**6. Antibacterial drug discovery by targeting menaquinone biosynthesis**

Menaquinone is the sole quinone in the electron transport chain in the majority of Gram‐ positive bacteria including *Mycobacterium* spp. The biosynthetic pathway leading to menaqui‐ none is absent in humans; therefore, the bacterial enzymes responsible for menaquinone biosynthesis are potential drug targets for development of novel antibacterial agents. It is speculated that dormant (non‐replicating) *M. tuberculosis* displays a less active metabolism and also diminished energy reserves; however, Adenosine triphosphate (ATP) synthesis during oxidative phosphorylation is active during the dormant state. Therefore, inhibition of the menaquinone biosynthesis might exert serious effects on the maintenance of dormancy in *M. tuberculosis*. This concept emphasized the reports that phenothiazines block the type II Nicotinamide adenine dinucleotide phosphate (NADH): menaquinone oxidoreductase (**Figure 4**) in the bacterial respiratory chain and also were effective in killing non‐replicating *M. tuberculosis*[27]. Interestingly, it was demonstrated that inhibition of MenA (1,4‐dihydroxy‐ 2‐naphthoate prenyltransferase) (**Figure 3**) showed significant growth inhibitory activities against drug‐resistant *Mycobacterium* spp. and Gram‐positive bacteria [28]. MenA inhibitors

**Figure 4.** Electron flow in *M. tuberculosis* and type II Nicotinamide adenine dinucleotide phosphate (NADH) dehydro‐ genase inhibitors.

effectively killed non‐replicating *M. tuberculosis in vitro*. Several other promising biological date were generated on MenA as a new antibacterial drug target; (1) *M. tuberculosis* growth *in vitro* could not be rescued by exogenous vitamin K2 supplementation, (2) all Gram‐positive bacteria tested (e.g., *Staphylococcus aureus*, *Enterococcus faecalis,* and *Clostridium difficile*) were susceptible to MenA inhibitors, whereas Gram‐negative bacteria (*E. coli*, *Klebsiella pneumoniae*, *Pseudomonas aeruginosa*, and *Acinetobacter baumannii*) were not susceptible under aerobic conditions, and (3) MenA inhibitors are effective in killing *E. coli* under anaerobic conditions [18]. To date, several menaquinone biosynthesis enzymes have been studies for the development of novel antibac‐ terial agents.

inhibitor **1** killed non‐replicating *M. tuberculosis* much faster than first‐line Tuberculosis (TB) drug, rifampicin at lower concentrations. Later, the same group developed selective antimy‐ cobacterial MenA inhibitor **3**. The optically pure **3** is the most active molecule in killing non‐ replicating Mtb. *In vitro* data with selective MenA inhibitors suggested that menaquinone biosynthesis is important in maintaining mycobacterial viability under conditions of restricted oxygen [18]. MenA inhibitors are likely to block the electron flow, consequently inhibiting the bacterial growth. The other group identified 7‐methoxy‐2‐naphthol‐based MenA inhibitors (e.g., **4**) that killed *M. tuberculosis* and Gram‐positive bacteria with the MIC level of 3–25 μg/mL

Vitamin K2 Biosynthesis: Drug Targets for New Antibacterials

http://dx.doi.org/10.5772/65487

289

One of at least seven enzymes in menaquinone biosynthesis, MenB (1,4‐dihydroxy‐2‐naph‐ thoyl‐CoA synthase) forms the bicyclic ring system by catalyzing the Dieckmann type reaction of *o*‐succinylbenzoyl‐coenzyme A to 1,4‐dihydroxy‐2‐naphthoic acid. A high‐ resolution co‐crystal structure of *E. coli* MenB with a stabilized analog of *o*‐succinylbenzoylCoA was successfully diffracted. The MenB X‐ray structure provides important insight into the catalytic mechanism. Similarly, the X‐ray structure of MenB from *M. tuberculosis* was charac‐ terized. A high‐throughput screen (HTS) against *M. tuberculosis* MenB led to the discovery of 2‐amino‐4‐oxo‐4‐phenylbutanoic acid (**5**) that inhibits MenB at low nanomolar concentrations [32]. Later, methyl 4‐(4‐chlorophenyl)‐4‐oxobut‐2‐enoate (**6**) was reported to inhibit MenB by forming the CoA adduct, **7**. The adduct **7** binds to the *S. aureus* MenB with a Kd value of 2 μM, and also killed drug‐sensitive and drug‐resistant *S. aureus* strains at 0.35–0.75 μg/mL concen‐ trations [33]. Bacterial growth inhibitory assays of **6** against a battery of bacteria concluded that **6** is effective only against bacteria that utilize menaquinone for respiration. *In vivo* efficacy of **1** using the mouse models of Methicillin‐resistant Staphylococcus aureus (MRSA) infection revealed that **6** increased survival in a systemic infection model and resulted in a dose‐ dependent decrease in bacterial load [34]. These *in vitro* and *in vivo* studies came to the conclusion that MenB is a valid target for the development of new anti‐Methicillin‐resistant Staphylococcus aureus drug and infections caused by Gram‐positive bacterial infections

MenD (2‐succinyl‐5‐enolpyruvyl‐6‐hydroxy‐3‐cyclohexadiene‐1‐carboxylate synthase) catal‐ yzes a thiamin diphosphate‐dependent decarboxylative carboligation of α‐ketoglutarate and isochorismate via a Stetter‐like conjugate addition. MenD is also essential for menaquinone

(**Figure 5**) [31].

(**Figure 6**).

**Figure 6.** MenB inhibitors.

**7.3. MenD inhibitors**

**7.2. MenB inhibitors**

#### **7. Menaquinone biosynthesis inhibitors**

#### **7.1. MenA inhibitors**

Among the menaquinone biosynthesis enzymes, MenA is a membrane‐associated protein that catalyzes prenylation of demethylmenaquinone (DMK), forming 1,4‐dihydroxy‐2‐naphtoate (DHNA). Analyses of the amino acid sequence of MenA were revealed that MenA displays five transmembrane segments, and that there exists highly conserved aspartate (D), which would be localized to the inner‐plasma membrane, which was being predicted by the aid of a prediction program (Sosui) [14]. The activity is totally dependent on the presence of divalent cations, such as Mg2+. It is therefore likely that these divalent cations produce ion pairs with Asp residues contained within the catalytic site within MenA. A library of DMMK mimics possessing the amino group(s) were generated and evaluated in an enzymatic assay *in vitro* (IC50) against *Mtb* MenA. Identified MenA inhibitors were evaluated in bacterial growth inhibitory assays (MIC). The *tertiary* or *secondary* amine‐containing benzophenone derivatives **1** and **2** are the first‐generation MenA inhibitors that exhibited bactericidal activities against *M. tuberculosis* (MIC 1–1.5 μg/mL for **1**) and Methicillin‐resistant Staphylococcus aureus (MRSA) (MIC 4.0 μg/mL for **2**), respectively. These molecules are inhibited growth of drug‐ resistant *Mycobacterium* spp. and drug‐resistant Gram‐positive bacteria (vancomycin‐resistant *S. aureus*, vancomycin‐resistant *E. faecalis*, and linezolid‐resistant Methicillin‐resistant Staphylococcus aureus (MRSA)) at low concentrations [29, 30]. Significantly, the MenA

**Figure 5.** MenA inhibitors.

inhibitor **1** killed non‐replicating *M. tuberculosis* much faster than first‐line Tuberculosis (TB) drug, rifampicin at lower concentrations. Later, the same group developed selective antimy‐ cobacterial MenA inhibitor **3**. The optically pure **3** is the most active molecule in killing non‐ replicating Mtb. *In vitro* data with selective MenA inhibitors suggested that menaquinone biosynthesis is important in maintaining mycobacterial viability under conditions of restricted oxygen [18]. MenA inhibitors are likely to block the electron flow, consequently inhibiting the bacterial growth. The other group identified 7‐methoxy‐2‐naphthol‐based MenA inhibitors (e.g., **4**) that killed *M. tuberculosis* and Gram‐positive bacteria with the MIC level of 3–25 μg/mL (**Figure 5**) [31].

#### **7.2. MenB inhibitors**

effectively killed non‐replicating *M. tuberculosis in vitro*. Several other promising biological date were generated on MenA as a new antibacterial drug target; (1) *M. tuberculosis* growth *in vitro* could not be rescued by exogenous vitamin K2 supplementation, (2) all Gram‐positive bacteria tested (e.g., *Staphylococcus aureus*, *Enterococcus faecalis,* and *Clostridium difficile*) were susceptible to MenA inhibitors, whereas Gram‐negative bacteria (*E. coli*, *Klebsiella pneumoniae*, *Pseudomonas aeruginosa*, and *Acinetobacter baumannii*) were not susceptible under aerobic conditions, and (3) MenA inhibitors are effective in killing *E. coli* under anaerobic conditions [18]. To date, several menaquinone biosynthesis enzymes have been studies for the development of novel antibac‐

Among the menaquinone biosynthesis enzymes, MenA is a membrane‐associated protein that catalyzes prenylation of demethylmenaquinone (DMK), forming 1,4‐dihydroxy‐2‐naphtoate (DHNA). Analyses of the amino acid sequence of MenA were revealed that MenA displays five transmembrane segments, and that there exists highly conserved aspartate (D), which would be localized to the inner‐plasma membrane, which was being predicted by the aid of a prediction program (Sosui) [14]. The activity is totally dependent on the presence of divalent cations, such as Mg2+. It is therefore likely that these divalent cations produce ion pairs with Asp residues contained within the catalytic site within MenA. A library of DMMK mimics possessing the amino group(s) were generated and evaluated in an enzymatic assay *in vitro* (IC50) against *Mtb* MenA. Identified MenA inhibitors were evaluated in bacterial growth inhibitory assays (MIC). The *tertiary* or *secondary* amine‐containing benzophenone derivatives **1** and **2** are the first‐generation MenA inhibitors that exhibited bactericidal activities against *M. tuberculosis* (MIC 1–1.5 μg/mL for **1**) and Methicillin‐resistant Staphylococcus aureus (MRSA) (MIC 4.0 μg/mL for **2**), respectively. These molecules are inhibited growth of drug‐ resistant *Mycobacterium* spp. and drug‐resistant Gram‐positive bacteria (vancomycin‐resistant *S. aureus*, vancomycin‐resistant *E. faecalis*, and linezolid‐resistant Methicillin‐resistant Staphylococcus aureus (MRSA)) at low concentrations [29, 30]. Significantly, the MenA

terial agents.

**7.1. MenA inhibitors**

288 Vitamin K2 - Vital for Health and Wellbeing

**Figure 5.** MenA inhibitors.

**7. Menaquinone biosynthesis inhibitors**

One of at least seven enzymes in menaquinone biosynthesis, MenB (1,4‐dihydroxy‐2‐naph‐ thoyl‐CoA synthase) forms the bicyclic ring system by catalyzing the Dieckmann type reaction of *o*‐succinylbenzoyl‐coenzyme A to 1,4‐dihydroxy‐2‐naphthoic acid. A high‐ resolution co‐crystal structure of *E. coli* MenB with a stabilized analog of *o*‐succinylbenzoylCoA was successfully diffracted. The MenB X‐ray structure provides important insight into the catalytic mechanism. Similarly, the X‐ray structure of MenB from *M. tuberculosis* was charac‐ terized. A high‐throughput screen (HTS) against *M. tuberculosis* MenB led to the discovery of 2‐amino‐4‐oxo‐4‐phenylbutanoic acid (**5**) that inhibits MenB at low nanomolar concentrations [32]. Later, methyl 4‐(4‐chlorophenyl)‐4‐oxobut‐2‐enoate (**6**) was reported to inhibit MenB by forming the CoA adduct, **7**. The adduct **7** binds to the *S. aureus* MenB with a Kd value of 2 μM, and also killed drug‐sensitive and drug‐resistant *S. aureus* strains at 0.35–0.75 μg/mL concen‐ trations [33]. Bacterial growth inhibitory assays of **6** against a battery of bacteria concluded that **6** is effective only against bacteria that utilize menaquinone for respiration. *In vivo* efficacy of **1** using the mouse models of Methicillin‐resistant Staphylococcus aureus (MRSA) infection revealed that **6** increased survival in a systemic infection model and resulted in a dose‐ dependent decrease in bacterial load [34]. These *in vitro* and *in vivo* studies came to the conclusion that MenB is a valid target for the development of new anti‐Methicillin‐resistant Staphylococcus aureus drug and infections caused by Gram‐positive bacterial infections (**Figure 6**).

**Figure 6.** MenB inhibitors.

#### **7.3. MenD inhibitors**

MenD (2‐succinyl‐5‐enolpyruvyl‐6‐hydroxy‐3‐cyclohexadiene‐1‐carboxylate synthase) catal‐ yzes a thiamin diphosphate‐dependent decarboxylative carboligation of α‐ketoglutarate and isochorismate via a Stetter‐like conjugate addition. MenD is also essential for menaquinone biosynthesis in some bacteria and has been recognized as an antibacterial drug target. A succinylphosphonate ester, 4‐(methoxyoxidophosphoryl)‐4‐oxobutanoate (**8**) was reported to be a competitive inhibitor of MenD (Ki values ∼700 nM) [35]. An analog of the cofactor, thiamine diphosphate, oxythiamine **9** was reported to exhibit MenD enzyme and *S. aureus* growth inhibitory activities (**Figure 7**) [36].

established drug targets for antibacterial and antiprotozoal infections. For example, Nicotina‐ mide adenine dinucleotide phosphate (NADH) hydrogenase is a sustainable drug target for the malaria parasite and is also a promising target for *M. tuberculosis* infections. On the other hand, menaquinone biosynthesis has recently been received attention for the development of novel antibacterial agents. Menaquinone is a key component of the electron transport systems in the majority of Gram‐positive bacteria including *M. tuberculosis*. As summarized in Chap‐ ter 6, inhibitors of menaquinone biosynthesis have been identified and several compounds are also effective inhibitors of bacterial growth. In development of new drugs for *M. tuberculosis* infections, it is the ultimate goal to discover a Tuberculosis (TB) drug that is effective against human latent tuberculosis infection. Among the menaquinone biosynthesis enzymes, MenA has been extensively studies as drug target for *M. tuberculosis* and Gram‐positive bacteria. Selective MenA inhibitors inhibit the growth of non‐replicating *M. tuberculosis*, suggesting that menaquinone is essential in maintaining the bacterial viability during conditions of restricted levels of oxygen. A large set of data suggest that the DosR/DosS/DosT signaling pathway is mandatory for *M. tuberculosis'* genetic response to hypoxic conditions and nitric oxide, in the adaptation of *M. tuberculosis* to conditions triggering a reversible bacteriostasis. In this way, the DosR/DosS/DosT signaling pathway may contribute to the latency seen *in vivo* [38]. MenA inhibitors display the ability to block or hamper the flow of electrons without inducing a dormancy response in *M. tuberculosis*. Consequently, menaquinone biosynthesis inhibitors have the potential to kill *M. tuberculosis* at any states by inhibiting Adenosine triphosphate

Vitamin K2 Biosynthesis: Drug Targets for New Antibacterials

http://dx.doi.org/10.5772/65487

291

Recently, a narrow‐spectrum antibiotic, siamycin I was reported to kill *Helicobacter* and *Campylobacter* by inhibiting the futalosine (alternative) pathway (**Figure 3**). Branched fatty acids (12‐ or 13‐methyltetradecanoic acids) also inhibit the biosynthesis of menaquinone biosynthesis of *H. pylori*. Because of advances of biological assays of menaquinone biosynthesis, new inhibitor molecules that possess drug‐like characteristics will be identified. It is import to prove the efficacy of menaquinone biosynthesis inhibitor using an appropriate infected animal model. To date, *in vivo* efficacy of a MenB inhibitor was demonstrated using the mouse model of Methicillin‐resistant Staphylococcus aureus (MRSA) infection. A Tuberculosis (TB) drug, SQ109, a strong inhibitor of trehalose monomycolate (TMM) transporter, was reported to exhibit inhibitory activities of MenA and MenG enzymes from *M. tuberculosis*. Discovery of a pharmacologically acceptable menaquinone biosynthesis inhibitor, which possesses a significant antibacterial activity against replicating and non‐ replicating *M. tuberculosis,* has been highlighted in Tuberculosis (TB) drug development. It is worthwhile mentioning that menaquinone biosynthesis inhibitors are also promising agents to kill Gram‐negative bacteria growing under oxygen‐depleted or anaerobic conditions in

I thank CORNET award (University of Tennessee Health Science Center) for generous financial

(ATP) synthesis non‐directory.

**Acknowledgements**

support.

which menaquinone is utilized for their respiration.

**Figure 7.** MenD inhibitors.

#### **7.4. MenE inhibitors**

MenE (*o*‐succinylbenzoate‐CoA synthetase) is an essential adenylate‐forming enzyme that is also a promising target for development of novel antibiotics in the menaquinone biosynthesis. Adenosylsulfonamide, adenosylsulfamate, and adenosylsulfamide analogs **10**, **11**, and **12** were developed as inhibitors of MenE enzymes based on the structure of *o*‐succinylbenzoate‐CoA. The vinyl sulfonamide **10** was found to be the most potent MenE inhibitor (IC50 ∼ 5.7 μM against *M. tuberculosis* MenE) [37]. The vinyl sulfonamide **10** is a competitive inhibitor of *M. tuberculosis* MenE with respect to Adenosine triphosphate (ATP) (Ki = 5.4 ± 0.1 nM) (**Figure 8**).

**Figure 8.** MenE inhibitors.

#### **8. Conclusion**

Bacterial Adenosine triphosphate (ATP) synthase, F1F0‐ATPase, is a viable target for treatment of *M. tuberculosis* infections. Diarylquinolone, an inhibitor of *M. tuberculosis* Adenosine triphosphate (ATP) synthase exhibited a remarkable activity against *Mycobacterium* spp. Electron transport systems associated with Adenosine triphosphate (ATP) synthases are established drug targets for antibacterial and antiprotozoal infections. For example, Nicotina‐ mide adenine dinucleotide phosphate (NADH) hydrogenase is a sustainable drug target for the malaria parasite and is also a promising target for *M. tuberculosis* infections. On the other hand, menaquinone biosynthesis has recently been received attention for the development of novel antibacterial agents. Menaquinone is a key component of the electron transport systems in the majority of Gram‐positive bacteria including *M. tuberculosis*. As summarized in Chap‐ ter 6, inhibitors of menaquinone biosynthesis have been identified and several compounds are also effective inhibitors of bacterial growth. In development of new drugs for *M. tuberculosis* infections, it is the ultimate goal to discover a Tuberculosis (TB) drug that is effective against human latent tuberculosis infection. Among the menaquinone biosynthesis enzymes, MenA has been extensively studies as drug target for *M. tuberculosis* and Gram‐positive bacteria. Selective MenA inhibitors inhibit the growth of non‐replicating *M. tuberculosis*, suggesting that menaquinone is essential in maintaining the bacterial viability during conditions of restricted levels of oxygen. A large set of data suggest that the DosR/DosS/DosT signaling pathway is mandatory for *M. tuberculosis'* genetic response to hypoxic conditions and nitric oxide, in the adaptation of *M. tuberculosis* to conditions triggering a reversible bacteriostasis. In this way, the DosR/DosS/DosT signaling pathway may contribute to the latency seen *in vivo* [38]. MenA inhibitors display the ability to block or hamper the flow of electrons without inducing a dormancy response in *M. tuberculosis*. Consequently, menaquinone biosynthesis inhibitors have the potential to kill *M. tuberculosis* at any states by inhibiting Adenosine triphosphate (ATP) synthesis non‐directory.

Recently, a narrow‐spectrum antibiotic, siamycin I was reported to kill *Helicobacter* and *Campylobacter* by inhibiting the futalosine (alternative) pathway (**Figure 3**). Branched fatty acids (12‐ or 13‐methyltetradecanoic acids) also inhibit the biosynthesis of menaquinone biosynthesis of *H. pylori*. Because of advances of biological assays of menaquinone biosynthesis, new inhibitor molecules that possess drug‐like characteristics will be identified. It is import to prove the efficacy of menaquinone biosynthesis inhibitor using an appropriate infected animal model. To date, *in vivo* efficacy of a MenB inhibitor was demonstrated using the mouse model of Methicillin‐resistant Staphylococcus aureus (MRSA) infection. A Tuberculosis (TB) drug, SQ109, a strong inhibitor of trehalose monomycolate (TMM) transporter, was reported to exhibit inhibitory activities of MenA and MenG enzymes from *M. tuberculosis*. Discovery of a pharmacologically acceptable menaquinone biosynthesis inhibitor, which possesses a significant antibacterial activity against replicating and non‐ replicating *M. tuberculosis,* has been highlighted in Tuberculosis (TB) drug development. It is worthwhile mentioning that menaquinone biosynthesis inhibitors are also promising agents to kill Gram‐negative bacteria growing under oxygen‐depleted or anaerobic conditions in which menaquinone is utilized for their respiration.
