Novel insights into the mode of inhibition of class A SHV-1 beta-lactamases revealed by boronic acid transition state inhibitors

Boronic acid transition state inhibitors (BATSIs) are potent class A and C β-lactamase inactivators and are of particular interest due to their reversible nature mimicking the transition state. Here, we present structural and kinetic data describing the inhibition of the SHV-1 β-lactamase, a clinically important enzyme found in Klebsiella pneumoniae, by BATSI compounds possessing the R1 side chains of ceftazidime and cefoperazone and designed variants of the latter, compounds 1 and 2. The ceftazidime and cefoperazone BATSI compounds inhibit the SHV-1 β-lactamase with micromolar affinity that is considerably weaker than their inhibition of other β-lactamases. The solved crystal structures of these two BATSIs in complex with SHV-1 reveal a possible reason for SHV-1's relative resistance to inhibition, as the BATSIs adopt a deacylation transition state conformation compared to the usual acylation transition state conformation when complexed to other β-lactamases. Active-site comparison suggests that these conformational differences might be attributed to a subtle shift of residue A237 in SHV-1. The ceftazidime BATSI structure revealed that the carboxyl-dimethyl moiety is positioned in SHV-1's carboxyl binding pocket. In contrast, the cefoperazone BATSI has its R1 group pointing away from the active site such that its phenol moiety moves residue Y105 from the active site via end-on stacking interactions. To work toward improving the affinity of the cefoperazone BATSI, we synthesized two variants in which either one or two extra carbons were added to the phenol linker. Both variants yielded improved affinity against SHV-1, possibly as a consequence of releasing the strain of its interaction with the unusual Y105 conformation.     

Evaluation of updated interpretative criteria for categorizing Klebsiella pneumoniae with reduced carbapenem susceptibility

We studied the accuracy of various susceptibility testing methods, including the 2009, 2010, and updated 2010 CLSI recommendations, to identify Klebsiella pneumoniae isolates with reduced susceptibility to carbapenems associated with different mechanisms of resistance. Forty-three wild-type (WT) strains, 42 extended-spectrum β-lactamase (ESBL) producers, 18 ESBL producers with outer membrane porin protein loss (ESBL/Omp strains), and 42 blaKPC-possessing K. pneumoniae (KPC-Kp) isolates were evaluated. Imipenem (IPM), meropenem (MEM), ertapenem (ERT), and doripenem (DOR) were tested by broth microdilution (BMD), Etest, and disk diffusion (DD), and the modified Hodge test (MHT) was performed using IPM and MEM disks. Results were interpreted according to original as well as recently updated interpretative criteria. MHT was positive for all 42 KPC-Kp isolates and 10 of 18 ESBL/Omp strains and therefore had poor specificity in differentiating between KPC-Kp and ESBL/Omp isolates. Based on the updated CLSI standards, phenotypic susceptibility testing by BMD and DD differentiated most carbapenem-susceptible from carbapenem-nonsusceptible K. pneumoniae isolates without the need for MHT, while the Etest method characterized many KPC-Kp isolates as susceptible, and breakpoints may need to be lowered for this method. However, both the original and updated CLSI criteria do not adequately differentiate between isolates in the KPC-Kp group, which are unlikely to respond to carbapenem therapy, and those in the ESBL/Omp group, which are likely to respond to carbapenem therapy if MICs are within pharmacokinetic/pharmacodynamic targets. Further studies are required to determine if there is a clinical need to differentiate between KPC-Kp and ESBL/Omp groups.     

Age-related changes in orolingual motor function in F344 vs F344/BN rats

Normal aging is associated with both locomotor and orolingual motor deficits. Preclinical studies of motor function in normal aging, however, have focused primarily on locomotor activity. The purpose of this study was to measure age-related changes in orolingual motor function and compare these changes between two rat strains commonly used in aging studies: Fischer 344 (F344) and Fischer 344/Brown Norway hybrid (F344/BN) rats. Rats (6-, 12-, 18- and 24-months of age) were trained to lick water from an isometric force-sensing operandum so that the number of licks per session, licking rhythm (licks/second) and lick force could be measured. In both strains, the number of licks per session was greatest in the oldest group, while this measure was greater for F344/BN rats at all ages. Peak tongue force increased with age in F344/BN rats, did not change with age in the F344 rats, and was greater for the F344/BN rats at all ages. Both strains exhibited an age-related slowing of licking rhythm beginning with the 18-month-old group. These findings suggest that despite lifespan differences between these two rat strains, diminished tongue motility emerges at the same age.     

Crystal structures of KPC-2 β-lactamase in complex with 3-nitrophenyl boronic acid and the penam sulfone PSR-3-226

Class A carbapenemases are a major threat to the potency of carbapenem antibiotics. A widespread carbapenemase, KPC-2, is not easily inhibited by β-lactamase inhibitors (i.e., clavulanic acid, sulbactam, and tazobactam). To explore different mechanisms of inhibition of KPC-2, we determined the crystal structures of KPC-2 with two β-lactamase inhibitors that follow different inactivation pathways and kinetics. The first complex is that of a small boronic acid compound, 3-nitrophenyl boronic acid (3-NPBA), bound to KPC-2 with 1.62-Å resolution. 3-NPBA demonstrated a K_m value of 1.0 ± 0.1 μM (mean ± standard error) for KPC-2 and blocks the active site by making a reversible covalent interaction with the catalytic S70 residue. The two boron hydroxyl atoms of 3-NPBA are positioned in the oxyanion hole and the deacylation water pocket, respectively. In addition, the aromatic ring of 3-NPBA provides an edge-to-face interaction with W105 in the active site. The structure of KPC-2 with the penam sulfone PSR-3-226 was determined at 1.26-Å resolution. PSR-3-226 displayed a K_m value of 3.8 ± 0.4 μM for KPC-2, and the inactivation rate constant (k_inact) was 0.034 ± 0.003 s⁻¹. When covalently bound to S70, PSR-3-226 forms a trans-enamine intermediate in the KPC-2 active site. The predominant active site interactions are generated via the carbonyl oxygen, which resides in the oxyanion hole, and the carboxyl moiety of PSR-3-226, which interacts with N132, N170, and E166. 3-NPBA and PSR-3-226 are the first β-lactamase inhibitors to be trapped as an acyl-enzyme complex with KPC-2. The structural and inhibitory insights gained here could aid in the design of potent KPC-2 inhibitors.     

Inhibitor development after changing FVIII/IX products in patients with haemophilia

The retrospective observational study surveys the relationship between development of inhibitors in the treatment of haemophilia patients and risk factors such as changing FVIII products. A total of 119 patients were included in this study, 198 changes of FVIII products were evaluated. Results: During the observation period of 12 months none of the patients developed an inhibitor, which was temporally associated with a change of FVIII products. A frequent change of FVIII products didn't lead to an increase in inhibitor risk. The change between plasmatic and recombinant preparations could not be confirmed as a risk factor. Furthermore, no correlation between treatment regimens, severity, patient age and comorbidities of the patients could be found.     

Morphometric analysis of the second through fifth metacarpal through posteroanterior X-Rays

Over the past 10 years, metacarpal fractures have had an annual incidence of 13.6 per 10,000 individuals. Literature has not reviewed anatomical variations through radiographic imaging, which may play a role in reducing postoperative complications. The purpose of this study was to use radiographic imaging to provide a detailed anatomy of the second through fifth metacarpals. This retrospective study measured length, neck width, narrowest body width, and narrowest medullary canal width of the second through fifth metacarpals through the use of posteroanterior X-rays. Patients who were ≥18 years and received hand radiographs from January 2015 to July 2019 were included in this study. Those with acute injury or fracture of the metacarpal were excluded. Five hundred and seventy-two metacarpals were included in this study, with 143 metacarpals measured each for the second through fifth metacarpal. The second metacarpal had the largest measured length, neck width, and narrowest body width at 68.72, 12.34, and 8.74 mm, respectively. The fifth metacarpal had the greatest average medullary canal width at 4.15 mm. This is the largest study in literature to comprehensively examine the anatomical variation of the second through fifth metacarpals. The second metacarpal had greatest dimensions except for canal width, which was the fifth metacarpal. Men almost consistently had greater metacarpal size when compared to women, and age was associated with second and third metacarpal canal width. The increased knowledge of metacarpal anatomy may potentially lay the foundation of further improvement of metacarpal implants and potentially reduce postoperative complications. Clin. Anat., 33:1014–1018, 2020. © 2019 Wiley Periodicals, Inc.