Structure-Based Ligand Design Targeting LpxA in Lipid A Biosynthesis.

Enzymes involved in lipid A biosynthesis are promising antibacterial drug targets in Gram-negative bacteria. In this study, we use a structure-based design approach to develop a series of novel tetrazole ligands with low μM affinity for LpxA, the first enzyme in the lipid A pathway. Aided by previous structural data, X-ray crystallography, and surface plasmon resonance bioanalysis, we identify 17 hit compounds.

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

The Asp233-Asp246 pair is highly conserved in Class A β-lactamases, which hydrolyze β-lactam antibiotics. Here, we characterize its function using CTX-M-14 β-lactamase. The D233N mutant displayed decreased activity that is substrate-dependent, with reductions in k /K ranging from 20% for nitrocefin to 6-fold for cefotaxime.

An Empirical Study of Amide-Heteroarene π-Stacking Interactions Using Reversible Inhibitors of a Bacterial Serine Hydrolase.

Compared to aryl-aryl π-stacking interactions, the analogous stacking of heteroarenes on amide π systems is less well understood and vastly underutilized in structure-based drug design. Recent theoretical studies have delineated the important geometric coordinates of the interaction, some of which have been confirmed with synthetic model systems based on Rebek imides. Unfortunately, a broadly useful and tractable protein-ligand model system of this interaction has remained elusive.

Active-Site Druggability of Carbapenemases and Broad-Spectrum Inhibitor Discovery.

Serine and metallo-carbapenemases are a serious health concern due to their capability to hydrolyze nearly all β-lactam antibiotics. However, the molecular basis for their unique broad-spectrum substrate profile is poorly understood, particularly for serine carbapenemases, such as KPC-2. Using substrates and newly identified small molecules, we compared the ligand binding properties of KPC-2 with the noncarbapenemase CTX-M-14, both of which are Class A β-lactamases with highly similar active sites.

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

CTX-M is the most prevalent family of extended-spectrum β-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 β-lactamases and Gram-negative bacteria.

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 β-lactamase, directly visualizing protonation state changes along the enzymatic pathway: apo protein at 0.79 Å, precovalent complex with nonelectrophilic ligand at 0.

Microfluidic Assembly of Cationic-β-Cyclodextrin:Hyaluronic Acid-Adamantane Host:Guest pDNA Nanoparticles.

Traditionally, transfection complexes are typically formed by bulk mixing, producing particles with high polydispersity and limited control over vector size. Herein, we demonstrate the use of a commercial micro-reactor to assemble pDNA:cationic cyclodextrin:pendant polymer nanoparticles using a layer-by-layer approach. Our studies reveal that the particles formulated via microfluidic assembly have much smaller sizes, lower polydispersity, lower ζ-potentials, and comparable cell viability and transfection profiles in HeLa cells than bulk mixed particles.

Effect of pendant group on pDNA delivery by cationic-β-cyclodextrin:alkyl-PVA-PEG pendant polymer complexes.

We have previously shown that cationic-β-cyclodextrin:R-poly(vinyl alcohol)-poly(ethylene glycol) (CD+:R-PVA-PEG) pendant polymer host:guest complexes are safe and efficient vehicles for nucleic acid delivery, where R = benzylidene-linked adamantyl or cholesteryl esters. Herein, we report the synthesis and biological performance of a family of PVA-PEG pendant polymers whose pendant groups have a wide range of different affinities for the β-CD cavity. Cytotoxicity studies revealed that all of the cationic-β-CD:pendant polymer host:guest complexes have 100-1000-fold lower toxicity than branched polyethylenimine (bPEI), with pDNA transfection efficiencies that are comparable to bPEI and Lipofectamine 2000.

Cationic α-cyclodextrin:poly(ethylene glycol) polyrotaxanes for siRNA delivery.

RNA interference has broad therapeutic potential due to its high specificity and ability to potentially evade drug resistance. Three cationic α-cyclodextrin:poly(ethylene glycol) polyrotaxanes derived from polymer axles of different sizes (MW 2,000, 3,400, and 10,000) have been synthesized for delivering siRNA. These polyrotaxanes are able to condense siRNA into positively charged particles that are <200 nm in diameter, enabling their facile internalization into mammalian cells.

Multi-armed cationic cyclodextrin:poly(ethylene glycol) polyrotaxanes as efficient gene silencing vectors.

A family of branched polyrotaxanes (bPRTx(+)), threaded with multiple cationic α-cyclodextrins (α-CDs) onto a multi-armed poly(ethylene glycol) (PEG) core, were synthesized and studied as gene silencing vectors. These bPRTx(+) formed stable, positively charged complexes with diameters of 150-250 nm at N/P ratios as low as 2.5.

Pendant polymer:amino-β-cyclodextrin:siRNA guest:host nanoparticles as efficient vectors for gene silencing.

A novel siRNA delivery vector has been developed, based on the self-assembly of monosubstituted cationic β-CD derivatives with a poly(vinyl alcohol)MW27kD (PVA) main-chain polymer bearing poly(ethylene glycol)MW2000 (PEG) and acid-labile cholesterol-modified (Chol) grafts through an acid-sensitive benzylidene acetal linkage. These components were investigated for their ability to form nanoparticles with siRNA using two different assembly schemes, involving either precomplexation of the pendant Chol-PVA-PEG polymer with the cationic β-CD derivatives before siRNA condensation or siRNA condensation with the cationic β-CD derivatives prior to addition of Chol-PVA-PEG to engage host:guest complexation. The pendant polymer:amino-β-CD:siRNA complexes were shown to form nanoparticles in the size range of 120-170 nm, with a slightly negative zeta potential.