A focused fragment library targeting the antibiotic resistance enzyme - Oxacillinase-48: Synthesis, structural evaluation and inhibitor design

Abstract

β-Lactam antibiotics are of utmost importance when treating bacterial infections in the medical community. However, currently their utility is threatened by the emergence and spread of β-lactam resistance. The most prevalent resistance mechanism to β-lactam antibiotics is expression of β-lactamase enzymes. One way to overcome resistance caused by β-lactamases, is the development of β-lactamase inhibitors and today several β-lactamase inhibitors e.g. avibactam, are approved in the clinic. Our focus is the oxacillinase-48 (OXA-48), an enzyme reported to spread rapidly across the world and commonly identified in Escherichia coli and Klebsiella pneumoniae. To guide inhibitor design, we used diversely substituted 3-aryl and 3-heteroaryl benzoic acids to probe the active site of OXA-48 for useful enzyme-inhibitor interactions. In the presented study, a focused fragment library containing 49 3-substituted benzoic acid derivatives were synthesised and biochemically characterized. Based on crystallographic data from 33 fragment-enzyme complexes, the fragments could be classified into R or R binders by their overall binding conformation in relation to the binding of the R and R side groups of imipenem. Moreover, binding interactions attractive for future inhibitor design were found and their usefulness explored by the rational design and evaluation of merged inhibitors from orthogonally binding fragments. The best inhibitors among the resulting 3,5-disubstituted benzoic acids showed inhibitory potential in the low micromolar range (IC = 2.9 μM). For these inhibitors, the complex X-ray structures revealed non-covalent binding to Arg250, Arg214 and Tyr211 in the active site and the interactions observed with the mono-substituted fragments were also identified in the merged structures.

19F NMR Monitoring of Reversible Protein Post‐Translational Modifications: Class D β‐Lactamase Carbamylation and Inhibition

“…The electron density corresponding to the hydrated TFA label is represented as a 2mFo‐DFc map, contoured at 1σ. (C) Overlay of the structure of the imipenem‐derived OXA‐48 complex (yellow sticks; PDB 5QB4) with the structure of OXA‐48 T213C* (cartoon, blue and orange sticks). The crystallographically observed position of the TFA label in the overlay was adjusted to represent the expected movement of the label resulting from substrate or inhibitor binding, as observed through changes in the 19 F NMR spectrum (Figure B, 3 C).…”

“…Comparison of the OXA‐48 T213C* structure with the structure of a carbapenem‐derived OXA‐48 acyl‐enzyme complex implies the overlap of the TFA label and elements of the carbapenem acyl‐enzyme complex (Figure C). Consequently, the 19 F label must be displaced from this position following substrate or inhibitor binding, as manifested by changes in the 19 F NMR spectrum (Figure B, C).…”

“…Comparison of the OXA-48 T213C*s tructure with the structure of ac arbapenem-derived OXA-48a cyl-enzymec omplex [20] implies the overlap of the TFAlabel and elements of the carbapenem acyl-enzyme complex ( Figure 4C). Consequently,t he 19 Fl abel must be displaced fromt his position following substrate or inhibitor binding, as manifested by changes in the 19 FNMR spectrum ( Figure 2B,3 C).A sa bove,i ti su nclear whethert he observed chemical shift differences relate to conformational changes associated with inhibitor binding, or if other interactions also contribute to the observed differences.…”

“…However,s ince these differences are relatively minor,a nd as the carbamylation of the 19 F-labelled enzyme behavess imilarly to what wasp reviously observed for wild-type OXA-48, [5] the presence of the label does not appear to have am ajor impact on carbamylation and enzymatic activity,s uggesting that the positioning of the label in OXA-48 T213C* is dynamic, and does not obstruct access to the active site. [20] with the structure of OXA-48 T213C* (cartoon,blue and orange sticks). The crystallographicallyobserved position of the TFAl abel in the overlay was adjusted to representthe expectedm ovement of the labelr esulting from substrate or inhibitor binding, as observed through changesi n the 19 FNMR spectrum( Figure2B, 3C).…”

Treatment of Infections by OXA-48-Producing Enterobacteriaceae

“…It has a 33-fold higher inhibition efficiency than tazobactam and binds OXA-48 very effectively (44). A focused fragment library has been set up to examine the in vitro inhibitory activity of 49 benzoic acid derivative fragments against OXA-48 as well as to observe their binding pockets and conformations (45). This approach shows promise in identifying new molecules for building novel BLIs.…”