ABSTRACT
Many drug candidates fail in clinical trials owing to a lack of efficacy from limited target engagement or an insufficient therapeutic index. Minimizing off-target effects while retaining the desired pharmacodynamic (PD) response can be achieved by reduced exposure for drugs that display kinetic selectivity in which the drug-target complex has a longer half-life than off-target-drug complexes. However, though slow-binding inhibition kinetics are a key feature of many marketed drugs, prospective tools that integrate drug-target residence time into predictions of drug efficacy are lacking, hindering the integration of drug-target kinetics into the drug discovery cascade. Here we describe a mechanistic PD model that includes drug-target kinetic parameters, including the on- and off-rates for the formation and breakdown of the drug-target complex. We demonstrate the utility of this model by using it to predict dose response curves for inhibitors of the LpxC enzyme from Pseudomonas aeruginosa in an animal model of infection.
Subject(s)
Amidohydrolases/antagonists & inhibitors , Anti-Bacterial Agents/pharmacology , Enzyme Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Threonine/analogs & derivatives , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacokinetics , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Hydroxamic Acids/chemistry , Hydroxamic Acids/pharmacokinetics , Kinetics , Mice, Inbred Strains , Microbial Sensitivity Tests , Models, Biological , Molecular Structure , Protein Binding , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/enzymology , Threonine/chemistry , Threonine/pharmacokinetics , Threonine/pharmacology , Time FactorsABSTRACT
UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is the first committed step to form lipid A, an essential component of the outer membrane of Gram-negative bacteria. As it is essential for the survival of many pathogens, LpxC is an attractive target for antibacterial therapeutics. Herein, we report the product-bound co-crystal structure of LpxC from the acheal Aquifex aeolicus solved to 1.6 Å resolution. We identified interactions by hydroxyl and hydroxymethyl substituents of the product glucosamine ring that may enable new insights to exploit waters in the active site for structure-based design of LpxC inhibitors with novel scaffolds. By using this product structure, we have performed quantum mechanical modeling on the substrate in the active site. Based on our results and published experimental data, we propose a new mechanism that may lead to a better understanding of LpxC catalysis and inhibition.
Subject(s)
Amidohydrolases/chemistry , Bacteria/enzymology , Bacterial Proteins/chemistry , Protein Structure, Tertiary , Acetylation , Amidohydrolases/metabolism , Bacterial Proteins/metabolism , Biocatalysis , Catalytic Domain , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Glucosamine/chemistry , Glucosamine/metabolism , Lipid A/biosynthesis , Models, Molecular , Molecular Structure , Protein Binding , Quantum Theory , Substrate Specificity , Zinc/chemistry , Zinc/metabolismABSTRACT
Avibactam is a ß-lactamase inhibitor that is in clinical development, combined with ß-lactam partners, for the treatment of bacterial infections comprising gram-negative organisms. Avibactam is a structural class of inhibitor that does not contain a ß-lactam core but maintains the capacity to covalently acylate its ß-lactamase targets. Using the TEM-1 enzyme, we characterized avibactam inhibition by measuring the on-rate for acylation and the off-rate for deacylation. The deacylation off-rate was 0.045 min(-1), which allowed investigation of the deacylation route from TEM-1. Using NMR and MS, we showed that deacylation proceeds through regeneration of intact avibactam and not hydrolysis. Other than TEM-1, four additional clinically relevant ß-lactamases were shown to release intact avibactam after being acylated. We showed that avibactam is a covalent, slowly reversible inhibitor, which is a unique mechanism of inhibition among ß-lactamase inhibitors.
Subject(s)
Anti-Bacterial Agents/pharmacology , Azabicyclo Compounds/pharmacology , Gram-Negative Bacterial Infections/drug therapy , beta-Lactamase Inhibitors , Acylation/drug effects , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Azabicyclo Compounds/chemistry , Azabicyclo Compounds/metabolism , Drug Discovery/methods , Kinetics , Magnetic Resonance Spectroscopy , Mass Spectrometry , Molecular Structure , beta-LactamasesABSTRACT
Avibactam is a non-ß-lactam ß-lactamase inhibitor with a spectrum of activity that includes ß-lactamase enzymes of classes A, C, and selected D examples. In this work acylation and deacylation rates were measured against the clinically important enzymes CTX-M-15, KPC-2, Enterobacter cloacae AmpC, Pseudomonas aeruginosa AmpC, OXA-10, and OXA-48. The efficiency of acylation (k2/Ki) varied across the enzyme spectrum, from 1.1 × 10(1) m(-1)s(-1) for OXA-10 to 1.0 × 10(5) for CTX-M-15. Inhibition of OXA-10 was shown to follow the covalent reversible mechanism, and the acylated OXA-10 displayed the longest residence time for deacylation, with a half-life of greater than 5 days. Across multiple enzymes, acyl enzyme stability was assessed by mass spectrometry. These inhibited enzyme forms were stable to rearrangement or hydrolysis, with the exception of KPC-2. KPC-2 displayed a slow hydrolytic route that involved fragmentation of the acyl-avibactam complex. The identity of released degradation products was investigated, and a possible mechanism for the slow deacylation from KPC-2 is proposed.
Subject(s)
Azabicyclo Compounds/chemistry , Escherichia coli/drug effects , beta-Lactamases/chemistry , Anti-Bacterial Agents/chemistry , Drug Resistance, Bacterial , Enterobacter cloacae/metabolism , Enzyme Inhibitors/chemistry , Escherichia coli/metabolism , Hydrolysis , Kinetics , Magnetic Resonance Spectroscopy , Mass Spectrometry , Microbial Sensitivity Tests , Plasmids/metabolism , Pseudomonas aeruginosa/metabolism , Time FactorsABSTRACT
In Gram-negative bacteria, the cell wall is surrounded by an outer membrane, the outer leaflet of which is comprised of charged lipopolysaccharide (LPS) molecules. Lipid A, a component of LPS, anchors this molecule to the outer membrane. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a zinc-dependent metalloamidase that catalyzes the first committed step of biosynthesis of Lipid A, making it a promising target for antibiotic therapy. Formation of soluble aggregates of Pseudomonas aeruginosa LpxC protein when overexpressed in Escherichia coli has limited the availability of high quality protein for X-ray crystallography. Expression of LpxC in the presence of an inhibitor dramatically increased protein solubility, shortened crystallization time and led to a high-resolution crystal structure of LpxC bound to the inhibitor. However, this approach required large amounts of compound, restricting its use. To reduce the amount of compound needed, an overexpression strain of E. coli was created lacking acrB, a critical component of the major efflux pump. By overexpressing LpxC in the efflux deficient strain in the presence of LpxC inhibitors, several structures of P. aeruginosa LpxC in complex with different compounds were solved to accelerate structure-based drug design.
Subject(s)
Amidohydrolases/metabolism , Bacterial Proteins/metabolism , Escherichia coli Proteins/genetics , Multidrug Resistance-Associated Proteins/genetics , Pseudomonas aeruginosa/enzymology , Amidohydrolases/antagonists & inhibitors , Amidohydrolases/genetics , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/genetics , Catalysis , Chromatography, Liquid , Crystallography, X-Ray , Escherichia coli , Gene Expression , Mass Spectrometry , Protein Conformation , Zinc/chemistry , Zinc/metabolismABSTRACT
In this work, we explore the potential of the metalloendopeptidase Lys-N for MALDI-MS/MS proteomics applications. Initially we digested a HEK293 cellular lysate with Lys-N and, for comparison, in parallel with the protease Lys-C. The resulting peptides were separated by strong cation exchange to enrich and isolate peptides containing a single N-terminal lysine. MALDI-MS/MS analysis of these peptides yielded CID spectra with clear and often complete sequence ladders of b-ions. To test the applicability for de novo sequencing we next separated an ostrich muscle tissue protein lysate by one-dimensional SDS-PAGE. A protein band at 42 kDa was in-gel digested with Lys-N. Relatively straightforward sequencing resulted in the de novo identification of the two ostrich proteins creatine kinase and actin. We therefore conclude that this method that combines Lys-N, strong cation exchange enrichment, and MALDI-MS/MS analysis provides a valuable alternative proteomics strategy.
Subject(s)
Lysine/metabolism , Metalloendopeptidases/metabolism , Peptides/chemistry , Sequence Analysis, Protein/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Actins/chemistry , Amino Acid Sequence , Animals , Cell Line , Chickens , Creatine Kinase/chemistry , Humans , Molecular Sequence Data , Proline/metabolism , Struthioniformes , Tandem Mass SpectrometryABSTRACT
Antibody drug conjugates (ADCs) are an emerging class of chemotherapeutic cancer treatment agents that combine the targeting specificity of antibodies with the efficient cell-killing potential of cytotoxic drugs. Unlike their protein and small-molecule therapeutic counterparts, the stability and degradation properties of ADCs are relatively unknown. Theoretically, ADC stability could be governed by properties and processes stemming from both the antibody and the linker-toxin chemistry. Recently, systematic studies of intrinsic ADC molecule stability have been presented in the primary literature. As there are burgeoning industrial and academic efforts aimed at developing optimized conjugation chemistries and antibody engineering approaches for next-generation ADCs, it is important to capture the current state of understanding of ADC stability. In this minireview, we discuss aspects of physical and chemical stability of ADCs gathered from a survey of the literature and illustrate how investigations into stability enable the development of more effective ADC molecules for the future.
Subject(s)
Antibodies/chemistry , Antineoplastic Agents/chemistry , Immunoconjugates/chemistry , Animals , Antibodies/analysis , Antineoplastic Agents/analysis , Drug Stability , Humans , Immunoconjugates/analysis , Immunotoxins/analysis , Immunotoxins/chemistryABSTRACT
In the search for novel Gram-negative agents, we performed a comprehensive search of the AstraZeneca collection and identified a tetrahydropyran-based matrix metalloprotease (MMP) inhibitor that demonstrated nanomolar inhibition of UDP-3-O-(acyl)-N-acetylglucosamine deacetylase (LpxC). Crystallographic studies in Aquifex aeolicus LpxC indicated the tetrahydropyran engaged in the same hydrogen bonds and van der Waals interactions as other known inhibitors. Systematic optimization of three locales on the scaffold provided compounds with improved Gram-negative activity. However, the optimization of LpxC activity was not accompanied by reduced inhibition of MMPs. Comparison of the crystal structure of the native product, UDP-3-O-(acyl)-glucosamine, in Aquifex aeolicus to the structure of a tetrahydropyran-based inhibitor indicates pathways for future optimization.
ABSTRACT
LpxA, the first enzyme in the biosynthetic pathway for the Lipid A component of the outer membrane lipopolysaccharide in Gram-negative bacteria, is a potential target for novel antibacterial drug discovery. A fluorescence polarization assay was developed to facilitate high-throughput screening for competitive inhibitors of LpxA. The assay detects displacement of a fluorescently labeled peptide inhibitor, based on the previously reported inhibitor peptide 920, by active site ligands. The affinity of the fluorescent ligand was increased ~10-fold by acyl carrier protein (ACP). Competition with peptide binding was observed with UDP-N-acetylglucosamine (IC(50) ~6 mM), UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine (IC(50) ~200 nM), and DL-3-hydroxymyristic acid (IC(50) ~50 µM) and peptide 920 (IC(50) ~600 nM). The IC(50)s were not significantly affected by the presence of ACP.
Subject(s)
Acyltransferases/antagonists & inhibitors , Drug Evaluation, Preclinical/methods , Enzyme Inhibitors/pharmacology , Escherichia coli/enzymology , Fluorescence Polarization/methods , High-Throughput Screening Assays/methods , Acyl Carrier Protein/metabolism , Acyltransferases/chemistry , Binding, Competitive , Catalytic Domain , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Escherichia coli/drug effects , Escherichia coli/metabolism , Inhibitory Concentration 50 , Ligands , Lipid A/metabolism , Myristic Acids/chemistry , Myristic Acids/metabolism , Peptides/chemistry , Peptides/metabolism , Uridine Diphosphate N-Acetylglucosamine/analogs & derivatives , Uridine Diphosphate N-Acetylglucosamine/chemistry , Uridine Diphosphate N-Acetylglucosamine/metabolismABSTRACT
Triply and doubly charged iTRAQ ( isobaric tagging for relative and absolute quantitation) labeled peptide cations from a tryptic peptide mixture of bovine carbonic anhydrase II were subjected to electron transfer ion/ion reactions to investigate the effect of charge bearing modifications associated with iTRAQ on the fragmentation pattern. It was noted that electron transfer dissociation (ETD) of triply charged or activated ETD (ETD and supplemental collisional activation of intact electron transfer species) of doubly charged iTRAQ tagged peptide ions yielded extensive sequence information, in analogy with ETD of unmodified peptide ions. That is, addition of the fixed charge iTRAQ tag showed relatively little deleterious effect on the ETD performance of the modified peptides. ETD of the triply charged iTRAQ labeled peptide ions followed by collision-induced dissociation (CID) of the product ion at m/ z 162 yielded the reporter ion at m/ z 116, which is the reporter ion used for quantitation via CID of the same precursor ions. The reporter ion formed via the two-step activation process is expected to provide quantitative information similar to that directly produced from CID. A 103 Da neutral loss species observed in the ETD spectra of all the triply and doubly charged iTRAQ labeled peptide ions is unique to the 116 Da iTRAQ reagent, which implies that this process also has potential for quantitation of peptides/proteins. Therefore, ETD with or without supplemental collisional activation, depending on the precursor ion charge state, has the potential to directly identify and quantify the peptides/proteins simultaneously using existing iTRAQ reagents.
Subject(s)
Electron Transport , Peptides/chemistry , Amino Acid Sequence , Peptide Mapping , Tandem Mass Spectrometry , Trypsin/chemistryABSTRACT
We describe here a multiplexed protein quantitation strategy that provides relative and absolute measurements of proteins in complex mixtures. At the core of this methodology is a multiplexed set of isobaric reagents that yield amine-derivatized peptides. The derivatized peptides are indistinguishable in MS, but exhibit intense low-mass MS/MS signature ions that support quantitation. In this study, we have examined the global protein expression of a wild-type yeast strain and the isogenic upf1Delta and xrn1Delta mutant strains that are defective in the nonsense-mediated mRNA decay and the general 5' to 3' decay pathways, respectively. We also demonstrate the use of 4-fold multiplexing to enable relative protein measurements simultaneously with determination of absolute levels of a target protein using synthetic isobaric peptide standards. We find that inactivation of Upf1p and Xrn1p causes common as well as unique effects on protein expression.