**6. Discovery of active compounds**

**5. Platform for the development of active principles in the treatment of TB**

Bothbasic andclinicalpharmacologyhave contributedto theprogress inthediscovery ofdrugs applyingtheirexperiencetothedevelopmentandvalidationofhypothesesofnewactiontargets in order to produce novel drugs. In this sense, researchers need to be innovative and they must haveawidevisionovertheinterpretationoftheresults[20].Thechoiceofatherapeuticcandidate is probably the most important decision to make in the discovery and development of a medication.Thechemicalstructureofadrugconfersitsbiologic,pharmacokinetic,physicochem‐ ical, andtoxicologicalproperties [22].Ontheotherhand,thediscoveryanddevelopmentofnew drugs is a complex and costly process requiring large amount of resources and time. The cost of launching a new drug to the market ranges from US\$ 800 million and 1000 million, and it may take between 8 and 17 years depending on the disease and the treatment (Figure 2) [23].

**Figure 2. TB drug pipeline.**From the discovery bench through preclinical and clinical studies for novel anti-TB agents,

The term "hit" is used to describe a small number of structurally related molecules possessing an established biologic (antituberculosis) activity [24,25]. The term "lead" is defined as a molecule belonging to a series, which shows a substantial structure – activity improvement around a determined *"hit"*, and from which other important factors have been obtained such

Once these terms are defined it is important to knowthe biochemical target on which a certain structural type of a chemical compound exerts a biological action. The determination of the mechanism can be carried out *in vitro* by generating drug-resistant mutants which are examined on their whole-genome sequences analysis. The transcriptional profile using cutting edge mycobacterial microarrays or *q*PCR) can be interrogated among the whole transcriptome for potentially distinguishing a defined set of genes involved in the response against chemical injury. Once a determined protein or receptor has been identified, cloning, over-expressing and purifying the proteins is usually performed with the aim of examining its biochemistry and its possibility of affinity or interaction in the tube test is always possible option. Gene deletions and over-expressing systems in *Mycobacterium* are also used for confirming the mechanism of action of a defined candidate [26].Ideally, an antibacterial agent must show bactericidal activity often impeding an essential function for the survival of the microorganism.

as evidence of selectivity and pharmacokinetic data or *in vivo* activity [24].

a process that could last more than 15 years.

336 Tuberculosis - Current Issues in Diagnosis and Management

The parameter most commonly determined to examine the in vitro antibacterial activity of a specific moleculeis the minimum inhibitory concentration (MIC) which represents the concentration required to inhibit 99.9% of the growth of bacilli The main limitations of these trials is that do not describe the percentage of dead bacteria (which critically depends on cell density) or the metabolic state of the bacteria, if we aim to examine the persistent antimicrobial effects of a certain drug [27]. Most publications include at least a compound with a MIC lower than 6.25 mg/Ll [24,26]. It is recommended that active compounds under a colorimetric assay (Resazurin, Alamar Blue, MTT) are reconfirmed usingagar-based techniques or MGIT. A simple and easy to use, agar-based method using Middlebrook 7H10 was introduced in 2004 by Bhakta et al for measuring MIC values [28,29]. The spot culture growth inhibition assay (SPOTi) has now being used to screen more at least more than 1000 compounds. Simultane‐ ously, the cytotoxicity in different type of mammalian and/or macrophages is carried out. The selectivity index (SI) is determined by dividing the growth inhibitory concentration 50 (GIC50) corresponding to the concentration of compound capable of killing half of the mammalian cellsby the MIC using the same concentration units. If the SI is larger than 10, infection of a macrophage with a selected strain of mycobacteria and treating with the drug candidate can help to determine its intracellular potential (Figure3)[26].

**Figure 3. Research and development of new TB active compounds.**In an attempt to promote quickly pre-clinical studies of early leads, Orme propose this rapid diagram based on the selectivity ration between a bacteria and mam‐ malian cell line [26].

The macrophage infectionmodel offers the possibility of evaluating the compound in a physiologically challenging intracellular space. By plotting a viability curve fordifferent concentrations of the active principle, The EC90, EC99 and EC99.9values of are determined verifying the concentration that is able to reduce the bacterial load by 1, 2 and 3 logarithmic units. MIC is most usually defined as EC99.(or EC95). Bactericidal compounds are generally associated with a 3-fold reduction in CFU logarithmic units. In addition the infection assay determines the activity of a compound in an intracellular medium which does not always correlate with *in vitro* media-based inhibition measurements. For instance, transport mecha‐ nisms in the cell may influence the intracellular concentration of the drug regardless of the external fluid concentration [30].

Targets existing in *M. tuberculosis*whileabsent in other bacteria would seem ideal since active compounds against this target will be harmless to bacteria beneficial for the human being. However, selecting targets complying with this requisite leads to restrict extensively the likely targets: for the most of it only the biosynthesis of the mycobacterial cell- wall or those implied

Research and Development of New Drugs Against Tuberculosis

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

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The validation of a target, involves the examination of bacterial viability when decreasingthe protein expression. If reducing the enzyme level,led to lose in bacterial viability, then the target is known as "vulnerable", and it is meant to be attacked [30]. The elimination or knockout of the gene that codifies an essential protein is difficult (or impossible) to produce by homologous recombination if the gene is essential in the conditions of growth, and therefore inducible promoters are a better chance to show the effect of tightly reducing its expression. Overexpression of the target is also possible, the growth of the over-expressed mutant being rescued under higher concentrations of inhibitor. These studied have led to many targets that have been identified and validated. The studies of Sasettiet al identified which enzymes were essential in vitro and in vivo using a transposon site hybridization analysis (TraSH) using both

Another approach is related to the genomics of virulence. Some mycobacterial genes are only expressed in granulomabut not inside the macrophages. Isocitratelyase enzyme is fundamen‐ tal in the persistence of bacilli in chronic infection in mice and its function is related to obtaining carbon during its persistence in the host [8,33]. The extracellular repetition protein (Erp) is another essential protein involved in *M. tuberculosis* virulence that was the first discovered virulence factor. The mutant Δ-erp that does not express correctly the extracellular repetition protein does not show any alteration in standard *in vitro* culture, but maintains an essential function for *in vivo* survival [34,35]. This protein is also a potential target for the development of anti-TB active principles. Two independent proteins (fadD28 and mmpL7) have been identified contributing to the early growth of *M. tuberculosis* in mice lungs and are related to the synthesis and transport of a complex lipid associated to the cell wall, i.e. phthioceroldi‐ mycocerosate (PDIM) [34]. Although the function of this lipid is unknown, it is suspected that itplays a role in the decrease of the host's immune response. There is no doubt about the remarkable progress that the sequencing of the*M. tuberculosis*genomehas brought to the anti-TB drug discovery area of research. The functional annotation of all these genes remains a considerable amount of experimental work. Sequencing of other related organisms such as *M. marinum*, *M. leprae*, *M. aurum*and othersoffer often clues about the essentiality of specific set of

**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

in specific mycobacterial process (virulence, detoxification, others).

in vitro or in vivo [31,32].

genes and operon distribution.

the cell wall biosynthesis.

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

The success of a discovery program of antibacterial principles is founded on three factors: identification of key elements contributing to pathogenicity of the microorganism, the understanding of the existing relationships between the microbe and the host, and important‐ ly, the properties of the chemical compound [30]. Two pathways have been traced with the aim of discovering active principles. One is the empirical pathway, mainly based on chemistry and phenotypic screening; and the more modern is the mechanistic, based on genomics, biochemistry and molecular biology. The former begins with the identification of an active principle with potent antimicrobial activity on *in vitro* conditions. The active principle is discovered by chance or by random screening. Then, it is subject to trial on rigoroustoxico‐ logical assays before using animal models. Some candidates may eventually beselected for human trials. The limitation of the empirical pathway is the lack of information on the specific target or the action mode, sometimes this lack of understanding can led to high failure rates mostly for toxicity problems [30]. On the other hand,the mechanistic pathway started with the age of molecular biology and genomics which allowed the identification of specific targets of the microbe, absent or structurally different in human hosts. This strategy can be upgradeto high-throughput screening (HTS) platform and to evaluate a large amount a substances in little time. Crystallization of the target proteins and X-ray diffraction spectroscopy, together with an analysis of the active site in the presence of the natural substrate and inhibitors allow the detailed study of the crucial structural interactions.

In the mechanistic approach discovery usually involves firstly the identification and validation of amycobacterial target macromolecule to be inhibited or interrupted. Obtain‐ ing the small molecules which inhibit such a target is another story. Large collections of compounds can be screened directly against the protein if a high-throughput method of assay is available. Alternatively if there is structural information it is possible to computationally interrogate the target against a defined set of computer-based compounds (docking). The preferred targets are generally the ones ocurring in *M. tuberculosis* and not represented in the human genome. By means of comparative genomics, the targets are present in the human genome. For example, nicotinamide adenine dinucleotide (NAD)is generated in humans either by *de novo*biosynthesis, or by DNA and RNA degradation. However*M. tuberculo‐ sis*can only synthesize NAD using the*de novo* synthesis. This allows to rationally explorequi‐ nolinatephosphoribosyltransferase (QAPRTase) inhibition (*de novo* pathway) for the developing of microbial selective inhibitors [30].

Targets existing in *M. tuberculosis*whileabsent in other bacteria would seem ideal since active compounds against this target will be harmless to bacteria beneficial for the human being. However, selecting targets complying with this requisite leads to restrict extensively the likely targets: for the most of it only the biosynthesis of the mycobacterial cell- wall or those implied in specific mycobacterial process (virulence, detoxification, others).

The macrophage infectionmodel offers the possibility of evaluating the compound in a physiologically challenging intracellular space. By plotting a viability curve fordifferent concentrations of the active principle, The EC90, EC99 and EC99.9values of are determined verifying the concentration that is able to reduce the bacterial load by 1, 2 and 3 logarithmic units. MIC is most usually defined as EC99.(or EC95). Bactericidal compounds are generally associated with a 3-fold reduction in CFU logarithmic units. In addition the infection assay determines the activity of a compound in an intracellular medium which does not always correlate with *in vitro* media-based inhibition measurements. For instance, transport mecha‐ nisms in the cell may influence the intracellular concentration of the drug regardless of the

The success of a discovery program of antibacterial principles is founded on three factors: identification of key elements contributing to pathogenicity of the microorganism, the understanding of the existing relationships between the microbe and the host, and important‐ ly, the properties of the chemical compound [30]. Two pathways have been traced with the aim of discovering active principles. One is the empirical pathway, mainly based on chemistry and phenotypic screening; and the more modern is the mechanistic, based on genomics, biochemistry and molecular biology. The former begins with the identification of an active principle with potent antimicrobial activity on *in vitro* conditions. The active principle is discovered by chance or by random screening. Then, it is subject to trial on rigoroustoxico‐ logical assays before using animal models. Some candidates may eventually beselected for human trials. The limitation of the empirical pathway is the lack of information on the specific target or the action mode, sometimes this lack of understanding can led to high failure rates mostly for toxicity problems [30]. On the other hand,the mechanistic pathway started with the age of molecular biology and genomics which allowed the identification of specific targets of the microbe, absent or structurally different in human hosts. This strategy can be upgradeto high-throughput screening (HTS) platform and to evaluate a large amount a substances in little time. Crystallization of the target proteins and X-ray diffraction spectroscopy, together with an analysis of the active site in the presence of the natural substrate and inhibitors allow the

In the mechanistic approach discovery usually involves firstly the identification and validation of amycobacterial target macromolecule to be inhibited or interrupted. Obtain‐ ing the small molecules which inhibit such a target is another story. Large collections of compounds can be screened directly against the protein if a high-throughput method of assay is available. Alternatively if there is structural information it is possible to computationally interrogate the target against a defined set of computer-based compounds (docking). The preferred targets are generally the ones ocurring in *M. tuberculosis* and not represented in the human genome. By means of comparative genomics, the targets are present in the human genome. For example, nicotinamide adenine dinucleotide (NAD)is generated in humans either by *de novo*biosynthesis, or by DNA and RNA degradation. However*M. tuberculo‐ sis*can only synthesize NAD using the*de novo* synthesis. This allows to rationally explorequi‐ nolinatephosphoribosyltransferase (QAPRTase) inhibition (*de novo* pathway) for the

external fluid concentration [30].

338 Tuberculosis - Current Issues in Diagnosis and Management

detailed study of the crucial structural interactions.

developing of microbial selective inhibitors [30].

The validation of a target, involves the examination of bacterial viability when decreasingthe protein expression. If reducing the enzyme level,led to lose in bacterial viability, then the target is known as "vulnerable", and it is meant to be attacked [30]. The elimination or knockout of the gene that codifies an essential protein is difficult (or impossible) to produce by homologous recombination if the gene is essential in the conditions of growth, and therefore inducible promoters are a better chance to show the effect of tightly reducing its expression. Overexpression of the target is also possible, the growth of the over-expressed mutant being rescued under higher concentrations of inhibitor. These studied have led to many targets that have been identified and validated. The studies of Sasettiet al identified which enzymes were essential in vitro and in vivo using a transposon site hybridization analysis (TraSH) using both in vitro or in vivo [31,32].

Another approach is related to the genomics of virulence. Some mycobacterial genes are only expressed in granulomabut not inside the macrophages. Isocitratelyase enzyme is fundamen‐ tal in the persistence of bacilli in chronic infection in mice and its function is related to obtaining carbon during its persistence in the host [8,33]. The extracellular repetition protein (Erp) is another essential protein involved in *M. tuberculosis* virulence that was the first discovered virulence factor. The mutant Δ-erp that does not express correctly the extracellular repetition protein does not show any alteration in standard *in vitro* culture, but maintains an essential function for *in vivo* survival [34,35]. This protein is also a potential target for the development of anti-TB active principles. Two independent proteins (fadD28 and mmpL7) have been identified contributing to the early growth of *M. tuberculosis* in mice lungs and are related to the synthesis and transport of a complex lipid associated to the cell wall, i.e. phthioceroldi‐ mycocerosate (PDIM) [34]. Although the function of this lipid is unknown, it is suspected that itplays a role in the decrease of the host's immune response. There is no doubt about the remarkable progress that the sequencing of the*M. tuberculosis*genomehas brought to the anti-TB drug discovery area of research. The functional annotation of all these genes remains a considerable amount of experimental work. Sequencing of other related organisms such as *M. marinum*, *M. leprae*, *M. aurum*and othersoffer often clues about the essentiality of specific set of genes and operon distribution.
