Unraveling the Gentamicin Drug Product Complexity Reveals Variation in Microbiological Activities and Nephrotoxicity.

The gentamicin drug product is a complex mixture of numerous components, many of which have not individually undergone safety and efficacy assessments. This is in contrast to the majority of medicines that require rigorous characterizations of trace impurities and are dosed as single components. In gentamicin, four components, known as gentamicin congeners C1, C1a, C2, and C2a, comprise the majority of the mixture.

Influence of the α-Methoxy Group on the Reaction of Temocillin with Pseudomonas aeruginosa PBP3 and CTX-M-14 β-Lactamase.

The prevalence of multidrug-resistant has led to the reexamination of older "forgotten" drugs, such as temocillin, for their ability to combat resistant microbes. Temocillin is the 6-α-methoxy analogue of ticarcillin, a carboxypenicillin with well-characterized antipseudomonal properties. The α-methoxy modification confers resistance to serine β-lactamases, yet temocillin is ineffective against growth.

Discovery of dual-activity small-molecule ligands of Pseudomonas aeruginosa LpxA and LpxD using SPR and X-ray crystallography.

The lipid A biosynthesis pathway is essential in Pseudomonas aeruginosa. LpxA and LpxD are the first and third enzymes in this pathway respectively, and are regarded as promising antibiotic targets. The unique structural similarities between these two enzymes make them suitable targets for dual-binding inhibitors, a characteristic that would decrease the likelihood of mutational resistance and increase cell-based activity.

Potent LpxC Inhibitors with Activity against Multidrug-Resistant Pseudomonas aeruginosa.

New drugs with novel mechanisms of resistance are desperately needed to address both community and nosocomial infections due to Gram-negative bacteria. One such potential target is LpxC, an essential enzyme that catalyzes the first committed step of lipid A biosynthesis. Achaogen conducted an extensive research campaign to discover novel LpxC inhibitors with activity against We report here the antibacterial activity and pharmacodynamics of ACHN-975, the only molecule from these efforts and the first ever LpxC inhibitor to be evaluated in phase 1 clinical trials.

Optimization and Mechanistic Characterization of Pyridopyrimidine Inhibitors of Bacterial Biotin Carboxylase.

A major challenge for new antibiotic discovery is predicting the physicochemical properties that enable small molecules to permeate Gram-negative bacterial membranes. We have applied physicochemical lessons from previous work to redesign and improve the antibacterial potency of pyridopyrimidine inhibitors of biotin carboxylase (BC) by up to 64-fold and 16-fold against and , respectively. Antibacterial and enzyme potency assessments in the presence of an outer membrane-permeabilizing agent or in efflux-compromised strains indicate that penetration and efflux properties of many redesigned BC inhibitors could be improved to various extents.

Optimization of LpxC Inhibitors for Antibacterial Activity and Cardiovascular Safety.

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a Zn deacetylase that is essential for the survival of most pathogenic Gram-negative bacteria. ACHN-975 (N-((S)-3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-(((1R,2R)-2-(hydroxymethyl)cyclopropyl)buta-1,3-diyn-1-yl)benzamide) was the first LpxC inhibitor to reach human clinical testing and was discovered to have a dose-limiting cardiovascular toxicity of transient hypotension without compensatory tachycardia. Herein we report the effort beyond ACHN-975 to discover LpxC inhibitors optimized for enzyme potency, antibacterial activity, pharmacokinetics, and cardiovascular safety.

Aminoglycoside Revival: Review of a Historically Important Class of Antimicrobials Undergoing Rejuvenation.

Aminoglycosides are cidal inhibitors of bacterial protein synthesis that have been utilized for the treatment of serious bacterial infections for almost 80 years. There have been approximately 15 members of this class approved worldwide for the treatment of a variety of infections, many serious and life threatening. While aminoglycoside use declined due to the introduction of other antibiotic classes such as cephalosporins, fluoroquinolones, and carbapenems, there has been a resurgence of interest in the class as multidrug-resistant pathogens have spread globally.

Tungstate as a Transition State Analog for Catalysis by Alkaline Phosphatase.

The catalytic mechanisms underlying Escherichia coli alkaline phosphatase's (AP) remarkable rate enhancement have been probed extensively. Past work indicated that whereas the serine nucleophile (Ser102) electrostatically repels the product phosphate, another oxyanion, tungstate, binds more strongly in the presence of Ser102. These results predict a covalent bond between the serine nucleophile and tungstate, a model that we test herein.

Structures of Pseudomonas aeruginosa LpxA Reveal the Basis for Its Substrate Selectivity.

In Gram-negative bacteria, the first step of lipid A biosynthesis is catalyzed by UDP-N-acetylglucosamine acyltransferase (LpxA) through the transfer of a R-3-hydroxyacyl chain from the acyl carrier protein (ACP) to the 3-hydroxyl group of UDP-GlcNAc. Previous studies suggest that LpxA is a critical determinant of the acyl chain length found in lipid A, which varies among species of bacteria. In Escherichia coli and Leptospira interrogans, LpxA prefers to incorporate longer R-3-hydroxyacyl chains (C14 and C12, respectively), whereas in Pseudomonas aeruginosa, the enzyme is selective for R-3-hydroxydecanoyl, a 10-hydrocarbon long acyl chain.

Probing the origins of catalytic discrimination between phosphate and sulfate monoester hydrolysis: comparative analysis of alkaline phosphatase and protein tyrosine phosphatases.

Catalytic promiscuity, the ability of enzymes to catalyze multiple reactions, provides an opportunity to gain a deeper understanding of the origins of catalysis and substrate specificity. Alkaline phosphatase (AP) catalyzes both phosphate and sulfate monoester hydrolysis reactions with a ∼10(10)-fold preference for phosphate monoester hydrolysis, despite the similarity between these reactions. The preponderance of formal positive charge in the AP active site, particularly from three divalent metal ions, was proposed to be responsible for this preference by providing stronger electrostatic interactions with the more negatively charged phosphoryl group versus the sulfuryl group.

Ground state destabilization by anionic nucleophiles contributes to the activity of phosphoryl transfer enzymes.

Enzymes stabilize transition states of reactions while limiting binding to ground states, as is generally required for any catalyst. Alkaline Phosphatase (AP) and other nonspecific phosphatases are some of Nature's most impressive catalysts, achieving preferential transition state over ground state stabilization of more than 10²²-fold while utilizing interactions with only the five atoms attached to the transferred phosphorus. We tested a model that AP achieves a portion of this preference by destabilizing ground state binding via charge repulsion between the anionic active site nucleophile, Ser102, and the negatively charged phosphate monoester substrate.

Isotope-edited FTIR of alkaline phosphatase resolves paradoxical ligand binding properties and suggests a role for ground-state destabilization.

Escherichia coli alkaline phosphatase (AP) can hydrolyze a variety of chemically diverse phosphate monoesters while making contacts solely to the transferred phosphoryl group and its incoming and outgoing atoms. Strong interactions between AP and the transferred phosphoryl group are not present in the ground state despite the apparent similarity of the phosphoryl group in the ground and transition states. Such modest ground-state affinity is required to curtail substrate saturation and product inhibition and to allow efficient catalysis.

Characterization of a novel bacterial arginine kinase from Desulfotalea psychrophila.

Phosphagen kinases are found throughout the animal kingdom and catalyze the transfer of a high-energy gamma phosphoryl-group from ATP to a guanidino group on a suitable acceptor molecule such as creatine or arginine. Recent genome sequencing efforts in several proteobacteria, including Desulfotalea psychrophila LSv54, Myxococcus xanthus, Sulfurovum sp. NBC37-1, and Moritella sp.