Dual Pharmacophore Pyrithione-Containing Cephalosporins Kill Both Replicating and Nonreplicating Mycobacterium tuberculosis

The historical view of β-lactams as ineffective antimycobacterials has given way to growing interest in the activity of this class against Mycobacterium tuberculosis (Mtb) in the presence of a β-lactamase inhibitor. However, most antimycobacterial β-lactams kill Mtb only or best when the bacilli are replicating. Here, a screen of 1904 β-lactams led to the identification of cephalosporins substituted with a pyrithione moiety at C3′ that are active against Mtb under both replicating and nonreplicating conditions, neither activity requiring a β-lactamase inhibitor. Studies showed that activity against nonreplicating Mtb required the in situ release of the pyrithione, independent of the known class A β-lactamase, BlaC. In contrast, replicating Mtb could be killed both by released pyrithione and by the parent β-lactam. Thus, the antimycobacterial activity of pyrithione-containing cephalosporins arises from two mechanisms that kill mycobacteria in different metabolic states.This work was supported by the Tres Cantos Open Lab Foundation, the Tri-Institutional TB Research Unit via NIH grants U19 AI109748 and AI111143, and the Abby and Howard Milstein Program in Chemical Biology and Translational Medicine

Activity-Based Protein Profiling Reveals That Cephalosporins Selectively Active on Non-replicating Mycobacterium tuberculosis Bind Multiple Protein Families and Spare Peptidoglycan Transpeptidases

This work is licensed under a Creative Commons Attribution 4.0 International License.As β-lactams are reconsidered for the treatment of tuberculosis (TB), their targets are assumed to be peptidoglycan transpeptidases, as verified by adduct formation and kinetic inhibition of Mycobacterium tuberculosis (Mtb) transpeptidases by carbapenems active against replicating Mtb. Here, we investigated the targets of recently described cephalosporins that are selectively active against non-replicating (NR) Mtb. NR-active cephalosporins failed to inhibit recombinant Mtb transpeptidases. Accordingly, we used alkyne analogs of NR-active cephalosporins to pull down potential targets through unbiased activity-based protein profiling and identified over 30 protein binders. None was a transpeptidase. Several of the target candidates are plausibly related to Mtb’s survival in an NR state. However, biochemical tests and studies of loss of function mutants did not identify a unique target that accounts for the bactericidal activity of these beta-lactams against NR Mtb. Instead, NR-active cephalosporins appear to kill Mtb by collective action on multiple targets. These results highlight the ability of these β-lactams to target diverse classes of proteins.NIH U19AI111143Milstein Program in Chemical Biology and Translational MedicineWilliam Randolph Hearst TrustWelch Foundation (A-0015

Dual-Pharmacophore Pyrithione-Containing Cephalosporins Kill Both Replicating and Nonreplicating Mycobacterium tuberculosis

The historical view of β-lactams as ineffective antimycobacterials has given way to growing interest in the activity of this class against Mycobacterium tuberculosis (Mtb) in the presence of a β-lactamase inhibitor. However, most antimycobacterial β-lactams kill Mtb only or best when the bacilli are replicating. Here, a screen of 1904 β-lactams led to the identification of cephalosporins substituted with a pyrithione moiety at C3′ that are active against Mtb under both replicating and nonreplicating conditions, neither activity requiring a β-lactamase inhibitor. Studies showed that activity against nonreplicating Mtb required the in situ release of the pyrithione, independent of the known class A β-lactamase, BlaC. In contrast, replicating Mtb could be killed both by released pyrithione and by the parent β-lactam. Thus, the antimycobacterial activity of pyrithione-containing cephalosporins arises from two mechanisms that kill mycobacteria in different metabolic states

Bactericidal disruption of magnesium metallostasis in Mycobacterium tuberculosis is counteracted by mutations in the metal ion transporter CorA

A defining characteristic of treating tuberculosis is the need for prolonged administration of multiple drugs. This may be due in part to subpopulations of slowly replicating or nonreplicating Mycobacterium tuberculosis bacilli exhibiting phenotypic tolerance to most antibiotics in the standard treatment regimen. Confounding this problem is the increasing incidence of heritable multidrug-resistant M. tuberculosis. A search for new antimycobacterial chemical scaffolds that can kill phenotypically drug-tolerant mycobacteria uncovered tricyclic 4-hydroxyquinolines and a barbituric acid derivative with mycobactericidal activity against both replicating and nonreplicating M. tuberculosis. Both families of compounds depleted M. tuberculosis of intrabacterial magnesium. Complete or partial resistance to both chemotypes arose from mutations in the putative mycobacterial Mg2+/Co2+ ion channel, CorA. Excess extracellular Mg2+, but not other divalent cations, diminished the compounds’ cidality against replicating M. tuberculosis. These findings establish depletion of intrabacterial magnesium as an antimicrobial mechanism of action and show that M. tuberculosis magnesium homeostasis is vulnerable to disruption by structurally diverse, nonchelating, drug-like compounds

Characterising HIV-associated Mycobacterium tuberculosis blood stream infection

Despite the success of antiretroviral therapy roll-out, one-million people still die with HIV-infection annually. In high-burden settings, tuberculosis remains the most common proximal cause of hospital admission and death in people living with HIV. In post-mortem series, 90% of fatal HIV-associated tuberculosis is ‘disseminated’. This is a form of tuberculosis which has been poorly characterised and, despite the high associated-mortality, never been the subject of interventional trials to define optimal treatment strategies. This thesis contends that the mode of severe HIV-associated tuberculosis is blood stream infection. First it is argued with reference to historical literature that blood stream dissemination is part of the natural history of post-primary tuberculosis infection, and that HIV-associated M. tuberculosis blood stream infection (MTBBSI) can be conceived of as a reversion to, and exaggerated form of this natural history. Using data from a large cohort (n=571) of HIV-infected inpatients with CD4 cell count <350 cells/mm3 and a new TB diagnosis from Khayelitsha Hospital, South Africa (the KDHTB study), the extent and magnitude of MTBBSI is shown to be a major determinant of clinical phenotype and mortality risk. Systematic, quantitative markers of blood stream dissemination, including TB blood culture, urine-lipoarabinomannan (uLAM), and urine GeneXpert MTB/RIF testing (uXpert), can be combined into a ‘disseminated TB score. KDHTB patients have high prevalence of abnormal sodium and fluid balance, metabolic acidosis associated with acute kidney injury, hyperlactataemia, infiltrative liver and splenic pathology, and anaemia. Each of these pathophysiologies in turn correlates to disseminated TB score, and to risk of death, suggesting bacterial burden and MTBBSI are central to the pathophysiology of severe HIV-associated tuberculosis. An individual patient data meta-analysis, with 20 independent data sets comprising over 6000 patients, is used to establish the prevalence of TB blood culture positive disease amongst critically unwell HIV-infected inpatients. This shows that MTBBSI is more common than previous estimates suggest, is a strong independent association with mortality risk, and is also associated with specific increased risk of death if empirical treatment is delayed. The development of tools to identify and measure MTBBSI is described, including Xpert-ultra testing of blood, and the use of a novel dye, DMN-trehalose, to perform direct microscopy on patient blood samples. These techniques are used to provide the first description of the pharmacodynamics of MTBBSI, by serially quantifying blood bacilli load over the first 72-hours of standard TB therapy, in 28 patients with high predicted probability of bacteraemia. In this cohort, risk of mortality is related to several summary measures of MTBBSI dynamics in the first 72-hours of therapy, suggesting this approach can be used to define biomarkers of treatment response. In conclusion, MTBBSI is a highly-specific diagnosis responsible for substantial mortality in hospitalised people living with HIV. Interventions with strengthened bacteriocidal activity, focussed on reducing bacterial burden, are warranted for MTBBSI. Tools developed in this thesis, including potential pharmacodynamic biomarkers, should facilitate such trials