ABSTRACT: QPX7728 is a new ultrabroad-spectrum inhibitor of serine and metallo-beta-lactamases (MBLs) from a class of cyclic boronates that gave rise to vaborbactam. The spectrum and mechanism of beta-lactamase inhibition by QPX7728 were assessed using purified enzymes from all molecular classes. QPX7728 inhibits class A extended-spectrum beta-lactamases (ESBLs) (50% inhibitory concentration [IC50] range, 1 to 3?nM) and carbapenemases such as KPC (IC50, 2.9?±?0.4?nM) as well as class C P99 (IC50 of 22?±?8?nM) with a potency that is comparable to or higher than recently FDA-approved beta-lactamase inhibitors (BLIs) avibactam, relebactam, and vaborbactam. Unlike those other BLIs, QPX7728 is also a potent inhibitor of class D carbapenemases such as OXA-48 from Enterobacteriaceae and OXA enzymes from Acinetobacter baumannii (OXA-23/24/58, IC50 range, 1 to 2?nM) as well as MBLs such as NDM-1 (IC50, 55?±?25?nM), VIM-1 (IC50, 14?±?4?nM), and IMP-1 (IC50, 610?±?70?nM). Inhibition of serine enzymes by QPX7728 is associated with progressive inactivation with a high-efficiency k 2/K ranging from 6.3?×?104 (for P99) to 9.9?×?105 M-1 s-1 (for OXA-23). This inhibition is reversible with variable stability of the QPX7728-beta-lactamase complexes with target residence time ranging from minutes to several hours: 5 to 20 min for OXA carbapenemases from A. baumannii, ?50 min for OXA-48, and 2 to 3 h for KPC and CTX-M-15. QPX7728 inhibited all tested serine enzymes at a 1:1 molar ratio. Metallo-beta-lactamases NDM, VIM, and IMP were inhibited by a competitive mechanism with fast-on-fast-off kinetics, with Ki s of 7.5?±?2.1?nM, 32?±?14?nM, and 240?±?30?nM for VIM-1, NDM-1, and IMP-1, respectively. QPX7728's ultrabroad spectrum of BLI inhibition combined with its high potency enables combinations with multiple different beta-lactam antibiotics.