Michelle S. DeStefano

Inhibition of mycobacterial alanine racemase activity and growth by thiadiazolidinones

The genus Mycobacterium includes non-pathogenic species such as M. smegmatis, and pathogenic species such as M. tuberculosis, the causative agent of tuberculosis (TB). Treatment of TB requires a lengthy regimen of several antibiotics, whose effectiveness has been compromised by the emergence of resistant strains. New antibiotics that can shorten the treatment course and those that have not been compromised by bacterial resistance are needed. In this study, we report that thiadiazolidinones, a relatively little-studied heterocyclic class, inhibit the activity of mycobacterial alanine racemase, an essential enzyme that converts l-alanine to d-alanine for peptidoglycan synthesis. Twelve members of the thiadiazolidinone family were evaluated for inhibition of M. tuberculosis and M. smegmatis alanine racemase activity and bacterial growth. Thiadiazolidinones inhibited M. tuberculosis and M. smegmatis alanine racemases to different extents with 50% inhibitory concentrations (IC50) ranging from <0.03 to 28μM and 23 to >150μM, respectively. The compounds also inhibited the growth of these bacteria, including multidrug resistant strains of M. tuberculosis. The minimal inhibitory concentrations (MIC) for drug-susceptible M. tuberculosis and M. smegmatis ranged from 6.25μg/ml to 100μg/ml, and from 1.56 to 6.25μg/ml for drug-resistant M. tuberculosis. The in vitro activities of thiadiazolidinones suggest that this family of compounds might represent starting points for medicinal chemistry efforts aimed at developing novel antimycobacterial agents.

Clinical isolates of Candida albicans, Candida tropicalis, and Candida krusei have different susceptibilities to Co(II) and Cu(II) complexes of 1,10-phenanthroline

The minimal inhibitory concentrations (MICs) of copper and cobalt based dimeric pyrophosphate complexes with capping 1,10-phenanthroline groups on clinical isolates of C. albicans (28 isolates), C. krusei (20 isolates) and C. tropicalis (20 isolates) are reported. C. albicans was inhibited by the cobalt complex better than by the copper complex, while C. krusei demonstrated the opposite results. C. tropicalis showed similar sensitivities to both metals in terms of calculated MIC50 values but was more sensitive to cobalt when MIC90 values were noted. Knockout strains of C. albicans that had the copper efflux protein P-type ATPase (CRP1), the copper binding metallothionein CUP1 or both CRP1/CUP1 removed clearly demonstrate that the origins of copper resistant in C. albicans lies primarily in the P-type ATPase, with the MT playing an important secondary role in the absence of the efflux protein. This study suggests that certain strains of Candida have evolved to protect against particular metal ions and that in the case of C. albicans, a primary invasive fungal species, cobalt may be a good starting-point for new therapeutic development.

A Modified Bacillus Calmette-Guérin (BCG) Vaccine with Reduced Activity of Antioxidants and Glutamine Synthetase Exhibits Enhanced Protection of Mice despite Diminished in Vivo Persistence

Early attempts to improve BCG have focused on increasing the expression of prominent antigens and adding recombinant toxins or cytokines to influence antigen presentation. One such modified BCG vaccine candidate has been withdrawn from human clinical trials due to adverse effects. BCG was derived from virulent Mycobacterium bovis and retains much of its capacity for suppressing host immune responses. Accordingly, we have used a different strategy for improving BCG based on reducing its immune suppressive capacity. We made four modifications to BCG Tice to produce 4dBCG and compared it to the parent vaccine in C57Bl/6 mice. The modifications included elimination of the oxidative stress sigma factor SigH, elimination of the SecA2 secretion channel, and reductions in the activity of iron co-factored superoxide dismutase and glutamine synthetase. After IV inoculation of 4dBCG, 95% of vaccine bacilli were eradicated from the spleens of mice within 60 days whereas the titer of BCG Tice was not significantly reduced. Subcutaneous vaccination with 4dBCG produced greater protection than vaccination with BCG against dissemination of an aerosolized challenge of M. tuberculosis to the spleen at 8 weeks post-challenge. At this time, 4dBCG-vaccinated mice also exhibited altered lung histopathology compared to BCG-vaccinated mice and control mice with less well-developed lymphohistiocytic nodules in the lung parenchyma. At 26 weeks post-challenge, 4dBCG-vaccinated mice but not BCG-vaccinated mice had significantly fewer challenge bacilli in the lungs than control mice. In conclusion, despite reduced persistence in mice a modified BCG vaccine with diminished antioxidants and glutamine synthetase is superior to the parent vaccine in conferring protection against M. tuberculosis. The targeting of multiple immune suppressive factors produced by BCG is a promising strategy for simultaneously improving vaccine safety and effectiveness.

Enhanced In Vitro Activity of Dihydrofolate Reductase and Dihydropteroase Synthase Inhibitors in Combination against Nocardia spp.

Common treatments for nocardial infections include the sulfonamides used alone or in combination, e.g., trimethoprim-sulfamethoxazole, with amikacin, imipenem, or ceftriaxone. Treatment is especially difficult in cases involving the central nervous system or disseminated disease or with drug-resistant species such as Nocardia farcinica and Nocardia otitidiscaviarum (5). Sulfamethoxazole and dapsone inhibit dihydropteroate synthase and hence prevent the conversion of bacterial para-aminobenzoic acid to dihydrofolic acid. The active putative metabolite of PS-22 is a dihydrotriazine, a sister compound of WR99210 (1). WR99210 is a dihydrofolate reductase inhibitor that has demonstrated in vitro activity against Nocardia species (3). Theoretically, the combination of a dihydrofolate reductase inhibitor with a dihydropteroate synthase inhibitor should lead to enhanced activity, as both these enzymes lie on the folate biosynthesis pathway. This was evaluated in vitro. The active putative metabolite of PS-22 and dapsone was provided by Jacobus Pharmaceutical Co., Princeton, N.J.; sulfamethoxazole was purchased from Sigma Chemical Co., St. Louis, Mo. Twenty strains of Nocardia spp. from the American Type Culture Collection and clinical isolates provided by the Clinical Microbiology Laboratory, Upstate Medical University (Betty Ann Forbes), Syracuse, N.Y., were used in this study. Experimental in vitro procedures were modeled after those listed in the paper of Meyer et al. (4). Varying concentrations of the active putative metabolite of PS-22 were tested against fixed concentrations of sulfamethoxazole and dapsone. The concentration of sulfamethoxazole at 10 μg/ml was physiologically achievable. The concentration of dapsone was fixed at 1 μg/ml, as following the administration of 100 mg of the drug orally serum drug concentrations range from 0.4 to 1.2 μg/ml (2). Table ​Table11 shows the results of the drug combinations against various Nocardia species. The in vitro activity of the active putative metabolite of PS-22 was generally enhanced by combination with dapsone or sulfamethoxazole. These results agree with the findings of McNeil et al. (3) and demonstrate that the rational drug combination of a dihydrofolate reductase inhibitor combined with a dihydropteroate synthase inhibitor provides good targets for the evaluation of agents that work at these sites. TABLE 1. MICs (μg/ml) of a WR99210 analogue alone and in combination with dapsone or sulfamethoxazole against various strains of Nocardia spp.

Therapy for Mycobacterium kansasii Infection: Beyond 2018

The current standard of care therapy for pulmonary Mycobacterium kansasii infection is isoniazid (300 mg/day), rifampin (600 mg/day), and ethambutol (15 mg/kg/day) for 12 months after achieving sputum culture negativity. Rifampin is the key drug in this regimen. The contribution of isoniazid is unclear since its in vitro MICs against M. kansasii are near the peak achievable serum levels and more than 100-fold greater than the MICs for Mycobacterium tuberculosis. Ethambutol likely decreases the emergence of rifampin resistant organisms. There are several new drug classes (e.g., quinolones, macrolides, nitroimidazoles, diarylquinolines, and clofazimine) that exhibit antimycobacterial activities against M. tuberculosis but have not yet been adequately studied against M. kansasii infections. The evaluation of in vitro activities of these agents as well as their study in new regimens in comparison to the standard of care regimen in mouse infection models should be undertaken. This knowledge will inform development of human clinical trials of new regimens in comparison to the current standard of care regimen. It is likely that shorter and more effective therapy is achievable with currently available drugs.