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

Novel Cephalosporins Selectively Active on Nonreplicating Mycobacterium tuberculosis

We report two series of novel cephalosporins that are bactericidal to Mycobacterium tuberculosis alone of the pathogens tested, which only kill M. tuberculosis when its replication is halted by conditions resembling those believed to pertain in the host, and whose bactericidal activity is not dependent upon or enhanced by clavulanate, a β-lactamase inhibitor. The two classes of cephalosporins bear an ester or alternatively an oxadiazole isostere at C-2 of the cephalosporin ring system, a position that is almost exclusively a carboxylic acid in clinically used agents in the class. Representatives of the series kill M. tuberculosis within macrophages without toxicity to the macrophages or other mammalian cells

Function of Macromolecular Serine Protease Inhibitors in the Human Blood Fluke Schistosoma mansoni

Serine protease inhibitors (serpins) are a superfamily of structurally conserved proteins that are key regulators of proteolysis in plants, animals, and certain viruses. The S. mansoni genome contains eight full-length serpins. Semi-quantitative PCRs performed on S. mansoni cDNA samples verified the transcription of these eight putative serpins. All identified serpin sequences had homology to other members of the serpin family and contained all the structural components needed for functionality. Some schistosome serpins are predicted to be non-inhibitory while others showed clear tryptic or chymotryptic protease specificity. Two of these serpins, SmSrpQ and SmSerp_C, were of particular interest owing to their secretion by the infective larva of S. mansoni. Little published data exists examining the function of SmSrpQ, SmSerp_C, or any other of the schistosome serpin. SmSrpQ and SmSerp_C are only expressed during larval development and, in the case of SmSrpQ, in the early stages of mammalian infection. 35S-SmSrpQ was able to form an SDS-stable complex with a component of the larval lysate but no complex was detected with several mammalian host proteases. Formation of a complex was sensitive to the protease active site inhibitors PMSF, Z-AAPF-CMK, and Z-AAPL-CMK. Similar experiments were carried out with 35S-SmSerp_C, which formed a complex with trypsin, human plasma kallikrein, and a molluscan protease, most likely the tryptases found in the intermediate host of S. mansoni, Biomphalaria glabrata.Invasion by S. mansoni larvae is facilitated by cercarial elastase (SmCE), a serine protease. The band detected between 35S-SmSrpQ and a protease in cercarial lysates was also detected in western blot analysis of parasite lysates. These blots showed that when active SmCE was present, a complex of comparable size to SmCE bound to SmSrpQ was observed. This data, together with the results from immunolocalization of these proteins in the parasite and in simulated infections, suggests that SmSrpQ carefully regulates SmCE degradation of skin tissue during larval invasion.SmSrpQ inhibition of SmCE and SmSerp_C inhibition of kallikrein and a snail protease confirm the inhibitory nature of these serpins and suggests a role in the pathogenesis/ biology of the parasite

Bactericidal Disruption of Magnesium Metallostasis in Mycobacterium tuberculosis Is Counteracted by Mutations in the Metal Ion Transporter CorA

Antimycobacterial agents might shorten the course of treatment by reducing the number of phenotypically tolerant bacteria if they could kill M. tuberculosis in diverse metabolic states. Here we report two chemically disparate classes of agents that kill M. tuberculosis both when it is replicating and when it is not. Under replicating conditions, the tricyclic 4-hydroxyquinolines and a barbituric acid analogue deplete intrabacterial magnesium as a mechanism of action, and for both compounds, mutations in CorA, a putative Mg2+/Co2+ transporter, conferred resistance to the compounds when M. tuberculosis was under replicating conditions but not under nonreplicating conditions, illustrating that a given compound can kill M. tuberculosis in different metabolic states by disparate mechanisms. Targeting magnesium metallostasis represents a previously undescribed antimycobacterial mode of action that might cripple M. tuberculosis in a Mg2+-deficient intraphagosomal environment of macrophages.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

Novel Cephalosporins Selectively Active on Nonreplicating Mycobacterium tuberculosis

[Image: see text] We report two series of novel cephalosporins that are bactericidal to Mycobacterium tuberculosis alone of the pathogens tested, which only kill M. tuberculosis when its replication is halted by conditions resembling those believed to pertain in the host, and whose bactericidal activity is not dependent upon or enhanced by clavulanate, a β-lactamase inhibitor. The two classes of cephalosporins bear an ester or alternatively an oxadiazole isostere at C-2 of the cephalosporin ring system, a position that is almost exclusively a carboxylic acid in clinically used agents in the class. Representatives of the series kill M. tuberculosis within macrophages without toxicity to the macrophages or other mammalian cells

A High-Throughput, High-Containment Human Primary Epithelial Airway Organ-on-Chip Platform for SARS-CoV-2 Therapeutic Screening

COVID-19 emerged as a worldwide pandemic in early 2020, and while the rapid development of safe and efficacious vaccines stands as an extraordinary achievement, the identification of effective therapeutics has been less successful. This process has been limited in part by a lack of human-relevant preclinical models compatible with therapeutic screening on the native virus, which requires a high-containment environment. Here, we report SARS-CoV-2 infection and robust viral replication in PREDICT96-ALI, a high-throughput, human primary cell-based organ-on-chip platform. We evaluate unique infection kinetic profiles across lung tissue from three human donors by immunofluorescence, RT-qPCR, and plaque assays over a 6-day infection period. Enabled by the 96 devices/plate throughput of PREDICT96-ALI, we also investigate the efficacy of Remdesivir and MPro61 in a proof-of-concept antiviral study. Both compounds exhibit an antiviral effect against SARS-CoV-2 in the platform. This demonstration of SARS-CoV-2 infection and antiviral dosing in a high-throughput organ-on-chip platform presents a critical capability for disease modeling and therapeutic screening applications in a human physiology-relevant in vitro system

Schistosome serine protease inhibitors: parasite defense or homeostasis?

Serpins are a structurally conserved family of macromolecular inhibitors found in numerous biological systems. The completion and annotation of the genomes of Schistosoma mansoni and Schistosoma japonicum has enabled the identification by phylogenetic analysis of two major serpin clades. S. mansoni shows a greater multiplicity of serpin genes, perhaps reflecting adaptation to infection of a human host. Putative targets of schistosome serpins can be predicted from the sequence of the reactive center loop (RCL). Schistosome serpins may play important roles in both post-translational regulation of schistosome-derived proteases, as well as parasite defense mechanisms against the action of host proteases.<br>Serpinas são uma família de inibidores macromoleculares estruturalmente conservados encontrados em inúmeros sistemas biológicos. O término e a anotação dos genomas de Schistosoma mansoni e de Schistosoma japonicum permitiram a identificação por análise filogenética de dois principais clados de serpinas. S. mansoni mostra uma multiplicidade maior de genes de serpinas, talvez refletindo uma adaptação à infecção de um hospedeiro humano. Alvos putativos das serpinas de esquistossomos podem ser preditos a partir da sequência do "loop" do centro reativo. Serpinas de esquistossomos podem ter importantes papeis tanto na regulação pós-traducional de proteases derivadas do esquistossoma, quanto nos mecanismos de defesa contra a ação de proteases do hospedeiro