ReviewChemical classes targeting energy supplying GyrB domain of Mycobacterium tuberculosis
Graphical abstract
Introduction
An infectious communicable disease, TB is caused by Mycobacterium tuberculosis (Mtb), which is a slow growing, acid fast bacillus that is capable of persisting in a semi dormant state of replication thereby withstanding invasion by human host macrophages [1]. Initially, DOTS (Directly Observed Treatment Strategy) was utilized to combat the problem of tuberculosis which involves intensive phase of four drugs (isoniazid, pyrazinamide, rifampicin and ethambutol) for first two months followed by continuation with isoniazid and rifampicin for next four months [2]. However, the development of MDR-TB (multidrug resistant tuberculosis) has significantly reduced the success rate of DOTS therapy [3]. In order to combat the problem of MDR-TB, the treatment with fluoroquinolones (levofloxacin, moxifloxacin etc.) or second line injectables such as amikacin, kanamycin and capreomycin has been suggested [4]. However, the emergence of XDR-TB (extensively drug resistant tuberculosis) has led to an alarming need of a new class of drug [5]. Fluoroquinolone based antibiotics act on GyrA domain of clinically validated target, DNA gyrase. However with the reports of mutation in GyrA domain and thereby failure of fluoroquinolones, different approaches are being explored by researchers to manage TB [6,7]. Out of all, targeting the energy-supplying GyrB domain appears to be a very lucrative and effective option [8]. Till date, there is only one approved antibiotic, novobiocin, discovered in 1955, is an aminocoumarin natural product, which targets gyrase B subunit [9]. Novobiocin was found to be a competitive inhibitor of ATPase activity and resulted in the inhibition of topoisomerase/gyrase [10]. Originally novobiocin was used to treat methicillin-resistant Staphylococcus aureus (MRSA) infections but was withdrawn from the market because it suffered with low efficacy and raised safety concerns [11]. Additionally, GyrB domain has also been previously demonstrated as a bactericidal drug target, but no effective therapeutics are yet developed against this target [12].
Mtb DNA gyrase is a heterotetramer (A2B2) consisting of two domains, gyrase A (encoded by gyrA gene) and gyrase B (encoded by gyrB gene) and is involved in maintaining the topology of DNA [13]. Domain A is involved in the breakage and reunion of the DNA, whereas domain B promote ATP hydrolysis [14]. The biochemical and structural studies suggested a “two-gate mechanism” model for the basic mechanism of action of DNA gyrase. The process of supercoiling via DNA gyrase involves formation of a complex between gyrase tetramer (dimeric gyrase A and dimeric gyrase B) and DNA, leading to the generation of three interfaces which can acquire both an open and a closed conformation. These interfaces include 1) The N-terminal domain of GyrB, which forms the N-gate, 2) The GyrA–GyrB–DNA interface, where the DNA actually interacts and get cleaved, and later forms DNA gate, and 3) The C-terminal area of GyrA, which forms the exit gate. The process of supercoiling begins with the initial interaction of the DNA G segment (region which gets cleaved) with the enzyme, i.e. with GyrA dimer at the N terminus and with GyrB at the TOPRIM domain and further, the DNA strand (G segment) of around 130 base pairs wrap around the enzyme (C-terminal) in a right-handed supercoil. This wrapping of DNA on the C-terminal eventually facilitates the movement of second segment (T segment or transported segment) of the DNA strand towards the N gate, which is situated over the G segment and leading to the formation of strand passage. In the next step, binding of ATP occurs, which results in closure of the N-terminal domain of GyrB i.e., the N-gate, trapping the T segment of the DNA. Following this, the enzyme attack on to the DNA backbone causing the cleavage of G segment, resulting in DNA– phosphotyrosyl bonds 4 bp apart, thus creating a double strand break. This catalytic reaction results in the covalent bond between GyrA and G segment of DNA. The mechanism of action of Mtb DNA gyrase is described in Fig. 1.
In the final step, T segment pass through the open DNA gate which is formed at the GyrA–GyrB–DNA interface upon catalytic cleavage of G segment and the broken G segment, then finally passes through the exit gate. The G and T segments are collinear and passage of the T segment is driven by the binding and hydrolysis of ATP. This strand-passage event leads to the introduction of negative supercoils, usually one gyrase supercoiling cycle introduces two negative supercoils into the DNA molecule at the expense of two ATPs. The hydrolysis of ATP and the release of ADP opens the N gate and resets the enzyme for the next supercoiling cycle [7].
The GyrB active site resembles typical tunnel shaped ATP-binding cleft with a bulky adenine pocket buried deep in the cleft. The lining of the catalytic domain is formed by key amino acid residues such as Asp79, Arg 82, Val 49, Val99, Arg 141 and Glu56. While the outer domain of the cleft is composed of solvent accessible region. Thus, the basic pharmacophore of GyrB inhibitor include a heterocyclic core similar to adenine (such as aminopyrimidine or aminoquinazoline) which could occupy the adenine pocket, coupled with substituted aryl ring which could form hydrophobic interactions with the cleft. Finally a terminal polar substituent such as amines are required to favourably interact with the solvent region [15].
Section snippets
Inhibitors targeting Mycobacterium tuberculosis gyrase B domain
Fluoroquinolones are the most researched anti-tubercular drugs that act through the inhibition of the gyrA domain of DNA gyrase by trapping the gyrase-DNA complex that results in oxidative damage eventually leading to bacterial cell death [13,16,17]. However, the emergence of fluoroquinolone resistance limits their use for long term. Developing gyrB inhibitors that target the ATPase activity and hinder the energy source of the bacteria required for maintaining the topological state of DNA,
Inhibitors targeting Mycobacterium tuberculosis gyrase B domain in clinical trials
DNA gyrase is a topoisomerase consisting of GyrA subunit which is a target of already marketed drugs, fluoroquinolones and GyrB inhibitors having antimycobacterial activity have been reported but none have yet reached the market. Initially, Vertex pharmaceuticals reported VXc-486, aminobenzimidazole analogue as preclinical candidate possessing GyrB inhibitory activity. The compound displayed antimycobacterial potency against drug-sensitive and drug-resistant strains of MTB with MICs from 0.05
Conclusion
TB related mortality in India has resurfaced as a major cause of mortality in past few years. High prevalence of HIV, low effectiveness, persistence of drug resistance and high risk of side effects have limited applicability of previously FDA approved therapies and thus highlighted the immediate need to develop the novel and less toxic drugs that can treat the emerging resistant TB. Second line therapy used for the management of MDR-TB includes fluoroquinolones, which act by binding to the GyrA
Conflicts of interest
Authors declare no conflict of interest.
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