Fatty acid biosynthetic enzymes exploit the reactivity of acyl- and malonyl-thioesters for catalysis. Here we synthesize acetyl/malonyl-CoA analogs with esters or amides in place of the thioester and characterize their behavior as substrates or inhibitors of the E. coli FabH ketosynthase. The acetyl- and malonyl-oxa(dethia)CoA analogs undergo extremely slow hydrolysis in the presence of FabH or C112Q mutant, which mimics the acyl-enzyme intermediate. Decarboxylation of malonyl-oxa(dethia)CoA by FabH or C112Q mutant was not detected. The amide analogs were completely stable to enzyme activity as expected. In enzyme assays, acetyl-oxa(dethia)CoA is surprisingly slightly activating, while acetyl-aza(dethia)CoA is a moderate inhibitor. The malonyl-oxa/aza(dethia)CoAs are inhibitors with Ki’s near the Km of malonyl-CoA. For comparison, we determine the FabH catalyzed decomposition rates for acetyl/malonyl-CoA, revealing some fundamental catalytic traits of FabH. The stable and inhibitory properties of the substrate analogs makes them promising for structure-function studies to undercover the basis of FabH cooperativity and enzyme:substrate interactions.

Background:The BCG vaccine induces trained immunity, an epigenetic-mediated increase in innate immune responsiveness. Therefore, this clinical trial evaluated if BCG-induced trained immunity could decrease coronavirus disease 2019 (COVID-19)-related frequency or severity. Methods:A double-blind, placebo-controlled clinical trial of healthcare workers randomized participants to vaccination with BCG TICE or placebo (saline). Enrollment included 529 healthcare workers randomized to receive BCG or placebo. Primary analysis evaluated COVID-19 disease frequency, while secondary analysis evaluated coronavirus immunity in a subset of participants. Study enrollment ceased early in December 2020 following introduction of COVID-19-specific vaccines. Results:Study enrollment was halted early, prior to reaching the targeted recruitment, and was not powered to detect a decrease in COVID-19 frequency. Symptomatic COVID-19 occurred in 21 of 263 and 10 of 266 participants in the BCG and placebo arms, respectively (P = .50, Fisher exact test). Participants vaccinated with BCG, but uninfected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), demonstrated increased coronavirus vaccine immunity (increase spike-inducible levels of tumor necrosis factor, interleukin 6, and interleukin 1β) 12 months after BCG vaccination compared to participants receiving placebo. Immune responsiveness to SARS-CoV-2 antigens correlated with BCG-induced DNA methylation changes. Conclusions:Due to early study closure, the study was not powered to evaluate COVID-19 frequency. Secondary analysis demonstrated that 12 months following vaccination, BCG increased coronavirus vaccine immunity compared to those who did not receive BCG. This increase in COVID-19 vaccine immunity correlated with BCG-induced DNA methylation changes.

Abstract Background The Bacillus Calmette-Guérin (BCG) vaccine induces trained immunity, an epigenetic-mediated increase in innate immune responsiveness. Therefore, this clinical trial evaluated if BCG-induced trained immunity could decrease COVID-19-related frequency or severity. Methods A double-blind, placebo-controlled clinical trial of healthcare workers randomized participants to vaccination with BCG TICE® or placebo (saline). Enrollment included 529 healthcare workers randomized to receive BCG or placebo. Primary analysis evaluated COVID-19 disease frequency, while secondary analysis evaluated coronavirus immunity in a subset of participants. Study enrollment ceased early in December 2020 following introduction of COVID-19-specific vaccines. Results Study enrollment was halted early, prior to reaching the targeted recruitment and was not powered to detect a decrease in COVID-19 frequency. Symptomatic COVID-19 occurred in 21 of 263 and 10 of 266 participants in the BCG and placebo arms, respectively (p= 0.50, Fisher’s exact test). Participants vaccinated with BCG, but uninfected with COVID-19, demonstrated increased coronavirus vaccine immunity (increase spike-inducible levels of TNF, IL6, and IL-1β) twelve months after BCG vaccination compared to participants receiving placebo. Immune responsiveness to COVID-19 antigens correlated with BCG-induced DNA methylation changes. Conclusion Due to early study closure, the study was not powered to evaluate COVID-19 frequency. Secondary analysis demonstrated that twelve months following vaccination, BCG increased coronavirus vaccine immunity compared to those who did not receive BCG. This increase in COVID-19 vaccine immunity correlated with BCG-induced DNA methylation changes.

The rise of drug resistance has become a global crisis, with >1 million deaths due to resistant bacterial infections each year. Pseudomonas aeruginosa, in particular, remains a serious problem with limited solutions due to complex resistance mechanisms that now lead to more than 32,000 multidrug-resistant (MDR) infections and over 2,000 deaths annually. While the emergence of resistant bacteria has become concerningly common, identification of useful new drug classes has been limited over the past 40+ years. We found that a potential novel therapeutic, the peptide-mimetic TM5, is effective at killing P. aeruginosa and displays sufficiently low toxicity for mammalian cells to allow for use in treatment of infections. Interestingly, TM5 kills P. aeruginosa more rapidly than traditional antibiotics, within 30-60 minutes in vitro , and is effective against a range of clinical isolates. In vivo , TM5 significantly reduced bacterial load in the lungs within 24 hours compared to untreated mice and demonstrated few adverse effects. Taken together, these observations suggest that TM5 shows promise as an alternative therapy for MDR P. aeruginosa respiratory infections.

New antiviral agents are essential to improving treatment and control of SARS-CoV-2 infections that can lead to the disease COVID-19. Antimicrobial peptoids are sequence-specific oligo-N-substituted glycine peptidomimetics that emulate the structure and function of natural antimicrobial peptides but are resistant to proteases. We demonstrate antiviral activity of a new peptoid (TM9) against the coronavirus, murine hepatitis virus (MHV), as a closely related model for the structure and antiviral susceptibility profile of SARS-CoV-2. This peptoid mimics the human cathelicidin LL-37, which has also been shown to have antimicrobial and antiviral activity. In this study, TM9 was effective against three murine coronavirus strains, demonstrating that the therapeutic window is large enough to allow the use of TM9 for treatment. All three isolates of MHV generated infection in mice after 15 min of exposure by aerosol using the Madison aerosol chamber, and all three viral strains could be isolated from the lungs throughout the 5-day observation period post-infection, with the peak titers on day 2. MHV-A59 and MHV-A59-GFP were also isolated from the liver, heart, spleen, olfactory bulbs, and brain. These data demonstrate that MHV serves as a valuable natural murine model of coronavirus pathogenesis in multiple organs, including the brain.

Background A large epidemic, such as that observed with SARS-CoV-2, seriously challenges available hospital capacity, and this would be augmented by infection of healthcare workers (HCW). Bacillus Calmette-Guérin (BCG) is a vaccine against tuberculosis, with protective non-specific effects against other respiratory tract infections in vitro and in vivo. Preliminary analyses suggest that regions of the world with existing BCG vaccination programs have lower incidence and mortality from COVID-19. We hypothesize that BCG vaccination can reduce SARS-CoV-2 infection and disease severity. Methods This will be a placebo-controlled adaptive multi-center randomized controlled trial. A total of 1800 individuals considered to be at high risk, including those with comorbidities (hypertension, diabetes, obesity, reactive airway disease, smokers), racial and ethnic minorities, elderly, teachers, police, restaurant wait-staff, delivery personnel, health care workers who are defined as personnel working in a healthcare setting, at a hospital, medical center or clinic (veterinary, dental, ophthalmology), and first responders (paramedics, firefighters, or law enforcement), will be randomly assigned to two treatment groups. The treatment groups will receive intradermal administration of BCG vaccine or placebo (saline) with groups at a 1:1 ratio. Individuals will be tracked for evidence of SARS-CoV-2 infection and severity as well as obtaining whole blood to track immunological markers, and a sub-study will include cognitive function and brain imaging. The majority of individuals will be followed for 6 months, with an option to extend for another 6 months, and the cognitive sub-study duration is 2 years. We will plot Kaplan-Meier curves that will be plotted comparing groups and hazard ratios and p -values reported using Cox proportional hazard models. Discussion It is expected this trial will allow evaluation of the effects of BCG vaccination at a population level in high-risk healthcare individuals through a mitigated clinical course of SARS-CoV-2 infection and inform policy making during the ongoing epidemic. Trial registration ClinicalTrials.gov NCT04348370. Registered on April 16, 2020.

Acetyl coenzyme A (acetyl-CoA) is a reactive metabolite that nonproductively hydrolyzes in a number of enzyme active sites in the crystallization time frame. In order to elucidate the enzyme-acetyl-CoA interactions leading to catalysis, acetyl-CoA substrate analogs are needed. One possible analog for use in structural studies is acetyl-oxa(dethia)CoA (AcOCoA), in which the thioester S atom of CoA is replaced by an O atom. Here, structures of chloramphenicol acetyltransferase III (CATIII) and Escherichia coli ketoacylsynthase III (FabH) from crystals grown in the presence of partially hydrolyzed AcOCoA and the respective nucleophile are presented. Based on the structures, the behavior of AcOCoA differs between the enzymes, with FabH reacting with AcOCoA and CATIII being unreactive. The structure of CATIII reveals insight into the catalytic mechanism, with one active site of the trimer having relatively clear electron density for AcOCoA and chloramphenicol and the other active sites having weaker density for AcOCoA. One FabH structure contains a hydrolyzed AcOCoA product oxa(dethia)CoA (OCoA), while the other FabH structure contains an acyl-enzyme intermediate with OCoA. Together, these structures provide preliminary insight into the use of AcOCoA for enzyme structure-function studies with different nucleophiles.

Fatty acid and polyketide biosynthetic enzymes exploit the reactivity of acyl- and malonyl-thioesters for catalysis. A prime example is FabH, which initiates fatty acid biosynthesis in many bacteria and plants. FabH performs an acyltransferase reaction with acetyl-CoA to generate an acetyl-S-FabH acyl-enzyme intermediate and subsequent decarboxylative Claisen-condensation with a malonyl-thioester carried by an acyl carrier protein (ACP). We envision that crystal structures of FabH with substrate analogues can provide insight into the conformational changes and enzyme/substrate interactions underpinning the distinct reactions. Here, we synthesize acetyl/malonyl-CoA analogues with esters or amides in place of the thioester and characterize their stability and behavior as Escherichia coli FabH substrates or inhibitors to inform structural studies. We also characterize the analogues with mutant FabH C112Q that mimics the acyl-enzyme intermediate allowing dissection of the decarboxylation reaction. The acetyl- and malonyl-oxa(dethia)CoA analogues undergo extremely slow hydrolysis in the presence of FabH or the C112Q mutant. Decarboxylation of malonyl-oxa(dethia)CoA by FabH or C112Q mutant was not detected. The amide analogues were completely stable to enzyme activity. In enzyme assays with acetyl-CoA and malonyl-CoA (rather than malonyl-ACP) as substrates, acetyl-oxa(dethia)CoA is surprisingly slightly activating, while acetyl-aza(dethia)CoA is a moderate inhibitor. The malonyl-oxa/aza(dethia)CoAs are inhibitors with Ki's near the Km of malonyl-CoA. For comparison, we determine the FabH catalyzed decomposition rates for acetyl/malonyl-CoA, revealing some fundamental catalytic traits of FabH, including hysteresis for malonyl-CoA decarboxylation. The stability and inhibitory properties of the substrate analogues make them promising for structure-function studies to reveal fatty acid and polyketide enzyme/substrate interactions.

AbstractThe rise of drug resistance has become a global crisis, with >1 million deaths due to resistant bacterial infections each year.Pseudomonas aeruginosa,in particular, remains a serious problem with limited solutions due to complex resistance mechanisms that now lead to more than 32,000 multidrug-resistant (MDR) infections and over 2,000 deaths annually. While the emergence of resistant bacteria has become concerningly common, identification of useful new drug classes has been limited over the past 40+ years. We found that a potential novel therapeutic, the peptide-mimetic TM5, is effective at killingP. aeruginosaand displays sufficiently low toxicity for mammalian cells to allow for use in treatment of infections. Interestingly, TM5 killsP. aeruginosamore rapidly than traditional antibiotics, within 30-60 minutesin vitro, and is effective against a range of clinical isolates.In vivo, TM5 significantly reduced bacterial load in the lungs within 24 hours compared to untreated mice and demonstrated few adverse effects. Taken together, these observations suggest that TM5 shows promise as an alternative therapy for MDRP. aeruginosarespiratory infections.

Methylmalonyl-CoA epimerase (MMCE) is proposed to use general acid-base catalysis, but the proposed catalytic glutamic acids are highly asymmetrical in the active site unlike many other racemases. To gain insight into the puzzling relationships between catalytic mechanism, structure, and substrate preference, we solved Streptomyces coelicolor MMCE structures with substrate or 2-nitropropionyl-CoA, an intermediate/transition state analogue. Both ligand bound structures have a planar methylmalonate/2-nitropropionyl moiety indicating a deprotonated C2 with >= 4 angstrom distances to either catalytic acid. Both glutamates interact with the carboxylate/nitro group, either directly or through other residues. This suggests the proposed catalytic acids sequentially catalyze proton shifts between C2 and carboxylate of the substrate with an enolate intermediate. In addition, our structures provide a platform to design mutations for expanding substrate scope to support combinatorial biosynthesis.

Staphylococcus aureus resistance is a consistent problem with a large impact on the health care system. Infections with resistant S. aureus can cause serious adverse effects and can result in death.

Tuberculosis (TB) is among the greatest public health and safety concerns in the 21st century, Mycobacterium tuberculosis, which causes TB, infects alveolar macrophages and uses these cells as one of its primary sites of replication. The current TB treatment regimen, which consist of chemotherapy involving a combination of 3-4 antimicrobials for a duration of 6-12 months, is marked with significant side effects, toxicity, and poor compliance. Targeted drug delivery offers a strategy that could overcome many of the problems of current TB treatment by specifically targeting infected macrophages. Recent advances in nanotechnology and material science have opened an avenue to explore drug carriers that actively and passively target macrophages. This approach can increase the drug penetration into macrophages by using ligands on the nanocarrier that interact with specific receptors for macrophages. This review encompasses the recent development of drug carriers specifically targeting macrophages actively and passively. Future directions and challenges associated with development of effective TB treatment is also discussed.

Tuberculosis is one of the most frequent causes of death in humans worldwide. One of the primary reasons tuberculosis remains a public health threat is that diagnosis can take weeks to months, is often not very sensitive and cannot be accomplished in many remote environments. A rapid, sensitive and inexpensive point-of-care (POC) diagnostic would have a major impact on tuberculosis eradication efforts. The tuberculosis diagnostic system REFtb is based on specific detection of the constitutively expressed β-lactamase (BlaC) in Mycobacterium tuberculosis using a custom fluorogenic substrate designated as CDG-3. REFtb has potential as a diagnostic for tuberculosis that could be very inexpensive (<USD 2.00/test), used at the POC and could provide definitive diagnosis within 10 min. However, the reagents for REFtb are currently in liquid form, making them more susceptible to degradation and difficult to transport. We evaluated the improvement in the stability of REFtb reagents by lyophilization under a variety of conditions through their effects on the performance of REFtb. We found that lyophilization of REFtb components produces an easily reconstituted powder that displays similar performance to the liquid system and that lactose represents one of the most promising excipients for use in a final POC REFtb diagnostic system. These studies provide the foundation for the production of a stable POC REFtb system that could be easily distributed worldwide with minimal or no requirement for refrigeration.

Pyruvate carboxylase (PC) is a central metabolic enzyme that produces oxaloacetate from pyruvate and bicarbonate in an ATP‐dependent manner. The catalytic activity of PC contributes to glucose homeostasis and has been implicated in enhanced tumorigenesis and metastasis in certain cancers. PC activity is allosterically activated by acetyl CoA, and allosterically inhibited by L‐aspartate. The binding of these effector molecules is mutually exclusive. The binding site for acetyl‐CoA has largely been identified through previous structural studies, but the exact binding site for the acetyl moiety is unknown, partly as a consequence of the enzyme‐catalyzed hydrolysis of acetyl CoA. The acetyl moiety is a particularly important feature of acetyl CoA activation, contributing to the significantly enhanced allosteric activation of acetyl CoA relative to CoA. Here, we have employed non‐hydrolysable and truncated analogs of acetyl‐CoA to more precisely probe the acetyl moiety binding site in PC. We have determined that the acetyl moiety contributes greatly to the binding of the molecule but that it does not alter the degree of activation. Additionally, kinetic studies strongly suggest that L‐aspartate competes for the acetyl moiety binding site with acetyl CoA. A definition of the acetyl moiety binding site further clarifies the mechanism of acetyl CoA binding and offers a more detailed molecular description of allosteric regulation in this central metabolic enzyme.

Malonyl-CoA and C2-subsituted variants are intermediates in a variety of biochemical pathways, including fatty acid polyketide biosynthesis. Due to the inherent reactivity of malonyl-CoA, a β-keto acid that can undergo spontaneous decarboxylation, molecular pictures of how enzymes catalyze this reaction remain elusive. In theory, malonyl-CoA decarboxylation is easy because an enol or enolate intermediate is generated that lowers the overall barrier. To speed up the reaction, enzymes must stabilize either the enol or enolate intermediate by interacting with the thioester ketone. Intriguingly other enzymes that catalyze transfer of malonyl-units by activating thioester ketone must find a way to avoid decarboxylation. To gain insight into various enzymes in malonyl-CoA metabolism we have synthesized malonyl-CoA analogs where the carboxylic acid is replaced by nitro or sulfonate groups. Capturing these analogs in the active sites of various enzymes by crystallization has allowed us elucidate catalytic molecular details, although with some interesting caveats. In all of our structures the nitro-bearing analog is bound as a nitronate species with the C2 being planar. While this raises some suspicions as to the appropriateness of the nitro group as a mechanistic analog, similar binding orientations of the sulfonate analog helps to solidify our conclusions. Support or Funding Information Startup Funds from Purdue University This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Natural products are a well‐established source of drugs and other commodity chemicals. Polyketide natural products have a high degree of chemical variability and play many roles in biology, making them targets for protein engineering to generate analogs. Polyketide synthases (PKS) exist as three different types, but all require the function of a ketoacyl synthase or ketosynthase (KS). The KS catalyzed reaction is carried out in three steps, transthiolation of an acyl‐chain onto an active site cysteine yielding an acyl‐enzyme intermediate, binding and decarboxylation of a malonyl‐group to yield an enolate, and a Claisen condensation of the enolate onto the acyl‐enzyme intermediate yielding a carbon‐carbon bond. The molecular details of decarboxylation and Claisen condensation are only poorly characterized, yet these details are needed for protein engineering efforts. Type III PKSs exist as an independently functioning KS, which primarily use coenzyme A (CoA) rather than acyl carrier proteins to carry the acyl‐chains and malonyl‐groups. As type III PKS are the simplest system we chose them as our starting point to elucidate the molecular details of KS catalysis. We have expressed and purified wild‐type and mutant forms of germicidin synthase (GCS) and tetrahydroxynaphthlene synthase (THNS) from Streptomyces coelicolor , and chalcone synthase‐2 (CHS‐II) from Arabidopsis thaliana in E. coli . Mutation of the active site cysteine to glutamine results in enzymes that mimic the acyl‐enzyme intermediate state, and can decarboxylate malonyl‐CoA. Interestingly, these mutants are much less stable than the wild‐type enzymes, suggesting that conformational changes occur upon formation of the acyl‐enzyme intermediate. We propose that using near natural substrate and intermediate mimics will allow us to capture the molecular details of catalysis. As such, X‐ray crystallography is underway with these enzymes in complex our synthetic analogs. Support or Funding Information We would like to thank the Department of Biochemistry, Purdue University for their sponsorship of the project. We would also like to thank Jeremy Lohman for providing the space needed to conduct our project.