ABSTRACT
Telavancin is a parenteral lipoglycopeptide antibiotic with a dual mechanism of action contributing to bactericidal activity against multidrug-resistant Gram-positive pathogens. It has been approved for the treatment of complicated skin and skin structure infections due to susceptible Gram-positive bacteria and hospital-acquired/ventilator-associated bacterial pneumonia due to Staphylococcus aureus when other alternatives are unsuitable. Telavancin has been demonstrated to be efficacious in multiple animal models of soft tissue, cardiac, systemic, lung, bone, brain and device-associated infections involving clinically relevant Gram-positive pathogens, including methicillin-resistant S. aureus, glycopeptide-intermediate S. aureus, heterogeneous vancomycin-intermediate S. aureus and daptomycin non-susceptible methicillin-resistant S. aureus. The AUC0-24h/MIC ratio is the primary pharmacodynamically-linked pharmacokinetic parameter. The preclinical data for telavancin supports further investigative clinical evaluation of its efficacy in additional serious infections caused by susceptible Gram-positive pathogens.
Subject(s)
Aminoglycosides/therapeutic use , Anti-Bacterial Agents/therapeutic use , Gram-Positive Bacterial Infections/drug therapy , Aminoglycosides/pharmacology , Animals , Anti-Bacterial Agents/pharmacology , Disease Models, Animal , Drug Resistance, Bacterial/drug effects , Drug Resistance, Multiple/drug effects , Gram-Positive Bacteria/drug effects , Lipoglycopeptides , MiceABSTRACT
Telavancin is approved in the United States, Canada, and Europe (At the time of submission, the telavancin European marketing authorization for nosocomial pneumonia was suspended until Theravance provides evidence of a new European Medicines Agency approved supplier) as an antibiotic to treat certain Gram-positive bacterial skin infections. Telavancin has been shown to prolong plasmatic prothrombin (PT) and activated partial thromboplastin (aPTT) clotting times in clinical diagnostic lab-based assays. In this study, we evaluated the potential for telavancin to prolong whole blood PT/International Normalized Ratio (INR) and aPTT tests on point-of-care (POC) instruments. Whole blood collected from 8 healthy subjects was supplemented with telavancin to final concentrations of 0, 10, 20, and 100 µg/ml. Final concentrations were selected to match trough, twice trough, and peak plasma levels following the approved 10 mg/kg dose. Four widely employed POC coagulation instruments were chosen to be representative of the POC platforms currently in use.. These systems were the Roche Coaguchek XS, the Abbott iSTAT, the ITC Hemochron SIG+, and the Alere INRatio2 POC devices. The PT/INR measured by the Coaguchek XS showed the greatest sensitivity to the presence of telavancin. The PT/INR measured by the Hemochron SIG+ and iSTAT were sensitive to telavancin but to a lesser extent. The INRatio2 was the least sensitive to the presence of telavancin when testing the whole blood PT/INR. Only the Hemochron SIG+ device was capable of measuring aPTT and showed a concentration-dependent increase in aPTT. This study supports the current recommendation that PT and aPTT monitoring be conducted immediately to the next dose of telavancin when coagulation parameters are tested using POC instrumentation.
Subject(s)
Aminoglycosides/administration & dosage , Anti-Bacterial Agents/administration & dosage , Blood Coagulation/drug effects , Prothrombin Time/instrumentation , Prothrombin Time/methods , Adolescent , Adult , Female , Humans , Lipoglycopeptides , Male , Middle Aged , Partial Thromboplastin Time/instrumentation , Partial Thromboplastin Time/methodsABSTRACT
The discovery and optimization of a novel class of quinolone small-molecules that inhibit NS5B polymerase, a key enzyme of the HCV viral life-cycle, is described. Our research led to the replacement of a hydrolytically labile ester functionality with bio-isosteric heterocycles. An X-ray crystal structure of a key analog bound to NS5B facilitated the optimization of this series of compounds to afford increased activity against the target enzyme and in the cell-based replicon assay system.
Subject(s)
Antiviral Agents/pharmacology , Chemistry, Pharmaceutical/methods , Hepacivirus/enzymology , Quinolones/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Allosteric Site , Antiviral Agents/chemical synthesis , Binding Sites , Crystallography, X-Ray/methods , Drug Design , Hydrogen Bonding , Hydrolysis , Inhibitory Concentration 50 , Models, Chemical , Molecular Conformation , Quinolones/chemical synthesis , Structure-Activity Relationship , Viral Nonstructural Proteins/chemistry , X-RaysABSTRACT
Hepatitis C virus (HCV) infection is treated with a combination of peginterferon alfa-2a/b and ribavirin. To address the limitations of this therapy, numerous small molecule agents are in development, which act by directly affecting key steps in the viral life-cycle. Herein we describe our discovery of quinolone derivatives, novel small-molecules that inhibit NS5b polymerase, a key enzyme of the viral life-cycle. A crystal structure of a quinoline analog bound to NS5B reveals that this class of compounds binds to allosteric site-II (non-nucleoside inhibitor-site 2, NNI-2) of this protein.
Subject(s)
Antiviral Agents/chemistry , Enzyme Inhibitors/chemistry , Hepacivirus/enzymology , Quinolones/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Allosteric Regulation , Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacology , Binding Sites , Computer Simulation , Crystallography, X-Ray , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Quinolones/chemical synthesis , Quinolones/pharmacology , Structure-Activity Relationship , Viral Nonstructural Proteins/metabolismABSTRACT
Telavancin is a bactericidal, semisynthetic lipoglycopeptide indicated in the United States for the treatment of complicated skin and skin structure infections caused by susceptible gram-positive bacteria and is under investigation as a once-daily treatment for nosocomial pneumonia. The related vanA and vanB gene clusters mediate acquired resistance to glycopeptides in enterococci by remodeling the dipeptide termini of peptidoglycan precursors from D-alanyl-D-alanine (D-Ala-D-Ala) to D-alanyl-D-lactate (D-Ala-D-Lac). In this study, we assessed the ability of telavancin to induce the expression of van genes in VanA- and VanB-type strains of vancomycin-resistant enterococci. Vancomycin, teicoplanin, and telavancin efficiently induced VanX activity in VanA-type strains, while VanX activity in VanB-type isolates was inducible by vancomycin but not by teicoplanin or telavancin. In VanA-type strains treated with vancomycin or telavancin, high levels of D-Ala-D-Lac-containing pentadepsipeptide were measured, while D-Ala-D-Ala pentapeptide was present at very low levels or not detected at all. In VanB-type strains, vancomycin but not telavancin induced high levels of pentadepsipeptide, while pentapeptide was not detected. Although vancomycin, teicoplanin, and telavancin induced similar levels of VanX activity in VanA-type strains, these organisms were more sensitive to telavancin, which displayed MIC values that were 32- and 128-fold lower than those of vancomycin and teicoplanin, respectively.
Subject(s)
Aminoglycosides/pharmacology , Bacterial Proteins/metabolism , Carbon-Oxygen Ligases/metabolism , Enterococcus/drug effects , Enterococcus/metabolism , Operon/drug effects , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Blotting, Western , Carbon-Oxygen Ligases/genetics , Drug Resistance, Bacterial/drug effects , Drug Resistance, Bacterial/genetics , Enterococcus/genetics , Gene Expression Regulation, Bacterial/drug effects , Gene Expression Regulation, Bacterial/genetics , Lipoglycopeptides , Operon/genetics , Teicoplanin/pharmacology , Vancomycin/pharmacology , Vancomycin Resistance/drug effects , Vancomycin Resistance/geneticsABSTRACT
Telavancin is an investigational lipoglycopeptide antibiotic currently being developed for the treatment of serious infections caused by gram-positive bacteria. The bactericidal action of telavancin results from a mechanism that combines the inhibition of cell wall synthesis and the disruption of membrane barrier function. The purpose of the present study was to further elucidate the mechanism by which telavancin interacts with the bacterial membrane. A flow cytometry assay with the diethyloxacarbocyanine dye DiOC(2)(3) was used to probe the membrane potential of actively growing Staphylococcus aureus cultures. Telavancin caused pronounced membrane depolarization that was both time and concentration dependent. Membrane depolarization was demonstrated against a reference S. aureus strain as well as phenotypically diverse isolates expressing clinically important methicillin-resistant (MRSA), vancomycin-intermediate (VISA), and heterogeneous VISA (hVISA) phenotypes. The cell wall precursor lipid II was shown to play an essential role in telavancin-induced depolarization. This was demonstrated both in competition binding experiments with exogenous D-Ala-D-Ala-containing ligand and in experiments with cells expressing altered levels of lipid II. Finally, monitoring of the optical density of S. aureus cultures exposed to telavancin showed that cell lysis does not occur during the time course in which membrane depolarization and bactericidal activity are observed. Taken together, these data indicate that telavancin's membrane mechanism requires interaction with lipid II, a high-affinity target that mediates binding to the bacterial membrane. The targeted interaction with lipid II and the consequent disruption of both peptidoglycan synthesis and membrane barrier function provide a mechanistic basis for the improved antibacterial properties of telavancin relative to those of vancomycin.
Subject(s)
Aminoglycosides/pharmacology , Anti-Bacterial Agents/pharmacology , Cell Wall/chemistry , Cell Wall/drug effects , Uridine Diphosphate N-Acetylmuramic Acid/analogs & derivatives , Aminoglycosides/chemistry , Anti-Bacterial Agents/chemistry , Cell Wall/metabolism , Flow Cytometry , Lipoglycopeptides , Membrane Potentials/drug effects , Methicillin Resistance/drug effects , Microbial Sensitivity Tests , Microscopy, Phase-Contrast , Staphylococcus aureus/cytology , Staphylococcus aureus/drug effects , Staphylococcus aureus/metabolism , Uridine Diphosphate N-Acetylmuramic Acid/chemistry , Vancomycin/pharmacologyABSTRACT
Tryptases are trypsin-like serine proteases whose expression is restricted to cells of hematopoietic origin, notably mast cells. gamma-Tryptase, a recently described member of the family also known as transmembrane tryptase (TMT), is a membrane-bound serine protease found in the secretory granules or on the surface of degranulated mast cells. The 321 amino acid protein contains an 18 amino acid propeptide linked to the catalytic domain (cd), followed by a single-span transmembrane domain. gamma-Tryptase is distinguished from other human mast cell tryptases by the presence of two unique cysteine residues, Cys(26) and Cys(145), that are predicted to form an intra-molecular disulfide bond linking the propeptide to the catalytic domain to form the mature, membrane-anchored two-chain enzyme. We expressed gamma-tryptase as either a soluble, single-chain enzyme with a C-terminal His tag (cd gamma-tryptase) or as a soluble pseudozymogen activated by enterokinase cleavage to form a two-chain protein with an N-terminal His tag (tc gamma-tryptase). Both recombinant proteins were expressed at high levels in Pichia pastoris and purified by affinity chromatography. The two forms of gamma-tryptase exhibit comparable kinetic parameters, indicating the propeptide does not contribute significantly to the substrate affinity or activity of the protease. Substrate and inhibitor library screening indicate that gamma-tryptase possesses a substrate preference and inhibitor profile distinct from that of beta-tryptase. Although the role of gamma-tryptase in mast cell function is unknown, our results suggest that it is likely to be distinct from that of beta-tryptase.
Subject(s)
Gene Expression , Recombinant Proteins/metabolism , Serine Endopeptidases/metabolism , Enteropeptidase/metabolism , Enzyme Activation , Humans , Kinetics , Molecular Structure , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Serine Endopeptidases/genetics , Serine Endopeptidases/isolation & purification , Substrate Specificity , TryptasesABSTRACT
The synthesis of novel [1,2,4]oxadiazoles and their structure-activity relationship (SAR) for the inhibition of tryptase and related serine proteases is presented. Elaboration of the P'-side afforded potent, selective, and orally bioavailable tryptase inhibitors.
Subject(s)
Enzyme Inhibitors/pharmacology , Serine Endopeptidases/drug effects , Administration, Oral , Biological Availability , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/chemistry , Models, Molecular , Structure-Activity Relationship , TryptasesABSTRACT
Beginning with the peptide sequence Cbz-Ile-Glu(OtBu)-Ala-Leu found in PSI (3), a series of vinyl sulfones (VS) were synthesized for evaluation as inhibitors of the chymotrypsin-like activity of the 20S proteasome. Variations at the key P3 position confirmed the importance of a long side chain capped with a hydrophobic group for optimal potency, consistent with a model of binding to the S3 subsite. The tert-butyl glutamic ester initially used at P3 gave plasma unstable, insoluble compounds and was replaced with the better isostere, N-beta-neopentyl asparagine. The inhibitors were shortened by replacing the N-terminal Cbz-isoleucine with a p-tosyl group without loss of potency. Small l-amino acids were used at P2, where d-substitution was not tolerated. The resulting optimized P4-P3-P2 sequence was grafted onto a novel proteasome inhibitor warhead, 2-keto-1,3,4-oxadiazoles (KOD), to produce reversible, subnanomolar proteasome inhibitors that were 1000-fold selective versus cathepsin B (CatB), cathepsin S (CatS), and trypsin-like as well as PGPH-like proteasome activity. A number of compounds in both the VS and the KOD series exhibited growth inhibitory effects against the human prostate cancer cell line PC3 at submicromolar concentrations.
Subject(s)
Oligopeptides/chemical synthesis , Oxadiazoles/chemical synthesis , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors , Sulfones/chemical synthesis , Vinyl Compounds/chemical synthesis , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cattle , Cell Proliferation/drug effects , Drug Stability , Humans , In Vitro Techniques , Models, Molecular , Oligopeptides/chemistry , Oligopeptides/pharmacology , Oxadiazoles/chemistry , Oxadiazoles/pharmacology , Proteasome Endopeptidase Complex/chemistry , Protein Subunits/antagonists & inhibitors , Protein Subunits/chemistry , Protein Subunits/metabolism , Solubility , Structure-Activity Relationship , Sulfones/chemistry , Sulfones/pharmacology , Vinyl Compounds/chemistry , Vinyl Compounds/pharmacologyABSTRACT
Improved peptide-based inhibitors of human beta tryptase were discovered using information gleaned from tripeptide library screening and structure-guided design methods, including fragment screening. Our efforts sought to improve this class of inhibitors by replacing the traditional Lys or Arg P1 element. The optimized compounds display low nanomolar potency against the mast cell target and several hundred-fold selectivity with respect to serine protease off targets. Thus, replacement of Lys/Arg at P1 in a peptide-like scaffold does not need to be accompanied by a loss in target affinity.
Subject(s)
Serine Endopeptidases/drug effects , Serine Proteinase Inhibitors/chemistry , Crystallography, X-Ray , Models, Molecular , Protein Conformation , Serine Proteinase Inhibitors/pharmacology , TryptasesABSTRACT
A series of novel alpha-keto-[1,2,4]-oxadiazoles has been synthesized as human tryptase inhibitors for evaluation as a new class of anti-asthmatic agent. The inhibitor design is focused on using a prime-side hydrophobic pocket and the S2 pocket of beta-tryptase to achieve inhibition potency and selectivity over other serine proteases.
Subject(s)
Oxazoles/pharmacology , Serine Endopeptidases/drug effects , Crystallography, X-Ray , Humans , Kinetics , Oxazoles/chemistry , TryptasesABSTRACT
We have prepared a series of cathepsin K inhibitors bearing the keto-1,3,4-oxadiazole warhead capable of forming a hemithioketal complex with the target enzyme. By modifying binding moieties at the P1, P2, and prime side positions of the inhibitors, we have achieved selectivity over cathepsins B, L, and S, and have achieved sub-nanomolar potency against cathepsin K. This series thus represents a promising chemotype that could be used in diseases implicated by imbalances in cathepsin K activity such as osteoporosis.
Subject(s)
Cathepsins/antagonists & inhibitors , Oxadiazoles/chemistry , Oxadiazoles/pharmacology , Protease Inhibitors/chemical synthesis , Protease Inhibitors/pharmacology , Animals , Cathepsin K , Cathepsins/metabolism , Molecular Structure , Oxadiazoles/chemical synthesis , Protease Inhibitors/chemistry , Rats , Structure-Activity RelationshipABSTRACT
Synthesis and biological data of a novel selective and efficacious factor IXa inhibitor are described along with its crystal structure in factor VIIa.
Subject(s)
Factor IXa/antagonists & inhibitors , Pyrazoles/pharmacology , Serine Proteinase Inhibitors/pharmacology , Factor IXa/chemistry , Humans , Models, MolecularABSTRACT
Efforts to improve the potency and pharmacokinetic properties of small molecule factor VIIa inhibitors are described. Small structural modifications to existing leads allow the modulation of half-life and clearance, potentially making these compounds suitable candidates for drug development.
Subject(s)
Anticoagulants/pharmacokinetics , Blood Coagulation/drug effects , Factor VIIa/antagonists & inhibitors , Serine Proteinase Inhibitors/pharmacokinetics , Animals , Drug Design , Half-Life , Humans , Molecular Structure , Structure-Activity RelationshipABSTRACT
Highly selective and potent factor VIIa-tissue factor (fVIIa.TF) complex inhibitors were generated through structure-based design. The pharmacokinetic properties of an optimized analog (9) were characterized in several preclinical species, demonstrating pharmacokinetic characteristics suitable for once-a-day dosing in humans. Analog 9 inhibited platelet and fibrin deposition in a dose-dependent manner after intravenous administration in a baboon thrombosis model, and a pharmacodynamic concentration-response model was developed to describe the platelet deposition data. Results for heparin and enoxaparin (Lovenox) in the baboon model are also presented.
Subject(s)
Factor VIIa/antagonists & inhibitors , Models, Animal , Serine Proteinase Inhibitors/pharmacology , Thrombosis/drug therapy , Animals , Chromatography, High Pressure Liquid , Crystallography, X-Ray , Models, Molecular , Papio , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/pharmacokinetics , Serine Proteinase Inhibitors/therapeutic useABSTRACT
Within the trypsin family of coagulation proteases, obtaining highly selective inhibitors of factor VIIa has been challenging. We report a series of factor VIIa (fVIIa) inhibitors based on the 5-amidino-2-(2-hydroxy-biphenyl-3-yl)-benzimidazole (1) scaffold with potency for fVIIa and high selectivity against factors IIa, Xa, and trypsin. With this scaffold class, we propose that a unique hydrogen bond interaction between a hydroxyl on the distal ring of the biaryl system and the backbone carbonyl of fVIIa lysine-192 provides a basis for enhanced selectivity and potency for fVIIa.
Subject(s)
Factor VIIa/antagonists & inhibitors , Binding Sites , Factor Xa Inhibitors , Humans , Hydrogen Bonding , Protein Binding , Prothrombin/antagonists & inhibitors , Structure-Activity Relationship , Trypsin/metabolismABSTRACT
Inhibition of coagulation proteases such as thrombin, fXa, and fVIIa has been a focus of ongoing research to produce safe and effective antithrombotic agents. Herein, we describe a unique zinc-mediated chelation strategy to streamline the discovery of potent inhibitors of fIIa, fXa, and fVIIa. SAR studies that led to the development of selective inhibitors of fXa will also be detailed.
Subject(s)
Anticoagulants/chemistry , Blood Coagulation/drug effects , Chelating Agents/chemistry , Protease Inhibitors/chemical synthesis , Zinc/chemistry , Anticoagulants/pharmacology , Blood Coagulation/physiology , Crystallography, X-Ray , Factor VII/antagonists & inhibitors , Factor Xa Inhibitors , Protease Inhibitors/pharmacology , Structure-Activity Relationship , Thrombin/antagonists & inhibitorsABSTRACT
We have prepared a series of achiral aminoacetonitriles, bearing tri-ring benzamide moieties and an aminocyclohexanecarboxylate residue at P2. This combination of binding elements resulted in sub-250 pM, reversible, selective, and orally bioavailable cathepsin K inhibitors. Lead compounds displayed single digit nanomolar inhibition in vitro (of rabbit osteoclast-mediated degradation of bovine bone). The best compound in this series, 39n (CRA-013783/L-006235), was orally bioavailable in rats, with a terminal half-life of over 3 h. 39n was dosed orally in ovariectomized rhesus monkeys once per day for 7 days. Collagen breakdown products were reduced by up to 76% dose-dependently. Plasma concentrations of 39n above the bone resorption IC50 after 24 h indicated a correlation between functional cellular and in vivo assays. Inhibition of collagen breakdown by cathepsin K inhibitors suggests this mechanism of action may be useful in osteoporosis and other indications involving bone resorption.
Subject(s)
Benzamides/chemical synthesis , Bone Density Conservation Agents/chemical synthesis , Cathepsins/antagonists & inhibitors , Nitriles/chemical synthesis , Thiazoles/chemical synthesis , Administration, Oral , Animals , Benzamides/chemistry , Benzamides/pharmacology , Biological Availability , Biomarkers/urine , Bone Density Conservation Agents/chemistry , Bone Density Conservation Agents/pharmacology , Bone Resorption/urine , Cathepsin K , Cathepsins/chemistry , Cattle , Collagen/antagonists & inhibitors , Collagen/metabolism , Crystallography, X-Ray , Drug Design , Humans , Kinetics , Macaca mulatta , Models, Molecular , Molecular Structure , Nitriles/chemistry , Nitriles/pharmacology , Rabbits , Rats , Structure-Activity Relationship , Thiazoles/chemistry , Thiazoles/pharmacologyABSTRACT
The synthesis of a series of highly potent and selective inhibitors of cathepsin K based on the 3,4-disubstituted azetidin-2-one warhead is reported. A high degree of potency and selectivity was achieved by introducing a basic nitrogen into the distal part of the P3 element of the molecule. Data from kinetic and mass spectrometry experiments are consistent with the interpretation that compounds of this series transiently acylate the sulfhydrile of cathepsin K.
Subject(s)
Azetidines/pharmacology , Cathepsins/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Azetidines/chemical synthesis , Azetidines/chemistry , Cathepsin K , Cathepsins/chemistry , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/chemistry , Drug Evaluation, Preclinical , Kinetics , Molecular Structure , Structure-Activity RelationshipABSTRACT
A site-directed mutant of the serine protease urokinase-type plasminogen activator (uPA), was produced to assess the contribution of the Ser190 side-chain to the affinity and selectivity of lead uPA inhibitors in the absence of other differences present in comparisons of natural proteases. Crystallography and enzymology involving WT and Ala190 uPA were used to calculate free energy binding contributions of hydrogen bonds involving the Ser190 hydroxyl group (O(gamma)(Ser190)) responsible for the remarkable selectivity of 6-halo-5-amidinoindole and 6-halo-5-amidinobenzimidazole inhibitors toward uPA and against natural Ala190 protease anti-targets. Crystal structures of uPA complexes of novel, active site-directed arylguanidine and 2-aminobenzimidazole inhibitors of WT uPA, together with associated K(i) values for WT and Ala190 uPA, also indicate a significant role of Ser190 in the binding of these classes of uPA inhibitors. Structures and associated K(i) values for a lead inhibitor (CA-11) bound to uPA and to five other proteases, as well as for other leads bound to multiple proteases, help reveal the features responsible for the potency (K(i)=11nM) and selectivity of the remarkably small inhibitor, CA-11. The 6-fluoro-5-amidinobenzimidzole, CA-11, is more than 1000-fold selective against natural Ala190 protease anti-targets, and more than 100-fold selective against other Ser190 anti-targets.