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NAMAN SHAH POLY ID-0396904

Discovery and Development of “Jainin” for the treatment of Pulmonary

Tuberculosis caused by Mycobacterium tuberculosis

Abstract/Summary: Pulmonary tuberculosis is the cause behind the death of 1.7 million people every year. The standard

treatment spans for 6 long months with stringent regimen. Patient compliance is difficult to achieve

which many times result in incomplete course of the medicine. This results in drug resistance in the

patients. As such Mycobacterium tuberculosis is a very hardy bacterium and drug resistance makes it

more difficult in treating the disease successfully. Moreover, the side effects of existence drugs are

unfathomable and give great mental and physical pain to the patient.

“Jainin” is the drug developed to counter all these and make the treatment of pulmonary tuberculosis

viable, successful and more generous on the patient. The treatment time reduces to about 4 months

with little or no side effects and virtually 100% curability.

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Contents

Sr. No. Title Page 1] Brief account on Pulmonary Tuberculosis 3

2] Medical need for a new drug for Pulmonary Tuberculosis 4

3] Target for new drug 5

4] Assay and structure of the lead 6

5] Lead Optimization 7

6] Pharmacological profiling of NCE 8

7] Biomarkers 11

8] Clinical Trials

Phase 1 12

Phase 2 13

Phase 3 14

9] Appendix 18

10] References 20

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[1] Introduction to TUBERCULOSIS

Tuberculosis is primarily caused by aerobic bacterium-Mycobacterium tuberculosis

SYMPTOMS AND PROGRESSION [21, 22]

Typical symptoms include:

• Fever, chills, chest pain, weakness

• Coughing up of blood

• Night sweats, appetite loss, weight loss

• Tuberculous pleuritis[1]

KIND OF PEOPLE AFFECTED & PREVALANCE OF TUBERCULOSIS [9]

• Annually 8 million people get infected with tuberculosis out of which 2.2 million people

develop tuberculosis.

• Tuberculosis is prevalent among all age groups but the age group of 25-44 has shown

the largest increase.

• Mostly people in the developing countries are at a greater risk.

TREATMENT [5]

• In the short treatment, isoniazid, rifampicin, pyrazinamide and ethambutol is given for

two monthsand then isoniazid and rifampicin are given for 4 months.

• The course runs for 6 months.

• Isoniazid is given for 9 months in latent tuberculosis.[21]

DIAGNOSIS [5]

• Chest X-ray gives a relevant idea of presence of tuberculosis.

• Microbial examinations of the sputum of the patient can also diagnose the disease. The

examinations include fluorescence microscopy.

• Bronchoscopy, biopsy and PCR can also be used to detect M.tuberculosis.[21]

• Tuberculin skin test like Mantoux test and Heaf test are used to detect the presence of

tuberculosis.

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[2] Is there a medical need to develop a new drug?

Table 1: Comparison of existing drugs in the market

DRUG [15,16,18]

MANUFACTURER[17]

MODE OF

ADMINISTRATION[19]

FREQUENCY[8]

SIDE EFFECTS[18]

ISONIAZID

1)Nydrazid

In

combination

with Rifampin

2)Rifamate

In

combination

with Rifampin

&

pyrazinamide

1)Rifadin

ETHAMBUTOL

1)Myambutol-

along with

isoniazid

Bristol-Myers

Squibb

Aventis

Pharmaceuticals

Aventis

Pharmaceuticals

Elan

pharmaceuticals

Oral

Oral

Oral or Injection IV

Oral

5 mg/kg up to 300mg

in a single dose; or 15

mg/kg up to 900

mg/day 2 or 3 times a

week

2 capsules once daily

10 mg/kg once a day

15mg/kg once a day

1)Severe/fatal

hepatitis

2)Peripheral

neuropathy

3)hypersensitivity

reactions

4)Nausea, vomiting

1)Ataxia, fatigue

2)Diarrhea, nausea

3)Hepatitis

4)Acute renal failure

1)Jaundice

2)Thrombocytopenia

3)Cerebral

hemorrhage

4)Visual

disturbances

1)Decrease in visual

acuity

2)Dermatitis

3)Hypersensitivity

4)Acute gout

MEDICAL NEED:- As it is evident from the side effects caused by the drugs there is an imminent need to develop a new

drug for treating tuberculosis. The standard treatment involves 6 months which is too long. Also, cases

of drug resistance are observed more often than not which compels us to think differently and devise

new drugs which are more effective and consume less time. A finding by WHO indicates that

tuberculosis falls under the neglected diseases as pharmaceutical companies are not that keen to devise

new drugs. Lastly, every year 1.7 million people die due to tuberculosis. So, there is absolutely very dire

need to develop new drug for treating tuberculosis.

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[3] TARGET for the new drug: My target is Isocitrate lyase which is the enzyme involved in the Glyoxylate cycle in

M.tuberculosis

Role of Isocitrate lyase:

• It has been found out through various transcriptional studies that M.tuberculosis

depends more on the fatty acids for survival rather than the carbohydrates in the host

body.

• The bacterium has several enzymes which are related to fatty acid metabolism-34 acyl-

CoA synthases, 34 acyl-CoA dehydrogenases, 21 enoyl CoA hydratases, six 3-

hydroxyacyl-CoA dehydrogenases and six acetyl-CoA C-acetyltransferases.

• It contains more than 250 lipid metabolizing enzymes which show the importance of

fatty acid metabolism in the bacterium.

• Isocitrate lyase is also required for the fatty acid catabolism and virulence in the

organism.

• Individual deletion of the genes coding for the 2 isoforms of the enzyme does not have

any effect on the catabolism but simultaneous deletion of the genes results in total

impairment of intracellular replication.

Isocitrate lyase as a target: • Glyoxylate cycle is very important in bacteria for their metabolism.

• A knockout of enzyme Isocitrate lyase can completely impair the glyoxylate cycle in the

organism.

• Due to this, there occurs a problem in the glyoxylate pathway which ultimately results in

the disruption of the metabolism of fatty acids.

• As a result, the organism cannot maintain its regular physiological processes and

ultimately dies.

• A drug can be devised which specifically targets this lyase.

• As a result the enzyme is denatured and the organism is killed.

Advantages of this drug as compared to the marketed drugs: • Currently available drugs in the market are highly efficient but lead to fatal side effects.

• Also, there is a high rise in the cases of resistance against these drugs which is posing a

big problem in successfully treating the disease.

• Also, current drugs find it difficult to treat the dormant bacteria in the granulomas.

• The new drug implies a new target which can successfully counter the resistance aspect.

• Moreover, it might be able to cure the disease in a short period as compared to current

drug time span as it is also effective on the dormant bacteria which are enclosed in the

granulomas. In fact it is more effective on the dormant bacteria as they live in hypoxic

conditions and hence need fatty acid metabolic pathways more than the bacteria in the

blood stream.

• Also, Isocitrate lyase is specific for the glyoxylate cycle which is not present in humans.

Due to this there is no effect on human beings. The drug will only act on ICL and nothing

else, so the possibility of side effects is greatly reduced.

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[4] Assay for evaluating Inhibitor of Isocitrate Lyase for developing anti-

tuberculosis drug:- Reagents and Materials required: M.tuberculosis culture, Pfu DNA Polymerase, DNA Ligase, dNTP,

Isocitrate Lyase, Inhibitory compound derived from Itaconic anhydride, 96-well plate, Biomek FX [1]

Assay for checking the inhibitory effect: The assay is divided into 2 parts-1) Checking the enzymatic

activity, 2) Checking the inhibitory effect of the compound under study. [1]

1) Checking the enzyme activity [1]

:

• The formation of glyoxylate-phenylhydrazone was checked in presence of phenylhydrazine and

isocitrate lyase when the enzyme was incubated at 370C.

• The reaction mixture of buffer and isocitrate lyase and phenyl hydrazine was kept for 10

minutes for incubation and then the absorbance was measured at 324 nm after the addition of

isocitrate.

• Buffer was composed of potassium phosphate, MgCl2, phenylhydrazine, and cysteine.

2) HTS checking of the inhibitory effect of the compound under study [1]

:

• 10 micro liter of the inhibitory substance was added into a 96-well plate. Into that, 178 micro

liters of reaction buffer, 10 micro liters compound solution, 2 micro liters recombinant protein

ICL solution and 10 micro liter of isocitrate solution was added. It was allowed to incubate for

some time. Then absorbance was measured at 324 nm.

• The activity of the enzyme was determined by change in the absorbance values.

Inhibitory substance+ Reaction buffer+ ICL +Isocitrate+ Phenyl hydrazine Glyoxylate-

Phenylhydrazone

Change in Absorbance

Lead Structure [1, 13]

:

OH

C NH2

H2C C O

CH O

HOOC

Figure 1

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[5] Lead Optimization and Selection of animal model:- The NCE structures are as follows:

OH

HO

CH2

NH2

OH

O

ClHO

O

HS

OH

NH2

Cl

CH2

NH2

OH

O

ClOH

Br

CH2

OH

O

ClHO

OH

N

Cl

Figure 2 (a, b, c, d)

Animal model [2, 3, 4]

:-

There are basically 4 models available for the study of Tuberculosis. They are-

Table 2: Animal model selection

MICE RABBIT GUINEA PIG RHESUS MONKEY

Mode of infection is

subcutaneous or

intravenous

Shows a spectrum of

disease-represent

specific stages of

human disease

Highly susceptible

towards the disease.

Susceptibility and

progression of disease

is like humans

Low cost Low-medium cost High cost High cost

Aggregation of the

lymphocytes is towards

the center in the

granuloma

The inbred are more

susceptible compared

to outbred.

They granulomas

exhibit many similarities

to human granulomas.

The granulomas are

similar to humans

Has innate resistance

towards tuberculosis

which is a negative

point for studies.

Has Innate resistance

towards tuberculosis

No resistance-Show

lung tissue necrosis,

lose weight, die-like

humans

Many reagents to study

pathological

parameters.

Easily available Large numbers required High rearing cost can be

countered by high

susceptibility

Can spread infection to

other monkeys.

Animal of choice: - Rhesus monkeys with SCID are best suited for the studies as they resemble the

humans very closely as far as the disease progression and symptoms are concerned. Also, there are

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many reagents to study the pathological parameters which are not available in other models. Other

close contender is the guinea pig which shows exceptional susceptibility towards the disease. But the

monkey is chosen ahead due to its similarity and efficacy of the drug in it. Mice and rabbits show innate

resistance which is not useful. Monkeys might spread infection but it can be dealt with. The cost is little

higher but the results are worth the cost. So, Rhesus monkey with SCID is the animal of choice.

[6] Pharmacological profiling of NCE [10]

:

LEAD

NH2

Cl

CH2

NH2

OH

O

ClHO

O

HS

OH

OH

HO

CH2

NH2

OH

O

ClHO

O

HS

OH

NCE1 NCE 2

NH2

Cl

CH2

NH2

OH

O

ClOH

Br

CH2

OH

O

ClHO

OH

N

Cl

NCE 3 NCE 4

Figure 3 (a, b, c, d, e)

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Table 3: Structural formula of NCEs

R1 R2 R3

Lead

NCE(1)

NCE(2)

NCE(3)

NCE (4)

-H

NH2

Cl

OH

OH

NH2

Cl

N

H

-H

-O-CH2-SH

-O-CH2-SH

Br

Cl

-H

-NH2

-NH2

-NH2

-H

Table 4: Pharmacological profiling

Absorption rate by

Caco-2 screening

assay (cm sec-1

)[4]

P450 Inhibition

(%)[4]

% GSH depletion[4]

IC50[16]

NCE 1 12 x 10-6

8 4 1.2 uM

NCE 2 9 x 10-6

40 27 7 uM

NCE 3 8 x 10-6

72 53 3.2 uM

NCE 4 6 x 10-6

24 37 4.1 uM

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Conclusion: From the pharmacokinetic values, NCE 1 is the best suited NCE to choose for animal

studies as it can be seen that, in all the 4 screens that are selected as testing parameters, the values of

NCE 1 are good. The absorption rate obtained by Caco 2 assays suggest that the absorption of NCE 1 is

better. The %P450 inhibition of NCE 1 is 8 which shows that it is better. The % GSH inhibition is also low

which suggests that NCE 1 is less toxic. The IC50 value is 1.2 uM which is good because at that low

concentration 50% of enzyme gets inhibited. In case of other NCEs the IC50 values are high which is not

good.

Also, the molecular weight is 459.34, LogP is 2.57 and N+O is 7 which follows the Lipinski rule.

The presence of –Cl, NH2, OH, COOH, SH groups makes it hydrophilic while the presence of 2 benzene

ring structures makes it hydrophobic. So stronger binding can be achieved.

Hence, it can be seen that NCE 1 is good enough and should proceed ahead with animal studies.

Route of Administration of drug:

Based on the studies performed on animal model, the route of administration of the drug will be oral

for all further studies.

Patent of NCE:

The most effective NCE determined through in vitro pharmacological profiling and animal studies is

patented under the patenting regulatory authority.

Summary of Pre-IND activities:

Duration: 3 years

� Research to generate Lead

� Efficacy and mechanism of action studies

� Animal model selection-SCID Rhesus monkey(dose exposure, metabolism) & route of

administration-oral

� Kinetic studies to check drug-drug metabolism, effect on enzymatic activity

� Safety pharmacology study (Pharmacological profiling-ADMET studies)

� Toxicology under GLP

� Single dose toxicity study

� Safety studies including range finding

� Genotoxicity study

� Drug impurity determination

� IND filing

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[7] Biomarkers used for clinical trials of pulmonary tuberculosis:

CD11c [7]

:

CD11c is involved in phagocytosis of M.tuberculosis by macrophages. Tuberculosis patients

have an elevated level of CD11c on blood monocytes as compared to normal and healthy

people. When anti-tuberculosis treatment is done then the levels of CD11c come down. So,

CD11c can be used as a biomarker for evaluating the efficacy of the drug.

sIL-2Rα, sTNF-R1, sTNF-R2 [8]

:

sIL-2Rα, sTNF-R1 and sTNF-R2 can be used as biomarkers for testing the efficacy of the

tuberculosis treatment. Before treatment the levels of all the 3 are relatively high in the

patients. After subsequent treatment, the levels of these biomarkers go down even in slow

treatment responders.

So, the above mentioned biomarkers can be used as efficacy biomarkers in monitoring the

efficacy of the drug for treatment of tuberculosis.

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[8]Phase 1 clinical trial for study of Isocitrate lyase inhibitor for treatment of

tuberculosis [15]

Purpose: This study is targeted to find out the safety and tolerability of an increasing dose of

Isocitrate lyase inhibitor by administering it into adult volunteers.

Condition: Tuberculosis

Intervention: Isocitrate lyase inhibitor

Phase: Phase-1

Study type: Interventional

Study design: Safety and efficacy study, Dose comparison, treatment

ELIGIBILITY

Age: 18 to 60 years

Gender: Both

Healthy Volunteers: No

Number of volunteers: 90

Doses taken of NCE:

From the results obtained from the trials on animal model the range of dose is selected. The

minimum effective dose was 10mg/kg and maximum tolerated dose was 200 mg/kg. 1/10th

of

this MTD was taken in the clinical trials which turned out to be 20 mg/kg.

Table 5: Results for Phase-1 trials

Dose (mg/kg) No of Subjects No of Adverse Events Average CD11c level (%) *

20

40

80

30

30

30

1-Nausea

2-Nausea, Fatigue,

3-Nuasea, Vomiting, Fatigue

60

40

6

*100% stands for total amount of CD11c receptors on the Phagocytes before the treatment of

the patient began.

Adverse event: Nausea is seen as a common side effect in majority of patients. It might be

resulting due to the creation of heaviness in the stomach as the drug may draw water from the

cells. This withdrawal of water might be due to the changes in osmotic pressure across the cells

of stomach. Also, vomiting and fatigue are observed.

Conclusion:

From the phase-1 trials the hMTD is 80mg/kg.

The drug is well tolerated as it is not giving major side effects.

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[9] Phase-2 clinical trial to determine the dosage and safety of Isocitrate lyase

inhibitor [15]

: Purpose: To evaluate safety of the drug and determine its effective dosage.

Study type: Interventional

Study design: Safety and efficacy study, placebo, treatment

Eligibility:

Age: 18 to 60 years

Gender: Both

Healthy volunteers: No

Number of volunteers: 180

Criteria [15]

:

Inclusion criteria:

• Sputum positive patients and patients previously treated for more than 1 month as per RNTCP/WHO

guidelines.

• Radiographic evidence of the disease.

• High WBC count. High WBC count symbolizes mycobacterial infection.

• Microbiological confirmation of Mycobacterial infection.

• Patients with treatment failure, disease relapse as it helps in establishing the superiority of the drug.

Exclusion criteria:

• Subjects below 18 years are not included as the drug might behave differently.

• Pregnant, breast feeding women in order to prevent the drug from going inside the baby.

• Patients with systematic cancer chemotherapy which may result in false side effects.

• Patients who are also infected with HIV, Hepatitis B or C as these infections make treatment difficult.

Table 6: Results for phase-2 trials

Dose (mg/kg) no of subjects No of Adverse events Average CD11c level (%) *

15

30

60

60

60

60

1-Nausea

3-Nuasea,Bowel upset, fatigue

4-Nausea, Indigestion, Fatigue,

Vomiting

65

45

7

*100% stands for total amount of CD11c receptors on the Phagocytes before the treatment of

the patient began.

Adverse events: Apart from the side effects observed in phase 1 trials which were also

observed in phase 2, bowel upset was observed in subjects. In some of the subjects, indigestion

was also observed.

Conclusion:

Phase 1 and 2 trials are giving sufficiently good results with fewer side effects to proceed ahead

with the phase 3 trials. The side effects are very marginal. Dose should be 50mg/kg.

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Phase 3 clinical trials for Isocitrate lyase inhibitor (Jainin):

Efficacy and safety study of Isocitrate Lyase inhibitor in Pulmonary Tuberculosis patients

Purpose of this study is to evaluate the efficacy, safety, side effects and decrease in daily dosage of drug

for treating pulmonary tuberculosis.

Study design: treatment, safety, efficacy, drug lowering

Study type: interventional

Placebo controlled trial [15]

: Currently there are no drugs being developed which inhibit Isocitrate Lyase.

So my study will tested against a placebo to get a clear idea of the effect of the drug. Drugs for

tuberculosis are known to give various side effects. Also, tuberculosis is a stubborn disease with large

amount of resistance. So a placebo controlled study would be ideal in order to study the side effects as

well as efficacy.

Primary endpoints [15]

:

Number of days required for the sputum culture to turn to negative.

Treatment success rate at the end of 4 months.

Adverse effects during the study

Secondary endpoints [15]

:

Tuberculosis cure rate

CD4 T Lymphocytes

Dose: 50mg/kg

Eligibility [15]

Age- 18 to 60 years

Sex- Both

Healthy volunteers- No

Inclusion criteria:

• Sputum positive patients and patients previously treated for more than 1 month as per RNTCP/WHO

guidelines.

• Radiographic evidence of the disease.

• High WBC count. High WBC count symbolizes mycobacterial infection.

• Microbiological confirmation of Mycobacterial infection.

• Patients with treatment failure, disease relapse as it helps in establishing the superiority of the drug.

• Ability and willingness to give written consent as it is a high risk study.

Exclusion criteria:

• Subjects below 18 years are not included as the drug might behave differently.

• Pregnant, breast feeding women in order to prevent the drug from going inside the baby.

• Patients with systematic cancer chemotherapy which may result in false side effects.

• Patients who are also infected with HIV, Hepatitis B or C as these infections make treatment difficult.

• Severely malnourished patients as proper nutrition is very essential especially proteins.

Results:

A total of 1200 patients went through phase 3 clinical trials for the drug JAININ against placebo

controlled trial. About 25% were given placebo.

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About 85% of the patients administered with JAININ showed relief in their tuberculosis symptoms. The

CD11c levels went down which showed successful treatment of tuberculosis.

There were side effects in about 10% of population.

Side Effects [17]

:

Anticipated side effects may not include any specific symptom as the drug targets isocitrate lyase which

is a bacterial specific enzyme and not present in humans. So it is highly unlikely that the drug may cause

any problems in humans. And this property is one of the high points of this drug.

Nausea, weakness or fatigue might be observed.

General energy drinks may be given to remove the weakness or fatigue.

Drug Administration Regimen:

Based on the clinical trials, the dosage for patients of 18 years and older is 50mg/kg tablet every day. For

patients under 18 years-the dosage should be determined by physician depending upon case to case

basis.

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Summary of Clinical Trials:

Table 7:

Key points for Phase1 Key points for Phase 2 Key points for Phase 3 NDA

Gateway to Phase

1Chemistry,Manufacturing

& Controls:

• Formulation of

drug

• Manufacturing

process on small

scale

• Bioassay, Assay for

purity and potency

• Stability Indicating

assay

Toxicology study

• Genotoxicity study

• Single and repeat

dose studies

• Reproductive &

developmental

toxicity studies.

Starting dose is defined

based on non clinical study

Phase 1 trialsStudy is done

on healthy

indivisuals.Phase 1 study is

randomized, dose

escalation study to

determine Maximum

Tolerated Dose(MTD)

,safety in human and safe

dose range in

human.Human

Pharmacokinetics and

exploratory end points are

also studied

Gateway to Phase 2

CMC-

• characterization of

drug

• Check impurities,

manufacturing

controls, product

release

specifications,

stability(at least 1

year)

Non clinical Toxicology study

• Single and repeat

dose studies

• Genotoxicity study

• Reproductive toxicity

Evaluation of potential for

delayed ventricular

repolarization

Phase 2 trials:

Study is randomized, placebo

controlled,double blind

efficacy study in the subjects

having asthma.

Gateway to Phase 3

CMC

• Manufacturing

process validation

• Product release

specification

validation

• Stability check-at

least for 2 years

Non clinical study:

Exposure of drug for 3

months-need 6 month

rodent & non rodent

Phase 3 trials:

Randomized, placebo

controlled, double blind

efficacy study in subjects

having asthma to know

efficacy and safety profile

on large scale.

Submission to

NDA & decision

2 years

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NDA Application:

Sufficient data is gathered after successful completion of phase 3 trials for filing of NDA approved

application form.

Life cycle management for JAININ:

� Patent for the chemical structure of the drug will be filed.

� Patent for the most effective NCE will be filed.

� The whole manufacturing process will be filed for patenting after the process is standardized in

the studies.

� Patent will be filed for the dosage after standardizing it in phase 2 studies.

� Application for FDA approval will be filed after successful completion of phase 3 studies.

After approval the drug is ready to be launched.

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Appendix:

APPENDIX-1

There have been many cases of drug resistance in tuberculosis but still the standard treatment regimen

include the above mentioned drugs as the resistance prevalence is not that much and these drugs can

successfully treat the disease. In some cases multiple drug resistance is observed which is also at a small

scale so the standard treatment is continued.

APPENDIX-2

The present drugs-Isoniazid, Rifampin, Pyrazinamide and Ethambutol are highly effective in treating

Tuberculosis but there is a problem with the side effects that they cause-liver problems being the most

severe.

Moreover, patient compliance is a big issue as the current treatment spans over 6 stringent months.

My target is a new one and currently under study. It is based on a hypothesis, the efficacy of which is

currently under experimental study.

There is a problem with redundancy as there are more than one synthase in the bacterium and

inhibiting one synthase might not be that much useful but a drug can be developed which selectively

inhibits all the synthases or the genes coding for the synthases.

Apart from the TAG synthase, isocitrate dehydrogense inhibition, pantothenate biosynthesis inhibition,

targeting the respiratory chain, ribonucleotide reductase and ATP synthase can be other possible

targets. They are all under experimental study.

APPENDIX-3

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