Homology Modeling of NAD ⁺ -Dependent DNA Ligase of the Wolbachia Endosymbiont of Brugia malayi and Its Drug Target Potential Using Dispiro-Cycloalkanones

Abstract

Lymphatic filarial nematodes maintain a mutualistic relationship with the endosymbiont Wolbachia. Depletion of Wolbachia produces profound defects in nematode development, fertility, and viability and thus has great promise as a novel approach for treating filarial diseases. NAD ؉ -dependent DNA ligase is an essential enzyme of DNA replication, repair, and recombination. Therefore, in the present study, the antifilarial drug target potential of the NAD ؉ -dependent DNA ligase of the Wolbachia symbiont of Brugia malayi (wBm-LigA) was investigated using dispiro-cycloalkanone compounds. Dispiro-cycloalkanone specifically inhibited the nick-closing and cohesive-end ligation activities of the enzyme without inhibiting human or T4 DNA ligase. The mode of inhibition was competitive with the NAD ؉ cofactor. Docking studies also revealed the interaction of these compounds with the active site of the target enzyme. The adverse effects of these inhibitors were observed on adult and microfilarial stages of B. malayi in vitro, and the most active compounds were further monitored in vivo in jirds and mastomys rodent models. Compounds 1, 2, and 5 had severe adverse effects in vitro on the motility of both adult worms and microfilariae at low concentrations. Compound 2 was the best inhibitor, with the lowest 50% inhibitory concentration (IC 50 ) (1.02 M), followed by compound 5 (IC 50 , 2.3 M) and compound 1 (IC 50 , 2.9 M). These compounds also exhibited the same adverse effect on adult worms and microfilariae in vivo (P < 0.05). These compounds also tremendously reduced the wolbachial load, as evident by quantitative real-time PCR (P < 0.05). wBm-LigA thus shows great promise as an antifilarial drug target, and dispiro-cycloalkanone compounds show great promise as antifilarial lead candidates. Human lymphatic filariasis (LF) continues to be a major public health problem as a vector-borne communicable disease in many tropical and subtropical areas of the world. It is caused by the nematode parasites Wuchereria bancrofti, Brugia malayi, and Brugia timori, affecting Ͼ1.3 billion people in 81 countries worldwide, and several millions more are at risk of infection. The mainstay of filarial disease control has been a limited number of drugs, such as diethylcarbamazine, albendazole, and ivermectin, which are principally microfilaricidal, thus necessitating the need for repeated administrations (1, 2). Furthermore, signs of emerging drug resistance are becoming increasingly apparent, especially against albendazole and ivermectin (3-5). There is an urgent need to develop novel drugs (particularly macrofilaricidal drugs) against LF, and also, renewed efforts are required to identify suitable antifilarial drug targets. Numerous lines of evidence, from both laboratory experiments and human trials, show that depletion of Wolbachia in filarial parasites by the use of antibiotics (e.g., doxycycline and tetracycline) can kill adult worms in addition to impairing embryogenesis, the output of microfilariae (MF), and worm development (6, 7). Fu...

Humans from Wuchereria bancrofti endemic area elicit substantial immune response to proteins of the filarial parasite Brugia malayi and its endosymbiont Wolbachia

“…We also cloned and characterized two Wolbachia proteins, Wol Tl IF-1 and w Bm-LigA [28–30]. In the current investigation, we undertook immune characterization of all the four recombinant proteins to assess both humoral and cellular immune response of human subjects staying in W. bancrofti -endemic areas.…”

“…Wolbachia translation initiation factor-1 (Wol Tl IF-1) is an excretory-secretory protein that also elicits protective immunity in rodent model [28]. We have also characterized and reported on NAD + -dependent DNA ligase ( w Bm-LigA), another vital enzyme of Wolbachia with an important role in DNA replication, transcription and repair [29, 30]. …”

Biochemical and structural characterization of DNA ligases from bacteria and archaea

“…By their very nature, structures from crystallographic studies capture only a particular snapshot of the enzyme, which may or may not be relevant for inhibitor binding. Several studies have undertaken basic modelling of the structures of NDLs and virtual screening of molecules that they interact with [ 8 , 53 , 55 , 61 , 63 , 87 , 91 , 98 – 100 ]. However, it is clear that detailed molecular dynamics studies of NDLs would assist with structure-based design of inhibitors.…”