Discovery of 1,4-Didydroxy-2-naphthoate Prenyltransferase Inhibitors

Abstract

Since utilization of menaquinone in the electron transport system is a characteristic of Gram-positive organisms, the 1,4-dihydroxy-2-naphthoate prenyltransferase (MenA) inhibitors 1a and 2a act as selective antibacterial agents against organisms such as methicillin-resistant Stapylococcus aureus (MRSA), Staphylococcus epidermidis (MRSE), and Mycobacterium spp. Growth of drug-resistant Gram-positive organisms was sensitive to the MenA inhibitors, indicating that menaquinone synthesis is a valid new drug target in Gram-positive organisms.

Antimicrobial resistance of pathogens is a global problem. Each year worldwide, more than 11 million people die from the major infectious killers (i.e., MDR^a tuberculosis, malaria, HIV, diarrhea diseases, and pneumonia).¹ The increasing drug resistance among Gram-positive bacteria is a significant problem because they are responsible for one-third of nosocomial infections; drug resistance in Gram-positive organisms (i.e., staphylococci, pneumococci, vancomycin resistance in enterococci, and mycobacteria) have achieved prominence in the past 15 years. Methicillin-resistant S. aureus (MRSA) is one of most frequent nosocomial pathogens in developed countries.² In addition, Mycobacterium tuberculosis (Mtb) is responsible for nearly 2 million deaths annually and one-third of the world population is infected with latent Mtb. In particular, people who are malnourished or have HIV-AIDS are susceptible to TB infection. Moreover, the emergence multidrug-resistant strains of Mtb (MDR-TB) seriously threatens TB control and prevention efforts.³ The results of over 10 years of screening of strains and molecular targets (existing and new) from traditional product sources (randomly generated library molecules, secondary metabolites, and drug libraries) have been disappointing.⁴ Therefore, identification of new molecular targets and mechanisms of action that involved identifying essential, ubiquitous bacterial genes in pathogens that are prokaryote and eukaryote selective to prevent side effects in the host has been studied.

The lipid-soluble electron carriers (lipoquinones) occupy a central and essential role in electron transport coupled ATP synthesis. The lipoquinones involved in the respiratory chains of bacteria consist of menaquinones and ubiquinones. From the taxonomic studies it is evident that a majority of Gram-positive bacteria including Mycobacterium spp. utilize only menaquinone in their electron transport systems,⁵ and menaquinone biosynthesis is essential for survival of nonfermenting Gram-positive bacteria.⁶ On the other hand, Gram-negative organisms such as E. coli utilize ubiquinone (CoQ) under aerobic conditions and utilize menaquinone under anaerobic conditions. Moreover, the electron transport chain in humans does not utilize menaquinone.⁷ Therefore, inhibitors of menaquinone biosynthesis have great potential for the development of novel and selective drugs against MDR Gram-positive pathogens.⁸ However, no study on the development of inhibitors for menaquinone biosynthetic enzymes has been reported. In this communication, we report that inhibition of 1,4-dihydroxy-2-naphthoate prenyltransferase (MenA), which catalyzes a formal decarboxylative prenylation of 1,4-dihydroxy-2-napthoate (DHNA) (Figure 1),⁹ showed significant growth inhibitory activities against drug-resistant Gram-positive bacteria.

Figure 1.

Schematic bacterial electron transport chain and menaquinone biosynthesis.

The MenA activity was characterized using membrane fractions prepared from M. tuberculosis as previously described.¹¹ MenA is predicted to have five transmembrane segments, and there are highly conserved Asp residues that would be located in the inner-plasma membrane.¹² The activity is absolutely dependent on the presence of the divalent cations such as Mg²⁺. Thus, it is likely that such divalent cations form ion pairs with Asp residues existing in the catalytic site of MenA. On the basis of the observation of this enzymatic activity and the structure of the MenA product, demethylmenaquinone (DMMK), we designed tertiary or secondary amine or hydrazine-containing DMMK mimics (1) in hope that the amine moiety would interact with Asp residue(s) directly or through the divalent cation(s) in the active site and (2) in which the chemically unstable 1,4-quinone system is replaced with the hydrophobicly substituted benzophenones. As illustrated in Scheme 1, the designed DMMK mimics were synthesized efficiently in four to six steps including (1) Friedel-Crafts acylation, (2) deprotection, (3) alkylation(s), (4) bromination, and (5) amination reactions.

Scheme 1.

Generation of a Library of Molecules in Solution¹⁰,^a

^a Reagents and conditions: (a) AlCl₃, PhNO₂ (75-90%); (b) (i) 48% HBr, AcOH (90%); (ii) 1,5-dibromopentane or 1,6-dibromohexane or 1,7-dibromoheptane or 1,8-dibromooctane, K₂CO₃, DMF (for 1) (80-95%); 1,4-dibromobutane, K₂CO₃, DMF; 1,3-propanediol, NaH, DMF; CBr₄, PPh₃, CH₂Cl₂ (for 2) (65%); 1,4-dibromobutene, K₂CO₃, DMF; 1,3-propanediol, NaH, DMF; CBr₄, PPh₃, CH₂Cl₂ (for 3) (65%); 1,4-dibromobutyne, K₂CO₃, DMF; 1,3-propanediol, NaH, DMF; CBr₄, PPh₃, zCH₂Cl₂ (for 4) (65%); (iii) R₅ (primary or secondary amines or hydrazines), NaHCO₃, DMF (50-98%); (iv) TFA, CH₂Cl₂ (for Boc-protected R₅) (100%).

We have synthesized 100 molecules in solution, and the library of molecules was evaluated in enzymatic assays in vitro (IC₅₀) against Mtb MenA¹¹ and in mycobacterial growth assays (MIC). More than 18 molecules exhibited MenA IC₅₀ and MIC values of less than 20 μM, and in all cases the MIC value was in good agreement with the IC₅₀ value. From these preliminary screenings it was shown that the shorter length of linker (C5-C7 in 1) between the phenolic oxygen and the nitrogen atom decreased the ability to inhibit MenA and the efficacy of growth inhibition. In addition, the structure of amine or hydrazine significantly influences the activity; α-substituted amine or bulky tertiary amine containing molecules did not show MenA inhibitory activity at lower concentrations.¹⁰ Identification of the effective substitution pattern (R₁, R₂, R₃, and R₄) in benzophenone moiety requires extensive SAR studies; however, the hydroxy group on R₃ (R₃ = OH in 1 and 3) seems to be superior to the others (R₃ = H or Cl) regardless of the structure of linker.¹³

Two molecules 1a and 2a, named allylaminomethanone-A and phenethylaminomethanone-A, respectively, showed MIC values of 1.5 and 12.5 μg/mL against Mtb (H37Rv, a common laboratory strain), respectively. We resynthesized 1a and 2a and determined MICs (μg/mL) against a variety of species and strains of Gram-positive (entries 1-19 in Table 1) and Gram-negative bacteria (entries 20-23 in Table 1). As summarized in Table 1, only Gram-positive bacterial growth was inhibited by 1a (for Mtb) and 2a (others), supporting the hypothesis that menaquinone synthesis is the target of these molecules.¹⁴ In addition, growth of drug-resistant Gram-positive organisms was sensitive to the MenA inhibitors (entries 2, 3, 14, 10-17), indicating that MenA is likely to be a valid drug target in Gram-positive pathogens involved in emerging diseases.

Table 1.

MICs of 1a, 2a, and Representative Antibacterial Agents (Clinically Used) for Gram-Positive Bacteria Including Mycobacterium spp

		MIC (μg/mL)a
entry	species and strain	1ab	2ac	RFPd	INHd	VCMd	LZDd
1	M. tuberculosis H37Rv	1.5	12.5	0.2	0.1
2	M. tuberculosis H37Rv INHr	1.5		0.2	>25
3	M. tuberculosis H37Rv RFPr	1.5		>25	0.05
4	M. bovis BCG Tokyo	3.1		0.1	0.1
5	M. avium Flamingo	6.3		3.1	>25
6	M. intracellulare ATCC15984	6.3		3.1	12.5
7	M. aurum	6.3		0.78	6.3
8	M. fortuitum NIHJ1615	12.5		>25	6.3
9	M. smegmatis Takeo	12.5		>25	12.5
10	MRSA high-resistance		4			1	2
11	VRSA 70		4			0.5	2
12	MRSA 92-1191		4			1	2
13	MRSA Mu50		8			1	2
14	MRSA LDZ-resistance06		8			1	32
15	S. aureus macrolide-resistance		4			1	2
16	E. faecalis NCTC12201 (VanA)		8			>128	2
17	E. faecalis NCTC12203 (VanA)		2			>128	4
18	S. aureus FDA209P		4			1	2
19	S. aureus Smith		8			1	2
20	E. coli NIHJ JC-2		>128			>128	128
21	K. pneumoniae NCTN9632		>128			>128	>128
22	P. mirabilis IFO3849		>128			>128	128
23	P. aeruginosa 46001		>128			>128	>128

^aThe agar plate dilution method was used (see Supporting Information).

^bMICs against Gram-positive bacteria were >12.5.

^cMICs of 2a against Mycobacterium spp. were >10-fold higher than those of 1a.

^dVCM: vancomycin. LZD: linezolid. RFP: rifampicin. INH: isoniazid.

In conclusion, we have shown, for the first time, that MenA inhibitors 1a and 2a inhibited growth of drug-resistant Mycobacterium spp. and other Gram-positive bacteria at low concentrations. The MenA inhibitors described here can be synthesized cost-effectively, and structural modifications to improve the inhibitory activity in vitro can be achieved in a time efficient manner. The results are expected to be of significance in terms of discovering new lead molecules that can be developed into new drugs to combat Gram-positive pathogens.