Structure, In Vivo Detection, and Antibacterial Activity of Metabolites of SQ109, an Anti-Infective Drug Candidate.

SQ109 is a drug candidate for the treatment of tuberculosis (TB). It is thought to target primarily the protein MmpL3 in Mycobacterium tuberculosis, but it also inhibits the growth of some other bacteria. SQ109 is metabolized by the liver, and it has been proposed that some of its metabolites might be responsible for its activity against TB. Here, we synthesized six potential P450 metabolites of SQ109 and used these as well as 10 other likely metabolites as standards in a mass spectrometry study of M. tuberculosis-infected rabbits treated with SQ109, in addition to testing all 16 putative metabolites for antibacterial activity. We found that there were just two major metabolites in lung tissue: a hydroxy-adamantyl analog of SQ109 and N'-adamantylethylenediamine. Neither of these, or the other potential metabolites tested, inhibited the growth of M. tuberculosis or of M. smegmatis, Bacillus subtilis, or E. coli, making it unlikely that an SQ109 metabolite contributes to its antibacterial activity. In the rabbit TB model, it is thus the gradual accumulation of nonmetabolized SQ109 in tissues to therapeutic levels that leads to good efficacy. Our results also provide new insights into how SQ109 binds to its target MmpL3, based on our mass spectroscopy results which indicate that the charge in SQ109 is primarily localized on the geranyl nitrogen, explaining the very short distance to a key Asp found in the X-ray structure of SQ109 bound to MmpL3.

N-Acetylglucosamine-1-Phosphate Transferase, WecA, as a Validated Drug Target in Mycobacterium tuberculosis.

The mycobacterial phosphoglycosyltransferase WecA, which initiates arabinogalactan biosynthesis in Mycobacterium tuberculosis, has been proposed as a target of the caprazamycin derivative CPZEN-45, a preclinical drug candidate for the treatment of tuberculosis. In this report, we describe the functional characterization of mycobacterial WecA and confirm the essentiality of its encoding gene in M. tuberculosis by demonstrating that the transcriptional silencing of wecA is bactericidal in vitro and in macrophages. Silencing wecA also conferred hypersensitivity of M. tuberculosis to the drug tunicamycin, confirming its target selectivity for WecA in whole cells. Simple radiometric assays performed with mycobacterial membranes and commercially available substrates allowed chemical validation of other putative WecA inhibitors and resolved their selectivity toward WecA versus another attractive cell wall target, translocase I, which catalyzes the first membrane step in the biosynthesis of peptidoglycan. These assays and the mutant strain described herein will be useful for identifying potential antitubercular leads by screening chemical libraries for novel WecA inhibitors.

Early phase evaluation of SQ109 alone and in combination with rifampicin in pulmonary TB patients.

OBJECTIVES: SQ109, an asymmetrical diamine, is a novel anti-TB drug candidate. This first study in patients was done to determine safety, tolerability, pharmacokinetics and bacteriological effect of different doses of SQ109 alone and in combination with rifampicin when administered over 14 days. PATIENTS AND METHODS: Smear-positive pulmonary TB patients were randomized into six groups of 15 to receive once-daily oral treatment with 75, 150 or 300 mg of SQ109, rifampicin (10 mg/kg body weight), rifampicin plus 150 mg of SQ109, or rifampicin plus 300 mg of SQ109 for 14 days. Patients were hospitalized for supervised treatment, regular clinical, biochemical and electrocardiographic safety assessments, pharmacokinetic profiling and daily overnight sputum collection. RESULTS: SQ109 was safe and generally well tolerated. Mild to moderate dose-dependent gastrointestinal complaints were the most frequent adverse events. No relevant QT prolongation was noted. Maximum SQ109 plasma concentrations were lower than MICs. Exposure to SQ109 (AUC0-24) increased by drug accumulation upon repeated administration in the SQ109 monotherapy groups. Co-administration of SQ109 150 mg with rifampicin resulted in decreasing SQ109 exposures from day 1 to day 14. A higher (300 mg) dose of SQ109 largely outweighed the evolving inductive effect of rifampicin. The daily fall in log cfu/mL of sputum (95% CI) was 0.093 (0.126-0.059) with rifampicin, 0.133 (0.166-0.100) with rifampicin plus 150 mg of SQ109 and 0.089 (0.121-0.057) with rifampicin plus 300 mg of SQ109. Treatments with SQ109 alone showed no significant activity. CONCLUSIONS: SQ109 alone or with rifampicin was safe over 14 days. Upon co-administration with rifampicin, 300 mg of SQ109 yielded a higher exposure than the 150 mg dose. SQ109 did not appear to be active alone or to enhance the activity of rifampicin during the 14 days of treatment.

Optimization of a nucleic acid-based reporter system to detect Mycobacterium tuberculosis antibiotic sensitivity.

We previously reported the development of a prototype antibiotic sensitivity assay to detect drug-resistant Mycobacterium tuberculosis using infection by mycobacteriophage to create a novel nucleic acid transcript, a surrogate marker of mycobacterial viability, detected by reverse transcriptase PCR (M. C. Mulvey et al., mBio 3: e00312-11, 2012). This assay detects antibiotic resistance to all drugs, even drugs for which the resistance mechanism is unknown or complex: it is a phenotypic readout using nucleic acid detection. In this report, we describe development and characteristics of an optimized reporter system that directed expression of the RNA cyclase ribozyme, which generated circular RNA through an intramolecular splicing reaction and led to accumulation of a new nucleic acid sequence in phage-infected bacteria. These modifications simplified the assay, increased the limit of detection from 10(4) to <10(2) M. tuberculosis cells, and correctly identified the susceptibility profile of M. tuberculosis strains exposed for 16 h to either first-line or second-line antitubercular drugs. In addition to phenotypic drug resistance or susceptibility, the assay reported streptomycin MICs and clearly detected 10% drug-resistant cells in an otherwise drug-susceptible population.

Therapeutic efficacy of SQ641-NE against Mycobacterium tuberculosis.

A phospholipid-based nanoemulsion formulation of SQ641 (SQ641-NE) was active against intracellular Mycobacterium tuberculosis in J774A.1 mouse macrophages, although SQ641 by itself was not. Intravenous (i.v.) SQ641-NE was cleared from circulation and reached peak concentrations in lung and spleen in 1 h. In a murine tuberculosis (TB) model, 8 i.v. doses of SQ641-NE at 100 mg/kg of body weight over 4 weeks caused a 1.73 log10 CFU reduction of M. tuberculosis in spleen and were generally bacteriostatic in lungs.

Discovery and development of SQ109: a new antitubercular drug with a novel mechanism of action.

Existing drugs have limited efficacy against the rising threat of drug-resistant TB, have significant side effects, and must be given in combinations of four to six drugs for at least 6 months for drug-sensitive TB and up to 24 months for drug-resistant TB. The long treatment duration has led to increased patient noncompliance with therapy. This, in turn, drives the development of additional drug resistance in a spiral that has resulted in some forms of TB being currently untreatable by existing drugs. New antitubercular drugs in development, particularly those with mechanisms of action that are different from existing first- and second-line TB drugs, are anticipated to be effective against both drug-sensitive and drug-resistant TB. SQ109 is a new TB drug candidate with a novel mechanism of action that was safe and well tolerated in Phase I and early Phase II clinical trials. We describe herein the identification, development and characterization of SQ109 as a promising new antitubercular drug.

Generation of a novel nucleic acid-based reporter system to detect phenotypic susceptibility to antibiotics in Mycobacterium tuberculosis.

UNLABELLED: We designed, constructed, and evaluated a prototype novel reporter system comprised of two functional cassettes: (i) the SP6 RNA polymerase gene under transcriptional control of a promoter active in mycobacteria and (ii) the consensus SP6 polymerase promoter that directs expression of an otherwise unexpressed sequence. We incorporated the reporter system into a mycobacteriophage for delivery into viable Mycobacterium tuberculosis, and introduction led to synthesis of an SP6 polymerase-dependent surrogate marker RNA that we detected by reverse transcriptase PCR (RT-PCR). The reporter confirmed the susceptibility profile of both drug-susceptible and drug-resistant M. tuberculosis strains exposed to first-line antitubercular drugs and required as little as 16 h of exposure to antibacterial agents targeting bacterial metabolic processes to accurately read the reaction. The reporter system translated the bacterial phenotype into a language interpretable by rapid and sensitive nucleic acid detection. As a phenotypic assay that works only on viable M. tuberculosis, it could be used to rapidly assess resistance to any drug, including drugs for which the mechanism of resistance is unknown or which result from many potential known (and unknown) genetic alterations. IMPORTANCE: The ability to detect antibiotic resistance of slow-growing bacteria (i.e., Mycobacterium tuberculosis) is hampered by two factors, the time to detection (weeks to months) and the resistance mechanism (unknown for many drugs), delaying the appropriate treatment of patients with drug-resistant or multidrug-resistant tuberculosis (TB). The novel technique described in this article uses a unique surrogate nucleic acid marker produced by phage that infects M. tuberculosis to record phenotypic antibiotic susceptibility in less than a day.

SQ109 and PNU-100480 interact to kill Mycobacterium tuberculosis in vitro.

OBJECTIVES: To investigate in vitro interaction between two compounds, SQ109 and PNU-100480, currently in development for the treatment of Mycobacterium tuberculosis (MTB). METHODS: The two-drug interactions between SQ109 and PNU-100480 and its major metabolite PNU-101603 were assessed by chequerboard titration, and the rate of killing and intracellular activity were determined in both J774A.1 mouse macrophages and whole blood culture. RESULTS: In chequerboard titration, interactions between SQ109 and either oxazolidinone were additive. In time-kill studies, SQ109 killed MTB faster than PNU compounds, and its rate of killing was further enhanced by both oxazolidinones. The order of efficacy of single compounds against intracellular MTB was SQ109 > PNU-100480 > PNU-101603. At sub-MIC, combinations of SQ109 + PNU compounds showed improved intracellular activity over individual drugs; at ≥MIC, the order of efficacy was SQ109 > SQ109 + PNU-100480 > SQ109 + PNU-101603. In whole blood culture, the combined bactericidal activities of SQ109 and PNU-100480 and its major metabolite against intracellular M. tuberculosis did not differ significantly from the sum of the compounds tested individually. CONCLUSIONS: SQ109 and PNU combinations were additive and improved the rate of MTB killing over individual drugs. These data suggest that the drugs may work together cooperatively to eliminate MTB in vivo.

Chemical modification of capuramycins to enhance antibacterial activity.

OBJECTIVES: To extend capuramycin spectrum of activity beyond mycobacteria and improve intracellular drug activity. METHODS: Three capuramycin analogues (SQ997, SQ922 and SQ641) were conjugated with different natural and unnatural amino acids or decanoic acid (DEC) through an ester bond at one or more available hydroxyl groups. In vitro activity of the modified compounds was determined against Mycobacterium spp. and representative Gram-positive and Gram-negative bacteria. Intracellular activity was evaluated in J774A.1 mouse macrophages infected with Mycobacterium tuberculosis (H37Rv). RESULTS: Acylation of SQ997 and SQ641 with amino undecanoic acid (AUA) improved in vitro activity against most of the bacteria tested. Conjugation of SQ922 with DEC, but not AUA, improved its activity against Gram-positive bacteria. In the presence of efflux pump inhibitor phenylalanine arginine ß-naphthyl amide, MICs of SQ997-AUA, SQ641-AUA and SQ922-DEC compounds improved even further against drug-susceptible and drug-resistant Staphylococcus aureus. In Gram-negative bacteria, EDTA-mediated permeabilization caused 4- to 16-fold enhancement of the activity of AUA-conjugated SQ997, SQ922 and SQ641. Conjugation of all three capuramycin analogues with AUA improved intracellular killing of H37Rv in murine macrophages. CONCLUSIONS: Conjugation of capuramycin analogues with AUA or DEC enhanced in vitro activity, extended the spectrum of activity in Gram-positive bacteria and increased intracellular activity against H37Rv.

In vitro antimicrobial activities of capuramycin analogues against non-tuberculous mycobacteria.

OBJECTIVES: To determine antibacterial activity of capuramycin analogues SQ997, SQ922, SQ641 and RKS2244 against several non-tuberculous mycobacteria (NTM). METHODS: In vitro antibiotic activities, i.e. MIC, MBC, rate of killing and synergistic interaction with other antibiotics, were evaluated. RESULTS: SQ641 was the most active compound against all the NTM species studied. The MIC of SQ641 was ≤0.06-4 mg/L for Mycobacterium avium complex (MAC; n = 20), 0.125-2 mg/L for M. avium paratuberculosis (MAP; n = 9), 0.125-2 mg/L for Mycobacterium kansasii (MKN;n = 2), 0.25-1 mg/L for Mycobacterium abscessus (MAB; n = 11), 4 mg/L for Mycobacterium smegmatis (MSMG; n = 1), and 1 and 8 mg/L for Mycobacterium ulcerans (MUL; n = 1), by microdilution and agar dilution methods, respectively. SQ641 was bactericidal against NTM, with an MBC/MIC ratio of 1 to 32, and killed all mycobacteria faster than positive control drugs for each strain. In chequerboard titrations, SQ641 was synergistic with ethambutol against both MAC and MSMG, and was synergistic with streptomycin and rifabutin against MAB. CONCLUSIONS: In vitro, SQ641 was the most potent of the capuramycin analogues against all NTM tested, both laboratory and clinical strains.

Anti-tuberculosis drug therapy in mice of different inbred strains.

Standard anti-tuberculosis (TB) drug therapy had distinct effects on the bacilli burden in mice of DBA/2, C3H, SWR/J, and C57BL/6 inbred strains. To standardize the TB infection process, susceptible DBA/2 mice were infected with 1/10 of the dose used for relatively resistant C57BL/6 mice, such that the lung CFUs were roughly identical 3 weeks after infection when therapy was initiated. We found that TB treatment was more effective in the susceptible DBA/2 mice than in the relatively resistant C57BL/6 mice.

In vitro interactions between new antitubercular drug candidates SQ109 and TMC207.

The in vitro interactions of two new antitubercular drugs, SQ109 and TMC207, with each other and with rifampin (RIF) were evaluated. The combination of SQ109 with TMC207 (i) improved an already excellent TMC207 MIC for M. tuberculosis H37Rv by 4- to 8-fold, (ii) improved the rate of killing of bacteria over the rate of killing by each single drug, and (iii) enhanced the drug postantibiotic effect by 4 h. In no instance did we observe antagonistic activities with the combination of SQ109 and TMC207. Rifampin activates cytochrome P450 genes to reduce the area under the curve (AUC) for TMC207 in humans. The presence of RIF in three-drug combinations did not affect the synergistic activities of SQ109 and TMC207, and SQ109 also dramatically decreased the MIC of RIF. SQ109 was active by itself, and both its activity was improved by and it improved the in vitro activities of both RIF and TMC207.

Activity of SQ641, a capuramycin analog, in a murine model of tuberculosis.

New delivery vehicles and routes of delivery were developed for the capuramycin analogue SQ641. While this compound has remarkable in vitro potency against Mycobacterium tuberculosis, it has low solubility in water and poor intracellular activity. We demonstrate here that SQ641 dissolved in the water-soluble vitamin E analogue alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) or incorporated into TPGS-micelles has significant activity in a mouse model of tuberculosis.

Preclinical study of new TB drugs and drug combinations in mouse models.

Tuberculosis (TB) remains one of the leading infectious killers in the world. New anti-TB drugs and more effective drug combinations are urgently needed, particularly given the increasing incidence of drug-resistant TB and HIV-TB co-infection. This review describes the available mouse models of TB and describes their utility in the evaluation of new TB drug candidates and in the evaluation of the efficacy of new TB drug combinations. Some of the most recent patents on promising TB drug-candidates are also mentioned here.

In vitro antimycobacterial activities of capuramycin analogues.

Translocase I inhibitor compounds derived from capuramycin demonstrated rapid bactericidal activity against several different mycobacterial species. SQ641 was the most active of the compounds, with a MIC of 0.12 to 8 microg/ml, a postantibiotic effect of 55 h, and interesting synergistic effects with other antitubercular drugs.

PrimaTB STAT-PAK assay, a novel, rapid lateral-flow test for tuberculosis in nonhuman primates.

Tuberculosis (TB) is the most important zoonotic bacterial disease in nonhuman primates (NHP). The current diagnostic method, the intradermal palpebral tuberculin test, has serious shortcomings. We characterized antibody responses in NHP against Mycobacterium tuberculosis to identify immunodominant antigens and develop a rapid serodiagnostic test for TB. A total of 422 NHP were evaluated, including 243 rhesus (Macaca mulatta), 46 cynomolgus (Macaca fascicularis), and 133 African green (Cercopithecus aethiops sabaeus) monkeys at five collaborative centers. Of those, 50 monkeys of the three species were experimentally inoculated with M. tuberculosis. Antibody responses were monitored every 2 to 4 weeks for up to 8 months postinfection by MultiAntigen Print ImmunoAssay with a panel of 12 recombinant antigens. All of the infected monkeys produced antibodies at various levels and with different antigen recognition patterns. ESAT-6 and MPB83 were the most frequently recognized proteins during infection. A combination of selected antigens which detected antibodies in all of the infected monkeys was designed to develop the PrimaTB STAT-PAK assay by lateral-flow technology. Serological evaluation demonstrated high diagnostic sensitivity (90%) and specificity (99%). The highest rate of TB detection was achieved when the skin test was combined with the PrimaTB STAT-PAK kit. This novel immunoassay provides a simple, rapid, and accurate test for TB in NHP.

In search of new cures for tuberculosis.

The last 10 years have seen resurgent industry activity in discovery and development of new drugs for the treatment of tuberculosis (TB), a growing widespread and devastating (more than 2 million deaths annually) bacterial infection that is of increasing concern in developing and developed nations alike. This review describes drugs currently being evaluated in the clinic for treatment of uncomplicated and drug resistant pulmonary TB, and updates the literature on 5 new drugs that entered clinical trials in the last 4 years.

Drug therapy of experimental tuberculosis (TB): improved outcome by combining SQ109, a new diamine antibiotic, with existing TB drugs.

Substitution of the new diamine antibiotic SQ109 for ethambutol in a mouse model of chronic tuberculosis (TB) improved efficacy of combination drug therapy with first-line TB drugs rifampin and isoniazid, with or without pyrazinamide: at 8 weeks, lung bacteria were 1.5 log10 lower in SQ109-containing regimens.

Synergistic interactions of SQ109, a new ethylene diamine, with front-line antitubercular drugs in vitro.

OBJECTIVES: The purpose of this study was to determine interactions of SQ109, a new asymmetric diamine tuberculosis (TB) drug candidate, with existing antitubercular drugs in vitro and assess its potential to improve combination drug activities against Mycobacterium tuberculosis. METHODS: Two-drug combinations at various concentrations below their MICs were tested for growth inhibition of M. tuberculosis using the BACTEC 460 system in vitro. Drug interactions were evaluated based on the quotient values that were derived numerically from the growth indices of cultures treated with a single antibiotic or combination treatment with two antibiotics. RESULTS: SQ109 at 0.5 of its MIC demonstrated strong Synergistic activity with 0.5 MIC isoniazid and as low as 0.1 MIC rifampicin in inhibition of M. tuberculosis growth. Additive effects were observed between SQ109 and streptomycin, but neither synergy nor additive effects were observed with the combination of SQ109 with ethambutol or pyrazinamide. The synergy between SQ109 and rifampicin was also demonstrated using rifampicin-resistant (RIFR) M. tuberculosis strains; SQ109 lowered the MIC of rifampicin for these drug-resistant strains. CONCLUSIONS: SQ109 interacts Synergistically with isoniazid and rifampicin, two of the most important front-line TB drugs. This finding supports efforts to further evaluate new combination therapies containing SQ109 in experimental animal models of TB that emulate future clinical trial studies in humans.