DEVELOPMENT OF INVIVO ACTIVE

COMPOUNDS TARGETING
DECAPRENYL PHOSPHORYL-β-D-
RIBOSE 2' - EPISOMER (DprE1)

A research proposal submitted to

charité-Universitätsmedizin Berlin

Prepared by:
Tsama Tekle (MD)
Date : 12/10/2018

1
 BACKGROUND

Mycobacterium Tuberculosis infection is one of the most important causes of death from
infectious diseases. 8.8 million incident cases of Tuberculosis worldwide and around 1.5 million
deaths occurred in the year 2010. (1)

Currently more than 150 species of Mycobacteria have been identified, the majority of which do
not cause disease in humans. (2)

Tuberculosis is almost always curable if patients are given sufficient uninterrupted therapy.
Despite the treatability of this infection, however, it has proved impossible to eliminate it, and
the number of drug-resistant cases has increased.(3)

Tuberculosis remains a leading cause of morbidity and mortality in developing countries -

particularly in sub-Saharan Africa, where the HIV epidemic rages.(2)

Ensuring the regular intake of drugs to achieve a cure is as important as making the diagnosis of
Tuberculosis.(3) Not only effective drug regimens are needed; without a well organized
infrastructure for diagnosis and treatment of Tuberculosis, therapeutic and control efforts are
severely hampered.(2)

The spread of Multidrug resistant Tuberculosis (MDR-TB), namely, resistant to both Isoniazid
(INH) and Rifampin (RIF), poses additional challenges to treatment with currently available
Anti-TB drugs. (1) Therefore, new drugs are required to counter the Tuberculosis pandemic.

2
 LITERATURE REVIEW

A new class of antimicrobial agents that kill Mycobacterium Tuberculosis in vitro, ex vivo and in
mouse models of TB have been the focus of recent studies. As has been identified using genetics
and biochemistry, 1,3-benzothiazine-4-ones (BZTs) targets the enzyme Decaprenyl-
phosphoribose epimerase (DprE1). Inhibition of this enzymatic activity abolishes the formation
of Decaprenylphosphoryl arabinose, a key precursor that is required for the synthesis of the cell
wall Arabinans, thus provoking cell lysis and bacterial death.(4)

The most advanced compound, BTZ043, is a candidate for inclusion in combination therapies for
both drug-sensitive and extensively drug-resistant tuberculosis.(4)

Studies have been conducted about the interaction profiles of BTZ043, the current lead compound,
with several anti- TB drugs against mycobacterium tuberculosis strain H37Rv, namely Rifampin,
Isoniazid, Ethambutol, TMC207, PA-824, Moxifloxacin, Meropenem with or without Clavulanate
and Ethylenediamine (SQ-109). No antagonism was found between BTZ 043 and the tested
compounds, and most of the interactions were purely additive.(5)

In fact TMC207 , a diarylquinoline which acts by inhibiting mycobacterial ATP synthase and has
been found to inhibit drug-sensitive and drug resistant mycobacterium TB in vitro(12), has lower
potency alone than in combination with BTZ043. The basis for their interaction is DprE1
inhibition by BZT043.(5)

The TB drug development pipeline now comprises several candidates that are in clinical trials or
soon will be.(5) BTZ043 displays similar activity against all clinical isolates of mycobacterium
tuberculosis yet tested, including MDR and XDR strains.(6)

What makes BZTs similar with other mycobacterial cell wall inhibitors is that, it appears to be
poorly active on non-replicating bacteria and require association with molecules that target this
reservoir of latent bacilli.(7)

The other important concern about these class of drugs is the reduction of an essential nitro
group to a nitroso derivative following their action. Endogenous formation of N-nitroso
compounds (NOC) from precursors (nitrite, nitrate, amino compounds) is the largest source of
exposure for the general population.(8)

Humans are exposed to a wide range of nitrogen-containing compounds which can react with
nitrosating agents to form NOC, a versatile class of carcinogens that produce tumors in 40
animal species.(8)

Nitrosation of certain phenolic compounds also results in the formation of C-nitroso, nitro and
diazo compounds, several of which have been reported to be mutagenic and/or carcinogenic.
Nitrite, nitrate and nitrosating agents can also be synthesized endogenously by reactions
mediated by bacteria and activated macrophages.(9)

3
Exposure to endogenously formed NOC has been associated with increased risk of cancers of the
stomach, esophagus and urinary bladder. But epidemiological evidence is still lacking mainly
because there are no appropriate methods measuring exposure. But if endogenously formed
nitrosamines contribute to the total burden of human cancer, preventive measures are clearly
feasible and justified.(10)

The chemistry of NOC formation has been studied extensively and a number of reviews have
been published. Its formation from amines and nitrosating species can be described in general by
equation:(10)

ˋ̗N-H + Y-NO ˋ̗N-NO + HY

The rate of nitrosation is PH-dependent, proportional to the square of the nitrite concentration
and to the amine concentration and is inversely related to the basicity of the amine.(10)

Rate = K [amine] [nitrite]2

A great variety of synthetic or naturally occurring compounds or mixtures have been shown to
inhibit the N- nitrosation reaction. The formation of NOC can be reduced, minimized or
completely prevented by the presence of reagents that rapidly destroy nitrosating agents or
reduce them to unreactive compounds.(11)

A number of studies have shown that regular consumption of fruits and vegetables is associated
with lower risk for cancers at a number of sites. Fruits and vegetables that are sources of Vitamin
C also contain β-carotene, Vitamin E and A and the non- nutritive compound, plant phenols.
Phenols are potent blocking agents of NOC formation.(8)

Generally, inhibitors of NOC formation involve compounds which either reduce Y-NO to N 2,
N2O, or NO or bind the NO+ group irreversibly. N2, N2O and NO2 are devoid of any nitrosating
activity, but NO is converted to nitrosating species in the presence of oxygen, inions and certain
metal salts.(11)

4
 OBJECTIVES

- To develop novel anti-TB compounds based on structure and chemistry, without the nitro
group liability when combined with DprE1.

- To assess the interactions of the new lead compound with other drugs in order to predict
which combinations have the best impact in the clinic.

- To investigate a completely new TB chemotherapy with innovative molecules in

combination to decrease the risk of drug resistance.

- To investigate ways for the reduction of nitro group liability by administration of potent
blocking agents of NOC formation.

5
 METHODOLOGY

The overall research methodology that will be followed for achieving the aforementioned
objectives are described in Figure 1.

Start

Literature review and bench marking

studies

Synthesis of Novel anti-TB compound

using structural chemistry

Characterization of the developed

Novel anti-TB compound and its
No
interaction with the enzyme DprE1 and
other drugs using Checkboard method
and cell viability assays
Yes
Investigations for the reduction of
nitro group liability by administration
of potent blocking agents of NOC
formation

Data analysis, interpretation and paper

writing

End

Further, in the typical experimental studies, a controlled experimental study design will be
conducted on laboratory animals which will be injected with active TB bacilli.

Clinical trials will also be conducted after the new lead compound targeting DprE1 is effectively
developed.

Trials involving human subjects will first be reviewed and approved by Ministry Of
Health(MOH) research and ethics committee. Participation will be on voluntary basis.
Participants will be fully informed about the study and possible risks if any.

6
 TIMELINE AND FUNDING

No 2019 2020
Research Activities
. J F M A MJ J A S O N D J F MA M J
Literature review and bench marking
1 studies.

Synthesis of Novel anti-TB *

2 compound.

Characterization of the developed *

Novel anti-TB compound and its
3 interaction with the enzyme DprE1
and other drugs.

Investigations for the reduction of *

nitro group liability by administration
4 of potent blocking agents of NOC
formation.

Data analysis, interpretation, journal *

5 writings and thesis writing.

Milestones and Dates

i. Completion of synthesis of the novel anti-TB compound October 2019

ii. Completion of the characterization studies March 2020
iii. Completion of investigation studies April 2020
iv. Completion of data analysis and thesis writing June 2020

The researcher would like to apply for any funding source (such as financial assistance and
laboratory facilities) at the University to undertake the overall research.

7
 REFERENCE

1. antimicrobial agents and chemotherapy. 2012 November; 56(11): 5790-5793

2. Harrison's principle of internal medicine, 18th edition, chapter 165, tuberculosis

3. uptodate, 19.3, Adherence of tuberculosis treatment, lee B.R, MD, MPH, Alfred A.L, MD

4. science 8 May 2009: vol.324 no. 5928 pp.801-804

5. -Cole ST, Riccard: G.211. New Tuberculosis drugs okn the horizon. Curr. Opin. Microbiol.
14: 570-576. [PubMed]

-Sala c, Hartkoorn RC. 2011. Tuberculosis drugs: New candidates and how to find more.
Future microbiol. 6: 617-633 [PubMed]

6. Pasca MR, et al. 2010. Clinical isolates of Micobacterium Tuberculosis in four European
hospitals are uniformly susceptible to benzothiazinones. Antimicrob. Agents chemother. 54:
1616-1618 [PMC free article] [PubMed]

7. Sala C, et al 2010. simple model for testing drugs against non-replicating Mycobacterium
Tuberculosis. Antimicrob. Agents chemother.54: 4150-4158 [PMC free articles] [PubMed]

8. National research council, 1981: Shephard et al., 1987: Wakabayashi et al, 1987

9. Steuehr and Marletla, 1985: Calmels et al., 1987: Mi wa et al., 1987

10. Mirvish, 1975: Digenis and Issi dorides, 1978: Douglas et al., 1979: Mergens and Newmark,
1980: Challis, 1981: Williams, 1983: Archer, 1984

11. Inhibitors of endogenous nitrosation mechanisms and implications in human cancer

prevention, Helmut B., Hiroshi O., and Brigitte P., Mutation research, 202(1988) 307-324

12. the new england journal okf medicine, JUNE 4, 2009, Vol. 360, page: 2397