Filling the Pipeline - New Drugs for an Old Disease

Send Orders for Reprints to [email protected] 110 Current Topics in Medicinal Chemistry, 2014, 14, 110-129 Filling the Pipeline - New Drugs for an Old Disease

Matthias Stehr1,2, Ayssar A. Elamin1 and Mahavir Singh1,3,*

1LIONEX Diagnostics and Therapeutics GmbH, D-38126 Braunschweig, Germany; 2Institute for Microbiology, De- partment of Infectious Diseases, University of Veterinary Medicine Hannover, D-30545 Hannover, Germany; 3Department of Genome Analytics, Helmholtz Centre for Infection Research, D-38124 Braunschweig, Germany

Abstract: Tuberculosis is a major global health problem. In the middle of the last century several laboratories identified, developed and synthesized several substances which were active against Mycobacterium tuberculosis, the causative agent of the disease. In the 1980s the standard oral treatment regimen was introduced with isoniazid, rifampicin, pyrazinamide, and ethambutol. In combination with the DOTS strategy it was possible treat TB within 6–8 months. But with the emergence of drug resistant strains, the formerly successful regiment became ineffective for MDR and XDR TB patients. Even more alarming, the rapidly increasing HIV epidemic also increases the number of HIV-related TB. Facing these facts, it became evident that novel strategies and antibiotics were needed to treat the new forms of TB. But over the last 60 years no novel TB drug was developed or even in the drug pipeline. But during the last ten years several novel substances have been developed to combat the deadly disease. For the first time in decades the TB drug pipeline is filled again with several promising compounds and many of them have reached Phase II and Phase III clinical trials. Several laboratories and companies all over the world currently are developing and evaluating these substances. This review presents novel substances, which were for the first time exclusively developed for TB such as bedaquilines, nitroimidazoles and the diamine SQ109. We also summarize the present knowledge about enzymes and biosynthesis pathways which offer potential targets for drug discovery against M. tuberculosis. Keywords: Bedaquiline, benzothiazinones, drug development, fluoroquinolones, Mycobacterium tuberculosis, nitroimidazoles, oxazolidinones, rifamycin.

INTRODUCTION potent substances that were effective against TB. The discovery of para-aminosalicylic acid (PAS) (1948) [4,5], Tuberculosis (TB) remains a major global health prob- thiacetazone (1951) [6], and isoniazid (1952) [7] led to the lem. Approximately 2 billion people are latently infected first TB treatment regimen including streptomycin, amino- with members of the Mycobacterium tuberculosis complex salicylic acid, and isoniazid. In the fifties of the 20th century (Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium caprae, Mycobacte- the TB regimen took 24 months of treatment but over the rium microti, Mycobacterium pinnipedii and Mycobacterium time it was improved and treatment could be shortened to 6- canettii) [1-3]. Despite the introduction of inexpensive and 9 months. In the 1980s the 6–8 months oral treatment regi- effective drug therapy regimen 40 years ago TB still contin- men was introduced with isoniazid, rifampicin, pyrazina- ues to spread. Economic and environmental issues such as mide, and ethambutol [8]. The regimen was regarded as great migration, crowded living conditions and malnutrition in- success, inexpensive and effective. After the discovery of crease the risk of transmission of TB. In 2011 there were 8.7 rifampicin, in the 1960s, almost 50 years of inactivity passed million new cases and 1.4 million deaths from TB, including without the development of any relevant TB drug. So far 350,000 deaths associated with HIV co-infection. Sub- rifampicin was the last novel class of antibiotics introduced Saharan Africa suffers most from the disease and has the for the ﬁrst-line treatment of tuberculosis [8-10]. The latest highest rates of TB. India, China, South Africa and the Rus- substances, which are recommended by World Health Orga- sian Federation have almost 60% of the world’s TB cases nization (WHO) to treat multidrug-resistant tuberculosis [3]. (MDR-TB) are fluoroquinolones [8,10]. These synthetic broad-spectrum antibacterial drugs were originally devel- Streptomycin was discovered in 1943 as first drug active oped in the 60s and 70s of the last century and their main use against tuberculosis. In the time period from 1950 to 1960 was the treatment of urinary tract infections [11]. several labs worked on TB drug research and identified With the emergence of strains resistant to multiple anti- *Address correspondence to this author at the Department of Gene Regula- biotics we are facing now a new global problem. Further- tion and Differentiation, Helmholtz Centre for Infection Research, Inhoffen- more one of the most potent TB drug, rifampicin, inactivates strasse 7, D-38124 Braunschweig, Germany; several antiviral drugs, making it difficult to use for TB-HIV Tel: +49(0) 531 6181 5302 / 5309; Fax: +49(0) 531 6181 5399; co-infections [12-17]. Email: [email protected]

It has been estimated that in 2011 half a million cases In the third section we discuss novel enzymes and path- were diagnosed with strains resistant to isoniazid and rifam- ways in M. tuberculosis and in the host, which are regarded picin (MDR-TB). Extensively drug-resistant TB (XDR-TB) as potential drug targets. is defined as MDR-TB + resistance against any fluoroquinolone and any of the second–line TB injectable drugs (ami- NOVEL SUBSTANCES kacin, kanamycin or capreomycin). By March 2013 XDR- TB has been reported in 84 countries. The frequency of Bedaquiline (ATP Synthase Inhibitor) MDR-TB varies substantially between countries. Belarus The diarylquinoline bedaquiline (TMC207; R207910) faces an alarming situation; MDR-TB is found in 32.3% and (Fig. 1) is a newly approved drug. Bedaquiline belongs to the 75.6% of the new and previously treated patients, respec- group of new chemical entities developed for novel TB tively. Approximately 12 % suffer from XDR-TB [18]. drugs and was discovered using phenotypic screening [12]. Worldwide about 4 % of newly diagnosed tuberculosis (TB) The substance is regarded as the first compound of a new patients have MDR-TB and 9% of MDR-TB cases are XDR- class of potent TB drugs [24]. Bedaquiline was found to tar- TB [19]. get the ATP synthase c subunit, but also binds to the epsilon Today we are facing an urgent need for the development subunit [25] (Fig. 7). of novel drugs, which are effective against drug resistant strains and compatible with antiretroviral therapy, as many patients are co-infected with HIV [20]. Novel regimens should be completed in a four-month period, because it has been shown that a short course treatment favors treatment completion, and consequently decreases the risk of developing MDR or XDR-TB. Finally, a key goal for drug development is to reduce serious toxicity and adverse effects [21]. Over the past 10 years several scientists, funding bodies, and the WHO’s Stop TB department boosted the discovery Fig. (1). Molecular structure of the diarylquinoline bedaquiline and development of novel TB drugs and TB treatment regi- (TMC207; R207910). mens. In 2001, the Working Group on New Drugs (WGND) was established as a Stop TB Partnership's working group in The solution structure of the gamma subunit of the M. tu- order to coordinate the development of new and affordable berculosis F-ATP synthase, which has been solved recently TB drugs. For the first time in decades the TB drug pipeline will help to elucidate the mechanism of inhibition [26]. is filled again with several diverse, novel, and promising compounds. The current drug pipeline can be monitored at Bedaquiline is supposed to interfere with the proton (http://www.newtbdrugs.org/pipeline.php). Currently there translocation step required for ATP production [27] and is are around fifty new treatment regimens with several new highly specific: Compared to the bacterial target the human therapeutic compounds under investigation, many of them in mitochondrial ATP synthase is 20,000-fold less sensitive to Phase II and Phase III clinical trials [22]. bedaquiline [28]. Extensive mutant studies suggested another target or resistance mechanism, such as drug efflux or de- These new compounds include the nitroimidazoles TBA- toxification [29]. De novo ATP synthesis is essential for the 354, PA-824 and delamanid, the fluoroquinolone DC-159a, viability of non-replicating mycobacteria and consequently, the dipiperidine SQ609, the capuramycin SQ641, the ben- bedaquiline shows activity against both replicating and dor- zothiazinone BTZ043, the caprazene nucleoside CPZEN-45 mant M. tuberculosis [30,31]. and the diamine SQ109. Delamanid (previously known as OPC67683) and bedaquiline (also known as TMC207 or Bedaquiline is highly effective against drug-susceptible R207910) are the most promising candidates. Bedaquiline and drug-resistant M. tuberculosis strains in vitro and has (Sirturo; Janssen Therapeutics) is currently part of a combi- bactericidal activity in patients who suffer from drug- nation therapy in the treatment of MDR-TB when other al- susceptible pulmonary tuberculosis. Moreover, when be- ternatives are not available [23]. Today most of the new daquiline is used in addition to standard therapy for MDR- drugs are mainly used in combination with existing TB drugs TB faster conversion to a negative sputum culture was ob- and regimens. This strategy offers hope for regimens that are served [32]. The strong sterilizing activity makes bedaquiline better tolerated, shorter in duration and with fewer drug-drug an attractive drug in the strategy of TB elimination. Recently interactions when compared with existing regimens. the US Food and Drug Administration (FDA) granted accel- erated approval to bedaquiline (Sirturo; Janssen Therapeu- This review is divided into three sections: In the first sec- tics) to treat MDR-TB in adults, despite serious safety con- tion we present novel substances, which were exclusively cerns. These include high mortality, induction of arrhythmia, developed for TB such as bedaquilines, nitroimidazoles (PA- as tissue accumulation and elevated transaminase levels 824 / OPC67683), and the diamine SQ109. [23,33,34]. Despite all concerns, it is hoped that the com- The second section, “Old TB drugs” reviews the devel- pound may be used in the near future to shorten treatment opment of drugs that are currently used for the treatment of regimen for drug-sensitive TB and the treatment of latent TB TB. [24,34]. 112 Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 1 Stehr et al.

Nitroimidazoles more potent than PA-824, thus TBA-354 has been selected by the TB Alliance for further development [49]. Nitroimidazole compounds have been used for around 50 years, e.g. the classic metronidazole, which is used against anaerobic microorganisms and amebiasis [35-38]. Metroni- SQ109 dazole and related compounds such as the nitrofurans nitro- Ethambutol is the weakest component of the drug therapy furantoin, furaltadone and nitrofurazone have been shown to regimen and should be replaced by a more potent substance. be active against tuberculosis [39,40]. Metronidazole kills Based on the 1,2-ethylenediamine core of ethambutol, over M. tuberculosis in vitro under hypoxic but not aerobic condi- 60,000 diamine analogs were synthesized and screened tions. Nevertheless it displays widely contrasting effects in against M. tuberculosis in vitro. The most potent hit was different animal models. Metronidazole seems to have no SQ109 (N-geranyl-N'-(2-adamantyl)ethane-1,2-diamine) efficacy in mice or guinea pig but other groups report that [50] (Fig. 3). SQ109 has been shown to target MmpL3, a metronidazole can prevent reactivation of latent M. tubercu- trehalose monomycolate (TMM) transporter (Fig. 7). SQ109 losis infection in macaques [41]. inhibits the synthesis of mycolic acids, such as the most The nitroimidazole CGI-17341 (Fig. 2), which was origi- toxic compound of the mycobacterial cell wall, the cord fac- nally derived from a series synthesized for the use in cancer tor [51,52]. The use of SQ109 in combination with bedaqui- chemotherapy, has been used as lead for the development of line is synergistic and improves the rate of bacterial killing in less mutagenic substances for the use in TB therapy. The vitro at an excellent minimal inhibitory concentration [53] derivate PA-824 (Fig. 2) shows potent bactericidal activity (Table 1). SQ109 is currently in Phase II clinical trials [22]. against multidrug-resistant M. tuberculosis and promising oral activity in animal infection models [42]. The derivate Benzothiazinones OPC-67683 (Now known as delamanid) (Fig. 2), a member Benzothiazinones were discovered among a series of sul- of the nitroimidazo-oxazole family is not mutagenic and has fur-containing heterocycles, which were synthesized and potent activity against drug–sensitive TB (DS-TB) and tested for activity against tuberculosis [54,55]. Benzothiazi- MDR-TB. Moreover, delamanid does not affect the activity nones have been found as the most potent inhibitors of of liver microsome enzymes, and may be used in combina- M. tuberculosis described to date. They are active at low tion with anti-retroviral agents, that induce or are metabo- nanomolar level and seen as a potential TB drug of the fu- lized by cytochrome P450 enzymes [43]. ture. It has been reported, that the efficacy of the most potent Both, PA-824 and the more potent delamanid are pro- candidate BTZ043 (Fig. 4) is comparable with isoniazid and drugs and have to be activated by the mycobacterial F420- rifampicin [54] (Table 1). Most importantly BTZ043 is ac- deazaflavin-dependent nitroreductase [44-46] (Fig. 7). The tive against DS, MDR and XDR clinical strains of M. active form is the corresponding des-nitroimidazole, which, tuberculosis and shows no interactions with other TB drugs in turn, can generate reactive nitrogen species [47]. It has [56]. BTZ043 targets DPR1, an enzyme, crucial for the been shown that nitroimidazoles finally inhibit mycolic acid biosynthesis of the mycobacterial cell wall. The details of the synthesis, but in a different manner as isoniazid [43]. mechanism of action of benzothiazinones are described in the chapter “Arabinose biosynthesis - decaprenylphosphoryl- PA-824 and delamanid are currently in Phase II and -D-ribose 2-epimerase”. Phase III clinical trials for the treatment of MDR-TB, respectively [22]. Delamanid it is currently developed by the Oxazolidinones Otsuka pharmaceutical company as a treatment for multi drug resistant TB and has been recently submitted to regula- Oxazolidinones belong to the three new novel structural tory agencies for approval [48]. PA-824 is currently being classes with antibacterial activity, which have been approved developed by the TB Alliance. TBA-354 (Fig. 2) is like PA- by the US Food and Drug Administration (FDA) in the last 824 an nitroimidazo-oxazole and was in preclinical studies

Fig. (2). Molecular structure of nitroimidazoles. Filling the Pipeline - New Drugs for an Old Disease Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 1 113

Fig. (3). Molecular structure of SQ109. Fig. (4). Molecular structure of the benzothiazinone BTZ043.

30 years. Oxazolidinones inhibit protein synthesis by binding [67]. Gatifloxacin and moxifloxacin belong to the a new to the 23S rRNA in the 50S ribosomal subunit of bacteria generation of fluoroquinolones [68]. Both substances are [57-59]. currently in Phase III clinical trials to establish whether DS- TB can be treated within a period of 4 months by substitut- In 1992, the Upjohn Company (Pfizer) synthesized dif- ing fluoroquinolones for ethambutol, or isoniazid [8]. An- ferent oxazolidinone analogs for the development of com- other fluoroquinolone, DC-159a, is currently in preclinical pounds with antibacterial activity. These studies led to the development. DC-159a displays pharmacokinetics similar to identification of the antimycobacterial compounds linezolid (PNU-100766, U-100766) and sutezolid (formerly known as those of moxifloxacin and shows higher activity against DS- and MDR-TB than gatifloxacin, moxifloxacin, levofloxacin, U-100480, PF-2341272 or PNU-100480) [57] (Fig. 5). Line- and rifamipicin [68-70]. Fluoroquinolones are widely avail- zolid was effective against MDR-TB and XDR-TB and the able, and used to treat many infectious diseases. Due to drug achieved culture conversion but more than 80 % of pa- widespread use of fluoroquinolones to treat several different tients showed serious side effects [59,60]. Linezolid also infectious diseases, resistant strains have emerged in several showed tuberculostatic activity in vitro experiments but only modest activity in murine models of TB [61]. countries, including Western Europe [71,72]. Especially in India high prevalence of ofloxacin resistance among MDR- Sutezolid (Fig. 5) also exhibited antimycobacterial activ- TB isolates is observed [73]. DNA replication is an impor- ity in vitro and studies in healthy volunteers revealed that is tant target and efforts are underway to identify novel DNA- was better tolerated by patients compared to linezolid gyrase inhibitors that are not based on the fluoroquinolone [62,63]. Moreover, combination studies revealed that the pharmacophore. application of SQ109 and sutezolid together improve the killing rate of M. tuberculosis over individual drugs [64]. Sutezolid has potent antibacterial activity against M. tuberculosis in animal models [62]. The substance has shown promising results in a Phase IIa early bactericidal activity (EBA) study and is currently in Phase II clinical trials [62,63,65]. Sequella recently licensed Pfizer´s exclusive worldwide rights to develop and commercialize sutezolid [66]. Oxazolidinones are regarded as promising candidates for TB therapy, despite serious side effects such as thrombocy- topaenia, optic neuritis, peripheral neuropathy and myelo- suppression [59,60]. Within the last years several oxazolidinones with antibacterial activity were discovered. But many of them turned out to have inadequate pharmacokinetics, and poor safety profile [57]. Nevertheless AstraZeneca has identified AZD5847 (AZD2563) (Fig. 5), a next-generation oxazolidinone, which also targets the 50S rRNA. AZD5847 is not antagonistic with other TB drugs and has completed Phase I trials [57].

OLD TB DRUGS Fig. (5). Molecular structure of different oxazolidinones. Fluoroquinolones Rifamycins Fluoroquinolones target the DNA gyrase and are fre- quently used as so called “second-line drugs” for the treat- Rifampicin, rifapentine, and rifabutin are members of the ment of MDR-TB. Some reports show that fluoroquinolones rifamycins. Rifampicin (international nonproprietary name) reduce the duration of therapy in murine models of TB and or rifampin (United States adopted name) is the most impor- thus may have the potential to reduce the duration of therapy tant sterilizing drug in the modern short-course regimen and 114 Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 1 Stehr et al. has been the most important drug for TB chemotherapy for Pyrazinamide and its Analogs more than 40 years. Rifamycins target the beta subunit of RNA polymerase, thereby preventing RNA transcription Pyrazinamide (PZA), a nicotinamide analog, is an impor- [74,75]. tant front line TB drug. It has sterilizing activity against semi-dormant tubercle bacilli inside the macrophage [87]. But rifampicin has only a short half-life of 2–4 h [76] and PZA itself is a prodrug which is converted into the active the current therapy, using 600-mg once to thrice-weekly is bactericidal form pyrazinoic acid (POA) by the bacterial suboptimal for TB treatment. High dose rifampicin-based enzyme pyrazinamidase (PZase). PZA plays a crucial role in regimens using 1,200 mg are more effective but result in an shortening the treatment duration from nine months to six influenza-like syndrome [77,78]. months when used in combination with rifampicin and isoni- To overcome the problem of the short half-life of rifam- azid [88-90]. Despite its importance in TB therapy, the picin, novel rifamycins were developed. Rifapentine is mar- mechanism of action of PZA is still unknown. Mutations in keted under the brand name Priftin by Sanofi-Aventis and is the pncA gene occur at a high frequency, which are highly a cyclopentyl-substituted rifamycin, which was developed as but not necessarily associated with PZA resistance [91]. a rifamycin for once-weekly administration [79]. Rifapentine Several mutations, scattered all over the gene have been re- has been approved for use against TB in the United States ported, which result in inactivation of PZase as a result of since 1998. thermodynamic instability [92]. PZase may not be the sole target of PZA and it has been suggested, that yet unidentified Rifapentine acts in the same way as rifampicin but has a transporter systems may play an additional role in PZA resis- much longer half-life than rifampicin, and has the potential tance [93]. to shorten duration of TB therapy [79], especially for the treatment of latent TB and DS-TB. Three-month short course In vitro both POA and PZA are effective against M. tu- treatment with rifapentine and isoniazid seems to be as effec- berculosis. The active derivative POA has no significant tive as treatment with only isoniazid for nine months. This activity in vivo, presumably due to poor absorption through short course regimen with rifapentine favors treatment com- the gastrointestinal tract and to serum binding [94]. It has pletion, and shows less liver toxicity. Many Phase II clinical been reported that derivates of POA are not dependent on the trials are in progress, in which rifampicin is replaced by activation by PZAse [95,96]. POA esters showed good an- high-dose rifapentine [80,81]. timycobacterial activity against M. tuberculosis [95,97], but Despite the efficiency of the rifamycins, the use of this have poor serum stability [98]. In addition to alkyl esters, other POA derivatives also have activity against PZA- compound class shows several difficulties. Rifamycins in- susceptible and PZA-resistant isolates of M. tuberculosis duce cytochromes P450 enzymes in the liver, which interact with other TB drug candidates such as bedaquiline and with [99]. antiretroviral agents [12-17]. Especially for the treatment of In order to increase serum stability, lipophilic POA esters patients suffering from HIV-TB co-infection new rifamycin- such as hexadecylpyrazinoate, tetradecylpyrazinoate and free regimens have to be developed. dodecylpyrazinoate have been synthesized by Simões (2009) [100,101]. Lipophilic POA esters show 5-20 times higher Clofazimine activity than POA against the inherently PZA-resistant M. Originally developed for the treatment of leprosy, the bovis BCG in vitro, but show still poor serum stability drug clofazimine was repurposed for TB treatment. The ex- [100,101]. Amide POA derivatives show higher stability in act mechanism of action of clofazimine is still unknown. The serum, but have only very low antibacterial activity [101]. putative targets of this agent are the bacterial respiratory Other examples are pyrazine thiocarboxamide and pyrazi- chain and ion transporters [82]. Recently clofazimine was noic acid pivaloxymethyl esters. Most of the reported com- found to be active against dormant bacilli and a meta analy- pounds showed an activity greater or comparable to that of sis concluded that clofazimine could be considered as an PZA. But unfortunately the most active derivates of POA are additional therapeutic option in the treatment of drug- not stable in serum [98]. resistant TB [83]. But clofazimine induces several well known adverse reactions including discoloration of the skin, TB DRUG TARGETS gastrointestinal dysfunction, severe and life-threatening ab- DXR dominal pain and organ damage [84]. An effective treatment regimen was reported, where M. tuberculosis synthesizes the essential isoprenoid pre- 87.9% among 206 patients were cured without a relapse. The cursor isopentenyl diphosphate via the 2-C-methyl-D- treatment required a minimum of nine months using a com- erythritol 4-phosphate (MEP) pathway rather than the classi- bination of clofazimine, gatifloxacin, ethambutol and pyrazi- cal mevalonate pathway typically found in humans. namide, supplemented with prothionamide, kanamycin and The 1-deoxy-D-xylulose 5-phosphate reductoisomerase high-dose isoniazid [85]. In order to reduce the serious side (DXR) carries out the second step in the MEP pathway. effects, it has been proposed to administer clofazimine via The efficient DXR inhibitor FR-900098 and fosmidomy- the aerosol route [86]. In summary, clofamizine seems to be cin (FR-31564) were isolated from Streptomyces strains dur- an inappropriate drug for the treatment of TB. The estima- ing several screening programs [102-104]. Both phospho- tion of the optimal dose of clofazimine, duration of treat- nates mimic the substrate and reaction product of DXR ment, and route of administration require further investiga- [102,105]. tion. Filling the Pipeline - New Drugs for an Old Disease Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 1 115

Table 1. MIC of Compounds Against M. tuberculosis.

Substance MW MICc (g/ml) MICc (M) Reference

Diarylquinolines

Bedaquiline (TMC207) 555.50 0.002–0.06 0.003 - 0.1 [264]

Nitroimidazoles

CGI-17341 183.16 0.06 – 0.3 0.003 - 1.6 [265]

0.01 d 0.04 d 0.81 e 2.28 e PA824 359.25 [42,43,266] 0.27 - 0.49 f 0.76 - 1.39 f 0.013 – 0.25 0.03 – 0.69

OPC-67683 534.48 0.006 – 0.024 0.01 – 0.04 [43]

0.02 d 0.006 d TBA-354 436.34 0.11 e 0.27 e [266] 0.01 – 0.15 f 0.034 - 0.364 f

Diamines

0.06 b 0.2b SQ109 330.55 [50] 0.2 a 0.63a

Benzothiazinones

BTZ043 431.38 0.001 0.0023 [54]

Oxazolidinones

Linezolid 337.34 0.99 g 2.96 g [267,268]

PNU-100480 (Sutezolid) 353.41 0.49 g 1.41 g [267,269]

AZD5847 465.40 1.00 g 2.15 g [267,270,271]

Fluoroquinolones

0.12 - 0.25 0.31 - 0.66 Gatifloxacin 375.39 [68,272] 0.007 - 0.12 g 0.001 - 0.31 g

0.18 - 0.5 0.44 - 1.24 Moxifloxacin 401.43 [68,272] 0.031 - 0.12 0.077 - 0.29

DC-159a 421.43 0.06 g 0.14 g [273]

Pyrazinoic acid esters

Pyrazinoic acid 123.11 200 1624 [101]

[101] Dodycylpyrazinoate 292.41 20 - 50 68 - 170 [100]

[101] Tetradecylpyrazinoate 320.46 10 - 25 31 - 78 [100]

[101] Hexadecylpyrazinoate 348.52 25 - 40 71 - 114 [100]

Old drugs

Pyrazinoic acid amides

N-Dodecylpyrazinamide, 291.43 N-Tetradecylpyrazinamide, 319.48 >800 >2300 [101] N-Hexadecylpyrazinamide 347.53

0.02 - 0.19 f 0.03 - 0.242 f [266] Rifampicin 822.94 0.04 - 0.24 0.04 - 0.29 [265] 1.02 g 1.25 g [267,274] 116 Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 1 Stehr et al.

(Table 1) contd….

Substance MW MICc (g/ml) MICc (M) Reference

Ethambutol 204.30 1.3 a – 1.8 b 7.00 a - 9.00 b [50]

[42,265] Isoniazid 137.13 0.02 – 0.48 0.153 - 3.50 [266,267] [274]

Clofazimine 473.39 0.12 - 2.0 0,25 - 4.2 [275] a microbroth dilution assay b BACTEC c values for susceptible M. tuberculosis d aerobic MIC (Microplate Alamar Blue assay (MABA)) e anaerobic MIC (low oxygen recovery assay) MIC90 (LORA)) f clinical strains g MIC90

Several crystal structures of DXR (Table 2) reveal that Aspartate/Semialdehyde Dehydrogenase the substrate-binding site is highly conserved in a number of During the last twenty years the complete chemical pathogens that use this pathway, so any new inhibitor that is mechanism of the aspartate/semialdehyde dehydrogenase designed for the M. tuberculosis enzyme is likely to exhibit (ASADH) has been well studied [121,122]. Substrate ana- broad-spectrum activity [106-108]. And indeed, both sub- logs such as methylene phosphonate, difluoromethylene stances show antibacterial activity against several Gram- phosphonate, phosphoramidate, cyclic phosphonate and un- negative and some Gram-positive bacteria [102], but fail to saturated and fluorinated analogs have also been shown to penetrate the lipophilic mycobacterial cell wall, thus show- inhibit the ASADH enzyme at micromolar concentrations ing no activity against Mycobacterium tuberculosis [108- [123-127]. Recently, several inhibitors have been identified 110]. through small-molecule fragment-library screening. Many of In order to improve cell wall penetration Deng et al. syn- these molecules such as D-2-aminoadipate, S-carbamoyl-L- thesized a set of lipophilic phosphonate-inhibitors of DXR cysteine, S-allyl-L-cysteine, aspartyl -difluorophosphonate and solved the crystal structure of six phosphonate-EcDXR and D-2,3-diaminopropionate have been co-crystallized with complexes (PDB ID code: 3RAS). The compound 5- ASADH from Streptomyces pneumoniae and Vibrio chol- phenylpyridin-2-ylmethylphosphonic acid showed good ac- erae [128]. tivity against E. coli DXR (Ki =420 nM), while the substance had only low affinity to MtDXR [111]. Dihydrodipicolinate Reductase In 2011, Andaloussi et al. synthesized a catechol- For the dihydrodipicolinate reductase (DapB) only a few containing phosphonic acid (“compound 8j”), which showed inhibitors are known. DapB is weakly inhibited by 2,6- an IC50 of 41 M against MtDXR in vitro. Anyway also this pyridinedicarboxylic acid (2,6-PDC) [129-131]. By an ap- phosphonate compound did not provide MIC value below proach using conventional screening in combination with 512 g/mL [112]. molecular modeling and docking inhibitor candidates of DapB have been identified [129]. A number of sulphona- Enzymes of the meso-DAP Pathway mides with promising Ki values (7 to 48 M) were identified however, the sulphonamide compounds lack good anti- Biosynthetic pathways that are critical for the survival of microbial activity [129]. the pathogen but absent in the host are good targets for novel drugs [113]. The lysine-biosynthetic pathway (Fig. 6) fulfils N-Succinyl-L,L-Diaminopimelic Acid Desuccinylase these criteria [114]. In mycobacteria L-lysine is synthesized by the meso-DAP biosynthesis pathway. The meso-DAP Two competitive inhibitors of the N-succinyl-L,L- biosynthesis pathway exists only in bacteria and is absent in diaminopimelic acid desuccinylase (DapE) from H influen- mammals [115-117]. zae are known: 2-carb-oxyethylphosphonic acid (CEPA) and 5-mercaptopentanoic acid (MSPA) [132]. A number of mi- The product L-lysine is required for protein production cromolar inhibitors of H. influenzae DapE were obtained by and its direct precursor meso-diaminopimelate (meso-DAP) screening compounds containing zinc-binding groups which plays a crucial role in cell-wall stability [118]. included thiols, carboxylic acids, boronic acids, phospho- L-lysine is synthesized in mycobacteria from aspartate in nates and hydroxamates [133]. nine steps (Fig. 6) [118]. L-lysine is obtained from meso- Diaminopimelic Acid Epimerase DAP by a single decarboxylation step [119]. Several of the enzymes of DAP biosynthesis are promising drug candi- DAP epimerase (DapF) is a unique member of the family dates. The structure of six enzymes of the DAP biosynthesis of pyridoxal phosphate–independent amino acid racemases. pathway have been solved yet (Table 2) [113,120]. The en- The active site of MtDapF contains two conserved catalyti- zymes for which inhibitors have been identified will be dis- cally active cysteines, Cys87 and Cys226, and is inactivated cussed below. in the presence of nanomolar concentrations of sulphydryl alkylating agents [134]. The active site contains a catalytic Filling the Pipeline - New Drugs for an Old Disease Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 1 117 active tyrosine (Tyr76), which is conserved among se- way converting meso-diaminopimelic acid (DAP) to L- quences of the suborder Corynebacterineae, and not preva- lysine. The lysA gene and the de novo biosynthesis of lysine lent in other bacterial species. This may facilitate the design are essential for in vivo viability of M. tuberculosis H37Rv of a species-specific inhibitor [135]. [136]. Thus DAPDC is an interesting target for efficient novel TB drugs. Crystal structures of MtDAPDC in complex with the reaction product lysine and PLP have elucidated the nature of the active sites of this enzyme [136]. Glutamine Synthetase Glutamine synthetase (GS, EC 6.3.1.2) catalyzes the synthesis of glutamine from glutamate and ammonia with con- current hydrolysis of adenosine 5´-triphosphate (ATP). GS has been recognized as potential target for tuberculosis drug development [137]. GS has central role in nitrogen metabolism is a major component of the cell wall [138,139]. By the consumption of ammonia the enzyme may influence the ammonia level within infected host cells and may contribute to the pathogen's capacity to inhibit phagosome-lysosome fusion and phagosome acidification [140]. The enzyme is released into the phagosome in infected human monocytes, a feature that is highly specific for pathogenic mycobacteria. Glutamine synthetase is essential for growth of Mycobacte- rium tuberculosis in human THP-1 and thus essential for virulence [141]. Several GS inhibitors are known [142-144]. L- methionine-(S)-sulfoximine (MSO) inhibits the growth of M. tuberculosis (IC50 = 51M) [138,142]. Like MSO several other GS inhibitors bind in the amino acid-binding site [142,145]. Unluckily the amino acid-binding site is highly conserved in the distantly related human enzyme [146]. The ATP-binding site seemed to offer far better prospects for obtaining selective inhibitors. Several novel classes of ATP-competitive MtGS inhibitors were identified by high-throughput screening [143]. One active class was the 3-amino-imidazo[1,2-]pyridines. Based on an imidazo[1,2-]pyridine several inhibitors were identified with single-digit micromolar potency and one with sub- micromolar potency. The crystal structure of an GS-inhibitor complex showed that the imidazopyridine {4-[6-bromo-3- (butylamino)imidazo [1,2-]pyridin-3-yl]phenoxy}acetic acid binds in the ATP-binding site [147]. Fatty Acid Biosynthesis Among procaryotes mycobacteria have an outstanding cell envelope, which is mainly composed of unique mycolic acids. The lipid rich cell wall plays central role for survival in the host. The fatty acids for lipid and mycolic acid synthesis are produced by two fatty acid synthesis enzyme complexes, FAS-I and FAS-II. The final step in the fatty acid biosynthesis by FAS-II is carried out mycobacterial FAS-II enoyl-acyl carrier protein (ACP) reductase (InhA). The FAS- I and FAS-II system synthesizes also the highly toxic cord factor [148]. Several inhibitors for the different enzymes of Fig. (6). Lysine biosynthesis: Enzymes and intermediates of the the FAS-I/FAS-II complex are known: KasA, the ketoacyl meso-DAP pathway. synthase is highly sensitive to cerulenin [149], thiolactomycin (TLM) [150] and platensimycin [151]. Since fatty acid Meso-Diaminopimelate Decarboxylase biosynthesis by mycobacteria has been described exhaus- The Mycobacterium tuberculosis lysA gene encodes the tively [52], the detailed steps will not be discussed further enzyme meso-diaminopimelate decarboxylase (DAPDC), a here. pyridoxal-5´-phosphate (PLP)-dependent enzyme. The en- Still InhA, the enzyme that catalyzes the last step of fatty zyme catalyzes the final step in the lysine biosynthetic path- acid biosynthesis is the prime target of the front line drugs 118 Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 1 Stehr et al.

Table 2. Structure Information of Protein Targets for Tuberculosis Treatment.

Enzyme PDB ID code Reference

Energy metabolism

F-ATP synthase 3OAAII, 1C17II [26]

Lysine biosynthesis

Aspartate/semialdehyde dehydrogenase 3TZ6, 3VOS [276]

Dihydrodipicolinate reductase 1YL5, 1YL6, 1YL7, 1C3V, 1P9L [277,278]

N-succinyl-L,L-diaminopimelic acid 3ISZI, 3IC1I [279] desuccinylase (DapE)

Diaminopimelic acid epimerase 1GQZI, 4IK0II [280,281]

Meso-diaminopimelate decarboxylase 1HKV, 1HKW [136]

Isoprenoid biosynthesis

DXR 4AIC, 4A03, 2Y1C, 2Y1D, 2Y1E, 2Y1F, 2Y1G, 2JCV, 2JCX, 2JCY, [106,108,112,282] 2JD0, 2JD1, 2JD2, 2C82

The glyoxylate cycle

Malate synthase 1N8I [176]

Isocitrate Lyase 1F8I, 1F8M, 1F61 [170]

D-arabinose biosynthesis

DprE1 4F4Q [195]

Cellular defense against reactive oxygen species (ROS)

Thiol peroxidase 1Y25 [232]

Mycothiol biosynthesis

MshC 3C8ZIII [235]

Phagosome-lysosome fusion inhibition

Protein kinase G 2PZI [205]

Glutamine biosynthesis

Glutamine synthetase (GS) 1FPYIV, 1HTO, 1HTQ, 2BVC, 2WHI, 2WGS [142,143,146,147,283]

Tetrahydrofolate biosynthesis

Dihydrofolate reductase (DHFR) 2C2TV, 2C2SV, 1DF7, 1DG5, 1DG7, 1DG8, [192,193]

Fatty acid biosynthesis

Enoyl-acyl carrier protein reductase 2X22, 2X23 [163] (InhA)

If not mentioned otherwise, PDB entries represent experimental structures of M. tuberculosis protein targets. PDB entries of structures from other organisms are indicated by roman numerals: I Haemophilus influenzae II E. coli III M. smegmatis IV Salmonella typhimurium V Homo sapiens isoniazid and ethionamide (ETH) [152]. Isoniazid is a key InhA component of short course chemotherapy for TB cure, which Still, the most potent known inhibitor for InhA is isoni- shortens TB therapy to six months. But the occurrence of azid [154,155]. Isoniazid is a prodrug that has to be activated multidrug-resistant strains, which show resistance to isoni- by KatG, the mycobacterial catalase-peroxidase, to form an azid, has become a significant threat for the public health in isoniazid-NAD adduct [154,156]. The isoniazid-NAD adduct several countries and an obstacle to effective global TB con- inhibits InhA, which in consequence leads to inhibition of trol [153]. mycolic acid biosynthesis, and ultimately to cell death [157]. Filling the Pipeline - New Drugs for an Old Disease Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 1 119

Extensive research is being carried out on the target, and up bromophenyl)-2,4-dioxobutanoic acid and 2-4-dioxo-4- to date there are 27 crystal structures of InhA available, phenylbutanoic acid [177,178]. many of them complexes with several inhibitors [158-160]. Other potent InhA inhibitors are triclosan, its derivates Leucyl-tRNA Synthetase [161,162] and 2-(o-tolyloxy)-5-hexylphenol (PT70) [52,163]. PT70 belongs to compounds that directly target Leucyl-tRNA synthetase (LeuRS) catalyzes the ATP- InhA and do not require activation by the mycobacterial dependent ligation of L-leucine to tRNA(Leu) [179]. LeuRS catalase-peroxidase KatG. These compounds are promising shows great structural divergence between prokaryotic and candidates for treating infections caused by isoniazid- eukaryotic enzymes and in the recent years several amino resistant strains. PT70 is a tight binding inhibitor of InhA acid tRNA synthetases (aaRSs) have been identified as drug with a Ki value of 22 pM. PT70 binds to the isoniazid-NAD targets [179-181]. Boron-containing agents have been shown complex. A 1.8 Å crystal structure of the ternary complex to inhibit LeuRS of Gram-negative bacteria [182]. Examples between InhA, NAD, and PT70 is available and can be used for potent boron containing compounds are the antifungal for rational drug design [163].(Table 2). agent, AN2690 [179,183,184], and the discovery of potent antitrypanosomal agents [185]. Boron-containing com- In 2009 a structure-based drug design approach identified pounds such as boromycin and aplasmomycin are examples two groups of triclosan derivatives with alkyl and aryl of macrocycles that exhibit antibiotic activity [186]. In addi- substituents, respectively, with dramatically enhanced tion synthetic boron-containing compounds are potent inhibi- potency against purified InhA. (IC50 value of 21 – 100 nM), tors of serine proteases by mimicking the transition state of which is 50-fold more potent than triclosan. The best the enzyme-catalyzed reaction by the boronyl group [187]. inhibitor is a triclosan derivative with an cyclohexane group The boron containing proteasom inhibitor Velcade® (Borte- at position 5, which shows an MIC value of 13 M, which zomib) is used in cancer therapy [188,189]. Oxaboroles are represents a tenfold improvement over the bacteriocidal attractive molecules for the design of novel enzyme inhibi- activity of triclosan [164]. tors for medical application [190]. The Glyoxylate Cycle - Isocitrate and Malate synthase Gorovoy et al. report the synthesis of highly selective antimicrobial boron-containing peptidomimetics which exhibit M. tuberculosis is dependent on fatty acids in acute and good activity against M. tuberculosis at 5 g/mL [191]. persistent infections and can grow on fatty acids as sole car- bon source [165,166]. M. tuberculosis oxidizes fatty acids Boron containing compounds can be used for several tar- via -oxidation, yielding acetyl -CoA. When acetate is util- gets. The crystal structure of two boron-containing, antifo- ized acetyl-CoA can enter the glyoxylate cycle, to produce lates has been solved with human dihydrofolate reductase TCA cycle intermediates and precursors for biosynthesis (hDHFR). The 2,4-diamino-6-methylpyrimidine with a 5- [167]. Both key enzymes of the glyoxylate cycle, the malate (1,2-closo-dicarbadodecarboran-1-yl)methyl substituent synthase and isocitrate lyase (ICL) are prominent drug target. showed modest antibacterial activity against M. tuberculosis ICL that catalyzes the cleavage of isocitrate to glyoxylate H37Ra, and three M. avium strains [192]. The dihydrofolate and succinate [167,168]. The M. tuberculosis ICL is encoded reductase from M. tuberculosis has been solved in complex by two genes, icl and icl2 [169], which are essential for the with the anticancer drug methotrexate, the antimicrobial fatty acid metabolism and jointly required for in vivo growth trimethoprim and Br-WR99210, an analog of the antimalar- and virulence. Disruption of both lyase genes results in com- ial agent WR99210 [193] (Table 2). plete impairment of intracellular replication and rapid elimination of bacteria from the lungs [165,166]. The structure of Arabinose Biosynthesis - Decaprenylphosphoryl--D- ICL from M. tuberculosis has been solved in complex with Ribose 2-Epimerase the inhibitors 3-nitropropionate and 3-bromopyruvate [170] The heteromeric decaprenylphosphoryl--D-ribose 2- (Table 2). epimerase (DPR1) is involved in the biosynthesis of D- Several groups regard MtICL as potential valuable target arabinose which is crucial for the synthesis of the mycobac- and presented the synthesis of inhibitors against MtICL [171- terial cell wall and essential for the pathogen’s survival. 174]. Other groups found that ICL has only a low potential DPR1 is encoded by the dprE1 gene (Rv3790). as drug target. The TB Alliance and GlaxoSmithKline DprE1 was identified as target of BTZ043 a new effec- (GSK) had performed an extensive high-throughput screen- tive antimycobacterial agent, belonging to the benzothiazi- ing aimed at identifying inhibitors of ICL. But ICL targets none (BTZ) class. BTZ043 was discovered by the New were found to have poor druggability [175]. It is assumed Medicines for Tuberculosis (NM4TB) Consortium that due to its deeper and more hydrophobic binding domain (http://www.mm4tb.org/) [54]. The flavoenzyme DprE1 malate synthase is a much more promising target[175,176]. works in concert with DprE2 and catalyses the epimerization The glcB gene from M. tuberculosis encodes malate syn- of deca-prenyl-phosphoryl-D-ribose (DPR) to decaprenyl- thase, and was crystallized in the presence of the substrate phosphoryl-D-arabinose (DPA). DPA is the sole and essen- glyoxylate as well as in complex with the products co- tial arabinose precursor for the biosynthesis of arabinogalac- enzyme A and malate [176] (PDB ID code: 1N8I). In vivo tan and lipoarabinomannan, a fundamental component of the the enzyme is inhibited by bromopyruvate, oxalate, and mycobacterial cell wall [194]. phosphoenolpyruvate [176]. A high-throughput screening using a 1.4 million compound library identified hits and fi- The crystal structures of MtDprE1 in complex with nally in 2012 Krieger et al. published a crystal structure of BTZ043 and its derivates CT325 and CT319 [195,196], re- Mt malate synthase in complex with the inhibitors 4-(2- vealed that BTZ043 forms a covalent bond to a cysteine 120 Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 1 Stehr et al. residue in the active site, and thus inactivates the enzyme tion and dormancy adaptation [215-219]. Several essential irreversibly. Recently, three other series of nitroaromatic molecules, proteins, coenzymes and virulence factors require compounds were reported to act as DprE1 inhibitors. These sulfur in its reduced form, such as cysteine, methionine are the dinitrobenzamides (DNB1) [197], the triazole 377790 [220], mycothiol [221-225] sulfolipid-1 [226], and coenzyme [22] and the benzoxyquinoxaline VI-9376 [198]. All these A [227]. In its oxidized form, sulfur is present as a sulfuryl - compounds target the same cysteine residue as BTZ043. moiety (SO3 ) that can modify hydroxyls and amines in Especially the dinitrobenzamides DNB1 and DNB2 have proteins, polysaccharides and lipids [228,229]. The myco- been shown to be highly active against multidrug-resistant bacterial sulfur metabolism has been well characterized dur- and extensively drug-resistant clinical isolates [197,199]. ing the last years. Several studies have revealed that strains, Due to the great diversity of inhibitor compounds DprE1 has carrying mutations in sulfur metabolism genes show de- turned out an very valuable antimycobacterial drug target creased ability to persist and cause disease [217,218,230]. [200]. Furthermore many crystal structures of various enzymes of several sulfur pathways have been solved during the last Protein Kinase G years and are available for structure-activity relationship Protein kinase G is a crucial virulence factor that pre- (SAR) studies [231,232]. vents phagosome-lysosome fusion and promotes intracellular Coates and Hu constructed a M. tuberculosis strain lack- survival [201,202], making the enzyme a highly potential ing the gene for the mycobacterial thiol peroxidase (tpx, drug target. M. tuberculosis protein kinase G has typically Rv1932). The tpx gene codes for the only thiol peroxidase the same eukaryotic domain which provides evidence of (Tpx) in M. tuberculosis. Tpx is the dominant redox enzyme horizontal gene transfer to mycobacterium genome in M. tuberculosis which protects the bacillus against oxida- [203,204]. The enzyme consists of a rubredoxin, kinase, and tive and nitrosative stresses [232-234]. The tpx strain tetratricopeptide repeat domain [204,205]. The cell- shows reduced peroxidase activity and is sensitive to H O permeable tetrahydrobenzothiophene AX20017 binds highly 2 2 and NO compared to the wild type strain. The mutant fails to specific the ATP-binding site of protein kinase G; IC = 390 50 grow and survive in macrophages. But most important the nM), while exhibiting only less or no activity against 8 my- tpx strain has attenuated virulence. These results demon- cobacterial and 25 human kinases [205]. AX20017 com- strate that Tpx is required for the survival in the host, and pletely inactivates Protein kinase G-mediated blockage of lysosomal transfer and degradation of M. bovis BCG in thus a promising drug target [233,234]. Despite the great macrophages at > 10M [202]. The crystal structure revealed importance of the mycobacterial sulfur metabolism only lit- that AX20017 is deeply buried in the adenosine-binding site tle research has been conducted so far. Anyway, several (PDB ID code: 2PZI) (Table 2). Remarkably the adenosine- studies have been performed to characterize mycothiol ligase binding site is shaped by a unique set of amino acid side (MshC), a key enzyme in the biosynthesis of mycothiol chains that are not found in any human kinase [205]. Dock- [235]. NTF1836 is a micromolar inhibitor of MshC which ing experiments with substances, isolated from Withania induces the reduction of mycothiol levels in the cell, and somnifera identified a set of withanferins and withanolides shows activity towards clinical strains of M. tuberculosis as potential inhibitors of Mt protein kinase G [206]. Withania [222]. Furthermore it has been shown that dequalinium chlo- somnifera, is known to produce a large number of steroidal ride binds in the ATP binding site of the enzyme and inhibits lactones with various pharmacological activities and antibac- MshC with an IC50 value of = 24 ±1 M. Additionally de- terial properties [207]. qualinium chloride inhibits the growth of M. tuberculosis under aerobic and anaerobic conditions [236]. MmpL3 HOST TARGETS As mentioned earlier in the text, MmpL3, an trehalose monomycolate (TMM) transporter, is inhibited by the dia- Macrophage Receptor GPR109A – Targeting Induction mine SQ109. MmpL3 seems to be the target for several of Foamy Macrophage other, structurally unrelated compounds, such as AU1235, which is no ethylenediamine such as SQ109, but shares the Upon infection, M. tuberculosis induces the formation of adamantyl residue. The adamantyl residue is part of several lipid droplets in the infected macrophage (foamy macro- pharmacological compounds [208,209]. But also other struc- phage). These lipid droplets are thought to be essential for turally unrelated compounds, such as the pyrrole derivative the survival of M. tuberculosis. The bacterium uses fatty BM212 and C215 are able to MmpL3 [210]. acids from these lipid droplets and produces lipid bodies (LBs), which serve as a source of nutrients and enable M. Mycobacterial Sulfur Metabolism tuberculosis to enter the dormant state [237,238]. Sulfur metabolic pathways in M. tuberculosis are essen- GPR109A was originally found to be a high affinity re- tial for bacterial survival and are largely absent in humans. ceptor for nicotinic acid (niacin) [239,240]. But recently it These features make them promising targets for therapeutic has been shown that the ketone body 3-hydroxy-butyrate intervention [211-214]. Macrophage infection and granu- (3HB) activates the lipolytic G protein-coupled receptor loma formation are crucial for the establishment of infection GPR109A [241,242]. It has been suggested that pathogenic in the host. It has been suggested that during granuloma for- M. tuberculosis stimulates glucose uptake and subsequent mation several genes of the bacterial sulfur metabolism are export of 3HB via an ESAT6 induced mechanism, which in up-regulated in response to oxidative stress, nutrient starva- turn leads to activation of GPR109A. Filling the Pipeline - New Drugs for an Old Disease Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 1 121

Fig. (7). Action of TB drugs, drug candidates, inhibitors and drug targets. Superscript numbers indicate compound classes. 1diarylquinoline, 2nitroimidazoles, 3ethylenediamine, 4benzothiazinones, 5oxazolidinones, 6rifamycins, 7fluoroquinolones, 8phosphonates, 9dinitrobenzamides, 10benzoxyquinoxaline, 11tetrahydrobenzothiophene, 12triazole. Ddn: F420-deazaflavin-dependent nitroreductase. LeuRS, Leucyl-tRNA synthetase. MS, malate synthase. DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase. DPR1, decaprenylphosphoryl--D-ribose 2- epimerase. GS, glutamine synthetase. MshC, mycothiol ligase. InhA, enoyl-acyl carrier protein (ACP) reductase. FASI/II, fatty acid synthase I/II. PknG, protein kinase G. Red circles marked with a yellow star indicate enzymes which activate prodrugs. P450, cytochromes P450 enzymes. Note: rifamycins induce cytochromes P450 enzymes in the liver (+).(The color version of the figure is available in the electronic copy of the article).

Activation of GPR109A causes inhibition of adenylyl cy- such as high throughput screening (HTS) has made possible clase activity, which leads to reduction in cellular cAMP and to increase the number of hits. Computational methodolo- attenuated protein kinase A activity and in an increased level gies, such as structure activity relationships (SAR) deal with of unphosphorylated perilipin. The non-phosphorylated per- the relationship between a drug's molecular structure and the ilipin forms a protective coating at the LB surface and stabi- drug's biological activity [248]. These studies in most of the lizes the lipid droplets of the macrophage, which can serve time followed by Quantitative Structure-Activity Relation- source for triglycerides for M. tuberculosis. The induction of ship (QSAR) methods which are modeling predictors to cor- lipid droplets in macrophages is a crucial virulence mecha- relate physico-chemical properties of chemicals from a data- nism of M. tuberculosis. The GPR109A-antagonist mepen- set of related compounds to their supposed relationship be- zolate bromide (MPN) has been shown to inhibit the induc- tween chemical structures and biological activity in a data- tion of lipid droplets in the macrophage [243]. set of chemicals [249,250]. QSAR shows great potential for modeling and designing novel drugs with robust properties Coronin 1 Inhibitors especially when the compounds being investigated is either Coronin-1, also termed as TACO or p57, is a tryptophan unknown or has not been structurally resolved [251,252]. aspartate-containing coat protein that prevents phagosome- Alternatively, computational methodologies based on free- lysosome fusion and thus degradation of mycobacteria cells alignment QSAR models can assist with the search for a [244,245]. Cholesterol accumulates at the mycobacterial wider chemical space, because they employ large and het- entry site and promotes mycobacterial uptake. For M. tuber- erogeneous datasets of compounds, in order to model in a culosis and M. leprae it has been shown that cholesterol also more efficient way a defined pharmacological profile [251- plays an important role in the recruitment of TACO from the 254]. In fact, nowadays, they can consider multiple experi- plasma membrane to the phagosome. The entering of myco- mental conditions such as biological targets (biomolecules, bacteria at cholesterol-rich domains and their subsequent cell lines, microorganisms, animals), measures of different uptake in TACO-coated phagosomes promotes intracellular biological effects (activity, toxicity, pharmacodynamic/ survival [245,246]. Upon mycobacterial infection, coronin 1 pharmacokinetic properties), and reliability of the assays is required for activation of the Ca2+-dependent phosphatase [255-258]. These methodologies are based on the calculation calcineurin. This mechanism blocks lysosomal delivery of of topological descriptors which cover a higher molecular mycobacteria and thus promotes infection [247]. space, and the principal condition is the search for efficient pharmacological agents. Quantitative Structure-Activity Relationship (QSAR) Studies of Antitubercular Drugs In this contest, descriptors of the chemical structures of N-1, C-7 and C-8 substituted quinolone derivatives with During years, drug discovery has been a random process promising antimycobacterial activities were generated be- based on test and error. Several experimental techniques 122 Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 1 Stehr et al. cause of the missing physicochemical data [259]. Ridge re- and the WHO has revived the tuberculosis drug discovery gression (RR), Principal component regression (PCR), and area of research. Today the TB drug pipeline is filled with partial least squares (PLS) regression were utilized to de- several promising compounds and many of them have velop predictive models of antimycobacterial activity [259]. reached Phase II and Phase III clinical trials. But anyway, A QSAR study on three diverse sets of structurally similar none of the compounds currently under investigation can be ﬂuoroquinolones has been performed and promising predic- regarded as new first line drug. So far research must be even tive models obtained. All models were useful for the design intensified to develop the urgently needed tuberculosis drugs of novel ﬂuoroquinolone analogs [260]. Moreover, a series in order to face future challenges. of nitrofuranyl compounds (nitrofuranylamide and related aromatic compounds) with potent in vitro activity against M. CONFLICT OF INTEREST tuberculosis has been investigated utilizing 3-dimensional Quantitative Structure-Activity Relationship (3D-QSAR) The author(s) confirm that this article content has no con- techniques [261]. flicts of interest. Using 3D-QSAR models and docking studies a plausible ACKNOWLEDGEMENT binding mode between thiolactomycin (TLM) analogs and beta-ketoacyl-acyl carrier protein synthase III (FabH) has Declared none. been proposed [262]. The 3D-QSAR models produced statis- tically significant results and highlights which modification REFERENCES enhance their activity especially compounds possessing hy- [1] Gutierrez, M.C.; Brisse, S.; Brosch, R.; Fabre, M.; Omais, B.; drogen bond acceptors attached to the end of side chains of Marmiesse, M.; Supply, P.; Vincent, V. Ancient origin and gene TLM [262]. The fosmidomycin and its derivatives are well mosaicism of the progenitor of Mycobacterium tuberculosis. PLoS defined inhibitors against E.coli DXR [111,263]. 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Received: August 09, 2013 Revised: August 30, 2013 Accepted: September 08, 2013