ABSTRACT
AIMS: The purpose of this study was to establish safety and tolerability of a single intravenous (IV) infusion of a p38 mitogen-activated protein kinase inhibitor, losmapimod, to obtain therapeutic levels rapidly for a potential acute coronary syndrome indication. Pharmacokinetics (PK) following IV dosing were characterized, and pharmacokinetic/pharmacodynamic (PK/PD) relationships between losmapimod and phosphorylated heat shock protein 27 (pHSP27) and high-sensitivity C-reactive protein were explored. METHODS: Healthy volunteers received 1 mg losmapimod IV over 15 min (n = 4) or 3 mg IV over 15 min followed by a washout period and then 15 mg orally (PO; n = 12). Pharmacokinetic parameters were calculated by noncompartmental methods. The PK/PD relationships were explored using modelling and simulation. RESULTS: There were no deaths, nonfatal serious adverse events or adverse events leading to withdrawal. Headache was the only adverse event reported more than once (n = 3 following oral dosing). Following 3 mg IV and 15 mg PO, Cmax was 59.4 and 45.9 µg l(-1) and AUC0-∞ was 171.1 and 528.0 µg h l(-1) , respectively. Absolute oral bioavailability was 0.62 [90% confidence interval (CI) 0.56, 0.68]. Following 3 mg IV and 15 mg PO, maximal reductions in pHSP27 were 44% (95% CI 38%, 50%) and 55% (95% CI 50%, 59%) occurring at 30 min and 4 h, respectively. There was a 17% decrease (95% CI 9%, 24%) in high-sensitivity C-reactive protein 24 h following oral dosing. A direct-link maximal inhibitory effect model related plasma concentrations to pHSP27 concentrations. CONCLUSIONS: A single IV infusion of losmapimod in healthy volunteers was safe and well tolerated, and may potentially serve as an initial loading dose in acute coronary syndrome as rapid exposure is achieved.
Subject(s)
Cyclopropanes/administration & dosage , Models, Biological , Protein Kinase Inhibitors/administration & dosage , Pyridines/administration & dosage , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Administration, Oral , Adult , Area Under Curve , Biological Availability , C-Reactive Protein/metabolism , Cohort Studies , Cyclopropanes/pharmacokinetics , Cyclopropanes/pharmacology , Female , HSP27 Heat-Shock Proteins/blood , Humans , Infusions, Intravenous , Male , Middle Aged , Phosphorylation , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/pharmacology , Pyridines/pharmacokinetics , Pyridines/pharmacology , Young AdultABSTRACT
DNA replication is fundamental to the propagation of cellular life. Remarkably, the bacterial replication machinery is distinct from that used by archaea and eukaryotes. In this article, we discuss the role that lateral gene transfer by extra-chromosomal elements might have had in shaping the replication machinery and even modulating the manner in which host cellular genomes are replicated.
Subject(s)
Biological Evolution , Chromosomes/genetics , DNA Replication , Gene Transfer, Horizontal/genetics , Archaeal Proteins/classification , Archaeal Proteins/genetics , Archaeal Proteins/metabolism , Bacterial Proteins/classification , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Eukaryotic Cells/physiology , Evolution, Molecular , Multigene Family , Phylogeny , Replication Origin , RepliconABSTRACT
The subunits of the presumptive replicative helicase of archaea and eukaryotes, the MCM complex, are members of the AAA+ (ATPase-associated with various cellular activities) family of ATPases. Proteins within this family harness the chemical energy of ATP hydrolysis to perform a broad range of cellular processes. Here, we investigate the function of the AAA+ site in the mini-chromosome maintenance (MCM) complex of the archaeon Sulfolobus solfataricus (SsoMCM). We find that SsoMCM has an unusual active-site architecture, with a unique blend of features previously found only in distinct families of AAA+ proteins. We additionally describe a series of mutant doping experiments to investigate the mechanistic basis of intersubunit coordination in the generation of helicase activity. Our results indicate that MCM can tolerate catalytically inactive subunits and still function as a helicase, leading us to propose a semisequential model for helicase activity of this complex.
Subject(s)
Adenosine Triphosphate/metabolism , Archaeal Proteins/chemistry , DNA Helicases/chemistry , Metalloendopeptidases/metabolism , Sulfolobus solfataricus/enzymology , Archaeal Proteins/genetics , Archaeal Proteins/metabolism , Binding Sites , Computer Simulation , DNA Helicases/genetics , DNA Helicases/metabolism , Hydrolysis , Metalloendopeptidases/chemistry , Metalloendopeptidases/genetics , Models, Chemical , Models, Molecular , Monte Carlo Method , Multiprotein Complexes/chemistry , Mutagenesis, Site-Directed , Protein Conformation , Protein Subunits , Sulfolobus solfataricus/geneticsABSTRACT
The mini-chromosome maintenance (MCM) complex is the principal candidate for the replicative helicase of archaea and eukaryotes. Here, we describe a functional dissection of the roles of the three principal structural modules of the homomultimeric MCM of the hyperthermophilic archaeon Sulfolobus solfataricus. Our results include the first analysis of the central AAA+ domain in isolation. This domain possesses ATPase and helicase activity, defining this as the minimal helicase domain. Reconstitution experiments show that the helicase activity of the AAA+ domain can be stimulated by addition of the isolated N-terminal half in trans. Addition of the N-terminus influences both the processivity of the helicase and the choice of substrate that can be melted by the ATPase domain. The degenerate helix-turn-helix domain at the C-terminus of MCM exerts a negative effect on the helicase activity of the complex. These results provide the first evidence for extensive regulatory inter-domain communication within the MCM complex.
Subject(s)
Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , DNA Helicases/chemistry , DNA Helicases/metabolism , Sulfolobus solfataricus/enzymology , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Archaeal Proteins/genetics , DNA/metabolism , DNA Helicases/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Protein Structure, Tertiary , Sequence Deletion , Substrate SpecificityABSTRACT
The mini-chromosome maintenance (MCM) complex is the presumptive replicative helicase in archaea and eukaryotes. In archaea, the MCM is a homo-multimer, in eukaryotes a heterohexamer composed of six related subunits, MCM 2-7. Biochemical studies using naked DNA templates have revealed that archaeal MCMs and a sub-complex of eukaryotic MCM 4, 6 and 7 have 3' to 5' helicase activity. Here, we investigate the influence of the major chromatin proteins, Alba and Sul7d, of Sulfolobus solfataricus (Sso) on the ability of the MCM complex to melt partial duplex DNA substrates. In addition, we test the effect of Sso SSB on MCM activity. We reveal that Alba represents a formidable barrier to MCM activity and further demonstrate that acetylation of Alba alleviates repression of MCM activity.
Subject(s)
Archaeal Proteins/metabolism , Chromatin/metabolism , DNA-Binding Proteins/metabolism , Multiprotein Complexes/metabolism , Sulfolobus solfataricus/metabolism , Acetylation , DNA Helicases/metabolism , Nucleic Acid Denaturation , Sulfolobus solfataricus/geneticsABSTRACT
The homomultimeric archaeal mini-chromosome maintenance (MCM) complex serves as a simple model for the analogous heterohexameric eukaryotic complex. Here we investigate the organization and orientation of the MCM complex of the hyperthermophilic archaeon Sulfolobus solfataricus (Sso) on model DNA substrates. Sso MCM binds as a hexamer and slides on the end of a 3'-extended single-stranded DNA tail of a Y-shaped substrate; binding is oriented so that the motor domain of the protein faces duplex DNA. Two candidate beta-hairpin motifs within the MCM monomer have partially redundant roles in DNA binding. Notably, however, conserved basic residues within these motifs have nonequivalent roles in the helicase activity of MCM. On the basis of these findings, we propose a model for the mechanism of the helicase activity of MCM and note parallels with SV40 T antigen.
