ABSTRACT
Human thymidine phosphorylase (HTP), also known as platelet-derived endothelial cell growth factor (PD-ECGF), is overexpressed in certain solid tumors where it is linked to poor prognosis. HTP expression is utilized for certain chemotherapeutic strategies and is also thought to play a role in tumor angiogenesis. We determined the structure of HTP bound to the small molecule inhibitor 5-chloro-6-[1-(2-iminopyrrolidinyl) methyl] uracil hydrochloride (TPI). The inhibitor appears to mimic the substrate transition state, which may help explain the potency of this inhibitor and the catalytic mechanism of pyrimidine nucleotide phosphorylases (PYNPs). Further, we have confirmed the validity of the HTP structure as a template for structure-based drug design by predicting binding affinities for TPI and other known HTP inhibitors using in silico docking techniques. This work provides the first structural insight into the binding mode of any inhibitor to this important drug target and forms the basis for designing novel inhibitors for use in anticancer therapy.
Subject(s)
Models, Molecular , Protein Binding , Protein Folding , Pyrrolidines/chemistry , Thymidine Phosphorylase/metabolism , Uracil/analogs & derivatives , Uracil/chemistry , Crystallization , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Protein Structure, Tertiary , Pyrrolidines/pharmacology , Uracil/pharmacologyABSTRACT
Inhibition of p38alpha MAP kinase is a potential approach for the treatment of inflammatory disorders. MKK6-dependent phosphorylation on the activation loop of p38alpha increases its catalytic activity and affinity for ATP. An inhibitor, BIRB796, binds at a site used by the purine moiety of ATP and extends into a "selectivity pocket", which is not used by ATP. It displaces the Asp168-Phe169-Gly170 motif at the start of the activation loop, promoting a "DFG-out" conformation. Some other inhibitors bind only in the purine site, with p38alpha remaining in a "DFG-in" conformation. We now demonstrate that selectivity pocket compounds prevent MKK6-dependent activation of p38alpha in addition to inhibiting catalysis by activated p38alpha. Inhibitors using only the purine site do not prevent MKK6-dependent activation. We present kinetic analyses of seven inhibitors, whose crystal structures as complexes with p38alpha have been determined. This work includes four new crystal structures and a novel assay to measure K(d) for nonactivated p38alpha. Selectivity pocket compounds associate with p38alpha over 30-fold more slowly than purine site compounds, apparently due to low abundance of the DFG-out conformation. At concentrations that inhibit cellular production of an inflammatory cytokine, TNFalpha, selectivity pocket compounds decrease levels of phosphorylated p38alpha and beta. Stabilization of a DFG-out conformation appears to interfere with recognition of p38alpha as a substrate by MKK6. ATP competes less effectively for prevention of activation than for inhibition of catalysis. By binding to a different conformation of the enzyme, compounds that prevent activation offer an alternative approach to modulation of p38alpha.
Subject(s)
MAP Kinase Kinase 6/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Enzyme Activation , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , MAP Kinase Kinase 6/chemistry , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitorsABSTRACT
Atmospheric nuclear weapons explosions and large-scale nuclear accidents may contaminate large areas of land with the long-lived radionuclides 137Cs and 90Sr. The mobility and bioavailability of these radionuclides in the environment is dependent primarily on soil characteristics and changes significantly over time after fallout (1-4). Radioisotope concentrations in different rivers and at different times after fallout vary over 2-3 orders of magnitude. Many previous studies have concentrated on the interactions of radiocesium and radiostrontium with various environmental components, but there are currently no operative models fortheirtransport over large spatial areas. We collected time-series measurements of 90Sr and 137Cs in 25 major European and Asian rivers and (using digital data sets with global coverage) determined characteristics of each of the rivers' catchments. This work has established, for the first time, a quantitative link between riverine transport of these radioisotopes and catchment and soil characteristics at a global scale. A generalized predictive model accounting for time changes in river concentrations and variation in catchment characteristics is developed. This can be used to predict the long-term riverine transport of these radiologically important radionuclides following any large-scale nuclear incident in North America, Europe, or (European and Asian) Russia.