Hypothesis
Treatment of tuberculosis using chemotherapeutic approach has become a greater challenge in the past. This is evident from the statics released in 2013 which indicated that the number of people who were infected with multidrug-resistant tuberculosis was around 480,000. This is attributed to resistance of Mtb to drugs used. The distinct components and effects of drugs used determine their effectiveness (W. H. Organization, 2014).
Invention of efficient vaccine against tuberculosis is challenging. Drugs developed are quickly resisted by bacteria. This research aims on interfering with metabolism of bacteria by destroying its semi-permeable cell wall.
This study was done under the guidance of the following objectives:
- To ascertain effects of inactivating biotin protein ligase inhibitors chemically.
- To determine the rate and mechanism of resistance of Mtbto inactivated biotin protein ligase inhibitors.
- To characterize metabolism of Mtb
World Health Organization (2017), reported that tuberculosis is the top infectious disease which has proven difficult to control. Various vaccines have been developed to manage an infection but they fail. (Kurth et al.,2009).
TB vaccine community in 2012 came up with a Blueprint for developing vaccine for TB. Among the attempts was on the use of cytomegalovirus (CMV) vector. CMV has an ability to slow down the rate of replication of Mtb but cannot stop its trasmission (Lyonnete et al.,2014).
Another attempt was on the use of mRNA vaccine. This vaccine was used to make any symptoms of the tuberculosis especially in latent type of tuberculosis to be expressed so that it can be treated. This vaccine presented a challenge in the way that it could be administered into the body (Nikonenko, Protopopova, Samala, Einck & Nacy,2007). The use of intravenous route was impossible since it could be integrated into non-functional vector (Galvão et al.,2014).
Due to this existing gap, this research focuses on use of biotinylation in fighting against the Mtb. (Moliva, Turner & Torrelles,2015).
To achieve the objectives of this research, the following materials were required and the following methods were followed (Srivastava & Gumbo,2011).:
-Four groups of female mice belonging to 4 CD-1, age: 4-6 weeks, 20-22 g weight, Rifampicin, Sucrose solution., Water-bath sonicator, Centrifuge, LC/MS-MS
Method: Rifampicin and doxycycline quantitation in mouse plasma.
-Water, Acetonitrile, Ascorbic acid, Methanol, Verapamil, Vertexing machine, Agilent and Mass selective detector.
Method: Use of Bio-AMS as a substrate for Rv3406 catalytic assay.
-Centrifuging, HPLC
Method: Identification of products generated by Rv3406 in Bio-AMS degradation using LC-MS/MS Analysis.
-LC-MS/MS, formic acid.
Method: Analysis of Bio-AMS and other metabolites using HPLC for analyses of intrabacterial metabolism.
– HPLC, mass spectrometry.
Method: Bio-AMS and metabolite 4 analyses in Mtb.
– Agar plates.
Method: Bio-AMS and biotin quantitation in mouse plasma.
– Acetonitrile, water, methanol, verapamil, mass spectrometer, LC/MS.
This section gives a detail on the procedures that were followed in carrying out the experiments using the above listed methods and their respective materials (Srivastava & Gumbo,2011).
Studies of Rifampicin-doxycycline interaction.
Selected four female mice were administered orally with 2000 ppm of rifampicin.
In preparing the rifampicin administered, 40% of sucrose was added in it and digested for ten minutes in water-bath sonicator and finally it was subjected to probe sonication for a period of three minutes.
40-50 mm were then collected from the veins of tails of the mice in both groups.
Different mice groups were given different doses.10 mg/kg of rifampicin was given to group one as a single dose. Doxycycline with chow was given consecutively for 7 days and a single of 10mg/kg of rifampicin was given on 7th day. For seven consecutive days, group three was given 10 mg/kg of rifampicin while group four was given for seven consecutive days 10 mg/kg of rifampicin and doxycycline having chow. Blood sample collection was done on seventh day in pre-dose,5 min, 30 min, 1 h, 3 h,5 h and 8 h after dose using heparin-made tubes. Plasma from blood were recovered by centrifugation and quantified for rifampicin and doxycycline using LC/MS-MS.
Objectives
To obtain the analytes for analysis,20 microliters of mouse plasma was diluted using 180 milliliters of methanol and acetonitrile having 20 ng/rifampicin in the ratio of 1:1, 5 microliters of 75 mg/mL ascorbic acid and 180 milliliters of methanol mixed with acetonitrile having 20ng/mL rifampicin and 10 ng/mL of verapamil to dissolve doxycycline. Vortexing and centrifugation was done to the mixture. 200 microliters of supernatant were retrieved and analyzed using LC/MS-MS which had 0.1% formic acid in pure water as mobile phase A and 0.1% formic acid in pure acetonitrile as mobile phase B. 2 mL was the volume of injection. m/z 823.50/791.60 ions for rifampicin and m/z 445.20/410.10 ions for doxycycline were detected by mass selective detector in MRM mode. 5 ng/mL and 50 ng/mL lower limits were set for rifampicin and doxycycline respectively. The mean of analyte concentrations from Microsoft excel were used to calculate pharmacokinetic parameters by rule of linear trapezoid excluding values of concentration below quantification lower limits. AUC values were compared using unpaired parametric t test (Bazet et al.,2014).
Rv3406 was added to the assay buffer containing1 mM Bio-AMS, 1 mM α-KG, 3 μM Rv3406
acetate buffer, pH 7.5, 50 mM NaCl, 0.2% triton X-100 and 100 μM FeCl2 to initiate reactions. 20 μL of solution were taken out at 0, 30, 60, 120, 240, 360and 1440 minutes and
20 μL of 10% TCA added for protein precipitation. The supernatant of 30 μL was analyzed using HPLC. Also, 7 diverse Bio-AMS concentrations were used and precipitated by addition of TCA solution, 10%, 20 μL. Metabolite 4’s concentration was quantified using LC-MS/MS to obtain Bio-AMS’ concentration s initial velocity.
Cs2CO3 was added to sulfamide and biotin-NHS ester in 10 mL DMF. () cooled to stirred at 0 ºC for 30 min followed by warming to 27 ºC and 15hrs of stirring and purification. The mixture was collected and lyophilized to obtain N-(biotinoyl) sulfamide 4.
LC-MS/MS set into Multiple Reaction Monitoring was used to analyze metabolite 4 by injecting 10 μL into positively ionized MS at a flow rate of 0.5 mL/min under 40 °C for 8 minutes. The precursor and ion fragments were detected at m/z 323 and 227.2 respectively. The area of peaks formed were calculated by adjusting them to normal internal standard peaks with standard curves of analytes whose area would be calculated with ease.
reverse phase HPLC was used for sample analysis with 1 mL/min as a flow rate. The mobile phase A and B had 20 mm of TEAB and acetonitrile respectively. These concentrations were increased to 20% after ten minutes and 95% after 13 minutes. The columns were allowed to re-equilibrate after resting to 5% for four minutes. 20 μL of the sample was injected and allowed to run for 21 minutes along with detection using ultra violate rays. The peaks formed were analyzed using the mass spectrometry.
Agar plates were used to grow Mtb laden filters for 5 days, exposed for 18 hours to Bio-AMS in GAST medium with25 μM of Bio-AMS then 24 hours incubation with antibiotic in GAST.
Gap Of Knowledge
Bio-AMS, N-(biotinoyl) sulfamide 4 and biotin after extraction using normal volumes of acetonitrile, methanol, verapamil and water from plasma of mouse were analyzed. The process involved protein precipitation, Vortexing and centrifugation and supernatant withdrawn and injected into mass spectrometer for quantification.
The outcomes of experiments were as follows.
Effects of inactivating BPL chemically on viability of Mtb in vitro
Mtb were killed by isoniazid Bio-AMS inhibitor as shown in fig. 1A (Kruh, Troudt, Izzo, Prenni & Dobos,2010).
However, resistance to Bio-AMS was displayed by Mtb after 10 days as the Bio-AMS reduced to below detection limit of 25 CFU/ml while Mtb colony increased as shown in fig.1A.
Change in carbon amounts impacted less on MIC of Bio-AMS and concentration of Mtb as in figures 1B and S1A respectively.
There was increase in MIC of Bio-AMS with addition of biotin. Fig. S1B
There was no reduction in Mtb viability with reduction of PBS. Fig S1C.
There was inhibition of Mtb growth which depended on concentration by Bio-AMS. Fig.1C.
There was no Bio-AMS mitochondrial (fig. S2) and Mouse macrophage (fig. S1D) toxicity in reduction of tetrazolium assay respectively.
Mtb resistance development to chemically inactivated BPL
There was expression of wild BPL by Mtb resistant to Bio-AMS when isolation frequency of drug resistant Mtb was retained while that of Bio-AMS reduced. Fig. S3A.
There were strains that inhibited mutation by inactivating rv3405c as shown in table S1.
There was a change in rv3406 when comparison of mRNAs and Mtb resistant to Bio-AMS were made as shown in table S2.
Controlling Mtb infection in mice by targeting BPL.
Bio-AMS prevents Mtb growth. There was no emergence of resistant strains of Mtb as well as shown in figure 3B.
The use of doxycycline or reducing BPL reduced the pathology of tissue in the lungs of mice. (fig.4D) (Karakousis et al.,2004).
Significance of biotinylating Mtb protein to the rifampicin, ethambutol and isonlazid activity.
After treatment of Mtb with Bio-AMS lethal dose followed by acid fast staining, it was discovered that there was reduction in protein biotinylation as shown in figure S10.
Use of rifampicin and ethambutol in combination with Bio-AMS for 20 days showed a 99.7% mortality rate of Mtb. This was as a result of destruction of the cell envelope permeability in the Mtb. Cell envelope was destroyed by increasing Bio-AMS which enhanced ability of rifampicin to penetrate it, interfering with its metabolism (figure S11).
The effect impacted on rifampicin activity by interference of protein biotinylation during Mtb infection.
Use of rifampicin and isoniazid killed 200 Mtb in 4 weeks.
Tuberculosis has the highest mortality rate up to date. Many drugs have been developed against the disease but without success because the bacteria causing the disease is sensitive to the drugs and quickly develop resistance towards the drugs. (Woong et al.,2011).
The research that has been carried out based on experiments indicate that Mtb BPL activities can be inhibited by chemically activated Bio-AMS. It disrupted on growth process of Mtb in vitro, a highly sensitive and resistant to drugs (Draper,1998).
Findings from research indicate that by chemically inhibiting BPL in Mtb, their growth significantly reduced resulting into decrease in number of Mtb strains resistant to drugs (figure 1, A -B & 2 A.)
Materials And Methods
It was confirmed that when BPL was depleted during either acute or chronic infection, there was s higher death of the Mycobacterium tuberculosis as shown in figures 4, C, D and S9.
Conclusion.
The experiments conducted demonstrated to greater extend that activities of MTB can be stopped by interfering with its protein biotinylation process by chemically inhibiting BPL using Bio-AMS. This method increases the Mtb’s susceptibility to rifampicin which in turn inhibits development of cell envelope resulting into loss of cell permeability hence lowering the drug resistance level.
The research experienced various challenges and limitations (Egelund & Alsultan,2015).
Among the limitations was in the difficulty in characterization of Mtb BPL. There are several strains of the bacteria with different BPL and therefore determining their appropriate inhibitors was difficult. In addition, it was challenging to improve on the bioavailability of Bio-AMS to Mtb BPL which is the target for the inhibition. This is because of cleavage that occur at the link between biotin and adenosine. This contributes to the bacterium’s ability to become sensitive and resistant to the drugs administered.
Research experimented on the possible ways of remedying this by improving on the bioavailability of Bio-AMS to BPL in the bacteria. This was achieved by enhancing the bond between acyl and sulfamide linked to molecules of nucleoside and biotin.
This approach did not only promote the Bio-AMS bioavailability but it also reduced the resistance and sensitivity of Mtb mutant strains to the drugs (Bhatt et al.,2007).
There was difficulty in identifying the enzyme that was to be used in while maintaining the viability if Bio-AMS. However, an enzyme was identified that could carry out the task, that is, alkyl sulfatase Rv3406 enzyme.
Finally, it is challenging relating animal symptoms with human (Galvão et al.,2014).
Treatment of tuberculosis using chemotherapeutic approach is difficult. Statics released in 2013 indicated that the number of people who were infected with multidrug-resistant tuberculosis was around 480,000(World Health Organization,2014). This is attributed to the constant resistance of the Mycobacterium tuberculosis to the drugs used. (Monteleone et al.,2013).
Having carried out research on existing vaccines, all drugs were resisted. This research aimed at breaking drug resistance in bacteria (Sogi et al.,2013).
The research and experiments carried out on the mouse demonstrated a successful use of BPL as the target in inhibiting the metabolic activities on the bacterium cell. This was done using the chemically activated Bio-AMS (Morris et al.,2005).
Various researches related to the use of protein biotinylation for tuberculosis treatment has been done. Among the articles that have covered the same include the work of Davya et al. (2018) in their work, “A new drug target for combatting TB”, demonstrate that the use of rifampicin to inhibit protein biotinylation in BPL enzyme was an effective method of killing Mtb.
Christopher, Barry and Aurelio (2015) in their article, “International journal of infectious diseases” agree that there is a challenge in vaccines for TB and there is a need to go out of common traditions into deep research to come up with effective vaccine.
Juan, Joanne and Jordi (2015) in their article, “Why does BCG fail to protect against tuberculosis?” agree that the use of BCG for TB control is only limited to infants but null in old people due to difference in Mtb strains. This calls for more effective vaccine that will overcome Mtb resistance.
In addition, Egelund, Alsutan and Peloquin (2015) in their work, “Optimizing the clinical pharmacology of tuberculosis medications”, suggest that pharmacokinetics and pharmacodynamics of rifampicin and isoniazid have proofed to be effective for TB treatment currently.
Explanations And Descriptions
This study was designed to carry out an in-depth evaluation of the past studies on Bio-AMS which is BPL inhibitor in the Mycobacterium tuberculosis. The study was also to prove that indeed BPL is the target part in the Mtb for drugs aimed at treating TB (Gill et al.,2009).
The characteristics of Bio-AMS were evaluated by subjecting it to different parameters which include diverse conditions for growth, mechanism of drug sensitivity and resistance, behaviour and the reaction with present drugs (Dartois & Barry,2010).
Several materials and methods were employed during the experiment such CFU calculation to determine bacterial activity. (Nuermberger,2011).
The test of Mtb ability to resist Bio-AMS was another method employed where the bacteria were cultured in agar plates with different drug concentrations. The rate at which resistance was developed by the bacteria was determined by measuring the CFU.
The dosage, environmental conditions and frequency of administration was changed in order to determine the effective dosage and period it could be effective (Jarlier & Nikaido,1994).
PK profiling was employed in selecting different groups of mice that were to be used in the experiment. This was aimed at establishing correct dosage that could be endured by an infected organism to the drug intended for TB treatment (Schnappinger, O’Brien & Ehrt,2015).
Van (2014) states that due to an increase in scientific research, there has been a great rise in the use of animals for this purpose. Making laws regarding protection of animal life is not enough.
In the use of any animal for research, an independent expert should be subjected to the research review in order to assess its viability (Grosset,2004).
This research used mice in testing drugs for tuberculosis treatment where some died in the process.
There are controversial ethical issues on use of animal pathological symptoms to come up with human medications because symptoms in animals are different in human. This trial and error are dangerous (John, 1991).
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