**7. Target or compound type in discovery stage**

**Analogues of thiolactomycin:** Thiolactomycinwas the first natural thiolactonedisplaying antibiotic activity. The compound showed moderate *in vitro* activity of 56 μM against *M. tuberculosis*[36]. Thiolactomycin analogues have been synthesized and some hits were found [5]. Analogues of thiolactomycin seem to inhibit mycolatesynthetase, an enzyme involved in the cell wall biosynthesis.

**Nitrofuranilamides:***M. tuberculosis*has been found to be susceptible to compounds containing a nitro group. Nitrofuranilamide was identified in a screening for inhibitors of UDP-galacto‐ semutase [5]. A set of compounds structurally related to nitrofuranilamides was synthesized and tested for antimicrobial activity. All resulted active both to sensitive and resistant strains with a MIC ranging from 0.0004 – 0.05 mg/L [37]. Four nitrofuranilamide type compounds showed significant activity in the tuberculous infection in mice models [37].

**Dihydrolipoamideacetyltransferase inhibitors:** Dihydrolipoamideacetyltransferase (dlaT) enzyme of *M. tuberculosis* is a potential target for TBdrug discovery [5]. This enzyme is a component of the pyruvate dehydrogenase sub-unit, an enzyme catalyzing acetyl-CoA synthesis and also contributes to peroxinitritereductase, adefense enzyme against oxidative/ nitrosative stress. Some heterocyclic compounds have been found to be inhibitors of the dlaT

Research and Development of New Drugs Against Tuberculosis

http://dx.doi.org/10.5772/54278

341

**"Focused Screening":** TB Alliance is helping to develop a set of projects identify chemical compounds which are active against specific molecular targets including DNA gyrase inhibitors (fluoroquinolones targets), peptidedeformylase inhibitors, and quinone-analogous

**InhA inhibitors:** InhA is the well-known enoyl-reductase of *M. tuberculosis*being an essential‐ biocatalyst forlong chain fatty acids biosynthesis (FAS-II) [41]. INH resistance is mainly mediated by mutations on KatG, the enzyme activating the prodrug. Consequently, InhA inhibitors that do not require activation by KatGcould be interesting candidates. The main goal of the screening is directInhA inhibition. Some compounds of the biphenylether type have proven to be inhibitory of InhAin a degree correlatingwith*in vitro* growth inhibition [42]. A possible limitation of this class is the possibility of cross resistance with INH and potentially

**Isocitratelyase inhibitors:** The isocitratelyase enzyme (ICL) has been found to be essential for the long term persistence of *M. tuberculosis* in mice but not in culture medium or under hypoxia conditions. McKinney and colleagues have proved recently that inhibition of the ICL1 and ICL2 isoforms block bacterial growth and survival in macrophages [8]. The absence of orthologues in mammals for this enzyme, makes it a good target for the development of inhibitors [5]. A screening of more than 900,000 compounds has been performed without satisfactory results. The potential of traditional Chinese medicine has also been researched in

**Pleuromutilins:** Pleuromutilins represent a new kind of antibiotics derived from pleuromu‐ tiline, a bioactive diterpene initially isolated from edible *Clitopilusscyphoides*fungus [44]. These molecules interfere with protein synthesis associating to the 23S rRNA unit. Despite the structural novelty of these compounds, recent studies have pointed out cross-resistance among pleuromutilins and oxazolidinones[5]. Pleuromutilins have proved to inhibit *M. tuberculosis*

**Macrolides:** This project aimed to optimize the anti-TB activity of the macrolide class through the synthesis of modified derivates of erythromycin [5]. Derivatives of erythromycin such as 11, 12-diol, 11, 12-carbamates, and 11, 12-carbazates have been found to be to most promissory

**Quinolones and DNA gyrase inhibitors**. The goal of this projectwasto synthesize and assess the potential of novel quinolones trying to decrease the time of treatment. More than 450 compounds were synthesized and assessed [5]. The 2-pyridones class has proven to be active DNA gyrase of *M. tuberculosis*, being KR1-10018 an interesting lead for the development of

enzyme, displaying non-replicative bacterial killing [40].

obtaining specific inhibitors of this enzyme [43].

electron transport inhibitors [5].

with ETH [5].

growth of *in vitro.*

anti-TBdrugs [46].

[45].

**Figure 4.** Development of new active compounds targeting *M. tuberculosis*

**Analogues of nitroimidazole:** While the PA-824 product is developed, the TB Alliance started a project to maximize the potential of this class, by identifying the improved versions of PA-824 [38,39].

**Dihydrolipoamideacetyltransferase inhibitors:** Dihydrolipoamideacetyltransferase (dlaT) enzyme of *M. tuberculosis* is a potential target for TBdrug discovery [5]. This enzyme is a component of the pyruvate dehydrogenase sub-unit, an enzyme catalyzing acetyl-CoA synthesis and also contributes to peroxinitritereductase, adefense enzyme against oxidative/ nitrosative stress. Some heterocyclic compounds have been found to be inhibitors of the dlaT enzyme, displaying non-replicative bacterial killing [40].

**Nitrofuranilamides:***M. tuberculosis*has been found to be susceptible to compounds containing a nitro group. Nitrofuranilamide was identified in a screening for inhibitors of UDP-galacto‐ semutase [5]. A set of compounds structurally related to nitrofuranilamides was synthesized and tested for antimicrobial activity. All resulted active both to sensitive and resistant strains with a MIC ranging from 0.0004 – 0.05 mg/L [37]. Four nitrofuranilamide type compounds

showed significant activity in the tuberculous infection in mice models [37].

340 Tuberculosis - Current Issues in Diagnosis and Management

**Figure 4.** Development of new active compounds targeting *M. tuberculosis*

[38,39].

**Analogues of nitroimidazole:** While the PA-824 product is developed, the TB Alliance started a project to maximize the potential of this class, by identifying the improved versions of PA-824

**"Focused Screening":** TB Alliance is helping to develop a set of projects identify chemical compounds which are active against specific molecular targets including DNA gyrase inhibitors (fluoroquinolones targets), peptidedeformylase inhibitors, and quinone-analogous electron transport inhibitors [5].

**InhA inhibitors:** InhA is the well-known enoyl-reductase of *M. tuberculosis*being an essential‐ biocatalyst forlong chain fatty acids biosynthesis (FAS-II) [41]. INH resistance is mainly mediated by mutations on KatG, the enzyme activating the prodrug. Consequently, InhA inhibitors that do not require activation by KatGcould be interesting candidates. The main goal of the screening is directInhA inhibition. Some compounds of the biphenylether type have proven to be inhibitory of InhAin a degree correlatingwith*in vitro* growth inhibition [42]. A possible limitation of this class is the possibility of cross resistance with INH and potentially with ETH [5].

**Isocitratelyase inhibitors:** The isocitratelyase enzyme (ICL) has been found to be essential for the long term persistence of *M. tuberculosis* in mice but not in culture medium or under hypoxia conditions. McKinney and colleagues have proved recently that inhibition of the ICL1 and ICL2 isoforms block bacterial growth and survival in macrophages [8]. The absence of orthologues in mammals for this enzyme, makes it a good target for the development of inhibitors [5]. A screening of more than 900,000 compounds has been performed without satisfactory results. The potential of traditional Chinese medicine has also been researched in obtaining specific inhibitors of this enzyme [43].

**Pleuromutilins:** Pleuromutilins represent a new kind of antibiotics derived from pleuromu‐ tiline, a bioactive diterpene initially isolated from edible *Clitopilusscyphoides*fungus [44]. These molecules interfere with protein synthesis associating to the 23S rRNA unit. Despite the structural novelty of these compounds, recent studies have pointed out cross-resistance among pleuromutilins and oxazolidinones[5]. Pleuromutilins have proved to inhibit *M. tuberculosis* growth of *in vitro.*

**Macrolides:** This project aimed to optimize the anti-TB activity of the macrolide class through the synthesis of modified derivates of erythromycin [5]. Derivatives of erythromycin such as 11, 12-diol, 11, 12-carbamates, and 11, 12-carbazates have been found to be to most promissory [45].

**Quinolones and DNA gyrase inhibitors**. The goal of this projectwasto synthesize and assess the potential of novel quinolones trying to decrease the time of treatment. More than 450 compounds were synthesized and assessed [5]. The 2-pyridones class has proven to be active DNA gyrase of *M. tuberculosis*, being KR1-10018 an interesting lead for the development of anti-TBdrugs [46].

**Survey of natural products:** Natural products represent an alternative for the search of new compounds. Different research institutescontinuously carry out screening of natural products (products from plants, fungi, and bacteria) with the hope of identifying compounds with anti-TB activity [5]. Some natural substances have shown significant anti-TB activity: saringosterol 24-epimers, esgosterol-5,8-endoperoxide, micromolide, ascididemin, the manzamines, and engelhardione, among others; however, there is lack of more research regarding selectivity and toxicity [47-50].

and in the post-therapy period. Post antibiotic effect, relapses, and resistance development are examined. Antagonists, additives, or synergistic effects are also evaluated when the compound is administered in combination with other active principles, as well as its capability to sterilize lesions in experimentally infected animals. Finally, toxicological studies, which must be highly controlled and documented, are carried out forthe determination of the safety window in order

Research and Development of New Drugs Against Tuberculosis

http://dx.doi.org/10.5772/54278

343

The drugs regime must be administered for several months, using commonly between 100 – 150 mice per test, therefore requiring large amounts of space and resources for maintaining the animals. Model in mice is more effective regarding the cost-benefit relation, and most of the data obtained can be reproduced in clinical studies. The model of infection by TB in mice has served to predict the sterilizing potential of new compounds, the effectiveness of the combination of drugs, success in intermittent therapy and the duration of therapy necessary to avoid relapse. The effectiveness of the active principle is measured mainly the reduction of the colony forming units (CFU) in the lung and spleen. Several varieties of mice have been used in laboratories conducting this type of test and, to this date, no comparisons have been

Genetically modified mice have been used in the in bioassay of compounds with antimycobacterial activity [62]. A mouse that does not express the interferon-γ gene (knock-out) is incapable of producing cytokine Th1 and therefore suffers a more acute infection. Bioassay with this mouse allows determining the initial efficacy of a chemical compound in six days. Because of their statistical power, substances with low antimycobacterial activity can be detected by a small decrease in the CFU count. The model has great usefulness in initial trials, when there is a limited amount of the chemical compound. Another model, still under development, has been proposed to study relapse. An animal that cannot produce the

Wayne's model, which indicates the effect of compounds against persistent bacilli, has also been used. Bacilli under anoxia conditions are used and they are directly inoculated in the mouse. The guinea pig model also allows observing the destruction of tissue by caseous necrosis where there is not oxygen contribution and bacteria go into a hypoxia state [61].

Pharmacokinetics and pharmacodynamics range from *in vitro* tests, *in vivo* tests in animal models, and finally clinical trials in humans [57]. The simplest pharmacodynamic measure is determining the MIC *in vitro,* used widely in the primary discovery of active principles. This measure can be roughly related to the maximum cut point of the active principle concentration in plasma (Cmax) and can aid in the prediction of *in vivo* pharmacodynamics among a series of structurally related agents. However, it does not represent the concentration at which the growth ceases, and, as we have already seen, does not allow distinguishing between bacteri‐ cide and bacteriostatic activity. Moreover, it does not allow obtaining information regarding the dynamic relationships *in vivo* either, since the growth conditions do not represent the ones

Animal models enable to evaluate the *in vivo* efficacy of novel active principles regimens. Protection experiments using monotherapyfor a certain amount of time and then performing

granulocyte-macrophage-colony stimulation factor (GM-CSF) is used.

of persistent organisms in the living tissue.

to perform the subsequent clinical trials in humans [60].

reported [61].

**Plants:** Drugs based on plants extracts have been used worldwide for the treatment of several diseases from ancient times.A great interest in phytomedicine and natural product structures are screened in order to measure their pharmacologic activity. In Colombia, there has been a resurgent interest in the discovery of novel natural anti-TB drugs [50-54].

**Natural sea products:** oceans are outstanding sources of natural products, not only in inverte‐ brate species such as sponges, mollusks, bryozoos, but also in marine bacteria and marine sediments. The alkaloid (+)-8-hydroxymanzamine A was initially isolated from the *Pachypelli‐ na*spsponge[55].Inthesameway,irciniolAwasfoundinspongesfromtheIndianPacificproving to be a good candidate for further studies [56]. Aerothiononine isolated from the marine sponge *Aplysinagerardogreeni*marine sponge was active against clinical isolates of MDR-TB, despite of the resistance patterns, with MIC from 6.5 to 25 mg/L [57]. The alkaloid (+)-8-hydroxymanza‐ mine A alkaloid showed potent inhibitory activity against *M. tuberculosis* H37Rv [58].

**Insects.** The immune system of invertebrates and vertebrates is made up by cytolitic peptides which act as antimicrobial agents during the invasion of eukaryotes and prokaryotes micro‐ organisms. Poison from arachnids (spiders and scorpions) contains toxic peptides of high molecular weight (2 – 12 kDa) with high specificity against prokaryote cells [59]. This type of compounds may be very promising as a drug in the treatment of tuberculosis.

**Microorganisms.**Most of the major antibiotics drugs have been isolated frommicroorganisms. Streptomycin,the first effective anti-TB drug was identified in *Streptomyces griseus*. Besides streptomycin, aminogyicosides kanamycin, amikacin, and capreomycinhave been very important therapeutically as second-line agents [59]. Other important anti-TB drugs in TB treatment are the rifamycins, which constitute a group of semi-synthetic antibiotics isolated from *Streptomyces mediterrani*[59].
