Engineering DUB-deficient Viral Proteases from FIPV and PEDV Coronaviruses

Coronaviruses form a class of viral pathogens lethal to humans and livestock. This issue is compounded by a lack of commercially available treatments or vaccines. In 2014, porcine epidemic diarrhea virus (PEDV) emerged in the United States and accounted for an estimated 7 million porcine deaths. Deaths of humans, companion animals, and livestock caused by coronaviruses highlight the need for therapeutic strategies to combat this devastating disease. One strategy involves engineering papain-like protease 2 (PLP2), an enzyme conserved among coronavirus species that is critical for virus replication and pathogenesis. PLP2’s de-ubiquitinating (DUB) activity aids in the suppression of the host’s innate antiviral immune response. By targeting and disrupting ubiquitin binding in PLP2 and thus its DUB activity, the virus would no longer be able to antagonize the innate immune response. To this end, we introduced informed single-point mutations in PEDV and in feline infectious peritonitis virus (FIPV) PLP2s using structure-guided mutagenesis. We then characterized the kinetic activity of the resulting mutants in vitro using fluorescent peptide and ubiquitin substrates. Through these studies, we were able to evaluate the relationship between PLP2-ubiquitin binding and DUB activity. Preliminary data analysis suggests that residues outside the active site of PLP2 and within the ubiquitin-binding interface are necessary for DUB activity; these residues can be selectively disrupted to abolish DUB activity relative to the wild-type. These results describe a series of DUB-deficient PLP2 mutants that can be leveraged as tools for use in future coronavirus research. Such tools will allow creation of an attenuated virus strain that could aid in vaccine and drug design

Acyl-intermediate Structures of an Extended Spectrum Clinically-Derived Class D β-lactamase Variant, OXA-160, in Complex with Cefotaxime, Ceftazidime, and Aztreonam

OXA-24 is a carbapenem-hydrolyzing class D β-lactamase (CHDL) that poses a serious medical threat by destroying carbapenem class antibiotics. OXA-160 is a clinically-derived OXA-24 variant with a Pro→Ser substitution. Previously, it was shown that OXA-160 has higher catalytic activity against third-generation cephalosporins compared to OXA-24 and is able to maintain normal activity against penicillins and carbapenems. To slow deacylation, we introduced a second mutation (Val130Asp) to allow us to capture a drug-complex structure. We examined the OXA-160/Val130Asp variant in complex with the substrates cefotaxime, ceftazidime, and aztreonam using X-ray crystallography. Our analysis shows that all three of these bulky antibiotics require β5-β6 and/or omega loop deviations, and we propose that these conformational changes are made possible by replacing the restricted proline with the more flexible serine. These crystallographic structures reveal that a Pro227Ser mutation enlarges the active site, better accommodating advanced cephalosporin drugs

Structural Basis of Activity against Aztreonam and Extended Spectrum Cephalosporins for Two Carbapenem-Hydrolyzing Class D β‑Lactamases from <i>Acinetobacter baumannii</i>

The carbapenem-hydrolyzing class D β-lactamases OXA-23 and OXA-24/40 have emerged worldwide as causative agents for β-lactam antibiotic resistance in <i>Acinetobacter</i> species. Many variants of these enzymes have appeared clinically, including OXA-160 and OXA-225, which both contain a P → S substitution at homologous positions in the OXA-24/40 and OXA-23 backgrounds, respectively. We purified OXA-160 and OXA-225 and used steady-state kinetic analysis to compare the substrate profiles of these variants to their parental enzymes, OXA-24/40 and OXA-23. OXA-160 and OXA-225 possess greatly enhanced hydrolytic activities against aztreonam, ceftazidime, cefotaxime, and ceftriaxone when compared to OXA-24/40 and OXA-23. These enhanced activities are the result of much lower <i>K</i>_m values, suggesting that the P → S substitution enhances the binding affinity of these drugs. We have determined the structures of the acylated forms of OXA-160 (with ceftazidime and aztreonam) and OXA-225 (ceftazidime). These structures show that the R1 oxyimino side-chain of these drugs occupies a space near the β5-β6 loop and the omega loop of the enzymes. The P → S substitution found in OXA-160 and OXA-225 results in a deviation of the β5-β6 loop, relieving the steric clash with the R1 side-chain carboxypropyl group of aztreonam and ceftazidime. These results reveal worrying trends in the enhancement of substrate spectrum of class D β-lactamases but may also provide a map for β-lactam improvement