Mutation of the conserved Asp-Asp pair impairs the structure, function, and inhibition of CTX-M Class A ß-lactamase.

The Asp233-Asp246 pair is highly conserved in Class A ß-lactamases, which hydrolyze ß-lactam antibiotics. Here, we characterize its function using CTX-M-14 ß-lactamase. The D233N mutant displayed decreased activity that is substrate-dependent, with reductions in kcat /Km ranging from 20% for nitrocefin to 6-fold for cefotaxime. In comparison, the mutation reduced the binding of a known reversible inhibitor by 10-fold. The mutant structures showed movement of the 213-219 loop and the loss of the Thr216-Thr235 hydrogen bond, which was restored by inhibitor binding. Mutagenesis of Thr216 further highlighted its contribution to CTX-M activity. These results demonstrate the importance of the aspartate pair to CTX-M hydrolysis of substrates with bulky side chains, while suggesting increased protein flexibility as a means to evolve drug resistance.

Antibacterial Spectrum of a Tetrazole-Based Reversible Inhibitor of Serine ß-Lactamases.

CTX-M is the most prevalent family of extended-spectrum ß-lactamases. We recently developed a tetrazole-derived noncovalent inhibitor of CTX-M-9. Here, we present the biochemical and microbiological activity of this inhibitor across a representative panel of serine ß-lactamases and Gram-negative bacteria. The compound displayed significant activity against all major subgroups of CTX-M, including CTX-M-15, while it exhibited some low-level inhibition of other serine ß-lactamases. Complex crystal structures with the CTX-M-14 S237A mutant and CTX-M-27 illustrate the binding contribution of specific active-site residues on the ß3 strand. In vitro pharmacokinetic studies revealed drug-like properties and positive prospects for further optimization. These studies suggest that tetrazole-based compounds can provide novel chemotypes for future serine ß-lactamase inhibitor discovery.

Correction to: Design and validation of an osteochondral bioreactor for the screening of treatments for osteoarthritis.

The original version of this article unfortunately contained a mistake.

Design and validation of an osteochondral bioreactor for the screening of treatments for osteoarthritis.

Bioreactors are systems that can be used to monitor the response of tissues and cells to candidate drugs. Building on the experience developed in the creation of an osteochondral bioreactor, we have designed a new 3D printed system, which allows optical access to the cells throughout testing for in line monitoring. Because of the use of 3D printing, the fluidics could be developed in the third dimension, thus maintaining the footprint of a single well of a typical 96 well plate. This new design was optimized to achieve the maximum fluid transport through the central chamber, which corresponds to optimal nutrient or drug exposure. This optimization was achieved by altering each dimension of the bioreactor fluid path. A physical model for optimized drug exposure was then created and tested.

Structural and Mechanistic Analysis of Drosophila melanogaster Agmatine N-Acetyltransferase, an Enzyme that Catalyzes the Formation of N-Acetylagmatine.

Agmatine N-acetyltransferase (AgmNAT) catalyzes the formation of N-acetylagmatine from acetyl-CoA and agmatine. Herein, we provide evidence that Drosophila melanogaster AgmNAT (CG15766) catalyzes the formation of N-acetylagmatine using an ordered sequential mechanism; acetyl-CoA binds prior to agmatine to generate an AgmNAT•acetyl-CoA•agmatine ternary complex prior to catalysis. Additionally, we solved a crystal structure for the apo form of AgmNAT with an atomic resolution of 2.3 Å, which points towards specific amino acids that may function in catalysis or active site formation. Using the crystal structure, primary sequence alignment, pH-activity profiles, and site-directed mutagenesis, we evaluated a series of active site amino acids in order to assign their functional roles in AgmNAT. More specifically, pH-activity profiles identified at least one catalytically important, ionizable group with an apparent pKa of ~7.5, which corresponds to the general base in catalysis, Glu-34. Moreover, these data led to a proposed chemical mechanism, which is consistent with the structure and our biochemical analysis of AgmNAT.

Ligand-Induced Proton Transfer and Low-Barrier Hydrogen Bond Revealed by X-ray Crystallography.

Ligand binding can change the pKa of protein residues and influence enzyme catalysis. Herein, we report three ultrahigh resolution X-ray crystal structures of CTX-M ß-lactamase, directly visualizing protonation state changes along the enzymatic pathway: apo protein at 0.79 Å, precovalent complex with nonelectrophilic ligand at 0.89 Å, and acylation transition state (TS) analogue at 0.84 Å. Binding of the noncovalent ligand induces a proton transfer from the catalytic Ser70 to the negatively charged Glu166, and the formation of a low-barrier hydrogen bond (LBHB) between Ser70 and Lys73, with a length of 2.53 Å and the shared hydrogen equidistant from the heteroatoms. QM/MM reaction path calculations determined the proton transfer barrier to be 1.53 kcal/mol. The LBHB is absent in the other two structures although Glu166 remains neutral in the covalent complex. Our data represents the first X-ray crystallographic example of a hydrogen engaged in an enzymatic LBHB, and demonstrates that desolvation of the active site by ligand binding can provide a protein microenvironment conducive to LBHB formation. It also suggests that LBHBs may contribute to stabilization of the TS in general acid/base catalysis together with other preorganized features of enzyme active sites. These structures reconcile previous experimental results suggesting alternatively Glu166 or Lys73 as the general base for acylation, and underline the importance of considering residue protonation state change when modeling protein-ligand interactions. Additionally, the observation of another LBHB (2.47 Å) between two conserved residues, Asp233 and Asp246, suggests that LBHBs may potentially play a special structural role in proteins.

Fragment-based inhibitor discovery against ß-lactamase.

The production of ß-lactamase is one of the primary resistance mechanisms used by Gram-negative bacterial pathogens to counter ß-lactam antibiotics, such as penicillins, cephalosporins and carbapenems. There is an urgent need to develop novel ß-lactamase inhibitors in response to ever evolving ß-lactamases possessing an expanded spectrum of ß-lactam hydrolyzing activity. Whereas traditional high-throughput screening has proven ineffective against serine ß-lactamases, fragment-based approaches have been successfully employed to identify novel chemical matter, which in turn has revealed much about the specific molecular interactions possible in the active site of serine and metallo ß-lactamases. In this review, we summarize recent progress in the field, particularly: the identification of novel inhibitor chemotypes through fragment-based screening; the use of fragment-protein structures to understand key features of binding hot spots and inform the design of improved leads; lessons learned and new prospects for ß-lactamase inhibitor development using fragment-based approaches.

Structure-based design of potent and ligand-efficient inhibitors of CTX-M class A ß-lactamase.

The emergence of CTX-M class A extended-spectrum ß-lactamases poses a serious health threat to the public. We have applied structure-based design to improve the potency of a novel noncovalent tetrazole-containing CTX-M inhibitor (K(i) = 21 µM) more than 200-fold via structural modifications targeting two binding hot spots, a hydrophobic shelf formed by Pro167 and a polar site anchored by Asp240. Functional groups contacting each binding hot spot independently in initial designs were later combined to produce analogues with submicromolar potencies, including 6-trifluoromethyl-3H-benzoimidazole-4-carboxylic acid [3-(1H-tetrazol-5-yl)-phenyl]-amide, which had a K(i) value of 89 nM and reduced the MIC of cefotaxime by 64-fold in CTX-M-9 expressing Escherichia coli . The in vitro potency gains were accompanied by improvements in ligand efficiency (from 0.30 to 0.39) and LipE (from 1.37 to 3.86). These new analogues represent the first nM-affinity noncovalent inhibitors of a class A ß-lactamase. Their complex crystal structures provide valuable information about ligand binding for future inhibitor design.