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

Another more classical possibility is monitoring of microbiologic parameters such as growth rate, CFU counting and chemical analysis of metabolites in the treatment with sib MIC, MIC and over-MIC values of the agent. Currently, many active principles are identified as the result of a rational design, supported by genomics inspired hypotheses or from another perspective,

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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

**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‐

by automated high-throughput screening (HTS) using compounds libraries [23].

**6. Discovery of active compounds**

help to determine its intracellular potential (Figure3)[26].

malian cell line [26].

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, a process that could last more than 15 years.

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

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. Another more classical possibility is monitoring of microbiologic parameters such as growth rate, CFU counting and chemical analysis of metabolites in the treatment with sib MIC, MIC and over-MIC values of the agent. Currently, many active principles are identified as the result of a rational design, supported by genomics inspired hypotheses or from another perspective, by automated high-throughput screening (HTS) using compounds libraries [23].
