Exploitation of a novel allosteric binding region in DNA gyrase and its implications for antibacterial drug discovery

By 2050, it is believed that approximately 10 million deaths will occur globally every year due to antimicrobial resistance [1]. This will cost the world economy around USD 100 trillion. There is no doubt that the continual evolution of antibiotic resistance is an existential threat to human beings. The discovery of penicillin in 1928 by Alexander Fleming yielded the start of the antibiotic era, revolutionizing the treatment of bacterial infections and preventing an inconceivable number of fatalities, notably during World War II. Antibiotics have since become a staple in modern medical procedures [2]. However, following what is known as the ‘golden period’ of antimicrobial drug discovery in the mid to late 20th century, a decline in new antibiotic approval by the US FDA, alongside a rise in antimicrobial resistance, has led to a staggering increase in untreatable bacterial infections [3]. This can also be partially attributed to the unfortunate withdrawal of investment from ‘Big Pharma’ companies in antibiotic drug discovery programmes. The total cost of development for an anti-infective drug is estimated at USD 500–800 million [4]. These hurdles illustrate why companies are hesitant to financially commit to such projects, where there are often more lucrative and viable options [3,5].

Bacterial type II topoisomerase enzymes, such as DNA gyrase and topoisomerase IV, are essential proteins that modulate the topology of DNA in bacteria during DNA transcription, replication and other DNA-associated processes [6,7]. The primary function of DNA gyrase is to introduce ‘negative supercoils’ into bacterial DNA through an ATP-dependent mechanism. Topoisomerase IV serves a different function, primarily eliminating entanglements that occur naturally in DNA during DNA replication.

Both enzymes are comprised of two proteins, coded for by the gyrA and gyrB genes for DNA gyrase and the parC and parE genes for topoisomerase IV [8,9]. These two proteins are composed of four subunits, forming heterotetrameric protein complexes: A₂B₂ for DNA gyrase and C₂E₂ for topoisomerase IV. They are well-validated targets for antibiotics; the fluoroquinolone antibiotics (FQs) being one of the most important clinically used classes of antibiotics available. This is in part because they are renowned for possessing a ‘dual-targeting’ mechanism whereby they can inhibit DNA gyrase and topoisomerase IV simultaneously, through the stabilization of the DNA cleavage complex.

The prospect of achieving dual-targeting is an immensely attractive one for the discovery of novel antimicrobial agents, as the inhibition of two important enzymes simultaneously presents bacteria with a more difficult route to evolving resistance [10,11]. Prominently, DNA gyrase and topoisomerase IV possess a high degree of structural and sequence similarity, but limited sequence similarity to human topoisomerase II, allowing for the design of inhibitors that are selective for bacterial topoisomerase enzymes over the human enzyme [12].

The well-established regions on bacterial type II topoisomerases are associated with their DNA- and ATP-binding sites [8,13]. FQs bind to the gyrase–DNA complex and ‘trap’ the bound DNA within gyrase by forming key interactions within the DNA binding site through a water–metal ion bridge [14]. Despite the success of dual-targeting inhibitors and the relatively slow rate at which bacterial resistance to these drugs has evolved, resistance is growing within the clinic, primarily through the development of point mutations [15]; for example, amino acid mutations of Ser83 to Phe and Leu, and Asp87 to Asn, in GyrA of DNA gyrase, and Glu84 to Lys modification in ParC of topoisomerase IV [8].

A promising approach to combating FQ resistance may lie in targeting allosteric binding sites present in DNA gyrase and topoisomerase IV. Allosteric sites are regions that are remote to the active site within a protein; that is, the site responsible for carrying out the protein’s catalytic function. In the case of topoisomerases, this is the DNA cleavage site.

Chan et al. report one such example within a Staphylococcus aureus DNA gyrase structure containing a thiophene carboxamide-based inhibitor [16,17] Upon examination of the co-crystal structure, it was discovered that the inhibitor was bound within a pocket between the GyrA and GyrB subunits. This region is remote from the FQ binding site, and as such is an allosteric inhibition site. The inhibitor was observed to be very potent toward Escherichia coli DNA gyrase (IC₅₀: 0.30 μM) and importantly is not cross-resistant with the FQs. However, it lacked any form of significant potency against topoisomerase IV (IC₅₀: >540 μM). While potent against DNA gyrase, the dual-targeting mechanism associated with the FQs was clearly not observed for this compound. Unfortunately, the development of this inhibitor was later terminated due to in vivo toxicity [16].

A later publication by the same team described examples of fused 5–6-heterocyclic inhibitors that incorporated or entirely replaced the thiophene moiety [17]. Their most potent inhibitor was determined against E. coli DNA gyrase (IC₅₀: 0.16 μM); this compound was fourfold less active than the parent thiophene inhibitor, but notably also displayed some mild E. coli topoisomerase IV inhibition (IC₅₀: ∼90 μM; inhibition of kinetoplast DNA decatenation). It was also observed to be weakly active against human topoisomerase IIα (IC₅₀: ∼210 μM) and was found to possess cytotoxicity and cardiac ion channel inhibition (hERG and Na_V1.5), which resulted in the termination of these compounds. These data, however, offer significant promise for the future development of dual allosteric DNA gyrase/topoisomerase IV inhibitors. These compounds were developed further by Thalji et al. [17], resulting in a novel inhibitor which was equipotent against DNA gyrase and more potent than the parent compound against topoisomerase IV. It remained weakly inactive against human topoisomerase II, showing the continual opportunity for selective targeting.

Given that there are two x-ray crystal structures (Protein Data Bank: 5NPP and 6QX1) of allosteric inhibitors bound to DNA gyrase, there is great potential for future structure-based molecular design strategies at this site. Given the challenges noted above in antibacterial drug discovery and antimicrobial resistance, there may even be an obligation for the scientific community to explore this site. Notably, with both the Chan et al. and Thalji et al. inhibitors, targeting residues Arg630 and Glu634 appears to be key to the inhibition of DNA gyrase. Currently, there is insufficient structural evidence for the presence of an allosteric site on topoisomerase IV. New structural data could validate the existence of a similar allosteric site present within topoisomerase IV and aid the development of allosteric inhibitors targeting both proteins. In this pursuit, the authors are using advanced computational modeling techniques to design and synthesize dual-targeting inhibitors tailored to this allosteric site.