Structure-Based Ligand Design Targeting Pseudomonas aeruginosa 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. Two of these hits were subsequently modified to optimize interactions with three regions of the LpxA active site. This strategy ultimately led to the discovery of ligand L13, which had a KD of 3.0 µM. The results reveal new chemical scaffolds as potential LpxA inhibitors, important binding features for ligand optimization, and protein conformational changes in response to ligand binding. Specifically, they show that a tetrazole ring is well-accommodated in a small cleft formed between Met169, the "hydrophobic-ruler" and His156, both of which demonstrate significant conformational flexibility. Furthermore, we find that the acyl-chain binding pocket is the most tractable region of the active site for realizing affinity gains and, along with a neighboring patch of hydrophobic residues, preferentially binds aliphatic and aromatic groups. The results presented herein provide valuable chemical and structural information for future inhibitor discovery against this important antibacterial drug target.

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.

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 ß-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 ß-lactamases with highly similar active sites. Notably, compared to CTX-M-14, KPC-2 was more potently inhibited by hydrolyzed ß-lactam products (product inhibition), as well as by a series of novel tetrazole-based inhibitors selected from molecular docking against CTX-M-14. Together with complex crystal structures, these data suggest that the KPC-2 active site has an enhanced ability to form favorable interactions with substrates and small molecule ligands due to its increased hydrophobicity and flexibility. Such properties are even more pronounced in metallo-carbapenemases, such as NDM-1, which was also inhibited by some of the novel tetrazole compounds, including one displaying comparable low µM affinities against both KPC-2 and NDM-1. Our results suggest that carbapenemase activity confers an evolutionary advantage on producers via a broad ß-lactam substrate scope but also a mechanistic Achilles' heel that can be exploited for new inhibitor discovery. The complex structures demonstrate, for the first time, how noncovalent inhibitors can be engineered to simultaneously target both serine and metallo-carbapenemases. Despite the relatively modest activity of the current compounds, these studies also demonstrate that hydrolyzed products and tetrazole-based chemotypes can provide valuable starting points for broad-spectrum inhibitor discovery against carbapenemases.

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. Here we employed a known inhibitor scaffold to study π-stacking of diverse heteroarene substituents on the amide face of Gly238 in the cephalosporinases CTX-M-14 and CTX-M-27. Biochemical inhibition constants (K i) and biophysical binding constants (K d) were determined for nineteen new analogues against both enzymes, while multiple high-resolution co-crystal structures revealed remarkably consistent placement of the probe heteroarene on Gly238. The data presented support the predicted importance of opposing dipoles in amide-heteroarene interactions and should be useful for evaluating other theoretical predictions concerning these interactions.

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.

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.

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

We have previously shown that cationic-ß-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 ß-CD cavity. Cytotoxicity studies revealed that all of the cationic-ß-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. Complexes formed with pDNA at N/P ratios greater than 5 produced particles with diameters in the 100-170 nm range and ζ-potentials of 15-35 mV. Gel shift and heparin challenge experiments showed that the complexes are most stable at N/P ≥ 10, with adamantyl- and noradamantyl-modified complexes displaying the best resistance toward heparin-induced decomplexation. Disassembly rates of fluoresceinated-pDNA:CD(+):R-PVA-PEG-rhodamine complexes within HeLa cells showed a modest dependence on host:guest binding constant, with adamantyl-, noradamantyl-, and dodecyl-based complexes showing the highest loss in FRET efficiency 9 h after cellular exposure. These findings suggest that the host:guest binding constant has a significant impact on the colloidal stability in the presence of serum and cellular uptake efficiency, whereas endosomal disassembly and transfection performance of cationic-ß-CD:R-poly(vinyl alcohol)-poly(ethylene glycol) pendant polymer complexes appears to be controlled by the hydrolysis rates of the acetal grafts onto the PVA main chain.

Microfluidic Assembly of Cationic-ß-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. The complexes also show a flow rate-dependent stability, with particles formed at slower flow rates giving rise to more stable complexes as determined by heparin challenge. Our findings suggest that microfluidic reactors offer an attractive method for assembling reproducible, size-controlled complexes from multi-component transfection complex assemblies.

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. The cationic polyrotaxanes display cytotoxicity profiles that are >10(2)-fold lower than the commercial standard bPEI and gene silencing efficiencies that are comparable to those of both Lipofectamine 2000 and bPEI. Our findings suggest that the cationic polyrotaxanes display a size-activity relationship, wherein the higher molecular weight polyrotaxanes (PEG3,400 and 10,000) are able to condense and deliver siRNA better than the lower molecular weight material (PEG2,000).

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. The bPRTx(+) materials were shown to have gene-silencing efficiencies comparable to those of Lipofectamine 2000 (L2k) and bPEI, while displaying similar toxicity profiles. The unique structure of these polyrotaxanes allows them to effectively condense and complex siRNA into nanoparticles at much lower N/P ratios than L2k or bPEI. These findings suggest that bPRTx(+) may be useful materials for gene therapy applications.

Pendant polymer:amino-ß-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 ß-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 ß-CD derivatives before siRNA condensation or siRNA condensation with the cationic ß-CD derivatives prior to addition of Chol-PVA-PEG to engage host:guest complexation. The pendant polymer:amino-ß-CD:siRNA complexes were shown to form nanoparticles in the size range of 120-170 nm, with a slightly negative zeta potential. Cell viability studies in CHO-GFP cells shows that these materials have 10(3)-fold lower cytotoxicities than 25 kD bPEI, while maintaining gene-silencing efficiencies that are comparable to those of benchmark transfection reagents such as bPEI and Lipofectamine 2000. These results suggest that the degradable Chol-PVA-PEG polymer is able to self-assemble in the presence of siRNA and cationic-ß-CD to form nanoparticles that are an effective and low-toxicity vehicle for delivering siRNA cargo to target cells.