Molecular Characterization of an rsmD-Like rRNA Methyltransferase from the Wolbachia Endosymbiont of Brugia malayi and Antifilarial Activity of Specific Inhibitors of the Enzyme

Abstract

The endosymbiotic organism Wolbachia is an attractive antifilarial drug target. Here we report on the cloning and expression of an rsmD-like rRNA methyltransferase from the Wolbachia endosymbiont of Brugia malayi, its molecular properties, and assays for specific inhibitors. The gene was found to be expressed in all the major life stages of B. malayi. The purified enzyme expressed in Escherichia coli was found to be in monomer form in its native state. The activities of the specific inhibitors (heteroaryl compounds) against the enzyme were tested with B. malayi adult and microfilariae for 7 days in vitro at various concentrations, and NSC-659390 proved to be the most potent compound (50% inhibitory concentration [IC₅₀], 0.32 μM), followed by NSC-658343 (IC₅₀, 4.13 μM) and NSC-657589 (IC₅₀, 7.5 μM). On intraperitoneal administration at 5 mg/kg of body weight for 7 days to adult jirds into which B. malayi had been transplanted intraperitoneally, all the compounds killed a significant proportion of the implanted worms. A very similar result was observed in infected mastomys when inhibitors were administered. Docking studies of enzyme and inhibitors and an in vitro tryptophan quenching experiment were also performed to understand the binding mode and affinity. The specific inhibitors of the enzyme showed a higher affinity for the catalytic site of the enzyme than the nonspecific inhibitors and were found to be potent enough to kill the worm (both adults and microfilariae) in vitro as well as in vivo in a matter of days at micromolar concentrations. The findings suggest that these compounds be evaluated against other pathogens possessing a methyltransferase with a DPPY motif and warrant the design and synthesis of more such inhibitors.

INTRODUCTION

Wolbachia is a Gram-negative bacterium classified in the alphaproteobacteria and has been observed in the majority of parasitic filarial nematodes, including human filarial species. These endosymbionts appear to play an important role in the fertility, development, and survival of filariids (1). The genome of the Wolbachia endosymbiont of Brugia malayi is represented by a single circular chromosome consisting of 1,080,084 nucleotides, and the G+C content is 34% (2). A total of 809 genes and open reading frames have been annotated, and these include many universal potential drug targets.

Lymphatic filariasis (LF), the second most common cause of long-term disability in tropical and subtropical countries, is caused by the lymphatic system-dwelling nematodes Wuchereria bancrofti, B. malayi, and B. timori through a mosquito vector. The disease globally affects ∼130 million people, with 1.3 billion people being at risk (3, 4), resulting in heavy socioeconomic losses to developing nations (5), and India accounts for about 40% of the cases. Recent results from sequencing of the genomes of B. malayi and its endosymbiont Wolbachia (2, 6) have made a major impact in filarial research with the discovery of the heme biosynthetic pathway in Wolbachia, which is absent in its host worm, B. malayi. This symbiotic pathway is on the front line of research in the field of filariasis for the identification of targets and inhibitors (1, 7, 8). The identification of small molecules which can hit this pathway or some other molecular targets in Wolbachia is necessary to develop drugs to eliminate this neglected tropical disease. The depletion of Wolbachia from B. malayi with the use of antibiotics or inhibitors leads to extensive apoptosis, which finally results in the death of the worm (9).

Recent advances in genome sequencing have made it possible to envision a molecular drug discovery method in which a bacterial enzyme target is first identified and characterized and new compounds that inhibit the enzyme are searched for. Successful compounds would be inhibitors of an essential or unique bacterial enzyme which has no mammalian homologue. The number of drug targets for helminthic diseases so far identified is few (63), and the threats are compounding with burgeoning resistance against the existing drugs (64, 65). The parasites and microorganisms are becoming increasingly resistant to the available antibiotics, which makes the search for novel antibiotic agents inevitable (66). The ribosome is one of the most common targets of antibiotics in the cell (11), and several mutations in the 23S and 16S rRNA genes, rRNA methyltransferases (MTases), and ribosomal proteins in various bacterial strains lead to drug resistance (12, 13). One of the resistance mechanisms employed by bacteria is the methylation of its rRNA in the ribosome through its specific methyltransferases (14) with the use of S-adenosyl-l-methionine (SAM) as a cofactor. Methylation of RNA by methyltransferases is a phylogenetically ubiquitous posttranscriptional modification that occurs most extensively in tRNA and rRNA (13). All the rRNA methyltransferases from Escherichia coli have been revealed, and many have been purified and biochemically characterized, but their exact functional role in bacteria is still to be explored (15). There are 23 methylated bases in E. coli, out of which the small ribosome subunit contains 10 sites with methylated bases distributed in important functional regions (15, 16). Methylation of the adenine base of RNA at a specific site is quite common in prokaryotic rRNA for regulation of 70S ribosome assembly. These modifications are performed at a rather significant metabolic cost to the cell (17). Numerous nucleotide modifications, mainly but not exclusively via methylation, are found on rRNAs, and their structural basis for ribosome assembly is quite evident, but the functional importance of these posttranscriptional modifications remains unclear (18–20).

The Wolbachia endosymbiont of B. malayi possesses five rRNA methyltransferases, viz., the housekeeping KsgA methyltransferase (rsmA, wBm0407), rRNA/tRNA methyltransferase (spoU, wBm0489), large-subunit methyltransferase E (rlmE, wBm0577), small-subunit methyltransferase H (rsmH, wBm0107), as well as another small rsmD-like methyltransferase (wBm0791). RsmD is an rRNA small-subunit methyltransferase D involved in catalyzing a single target nucleotide, G966, of the 16S rRNA in helix 31 (21). RsmD acts late in the assembly process and is able to modify a completely assembled 30S subunit (22). Due to its high level of importance in ribosome translation events, this enzyme has now been considered a novel drug target (11, 23). Here we report on the molecular characterization of an rsmD-like methyltransferase from the Wolbachia endosymbiont of B. malayi (the wBmrRNA MTase) and the use of specific inhibitors against its catalytic motif, DPPY (24–26). Methyltransferases with the DPPY motif are not present in higher eukaryotes; rather, most of their methyltransferases have the CCWGG motif for C-5 cytosine methylation (27–30). The presence of the DPPY catalytic motif in prokaryotes and its absence in eukaryotes make this an ideal drug target, similar to the Dam methylase of E. coli (31).

We used three specific inhibitors (32) which are heteroaryl compounds procured from the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health (NIH), Bethesda, MD. As antifilarial compounds, their activities are directed against the DPPY motif. The activities of these compounds against B. malayi worms were tested in vitro as well as in vivo. The affinity and specificity of the inhibitors against the wBmrRNA MTase enzyme were calculated through a molecular docking study as well as a tryptophan fluorescence-quenching experiment. This enzyme has been preliminarily characterized and explored as a drug target for combating human LF.

MATERIALS AND METHODS

Experimental maintenance of B. malayi.

Young mosquitoes were fed on B. malayi-infected donor mastomys (Mastomys coucha; microfilarial [mf] density, 100 to 200/10 μl of tail blood). Within 9 ± 1 days of a blood meal, ingested mf develop into infective larvae (L3) inside the mosquito after 2 successive molts. On day 9, the fed mosquitoes were gently crushed, and the crushed material was placed in a Baermann apparatus to collect L3 in sterile Ringer solution. L3 were repeatedly washed and counted, and 150 to 200 L3 each were given intraperitoneally to 8-week-old male jirds (Meriones unguiculatus). It takes about 3 to 4 months for L3 to develop into sexually mature adult B. malayi worms in the peritoneal cavity of jirds.

Studies with all the animals used in the study, viz., jirds (Meriones unguiculatus) and mastomys (Mastomys coucha), were approved by the Institutional Animal Ethics Committee (IAEC) of the Central Drug Research Institute (CDRI), Lucknow, India, duly constituted under the provision of the 1998 rules and guidelines of the Committee for the Purpose of Control and Supervision of Animals (CPCSEA), government of India. The study bears approval no. 129/8/PARA/IAEC/RENEW03 (111/10), dated 7 March 2010, for jirds and mastomys. The study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals (33). All surgery in jirds was performed while the animals were under ketamine anesthesia (100 mg/kg of body weight), and efforts were made to minimize suffering.

The animals were housed at the National Laboratory Animal Centre (NLAC) at CDRI, Lucknow, India, under controlled conditions of temperature (23 ± 1°C), relative humidity (55% ± 10%), and photoperiod (12-h light, 12-h dark cycle). They were given a standard rodent pellet diet and drinking water ad libitum.

Isolation of adult B. malayi worms and preparation of genomic DNA.

Adult worms of B. malayi were harvested from euthanized jirds by washing the jird peritoneal cavity, washed repeatedly, and subjected to genomic DNA isolation as per the protocol of a standard laboratory manual (34), with a slight modification, in which lysozyme was used in the extraction buffer. The genomic DNA of B. malayi also contains the genomic DNA of its endosymbiont, Wolbachia.

Cloning of rRNA methyltransferase gene of Wolbachia.

The ∼546-bp rRNA methyltransferase gene of Wolbachia (AN-wbm0791) was amplified from B. malayi genomic DNA using forward sense primer 5′-GGATCCTTACGTATTATTGCAGGAAAGTATCGT-3′ and reverse antisense primer 5′-CTCGAGAGTTGATAGAGAAAGAAAAATTATTCG-3′ containing flanking BamHI and XhoI restriction sites (underlined), respectively. Amplification of the gene was carried out by mixing 1 μM each primer, 200 μM each deoxynucleoside triphosphate (MBI Fermentas), 0.5 unit Taq DNA polymerase, 1× PCR buffer, and 1.5 μM MgCl₂ under conditions of initial denaturation at 95°C for 4 min, 29 cycles at 95°C for 45 s, 55°C for 1.30 min, and 72°C for 1 min, and 1 cycle at 72°C for 20 min. The amplified product was subcloned into the cloning InsTA vector (MBI Fermentas) and subjected to automated sequencing.

Protein expression and purification.

The clone from the InsTA vector (MBI Fermentas) was initially transferred into the pET28a(+) (Novagen) expression vector. The protein expressed in E. coli cells went completely into the insoluble fraction, in spite of several attempts. The overexpressed insoluble protein was, however, purified by preparatory SDS-PAGE with negative staining (using 4 M sodium acetate) and electroelution in water and was used for raising antibody in BALB/c mice (35). To get native and active methyltransferase, the InsTA clone was again transferred to the pET41a(+) vector for expression with a glutathione S-transferase (GST) fusion tag. The pET41a(+) vector containing the wBmrRNA MTase gene was transformed into expression host cells, viz., cells of the Rosetta(pLys) strain of E. coli. The cells were cultured at 37°C in LB medium with a 1% inoculum prepared overnight and induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) after reaching an optical density of 0.6 (measured at 600 nm). The cells were harvested, pelleted, and immediately suspended in chilled lysis buffer (20 mM Tris, pH 7.5, 250 mM NaCl, 1% Triton X-100, 1 mM EDTA, 0.5 mM phenylmethanesulfonyl fluoride[PMSF]) for 1 h. The cells were subjected to sonication with a 750-W ultrasonic processor at 40% amplitude with a pulse cycle of 10 s on and 30 s off in ice for 10 min. The lysate was then centrifuged for 30 min at 12,500 × g at 4°C, and the supernatant was loaded on a Ni-nitrilotriacetic acid (NTA)-agarose bead-packed column that had been preequilibrated with lysis buffer for 1 h at 4°C. The beads were washed with 5 column volumes (CVs) of wash buffer (20 mM Tris, pH 7.5, 250 mM NaCl, 1 mM EDTA, 200 μM PMSF, 30 mM imidazole). The wBmrRNA MTase was finally eluted with wash buffer containing 300 mM imidazole. The eluted fraction was concentrated with an Amicon ultracentrifugal filter device (Millipore). wBmrRNA MTase was finally purified on a Superdex 200 HR 10/300 column on an Akta fast-performance liquid chromatograph (FPLC; GE Healthcare). The peak fraction was tested for purity on a 10% SDS-polyacrylamide gel and was found to be of 95% purity. The recombinant fusion protein was immediately used (within a day or two) for the experiments.

Expression of the wBmrRNA MTase gene in various stages of B. malayi.

Adult worms and mf of B. malayi were recovered from the peritoneal cavities of infected jirds. Adult parasites were made free of host tissue under a dissecting microscope. mf were isolated by passing the peritoneal wash through a 5.0-μm-pore-size membrane filter and pelleting the mf suspension after dispensing the filter in sterile phosphate-buffered saline (PBS). L3 of B. malayi (500 to 700) were recovered from the experimentally infected laboratory-bred mosquitoes (Aedes aegypti) by use of a Baermann apparatus and washed several times in sterile PBS. RNA was extracted from all three life stages using the TRIzol reagent (Invitrogen) and quantified with a GeneQuant apparatus (Bio-Rad). After treatment with DNase I to eliminate genomic DNA contamination, 2 μg of total RNA from each life stage was used for the first cDNA synthesis using a first-strand cDNA synthesis kit (Sigma-Aldrich, USA). cDNAs were amplified with specific primer pairs under the conditions mentioned above.

rRNA MTase multiple-amino-acid sequence alignments and phylogeny.

Twenty-three methyltransferase sequences available in the GenBank database (http://www.ncbi.nlm.nih.gov) were retrieved for the current study. The amino acid sequences of methyltransferases from the following organisms were aligned using Clustal W software (36) (GenBank accession numbers are given in parentheses, unless indicated otherwise): Clostridium botulinum rsmD MTase (YP_001787809.1), Lactobacillus crispatus rRNA MTase (ZP_05548400.1), Rhodobacteraceae bacterium rsmD MTase (ZP_05124118.1), Shigella flexneri rsmD MTase (NP_709235.1), Streptococcus pneumoniae rsmD RNA MTase (NP_346396.1), Streptococcus cristatus rsmD mG7 MTase (ZP_08059562.1), Caulobacter crescentus 16S rRNA MTase (ZP_10750229.1), C. crescentus 23S rRNA MTase (NP_421117.1), Caulobacter crescentus M.CcrMI (NP_419197.1), E. coli rRNA N6A MTase (YP_003937705.1), E. coli M.EcoDam (2G1P_A), E. coli rsmD MTase (Protein Data Bank [PDB] accession number 2FPO_A), Brucella abortus 16S rRNA m7G MTase (B2S959.1), Rickettsia felis N6A MTase (YP_246881.1), Mycobacterium tuberculosis rsmD MTase (3P9N_A), Rhodobacter sphaeroides M.RsrI (P14751.1), Staphylococcus aureus rRNA N6A MTase (P13957.1), Wolbachia endosymbiont of Drosophila melanogaster RNA (wMerRNA) MTase (NP_966602.1), wBmrRNA spoU MTase (YP_198319.1), wBmrRNA rsmA MTase (YP_198237.1), wBmrRNA rlmE MTase (YP_198407.1), wBmrRNA rsmH MTase (YP_197941.1), and wBmrRNA MTase (YP_198621.1). Neighbor-joining analysis was carried out with the NJ program using the input order of sequences with 1,000 bootstrap replicates. A phylogenetic tree was drawn with the MEGA program. The Clustal W alignment of related sequences (rsmD methyltransferases) was performed and drawn for analysis of highly conserved motifs.

Biophysical characterization of wBmrRNA MTase.

Gel filtration experiments were performed at room temperature using a Tricon 30/100 Superdex 200 column with a separation range of 10 to 600 kDa connected to an FPLC system (Akta). The absorbance was monitored at 280 nm using a flow rate of 0.5 ml/min. The column was calibrated using a standard curve of the elution volume versus the log of the molecular mass made with the following molecular mass standards: RNase (13.7 kDa), chymotrypsinogen A (25 kDa), ovalbumin (44 kDa), bovine serum albumin (66 kDa), and ferritin (443 kDa). To determine the exact mass of the wBmrRNA MTase, the protein was dialyzed in water overnight at 4°C to remove any buffer content, put in a 30% acetonitrile and 0.1% paraformaldehyde solution in a matrix of sinapinic acid, and finally, spotted on a plate for matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry. The fluorescence spectrum of the wBmrRNA MTase was recorded on a PerkinElmer LS50b luminescence spectrometer at 25°C using an excitation wavelength of 280 nm and emission wavelengths ranging from 300 to 400 nm. Slit widths for both excitation and emission were kept at 5 nm, and the photomultiplier voltage was 900 V. The concentration of wBmrRNA MTase was 2 μM in buffer containing 20 mM Tris-HCl, pH 7.4, and 250 mM NaCl. Circular dichroism (CD) experiments were performed with a Jasco J810 spectropolarimeter in a 0.2-cm cell at 25°C. The CD spectrum was measured at an enzyme concentration of 20 μM in 20 mM phosphate buffer (pH 7.4) and 250 mM sodium sulfate for far-UV measurements. The spectrum obtained was normalized by subtracting the baseline observed for the buffer under similar conditions. CD data were analyzed using K2D2 software and verified by the Chau and Fasman method of secondary structure prediction (37, 38).

Methods and evaluations of wBmrRNA MTase inhibitors as potential antifilarial agents against B. malayi. (i) Compounds.

Three specific inhibitors of Dam DNA methyltransferase were procured from the Diversity Set, National Cancer Institute, NIH. These are heteroaryl compounds 3-[2-[4-(3,4-dichlorophenyl)-1,3-thiazol-2-yl]hydrazinyl]indol-2-one (NSC-657589); 3-[[(4-phenyl-1,3-thiazol-2-yl)amino]methylidene]chromene-2,4-dione (NSC-658343), and 3-[[[4-(3,4-dihydroxyphenyl)-1,3-thiazol-2-yl]amino]methylidene]chromene-2,4-dione (NSC-659390). Four nonspecific inhibitors purchased from Sigma-Aldrich were S-(5′-adenosyl)-l-homocysteine (SAH), sinefungin, 5-azacytidine, and 5-aza-2′-deoxycytidine.

For in vitro studies, stocks of 1 mM solutions were prepared in dimethyl sulfoxide (DMSO) and subsequently diluted in RPMI 1640 medium. For in vivo studies, compounds were dissolved in 5% DMSO-water solution.

(ii) Parasites.

Jirds were housed under specific-pathogen-free conditions at a temperature of 23 ± 1°C and a relative humidity of 55% ± 10% with a 12-h light, 12-h dark cycle at the National Laboratory Animal Centre (NLAC) of the Central Drug Research Institute, Lucknow, India, and received a standard pellet diet and water ad libitum. All the animals and animal handling protocols were duly approved by the Institutional Animal Ethics Committee. Adult worms and mf of B. malayi were recovered aseptically from the peritoneal cavity of infected jirds and washed, while mf were isolated by passing the suspension through a 5.0-μm-pore-size membrane filter and thereafter pelleting, as described previously (39).

(iii) In vitro testing.

The inhibitors described above were tested in duplicate at different concentrations between 25 and 0.15 μM (2-fold dilutions) on actively motile female worms in a 24-well culture plate (Nunc) containing 1,000 μl medium and one female parasite per well. The compound dilutions were prepared from the stock solutions prepared in DMSO, as mentioned above. RPMI 1640 medium containing antibiotics (penicillin, 100 units/ml; streptomycin sulfate, 100 μg/ml; neomycin mixture; Sigma-Aldrich) and fortified with 10% fetal bovine serum was used. The worms were exposed to a test sample for 7 days at 37°C in a CO₂ incubator. The motility of the worms was recorded microscopically every day after transfer of the worms to fresh medium containing the same concentration of each inhibitor. After cessation of adult parasite motility, parasites were processed for the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay as described earlier (40). The activities of the test samples containing inhibitors at the various concentrations used in the case of the adult worms were also assessed in duplicate against mf (20 live mf per well) using a 96-well plate containing 200 μl medium. The incubation conditions were the same as those used for the adult parasites, and mf motility was assessed microscopically. The antifilarial effects of the compounds were ascertained in terms of the percent decrease in the motility of the parasite over that of an untreated control worm, as denoted in Table 1. The decrease in motility of both adult female worms and mf and the percent inhibition of MTT reduction in treated adult parasites over that in the respective untreated controls were evaluated. The minimum lethal concentrations of compounds causing total (100%) irreversible immobility (LC₁₀₀s) were determined, and the motility score and percent antifilarial action on adult worms as well as mf were evaluated as follows: 0% motility reduction was given a score of 4+, 1 to 49% motility reduction was given a score of 3+, 50 to 74% motility reduction was given a score of 2+, 75 to 99% motility reduction was given a score of 1+, and 100% motility reduction was given a score of D (for death). Ivermectin was used as a standard filaricide (50% inhibitory concentration [IC₅₀] for adult worms, 1.86 μM; IC₅₀ for mf, 4.2 μM). The criterion for adulticidal activity was 100% irreversible immobility of adult worms with a ≥50% inhibition of MTT reduction for the treated parasite over that for the untreated control (40). In the case of mf, only motility data were considered for assessing antifilarial activity.

Table 1.

Concentration-dependent in vitro activity of specific inhibitors against B. malayi adult and microfilaria

Specific inhibitor no.	Inhibitor	Concn (μM)	Adult motility scorea	% inhibition of MTT reduction over control	mf motility scorea
1	NSC-659390	3.50	D	98.2	D
		1.75	D	77.5	D
		0.75	D	69.4	D
		0.35	D	63.0	D
		0.25	1+	53.0	D
		0.15	3+	41.3	2+
2	NSC-658343	3.50	D	90.1	D
		1.75	D	73.9	D
		0.75	D	56.2	D
		0.50	1+	35.7	D
		0.25	2+	23.5	1+
3	NSC-657589	6.50	D	80.3	D
		3.50	D	44.8	D
		1.75	3+	21.2	2+
		0.75	4+	10.9	4+

^aReduction in motility of parasite: 4+, 0%; 3+, 1 to 49%; 2+, 50 to 74%; 1+, 75 to 99%; D (death), 100%.

(iv) Secondary in vitro screening of compounds for determining IC₅₀s and SIs.

For assessing IC₅₀ values, 2-fold dilutions of each test material were tested starting from the LC₁₀₀. The IC₅₀ was determined by use of an Excel software-based line graphic template after plotting the concentration value of each sample against percent inhibition of motility of the parasite on the x and y axes, respectively (41). The motility data were considered for evaluating IC₅₀s. An in vitro cytotoxicity assay with all the test samples was carried out for assessment of the 50% cytotoxicity concentration (CC₅₀), as described earlier (42). The selectivity index (SI) was calculated as CC₅₀/IC₅₀ to determine the therapeutic window for safety for the use of the compounds in animals. The compounds with SIs of ≥10 were considered safe and further used for in vivo evaluation in animal models.

(v) Evaluation of antifilarial activity of inhibitors in primary in vivo model (jirds into which adult B. malayi worms were transplanted i.p.).

Ten female and five male adult B. malayi worms recovered from jirds infected 4 to 6 months earlier by intraperitoneal (i.p.) inoculation of 150 to 250 L3 were transplanted into 6- to 8-week-old male jirds. The worms were introduced into the peritoneal cavity of the recipient jird after making a small incision in the lateroventral abdominal wall while the jird was under ketamine anesthesia (50 mg/kg, i.p.). On day 4 or 5, a drop of peritoneal fluid was aspirated and checked under a microscope for the presence of live mf to ensure a successful transplant.

At 5 to 6 days posttransplant, the respective compounds were administered at 5 mg/kg of body weight once daily for 7 consecutive days into the peritoneal cavity of the transplanted jirds, with groups of three jirds used for each inhibitor. Subcutaneous (s.c.) administration of 100 mg/kg diethylcarbamazine (DEC) to the transplanted jirds served as a positive control. At the end of the observation period (on day 42), the treated and control jirds were euthanized and worms were recovered from the peritoneal cavity after vigorous washing and observed for their numbers, motility, death, or encapsulation, if any. All the surviving females were teased individually in a drop of PBS (pH 7.2) to examine the condition of the intrauterine contents (43). Any abnormality or death/distortion detected in the uterine stages, including oocytes, eggs, embryos, or mf, was considered an embryostatic/sterilization effect of the compound on the female worm, and the percentage of sterile females was assessed (42).

(vi) Evaluation of antifilarial activity of inhibitors in a secondary in vivo model (M. coucha infected with B. malayi L3 s.c.).

Mastomys were infected by s.c. inoculation of 100 infective larvae (L3) recovered from A. aegypti mosquitoes fed on donor mastomys 8 or 9 days earlier, as described previously (44). Animals were monitored for microfilaremia in 10 μl tail blood at between 12:00 and 12:45 h starting from day 90 onwards (completion of the incubation period). Mastomys that had been infected 5 to 8 months earlier and that showed a progressive rise in microfilaremia were selected for inhibitor treatment.

Mastomys in groups of four each were i.p. treated with each inhibitor at 5 mg/kg (NSC-657589, NSC-658343) or 3 mg/kg (NSC-659390) once daily for 7 consecutive days. Animals infected under identical conditions received only vehicle (PBS) to serve as controls. A group of mastomys was administered DEC (the standard filaricide) at 50 mg/kg for 7 days by the oral route. The microfilaricidal as well as adulticidal (macrofilaricidal) activities of the inhibitors were evaluated as described previously (43). Thick smears of 10 μl tail blood were made from the treated and untreated animal groups just before the start of treatment, i.e., on day 0, and on day 15 after the initiation of treatment and thereafter continued at fixed intervals until day 90. Microfilaricidal activity was assessed by the percent change in mf density from the pretreatment (day 0) level at each time point. At the end of the observation period (on day 90), the treated and control mastomys were euthanized and various tissues (lungs, heart, testes, lymph nodes) were isolated and teased gently in PBS to recover the adult parasites, which were subsequently examined to assess macrofilaricidal and embryostatic effects (43).

Homology modeling of wBmrRNA MTase.

The enzyme three-dimensional (3D) structure was predicted by an homology modeling approach that was carried out using the MODELLER (version 9.9) program (45), and the predicted 3D structure was evaluated using the PROCHECK program (46). All the analyses and visualization of the structure files were carried out using Chimera software (47). The Clustal W program was used to produce the alignment between the wBmrRNA MTase sequence and the sequence of the best available template (PDB accession number 2FPO_A) chosen from a PDB BLAST hit (36).

Molecular docking study.

AutoDock (version 4.2) docking software and the AutoDock tool were used to perform docking experiments. AutoDock is a suite of automated docking tools designed to predict how small molecules/ligands bind to a receptor/protein of known 3D structure (48). The docking study at the conserved catalytic DPPY motif (29) of the wBmrRNA MTase protein was carried out for SAM, NSC-659390, NSC-658343, and NSC-657589, as well as the nonspecific inhibitors SAH, sinefungin, 5-azacytidine, and 5-aza-2′-deoxycytidine. Two-dimensional (2D) structures of these molecules were retrieved from the Pubchem database. Conversion of the 2D to the 3D structure and energy minimization of these structures were done by use of the Insight II 2000.1 program (49). The best conformation was chosen as the one with the lowest docked energy, after the docking search was completed. Docking of these compounds was compared with that of SAM, a cofactor for all methyltransferases.

Study of wBmrRNA MTase interaction with specific inhibitors by intrinsic fluorescence quenching.

Quenching of the fluorescence of tryptophan at position 138 in wBmrRNA MTase was exploited in a study of the interaction of various inhibitors with this enzyme. Fluorescence spectra were recorded on a PerkinElmer LS50b luminescence spectrometer at 25°C. A scan was recorded after excitation at 280 nm and recording of the emission wavelength at between 300 and 400 nm. Slit widths for both excitation and emission were kept at 5 nm, and the photomultiplier voltage was 900 V. An enzyme concentration of 0.74 μM in a 500-μl volume in 20 mM Tris HCl buffer (pH 7.5) with 200 mM NaCl and 1 mM EDTA was used in the study. Quenching of the fluorescence intensity with increasing inhibitor concentrations (0 nm to 400 nm) was exploited in calculating the interaction constant, which is given by a Stern-Volmer plot (50). The Stern-Volmer relationship is represented by F₀/F = 1 + K_SV [Q], where K_SV is the collisional Stern-Volmer quenching constant; F₀ and F are the fluorescence intensities in the absence and presence of quencher, respectively; and [Q] is the quencher concentration. A plot of F₀/F as a function of [Q] yields a linear plot for homogeneous fluorescence emitters, all of which are equally accessible to the quencher, whose slope is equal to K_SV (51).

RESULTS

The enzyme is an rsmD-like RNA MTase.

The findings demonstrate that Wolbachia endosymbiont B. malayi possesses an active rRNA MTase (wBm0791) which is phylogenetically related to rsmD-like methyltransferases from the prokaryotic lineage. The gene is 549 bp and codes for a protein of ∼182 amino acids resembling a small rsmD-like rRNA MTase. Apart from this methyltransferase, Wolbachia possesses four more rRNA MTases, the housekeeping KsgA methyltransferase (rsmA, wBm0407), a tRNA/rRNA methyltransferase (spoU, wBm0489), a large-subunit methyltransferase E (rlmE, wBm0577), and a small-subunit methyltransferase H (rsmH, wBm0107).

A phylogram based on neighbor-joining analysis was generated by comparing various available rRNA MTase sequences and a few DNA MTases from different organisms, including the wBmrRNA MTase, using MEGA (version 5.01) phylogenetic analysis software (52). The analysis suggests that wBmrRNA MTase shares a strong similarity with the methylase from the Wolbachia endosymbiont of Drosophila melanogaster but very weak similarity with other rRNA MTases. Figure 1A shows the tree obtained by neighbor-joining analysis with 1,000 bootstrap replicates. The resulting phylogenetic tree also shows that wBmrRNA MTase is evolutionarily more closely related to rsmD methyltransferases from various genera but distantly related to other rRNA MTases. Amino acid sequence alignment of the wBmrRNA MTase enzyme with homologous sequences of other genera and species retrieved from the NCBI database revealed that it shares approximately 86% identity with Wolbachia pipientis of Drosophila melanogaster (GenBank accession number NP_966602.1), 38% identity with Rickettsia felis (GenBank accession number YP_246881.1), 33% identity with E. coli rsmD (GenBank accession number NP_417922.1), 31% identity with Mycobacterium tuberculosis (GenBank accession number NP_217482.1), 28% identity with Streptococcus cristatus (GenBank accession number ZP_08059562.1), and less than 25% identity with the sequences of other genera (Fig. 1B). There are two important motifs conserved in the wBmrRNA MTase sequence. One of these is the FXGXG motif (positions 52 to 56) at the N terminus, which is essential for binding of the cofactor SAM, and the other one is the DPPY motif (positions 118 to 121) at the C terminus, which is involved in the catalytic activity to transfer a methyl group to the RNA substrate (53).

Fig 1.

(A) Phylogenetic tree showing relationships among various methyltransferases. The amino acid sequences of 23 different methyltransferases (mainly rRNA MTases and a few DNA MTases) from various organisms were aligned using Clustal W software. Neighbor-joining analysis was carried out with the NJ program using the input order of sequences with 1,000 bootstrap replicates. The phylogenetic tree was drawn with the MEGA program. (B) Clustal W analysis of the amino acid sequences of rsmD-like rRNA MTases. Sequences from the following various organisms were aligned using Clustal W software (GenBank accession numbers are given in parentheses, unless indicated otherwise): wBmrRNA MTase (YP_198621.1), wMerRNA MTase (NP_966602.1), Rickettsia felis N6A MTase (YP_246881.1), Clostridium botulinum rsmD MTase (YP_001787809.1), Lactobacillus crispatus rRNA MTase (ZP_05548400.1), Mycobacterium tuberculosis (Mtb) rsmD RNA MTase (3P9N_A), and E. coli rsmD MTase (PDB accession number 2FPO_A). Conserved motifs are marked and boxed. White letters with a black background indicate identical amino acid positions/sequence from various organisms.

The wBmrRNA MTase was cloned, overexpressed, purified, and confirmed by Western blotting.

The designated gene of Wolbachia of B. malayi was successfully PCR amplified and ligated into the pTZ57R/T cloning vector. The construct was double digested and finally ligated in the pET28a(+) expression vector. The wBmrRNA MTase was overexpressed as an insoluble protein with a 6× His tag. The use of various E. coli host expression systems, such as Rosetta(pLysS), BL21(DE3), and C41(DE3), was tried; however, the protein could not be brought into the supernatant even after the temperature or the IPTG concentration was lowered or glucose was supplemented in the growing culture. Several attempts were made but without any success. However, it was solubilized as well as purified by using the pET41a(+) vector and attachment of the GST tag to the protein (Fig. 2A). The fusion protein was purified using Ni-NTA affinity chromatography, and the GST fusion was confirmed with enterokinase cleavage (Fig. 2B). The expressed protein contained the GST fusion tag at the N terminus as well as one His tag at both ends. Anti-His antibody reacted with the GST fusion protein in the E. coli lysate due to the presence of His tags (Fig. 2C). One hundred milliliters of culture yielded ∼0.5 mg of fusion protein after purification. The antibodies against the wBmrRNA MTase (without the GST tag) raised in mouse reacted with the GST fusion protein in an induced whole-cell lysate of E. coli (Fig. 2D).

Fig 2.

Purification, Western blotting, and life-stage expression of recombinant wBmrRNA MTase. (A) A Coomassie-stained SDS-polyacrylamide gel of recombinant wBmrRNA MTase overexpressed in E. coli with a GST tag fusion protein using the pET41a(+) vector. Lane 1, molecular mass markers; lane 2, uninduced E. coli lysate; lane 3, E. coli lysate after 4 h induction with 0.5 mM IPTG; lane 4, pellet fraction after sonication; lane 5, supernatant fraction after sonication; lane 6, flowthrough after passing the supernatant through an Ni-NTA column; lane 7, fraction from 10 CVs eluted with wash buffer containing 20 mM imidazole; lane 8, fraction from the next 10 CVs eluted with wash buffer containing 30 mM imidazole; lane 9, purified recombinant fusion protein GST-wBmrRNA MTase eluted through an Ni-NTA column with wash buffer containing 300 mM imidazole. (B) GST-wBmrRNA MTase fusion protein purified by Ni-NTA affinity chromatography (lane 2). rwBmN6A MTase was partially cleaved from the fusion protein as a 22.0-kDa protein with enterokinase (lane 3) (cleavage reaction for 8 h at 25°C). Lane 1, molecular mass markers. (C) Western blot developed using the diaminobenzidine method with mouse anti-His monoclonal antibodies, followed by HRP-conjugated anti-mouse IgG (lane 2). Lane 1, molecular mass markers. (D) Western blot developed using the diaminobenzidine method with mouse anti-wBmrRNA MTase polyclonal antibodies, followed by HRP-conjugated anti-mouse IgG (lane 2). Lane 1, molecular mass markers. (E) Stage-specific expression of the wBmrRNA MTase gene. The full-length DNA (550 bp) was amplified from cDNA of three major life stages of B. malayi using primers specific for the gene. Lane 1, molecular size markers; lane 2, mf; lane 3, L3; lane 4, adult worms (both sexes).

The wBmrRNA MTase gene is expressed in all the major life stages of B. malayi.

As shown in Fig. 2E, wBmrRNA MTase gene expression (through cDNA amplification) was observed in all the major life stages of B. malayi, viz., microfilariae (mf), infective larvae (L3), and adult worms.

Biophysical properties of wBmrRNA MTase.

Size-exclusion chromatography of the wBmrRNA MTase gave a single peak of monomer size, which indicates that in the native state this enzyme exists as a monomer to catalyze rRNA methylation (Fig. 3A). The purified protein was further subjected to MALDI-TOF spectroscopy, which gave a molecular mass of ∼62 kDa for the fusion protein, corroborating the data presented above (Fig. 3B). Fluorescence spectroscopy yielded a native form of the enzyme with a fluorescence maximum at 333 nm when it was excited at 280 nm (Fig. 3C). Circular dichroism (CD) spectroscopy of the enzyme revealed the presence of 50% α helix and 35% β sheet; the other 15% was a random coil (Fig. 3D).

Fig 3.

Biophysical characterization of wBmrRNA MTase. (A) Size-exclusion profile of the GST-wBmrRNA MTase fusion protein after Ni-NTA column affinity chromatography. The single peak of purified wBmrRNA MTase eluted near its molecular mass. Arrows mark peak positions of the marker proteins ferritin (443 kDa), albumin (66 kDa), ovalbumin (44 kDa), chymotrypsinogen (25 kDa), and RNase A (13.7 kDa). (B) MALDT-TOF mass spectrometry of purified wBmrRNA MTase showing a molecular mass of 62,000 Da. (C) Fluorescence spectrum maxima of wBmrRNA MTase emitted at 333 nm when excited at 280 nm. (D) Far-UV CD spectrum (190 nm to 250 nm) of wBmrRNA MTase showing the characteristic content of an α helix and a β sheet. A.U., absorbance units; θ, mean residue ellipticity.

In vitro antifilarial activities of inhibitors on B. malayi adult worms and mf.

The specific inhibitors of wBmrRNA MTase (NSC-659390, NSC-658343, and NSC-657589) were found to be effective (LC₁₀₀) at killing adults and mf of B. malayi at different concentrations (concentration range, 25.0 to 0.15 μM). NSC-659390 killed both adult parasites and mf at the minimum lethal concentrations of 0.35 and 0.25 μM, respectively, while NSC-658343 did so at 0.75 and 0.50 μM, respectively, and NSC-657589 did so at 3.5 and 3.5 μM, respectively. The IC₅₀ values were 0.32 and 0.30 μM for adult worms and mf, respectively, in the case of NSC-659390. IC₅₀ values for NSC-658343 were 4.13 μM for adult worms and 3.8 μM for mf, while the 3rd inhibitor (NSC-657589) possessed IC₅₀ values of 7.5 and 7.5 μM for adult worms and mf of B. malayi, respectively, on the basis of inhibition of the motility of the parasites (Table 1 and 2). The standard in vitro antifilarial drug ivermectin killed adult worms at a concentration of 9.0 μM (IC₅₀ = 1.86 μM) and mf at a concentration of 140.0 μM (IC₅₀ = 4.2 μM), while DEC was inactive against both in vitro.

Table 2.

In vitro activities of the specific inhibitors against B. malayi

Specific inhibitor no.	Inhibitor	Adult worm				Microfilaria
		LC100a (μM)	IC50b (μM)	CC50c (μM)	SId	LC100 (μM)	IC50 (μM)	SI
1	NSC-659390	0.35	0.32	19.5	60.93	0.25	0.30	63.30
2	NSC-658343	0.75	4.13	50.0	12.10	0.50	3.8	13.15
3	NSC-657589	3.50	7.50	>100	13.33	3.5	7.5	13.33
4	Ivermectin	9.00	1.86	60.3	32.41	140	4.2	14.28

^aThe LC₁₀₀s of adult B. malayi worms and mf were determined as the minimum concentration of test sample causing total irreversible immobility (death).

^bThe IC₅₀s were determined by use of an Excel software-based line graphic template after plotting the concentrations of each sample against percent inhibition of parasite motility on the x and y axes, respectively.

^cThe CC₅₀ of each inhibitor was determined on Vero cells.

^dSI = CC₅₀/IC₅₀.

Antifilarial activity of specific inhibitors of B. malayi in primary in vivo screen (jird model of adult worm transplanted i.p.).

Since all three specific inhibitors demonstrated profound antifilarial activity against B. malayi adult worms and mf in vitro, these were further tested in a primary in vivo screen with the jird model, in which adult B. malayi worms were transplanted at 5 mg/kg of body weight for 7 consecutive days by the intraperitoneal route. NSC-659390 caused an 83.4% decrease of the transplanted adult worm load, while NSC-658343 and NSC-657589 brought about, respectively, 70% and 80% decreased recovery of transplanted adult worms in comparison to the results for the control. In NSC-658343- and NSC-657589-treated jirds, the surviving female worms were observed to have encapsulation and signs of calcification with impaired embryogenesis, though they were alive. DEC administered by the s.c. route at 100 mg/kg for 7 days revealed only 30% adulticidal activity without female worm sterilization (Table 3).

Table 3.

In vivo activity of specific inhibitors against transplanted adult B. malayi worms in M. unguiculatus jirds^a

Inhibitor	Dose (mg/kg) for 7 days	No. of animals	Avg no. of worms recovered			% change in worm recovery	Fecundity/motility/texture
			Female	Male	Total
Control (5% DMSO)	0	3	6.4	2.6	9.0		Fertile/3+/normal
NSC-659390	5	3	0.33	0.3 3	0.66	−83.4**	NA/2+/normal
NSC-658343	5	3	2.0	0.0	2.0	−70.0**	Infertile/1+/shredded
NSC-657589	5	3	1.0	0.0	1.0	−80.0**	Infertile/1+/calcified
DEC	100	3	4.5	1.5	6.0	−30.0*	Fertile/3+/normal

^aThe percent reduction in worm recovery was assessed by comparing the mean values of the treated group with those of the respective untreated control group. Statistical analysis was done by comparing each treated group and the respective control group using one-way analysis of variance (nonparametric) and Dunnett's multiple-comparison test. Statistical significance was as follows: a P value of <0.05 (*) was of low significance, and a P value of <0.01 (**) was highly significant.

Antifilarial activity of specific inhibitors against B. malayi in the secondary in vivo mastomys model (s.c. L3-induced infection).

The microfilarial density in the tail blood of mastomys was monitored 7 days after the commencement of treatment with 3 mg/kg or 5 mg/kg inhibitor. The mf density of the inhibitor-treated groups was lower than that of the untreated control group. In the case of mastomys treated with NSC-659390 and NSC-658343, there was a progressive decrease in mf density, while the NSC-657589-treated group had a progressive increase in mf density which was comparable to that in the control group (Fig. 4).

Fig 4.

mf concentration in blood of mastomys treated with inhibitors and control groups. Mastomys were tested in groups of 4 animals, viz., three inhibitor-treated groups (NSC-659390, NSC-658343, NSC-657589), as well as a PBS-treated negative-control group and a DEC-treated positive-control group, for observation of mf in blood following treatment. There was a marked decrease in the mf density in the NSC-659390 and NSC-658343 groups, while the NSC-657589 group showed an mf density similar to that of the control group at the given dose.

Worm recovery was assessed after 3 months of observation of microfilaremia. The two inhibitors NSC-659390 and NSC-658343 exerted 92.3% and 76.9% adulticidal action at doses of 3 mg/kg and 5 mg/kg, respectively, while the third inhibitor, NSC-657589, proved inactive at 5 mg/kg and did not cause any change in adult parasite recovery over that for the control (Table 4). DEC, which is principally microfilaricidal, exhibited 53.8% adulticidal activity and sterilized 34.31% of the recovered live females.

Table 4.

In vivo activity of specific inhibitors against adult B. malayi worms in infected M. coucha mastomys^a

Inhibitor	Dose (mg/kg) for 7 days	No. of animals taken	Avg no. of worms recovered			% change in worm recovery	Fecundity/motility/texture
			Female	Male	Total
Control (5% DMSO)	0	4	9.0	4.0	13.0		Fertile/3+/normal
NSC-659390	3	4	1.0	0.0	1.0	−92.0**	Infertile/2+/normal
NSC-658343	5	4	2.0	1.0	3.0	−76.9**	Fertile/2+/fertile
NSC-657589	5	4	8.0	4.0	12.0	−7.69	Fertile/3+/normal
DEC	50	4	4.0	2.0	6.0	−53.8*	Partially fertile/3+/normal

^aThe percent reduction in worm recovery was assessed by comparing the mean values of the treated group with those of the respective untreated control group. Statistical analysis was done by comparing each treated group and the respective control group using one-way analysis of variance (nonparametric) and Dunnett's multiple-comparison test. Statistical significance was as follows: a P value of <0.05 (*) was of low significance, and a P value of <0.01 (**) was highly significant.

Specific inhibitors interact strongly with wBmrRNA MTase: fluorescence spectroscopy study.

Tryptophan quenching of wBmrRNA MTase was exploited to study the interaction of the specific inhibitors with this enzyme. As per the in vitro activity, the three inhibitors quenched the fluorescence with Stern-Volmer constants of 4.60, 2.21, and 0.85 μM⁻¹ for NSC-659390, NSC-658343, and NSC-657589, respectively, whereas the nonspecific inhibitor S-adenosyl-l-homocysteine had a Stern-Volmer constant of 0.55 μM⁻¹ (Fig. 5 and 6). The fluorescence quenching was linear with increasing concentrations of inhibitors due to the similar accessibility of tryptophan. The Stern-Volmer quenching constant is directly related to the extent of interaction (binding strength) of these inhibitors with the enzyme.

Fig 5.

Study of interaction of inhibitors with wBmrRNA MTase with the help of fluorescence spectroscopy. The wBmrRNA MTase fluorescence-quenching spectra in the presence of specific inhibitors as well as nonspecific SAH inhibitor are shown. Inhibitor concentrations were 0, 40, 80, 120, 160, 200, 240, and 400 nM; the wBmrRNA MTase concentration was 0.74 μM. (A to D) Quenching of enzyme with the inhibitors NSC-659390 (A), NSC-658343 (B), NSC-657589 (C), and SAH (D); (E) buffer conditions and the various inhibitor concentrations used in the experiment; (F) fluorescence spectra of the highest concentration of all the inhibitors (400 nM) used in the experiment.

Fig 6.

Stern-Volmer plot and calculation of SV quenching constant. (A) Stern-Volmer plots of specific inhibitors as well as SAH show linear quenching of intrinsic tryptophan fluorescence. The plot of F₀/F as a function of the inhibitor concentration yields a linear graph indicating that the tryptophan residue is evenly accessible to all the inhibitors, including SAH. (B) The Stern-Volmer constant for the different inhibitors was calculated from the equation F₀/F = 1 + K_SV [Q].

Homology modeling, molecular docking and binding mode analysis of wBmrRNA MTase inhibitors.

The wBmrRNA MTase possesses 33% sequence identity with the template from E. coli (PDB accession number 2FPO_A), which is an rsmD MTase, and the superimposition of the modeled complex with the template showed a root mean square deviation of 0.442 Å (Fig. 7). The models generated in the Modeler (version 9.9) program were analyzed online by submitting data to the SAVES server. For the final selected model, the Ramachandran plot generated by PROCHECK showed that 92.1% of the residues were in the most favored region and 6.1%, 1.8%, and 0.0% of the residues lay in an additional allowed region, a generously allowed region, and a disallowed region, respectively.

Fig 7.

3D structure modeling and sequence alignment with E. coli rsmD rRNA MTase. (A) Superimposed structure of wBmrRNA MTase (cyan) and the E. coli rsmD MTase (PDB accession number 2FPO_A) template (magenta). The suitable template was chosen from a PDB BLAST hit after predicting various 3D structures using the PROCHECK program. (B) On sequence alignment, the structures share 33% identity and 54% similarity.

The multiple-sequence alignment (Fig. 1B) and previous knowledge helped with selection of active-site residues involved in the interaction of SAM, SAH, NSC-657589, NSC-658343, and NSC-659390. The key residues selected for docking included Asp119, Pro120, Arg151, Tyr122, Glu149, and Met26. These residues are conserved in all methyltransferases which form the catalytic pocket (29).

To identify the binding mode of the cofactor SAM and a possible mechanism of inhibitor action, the docking modes of the docked ligands in the active site of wBmrRNA MTase were analyzed. The top-scoring docked conformations of the compounds NSC-659390, NSC-658343, NSC-657589, and SAH and the cofactor SAM in the active site of the homology model of wBmrRNA MTase were analyzed in terms of the key residues involved in the interaction and their preferred mode of binding. The binding mode was found to be very similar for all these compounds. All compounds and SAM were fairly well accommodated inside the active site of the wBmrRNA MTase.

NSC-659390, NSC-658343, and NSC-657589 were docked into the active site of the wBmrRNA MTase with the AutoDock (version 4.2) program. The binding energies of the three inhibitors were −9.6, −9.01, and −8.95 kcal/mol, respectively. The residues forming hydrogen bonds at the active site with NSC-659390 are Pro120, Glu149, and Arg151, those forming hydrogen bonds at the active site with NSC-658343 are Pro120 and Asp119, and those forming hydrogen bonds at the active site with NSC-657589 are Asp119 and Pro120 (Fig. 8A to C). Finally, the substrate SAM and other nonspecific inhibitors, including the analogue SAH, were docked into the active site of the wBmrRNA MTase. SAM and SAH have binding energies of −7.74 kcal/mol and 7.72 kcal/mol, respectively, and the H bonds formed between the residues in the active site are at Asp119, Pro120, Glu149, and Met26 (Fig. 8D). The binding energies of other nonspecific inhibitors of methyltransferase are shown Table 5.

Fig 8.

Interaction study and binding energy of inhibitors/substrate in the catalytic site of wBmrRNA MTase. All three specific inhibitors and the inhibitors SAH and SAM (all in magenta) were docked at the active site of wBmrRNA MTase (cyan) with AutoDock (version 4.2) software. (A) NSC-659390 showed a binding energy of −9.60 kcal/mol. The residues involved in H-bond formation between the enzyme's active site and NSC-659390 are Pro120, Arg151, and Glu149. (B) NSC-658343 has a binding energy of −9.01 kcal/mol, and the residues involved in H-bond formation are Pro120 and Asp129. (C) NSC-657589 has a binding energy of −8.95 kcal/mol, and H bonds were formed with Asp119 and Pro120. (D) Finally, SAM, including other nonspecific inhibitors (not shown), were docked into the active site of wBmrRNA MTase. SAM showed a binding energy of −7.74 kcal/mol, while SAH had a binding energy of −7.72 kcal/mol, and the residues that participated in H-bond formation were Pro120, Glu149, and Met26.

Table 5.

Affinity of inhibitors at the catalytic site of wBmrRNA MTase^a

Specific inhibitor no.	Inhibitor	Affinity (kcal/mol)
1	NSC-659390	−9.60
2	NSC-658343	−9.01
3	NSC-657589	−8.95
4	SAM	−7.74
5	SAH	−7.72
6	Sinefungin	−8.83
7	5-Azacytidine	−5.76
8	5-Aza-2′-deoxycytidine	−6.06

^awBmrRNA MTase was modeled against the best PDB structure (PDB accession number 2FPO_A), and inhibitors were docked and screened for their binding. Specific inhibitors (1 to 3) showed very high affinities of binding at the catalytic site of the wBmrRNA MTase.

Correlation among docking data, biochemical activity, and in vitro and in vivo efficacy.

The inhibitors demonstrated in vitro antifilarial activity in the order of NSC-659390 > NSC-658343 > NSC-657589. These data correlate well with the fluorescence quenching of the enzyme with specific inhibitors as well as the findings of the study of the docking of these inhibitors at the catalytic site. In the case of in vivo antifilarial testing of the inhibitors in the jird model of transplanted B. malayi, the activity trend was somewhat altered and NSC-659390 was found to be the most active, followed by NSC-657589 and NSC-658343, in that order. In mastomys, only NSC-659390 and NSC-658343 were found to be highly active, while NSC-657589 was almost inactive against adult parasites. All these data are presented in a unified form in Table 6. Nonspecific inhibitors were inactive in vitro even at 100 μM (data not shown) and therefore were neither followed for their IC₅₀/CC₅₀ nor evaluated for their in vivo antifilarial efficacy.

Table 6.

Comparative analysis of activities of specific inhibitors

^aData are for mastomys/jirds. The transplanted jirds were given an intraperitoneal dose of NSC-659390 inhibitor at 5.0 mg/kg of their body weight, while infected mastomys were intraperitoneally treated with 3.0 mg/kg of the same inhibitor.

DISCUSSION

In the current study, attempts have been made to characterize the rsmD-like rRNA MTase from the Wolbachia endosymbiont of the lymphatic filarial parasite B. malayi and the use of specific inhibitors of the enzyme as antifilarial agents. The study utilized enzyme-specific inhibitors whose activity was directed against the highly conserved catalytic motif DPPY for further exploitation as potential antifilarial compounds. The enzyme was successfully cloned, expressed, and purified to homogeneity for biochemical activity. The rsmD-like rRNA MTase was found to exist as a monomer form in its native state and possesses nucleic acid binding property, predominantly RNA (see Fig. S1 in the supplemental material). The gene was found to be expressed in all the major stages of B. malayi, and therefore, it can be inferred that it is an important enzyme for Wolbachia maintenance, which is involved in ribosomal modification. The enzyme structure was predicted with homology modeling, and a search for the presence of motifs was performed. Two motifs, viz. the SAM binding motif (FXGXG) as well as the catalytic motif (DPPY), are present in the enzyme sequence for proper catalysis of the methylation reaction (24–26), with the RNA recognition sequence being located in the stretch of 8 to 26 amino acids at the N terminus, as predicted (54). The latter information conforms to the fact that the naked RNA is copurified along with the enzyme, which gets methylated on addition of SAM-3H (data not shown); however, there is still a possibility that it has methylation activity on the assembled 30S subunit from an rsmD mutant strain, and this possibility should be examined. The intriguing conclusion is that the genome of the Wolbachia endosymbiont of B. malayi seems to be devoid of DNA methyltransferase activity; however, the genome of the prophage harbored by Wolbachia, such as the Wolbachia endosymbiont of D. melanogaster, does possess DNA adenine methyltransferases (55). DNA methylation is required in prokaryotes for fast and faithful replication and for maintaining DNA integrity by the use of repair mechanisms (56–58). It is quite possibly not required in the case of these alphaproteobacteria, since these have coadapted and optimized themselves to grow with eukaryotic host worm or insect cells. Moreover, these are the most primitive bacteria, and the evolution of RNA methylation prior to DNA methylation was supposedly the prime objective of nature to save the prokaryotic world evolving from RNA.

According to the ribosomal substrate specificity, rRNA MTases can basically be categorized into two groups. The relatively large enzymes RlmG and RlmL possess an additional RNA-binding domain that acts on naked rRNA or early assembly intermediates in the cell. The smaller RsmC and RsmD methyltransferases utilize the assembled 30S small subunit or its late-assembly intermediates as the substrate (11, 22). The progress in the characterization of the rsmD-like rRNA methyltransferase gene of Wolbachia in the present study will certainly boost the effort to explore other RNA-modifying enzymes in the coming years. There are five different rRNA MTases in Wolbachia which can be identified and explored on the basis of their homology with other bacterial rRNA MTases, since knockout of endosymbiotic bacteria was very difficult to perform here. The rRNA MTases that transfer a methyl group from SAM on a different base are highly specific (however, there are some exceptions, including an rRNA MTase that methylates at many positions in the rRNA sequence, even when its sequence is homologous to the sequences of other MTases [59]). Decoding the specificity and functions of rRNA MTases through their sequence alignment and identifying highly conserved motifs and cellular and biological function could help with the rapid research on these biologically important rRNA methyltransferases.

This is the first in vitro and in vivo preclinical study involving the use of inhibitors against one of the enzymes of the symbiotic Gram-negative intracellular bacterium Wolbachia of B. malayi, the causative agent of filarial clinical manifestations (7, 8, 60–62). The three specific inhibitors used were found to kill the parasite in a few days or a week at a very low concentration (0.15 to 3.5 μM). All these inhibitors were tested in vitro to determine the therapeutic window for safety by evaluating the selectivity index (SI) by cytotoxicity testing in Vero cells before administering them to animals in vivo. These inhibitors were used in the primary in vivo screening model (with jirds into which adult B. malayi worms were transplanted i.p.), and those demonstrating good antifilarial activity in the primary jird screen were further evaluated against B. malayi infection in a secondary in vivo screening model (s.c. L3-induced infection in mastomys). NSC-659390 and NSC-658343 treatment of jirds and mastomys at the effective concentrations given above led to the overall death of 80% and 75% of the transplanted adult B. malayi parasites, respectively. Thus, the three specific inhibitors demonstrated high adulticidal action in vivo in the two screening models at a very low concentration. The third inhibitor (NSC-657589) was also tested at a higher concentration in mastomys (>8 to <15 mg/kg of body weight) to evaluate its effective concentration and activity.

A molecular docking study of the enzyme with the various inhibitors was carried out in order to assess their specificity toward the target enzyme (wBmrRNA MTase). The three specific inhibitors had far better affinities for the enzyme than the nonspecific inhibitors, and this was corroborated and confirmed by the results of the fluorescence-quenching experiment.

A further possibility for the evaluation of these inhibitors is in an in vivo model using various combinations of these inhibitors as well as combinations of these inhibitors and existing drugs. The specific inhibitors employed in the current investigation may also be useful for controlling several other microbial infections where DNA/RNA methyltransferases with the DPPY catalytic motif are present in the pathogens. The enzyme is prokaryotic in origin, and the catalytic motif of this enzyme is also not known to exist in the eukaryotic nucleic acid methyltransferases; therefore, the enzyme appears to be an ideal drug target. More research is still needed to further narrow the range of drugs with activities against these pathogens.