Targeting Protein Biotinylation for Enhanced Tuberculosis Therapy

This report investigates the potential of targeting protein biotinylation to enhance tuberculosis chemotherapy, addressing the growing challenge of drug-resistant Mycobacterium tuberculosis (Mtb). The research focuses on chemically inhibiting biotin protein ligase (BPL) using Bio-AMS to disrupt Mtb's metabolism and cell envelope permeability. The study details the materials and methods used, including experiments on rifampicin-doxycycline interaction, Bio-AMS catalytic assays, and LC-MS/MS analysis. Results indicate that Bio-AMS inhibits Mtb growth and reduces drug resistance, particularly when combined with rifampicin. The conclusion highlights the potential of this approach to increase Mtb's susceptibility to rifampicin, offering a promising strategy to combat tuberculosis, while acknowledging limitations in characterizing Mtb BPL and improving Bio-AMS bioavailability.

TARGETING PROTEIN BIOTINYLATION ENHANCES TUBERCULOSIS
CHEMOTHERAPY
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INTRODUCTION.
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).
HYPOTHESIS
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.
OBJECTIVES.
This study was done under the guidance of the following objectives:
1. To ascertain effects of inactivating biotin protein ligase inhibitors
chemically.
2. To determine the rate and mechanism of resistance of Mtb to inactivated
biotin protein ligase inhibitors.
3. To characterize metabolism of Mtb
GAP OF KNOWLEDGE.
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).
MATERIALS AND METHODS.
To achieve the objectives of this research, the following materials were required and the
following methods were followed (Srivastava & Gumbo,2011).:
Method: Studies of Rifampicin-doxycycline interaction.
-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.
EXPLANATIONS AND DESCRIPTIONS.
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.
Rifampicin and doxycycline quantitation in mouse plasma.
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).
Use of Bio-AMS as a substrate for Rv3406 catalytic assay.
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.
Identification of products generated by Rv3406 in Bio-AMS degradation using LC-
MS/MS Analysis.
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.

Analysis of Bio-AMS and other metabolites using HPLC for analyses of
intrabacterial metabolism.
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.
Bio-AMS and metabolite 4 analyses in Mtb.
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.
Bio-AMS and biotin quantitation in mouse plasma.
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.
RESULTS.
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.
DISCUSSION.
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.)
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).
SUMMARY
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).
ARTICLES WITH RELATED WORK.
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.
CRITIQUE.

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).
CONTROVERSIES IN THE RESEARCH.
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).

REFERENCES
Bazet, L., Diacovich, L., Cabruja, M., Bardou, F., Quémard, A., Gago, G., & Gramajo, H.
(2014). Pleiotropic effect of AccD5 and AccE5 depletion in acyl-coenzyme A
carboxylase activity and in lipid biosynthesis in mycobacteria. PLOS ONE (9), e99853.
Bhatt, A., Fujiwara, N., Bhatt, K., Gurcha, S.S., Kremer, L., & Chen, B. (2007). Deletion of
kasB in Mycobacterium tuberculosis causes loss of acid-fastness and subclinical latent
tuberculosis in immunocompetent mice. Proc. Natl. Acad. Sci. U.S.A. (104), 5157–5162.
Dartois, V., & Barry, C.E., (2010). Clinical pharmacology and lesion penetrating properties of
second- and third-line antituberculous agents used in the management of multidrug-
resistant (MDR) and extensively-drug resistant (XDR) tuberculosis. Curr. Clin.
Pharmacol. (5), 96–114.
Draper, P. (1998). The outer parts of the mycobacterial envelope as permeability barriers. Front.
Biosci. (3), D1253–D1261.
Egelund, E.F., & Alsultan, A. (2015). Peloquin, Optimizing the clinical pharmacology of
tuberculosis medications. Clin. Pharmacol. Ther (98),387–393.
Galvão, C. R., Lima, L. H., Gomes, T. D, Pagani, M. A., Ferreira, A. S. & Gonçalves, T.T.
(2014). The BCG Moreau RD16 deletion inactivates a repressor reshaping transcription
of an adjacent gene. Tuberculosis 94,26–33.
Gill, W.P., Harik, P.N., Whiddon, M.R., Liao, R. P., Mittler, D., & Sherman, D.R. (2009). A
replication clock for Mycobacterium tuberculosis. Nat. Med. (15), 211–214.
Jarlier, V., & Nikaido, H. (1994). Mycobacterial cell wall: Structure and role in natural resistance
to antibiotics. FEMS Microbiol. Lett. (123), 11–18.
Karakousis, P.C., Yoshimatsu, T., Lamichhane, G., Woolwine, S. C., Nuermberger, E.L., &
Grosset, J. (2004). Dormancy phenotype displayed by extracellular Mycobacterium
tuberculosis within artificial granulomas in mice. J. Exp. Med. (200) 647–657.
Kruh, N., Troudt, J., Izzo, A., Prenni, J., & Dobos, K.M. (2010). Portrait of a pathogen: The
Mycobacterium tuberculosis proteome in vivo. PLOS ONE (5), e13938.
Nikonenko, B. V., Protopopova, M., Samala, R., Einck, L., & Nacy, C.A., (2007). Drug therapy
of experimental tuberculosis (TB): Improved outcome by combining SQ109, a new
diamine antibiotic, with existing TB drugs. Antimicrob. Agents Chemother. (51) 1563–
1565.

Moliva, J.I., Turner, J., & Torrelles, J.B. (2015). Prospects in Mycobacterium bovis Bacille
Calmette et Guérin (BCG) vaccine diversity and delivery: Why does BCG fail to protect
against tuberculosis? Vaccine (33), 5035–5041.
Monteleone, M., Wilson, D.J., Aldrich, C.C., Barry, C.C., Rhee, K.Y., Ehrt, S., & Schnappinger,
D. (2013). A genetic strategy to identify targets for the development of drugs that prevent
bacterial persistence. Proc. Natl. Acad. Sci. U.S.A. (110), 19095–19100.
Morris, R.P., Nguyen, L., Gatfield, J., Visconti, K., Nguyen, J., & Schnappinger, D (2005).
Ancestral antibiotic resistance in Mycobacterium tuberculosis. Proc. Natl. Acad. Sci.
U.S.A. (102), 12200–12205.
Nuermberger, E.L. (2011). The role of the mouse model in the evaluation of new antituberculosis
drugs, in Antituberculosis Chemotherapy, Donald, P.R., Helden, P.D., chap. 15, pp. 145–
152.
Nguyen, L., & Pieters, J. (2009). Mycobacterial subversion of chemotherapeutic reagents and
host defense tactics: Challenges in tuberculosis drug development. Annu. Rev.
Pharmacol. Toxicol. (49), 427–453.
Nguyen, L., & Thompson, C.J. (2006). Foundations of antibiotic resistance in bacterial
physiology: The mycobacterial paradigm. Trends Microbiol. (14) 304–312.
John, G. (1991). Ethics and Behaviour. (1), 200.
Kurth, D.G., Gago, G.M., de la Iglesia, G., Bazet, L., Lin, T. W., Morbidoni, T. (2009). ACCase
6 is the essential acetyl-CoA carboxylase involved in fatty acid and mycolic acid
biosynthesis in mycobacteria. Microbiology (155), 2664–2675.
Schnappinge, R, D., O’Brien, K.M., & Ehrt, S. (2015). Construction of conditional knockdown
mutants in mycobacteria. Methods Mol. Biol. (1285) 151–175.
Sogi, K. M., Gartner, Z. J., Breidenbach, M.A, Appel, M. J., Schelle., J & Bertozzi, C.R. (2013).
Mycobacterium tuberculosis Rv3406 is a type II alkyl sulfatase capable of sulfate
scavenging.
Srivastava, S., & Gumbo, T. (2011). In vitro and in vivo modeling of tuberculosis drugs and its
impact on optimization of doses and regimens. Curr. Pharm. Des. (17), 2881–2888.
World Health Organization. (2014). Antimicrobial Resistance: Global Report on Surveillance.