Bacterial Resistance to Leucyl-tRNA Synthetase Inhibitor GSK2251052 Develops during Treatment of Complicated Urinary Tract Infections

GSK2251052, a novel leucyl-tRNA synthetase (LeuRS) inhibitor, was in development for the treatment of infections caused by multidrug-resistant Gram-negative pathogens. In a phase II study (study LRS114688) evaluating the efficacy of GSK2251052 in complicated urinary tract infections, resistance developed very rapidly in 3 of 14 subjects enrolled, with ≥32-fold increases in the GSK2251052 MIC of the infecting pathogen being detected. A fourth subject did not exhibit the development of resistance in the baseline pathogen but posttherapy did present with a different pathogen resistant to GSK2251052. Whole-genome DNA sequencing of Escherichia coli isolates collected longitudinally from two study LRS114688 subjects confirmed that GSK2251052 resistance was due to specific mutations, selected on the first day of therapy, in the LeuRS editing domain. Phylogenetic analysis strongly suggested that resistant Escherichia coli isolates resulted from clonal expansion of baseline susceptible strains. This resistance development likely resulted from the confluence of multiple factors, of which only some can be assessed preclinically. Our study shows the challenges of developing antibiotics and the importance of clinical studies to evaluate their effect on disease pathogenesis. (These studies have been registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT01381549","term_id":"NCT01381549"}}NCT01381549 for the study of complicated urinary tract infections and registration no. {"type":"clinical-trial","attrs":{"text":"NCT01381562","term_id":"NCT01381562"}}NCT01381562 for the study of complicated intra-abdominal infections.)     

A Broad-Spectrum Microbicide with Virucidal Activity against Sexually Transmitted Viruses

Sodium dodecyl sulfate (SDS), an alkyl sulfate surfactant derived from an organic alcohol, possesses surfactant properties but also denatures and unfolds both monomeric and subunit proteins. In preliminary experiments, we demonstrated that SDS is a potent inactivator of herpes simplex virus type 2 and human immunodeficiency virus type 1 at concentrations comparable to those used for the surfactant nonoxynol-9. We hypothesized that SDS might be capable of denaturing the capsid proteins of nonenveloped viruses. In this report, we demonstrate inactivation of rabbit, bovine, and human papillomaviruses after brief treatment with dilute solutions of SDS. Effective concentrations were nontoxic to rabbit skin and to split-thickness grafts of human foreskin epithelium. This is the first report of a microbicidal surfactant that will inactivate papillomaviruses. We propose that SDS is now a candidate microbicide for formulation and testing with humans.     

Cryptosporidium and Toxoplasma Parasites Are Inhibited by a Benzoxaborole Targeting Leucyl-tRNA Synthetase

The apicomplexan parasites Cryptosporidium and Toxoplasma are serious threats to human health. Cryptosporidiosis is a severe diarrheal disease in malnourished children and immunocompromised individuals, with the only FDA-approved drug treatment currently being nitazoxanide. The existing therapies for toxoplasmosis, an important pathology in immunocompromised individuals and pregnant women, also have serious limitations. With the aim of developing alternative therapeutic options to address these health problems, we tested a number of benzoxaboroles, boron-containing compounds shown to be active against various infectious agents, for inhibition of the growth of Cryptosporidium parasites in mammalian cells. A 3-aminomethyl benzoxaborole, AN6426, with activity in the micromolar range and with activity comparable to that of nitazoxanide, was identified and further characterized using biophysical measurements of affinity and crystal structures of complexes with the editing domain of Cryptosporidium leucyl-tRNA synthetase (LeuRS). The same compound was shown to be active against Toxoplasma parasites, with the activity being enhanced in the presence of norvaline, an amino acid that can be mischarged by LeuRS. Our observations are consistent with AN6426 inhibiting protein synthesis in both Cryptosporidium and Toxoplasma by forming a covalent adduct with tRNA^Leu in the LeuRS editing active site and suggest that further exploitation of the benzoxaborole scaffold is a valid strategy to develop novel, much needed antiparasitic agents.     

Pirbenicillin, a New Semisynthetic Penicillin with Broad-Spectrum Activity

Pirbenicillin is a new semisynthetic penicillin which inhibited 67% of isolates of Proteus aeruginosa tested in our laboratory, 93% of P. mirabilis, 31% of Enterobacter spp., 41% of Serratia spp., and 58% of Escherichia coli at a concentration of 6.25 μg/ml. Its activity appeared to be inoculum dependent and it was virtually inactive against 10⁷ inocula of P. aeruginosa. It was more active than carbenicillin or ticarcillin, but less active than BL-P1654 against P. aeruginosa. Carbenicillin and ticarcillin appeared to be more active than pirbenicillin against Proteus spp., but pirbenicillin was active against some isolates of Klebsiella spp.     

Thienamycin: New Beta-Lactam Antibiotic with Potent Broad-Spectrum Activity

Thienamycin, a new beta-lactam antibiotic, exhibited potent, broad-spectrum activity in vitro against gram-negative bacilli and gram-positive cocci, including many isolates resistant to currently available antibiotics. All isolates were inhibited at concentrations less than or equal to 25 mug/ml, with the exception of 12% of isolates of Enterobacter spp. and 3% of isolates of Serratia marcescens. Its activity decreased with an increase in inoculum concentration of from 10(5) to 10(7) cells per ml.     

Discovery of Novel Oral Protein Synthesis Inhibitors of Mycobacterium tuberculosis That Target Leucyl-tRNA Synthetase

The recent development and spread of extensively drug-resistant and totally drug-resistant resistant (TDR) strains of Mycobacterium tuberculosis highlight the need for new antitubercular drugs. Protein synthesis inhibitors have played an important role in the treatment of tuberculosis (TB) starting with the inclusion of streptomycin in the first combination therapies. Although parenteral aminoglycosides are a key component of therapy for multidrug-resistant TB, the oxazolidinone linezolid is the only orally available protein synthesis inhibitor that is effective against TB. Here, we show that small-molecule inhibitors of aminoacyl-tRNA synthetases (AARSs), which are known to be excellent antibacterial protein synthesis targets, are orally bioavailable and effective against M. tuberculosis in TB mouse infection models. We applied the oxaborole tRNA-trapping (OBORT) mechanism, which was first developed to target fungal cytoplasmic leucyl-tRNA synthetase (LeuRS), to M. tuberculosis LeuRS. X-ray crystallography was used to guide the design of LeuRS inhibitors that have good biochemical potency and excellent whole-cell activity against M. tuberculosis. Importantly, their good oral bioavailability translates into in vivo efficacy in both the acute and chronic mouse models of TB with potency comparable to that of the frontline drug isoniazid.     

In Vitro Antimycobacterial Activity of 5-Chloropyrazinamide

5-Chloropyrazinamide and 5-chloropyrazinoic acid were evaluated for in vitro activity against Mycobacterium tuberculosis, Mycobacterium bovis, and several nontuberculous mycobacteria by a broth dilution method. 5-Chloropyrazinamide was more active than pyrazinamide against all organisms tested. It is likely that this agent has a different mechanism of action than pyrazinamide.     

The Efflux Pump Inhibitor Timcodar Improves the Potency of Antimycobacterial Agents

Previous studies indicated that inhibition of efflux pumps augments tuberculosis therapy. In this study, we used timcodar (formerly VX-853) to determine if this efflux pump inhibitor could increase the potency of antituberculosis (anti-TB) drugs against Mycobacterium tuberculosis in in vitro and in vivo combination studies. When used alone, timcodar weakly inhibited M. tuberculosis growth in broth culture (MIC, 19 μg/ml); however, it demonstrated synergism in drug combination studies with rifampin, bedaquiline, and clofazimine but not with other anti-TB agents. When M. tuberculosis was cultured in host macrophage cells, timcodar had about a 10-fold increase (50% inhibitory concentration, 1.9 μg/ml) in the growth inhibition of M. tuberculosis and demonstrated synergy with rifampin, moxifloxacin, and bedaquiline. In a mouse model of tuberculosis lung infection, timcodar potentiated the efficacies of rifampin and isoniazid, conferring 1.0 and 0.4 log₁₀ reductions in bacterial burden in lung, respectively, compared to the efficacy of each drug alone. Furthermore, timcodar reduced the likelihood of a relapse infection when evaluated in a mouse model of long-term, chronic infection with treatment with a combination of rifampin, isoniazid, and timcodar. Although timcodar had no effect on the pharmacokinetics of rifampin in plasma and lung, it did increase the plasma exposure of bedaquiline. These data suggest that the antimycobacterial drug-potentiating activity of timcodar is complex and drug dependent and involves both bacterial and host-targeted mechanisms. Further study of the improvement of the potency of antimycobacterial drugs and drug candidates when used in combination with timcodar is warranted.     

Laboratory Evaluation of BL-S786, a Cephalosporin with Broad-Spectrum Antibacterial Activity

Biological and physicochemical properties of BL-S786 were compared with those of cephalothin, cephaloridine, and cefazolin. With few exceptions, BL-S786 was more active than the reference compounds against major gram-negative pathogenic species and its antibacterial spectrum was broader than that of cephalosporins currently available for clinical use. Although BL-S786 was generally less active than the control cephalosporins against gram-positive pathogens, it inhibited their growth at concentrations that should readily be achieved in humans after standard parenteral dosage. Streptococcus faecalis, a species relatively unsusceptible to cephalosporins in general, was an exception. BL-S786 was an effective bactericidal agent for strains of various gram-negative organisms. After intramuscular administration to mice, BL-S786 achieved high concentrations in blood, and its biological half-life was longer than that of the other three cephalosporins.     

Antimycobacterial Activity and Mechanism of Action of NAS-91

The antimalarial agents NAS-91 and NAS-21 were found to express potent antimycobacterial activity, NAS-91 being more active than NAS-21. They partially inhibited mycolic acid biosynthesis and profoundly altered oleic acid production. The development of a cell-free assay for Delta 9-desaturase activity allowed direct demonstration of the inhibition of oleic acid biosynthesis by these compounds.     

Inhibition of Antibiotic-Resistant Staphylococcus aureus by the Broad-Spectrum Dihydrofolate Reductase Inhibitor RAB1

The bacterial burden on human health is quickly outweighing available therapeutics. Our long-term goal is the development of antimicrobials with the potential for broad-spectrum activity. We previously reported phthalazine-based inhibitors of dihydrofolate reductase (DHFR) with potent activity against Bacillus anthracis, a major component of Project BioShield. The most active molecule, named RAB1, performs well in vitro and, in a cocrystal structure, was found deep within the active site of B. anthracis DHFR. We have now examined the activity of RAB1 against a panel of bacteria relevant to human health and found broad-spectrum applicability, particularly with regard to Gram-positive organisms. RAB1 was most effective against Staphylococcus aureus, including methicillin- and vancomycin-resistant (MRSA/VRSA) strains. We have determined the cocrystal structure of the wild-type and trimethoprim-resistant (Phe 98 Tyr) DHFR enzyme from S. aureus with RAB1, and we found that rotational freedom of the acryloyl linker region allows the phthalazine moiety to occupy two conformations. This freedom in placement also allows either enantiomer of RAB1 to bind to S. aureus, in contrast to the specificity of B. anthracis for the S-enantiomer. Additionally, one of the conformations of RAB1 defines a unique surface cavity that increases the strength of interaction with S. aureus. These observations provide insights into the binding capacity of S. aureus DHFR and highlight atypical features critical for future exploitation in drug development.Despite the introduction of antibiotics such as penicillin in the 1940s and approval of new classes, such as cyclic lipopeptides like daptomycin, infectious disease continues worldwide. Antibiotic resistance is increasing, and reliance on existing scaffolds is not sufficient to combat multidrug resistance (23, 33). Staphylococcus aureus has become a primary concern among antibiotic-resistant infectious disease agents, with methicillin-resistant S. aureus (MRSA) infections claiming 19,000 lives in the United States per year and costing the United States $3 to 4 billion dollars per year (18). As the incidence of MRSA has shifted from hospital settings to the community at large, treatment preferences have moved to oral formulations and have impacted drug resistance profiles. Community-acquired MRSA is showing increasing resistance to fluoroquinolones and clindamycin, and in response clinicians are shifting to doxycycline, linezolid, or trimethoprim-sulfamethoxazole as the best options for outpatient treatment (21, 30).While treatment preferences are adjusting to isolated outbreaks, S. aureus is increasingly resistant to trimethoprim (TMP) (13). TMP targets the enzyme dihydrofolate reductase (DHFR), a critical component of folate metabolism, and is required for continued nucleic acid synthesis (6). DHFR is a good target because of the specificity that can be achieved; however, bacteria have developed resistance mechanisms that include acquisition of plasmid-derived (though catalytically inferior) versions of DHFR, leading to high-level resistance. Alternatively, point mutations of the chromosomal DHFR have been demonstrated to confer intermediate resistance, such as the S. aureus DHFR (saDHFR) residue Phe 98 to Tyr (F98Y) mutation (13). While other mutations are frequently found with F98Y, it alone can increase the MIC of TMP by at least 1 order of magnitude. In efforts to compensate for a lower inhibitory action, TMP is frequently combined with a sulfa inhibitor of the enzyme preceding DHFR, termed dihydropterin synthase (DHPS). Unfortunately, sulfa drugs have notoriously poor pharmacokinetics and can have unpleasant, and sometimes fatal, side effects (15).RAB1 is a lead compound under investigation for treatment of inhalation anthrax. RAB1''s structure is modeled on trimethoprim but is extended by addition of an acryloyl linker and phthalazine moiety, including a propyl group at a stereogenic carbon. This results in approximately 40% more surface area than TMP, which provides critical contact regions for further interaction with the binding site. Specific structural features of RAB1 allow unique contacts with the protein that can overcome natural and induced resistance. For B. anthracis, the half-maximal inhibitory concentration (IC₅₀) of purified DHFR enzyme with TMP is 77 μM, indicating a natural resistance, while with RAB1 it is ≈60 nM (3). Selectivity for the binding site in B. anthracis DHFR is controlled by the large hydrophobic phthalazine moiety, which is embedded within and causes extension of the binding site (4). Concomitant with the current work, the broad-spectrum activities of related RAB1-like molecules have been demonstrated by Basilea Pharmaceutica International AG. These studies also highlighted the difficulty in generating spontaneous resistant mutations of chromosomal DHFR to inhibitors in the dihydrophthalazine series (7, 9).We have characterized the broad-spectrum applicability of RAB1 and its particular effectiveness against S. aureus, including MRSA and vancomycin-resistant S. aureus (VRSA) strains. Both enantiomers of RAB1 show potent antimicrobial activities, as indicated by favorable MICs and IC₅₀s. The impact of the TMP resistance-conferring mutation F98Y is negligible on RAB1 activity and preferentially decreases binding of the R-enantiomer. The X-ray crystal structures of RAB1 complexed to S. aureus DHFR reveal two conformations for the large phthalazine group. While one conformation resides in the known binding site, the other sits in a shallow surface cavity that has been previously undocumented as susceptible to inhibitor binding. This allows an increase in binding strength and provides an explanation for the lack of enantiomeric preference. These observations provide insights into the binding capacity of S. aureus DHFR and highlight atypical features critical for future exploitation in drug development, as well as insights into the mechanism of action of phthalazine-based inhibitors.     

Biological Activity of Netilmicin, a Broad-Spectrum Semisynthetic Aminoglycoside Antibiotic

Netilmicin (Sch 20569) is a new broad-spectrum semisynthetic aminoglycoside derived from sisomicin. Netilmicin was compared to gentamicin, tobramycin, and amikacin in a variety of in vitro test systems as well as in mouse protection tests. Netilmicin was found to be similar in activity to gentamicin against aminoglycoside-susceptible strains in both in vitro and in vivo tests. Netilmicin was also active against many aminoglycoside-resistant strains of gram-negative bacteria, particularly those known to possess adenylating enzymes (ANT 2′) or those with a similar resistance pattern. Netilmicin was found to be markedly less toxic than gentamicin in chronic studies in cats, although gentamicin appeared less toxic in acute toxicity tests in mice. The concentrations of netilmicin and gentamicin in serum were compared in dogs after intramuscular dosing, and the pharmacokinetics including peak concentrations in serum were found to be similar.     

Moxifloxacin Retains Antimycobacterial Activity in the Presence of gyrA Mutations

Moxifloxacin-resistant Mycobacterium tuberculosis mutants were selected in vitro using different concentrations of moxifloxacin. gyrA mutations at codons 88 and 94 were associated with resistance (defined as an MIC of ≥2 μg/ml) (P < 0.0001 and P = 0.0053, respectively). Despite the presence of gyrA mutations, moxifloxacin significantly impedes bacterial growth, supporting its use for the treatment of ofloxacin-resistant M. tuberculosis.     

Decreased Haem Synthetase Activity in Blood Cells of Patients with Erythropoietic Protoporphyria

Abstract. A constant finding in erythropoietic protoporphyria (E. P. P.) is a raised protoporphyrin content of the erythrocytes. It has been shown before that the biosynthesis of this protoporphyrin takes place in the erythroid cell. In circulating blood cells of patients with E. P. P. the haem synthetase activity is significantly decreased. This observation and the increased protoporphyrin concentration in the cells can be explained by assuming a decreased stability of haem synthetase in the red blood cells of E. P. P. patients.