Inhibitory Compounds Targeting Plasmodium falciparum Gyrase B

Abstract

Malaria persists as a major health problem due to the spread of drug resistance and the lack of effective vaccines. DNA gyrase is a well-validated and extremely effective therapeutic target in bacteria, and it is also known to be present in the apicoplast of malarial species including Plasmodium falciparum . This raises the possibility that it could be a useful target for novel antimalarials. To date, characterisation and screening of this gyrase has been hampered by difficulties in cloning and purification of the GyrA subunit, which is necessary together with GyrB for reconstitution of the holoenzyme. To overcome this, we employed a library of compounds with specificity for P. falciparum GyrB and assessed them in activity tests utilising P. falciparum GyrB together with E. coli GyrA to reconstitute a functional hybrid enzyme. Two inhibitory compounds were identified that preferentially inhibited the supercoiling activity of the hybrid enzyme over the E. coli enzyme. Of these, purpurogallin (PPG) was found to disrupt DNA binding to the hybrid gyrase complex and thus reduce the DNA-induced ATP hydrolysis of the enzyme. Binding studies indicated that PPG showed higher affinity binding to P. falciparum GyrB compared to the E. coli protein. We suggest that PPG achieves its inhibitory effect on gyrase through interaction with P. falciparum GyrB leading to disruption of DNA binding and, consequently reduction of DNA-induced ATPase activity. The compound also showed an inhibitory effect against the malaria parasite in vitro and maybe of interest for further development as an antimalarial agent.

Malaria parasites require a divergent heme oxygenase for apicoplast gene expression and biogenesis

“…The apicoplast-targeted gyrase A, gyrase B, DDX21 DEAD-box helicase, ligase I, the helicase domain of PREX, histone-like protein (HU) 82 , and single-strand DNA binding protein 71 all co-purify with PfHO in our IP/MS experiments ( Figure 5A ). Of these proteins, DNA gyrase A 83 , gyrase B 84 , PREX 85 , and HU 86 have been directly tested as essential apicoplast biogenesis factors in Plasmodium parasites. The relationship of most of these proteins with apicoplast transcription is unknown, but their activity in other organisms may shed light on a functional pathway in Plasmodium .…”

Apicoplast-Resident Processes: Exploiting the Chink in the Armour of Plasmodium falciparum Parasites

“…Te type II topoisomerases gyrase A (GyrA) and gyrase B (GyrB), which introduce negative supercoils in DNA that are necessary for replication and transcription, are present in the apicoplast [27]. Tis reduces the strain during the replication process by employing the C-terminal domains of GyrA (which forms a six-bladed propeller) to wrap the DNA with positive handedness and a "DNA clamp" at the N terminus of GyrB (which dimerises when ATP is bound), which seizes another chunk of the same doublestranded DNA (dsDNA) [28].…”

“…All the apicoplast-resident processes, including housekeeping processes such as DNA replication, transcription, and translation, can be potential antimalarial targets and have been exploited in drug discovery against malaria. Te DNA replication of the circular apicoplast genome requires the nuclear-encoded tetrameric DNA gyrase, an ATP-dependent type II topoisomerase that helps resolve topological problems during replication and transcription [28]. Earlier studies reporting bacterial gyrase as a potent antibiotic target have propelled studies into the malaria parasite DNA gyrase as a novel antimalarial target.…”

“…Earlier studies reporting bacterial gyrase as a potent antibiotic target have propelled studies into the malaria parasite DNA gyrase as a novel antimalarial target. Several successes have been recorded in targeting gyrase and inhibiting parasite development, including fuoroquinolones (e.g., ciprofoxacin), purpurogallin (PPG), aminocoumarin (e.g., novobiocin), and clindamycin [28,36,74]. Also, nuclear-encoded apicoplast DNA polymerase (apPOL) replicates and repairs the apicoplast genome.…”

A tale of topoisomerases and the knotty genetic material in the backdrop of Plasmodium biology

“…Recently, a chemical library that showed specific binding to PfGyrB but not to EcGyrB, was used to test its efficacy against the ATPase activity, supercoiling, and cleavage activity of the hybrid enzyme, PfGyrB-EcGyrA. This experiment helped in the identification of a novel inhibitor, purpurogallin (PPG) [ 81 ]. PPG inhibits the DNA binding activity of both PfGyrB and EcGyrA-PfGyrB as well as the DNA supercoiling activity and ATP hydrolysis of the hybrid enzyme.…”