Molecular docking and simulation study for synthesis of alternative dapsone derivative as a newer antileprosy drug in multidrug therapy.

Leprosy (causative, Mycobacterium leprae) continues to be the persisting public health problem with stable incidence rates, owing to the emergence of dapsone resistance that being the principal drug in the ongoing multidrug therapy. Hence, to overcome the drug resistance, structural modification through medicinal chemistry was used to design newer dapsone derivative(s) (DDs), against folic acid biosynthesis pathway. The approach included theoretical modeling, molecular docking, and molecular dynamic (MD) simulation as well as binding free energy estimation for validation of newly designed seven DDs, before synthesis. Theoretical modeling, docking, and MD simulation studies were used to understand the mode of binding and efficacy of DDs against the wild-type and mutant dihydropteroate synthases (DHPS). Principal component analysis was performed to understand the conformational dynamics of DHPS-DD complexes. Furthermore, the overall stability and negative-binding free energy of DHPS-DD complexes were deciphered using Molecular Mechanics/Poisson-Boltzmann Surface Area technique. Molecular mechanics study revealed that DD3 possesses higher binding free energy than dapsone against mutant DHPS. Energetic contribution analysis portrayed that van der Waals and electrostatic energy contributes profoundly to the overall negative free energy, whereas polar solvation energy opposes the binding. Finally, DD3 was synthesized and characterized using Fourier-transform infrared spectroscopy, UV, liquid chromatography-mass spectrometry, and proton nuclear magnetic resonance techniques. This study suggested that DD3 could be further promoted as newer antileprosy agent. The principles of medicinal chemistry and bioinformatics tools help to locate effective therapeutics to minimize resources and time in current drug development modules.

Discovery of a potential lead compound for treating leprosy with dapsone resistance mutation in M. leprae folP1.

Dapsone resistance is a serious impediment to the implementation of the present leprosy control strategies. In the recent past, many studies have been undertaken to address the antibiotic activity and binding pattern of dapsone against both native and mutant (Pro55Leu) folP1. Yet, there is no well-developed structural basis for understanding drug action and there is dire need for new antibacterial therapies. In the present study, molecular simulation techniques were employed alongside experimental strategies to address and overcome the mechanism of dapsone resistance. In essence, we report the identification of small molecule compounds to effectively and specifically inhibit the growth of M. leprae through targeting dihydropteroate synthase, encoded by folP1 which is involved in folic acid synthesis. Initially, ADME and toxicity studies were employed to screen the lead compounds, using dapsone as standard drug. Subsequently, molecular docking was employed to understand the binding efficiency of dapsone and its lead compounds against folP1. Further, the activity of the screened lead molecule was studied by means of molecular dynamics simulation techniques. Furthermore, we synthesized 4-(2-fluorophenylsulfonyl)benzenamine, using (2-fluorophenyl)boronic acid and 4-aminobenzenesulfonyl chloride, and the compound structure was confirmed by (1)H NMR and (13)C NMR spectroscopic techniques. Most importantly, the antibacterial activity of the compound was also examined and compared against dapsone. Overall, the result from our analysis suggested that CID21480113 (4-(2-fluorophenylsulfonyl)benzenamine) could be developed into a promising lead compound and could be effective in treating dapsone resistant leprosy cases.

Computational Modelling of Dapsone Interaction With Dihydropteroate Synthase in Mycobacterium leprae; Insights Into Molecular Basis of Dapsone Resistance in Leprosy.

The molecular basis for determination of resistance to anti-leprosy drugs is the presence of point mutations within the genes of Mycobacterium leprae (M. leprae) that encode active drug targets. The downstream structural and functional implications of these point mutations on drug targets were scarcely studied. In this study, we utilized computational tools to develop native and mutant protein models for 5 point mutations at codon positions 53 and 55 in 6-hydroxymethyl-7, 8-dihydropteroate synthase (DHPS) of M. leprae, an active target for dapsone encoded by folp1 gene, that confer resistance to dapsone. Molecular docking was performed to identify variations in dapsone interaction with mutant DHPS in terms of hydrogen bonding, hydrophobic interactions, and energy changes. Schrodinger Suite 2014-3 was used to build homology models and in performing molecular docking. An increase in volume of the binding cavities of mutant structures was noted when compared to native form indicating a weakening in interaction (60.7 Å(3) in native vs. 233.6 Å(3) in Thr53Ala, 659.9 Å(3) in Thr53Ile, 400 Å(3) for Thr53Val, 385 Å(3) for Pro55Arg, and 210 Å(3) for Pro55Leu). This was also reflected by changes in hydrogen bonds and decrease in hydrophobic interactions in the mutant models. The total binding energy (ΔG) decreased significantly in mutant forms when compared to the native form (-51.92 Kcal/mol for native vs. -35.64, -35.24, -46.47, -47.69, and -41.36 Kcal/mol for mutations Thr53Ala, Thr53Ile, Thr53Val, Pro55Arg, and Pro55Leu, respectively. In brief, this analysis provided structural and mechanistic insights to the degree of dapsone resistance contributed by each of these DHPS mutants in leprosy.

Detection of mutations in folp1, rpoB and gyrA genes of M. leprae by PCR- direct sequencing--a rapid tool for screening drug resistance in leprosy.

UNLABELLED: Conventional Mouse foot-pad (MFP) assay for screening drug resistance in M. leprae is cumbersome and time-consuming (approximately 6 to 12 months). Molecular targets for different anti-leprosy drugs have been well defined. Molecular tools for rapid detection of drug resistance in M. leprae have been standardised. A study to compare molecular methods with MFP assay in determining the drug susceptibility of M. leprae was carried out. METHODS: Forty Bacteriological Index (BI) positive patients of leprosy with clinical features of relapse (25), new cases (11) and defaulters (4) were included in the study. A skin biopsy was done and the samples were processed using both MFP assay and Molecular method. PCR assays were carried out to amplify, 388 bp of folP1 gene for dapsone resistance, 305 bp of rpoB gene for rifampicin resistance and 342 bp of gyrA gene for ofloxacin resistance, followed by direct DNA sequencing. RESULTS: Significant growth in the MFP test was obtained in only 28 out of 40 biopsies processed (70%). Ten of these isolates were dapsone resistant; one isolate showed combined resistance against dapsone, rifampicin and clofazimine. Amplification for all three genes was successful in all the 40 (100%) samples. Among folP1 products sequenced, six isolates showed mutations at 53 (or) 55 amino acid positions. Those strains which showed high-level resistance with two log growth in MFP test, and/or showed growth in passage had mutations in folp1 gene. No mutation was detected in rpoB and gyrA products. Thus no molecular evidence of Rifampicin resistance was found in the DNA isolated from biopsies. CONCLUSION: Thus PCR-direct sequencing--the rapid and high sensitive molecular technique can be applied for detection of resistance against dapsone, rifampicin and ofloxacin in M. leprae, to over come the limitations of the conventional MFP assay.

Mutation analysis of the Mycobacterium leprae folP1 gene and dapsone resistance.

Diaminodiphenylsulfone (dapsone) has long been used as a first-line drug worldwide for the treatment of leprosy. Diagnosis for dapsone resistance of Mycobacterium leprae by DNA tests would be of great clinical value, but the relationship between the nucleotide substitutions and susceptibility to dapsone must be clarified before use. In this study, we constructed recombinant strains of cultivable Mycobacterium smegmatis carrying the M. leprae folP1 gene with or without a point mutation, disrupting their own folP gene on the chromosome. Dapsone susceptibilities of the recombinant bacteria were measured to examine influence of the mutations. Dapsone MICs for most of the strains with mutations at codon 53 or 55 of M. leprae folP1 were 2 to 16 times as high as the MIC for the strain with the wild-type folP1 sequence, but mutations that changed Thr to Ser at codon 53 showed somewhat lower MIC values than the wild-type sequence. Strains with mutations at codon 48 or 54 showed levels of susceptibility to dapsone comparable to the susceptibility of the strain with the wild-type sequence. This study confirmed that point mutations at codon 53 or 55 of the M. leprae folP1 gene result in dapsone resistance.

Dihydropteroate synthase mutations in the folP1 gene predict dapsone resistance in relapsed cases of leprosy.

Molecular detection was compared with the mouse footpad inoculation test for detection of dapsone resistance in 38 strains of Mycobacterium leprae. Mutations of the folP1 gene (at codons 53 or 55) were found in 6 of 6 strains with high-level resistance, in 3 of 4 strains with intermediate-level resistance, and in 1 of 6 strains with low-level resistance, but not in 22 dapsone-susceptible strains. In cases of infection with strains of M. leprae carrying the folP1 mutation, therapy with dapsone may be replaced by therapy with a fluoroquinolone.

A drug-resistant leprosy case detected by DNA sequence analysis from a relapsed Mexican leprosy patient.

A skin biopsy sample was obtained from a relapsed lepromatous leprosy patient from the central area of Mexico. Genes associated with resistance to anti-leprosy drugs were analyzed by DNA sequence assay. A single nucleotide substitution was found at codon 53 (ACC-->GCC) in the folP gene, which is known to confer dapsone resistance. No mutations in the rpoB and gyrA, which indicate resistance to rifampicin and fluoroquinoles, were detected. This is the first reported case of dapsone resistant leprosy in Mexico in which the cause of the resistance is shown at genomic level. Evaluation of drug resistance by identifying known mutations in these genes by PCR is simple and reliable. Testing for resistance to anti-leprosy drugs should be performed in relapses or intractable cases for a better outcome.

[Diaminodiphenylsulfone resistance of Mycobacterium leprae due to mutations in the dihydropteroate synthase gene].

The relation between diaminodiphenylsulfone (called dapsone)-resistance and point mutations of the dihydropteroate synthase (DHPS) gene was analyzed using dapsone resistant Mycobacterium leprae isolates derived from Japanese leprosy patients. The mutation was found at amino acid residues 53 or 55 of the DHPS. This finding suggests that two specific mutations in the DHPS gene involved in dapsone resistance of M. leprae.

Dihydropteroate synthase of Mycobacterium leprae and dapsone resistance.

Two Mycobacterium leprae genes, folP1 and folP2, encoding putative dihydropteroate synthases (DHPS), were studied for enzymatic activity and for the presence of mutations associated with dapsone resistance. Each gene was cloned and expressed in a folP knockout mutant of Escherichia coli (C600DeltafolP::Km(r)). Expression of M. leprae folP1 in C600DeltafolP::Km(r) conferred growth on a folate-deficient medium, and bacterial lysates exhibited DHPS activity. This recombinant displayed a 256-fold-greater sensitivity to dapsone (measured by the MIC) than wild-type E. coli C600, and 50-fold less dapsone was required to block (expressed as the 50% inhibitory concentration [IC(50)]) the DHPS activity of this recombinant. When the folP1 genes of several dapsone-resistant M. leprae clinical isolates were sequenced, two missense mutations were identified. One mutation occurred at codon 53, substituting an isoleucine for a threonine residue (T53I) in the DHPS-1, and a second mutation occurred in codon 55, substituting an arginine for a proline residue (P55R). Transformation of the C600DeltafolP::Km(r) knockout with plasmids carrying either the T53I or the P55R mutant allele did not substantially alter the DHPS activity compared to levels produced by recombinants containing wild-type M. leprae folP1. However, both mutations increased dapsone resistance, with P55R having the greatest affect on dapsone resistance by increasing the MIC 64-fold and the IC(50) 68-fold. These results prove that the folP1 of M. leprae encodes a functional DHPS and that mutations within this gene are associated with the development of dapsone resistance in clinical isolates of M. leprae. Transformants created with M. leprae folP2 did not confer growth on the C600DeltafolP::Km(r) knockout strain, and DNA sequences of folP2 from dapsone-susceptible and -resistant M. leprae strains were identical, indicating that this gene does not encode a functional DHPS and is not involved in dapsone resistance in M. leprae.

Cloning and expression of Mycobacterium tuberculosis and Mycobacterium leprae dihydropteroate synthase in Escherichia coli.

The genes for dihydropteroate synthase of Mycobacterium tuberculosis and Mycobacterium leprae were isolated by hybridization with probes amplified from the genomic DNA libraries. DNA sequencing revealed an open reading frame of 840 bp encoding a protein of 280 amino acids for M. tuberculosis dihydropteroate synthase and an open reading frame of 852 bp encoding a protein of 284 amino acids for M. leprae dihydropteroate synthase. The dihydropteroate synthases were expressed under control of the T5 promoter in a dihydropteroate synthase-deficient strain of Escherichia coli. Using three chromatography steps, we purified both M. tuberculosis and M. leprae dihydropteroate synthases to >98% homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed molecular masses of 29 kDa for M. tuberculosis dihydropteroate synthase and 30 kDa for M. leprae dihydropteroate synthase. Gel filtration of both enzymes showed a molecular mass of ca. 60 kDa, indicating that the native enzymes exist as dimers of two identical subunits. Steady-state kinetic parameters for dihydropteroate synthases from both M. tuberculosis and M. leprae were determined. Representative sulfonamides and dapsone were potent inhibitors of the mycobacterial dihydropteroate synthases, but the antimycobacterial agent p-aminosalicylate, a putative dihydropteroate synthase inhibitor, was a poor inhibitor of the enzymes.

Diaminodiphenylsulfone resistance of Mycobacterium leprae due to mutations in the dihydropteroate synthase gene.

The nucleotide sequence analysis of the dihydropteroate synthase (DHPS) gene of six diaminodiphenylsulfone-resistant Mycobacterium leprae strains revealed that the mutation was limited at highly conserved amino acid residues 53 or 55. Though the mutation at amino acid residue 55 or its homologous site has been reported in other bacteria, the mutation at residue 53 is the first case in bacteria. This is the first paper which links the mutations in DHPS and sulfonamide resistance in M. leprae. This finding is medically and socially relevant, since leprosy is still a big problem in certain regions.

Dapsone resistance does not appear to be associated with a mutation in the dihydropteroate synthase-2 gene of Mycobacterium leprae.

Evidence suggests that resistance to dapsone (DDS) in Mycobacterium leprae is related to the enzyme dihydropteroate synthase (DHPS). Two M. leprae genes (folP-1 and folP-2) encoding DHPS-1 and DHPS-2, respectively, have been identified through the M. leprae genome project. We have studied DDS-susceptible and resistant strains of M. leprae to determine whether the DDS-resistant phenotype is associated with a mutation(s) in folP-2 and to establish the number of genomic copies of the gene encoding DHPS-2 (folP-2). RFLP analysis of genomic DNA from DDS-susceptible and resistant strains of M. leprae exhibited a unique 4.2 kb restriction fragment consistent with a single genomic copy of folP-2 in both phenotypes. DNA encoding folP-2 was amplified by PCR and sequenced from two susceptible and two resistant strains of M. leprae. The folP-2 sequences from these strains were identical indicating that resistance to DDS was not associated with mutation(s) in the gene encoding DHPS-2.