two further residues, Gln335 and Lys337, found in the GyrB-CTD are also involved in ATP binding. To the date, there are hundreds of potential novel inhibitors which have been identified to inhibit the activity of GyrB. Inhibitors could be design to target the ATP-binding site or the non-ATP-binding site, however, little is known about structure of the non-ATP binding site. Novobiocin is the only approved antibiotic which has shown to inhibit the activity of GyrB. However, novobiocin has been withdrawn from the market because it is extremely toxic and has low permeability. Another drug class of drugs, aminobenzimidazole, are another strong candidate for inhibiting GyrB due to their excellent efficacy against MDR-TB strains (Chaudhari et al., 2016).
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