Chemical Genetic Analysis and Functional Characterization of Staphylococcal Wall Teichoic Acid 2-Epimerases Reveals Unconventional Antibiotic Drug Targets

Here we describe a chemical biology strategy performed in Staphylococcus aureus and Staphylococcus epidermidis to identify MnaA, a 2-epimerase that we demonstrate interconverts UDP-GlcNAc and UDP-ManNAc to modulate substrate levels of TarO and TarA wall teichoic acid (WTA) biosynthesis enzymes. Genetic inactivation of mnaA results in complete loss of WTA and dramatic in vitro β-lactam hypersensitivity in methicillin-resistant S. aureus (MRSA) and S. epidermidis (MRSE). Likewise, the β-lactam antibiotic imipenem exhibits restored bactericidal activity against mnaA mutants in vitro and concomitant efficacy against 2-epimerase defective strains in a mouse thigh model of MRSA and MRSE infection. Interestingly, whereas MnaA serves as the sole 2-epimerase required for WTA biosynthesis in S. epidermidis, MnaA and Cap5P provide compensatory WTA functional roles in S. aureus. We also demonstrate that MnaA and other enzymes of WTA biosynthesis are required for biofilm formation in MRSA and MRSE. We further determine the 1.9Å crystal structure of S. aureus MnaA and identify critical residues for enzymatic dimerization, stability, and substrate binding. Finally, the natural product antibiotic tunicamycin is shown to physically bind MnaA and Cap5P and inhibit 2-epimerase activity, demonstrating that it inhibits a previously unanticipated step in WTA biosynthesis. In summary, MnaA serves as a new Staphylococcal antibiotic target with cognate inhibitors predicted to possess dual therapeutic benefit: as combination agents to restore β-lactam efficacy against MRSA and MRSE and as non-bioactive prophylactic agents to prevent Staphylococcal biofilm formation.publishe

Blood glucose as a predictor of mortality in children admitted to the hospital with febrile illness in Tanzania.

Data from a prospective study of 3,319 children ages 2 months to 5 years admitted with febrile illness to a Tanzanian district hospital were analyzed to determine the relationship of blood glucose and mortality. Hypoglycemia (blood sugar 5 mmol/L, the adjusted odds of dying were 3.3 (95% confidence interval = 2.1-5.2) and 9.8 (95% confidence interval = 5.1-19.0) among children with admission blood glucose 2.5-5 and < 2.5 mmol/L, respectively. Receiver operating characteristic (ROC) analysis suggested an optimal cutoff for admission blood sugar of < 5 mmol/L in predicting mortality (sensitivity = 57.7%, specificity = 75.2%). A cutoff for admission blood glucose of < 5 mmol/L represents a simple and clinically useful predictor of mortality in children admitted with severe febrile illness to hospital in resource-poor settings

Conserved allosteric inhibitory site on the respiratory syncytial virus and human metapneumovirus RNA-dependent RNA polymerases

Abstract Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are related RNA viruses responsible for severe respiratory infections and resulting disease in infants, elderly, and immunocompromised adults1–3. Therapeutic small molecule inhibitors that bind to the RSV polymerase and inhibit viral replication are being developed, but their binding sites and molecular mechanisms of action remain largely unknown4. Here we report a conserved allosteric inhibitory site identified on the L polymerase proteins of RSV and HMPV that can be targeted by a dual-specificity, non-nucleoside inhibitor, termed MRK-1. Cryo-EM structures of the inhibitor in complexes with truncated RSV and full-length HMPV polymerase proteins provide a structural understanding of how MRK-1 is active against both viruses. Functional analyses indicate that MRK-1 inhibits conformational changes necessary for the polymerase to engage in RNA synthesis initiation and to transition into an elongation mode. Competition studies reveal that the MRK-1 binding pocket is distinct from that of a capping inhibitor with an overlapping resistance profile, suggesting that the polymerase conformation bound by MRK-1 may be distinct from that involved in mRNA capping. These findings should facilitate optimization of dual RSV and HMPV replication inhibitors and provide insights into the molecular mechanisms underlying their polymerase activities

Discovery and multimerization of cross-reactive single-domain antibodies against SARS-like viruses to enhance potency and address emerging SARS-CoV-2 variants

Abstract Coronaviruses have been the causative agent of three epidemics and pandemics in the past two decades, including the ongoing COVID-19 pandemic. A broadly-neutralizing coronavirus therapeutic is desirable not only to prevent and treat COVID-19, but also to provide protection for high-risk populations against future emergent coronaviruses. As all coronaviruses use spike proteins on the viral surface to enter the host cells, and these spike proteins share sequence and structural homology, we set out to discover cross-reactive biologic agents targeting the spike protein to block viral entry. Through llama immunization campaigns, we have identified single domain antibodies (VHHs) that are cross-reactive against multiple emergent coronaviruses (SARS-CoV, SARS-CoV-2, and MERS). Importantly, a number of these antibodies show sub-nanomolar potency towards all SARS-like viruses including emergent CoV-2 variants. We identified nine distinct epitopes on the spike protein targeted by these VHHs. Further, by engineering VHHs targeting distinct, conserved epitopes into multi-valent formats, we significantly enhanced their neutralization potencies compared to the corresponding VHH cocktails. We believe this approach is ideally suited to address both emerging SARS-CoV-2 variants during the current pandemic as well as potential future pandemics caused by SARS-like coronaviruses

Novel Pan-Coronavirus 3CL Protease Inhibitor MK-7845: Biological and Pharmacological Profiling

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) continues to be a global threat due to its ability to evolve and generate new subvariants, leading to new waves of infection. Additionally, other coronaviruses like Middle East respiratory syndrome coronavirus (MERS-CoV, formerly known as hCoV-EMC), which first emerged in 2012, persist and continue to present a threat of severe illness to humans. The continued identification of novel coronaviruses, coupled with the potential for genetic recombination between different strains, raises the possibility of new coronavirus clades of global concern emerging. As a result, there is a pressing need for pan-CoV therapeutic drugs and vaccines. After the extensive optimization of an HCV protease inhibitor screening hit, a novel 3CLPro inhibitor (MK-7845) was discovered and subsequently profiled. MK-7845 exhibited nanomolar in vitro potency with broad spectrum activity against a panel of clinical SARS-CoV-2 subvariants and MERS-CoV. Furthermore, when administered orally, MK-7845 demonstrated a notable reduction in viral burdens by >6 log orders in the lungs of transgenic mice infected with SARS-CoV-2 (K18-hACE2 mice) and MERS-CoV (K18-hDDP4 mice)

Whole-genome sequencing of L638^R mutants.

<p>Heat map summary of all non-synonymous mutations identified by Illumina-based whole-genome sequencing (100X genome coverage) of L638^R mutants in MRSE CLB26329 (A) or MRSA COL (B). Red, non-synonymous mutation; grey, no change versus parental genome sequence; yellow, non-synonymous mutations in genes other than <i>mnaA</i>. Genome position, base pair change, and resulting amino acid residue substitution are highlighted. Note: with only one exception (<i>Δcap5P mnaA</i>_<i>Sa</i>^<i>D281Y</i>), no additional non-synonymous mutations besides the indicated <i>mnaA</i> mutation were identified in each of the drug resistant strains examined.</p

MRSA and MRSE MnaA LOF mutants are highly susceptible to imipenem in a murine thigh infection.

<p>Immune-suppressed CD-1 mice (5 per group) were challenged intramuscularly with the parental MRSA COL strain, MRSA <i>Δcap5P</i>, or MRSA <i>Δcap5P mnaA</i>_<i>Sa</i> LOF mutants (A) or with the parental MRSE strain versus <i>mnaA</i>_<i>Se</i>, <i>tarO</i>_<i>Se</i> and <i>tarA</i>_<i>Se</i> LOF mutants (B) and treated three times daily (TID) with imipenem (IPM). Thighs were harvested at 24hrs, homogenized and plated to determine CFU per thigh. (A) Restored efficacy of IPM (10 mg kg^-1) against MRSA <i>Δcap5P mnaA</i>_<i>Sa</i>^<i>P12L</i>, <i>Δcap5P mnaA</i>_<i>Sa</i>^<i>Y194</i>*, and <i>Δcap5P mnaA</i>_<i>Sa</i>^<i>D281Y</i>. Following IPM treatment, bacterial burden amongst mice infected with <i>Δcap5P mnaA</i>_<i>Sa</i>^<i>P12L</i>, <i>Δcap5P mnaA</i>_<i>Sa</i>^<i>Y194</i>*, and <i>Δcap5P mnaA</i>_<i>Sa</i>^<i>D281Y</i> is reduced approximately 2–3 log at 24 hrs versus those infected with MRSA COL or <i>Δcap5P</i> controls. * p<0.01 versus parent at 24 hr; $ p<0.05 versus respective 24 hr vehicle. (B) Restored efficacy of IPM (2.5 mg kg^-1) against MRSE <i>mnaA</i>, <i>tarO</i>, and <i>tarA</i> LOF mutants. Reduction in bacterial burden of mice infected with the <i>mnaA</i>_<i>Se</i>^<i>G171D</i> is comparable to those infected with <i>tarO</i>_<i>Se</i>^<i>G84</i>* or <i>tarA</i>_<i>Se</i>^<i>G129R</i> mutants, yielding an approximate 3 log reduction in 24 hr IPM treatment versus the wild-type control. Note, as MRSE CLB26329 is more susceptible to IPM than MRSA COL, its dose was reduced to 4-fold versus the MRSA efficacy study (A).</p

Mapping of MnaA LOF mutations into the MnaA crystal structure reveal key residues for substrate binding site stability and charge.

<p>(A) Overall MRSA COL MnaA crystal structure. The molecular surface is shown in grey. The protein is represented as a cartoon. In all figures one monomer is consistently colored in orange and the other in cyan, and the bound UDP molecules are shown as sticks, methyl groups colored in light blue. Nitrogen, oxygen and phosphor atoms are in blue, red or orange, respectively. (B,C,D,E) Comparison with the <i>M</i>. <i>jannaschii</i> structure in “opened” form (PDB 3NEQ) or “closed” form (PDB 3NES). Both structures are represented as ribbons, one monomer at a time, and UDP as sticks. (B) and (C) compares the opened form, in grey, with each monomer, while the superposition is with the closed form, in (D) and (E), drawn in purple. The RMS deviation in Cα positions are 1.6Å for 262 atoms, 1.6Å for 256 atoms, 1.5Å for 321 atoms, and 1.3Å for 336 atoms, for the superpositions in cartoon (B), (C), (D) and (E), respectively. (F) Mapping MRSE LOF mutants. Eight mutation sites are mapped onto the X-ray crystal structure of UDP bound MRSA COL MnaA. The allosteric site ligand UDP-GlcNAc was taken from the structure of UDP-GlcNAc bound <i>B</i>. <i>anthracis</i> 2-epimerase (PDB ID 3BEO). UDP and UDP-GlcNAc are displayed as thin lines with the carbon atoms colored in light blue. One monomer of MnaA dimer is colored in cyan and the other in white. The mutation sites are highlighted in stick. The carbon atoms of the wild-type residues are colored in yellow; those of the mutant residues are in green. (G) Mapping MRSA <i>mnaA</i> LOF mutants. LOF mutations isolated in MRSA COL MnaA are highlighted. All coloring as in (C), but for simplicity, only the original sequence is shown.</p