Preclinical safety and efficacy characterization of an LpxC inhibitor against Gram-negative pathogens.

The UDP-3--(-3-hydroxyacyl)--acetylglucosamine deacetylase LpxC is an essential enzyme in the biosynthesis of lipid A, the outer membrane anchor of lipopolysaccharide and lipooligosaccharide in Gram-negative bacteria. The development of LpxC-targeting antibiotics toward clinical therapeutics has been hindered by the limited antibiotic profile of reported non-hydroxamate inhibitors and unexpected cardiovascular toxicity observed in certain hydroxamate and non-hydroxamate-based inhibitors. Here, we report the preclinical characterization of a slow, tight-binding LpxC inhibitor, LPC-233, with low picomolar affinity.

Curative Treatment of Severe Gram-Negative Bacterial Infections by a New Class of Antibiotics Targeting LpxC.

The infectious diseases caused by multidrug-resistant bacteria pose serious threats to humankind. It has been suggested that an antibiotic targeting LpxC of the lipid A biosynthetic pathway in Gram-negative bacteria is a promising strategy for curing Gram-negative bacterial infections. However, experimental proof of this concept is lacking.

High susceptibility of MDR and XDR Gram-negative pathogens to biphenyl-diacetylene-based difluoromethyl-allo-threonyl-hydroxamate LpxC inhibitors.

Objectives: Inhibitors of uridine diphosphate-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC, which catalyses the first, irreversible step in lipid A biosynthesis) are a promising new class of antibiotics against Gram-negative bacteria. The objectives of the present study were to: (i) compare the antibiotic activities of three LpxC inhibitors (LPC-058, LPC-011 and LPC-087) and the reference inhibitor CHIR-090 against Gram-negative bacilli (including MDR and XDR isolates); and (ii) investigate the effect of combining these inhibitors with conventional antibiotics.

Methods: MICs were determined for 369 clinical isolates (234 Enterobacteriaceae and 135 non-fermentative Gram-negative bacilli).

A Scalable Synthesis of the Difluoromethyl-allo-threonyl Hydroxamate-Based LpxC Inhibitor LPC-058.

The difluoromethyl-allo-threonyl hydroxamate-based compound LPC-058 is a potent inhibitor of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) in Gram-negative bacteria. A scalable synthesis of this compound is described. The key step in the synthetic sequence is a transition metal/base-catalyzed aldol reaction of methyl isocyanoacetate and difluoroacetone, giving rise to 4-(methoxycarbonyl)-5,5-disubstituted 2-oxazoline.

Drug design from the cryptic inhibitor envelope.

Conformational dynamics plays an important role in enzyme catalysis, allosteric regulation of protein functions and assembly of macromolecular complexes. Despite these well-established roles, such information has yet to be exploited for drug design. Here we show by nuclear magnetic resonance spectroscopy that inhibitors of LpxC--an essential enzyme of the lipid A biosynthetic pathway in Gram-negative bacteria and a validated novel antibiotic target--access alternative, minor population states in solution in addition to the ligand conformation observed in crystal structures.

Specific binding at the cellulose binding module-cellulose interface observed by force spectroscopy.

The need for effective enzymatic depolymerization of cellulose has stimulated an interest in interactions between protein and cellulose. Techniques utilized for quantitative measurements of protein-cellulose noncovalent association include microgravimetry, calorimetry, and atomic force microscopy (AFM), none of which differentiate between specific protein-cellulose binding and nonspecific adhesion. Here, we describe an AFM approach that differentiates nonspecific from specific interactions between cellulose-binding modules (CBMs) and cellulose.

Photo-activated psoralen binds the ErbB2 catalytic kinase domain, blocking ErbB2 signaling and triggering tumor cell apoptosis.

Photo-activation of psoralen with UVA irradiation, referred to as PUVA, is used in the treatment of proliferative skin disorders. The anti-proliferative effects of PUVA have been largely attributed to psoralen intercalation of DNA, which upon UV treatment, triggers the formation of interstrand DNA crosslinks (ICL) that inhibit transcription and DNA replication. Here, we show that PUVA exerts antitumor effects in models of human breast cancer that overexpress the ErbB2 receptor tyrosine kinase oncogene, through a new mechanism.

Structural basis of the promiscuous inhibitor susceptibility of Escherichia coli LpxC.

The LpxC enzyme in the lipid A biosynthetic pathway is one of the most promising and clinically unexploited antibiotic targets for treatment of multidrug-resistant Gram-negative infections. Progress in medicinal chemistry has led to the discovery of potent LpxC inhibitors with a variety of chemical scaffolds and distinct antibiotic profiles. The vast majority of these compounds, including the nanomolar inhibitors L-161,240 and BB-78485, are highly effective in suppressing the activity of Escherichia coli LpxC (EcLpxC) but not divergent orthologs such as Pseudomonas aeruginosa LpxC (PaLpxC) in vitro.

Synthesis, structure, and antibiotic activity of aryl-substituted LpxC inhibitors.

The zinc-dependent deacetylase LpxC catalyzes the committed step of lipid A biosynthesis in Gram-negative bacteria and is a validated target for the development of novel antibiotics to combat multidrug-resistant Gram-negative infections. Many potent LpxC inhibitors contain an essential threonyl-hydroxamate headgroup for high-affinity interaction with LpxC. We report the synthesis, antibiotic activity, and structural and enzymatic characterization of novel LpxC inhibitors containing an additional aryl group in the threonyl-hydroxamate moiety, which expands the inhibitor-binding surface in LpxC.

Effect of compressive force on unbinding specific protein-ligand complexes with force spectroscopy.

Atomic force microscopy (AFM) is used extensively for the investigation of noncovalent molecular association. Although the technique is used to derive various types of information, in almost all instances the frequency of complex formation, the magnitude of rupture forces, and the shape of the force-distance curve are used to determine the behavior of the system. We have used AFM to consider the effect of contact force on the unbinding profiles of lactose-galectin-3, as well as the control pairs lactose-KDPG aldolase, and mannose-galectin-3, where the interacting species show negligible solution-phase affinity.

Enthalpic signature of methonium desolvation revealed in a synthetic host-guest system based on cucurbit[7]uril.

Methonium (N(+)Me3) is an organic cation widely distributed in biological systems. As an organic cation, the binding of methonium to protein receptors requires the removal of a positive charge from water. The appearance of methonium in biological transmitters and receptors seems at odds with the large unfavorable desolvation free energy reported for tetramethylammonium (TMA(+)), a frequently utilized surrogate of methonium.

An enthalpic basis of additivity in biphenyl hydroxamic acid ligands for stromelysin-1.

Fragment based drug discovery remains a successful tool for pharmaceutical lead discovery. Although based upon the principle of thermodynamic additivity, the underlying thermodynamic basis is poorly understood. A thermodynamic additivity analysis was performed using stromelysin-1 and a series of biphenyl hydroxamate ligands identified through fragment additivity.

Multicomponent patterning of indium tin oxide.

We report a versatile functionalization and pattering technique that permits multicomponent pattern-specific modification of indium tin oxide (ITO) with organic species. The method relies on a bilayered molecular system that simultaneously protects ITO from degradation and provides uniform chemical functionality suitable for further elaboration. Pattern-specific modification is achieved via specific reaction between functionality on an elastomeric stamp and functionality of cognate reactivity at the surface of a bilayered molecular substrate.

A general and efficient cantilever functionalization technique for AFM molecular recognition studies.

Atomic force microscopy (AFM) is a versatile technique for the investigation of noncovalent molecular associations between ligand-substrate pairs. Surface modification of silicon nitride AFM cantilevers is most commonly achieved using organic trialkoxysilanes. However, susceptibility of the Si−O bond to hydrolysis and formation of polymeric aggregates diminishes attractiveness of this method for AFM studies.

Improving upon nature: active site remodeling produces highly efficient aldolase activity toward hydrophobic electrophilic substrates.

The substrate specificity of enzymes is frequently narrow and constrained by multiple interactions, limiting the use of natural enzymes in biocatalytic applications. Aldolases have important synthetic applications, but the usefulness of these enzymes is hampered by their narrow reactivity profile with unnatural substrates. To explore the determinants of substrate selectivity and alter the specificity of Escherichia coli 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, we employed structure-based mutagenesis coupled with library screening of mutant enzymes localized to the bacterial periplasm.

Soft lithographic functionalization and patterning oxide-free silicon and germanium.

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques.

Derivatives of plant phenolic compound affect the type III secretion system of Pseudomonas aeruginosa via a GacS-GacA two-component signal transduction system.

Antibiotic therapy is the most commonly used strategy to control pathogenic infections; however, it has contributed to the generation of antibiotic-resistant bacteria. To circumvent this emerging problem, we are searching for compounds that target bacterial virulence factors rather than their viability. Pseudomonas aeruginosa, an opportunistic human pathogen, possesses a type III secretion system (T3SS) as one of the major virulence factors by which it secretes and translocates T3 effector proteins into human host cells.

Directed evolution of a pyruvate aldolase to recognize a long chain acyl substrate.

The use of biological catalysts for industrial scale synthetic chemistry is highly attractive, given their cost effectiveness, high specificity that obviates the need for protecting group chemistry, and the environmentally benign nature of enzymatic procedures. Here we evolve the naturally occurring 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolases from Thermatoga maritima and Escherichia coli, into enzymes that recognize a nonfunctionalized electrophilic substrate, 2-keto-4-hydroxyoctonoate (KHO). Using an in vivo selection based on pyruvate auxotrophy, mutations were identified that lower the K(M) value up to 100-fold in E.

A multidisciplinary approach to probing enthalpy-entropy compensation and the interfacial mobility model.

In recent years, interfacial mobility has gained popularity as a model with which to rationalize both affinity in ligand binding and the often observed phenomenon of enthalpy-entropy compensation. While protein contraction and reduced mobility, as demonstrated by computational and NMR techniques respectively, have been correlated to entropies of binding for a variety of systems, to our knowledge, Raman difference spectroscopy has never been included in these analyses. Here, nonresonance Raman difference spectroscopy, isothermal titration calorimetry, and X-ray crystallography were utilized to correlate protein contraction, as demonstrated by an increase in protein interior packing and decreased residual protein movement, with trends of enthalpy-entropy compensation.

Lipooligosaccharide is required for the generation of infectious elementary bodies in Chlamydia trachomatis.

Lipopolysaccharides (LPS) and lipooligosaccharides (LOS) are the main lipid components of bacterial outer membranes and are essential for cell viability in most Gram-negative bacteria. Here we show that small molecule inhibitors of LpxC [UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase], the enzyme that catalyzes the first committed step in the biosynthesis of lipid A, block the synthesis of LOS in the obligate intracellular bacterial pathogen Chlamydia trachomatis. In the absence of LOS, Chlamydia remains viable and establishes a pathogenic vacuole ("inclusion") that supports robust bacterial replication.

Soft-lithographic approach to functionalization and nanopatterning oxide-free silicon.

We report a simple, reliable high-throughput method for patterning passivated silicon with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The approach completely protects silicon from chemical oxidation, provides precise control over the shape and size of the patterned features in the 100 nm domain, and gives rapid, ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules.

Patterning NHS-terminated SAMs on germanium.

Here we report a simple, robust approach to patterning functional SAMs on germanium. The protocol relies on catalytic soft-lithographic pattern transfer from an elastomeric stamp bearing pendant immobilized sulfonic acid moieties to an NHS-functionalized bilayer molecular system comprising a primary ordered alkyl monolayer and a reactive ester secondary overlayer. The catalytic polyurethane-acrylate stamp was used to form micrometer-scale features of chemically distinct SAMs on germanium.

Syntheses, structures and antibiotic activities of LpxC inhibitors based on the diacetylene scaffold.

Compounds inhibiting LpxC in the lipid A biosynthetic pathway are promising leads for novel antibiotics against multidrug-resistant Gram-negative pathogens. We report the syntheses and structural and biochemical characterizations of LpxC inhibitors based on a diphenyl-diacetylene (1,4-diphenyl-1,3-butadiyne) threonyl-hydroxamate scaffold. These studies provide a molecular interpretation for the differential antibiotic activities of compounds with a substituted distal phenyl ring as well as the absolute stereochemical requirement at the C2, but not C3, position of the threonyl group.