Oral bemnifosbuvir (AT-527) vs placebo in patients with mild-to-moderate COVID-19 in an outpatient setting (MORNINGSKY).

Aim: This phase III study assessed the efficacy/safety/antiviral activity/pharmacokinetics of bemnifosbuvir, a novel, oral nucleotide analog to treat COVID-19. Patients & methods: Outpatient adults/adolescents with mild-to-moderate COVID-19 were randomized 2:1 to bemnifosbuvir/placebo. Time to symptom alleviation/improvement (primary outcome), risk of hospitalization/death, viral load and safety were evaluated. Results: Although the study was discontinued prematurely and did not meet its primary end point, bemnifosbuvir treatment resulted in fewer hospitalizations (71% relative risk reduction), COVID-19-related medically attended hospital visits, and COVID-19-related complications compared with placebo. No reduction in viral load was observed. The proportion of patients with adverse events was similar; no deaths occurred. Conclusion: Bemnifosbuvir showed hospitalization reduction in patients with variable disease progression risk and was well tolerated. Clinical Trial Registration: NCT04889040 (ClinicalTrials.gov).

Benzothioxalone derivatives as novel inhibitors of UDP-N-acetylglucosamine enolpyruvyl transferases (MurA and MurZ).

OBJECTIVES: We sought to identify and characterize new inhibitors of MurA and MurZ, which are enzymes involved in the early stages of bacterial peptidoglycan synthesis. METHODS: A library of ∼650 000 compounds was screened for inhibitors of Escherichia coli MurA in an endpoint assay measuring release of inorganic phosphate from phosphoenolpyruvate. Hits were validated by determining the concentrations required for 50% inhibition (IC(50)) of MurA from E. coli and MurA/MurZ from Staphylococcus aureus. The mode of action of selected inhibitors was explored by examining the reversibility of MurA inhibition, the binding of a radiolabelled inhibitor to MurA proteins and through docking studies. Inhibitors were further characterized by determining their antibacterial activity against E. coli and S. aureus. RESULTS: Benzothioxalone derivatives were identified that inhibited MurA from E. coli and MurA/MurZ from S. aureus with IC(50) values between 0.25 and 51 µM. Several inhibitors also exhibited activity against S. aureus with MICs in the range 4-128 mg/L. Inhibition of MurA was irreversible and a radiolabelled inhibitor from this compound class displayed stoichiometric binding to the enzyme, which was displaced by dithiothreitol. Binding was undetectable with a C115D mutant MurA protein. CONCLUSIONS: The results suggest a mode of action for the benzothioxalones that involves the formation of a disulfide bond with MurA/MurZ, via attack from an active site cysteine on the thioxalone ring carbonyl group, followed by ring opening to yield an S-acylated protein. The proposed covalent mode of action may prove useful in the design of new antibacterial agents.

2-Aminotetralones: novel inhibitors of MurA and MurZ.

Several 2-aminotetralones were identified as novel inhibitors of the bacterial enzymes MurA and MurZ. A number of these inhibitors demonstrated antibacterial activity against Staphylococcus aureus and Escherichia coli with MICs in the range 8-128 microg/ml. Based on structure-activity relationships we propose that the alpha-aminoketone functionality is responsible for the inhibitory activity and evidence is provided to support a covalent mode of action involving the C115 thiol group of MurA/MurZ.

Antistaphylococcal activity of the novel cephalosporin CB-181963 (CAB-175).

OBJECTIVES: We examined the antistaphylococcal activity of the novel cephalosporin CB-181963 (formerly known as CAB-175), with emphasis on its microbiological activity and penicillin-binding protein specificities. METHODS: Using established procedures, we examined the activity of CB-181963 against methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) strains of Staphylococcus aureus in both planktonic and biofilm culture. We also determined whether CB-181963 exhibited a post-antibiotic effect (PAE). A radioactive competition assay with (3)H-labelled benzylpenicillin was used to determine penicillin-binding protein (PBP) affinities of CB-181963, including binding to PBP2a from MRSA. The potential for emergence of CB-181963-resistant mutants in MSSA and MRSA strains was examined using plating procedures. RESULTS: CB-181963 showed excellent activity against MRSA strains resistant to other cephalosporins in both planktonic and biofilm cultures. However, in common with other cephalosporins it was unable to eradicate biofilms. CB-181963 had a short PAE compared with other beta-lactam antibiotics. CB-181963 retained activity against a strain expressing type A beta-lactamase and demonstrated affinity for PBP2a of MRSA. Mutants resistant to CB-181963 were not recovered in either MSSA or MRSA. CONCLUSIONS: CB-181963 is a potent antistaphylococcal agent with better activity against MRSA than other cephalosporins. The anti-MRSA activity is correlated with elevated binding to PBP2a. CB-181963 may have a role in the treatment of staphylococcal infections, including those caused by MRSA and in the prophylaxis of biofilm-associated MSSA and MRSA infections. However, because of its short PAE, CB-181963 may have to be administered more frequently than other beta-lactam antibiotics, or given via prolonged infusion.

Assessment of a microplate method for determining the post-antibiotic effect in Staphylococcus aureus and Escherichia coli.

OBJECTIVES: The post-antibiotic effect (PAE) is an important parameter of antibiotic action that is widely used as a predictor of pharmacodynamic activity. Traditionally, PAE has been determined by a labour-intensive method involving determination of viable cell numbers. New methods using spectrophotometric procedures could offer significant advantages for PAE determinations, particularly in terms of speed. A number of such methods have been described in the literature, but extensive comparison with the classical procedure for determining PAEs has not been carried out. We have now compared PAE values obtained using a rapid microplate method with those achieved by the classical viable count procedure. METHODS: We determined PAE values for a variety of antibiotics against Staphylococcus aureus and Escherichia coli following exposure to 5 x MIC drug concentrations for 60 min in Mueller-Hinton Broth (MHB). The duration of the PAE was obtained by following the recovery of bacterial growth in antibiotic-free MHB measured either as colony forming units on Mueller-Hinton agar, or as culture absorbance (600 nm) in a microplate reader. RESULTS: For bacteriolytic agents there was poor correlation between the two methods for both S. aureus (R2=0.096) and E. coli (R2=0.5456). However, when PAEs for bacteriostatic agents and non-lytic bactericidal agents were compared, correlation between the two methods was high for both S. aureus (R2=0.7529) and E. coli (R2=0.7687). CONCLUSIONS: The spectrophotometric microplate method for determining PAEs may be a suitable alternative to the classical method for those antibiotics that do not induce bacterial cell lysis.