A covalent inhibitor targeting the papain-like protease from SARS-CoV-2 inhibits viral replication.

Covalent inhibitors of the papain-like protease (PLpro) from SARS-CoV-2 have great potential as antivirals, but their non-specific reactivity with thiols has limited their development. In this report, we performed an 8000 molecule electrophile screen against PLpro and identified an α-chloro amide fragment, termed compound 1, which inhibited SARS-CoV-2 replication in cells, and also had low non-specific reactivity with thiols. Compound 1 covalently reacts with the active site cysteine of PLpro, and had an IC50 of 18 µM for PLpro inhibition. Compound 1 also had low non-specific reactivity with thiols and reacted with glutathione 1-2 orders of magnitude slower than other commonly used electrophilic warheads. Finally, compound 1 had low toxicity in cells and mice and has a molecular weight of only 247 daltons and consequently has great potential for further optimization. Collectively, these results demonstrate that compound 1 is a promising lead fragment for future PLpro drug discovery campaigns.

A self-immolative linker that releases thiols detects penicillin amidase and nitroreductase with high sensitivity via absorption spectroscopy.

This article reports the synthesis and characterization of a novel self-immolative linker, based on thiocarbonates, which releases a free thiol upon activation via enzymes. We demonstrate that thiocarbonate self-immolative linkers can be used to detect the enzymes penicillin G amidase (PGA) and nitroreductase (NTR) with high sensitivity using absorption spectroscopy. Paired with modern thiol amplification technology, the detection of PGA and NTR were achieved at concentrations of 160 nM and 52 nM respectively. In addition, the PGA probe was shown to be compatible with both biological thiols and enzymes present in cell lysates.

Dimensionless parameter predicts bacterial prodrug success.

Understanding mechanisms of antibiotic failure is foundational to combating the growing threat of multidrug-resistant bacteria. Prodrugs-which are converted into a pharmacologically active compound after administration-represent a growing class of therapeutics for treating bacterial infections but are understudied in the context of antibiotic failure. We hypothesize that strategies that rely on pathogen-specific pathways for prodrug conversion are susceptible to competing rates of prodrug activation and bacterial replication, which could lead to treatment escape and failure. Here, we construct a mathematical model of prodrug kinetics to predict rate-dependent conditions under which bacteria escape prodrug treatment. From this model, we derive a dimensionless parameter we call the Bacterial Advantage Heuristic (BAH) that predicts the transition between prodrug escape and successful treatment across a range of time scales (1-104 h), bacterial carrying capacities (5 × 104 -105 CFU/µl), and Michaelis constants (KM = 0.747-7.47 mM). To verify these predictions in vitro, we use two models of bacteria-prodrug competition: (i) an antimicrobial peptide hairpin that is enzymatically activated by bacterial surface proteases and (ii) a thiomaltose-conjugated trimethoprim that is internalized by bacterial maltodextrin transporters and hydrolyzed by free thiols. We observe that prodrug failure occurs at BAH values above the same critical threshold predicted by the model. Furthermore, we demonstrate two examples of how failing prodrugs can be rescued by decreasing the BAH below the critical threshold via (i) substrate design and (ii) nutrient control. We envision such dimensionless parameters serving as supportive pharmacokinetic quantities that guide the design and administration of prodrug therapeutics.

Maltohexaose-indocyanine green (MH-ICG) for near infrared imaging of endocarditis.

Infectious endocarditis is a life-threatening disease, and diagnostics are urgently needed to accurately diagnose this disease especially in the case of prosthetic valve endocarditis. We show here that maltohexaose conjugated to indocyanine green (MH-ICG) can detect Staphylococcus aureus (S. aureus) infection in a rat model of infective endocarditis. The affinity of MH-ICG to S. aureus was determined and had a Km and Vmax of 5.4 µM and 3.0 X 10-6 µmol/minutes/108 CFU, respectively. MH-ICG had no detectable toxicity to mammalian cells at concentrations as high as 100 µM. The in vivo efficiency of MH-ICG in rats was evaluated using a right heart endocarditis model, and the accumulation of MH-ICG in the bacterial vegetations was 2.5 ± 0.2 times higher than that in the control left ventricular wall. The biological half-life of MH-ICG in healthy rats was 14.0 ± 1.3 minutes, and approximately 50% of injected MH-ICG was excreted into the feces after 24 hours. These data demonstrate that MH-ICG was internalized by bacteria with high specificity and that MH-ICG specifically accumulated in bacterial vegetations in a rat model of endocarditis. These results demonstrate the potential efficacy of this agent in the detection of infective endocarditis.