Sequencing, Comparative Modelling and Docking Analysis of Drug Target Genes

Transcription

1 CHAPTER 5 Sequencing, Comparative Modelling and Docking Analysis 5.1 Introduction: D rug treatment is fundamental for managing and controlling TB, promoting the cure of patients and breaking the chain of transmission. According to World Health Organization, resistance to at least two drugs, namely, Isoniazid (INH) and Rifampicin (RIF), causes multi-drug resistant tuberculosis (MDR-TB). The drug resistance in M. tuberculosis is mostly caused by mutations in specific regions in drug target genes and causes reduction in the sensitivity of M. tuberculosis to anti-tuberculosis chemotherapy. The treatment of tuberculosis involves DOTS, a multi-drug therapy approach. Initially, when mono therapy with p-aminosalicylic acid or streptomycin was started in 1940s, high treatment failure rates were observed. But, later on, this was controlled by combination of two or more drugs (Pyle, 1947). Molecular genetic studies during 1970s showed that resistance to anti-tb drugs occurred due to naturally occurring mutations in the genome of M. tuberculosis (David, 1970). Subsequent studies demonstrated that within a population of M. tuberculosis there are mutants that arise due to spontaneous point or deletion mutations (Zhang & Telenti, 2000). Mutations in genes encoding drug targets or drug activating enzymes are responsible for resistance, and such mutations have been found for all first-line drugs and some second line drugs (Ramaswamy & Musser, 1998). Mutants drug resistant to a particular drug occur approximately one in every 10 7 to cells (Parsons et al., 2004; Rattan et al., 1998). Therefore, mono drug therapy results into rapid selection of drug resistant mutants, which dominate the lesions in patients. Thus, combining two or more anti-tuberculosis drugs effectively reduces the [144]

2 risk of selecting resistant mutants in a predominantly susceptible population of M. tuberculosis. Therefore, by combining p-aminosalicylic acid and streptomycin during 1940s, the treatment failure rate was reduced to 9% (Canetti et al., 1963). Since then, combined chemotherapy has remained the cornerstone for tuberculosis treatment. The treatment of tuberculosis at public health level involves DOTS strategy through a multi drug therapy approach. Multi drug resistant and extensively drug resistant TB strains arise by sequential accumulation of resistant mutants to individual drugs until the strain is resistant to drugs that define these forms of resistance. M. tuberculosis use various mechanisms to avoid killing by drugs, including mutations in genes that code for drug target proteins, a complex cell wall, which blocks drug entry and membrane proteins that act as drug efflux pumps (Ramaswamy & Musser, 1998). Earlier studies on molecular mechanisms of katg, encoding catalase peroxidase and rpob, encoding β-subunit of RNA polymerase gene emphasized them as major targets, conferring resistance to M. tuberculosis against isoniazid and rifampicin, respectively. A better understanding of mechanisms involved in drug resistance of M. tuberculosis is essential for the development of new diagnostic tests as well as new anti-tb drugs or identify new drug targets to treat MDR-TB/XDR-TB patients. The systematic probing of these drug target genes through sequencing and bioinformatics based approach in clinical isolates may offer great potential in terms of identification of new drug targets and development of new anti-bacterial agents. Various reports are available on molecular epidemiology and characterization of multi-drug resistant tuberculosis isolates from Europe, America and other regions of the world. The World Health Organization has identified India as a major hot spot for Mycobacterium tuberculosis infection. From India, few reports on the prevalence of drug resistant strain are available, especially from North India. The drug susceptibility profile, molecular characterization of drug resistance genotypes or their prevalence in the community has been explored by various research groups in North India (Gupta et al., 2014; Singhal et al., 2014; Sharma & Madan, 2014; Sagar et al., 2013; Dave et al., 2009; Siddiqi et al., 2002). But, report from tribal population, such as Sahariya, or from North Central India on the characterization of drug resistance genotypes is still lacking. Therefore, the present study was undertaken to characterize the mutations prevalent in M. tuberculosis isolates from Sahariya tribe and their non-tribal [145]

3 neighbours for two most important drug target genes, katg and rpob. Further, bioinformatics approach was used to forecast the structures of mutant katg and rpob proteins along with docking studies to elucidate the mechanism of drug resistance and reveal the drug-drug target interactions at structural level. [146]

4 5.2 Results: PCR Results for katg and rpob: All 27 multi-drug resistant isolates obtained in this study were sequenced. The PCR products for katg and rpob genes had targeted amplicons of 414 bp and 350 bp, respectively (Fig. 5.1 & 5.2). Fig. 5.1: Gel photograph showing amplicon of 414 bp for katg gene, M: 50 bp Molecular Weight Marker Fig. 5.2: Gel photograph showing amplicon of 350 bp for rpob gene M: 50 bp Molecular Weight Marker Sequencing : The chromatograms of DNA sequence for both the genes i.e., katg and rpob, were generated from DNA analyser (ABI3730XL) using Sanger di-deoxy nucleotide sequencing method that were used for further analysis (Fig. 5.3 A-D). The sequences were confirmed for their identity using BLAST ( (Altschul et al., 1990) (Fig. 5.4 & 5.5). The variant alleles observed in the present [147]

5 study are submitted to NCBI with accession numbers: KR424777, KR424778, KR and KR Fig. 5.3 A-D: Chromatograms generated showing representative mutations Fig. 5.4: Nucleotide Sequence Alignment of katg gene from representative isolates of Mycobacterium tuberculosis from Sahariya tribe and non-tribe population. Nucletoide changes are shown in the representative isolates with respect to reference gene M. tuberculosis H37Rv at nucelotide position 1388 and [148]

6 Fig. 5.5: Nucleotide Sequence Alignment rpob gene from representative isolates of Mycobacterium tuberculosis from Sahariya tribe and non-tribe population. Nucletoide change is shown in representative isolate with respect to reference gene M. tuberculosis H37Rv at nucelotide position 1349 The nucleotide sequences were converted to putative amino acids using Expasy Translate ( Multiple sequence alignments were performed using Clustal Omega ( (Sievers et al., 2011) Multiple Sequence Alignment: In all 27 multi-drug resistant isolates, three types of mutations in katg gene were observed. The most common mutation in all of the isolates was at codon 463 (Arginine 463 Leucine), the second was a combination of amino acid changes in two isolates at codon 463 (Arginine 463 Leucine) and codon 529 (Asparagine 529 Threonine), and the third was the amino acid change in two isolates at codon 463 (Arginine 463 Leucine) and codon 529 (Asparagine 529 Histidine). The amino acid sequence change, Arginine 463 Leucine, has been observed in other studies also. In rpob gene, only one type of mutation was observed at codon 531 (Serine 531 Leucine). [149]

7 For M. tuberculosis isolates from Sahariya tribe, the katg gene was observed to have variation at codon 463 (Arginine 463 Leucine) in all the drug-resistant isolates (100%), while the amino acid variations in two different combinations (Arginine 463 Leucine & Asparagine 529 Threonine and Arginine 463 Leucine & Asparagine 529 Histidine) was observed in about 10% of the isolates. For isolates from non-tribes, the variation at codon 463 (Arginine 463 Leucine) was again observed in all the drugresistant isolates. For rpob gene, out of 21 drug resistant isolates from Sahariya tribe, 81% isolates were observed to have variation at codon 531 (Serine 531 Leucine), while the other 19% isolates had none. In non-tribes, out of 6 MDR-TB isolates, 50% were observed to have the same mutation like that of isolates from Sahariya tribe (Serine 531 Leucine), one isolate showed change for any amino acid at the same position, whereas, rest of the two isolates (33%) did not show any mutation or variation (Table 5.1). Table 5.1: Molecular characteristics of katg and rpob gene resistant Mycobacterium tuberculosis isolates of Sahariya tribe and non-tribal population Isolate No. Base Change Nucleotide Position katg Gene Amino acid Change NCBI Accession No. GWL-82 CGG-CTG 1388 Arg463Leu KR GWL-114 AAC-ACA 1586 Asp529Thr KR GWL-250 AAC-CAC 1586 Asp529His KR rpob Gene GWL-138 TCG-TTG 1349 Ser531Leu KR GWL-360 TCG-TTG 1349 Ser531Leu NS NS, Not submitted to NCBI The multiple sequence alignment for all the 21 isolates belonging to Sahariya tribe and 6 MDR-TB isolates from non-tribal population for both the drug target genes revealed sequence variations in different isolates at amino acid level (Fig ). [150]

8 Chapter-5 [151]

9 Fig. 5.6: Multiple Sequence Alignment of 21 MDR isolates of M. tuberculosis for Sahariya tribal population. Changes in amino acids at two positions (Arginine 463 Lysine and Asparagine 529 Histidine as well as Asparagine 529 Threonine) are shown. [152]

10 Fig. 5.7: Multiple Sequence Alignment for rpob gene from 21 MDR isolates of M. tuberculosis for Sahariya tribal population showing change of amino acid (Serine 531 Leucine). Mutations were screened according to E. coli numbering system. The codon 531 position in E. coli and in respective isolates corresponds to 450 position in M. tuberculosis. [153]

11 Chapter-5 Fig. 5.8: Multiple Sequence Alignment for katg gene from 6 MDR isolates of M. tuberculosis from non-tribal population showing change of amino acid (Arginine 463 Lysine). Fig. 5.9: Multiple Sequence Alignment for rpob gene of 6 MDR isolates of M. tuberculosis for non-tribal population and change of amino acids (S531L). Mutations were screened according to E. coli numbering system. The codon 531 position in E. coli and in respective isolates corresponds to 450 position in M. tuberculosis. [154]

12 5.2.4 Template Selection: Molecular Modelling of katg and rpob mutant proteins was done by comparative modelling using restraint based modelling implemented in Modeller 9.14 programme ( The sequences used to build up the proteins for katg and rpob mutants were taken after sequencing of drug target genes (i.e., katg and rpob) from clinical isolates of pulmonary tuberculosis patients and these nucleotide sequences were converted to putative amino acids using Expasy Translate ( The amino acid sequences of mutant katg and rpob proteins in the clinical isolates were used for template selection for protein modelling using Modeller 9.14 software and comparing the sequence similarity with the proteins with PDB ID s. The template selection for 3 mutants (Arginine 463 Leucine, Asparagine 529 Threonine & Asparagine 529 Histidine) of katg protein and one mutant (Serine 531 Leucine) of rpob protein in our clinical isolates was matched with the 1SJ2 A and 1YNJ C (PDB) as a template for katg and rpob, respectively. The E value, percent similarity and bit scores for the three katg mutants (Arginine 463 Leucine, Asparagine 529 Threonine & Asparagine 529 Histidine) as well as one rpob mutant are shown in Table 5.2. Table 5.2: E value, Percent Similarity and Bit score for katg as well as rpob mutant proteins used for the template selection S. No. PDB IDs Bit Score Percent Similarity E Value katg (Arginine 463 Leucine; Arginine 463 Leucine & Asparagine 529 Threonine; Arginine 463 Leucine & Asparagine 529 Histidine) 1. 1zbyA % itkA % mwvA % sj2A % u2kA % ub2A % 0.0 rpob (Serine 531 Leucine) 1. 1ynjC % twfB % iw7C % 0.0 [155]

13 5.2.5 Molecular Modelling: The target alignment was constructed for all the mutant katg and rpob proteins using Modeller Five different models were constructed from Modeller for each mutant by using model single command (model-sinlge.py). Successful models were generated, each with a Molpdf, GA341 and DOPE score. Molecular PDF (molpdf) is the standard Modeller scoring function and is the sum of all restraints. DOPE or Discrete Optimized Protein Energy is a statistical potential and is used to assess the energy of the protein model generated through many iterations by MODELLER, which produces homology models by the satisfaction of spatial restraints. DOPE is based on an improved reference state that corresponds to non-interacting atoms in a homogenous sphere with the radius dependent on a sample native structure; it thus, accounts for the finite and spherical shape of the native structures. The DOPE method is generally used to assess the quality of a structure model as a whole. Alternatively, DOPE can generate a residue-by-residue energy profile for the input model, making it possible for the user to spot the problematic region in the structure model. The DOPE model score is designed for selecting the best structure from a collection of models built by MODELLER. The molpdf and DOPE scores are not absolute measures, in the sense that they can only be used to rank models (molpdf is specific for a given set of restraints and DOPE for a given target sequence). Other scores are transferrable. For example GA341 score always range from 0.0 (worst) to 1.0 (native like); however, GA341 is not as good as DOPE at distinguishing good models from bad models. A good model will have a GA341 score near 1.0 (larger is better) but a small DOPE core (DOPE scores, like molpdf, are energies, so small or negative) is better. The models selected on the basis of lowest DOPE score for katg as well as rpob mutants are summarized in Table 5.3. [156]

14 Table 5.3: Values of Molpdf, DOPE score and GA341 score for the possible PDB models built for various katg and rpob mutants (The selection of the best model is shown in red colour) katg (Arginine 463 Leucine) File Name Molpdf DOPE Score GA341 Score TvLDH.B pdb TvLDH.B pdb TvLDH.B pdb TvLDH.B pdb TvLDH.B pdb katg (Arginine 463 Leucine; Asparagine 529 Threonine) File Name Molpdf DOPE Score GA341 Score TvLDH.B pdb TvLDH.B pdb TvLDH.B pdb TvLDH.B pdb TvLDH.B pdb katg (Arginine 463 Leucine; Asparagine 529 Histidine) File Name Molpdf DOPE Score GA341 Score TvLDH.B pdb TvLDH.B pdb TvLDH.B pdb TvLDH.B pdb TvLDH.B pdb rpob (Serine 531Leucine) File Name Molpdf DOPE Score GA341 Score TvLDH.B pdb TvLDH.B pdb TvLDH.B pdb TvLDH.B pdb TvLDH.B pdb Modelled Structures: The structures modelled for various katg and rpob mutant proteins are shown in Figs & 5.11, respectively. The wild type structure of katg protein is shown in Fig. [157]

15 5.10 A, whereas, the crystal structure for rpob protein in the database is not available. In the present study, the three dimensional structure for the amino acid sequence of rpob M. tuberculosis H37Rv was obtained from NCBI database (Accession No. NP_ ) and the query sequence were searched against PDB to find out protein structure of related family in PDB database ( (Natraj et al., 2008). The modelled mutant protein structures were submitted to I-TASSER online server for protein energy minimization. The root mean square deviation values obtained for the three mutants katg, Arg463Leu (0.899), Arg463Leu; Asp529Thr (1.959), Arg463Leu; Asp529His (0.807) as well as one rpob Ser531Leu (2.763) mutant structures were observed higher than wild type katg (0.703) and rpob (1.167) protein structures, respectively. Fig A-D: Structures of wild type as well as mutant katg proteins A: Wild type katg protein; B: Mutant kat G (R463L) C: Mutant katg (R463L, N529T); D: Mutant katg (R463L, N529H) [158]

16 Fig A-B: Structures of wild type and mutants rpob proteins. A: Wild type rpob protein; B: Mutant rpob (S531L) Ramachandran Plot for various katg and rpob mutants: The 3D structures of various mutants for katg (R463L, R463L & N529T, R463L & N529H) and rpob (S531L) obtained from homology modelling were submitted to RAMPAGE (www. for analysis (Fig & 5.13). The Φ and Ψ distributions for non-glycine non-proline residues in mutant structures for katg and rpob proteins are summarized in Table 5.4. Fig A-C: Ramachandran plots generated for various katg mutant proteins. A: R463 L; B: R463L, N529T; C: R463L, N529H [159]

17 Fig. 5.13: Ramachandran plot generated for rpob mutant (S531L) Table 5.4: Ramachandran Plot calculations for katg and rpob mutants Mutants Number of residues in favoured region N (%) [160] Number of residues in allowed region N (%) Number of residues in outlier region N (%) katg Mutant (R463L) 709 (96.1%) 22 (3.0%) 7 (0.9%) katg Mutant (R463L & 709 (96.1%) 21 (2.8%) 8 (1.1%) N529T) katg Mutant (R463L & 708 (95.9%) 20 (2.7%) 10 (1.4%) N529H) rpob Mutant (S531L) 1080 (92.3%) 70 (6.0%) 20 (1.7%) Docking: The root mean square deviation values (RMSD) obtained after protein energy minimization in I-TASSER online server ( for wild type and mutant structures of katg and rpob proteins are shown in Table 5.5. The docked complexes were analysed and the interactions between drug and wild type as well as mutant proteins were visualized through PyMOL. The best conformations were measured on the basis of hydrogen bond interactions along with bonding distance.

18 Docking of katg wild type as well as mutant proteins with Isoniazid: The computational analysis of docking score and interaction energy between katg wild type as well as mutant modelled proteins of Mycobacterium tuberculosis and isoniazid were carried out through AutoDock4.0. The docking results showed that isoniazid binds to wild type katg protein at one of its active site residue HIS108 with one hydrogen bond formation, whereas, the drug binds to different positions for two of its mutant forms, viz., Arg463Leu and Arg463Leu & Asp529Thr, with formation of one hydrogen bond at two different residues, LYS274 and HIS270, respectively (Fig A-C). The drug binds to third mutant (Arg463Leu & Asp529His, a semi conservative substitution) at TYR229 and VAL230 residues forming 2 hydrogen bonds (Fig D). The residues HIS108 and VAL230 are also one of the proven active site residues for isoniazid interaction (Tally et al., 2008). Fig A-D: Docking of isoniazid with wild type and mutant katg proteins A: Docking of isoniazid with wild type katg protein B: Docking with mutant katg (R463L) C: Docking with mutant katg (R463L & N529T) D: Docking with mutant katg (R463L & N529T). [161]

19 Docking of rpob wild type as well as mutant proteins with Rifampicin: The docking of rifampicin with wild type and mutant proteins also showed variation in binding energies, formation of hydrogen bonds and their distances. Interaction of rifampicin with wild type rpob protein revealed formation of 1 hydrogen bond at LYS799 residue along with the formation of 1 oxygen bond at THR829 residue, a hypothesized active site for rifampicin binding, since no crystal structure is available for rpob protein till date. Whereas, its interaction with mutant protein (one of the most common mutation in 81bp region, RRDR) resulted in the formation of 1 hydrogen bond at ILE522 residue with bond distance of 2.23 Å, smaller than bond distance at THR829 residue 2.46 Å in the wild type (Fig A & B). The binding energies were also observed varying. Fig A-B: Docking of rifampicin with wild type and mutant rpob proteins A: Docking of rifampcin with wild type rpob protein B: Docking with mutant rpob (S531L) The binding energies as well as hydrogen bond distances calculated for wild type and mutant proteins of katg and rpob genes are shown in Table 5.5. [162]

20 Table 5.5: Molecular interactions of wild type and mutant structures of katg and rpob proteins Structure Number of Hydrogen Bonds Interactive Residues H-Bond Distance Binding Affinity (Kcal/mol) RMSD Value katg Wild Type 1 HIS108:HE Å katg Mutant KR (R463L) katg Mutant KR (R463L, N529T) 1 LYS274: HN 2.0 Å HIS270:HE2 1.9 Å katg Mutant KR (R463L, N529H) 2 TYR229:HN VAL230:HN 2.0 Å 1.9 Å rpob Wild Type 1 LYS799: HZ THR829: O 2.19 Å 2.46 Å rpob Mutant 1 ILE522:HN 2.23 Å KR (S531L) HN: Hydrogen Nitrogen; O: Oxygen [163]

21 5.3 Discussion: The molecular basis of drug resistance in multi-drug resistant tuberculosis isolates is well studied (Gupta et al., 2014; Singhal et al., 2014; Sharma & Madan, 2014; Sagar et al., 2013; Dave et al., 2009; Siddiqi et al., 2002; Ramaswamy & Musser, 1998; Rattan et al., 1998). Recently, Bhat et al () reported the situation of MDR-TB in Sahariya tribe from Gwalior region. But, till date, no data is available on the mutations in M. tuberculosis isolates responsible for causing drug resistance in Sahariya tribe and their non-tribal neighbours. To the best of our knowledge this is the first ever investigation on sequencing of drug target genes and drug-drug target interactions in mycobacterial isolates from Sahariya tribe and their non-tribal populations from North Central India. Mycobacterium tuberculosis uses various mechanisms, including mutations in genes that code for drug target genes and become resistant to particular drugs. In present study, the sequencing of katg gene revealed common mutation Arg463Leu in all the isolates of Sahariya tirbe and non-tribes, whereas mutation at codon 529 (Asp529Thr & Asp529His) was observed only in two isolates of Sahariya tribe. In rpob gene, the most common mutation Ser531Leu was observed in the 81 bp Rifampicin Resistance Determining Region (RRDR) in Sahariya tribe as well as nontribe isolates. A bioinformatics approach was used to forecast the structure of mutant katg and rpob proteins along with docking studies to elucidate the mechanism of drug resistance. The present investigation on drug-drug target interactions of wild type and mutant structures of katg protein, revealed variation in their interactive residues, is being HIS108 in wild type katg protein, already proved interactive site for INH (Tally et al., 2008) and LYS274, HIS270 and TYR229 & VAL230 in katg mutant structures. The docking analysis of wild type and mutant structure of rpob protein affirmed LYS799 and THR829 binding residues for wild type protein and ILE522 for mutant protein. Studies in North Indian population have documented the mutation in katg at codon Arg463Leu (Dave et al., 2009; Siddiqi et al., 2002). However, other variations, like Thr275Ala, Arg409Ala and Asp695Ala along with change at Arg463Leu were observed in isoniazid resistant clinical isolates from South Africa, Switzerland and USA (Pretorius et al., 1995). Large number of studies have shown a most common [164]

22 mutation at Ser315Thr position (Gupta et al., 2014; Samad et al., 2014; Poudel et al., 2012; Yuan et al., 2012; Ajbani et al., 2011; Aslan et al., 2008). In South India, a novel mutation at Gly297Pro was observed along with Ser315Thr change (Ravibalan, 2014). Variable percentage of mutations in rpob gene has been reported by different groups. The locus Ser531Leu is suggested to confer resistance to rifampicin at high frequency, ranging from 30% (Siddiqi et al., 2002), 44.2% (Yuan et al., 2012), 52% (Khan et al., 2013), 58.7% (Poudel et al., 2012), 64% (Aslan et al., 2008), 72% (Ravibalan, 2014), 97% (Ajbani et al., 2011), 98% (Wang et al., 2013), to 100% of the isolates (Dave et al., 2009). In rpob gene, mutations in some other loci, such as His526Tyr and Asp516Val, have been reported (Sharma & Madan, 2014; Ravibalan, 2014; Khan et al., 2013; Aslan et al., 2008; Somoskovi et al., 2006; Hwang et al., 2003; Siddiqi et al., 2002, Bodmer et al., 1995). It was observed that mutations at Arg463Leu and Ser531Leu in katg and rpob genes, respectively, of Sahariya tribe and non-tribes are not different from those studied in various parts of the country. Thus, the control of MDR-TB is critical, while the level of MDR-TB is increasing, expanding into clones (Kritski et al., 1997). This stresses the need of early detection of MDR-TB to control tuberculosis. Studies on the structural basis of drug resistance for isoniazid and rifampicin in MDR- TB isolates with various mutations have revealed decreased stability and flexibility of proteins at isoniazid and rifampicin binding sites, which lead to impaired action (Kumar & Jena, 2014; Samad et al., 2014; Ubyaan et al., 2012; Ramasubban et al., 2011; Purohit et al., 2011). Through docking studies it was observed that the mutant protein with positive binding energy fails to cause rifampicin inhibition and thus, provides resistance (Kumar & Jena, 2014). The docking studies on mutant proteins of both genes revealed that these mutations have the potential to alter the interaction of their respective drugs to their active residues. The present study indicates that these mutations make the protein unstable and interactive sites rigid. It is suggested that in wild type protein the binding sites are flexible and favours drug binding (Ramasubban et al., 2011). Thus, mutations observed in MDR-TB isolates in the present study might be causing impaired activity of the active binding sites. [165]

23 5.4 Conclusion: The data obtained in the present study may be considered as a representation of molecular characteristics of MDR-TB isolates in Sahariya tribe and non-tribes. The study also explores the possible functional variation in isoniazid and rifampicin resistance with respect to the mutations identified. Already reported mutations, Arg463Leu and Ser531Leu for katg and rpob genes, respectively, were observed in most of the isolates. Only two novel mutations, Asp529Thr and Asp529His for katg gene, were identified in only two different isolates of Sahariya tribe. The knowledge of structural mechanism of drug resistance may pave the way for designing of new drug targets to tackle the situation of multi-drug resistant tuberculosis in near future. Thus, the application of molecular methods to detect drug resistance in tuberculosis is important and rapid. As a result, suitable therapy can be initiated to break the transmission of MDR-TB in this population. [166]

24 , , , ,148, , [167]