KWARA STATE UNIVERSITY, MALETE

The University for Community Development

Faculty of Pure and Applied Sciences

IN VITRO INHIBITION OF Mycobacterium tuberculosis PROTEINS

TYROSINE PHOSPHATASE B BY AGMATINE SULFATE.

BY

ISLAMIAT OMOLARA MOLIK

20/57BC/01140

AUGUST, 2024
IN VITRO INHIBITION OF Mycobacterium tuberculosis PROTEINS

TYROSINE PHOSPHATASE B BY AGMATINE SULFATE.

BY

ISLAMIAT OMOLARA MOLIK

20/57BC/01140

A RESEARCH PROJECT SUBMITTED TO DEPARTMENT

OF BIOCHEMISTRY, FACULTY OF PURE AND APPLIED
SCIENCES, KWARA STATE UNIVERSITY, MALETE.
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR
THE AWARD OF BACHELOR OF SCIENCE ([Link].)
DEGREE IN BIOCHEMISTRY.

AUGUST, 2024
 DECLARATION

I hereby declare that this research project titled “IN VITRO INHIBITION OF Mycobacterium

Tuberculosis PROTEINS TYROSINE PHOSPHATASE B BY AGMATINE SULFATE” is

my own work and as not been submitted by other person for any degree or qualification at any

higher institution. I also declare that the information provided therein are mine and those that are

not mine are properly acknowledge.

ISLAMIAT OMOLARA MOLIK _______________________

Name of Student Signature and Date

 CERTFICATION

This is to certify that this research project titled “IN VITRO INHIBITION OF Mycobacterium

Tuberculosis PROTEINS TYROSINE PHOSPHATASE B BY AGMATINE SULFATE”

was carried out by “ISLAMIAT OMOLARA MOLIK”. The project has been read and

approved as meeting the requirements for the award of Bachelor of Science (B. Sc.) Degree in

biochemistry in the Department of biochemistry, Faculty of Pure and Applied Sciences, Kwara

State University, Malete.

__________________________ ________________________
Date

Supervisor

Dr. Sulyman O.A

________________________ ________________________

Dr. Irondi Emmanuel Date

(Head of Department)

________________________ ________________________

External Examiner Date

 DEDICATION

This project is dedicated to Almighty God.

 ACKNOWLEDGMENTS

All praises, adoration and thanks goes to Almighty God, the Seer of the beginning and end of

everything, for His infinite mercies, love, grace and divine favor towards me.

Special appreciation goes to my able supervisor, who is always like a Father, Dr. Sulyman

O.A for his unflinching support and guidance, which culminated in the success of this research

work.

Also, I express my deep appreciation to Dr. Irondi Emmanuel, the Head of Biochemistry

Department, Faculty of Pure and Applied Science for his support and teachings.

Outstanding thanks goes to my parents, Mr and Mrs Molik for their sound and flawless training

and for leading me thus far in my academic pursuit. May God satisfy you with peace, long-life

and sound health to enjoy the rewards of your labour.

 CHAPTER ONE

INTRODUCTION

1.0 Background on Tuberculosis (TB)

Tuberculosis (TB) is an airborne infectious disease caused by the intracellular pathogen.

Mycobacterium tuberculosis (Mtb). TB is one of the top 10 causes of human death worldwide

and the leading cause of death from a single infectious agent. According to the World Health

Organization (WHO, 2018 tuberculosis report), an estimated 10 million people developed TB,

and over 1.2 million people succumbed to it in 2018. It is also estimated that about a quarter of

the global population has latent TB. Current TB treatment is based on combination

chemotherapy and requires 6 to 9 months of treatment. However, due to increasing drug

resistance, antibiotics are losing efficacy in treating TB. Inadequate diagnosis, lack of

compliance from patients, irregular drug supply, poor bioavailability of drugs, and mutation/gene

transfer are some of the factors contributing to drug resistance. According to the WHO, there are

currently 50 antibiotics in the clinical pipeline. However, this clinical pipeline is insufficient to

tackle the problem of increasing antibiotic resistance. Since July 2017, eight new antibiotics have

been approved, but many have limited clinical benefits compared to the existing treatments. The

occurrence of multidrug-resistant and extensively drug-resistant tuberculosis demands the

development of therapeutic agents with novel mechanisms of action. Since early interactions

between Mtb and the host innate immune system play essential roles in the establishment of TB

infection and disease development, alternative therapeutic approaches such as antivirulence

strategies are gaining interest among researchers in the development of more effective TB

therapies and in combating antibiotic resistance (Menzies et al., 2018).

1.1 Global Impact and Prevalence

Tuberculosis (TB) is a preventable and usually curable disease. Yet in 2022, TB was the world’s

second leading cause of death from a single infectious agent, after coronavirus disease (COVID-

19), and caused almost twice as many deaths as HIV/AIDS. More than 10 million people

continue to fall ill with TB every year. Urgent action is required to end the global TB epidemic

by 2030, a goal that has been adopted by all Member States of the United Nations (UN) and the

World Health Organization (WHO) (SDG, 2022). TB is caused by the bacillus Mycobacterium

tuberculosis, which is spread when people who are sick with TB expel bacteria into the air (e.g.

by coughing). About a quarter of the global population is estimated to have been infected with

TB (Houben et al., 2016). Following infection, the risk of developing TB disease is highest in the

first 2 years (approximately 5%), after which it is much lower (Menzies et al., 2018). Some

people will clear the infection (Emery et al., 2021). Of the total number of people who develop

TB disease each year, about 90% are adults, with more cases among men than women. The

disease typically affects the lungs (pulmonary TB) but can affect other sites as well. Basic facts

about TB are provided in Annex 1. Without treatment, the death rate from TB disease is high

(about 50%) (Tiemersma et al., 2011). With treatments currently recommended by WHO (a 4-6

months course of anti-TB drugs), about 85% of people with TB can be cured. Regimens of 1-6

months are available to treat TB infection. Universal health coverage (UHC) is necessary to

ensure that all people who need treatment for TB disease or infection can access these

treatments. The number of people acquiring infection and developing disease (and in turn the

number of deaths caused by TB) can also be reduced through multisectoral action to address TB
determinants such as poverty, undernourishment, HIV infection, smoking and diabetes. Some

countries have already reduced their burden of TB disease to fewer than 10 cases and less than

one death per 100 000 population per year. Research breakthroughs (e.g. a new vaccine) are

needed to rap- idly reduce the number of new cases each year (i.e. TB incidence) worldwide to

the levels already achieved in these low-burden countries. Political commitment to ending the

TB epidemic has stepped up in recent years. The UN held its first-ever high-level meeting on TB

in 2018. With global progress off track and in the wake of setbacks during the COVID-19

pandemic, a second UN high-level meeting on TB was held on 22 September 2023. The resulting

political declaration reaffirms existing commitments and tar- gets and includes new ones for the

period 2023-2027. This WHO global TB report follows just over 1 month later. It provides a

comprehensive and up-to-date assessment of the status of the TB epidemic and progress in the

response at global, regional and national levels, in the context of global commitments, strategies

and targets. As with previous WHO global TB reports, the 2023 edition is based primarily on

data gathered by WHO from national ministries of health in annual rounds of data collection

(Menzies et al., 2018).

In 2023, 192 countries and areas (out of 215) with more than 99% of the world’s population and

TB cases reported data (Annex 2), including all high TB burden countries. The report also draws

on monthly or quarterly national TB case notification data, which have been collected since the

start of the COVID-19 pandemic, and databases maintained by other UN agencies and the World

Bank. The report has three main components. There is a short main report that focuses on key

findings and messages (this document); webpages that provide more detailed and digitized

information, including many interactive graphics and an app that contains country, regional and

global profiles as well as two slide sets. All components can be accessed from the report landing
page on the WHO website, and all data can be downloaded from WHO’s online global TB data-

base. The report format ensures web or app-based availability of all content in relatively small

and “bite- sized” chunks, which facilitates navigation, reading and use.

1.1.2 Challenges in current TB treatment, including drug resistance

Mycobacterium tuberculosis (Mtb), the causative agent of TB, is an obligate pathogen that is

thought to have co- existed with its human host for millions of years. The same features of the

metabolism and physiology of Mtb that enable it to persist quiescently for years within the

human host present formidable challenges for effective chemotherapy (Hartman et al., 2017).

For the purposes of this review, it is important to distinguish drug resistance the heritable ability

of an organism to resist the effects of an antibiotic to which its parent was susceptible from drug

tolerance and persistence, which allow transient survival of an organism at concentrations of an

antibiotic that would otherwise be lethal (Brauner et al., 2016).

In many bacterial pathogens, horizontal gene transfer (HGT) plays a major role in the acquisition

of drug resistance determinants. However, HGT is thought to be negligible in Mtb (Yadon et al.,

2017). instead, drug resistance is mediated by single nucleotide polymorphisms (SNPs),

multinucleated polymorphisms, indels and rearrangements in chromosomal genes that encode

drug targets; enzymes that metabolize prod rugs to their active forms, or drug efflux systems

(Hartman et al., 2017). For some TB drugs, such as RIF, the genotype phenotype relationship

with respect to resistance is well-established whereas for others, the association is less clear.

Moreover, in the case of RIF, the range of resistance-conferring mutations is quite restricted,

whereas a much wider range of mutations can confer resistance to pyrazinamide (PZA), being

scattered across the entire pncA gene (Hartman et al., 2017).

Cross-resistance can occur to TB drugs within the same class as well as between classes. For

example, mutations in gyrA and gyrB can result in cross-resistance to multiple fluoroquinolones

(Willby et al., 2015). Likewise, mutations in rpoB that confer resistance to RIF can result in

cross-resistance to other rifamycins. In a sobering example of cross-resistance with potential

implications for the management of DR-TB, a mutation in a transcriptional regulator, Rv0678,

was shown to result in cross-resistance of Mtb to clofazimine a leprosy drug used in DR-TB

treatment and bedaquiline, a drug recently approved for the treatment of MDR-TB, through up

regulation of a multi substrate efflux pump (Hartkoorn et al., 2014). Curiously, a markedly

higher prevalence of resistance-associated variants in Rv0678 was found in MDR-TB patients

with no evidence of prior use of clofazimine or bedaquiline than in non- MDR-TB patients

suggesting an association with prior TB drug exposure. Although the underlying driver/s remains

unclear, these findings highlight the formidable range of mechanisms that Mtb can engage to

evade drug pressure which complicates the design new therapeutic regimens for DR-TB.

1.2 Significance of Mycobacterium tuberculosis protein tyrosine phosphatase A (PtpA)

The etiological agent of tuberculosis (TB), Mycobacterium tuberculosis, is one of the most

devastating infectious agents in the world. One-third of the world’s population is exposed to

Mtb, which causes nearly 3 million deaths annually, and is expected to cause an estimated 1

billion new infections by 2020 (Dye et al., 2010). Mtb primarily infects alveolar macrophages

that provide the first line of defense against microbial invasion. Normally, macrophages’

engulfment of foreign bodies results in the formation of phagosome, which matures in a process

that remodels its membrane and luminal contents through interaction and fusion with the

endosomal network (Hartman et al., 2017). These membrane fusions allow the phagosome to

acquire antimicrobial properties, including a profoundly acidic lumen, the hallmark of the
macrophage maturation process. Macrophages acidify the phagosomal lumen by recruiting V-

ATPase, a multisubunit protein-pump complex that actively transports protons across

membranes using energy from ATP hydrolysis. Structurally similar to ATP synthase, the V-

ATPase consists of a membrane-bound domain with five different subunits and a cytosolic

domain composed of subunits A-H. Delivery of the V-ATPase to the phagosome results in a pH

decrease from 6.5 to 5.0 within minutes of phagosome maturation (Hartman et al., 2017). This

acidic environment inhibits bacterial growth and enhances the activities of antimicrobial

hydrolases. Acidic pH is also crucial for proper vesicular trafficking, directing the fusion of

phagosomes with lysosomes or vesicles harboring antimicrobial molecules. Therefore,

phagosome acidification is a critical event that prompts the destruction of invading particles into

constituents for antigen presentation and the initiation of adaptive immune responses.

Intracellular pathogens that enter host cells through the phagocytic or endocytic pathway have

developed mechanisms to counter the acidic environment within phagosomes. For instance,

Salmonella enterica adapts to lower pH by activating acid tolerance genes. Yersinia

pseudotuberculosis blocks phagosome acidification by directly inhibiting V-ATPase activity in

mouse macrophages, and Legionella pneumophila secretes substrates into the host phagocyte to

inhibit vacuole acidification through interaction with V-ATPase subunit A (Xu et al., 2010). In

the case of Mtb, lack of acidification in the mycobacterial phagosome is mainly because of the

absence of the V-ATPase on the phagosomal membrane. However, the mechanism by which

Mtb accomplishes this remains undefined. Mtb is known to be capable of sensing engulfment by

macrophages and subsequently interferes with host signaling pathways to promote its

intracellular survival. Mtb possesses a wide repertoire of signal transduction systems, including

11 two-component systems, 11 eukaryotic-like serine/threonine protein kina- ses (PknA-PknL),

two protein tyrosine phosphatases (PtpA and PtpB), and the newly identified protein tyrosine

kinase (PtkA) (Hartman et al., 2017). These signaling proteins play key roles in bacterial

adaptation and response to host defense mechanisms. PtpA, a secreted protein phosphatase, is

essential for Mtb pathogenicity, participating in the arrest of phagosome maturation within the

host macrophages. Earlier, we identified the host vacuolar protein sorting 33B (VPS33B) as the

cognate substrate of PtpA. VPS33B is a member of the class C VPS complex that regulates

membrane fusion within the endocytic pathway. PtpA dephosphorylation of VPS33B inactivates

this host protein, leading to inhibition of phagosome lysosome fusion (Hartman et al., 2017).

1.2.1 Role of PtpA in Mtb Virulence and Immune evasion

Protein Virulence Factors Along with abundant lipids, the Mtb cell wall also contains proteins

that directly affect the host’s immune response. Various reports have shown that protein families,

such as PE, PPE, and lipoproteins, alter the host immune response. The effects include odulating

cytokine production and arresting phagosome maturation, phagosome escape, autophagy, and

cell death (Echeverria-Valencia et al., 2017).

In Major Proteins Virulence Factors from Mtb Mtb possesses an early secretory antigenic target

(ESAT-6) secretion (ESX) system, also known as the type VII secretion system. Mtb has five

secretion systems, or ESX, ranging from ESX-1 to ESX-5. The ESX-1, -3, and -5 are essential

for mycobacterial virulence and regulate protein secretion and transport across the cytoplasmic

membrane and complex mycobacterial cell wall. ESX genes encode the ESX-type proteins

EspA, EspB, EspC, and EspG, the secreted proteins ESAT-6 and CFP-10, PE-PPE family

proteins, and the conserved components EccB, EccC, EccD, and MycP (Echeverria-Valencia et

al., 2017). ESAT-6, delivered through the ESX system, is also known as EsxA or Rv3875. It is

either secreted alone or in a complex with the chaperone CFP-10 (EsxB). It regulates Mtb’s
colonization, survival, pathogenesis, and granuloma formation. Additionally, it has been

implicated in inhibiting phagosome maturation and modulation of host cell death (Peng et al.,

2016). PE and PPE proteins are transported across the bacterial inner membranes by the ESX-1,

ESX-3, and ESX-5 secretion systems through the PE and PPE domains. The PE and PPE protein

families have conserved Pro-Glu and Pro-Pro-Glu motifs in their N-terminal regions and are

named after these conserved amino acid residues. It has been estimated that 10% of the Mtb

genome encodes PE/PPE proteins, which participate in infection, antigenic variation, and host

pathogen interactions (Echeverria-Valencia et al., 2017). In addition, some PE-PPE members,

such as PPE34, PE-PGRS11, PE-PGRS17, PE-PGRS33, PPE26, PPE57, and PPE60, interact

with TLR2 to modulate the host innate immune response by inducing cytokine secretion,

necrosis, and apoptosis, and enhancing mycobacterial survival (Palucci et al., 2016).

Lipoarabinomannan carrier protein (LprG) is a 27 kDa triacylated lipoprotein of the Mtb cell

wall, which is involved in cell wall biosynthesis, and is essential for the expression of surface

LAM. LprG displays TLR2 agonist activity; it binds to LAM, PIMs, and LM, enhancing their

recognition by TLRs. It also inhibits antigen processing in human macrophage MHC II through

TLR-2 signaling and inhibits and controls phagosome lysosome fusion (Shukla et al., 2014).

LpqH is a 19 kDa O-glycosylated and acylated glycolipoprotein and a primary cell wall antigen

recognized by T cells. It induces T cell proliferation in vitro, stimulates DC maturation and

autophagy, activates TLR-2, and can affect the expression and antigen presentation of MHCII

(Su et al., 2016).

1.3. Overview of Agmatine sulphate and its Pharmacological Properties

In general, natural substances have been a recent target of research for their numerous health

benefits and also for their potential as the basis for new drugs (Seidel, 2020). The use of plants
and herbs is documented by numerous authors both in Europe and elsewhere (Singh et al., 2012).

The aim of such research is two-pronged, both to explore new opportunities for effective

therapeutical agents, and also to elucidate the correlation between a decreased incidence of

health problems and the consumption of certain food types. Regarding this last aim, it is the

logical course of action, since certain diets are correlated with negative mortality and morbidity

incidence rates in addition, based on the research of specific diet choices after the diagnosis of

cancer may improve survival rates. The focus of this paper is Agmatin sulphate, a flavonoid

with many promising health benefits found in a variety of plants. Agmatine sulphate's , a German

doctor, naturalist, and historian who lived during the 17th century and made a significant

contribution to transporting medical knowledge from Japan to the West (Periferakis et al., 2020).

Agmatine sulphate, as a chemical compound, was discovered in Camelia sinensis (tea tree) and

exhibits a host of different positive health-related effects.

1.3.1 Previous studies on Agmatin sulphate’s antimicrobial effects

The antibacterial properties of the secondary metabolites of plants have been in the foreground of

research in the last two decades (Bhatia et al., 2021). Such research is even more important

considering the emergence of numerous resistant and multi-drug resistant (MRD) bacteria [198].

Agmatine-containing extracts and preparations, as well as pure kaempferol compounds, have

been tested as possible antibacterials for quite some time (Bhatia et al., 2021). The investigation

into the action mechanisms behind the antibacterial activity of Agmatine has proven difficult due

to the large variety within the family of Agmatine derivatives but also due to the diversity in

morphology and functions between the numerous species of bacteria. However, some theories

have been advanced and validated regarding the potential action mechanisms in specific bacteria.

For instance, Li et al., 2015 have shown that a mixture of Agmatine 3-O-b-(200-acetyl)
galactopyranoside and quercetin exerts antibacterial effects through cell membrane disruption,

followed by activation of apoptosis and DNA fragmentation in M. luteus cells. Agmatine was

also the most effective tested flavonoid in damaging the cell membrane of Escherichia coli in a

study by He et al., 2014, where the findings were objectified by showing bacterial protein

leakage into the extracellular environment. Moreover, Agmatine and quercetin interact with 3-

oxyacyl-[acyl carrier protein] reductase (FabG) and enoyl-acyl carrier protein reductase therefore

inhibiting the biosynthesis of fatty acids by Mycobacterium, Pseudomonas aeruginosa, and

Vibrio cholerae thus hindering the function of the cell envelope as well as the impeding creation

of bacterial biofilms (Al-Nour et al., 2019). Agmatine inhibited the DNA gyrase in methicillin-

resistant Staphylococcus aureus. Agmatine was also able to inhibit DNA helicases, more

specifically SAPriA in Staphylococcus aureus, as shown by (Huang et al., 2015). Actions of

Agmatine compounds against Porphyromonas gingivalis, Prevotella intermedia, and

Cutibacterium acnes have been described by. The research of had already indicated the

antibacterial effect of the extract of S. hymettia against Enterobacter cloacae, and also other

bacteria, as will be presented below. The extract of Helichrysum compactum, which contained

pure Agmatine and also Agmatine-3-O-glucoside, proved to have a degree of antibacterial

activity (Bhatia et al., 2021). It is also possible, that the extract from Nephelium lappaceum,

which contains kaempferol compounds, has antimicrobial activity. A local Malaysian herb, kacip

Fatimah, i.e., the plant Labisa pumila Benth, which contains Agmatine, was found to have some

antibacterial activity against Micrococcus luteus, Bacillus subtillis, Bacillus cereus,

Staphylococcus aureus, Enterobacter aerogenes, Klebsiella pneumoniae, Escherichia coli and

Pseudomonas aeruginosa, albeit at relatively low bacterial loads. The extract of Uapaca

heudelotti proved effective against S. pneumoniae, as well as against other pathogens. It is also
important to note that while some Agmatine containing extracts may not have significant

antibacterial action on their own, they may potentiate the action of some antibiotics (Bhatia et

al., 2021).
From the Mycobacterium genus, the most well-known and dangerous pathogen is

Mycobacterium tuberculosis, which is the causative agent of tuberculosis, one of the oldest

human diseases (Bhatia et al., 2021). Although a vaccine against the disease exists, it is of

varying efficiency and has proven incapable of stopping the global epidemic (Scriba et al.,

2020). While there exist antibiotics effective against tuberculosis during the last few years, the

increase in antibiotic resistance of M. tuberculosis has led to the emergence of multi (MDR),

extensively (XDR), extremely (XXDR) and total (TDR) drug-resistant strains; these are

estimated to kill about 75 · 106 people, in the next three decades (Allué-Guardia et al., 2021). M.

bovis infects primarily cattle but can also spread to humans; however, it is not of particular

importance as a human pathogen. Rather, its study is of interest in understanding the

pathogenetic mechanism of M. tuberculosis (Allué-Guardia et al., 2021). Based on the research

of, a leaf and hardwood extract from Vatairea macrocarpa, a plant used in Brazilian folk

medicine, exhibited antibacterial action, in an in vivo model, in rat paws infected with M. bovis.

The action of Agmatine-3-O-rhamnopyranoside was supplemented by that of other flavonoids in

the extract. The extract was also found to have significant anti-inflammatory parameters. The

extract of Argyreia speciosa was found to have antibacterial properties against M. tuberculosis.

Another medicinal plant, Doliocarpus dentatus, proved to be effective in a rat model, as an

antimycobacterial agent; the phenolic extract of its leaves contains Agmatine3-O-α-L-

rhamnopyranoside. The extract of Pluchea indica, which contained Agmatine, was identified as a

potent inhibitor of the M. tuberculosis CYP121 in a recent study by. Finally, pure Agmatine

sulphate from Bauhinia vahlii, was found, along with other flavonols, to be effective against M.

tuberculosis (Allué-Guardia et al., 2021).

1.4 Objectives of the Study
1. To investigate the inhibitory effect of agmatine sulfate on Mycobacterium tuberculosis protein

phosphatase B (MtbPPB) activity in vitro.

2. To determine the IC50 (half-maximal inhibitory concentration) of agmatine sulfate against

MtbPPB.

3. To explore the mechanism of inhibition of MtbPPB by agmatine sulfate.

4. To evaluate the specificity of agmatine sulfate towards MtbPPB compared to other protein

phosphatases.

5. To assess the potential of agmatine sulfate as a lead compound for the development of novel

anti-tuberculosis drugs targeting MtbPPB.

6. To investigate the synergistic effect of agmatine sulfate with existing anti-tuberculosis drugs

on MtbPPB activity.

7. To determine the structural requirements for the inhibition of MtbPPB by agmatine sulfate

through structure-activity relationship (SAR) studies.

8. To explore the potential of agmatine sulfate to inhibit the growth of Mycobacterium

tuberculosis in vitro.
 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Overview of TB and current therapeutic strategies

WHO uses five categories to classify cases of drug-resistant TB: isoniazid-resistant TB; RR-TB

and MDR-TB (defined above); extensively drug-resistant TB (XDR- TB); and pre-XDR-TB.

Pre-XDR-TB is TB that is resistant to rifampicin and any fluoroquinolone (a class of second-line

anti-TB drug). XDR-TB is TB that is resistant to rifampicin, plus any fluoroquinolone, plus at

least one of either bedaquiline or linezolid. Detection of drug resistance requires bacteriological

confirmation of TB and testing for drug resistance using rapid molecular diagnostic tests, culture

methods or sequencing technologies. Since 2018, WHO has recommended all-oral regimens for

the treatment of MDR/RR-TB, marking a major advance compared with previous regimens that

included injectable agents. The latest guidelines for treatment of DR-TB, updated in 2022,

include three major categories of regimen (WHO, 2022). The first is a short 6-month all-oral

regimen for people with MDR/RR-TB (which may be extended by 3 months if necessary)

consisting of bedaquiline, pretomanid, linezolid and moxifloxacin, referred to as BPaLM; for

people with pre-XDR-TB, the regimen can be used without moxifloxacin and is referred to as

BPaL.

The second category is all-oral short regimens of 9 months for people with MDR/RR-TB (which

may be extended by 2 months if necessary). The third category is longer regimens of 18–20

months that may include an injectable drug (amikacin). The short 6-month regimen is prioritized
for use and is recommended for people aged 14 years and older who have MDR/RR-TB or pre-

XDR-TB. Globally in 2022, 73% of people (2.9/4.0 million) diagnosed with bacteriologically

confirmed pulmonary TB were tested for rifampicin resistance, up from 69% (2.4/3.5 million) in

2021 and above the pre-pandemic level of 62% (2.2/3.6 million) in 2019. Among those tested,

149 511 people with MDR/RR-TB and 27 075 people with pre-XDR-TB or XDR-TB were

detected, giving a combined total of 176 586 (4.4% of those tested). Despite increased testing

coverage and an increase in the absolute number of people tested, the number of people detected

with MDR/RR-TB was lower in 2022 than in 2019 (when the total was 202 009, 5.6% of those

tested). This is consistent with the estimated decline in the proportion of people with TB who

have MDR/RR-TB. Worldwide, 175 650 people with MDR/RR-TB were enrolled on treatment

in 2022, up 8.5% from 161 843 in 2021 and up 17% from 150 510 in 2020 but still below the

pre-pandemic level of 181 533 in 2019 This level of enrolment is equivalent to about 43% of the

estimated number of people who develop MDR/RR-TB each year. The global targets set at the

UN high-level meeting in 2018 for the number of people to be treated for drug-resistant TB were

not reached. The cumulative number of people with MDR/RR-TB who were reported as being

enrolled on treatment from 2018 to 2022 was 825 000, only 55% of the 5-year target (2018–

2022) of 1.5 million. For children specifically, the cumulative number was 21 600, only 19% of

the 5-year target of 115 000. Ten countries account for about 70% of the global gap between the

estimated global number of people who develop MDR/RR-TB each year (incident cases of

MDR/RR-TB) and the global number of people enrolled on treatment in 2022: China, the

Democratic People’s Republic of Korea, India, Indonesia, Myanmar, Nigeria, Pakistan, the

Philippines, Ukraine and Viet Nam. To make substantial progress in closing this gap,
improvements in the coverage of testing for drug resistance and access to treatment are needed in

these countries.

2.2 Role and mechanism of PtpA in Mycobacterium Tuberculosis pathogenicity

Macrophages provide the first line of immune defense against invading pathogens, including

Mycobacterium Tuberculosis. Macrophages engulf every microorganism or foreign particle into

phagosomes that consequently interact with the endocytic pathway, causing changes to

membranes that allow the phagosome to recruit the vacuolar H+ATPase (V-ATPase) and

hydrolases (Zhou et al., 2010). The presence of V-ATPase on the phagosome membrane creates

an acidic compartment of pH 4.5-5.0, which indicates phagosome maturation. Acid activated

hydro- lases then mediate the killing of the invading microorganism or foreign particle.

Available evidence suggests that mPTPA inhibits phagosome acidification and therefore

prevents Mycobacterium Tuberculosis phagocytosis by the macrophage (Zhou et al., 2010).

Upon Mycobacterium Tuberculosis infection, macrophages also activate various host innate

immune surveillance pathways. To avoid host immune clearance, mPTPB decreases the

secretion of inflammatory cytokines and suppresses macrophage apoptosis. Indeed, both mPTPA

and mPTPB are indispensable for Mycobacterium Tuberculosis intracellular survival (Zhou et

al., 2010). mPTPA was first identified by Koul et al. from the genome sequence of Mtb H37Rv

due to its homology with low molecular weight PTPs (LMW-PTPA). Similar to other PTPs, it

contains the conserved C(X) 5R(S/T) motif with the Cys11 serving as the nucleophile that

attacks the phosphotyrosine residue on the substrate. Residues Cys11, Arg17, and Asp126 play a
critical role in phosphatase activity of mPTPA. In addition to Cys11, a second cysteine (Cys16)

is present in the active site of the enzyme and protects the Cys11 from oxidative inactivation by

forming a reversible disulfide bond. The sequence comparison of mPTPA and LMW- PTPA

shows conservation of residues in the DPYY loop of the active site and the overall 3D fold is

similar. However, the surface of mPTPA shares little shape and sequence similarity with the

LMW-PTPA. The differences in surface-charge distribution can be observed by analyzing the

electrostatic surface diagrams of these proteins. The residues Trp48 and His49 in mPTPA are

substituted by Tyr49 and Glu50 in LMW-PTPA, which creates a negatively charged patch near

the opening of the active site. Moreover, the α4 region also showed significant differences due to

the replacement of charged Asp98 and Lys102 in LMW-PTPA by the hydrophobic Leu96 and

Leu100 in mPTPA

2.3 Natural compounds as potential TB treatments

2.3.1 Potential of natural compounds in drug discovery
Natural products (NPs) have played and continue to play a significant role in the drug discovery

process. For a long period of human existence, NPs were the only form of therapy available for

use by sick people or to maintain health. Drugs of natural origin have been classified as: i)

natural products, ii) products derived semi-synthetically from natural products, and iii) synthetic

products based on natural product models (Cragg et al., 1997). Natural product chemistry and

organic synthesis are powerful tools for optimising leads and for generating new diversity from

natural scaffolds. The amalgamation of both is an important strategy in rational drug design.

Statistics from studies carried out by different workers continue to emphasize the potentials and

untapped reservoir of molecules with therapeutic interest. Eighty percent of the world’s

population depends mainly on NPs for their health care and sixty percent of the orthodox drugs

currently in use have their origin from NPs (Cragg and Newman, 2005). Evidence of the
importance of NPs is provided by the fact that close to half of the bestselling pharmaceuticals in

1991 were either NPs or their derivatives (O’Neill and Lewis, 1993). Newman and co- workers

(2003) reported that 61% of the 877 new chemical entities (with low molecular weights)

registered as drugs worldwide during the period of 1981-2002 were or have been inspired by

NPs. A total of 29 new NPs and NP-derived drugs were introduced in the United States, Europe

and Japan between 2000 and 2003 (Burtler, 2004). More recent studies revealed that between

2005 and April 2010, some 19 NP-based drugs were approved for marketing worldwide (Mishra

and Tiwari, 2011). There is an urgent need to identify novel, active chemotypes as leads for

effective drug development, and, as was dramatically illustrated by the discovery of the

“wonder” antibiotics of the 1940s and 1950s, nature is the prime source of such lead discoveries.

It has however been estimated that only 5 to 15% of the approximately 250 000 species of higher

plants have been systematically investigated for the presence of bioactive compounds (Balandrin

et al., 1993). Norman Farnsworth stated in his closing remarks in his guest editorial on “An old

source for new drugs” in the August 1995 issue of Pharmaceutical Technology that “the world of

plants represents a virtually untapped reservoir of novel drugs awaiting imaginative and

progressive organisations” (Farnsworth, 1995).

Despite the successes recorded, there was a global decline of the NP discovery programme by

pharmaceutical companies in the 1990s. This was replaced by combinatorial chemistry, which,

with the introduction of High throughput screening (HTS), became the preferred choice in drug

discovery and development (Lee and Breitenbucher, 2003). This was based on the premise that

combinatorial chemistry would generate libraries consisting of millions of compounds, which

would be screened by HTS and produce drug leads by sheer number of molecules. In addition, it

would also take care of intellectual property (IP) issues generated with NPs. This technology did
not prove successful as results from early combinatorial libraries were often disappointing and

only few drugs have been discovered by the combination of HTS and combinatorial chemistry

(Kingston, 2011).

The use of plants and plant preparations for the treatment of diseases has been in existence since.

Some of the earliest records of usage of plants as drugs are found in Artharvaveda, which is the

basis for Ayurvedic medicine in India, dating back to 2000 BCE; the clay tablets in

Mesopotamia, dating to 1700 BCE; and the Eber Papyrus in Egypt, dating to 1550 BCE

(Sneader, 2005). In developing countries particularly in Africa, the population continues to rely

on traditional medicine (TM) for their primary healthcare. It is estimated that up to 80% of the

populations in Africa, Asia and Latin America depend mainly on TM for their healthcare needs,

involving mainly the use of plant extracts (WHO, 2003). These extracts are used as herbal drugs

in form of powders, concoctions, ointment, decoctions and infusions. The limitations of these

herbal drugs revolve around lack of documentation, lack of standardization and quality control,

dosage, and the common tendency to describe diseases and ailments vaguely (Okogun, 2002).

2.3.2 Previous studies on flavonoids and their antimicrobial activities

Kaempferol is a flavonoid; flavonoids are regarded as the largest group of secondary plant

metabolites. They are polyphenolic compounds of low molecular weight and are used by plants

to stimulate and regulate their growth and for defense purposes (Singh et al., 2012). Flavonoids

are divided into a number of groups based on their chemical composition, namely flavones,

flavonols, flavanones, isoflavonoids, neoflavonoids, catechins (flavanols), anthocyanins and

chalcones (Panche et al., 2016). The antioxidant properties of polyphenols flavonoids are such

compounds are already well known; more than 104 types of flavonoids are estimated to exist

(Amawi et al., 2017). Other proven effects of flavonoids include hepatoprotective, antimicrobial,
renoprotective, antidiabetic, cardioprotective, anti-arthritic, neuroprotective, gastroprotective and

anti-mutagenic, among others. Recently, there has been an increasing amount of research interest

in the anti-carcinogenic potential of kaempferol, as a positive correlation between its

consumption and reduced cancer incidence has been documented this is in addition to existing

epidemiological studies linking increased flavonoid consumption with reduced cancer incidence

(Petrick et al., 2015). The anti-inflammatory role of agmatine has also been concisely presented

by, while even its anti-adipogenic potential has come under investigation (Park et al., 2022). The

basic structure of all flavonoids, regardless of their subclass, is a 15-carbon benopyranone or

benzopyran in which the three-carbon bridge between the phenyl groups is commonly cyclized

with oxygen forming a C6-C3-C6 flavan nucleus (Kumar et al., 2013;Park et al., 2022).
 REFERENCES

Allué-Guardia, A.; García, J.I.; Torrelles, J.B. Evolution of Drug-Resistant Mycobacterium

tuberculosis Strains and Their Adaptation to the Human Lung Environment. Front.

Microbiol. 2021, 12, 612675.

Al-Nour, M.Y.; Ibrahim, M.M.; Elsaman, T. Ellagic Acid, Kaempferol, and Quercetin from

Acacia nilotica: Promising Combined Drug With Multiple Mechanisms of Action. Curr.

Pharmacol. Rep. 2019, 5, 255–280.

Amawi, H.; Ashby, C.R.; Tiwari, A.K. Cancer chemoprevention through dietary flavonoids:

What’s limiting? Chin. J. Cancer 2017, 36, 50.

Behr MA, Edelstein PH, Ramakrishnan L. Is Mycobacterium tuberculosis infection life long?

BMJ. 2019;367:l5770 ([Link]

Bhatia, P.; Sharma, A.; George, A.J.; Anvitha, D.; Kumar, P.; Dwivedi, V.P.; Chandra, N.S.

Antibacterial activity of medicinal plants against ESKAPE: An update. Heliyon 2021, 7,

e06310.
Brauner A, Fridman O, Gefen O, Balaban NQ: Distinguishing between resistance, tolerance and

persistence to antibiotic treatment. Nat Rev Microbiol 2016, 14:320-330.

Dye C, Williams BG (2010). The population dynamics and control of tuberculosis. Sci- ence

328:856–861.

Echeverria-Valencia, G.; Flores-Villalva, S.; Espitia, C.I. Virulence Factors and Pathogenicity of

Mycobacterium; Ribón, W., Ed.; IntechOpen: Rijeka, Croatia, 2017; p. Ch. 12. ISBN

978-1-78923-211-0.

Emery JC, Richards AS, Dale KD, McQuaid CF, White RG, Denholm JT, Houben RM. Self-

clearance of Mycobacterium tuberculosis infection: implications for lifetime risk and

population at-risk of tuberculosis disease. Proc Biol Sci. 2021; 288(1943):20201635.

([Link]

Global strategy and targets for tuberculosis prevention, care and control after 2015 (Resolution

WHA67.1, Agenda item 12.1). Geneva: World Health Assembly; 2014

([Link]

Hartkoorn RC, Uplekar S, Cole ST: Cross-resistance between clofazimine and bedaquiline

through upregulation of MmpL5 in Mycobacterium tuberculosis. Antimicrob Agents

Chemother 2014, 58:2979-2981.

Hartman TE, Wang Z, Jansen RS, Gardete S, Rhee KY: Metabolic perspectives on persistence.

Microbiol Spectr 2017, 5.

He, M.; Wu, T.; Pan, S.; Xu, X. Antimicrobial mechanism of flavonoids against Escherichia coli

ATCC 25922 by model membrane study. Appl. Surf. Sci. 2014, 305, 515–521.
Houben RM, Dodd PJ. The global burden of latent tuberculosis infection: a re-estimation using

mathematical modelling. PLoS Med. 2016;13(10):e1002152

([Link]

Huang, Y.H.; Huang, C.C.; Chen, C.C.; Yang, K.J.; Huang, C.Y. Inhibition of Staphylococcus

aureus PriA Helicase by Flavonol Agmatine. Protein. J. 2015, 34, 169–172.

In this article, the authors describe a useful experimental framework for distinguishing between

drug resistance, phenotypic tolerance and per- sistence phenotypes in bacteria.

Kumar, S.; Pandey, A.K. Chemistry and Biological Activities of Flavonoids: An Overview. Sci.

World J. 2013, 2013, 162750.

Li, X.-M.; Luo, X.-G.; Si, C.-L.; Wang, N.; Zhou, H.; He, J.-F.; Zhang, T.-C. Antibacterial

active compounds from Hypericum ascyron L. induce bacterial cell death through

apoptosis pathway. Eur. J. Med. Chem. 2015, 96, 436–444.

Menzies NA, Wolf E, Connors D, Bellerose M, Sbarra AN, Cohen T et al. Progression from

latent infection to active disease in dynamic tuberculosis transmission models: a

systematic review of the validity of modelling assumptions. Lancet Infect Dis.

2018;18(8):e228–e38 ([Link]

Palucci, I.; Camassa, S.; Cascioferro, A.; Sali, M.; Anoosheh, S.; Zumbo, A.; Minerva, M.;

Iantomasi, R.; De Maio, F.; Di Sante, G.; et al. PE-PGRS33 Contributes to

Mycobacterium tuberculosis Entry in Macrophages through Interaction with TLR2. PLoS

ONE 2016, 11, e0150800. [CrossRef]

Panche, A.N.; Diwan, A.D.; Chandra, S.R. Flavonoids: An overview. J. Nutr. Sci. 2016, 5, e47.
Park, U.H.; Hwang, J.T.; Youn, H.; Kim, E.J.; Um, S.J. Kaempferol antagonizes adipogenesis by

repressing histone H3K4 methylation at PPARγ target genes. Biochem. Biophys. Res.

Commun. 2022, 617, 48–54.

Peng, X.; Sun, J. Mechanism of ESAT-6 Membrane Interaction and Its Roles in Pathogenesis of

Mycobacterium tuberculosis. Toxicon 2016, 116, 29–34.

Periferakis, A.; Periferakis, K. On the Dissemination of Acupuncture to Europe. JournalNX

2020, 6, 201–209.

Petrick, J.L.; Steck, S.E.; Bradshaw, P.T.; Trivers, K.F.; Abrahamson, P.E.; Engel, L.S.; He, K.;

Chow, W.H.; Mayne, S.T.;

Risch, H.A.; et al. Dietary intake of flavonoids and oesophageal and gastric cancer: Incidence

and survival in the United States of America (USA). Br. J. Cancer 2015, 112, 1291–

1300.

Scriba, T.J.; Netea, M.G.; Ginsberg, A.M. Key recent advances in TB vaccine development and

understanding of protective immune responses against Mycobacterium tuberculosis.

Semin. Immunol. 2020, 50, 101431.

Seidel, V. Plant-Derived Chemicals: A Source of Inspiration for New Drugs. Plants 2020, 9,

1562.

Shukla, S.; Richardson, E.T.; Athman, J.J.; Shi, L.; Wearsch, P.A.; McDonald, D.; Banaei, N.;

Boom, W.H.; Jackson, M.; Harding, C.V. Mycobacterium tuberculosis Lipoprotein LprG

Binds Lipoarabinomannan and Determines Its Cell Envelope Localization to Control

Phagolysosomal Fusion. PLoS Pathog. 2014, 10, e1004471.

Significance of Mycobacterium tuberculosis protein tyrosine phosphatase A (PtpA)

Singh, N.; Pandey, B.; Verma, P.; Bhalla, M.; Gilca, M. Phyto-pharmacotherapeutics of Cyperus

rotundus Linn.(Motha): An overview. Indian J. Nat. Prod. Res. 2012, 3, 467–476.

Su, H.; Zhu, S.; Zhu, L.; Huang, W.; Wang, H.; Zhang, Z.; Xu, Y. Recombinant Lipoprotein

Rv1016c Derived from Mycobacterium tuberculosis Is a TLR-2 Ligand That Induces

Macrophages Apoptosis and Inhibits MHC II Antigen Processing. Front. Cell Infect.

Microbiol. 2016, 6, 147.

Sustainable Development Goals [website]. New York: United Nations; 2022

([Link]

Tiemersma EW, van der Werf MJ, Borgdorff MW, Williams BG, Nagelkerke NJ. Natural

history of tuberculosis: duration and fatality of untreated pulmonary tuberculosis in HIV

negative patients: a systematic review. PLoS One. 2011;6(4):e17601.

([Link]

Willby M, Sikes RD, Malik S, Metchock B, Posey JE: Correlation between GyrA substitutions

and ofloxacin, levofloxacin, and moxifloxacin cross-resistance in Mycobacterium

tuberculosis. Antimicrob Agents Chemother 2015, 59:5427-5434.

Xu L, et al. (2010) Inhibition of host vacuolar H+-ATPase activity by a Legionella pneumophila

effector. PLoS Pathog 6:e1000822.

Yadon AN, Maharaj K, Adamson JH, Lai YP, Sacchettini JC, Ioerger TR, Rubin EJ, Pym AS: A

comprehensive characterization of PncA polymorphisms that confer resistance to

pyrazinamide. Nat Commun 2017, 8:588.

Zhou, B.; He, Y.; Zhang, X.; Xu, J.; Luo, Y.; Wang, Y.; Franzblau, S.G.; Yang, Z.; Chan, R.J.;

Liu, Y.; et al. Targeting mycobacterium protein tyrosine phosphatase B for

antituberculosis agents. Proc. Natl. Acad. Sci. USA 2010, 107, 4573–4578.