NEW INHIBITORS AGAINST accD3 GENE OF MYCOBACTERIUM TUBERCULOSIS, IN-SILICO STUDY AND IN -VITRO APPLICATION

NEW INHIBITORS AGAINST accD3 GENE OF MYCOBACTERIUM TUBERCULOSIS, IN-SILICO STUDY AND IN -VITRO APPLICATION

Asra’a A. Abdul-Jalil ^*1 and Zahra M. Al-Khafaji ²

College of Science ¹, University of Anbar, Baghdad, Iraq.

Genetic Engineering and Biotechnology Institute ², Baghdad University, Baghdad, Iraq.

ABSTRACT: Mycobacterium tuberculosis (Mtb) is a pathogenic bacteria species in the genus Mycobacterium and the causative agent of most cases of tuberculosis. Tuberculosis (TB) is the leading cause of death in the world from a bacterial infectious disease. This antibiotic resistance strain lead to development of the new antibiotics or drug molecules which can kill or suppress the growth of Mycobacterium tuberculosis. The aim of  this study  is  discovery  a  new  drug molecules against  the  product of  accD3 gene which play a vital role in mycolic  acid  biosynthesis  pathway (MAP). As a first step  accD3  gene amplified  from the  complete genome of  Mycobacterium  tuberculosis Iraqi isolates and sequenced, eight  out of  twenty of  the  resulted  sequences were chosen to achieve the in silico studies, these  sequences  have  deposited in GenBank database with strain  names (ALQM1,  ALQM2,  ALQM3, ALQM4, ALQM5, ALQM6, ALQM7,  ALQM8 ) and  Accession  numbers  (LC006979, LC008196, LC009412, LC009414,  LC034168,  LC038020,  LC041163,  LC041368) respectively, two drug molecules  resulted from in silico studies and  used in the practical  application  to  prove  the  in vitro inhibitory  ability  of  these  molecules on  viable cells  of  M. tuberculosis, REMA which is a colorimetric assay was used in this research to estimate the inhibitory application of these molecules.

Tuberculosis, REMA, Homology modeling, accD3 gene

INTRODUCTION: Tuberculosis (TB) proved to be a controllable, preventable and curable disease, but it still remains as a leading cause for mortality¹. It was declared a global health emergency in 1993 by the World Health Organization (WHO) when approximately 8 million Tb cases were estimated and 1.3-1.6 million deaths were occurred from the disease each year, now TB exacerbated by the spread of AIDS.

Iraq  has  a high  burden  of  TB and  ranks  44^th  worldwide  among countries  with  a high TB  burden  and 7^th among  the  countries of  the  Eastern  Mediterranean  region.  Anti - tuberculosis (TB) drug resistance is a major public health problem that threatens progress made in TB care and control worldwide. Rational  Drug  Design (RDD) is basically a computer-aided  molecular  modeling,  it is an iterative process,  If  it is based  on  the  knowledge of  three - dimensional (3-D) structure of  the target proteins of  interest; it is called “structure based drug design” or “target based drug design”. Eight nucleotide sequences were extracted from Iraqi Mtb isolates were used in this study, two molecules gave a good results  in  the  inhibition of  M. tuberculosis growth  by  using  REMA test  which is a colorimetric assay ².

The main aim of this study is the discovery of new drugs against Tuberculosis.

MATERIALS AND METHODS: Sputum  samples were  collected from 200 Iraqi suspected patients who attended the  Institute  of  Chest and  Respiratory diseases / Baghdad,  for the period from 1st of  May 2013 to  31st  July  2013. Their ages were ranging from 8 years up to 85 years, 50 of the total 86 positive cultures were randomly chosen to complete this study.

DNA Extraction from M. tuberculosis isolates and primer design: DNA was extracted from pre diagnosed  mycobacterial  cultures and purified by CTAB method ^{3, 4}. M. tuberculosis isolates were characterized at molecular level using  LprM  gene, primer  pairs  were used  to  amplify  this gene  to differentiate M. tuberculosis from  the  other  types  of  mycobacteria ^{5, 6}. The results  showed that  43 isolates (86%) among  of  50 isolate were M. tuberculosis.

DNA sequences of accD3(1488 bp)  was  retrieved  from puplic database, primers were designed for the gene at two position, segment I from (38-831) to give PCR product  793 bp. Segment II from (631-1482) to give PCR product 851 bp. There was an overlapped sequence about 300 bp.  Which were curated manually. The  resulted  sequence covered the region from(39-1482), these were aligned using Clustal W, and phylogenetic tree was built using NJ method, all these sequences were  deposited  at  NCBI/nucleotide (GenBank database)  with Accession numbers (LC006979, LC008196, LC009412, LC009414, LC034168, LC038020, LC041163,  LC041368). The primers sequences used in PCR experiments are shown in Table 1.

TABLE 1:  PRIMER PAIRS FOR accD3 GENE SEGMENTS

Amplification of Target gene: AccD3 gene segments (SI and SII) were amplified separately. Each PCR mixture was prepared with 25 µl of Green Master  Mix 2x(Promega), 17µl  of  nuclease  free  water, 2µl of each  primer ( F,R)  at 10µM,  and  4µl of  DNA (equaling 25 to 250 ng). Multiple PCR programs were used until reach to  the  optimum  program  which  gives  a good  PCR product, the  thermocycling  conditions for  SI and SII  were  94 °C for 5 minutes,  followed by 35 cycles of  denaturation  at 94 °C for 1 minute , and  annealing  at  59 °C  for 1.30 minute and 72 °C for 1 minute, then the final extension  steps at 72 °C for 10 minutes. PCR amplified  products (10µl)  and  DNA molecular –weight marker (Ladder) were  electrophorized  on  1% Agarose gel with ethidium bromide staining to verify the size of  the amplicon, the resulted PCR product were sequenced (NICEM-USA, Apparatus: Applied Biosystem).

Computer aided drug design and Discovery steps:

Target Determination: The following programs and databases were used for target determination:

NCBI Database ⁷

(http://www.ncbi.nlm.nih.gov/)

Uniprot Database ⁸

(http://www.uniprot.org/)   

KEGG Database ⁹

(http://www.genome.jp/kegg/)

TMHMM server v.2.0 ¹⁰

(http://www.cbs.dtu.dk/services/TMHMM/

Tuberculist Database ¹¹

(http://tuberculist.epfl.ch/)

Mega6 software v 6.1 ¹²

Homology Modelling: The following servers were used for protein modeling:

Swiss-Model Server ^{13, 14}

(http://swissmodel.expasy.org/)

QMEAN server:

(http://swissmodel.expasy.org/qmean/cgi/index.cgi) for the best protein model estimation. ¹⁵

 Ligand search and Pharmacophore building:

A. Zinc database ¹⁶

(http://zinc.docking.org/)

Zinc Pharmer ¹⁷

(http://zincpharmer.csb.pitt.edu/).

Discovery Studio software v.2.5: ¹⁸

Toxicity and Mutagenicity Estimation:

Lazar software

(http://lazar.in-silico.ch/predict) ¹⁹

T.E.S.T software v.4.1 ²⁰

In vitro application:

Resazurin Microtiter Assay (REMA) is a Colorimetric Assay was used in this study to test the activity of drugs on the viable cells of M. tuberculosis. ^{21, 22}

RESULTS AND DISCUSSION: M. Tuberculosis contain multiple versions of accD genes that encode α and β subunits of at least three distinct multifunctional acyl CoA Carboxylase Complexes ^{23, 24}. The function of a number of genes involved in fatty acid  and  mycolic  acid biosynthesis  are  known  for their role  in the survival  of  pathogenic  M. tuberculosis ^{25, 26}. In this study, (Rv1970) gene was used to differentiate M. tuberculosis from the other of M. tuberculosis subspecies, LprM (Rv1970) is a gene lies in the RD7 region ^{27, 28}. In prior studies, PCR analysis revealed RD7 to be present in all of the tested M. tuberculosis but not in the evaluated M. microti, M. bovis, M. bovis BCG, and M. caprae strains ^{29, 30}. Primers were used to amplify Rv1970 (mce3E) and the PCR results were positive for 40(80%) of isolates (Figure not shown).

Two PCR products of typical M. tuberculosis phenotype namely Asraa 1 and Asraa 2 were sequenced and deposited in NCBI with Accession numbers KJ809110, KJ809111 respectively and at DDBJ database with Accession numbers LC009880, LC009881.

In  this  study,  accD3 (Rv0904c)  was used as a target protein  for  Computer  Aided  Drug  Design  because  the  product  of  this gene  is acetyl-CoA carboxylase  which  catalyzes the first step in the pathway of fatty acid biosynthesis in M. tuberculosis. Acetyl - CoA is coupled  with (Carbon mono Oxide ) CO to form  malonyl - CoA,  which  is used  solely  for  the  production of fatty acids and related  compounds,  this  step is the main point for regulation of the entire pathway, thus  making it an attractive  target  for the development of new drugs ^{31, 32}.

H37Rv strain was  used as the reference strain in this study, accD3 (Rv0904)  sequence  of  H37Rv  was  retrieved  from  NCBI  and  aligned with other  M. tuberculosis strains to find  the conserved regions in the accD3 sequence  among  the  different  strains.  Fig. 1 shows the alignment results using Clustal W incorporated in Bio Edit program.

FIG. 1: AccD3 SEQUENCES OF DIFFERENT M. TUBERCULOSIS STRAINS

The information about the target gene (accD3) were collected from VFDB (Virulence Factor of Pathogenic Bacteria) and Tuberculosis databases, Rv0904c (accD3) is a gene located at 1006693 – 1008180 from M. tuberculosis (H37Rv) genome and considered a relatively long gene (Fig. 2).

FIG. 2: GENOMIC LOCATION OF Rv0904c. TUBERCULIST DATABASE

Due to the long length of the gene, it was divided in to two segments (SI and SII).  Primer  pairs  for SI able to amplify the region on genome at location (38-831) with  PCR  product 793 bp which represent the first half of target gene and primers pair for SII able to amplify the region (631-1482) with PCR product 851 bp which  represent  the  second  half  of the target gene as shown in Table 2. 

TABLE 2: PRIMER PAIRS FOR accD3 GENE SEGMENTS DESIGNED WITH PRIMER-BLAST PROGRAM

The  forty  positive  for  M. tuberculosis isolates were subjected to  PCR  amplification  and the designed  primer  pairs for the two segments of accD3 gene were  used to amplify the  region  of  accD3 gene  at  location 1006693-1008180 on the M. tuberculosis  genome. (Figures not shown). The resulted products were sequenced. BLASTn at NCBI showed that these  sequences were matched with M. tuberculosis at Query cover 98% for SI and  97%  for  SII  and  identity percentage  reached  to 99% at E-value 0.00 as shown  in  Fig. 3 for  SI  and  Fig. 4  for SII.

FIG. 3: ALIGNMENT RESULTS OF accD3 (SI)

FIG. 4:  ALIGNMENT RESULTS OF accD3 (SII)

There  was  an  overlap  sequence  with  length  about 300 bp between SI and SII, this was removed  and  merged using Mega 6 and merger tools at EMBOSSpackage(http://emboss.bioinformatics.nl/cgi-bin/emboss/merger) to obtain the whole  sequence. The  obtained  sequences of accD3 were BLASTed with  other  strains  in  GenBank  database  at E.value 0.00 and the results were showed  that  the query  sequences  were  matched  the accD3 sequences in the databases  with  Query  cover  100%  and  identity  percentage  99%. Table  3 shows  the  results of  BLAST  program  (nucleotide  Blast)  for the eight obtained accD3 sequences which were designated as (ALQM1, ALQM2, ALQM3,  ALQM4,  ALQM5,  ALQM6,  ALQM7,  ALQM8)

TABLE 3: BLASTING RESULTS OF accD3 SEQUENCES

The whole sequences of accD3 of different strains of this study (ALQM1, ALQM2, ALQM3, ALQM4, ALQM5, ALQM6, ALQM7, ALQM8) were deposited at NCBI with Accession numbers (LC006979, LC008196, LC009412, LC009414, LC034168, LC038020, LC041163, LC041368) respectively.

The eight nucleotide sequences were translated in to amino acids (495 amino acids) sequences  using EMBOSS software (http://www.ebi.ac.uk/Tools/ emboss/) and the resulted protein sequences were  subjected  for confirmation, then the eight protein sequences modeled by using SWISS-MODEL server (http://swissmodel.expasy.org/) ¹³ with  three  templates 2f9y, 2f9i and 1xou proteins, Fig. 5 shows the best model for accD3 model.

FIG. 5: AccD3 PROTEIN (H37Rv STRAIN) 3D MODEL OF M. TUBERCULOSIS

All old and new drugs which were used in TB treatment and some natural products were collected from literatures and research papers and used as ligands in this study. These ligands are:

Old drugs: ^{33, 34}

Isoniazid (INH)

Rifampin (RIF)

Fluoroquinolone

Pyrazinamide (PZA)

Ethambutol (EMB)

Streptomycin (SM)

Cycloserine

Capreomycin

ρ-Aminosalicylic acid

Levofloxacin

Moxifloxacin

Gatifloxacin

Amikacin/Kanamycin

Ethionamide

Macrolides

New Drugs: ³⁵

Nitroimidazoles

PA-824

OPC-67683

TMC207

Linezolid 

III. Natural products: ³⁶

Pyridomycin

Vitamin D3

Docking: Many softwares are available to predict the molecular interactions that may occur between a target protein and small molecule. Among these Swiss Dock, which is a web server dedicated for docking of small molecules on target proteins ³⁷.

All 22 molecules and models of this study were entered to EADockDSS engine in the Swiss Dock server and the results were visualized with UCSF Chimera. Five molecules have a high negative free binding energy (lowest binding energy) and these were chosen for pharmacophore building ³⁸.

TABLE 4: MOLECULES BINDING ENERGY

The characterization of the structure and the energetics of molecular complexes are a key factor for understanding biological functions and the energetics often  provides  the most important and useful link between structure and function of  biomolecular  systems. Furthermore, the prediction and design of ligands that can reversibly bind to pharmaceutical targets (enzyme inhibitors, receptor agonists and antagonists etc.) is at the heart of structure-based drug design. To be able to predict the strength of noncovalent associations, as well as the structures of molecular complexes, has therefore been an important objective in computational chemistry ³⁹.

Virtual  screening (VS)  is  the  process  of evaluating a library of compounds  using  a computational  model  in order to rank, and  thus  screen for molecules that exhibit desired characteristics, LBVS (Ligand based virtual screening)  was  adopted in this  study  and  pharmacophore  model  generated from a set of known ligands which was used in the virtual screening to elicit specific inhibitors against accD3 protein ^{40, 41}.

In this study,  Discovery Studio program ⁴² was used for  pharmacophore  modeling  and  the five molecules  from the previous step were  entered  to  the  Hip  Hop  generator  at  Discovery  Studio  program and gave two hypothetical  pharmacophores (pictures not shown).

These pharmacophores were used in the Zinc Pharmer program (pictures not shown). Zinc Pharmer provides tools for constructing and refining pharmacophore hypotheses directly from molecular structure ⁴³. One thousand molecules were obtained from Zinc database after filtering through Lipinskie’s rules of five, All molecules tested for their mutagenicity and toxicity with LAZAR and T.E.S.T softwares.

All molecules were used in the Swiss Dock to estimate their binding affinity to the proteins of all nine strain depending on the binding free energy Fig. (6A, B) shows the protein-ligand docking results with minimum binding energy visualized with chimera UCSF program (Data table not shown)

A 

B

FIG. 6 A, B: PROTEIN-LIGAND DOCKING RESULTS

For  accD3 modeled  protein ( H37Rv strain), All molecules gave a negative binding energy  (positive  docking results)  ranged  between -2.3 as a lowest value  and  -36.7  as  a highest  value. Twenty nine molecules  gave  a negative  binding  energy  with  accD3 modeled  protein (ALQM1 strain)  ranged  between -1.15  as a lowest  value  and - 43.6 as a highest value. The  same  molecules  gave  a negative  binding energy with the model of  ALQM2  strain  with -2.3 as  a lowest  value  and  -46.3  as  a highest  value. Twenty seven  out of  30  gave a negative binding energy with the modeled protein  of ALQM3 strain  and  values  ranged  between -1.4  as a lowest value and -43.5 as a  highest  value.  Twenty nine  out of  30 gave  a negative  binding  energy with the  modeled  protein  of  ALQM4  ranged  between -0.1 as a lowest value and -43.5 as a highest  value.

All 30 molecules gave a negative binding energy with the model  of  ALQM5 strain  and  the value  ranged  between -3.34 as a lowest value and  -42.08  as  a highest  value. Twenty seven out of 30 molecules gave a negative binding energy with the modeled protein of ALQM6 and the values ranged between -1.45 as a lowest value and -43.9 as a highest value. The same molecules gave a negative binding energy with the modeled protein of ALQM7 strain and the values ranged between -0.6 as a lowest value and -43.7 as a highest value. All molecules gave a negative binding energy with the modeled protein of ALQM8 and the values ranged between -1.9 as a lowest value and -38.8 as a highest value.

Two molecules with Zinc ID (391093, 16051516) are commercial available, the chemical structures of these molecules are shown in Fig. (7 A, B).

2'Hydroxypropiophenone        trans-Cinnamic acid

FIG. 7: A, B: THE CHEMICAL STRUCTURES OF THE SELECTED LIGAND MOLECULES

In vitro Application of  Drugs: In this study,  Resazurin  Microtiter  Assay  Plate (REMA) was used to prove the  inhibitory  activity  of  ligands against the viable cells of  different strains of M. tuberculosis, this test based on Resazurin which is a blue - purple, non-toxic, oxidation-reduction indicator that becomes pink when reduced to resorufin by cellular oxidoreductases ⁴⁴. This assay is colorimetric and the rate of color change is directly proportional to the number of viable cells in the initial suspension. In other words, the concentration of viable cells in a suspension containing resazurin directly determines the visible conversion from purple to a pink color ^{44, 45}.

The wells  with  dark  purple color are  more sensitive to the  added molecule suspension when compared with wells of  the control,  and this match with the study of ⁴⁵, in this study, six different  concentrations  (10 µg/ml, 50 µg/ml, 75 µg/ml, 100 µg/ml, 150µg/ml, 200µg/ml) of Ligand I (trans-Cinnamic acid), Ligand II (2'-Hydroxypropiophenone) and Ligand III (as a control) (4-Amino-2-hydroxybenzoic acid)  were applied on sensitive and MDR strains of M. tuberculosis and the inhibitory activity of  these ligands determined depending on  color change from  blue - purple (resazurin color ) to pink color as shown in Fig. 8 for ligand 1 which have been inspect visually the effect on two sensitive  strains of M. tuberculosis (sensitive to the routine drugs regime in TB treatment), the results described as high sensitive to the new ligands at concentration above 10µg/ml.

A (Sensitive I)                                    B (Sensitive II)

FIG. 8:  EFFECT OF LIGAND MOLECULE I (TRANS-CINNAMIC ACID) ON TWO DIFFERENT SENSITIVE STRAINS (I, II), FROM LEFT TO RIGHT, POSITIVE CONTROL, LIGAND1, NEGATIVE CONTROL

Fig. 9 shows the effect of ligand I on two MDR strains of M. tuberculosis, the results described as high sensitive to the new ligands at concentration above 75µg/ml.

A (MDR I)                                               B (MDR II)

FIG. 9: EFFECT OF LIGAND MOLECULE I (TRANS-CINNAMIC ACID) ON TWO DIFFERENT MDR STRAINS (I, II), FROM LEFT TO RIGHT: POSITIVE CONTROL, LIGAND1, NEGATIVE CONTROL

Fig. 10 shows the effect of ligand II on two sensitive strains of M. tuberculosis and the results described as high sensitive at all concentrations.

A (sensitive I)                                     B (Sensitive II)

FIG. 10: EFFECT OF LIGAND MOLECULE II (2'-HYDROXYPROPIOPHENONE) ON TWO DIFFERENT SENSITIVE STRAINS (I, II), FROM LEFT TO RIGHT: POSITIVE CONTROL, LIGAND II, NEGATIVE CONTROL

Fig. 11 shows the effect of ligand II on two MDR strains of M. tuberculosis and the results described as high sensitive at concentration above 75µg/ml.

A (MDR I)                                      B (MDR II)

FIG. 11: EFFECT OF LIGAND MOLECULE II (2'-HYDROXYPROPIOPHENONE) ON TWO DIFFERENT MDR STRAINS (I, II), FROM LEFT TO RIGHT: POSITIVE CONTROL, LIGANDII, NEGATIVE CONTROL

Fig. 12  shows  the  effect of  ligand III on  two  different  sensitive  strains of M. tuberculosis  and  the  results  described  as high  sensitive  at all concentrations.

A (Sensitive I)                                  B (Sensitive II)

FIG. 12: EFFECT OF LIGAND MOLECULE III (4-AMINO-2-HYDROXYBENZOIC ACID) AS A CONTROL ON TWO DIFFERENT SENSITIVE STRAINS (I, II), FROM LEFT TO RIGHT: POSITIVE CONTROL, LIGAND III, NEGATIVE CONTROL

From in vitro studies, it could concluded that:

Sensitive isolate: Trans-Cinnamic acid is effective at 100µg/ml, 150µg/ml & 200µg/ml.

It is obtained from oil of cinnamon, it is also found in shea butter. ⁴⁶ used trans - cinnamic acid as anti-hepatitis virus agent.

2'-Hydroxypropiophenone is effective at all concentrations.  It is used as a flavorant to enhance the flavor of wintergreen-flavored products ⁴⁷.

4-Amino-2-hydroxybenzoic acid is effective at all concentrations.

MDR isolate:

Trans-Cinnamic acid is very effective at 150µg/ml, 200µg/ml.

2'-Hydroxypropiophenone is very effective at all concentration.

The  difference  of   effects  attributes  to the variations of  sensitive strains, and due  to  the changes occurred in  mutated  strains, looking  for  the real reasons need   more  studies  at least  at  the  molecular level. It might be expected to be something related to permeability and pump effluxes.

ACKNOWLEDGMENT: A special thanks for the NRL (National Reference Laboratory) at the Institute of Chest and Respiratory diseases / Baghdad for their cooperation in the achievement of this research.

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    Abdul-Jalil AA and Al-Khafaji ZM: New inhibitors against accD3 gene of Mycobacterium tuberculosis, in-silico study and in -vitro application. Int J Pharm Sci Res 2017; 8(7): 2839-48.doi: 10.13040/IJPSR.0975-8232.8(7).2839-48.

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