Baojie Wan

Low-Oxygen-Recovery Assay for High-Throughput Screening of Compounds against Nonreplicating Mycobacterium tuberculosis

ABSTRACT Screening for new antimicrobial agents is routinely conducted only against actively replicating bacteria. However, it is now widely accepted that a physiological state of nonreplicating persistence (NRP) is responsible for antimicrobial tolerance in many bacterial infections. In tuberculosis, the key to shortening the 6-month regimen lies in targeting this NRP subpopulation. Therefore, a high-throughput, luminescence-based low-oxygen-recovery assay (LORA) was developed to screen antimicrobial agents against NRP Mycobacterium tuberculosis. M. tuberculosis H37Rv containing a plasmid with an acetamidase promoter driving a bacterial luciferase gene was adapted to low oxygen conditions by extended culture in a fermentor with a 0.5 headspace ratio. The MICs of 31 established antimicrobial agents were determined in microplate cultures maintained under anaerobic conditions for 10 days and, for comparative purposes, under aerobic conditions for 7 days. Cultures exposed to drugs under anaerobic conditions followed by 28 h of “recovery” under ambient oxygen produced a luminescent signal that was, for most compounds, proportional to the number of CFU determined prior to the recovery phase. No agents targeting the cell wall were active against NRP M. tuberculosis, whereas drugs hitting other cellular targets had a range of activities. The calculated Z′ factor was in the range of 0.58 to 0.84, indicating the suitability of the use of LORA for high-throughput assays. This LORA is sufficiently robust for use for primary high-throughput screening of compounds against NRP M. tuberculosis.

Structure−Activity Relationships for a Series of Quinoline-Based Compounds Active against Replicating and Nonreplicating Mycobacterium tuberculosis

Tuberculosis (TB) remains as a global pandemic that is aggravated by a lack of health care, the spread of HIV, and the emergence of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) strains. New anti-TB drugs are urgently required to shorten the long 6-12 month treatment regimen and to battle drug-resistant Mtb strains. We have identified several potent quinoline-based anti-TB compounds, bearing an isoxazole containing side-chain. The most potent compounds, 7g and 13, exhibited submicromolar activity against the replicating bacteria (R-TB), with minimum inhibitory concentrations (MICs) of 0.77 and 0.95 microM, respectively. In general, these compounds also had micromolar activity against the nonreplicating persistent bacteria (NRP-TB) and did not show toxicity on Vero cells up to 128 microM concentration. Compounds 7g and 13 were shown to retain their anti-TB activity against rifampin, isoniazid, and streptomycin resistant Mtb strains. The results suggest that quinoline-isoxazole-based anti-TB compounds are promising leads for new TB drug development.

Synthesis, antimalarial and antitubercular activity of acetylenic chalcones

A series of acetylenic chalcones were evaluated for antimalarial and antitubercular activity. The antimalarial data for this series suggests that growth inhibition of the W2 strain of Plasmodium falciparum can be imparted by the introduction of a methoxy group ortho to the acetylenic group. Most compounds were more active against non-replicating than replicating cultures of Mycobacterium tuberculosis H(37)Rv, an unusual pattern with respect to existing anti-TB agents.

5-tert-butyl-N-pyrazol-4-yl-4,5,6,7-tetrahydrobenzo[d]isoxazole-3-carboxamide derivatives as novel potent inhibitors of Mycobacterium tuberculosis pantothenate synthetase: initiating a quest for new antitubercular drugs.

Pantothenate synthetase (PS) is one of the potential new antimicrobial targets that may also be useful for the treatment of the nonreplicating persistent forms of Mycobacterium tuberculosis. In this Letter we present a series of 5- tert-butyl- N-pyrazol-4-yl-4,5,6,7-tetrahydrobenzo[ d]isoxazole-3-carboxamide derivatives as novel potent Mycobacterium tuberculosis PS inhibitors, their in silico molecular design, synthesis, and inhibitory activity.

Synthesis and structure-activity studies of biphenyl analogues of the tuberculosis drug (6S)-2-nitro-6-{[4-(trifluoromethoxy)benzyl]oxy}-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (PA-824).

A series of biphenyl analogues of the new tuberculosis drug PA-824 was prepared, primarily by coupling the known (6S)-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-ol with iodobenzyl halides, followed by Suzuki coupling of these iodides with appropriate arylboronic acids or by assembly of the complete biaryl side chain prior to coupling with the above alcohol. Antitubercular activity was determined under both replicating (MABA) and nonreplicating (LORA) conditions. para-Linked biaryls were the most active, followed by meta-linked and then ortho-linked analogues. A more detailed study of a larger group of para-linked analogues showed a significant correlation between potency (MABA) and both lipophilicity (CLOGP) and the electron-withdrawing properties of terminal ring substituents ( summation operatorsigma). Selected compounds were evaluated for their efficacy in a mouse model of acute Mycobacterium tuberculosis infection. In vivo activity correlated well with the stability of compounds to microsomal metabolism. Three compounds bearing combinations of lipophilic, electron-withdrawing groups achieved >200-fold higher efficacies than the parent drug.

Design, Synthesis, and SAR Studies of Mefloquine-Based Ligands as Potential Antituberculosis Agents

Tuberculosis (TB), a chronic infectious disease caused by Mycobacterium tuberculosis, is one of the leading causes of death in the world. According to the World Health Organization (WHO), one-third of the world’s population is currently infected with the TB bacillus. It is estimated that 1.75 million deaths resulted from TB in 2003. One-third of the increase in the incidence of TB in the past five years can be attributed to its co-infection with HIV, as this weakens the immune system and facilitates the development of multi-drug-resistant TB (MDR-TB). It has been estimated that 50 million people have been infected with MDR-TB. As no new drugs have been introduced for TB over the last 40 years, there is an urgent need to identify improved medications that are able to combat MDR-TB and to decrease the current six-month treatment protocol that is required for drugs in current use. To achieve this goal, we need to identify potent and nontoxic antituberculosis agents that kill both the replicating (R-TB) and nonreplicating persistent (NRPTB) phenotypes; eradicating the latter is implicated as a necessity for shortening treatment. As part of our drug-discovery efforts aimed at developing novel antituberculosis agents, we report herein the design, synthesis, and structure–activity studies of mefloquine analogues. Mefloquine is a well-known antimalarial drug still used today in spite of its neuropsychiatric side-effects that include dizziness, headache, insomnia, and vivid dreams. Mefloquine and several of its analogues have been reported to have antibacterial activity. From a screening program carried out at the Walter Reed Army Institute of Research, a series of mefloquine-related compounds with various substitutions on the quinoline ring were found to be more active than mefloquine itself against gram-positive bacteria. Although mefloquine derivatives are also known to act as purine receptor antagonists, the only prokaryotic target of mefloquine identified to date is an F0F1H + ATPase in Streptococcus pneumoniae. The crystal structures of the rotor of the V-type and F-type Na ATPases were disclosed recently. In this context, it is important to note that the recently described diarylquinoline R207910, a potent antituberculosis agent, is also believed to owe its activity to interaction with a proton pump of the ATP synthase of M. tuberculosis. From our own in-house screening of the Gen-Plus 960 compound library (MicroSource), mefloquine was found to have relatively potent activity against NRP-TB, which prompted us to choose it as a lead candidate for TB drug discovery. The results of biological assays performed on the (+) and ( ) forms of the erythro and threo isomers of mefloquine are shown in Table 1. The mefloquine isomers were tested against NRP-TB in the luciferase-based low oxygen recovery assay (LORA), and these data were confirmed by the quantification of colony-forming units (cfu) immediately following the hypox-

Design, Synthesis, and Pharmacological Evaluation of Mefloquine‐Based Ligands as Novel Antituberculosis Agents

Tuberculosis (TB) is presently regarded as one of the most dangerous infective diseases worldwide and one of the major AIDS‐associated infections. To shorten the current treatment regimen, there is an urgent need to identify new anti‐TB agents which are active against both replicating TB (R‐TB) and nonreplicating TB (NRP‐TB). Mefloquine, a well‐known antimalarial drug was found to possess reasonable activity against NRP‐TB, and accordingly, 30 new analogues were synthesized and evaluated for their anti‐TB activity against Mycobacterium tuberculosis H37Rv. As the target of mefloquine in Mycobacterium tuberculosis remains unknown, we resorted to modifying mefloquine in a variety of chemically convenient ways, which led us in turn to the active hydrazone 10 a. Further modifications of 10 a led to compound 7 f, with an improved anti‐TB activity/selectivity profile with both less cytotoxicity and less predicted CNS side effects compared with mefloquine. The clear structure–activity relationships (SARs) derived from this study should facilitate our ultimate goal of identifying improved anti‐TB agents.

Synthesis and Structure−activity Relationships of Antitubercular 2-Nitroimidazooxazines Bearing Heterocyclic Side Chains

Recently described biphenyl analogues of the antituberculosis drug PA-824 displayed improved potencies against M. tuberculosis but were poorly soluble. Heterobiaryl analogues of these, in which the first phenyl ring was replaced with various 5-membered ring heterocycles, were prepared with the aim of identifying potent new candidates with improved aqueous solubility. The compounds were constructed by coupling the chiral 2-nitroimidazooxazine alcohol with various halomethyl-substituted arylheterocycles, by cycloadditions to a propargyl ether derivative of this alcohol, or by Suzuki couplings on haloheterocyclic methyl ether derivatives. The arylheterocyclic compounds were all more hydrophilic than their corresponding biphenyl analogues, and several showed solubility improvements. 1-Methylpyrazole, 1,3-linked-pyrazole, 2,4-linked-triazole, and tetrazole analogues had 3- to 7-fold higher MIC potencies against replicating M. tb than predicted by their lipophilicities. Two pyrazole analogues were >10-fold more efficacious than the parent drug in a mouse model of acute M. tb infection, and one displayed a 2-fold higher solubility.

Facile transformation of Biginelli pyrimidin-2(1H)-ones to pyrimidines. In vitro evaluation as inhibitors of Mycobacterium tuberculosis and modulators of cytostatic activity

A series of pyrimidine derivatives bearing amine substituents at C-2 position were obtained from Biginelli 3,4-dihydropyrimidin-2(1H)-ones and the effect of structural variation on anti-TB activity against Mycobacterium tuberculosis H37Rv strain and antiviral activity in a series of cell cultures was evaluated. While the compounds were found to possess structure dependent cytostatic activity, these were not found to be efficient inhibitors of M. tuberculosis nor did they inhibit a broad variety of DNA or RNA viruses in cell culture.

Evaluation of gyrase B as a drug target in Mycobacterium tuberculosis

OBJECTIVES New classes of drugs are needed to treat tuberculosis (TB) in order to combat the emergence of resistance to existing agents and shorten the duration of therapy. Targeting DNA gyrase is a clinically validated therapeutic approach using fluoroquinolone antibiotics to target the gyrase subunit A (GyrA) of the heterotetramer. Increasing resistance to fluoroquinolones has driven interest in targeting the gyrase subunit B (GyrB), which has not been targeted for TB. The biological activities of two potent small-molecule inhibitors of GyrB have been characterized to validate its targeting as a therapeutic strategy for treating TB. MATERIALS AND METHODS Novobiocin and aminobenzimidazole 1 (AB-1) were tested for their activity against Mycobacterium tuberculosis (Mtb) H37Rv and other mycobacteria. AB-1 and novobiocin were also evaluated for their interaction with rifampicin and isoniazid as well as their potential for cytotoxicity. Finally, AB-1 was tested for in vivo efficacy in a murine model of TB. RESULTS Novobiocin and AB-1 have both been shown to be active against Mtb with MIC values of 4 and 1 mg/L, respectively. Only AB-1 exhibited time-dependent bactericidal activity against drug-susceptible and drug-resistant mycobacteria, including a fluoroquinolone-resistant strain. AB-1 had potent activity in the low oxygen recovery assay model for non-replicating persistent Mtb. Additionally, AB-1 has no interaction with isoniazid and rifampicin, and has no cross-resistance with fluoroquinolones. In a murine model of TB, AB-1 significantly reduced lung cfu counts in a dose-dependent manner. CONCLUSIONS Aminobenzimidazole inhibitors of GyrB exhibit many of the characteristics required for their consideration as a potential front-line antimycobacterial therapeutic.