Darren Thompson

Targeting C-reactive protein for the treatment of cardiovascular disease

Complement-mediated inflammation exacerbates the tissue injury of ischaemic necrosis in heart attacks and strokes, the most common causes of death in developed countries. Large infarct size increases immediate morbidity and mortality and, in survivors of the acute event, larger non-functional scars adversely affect long-term prognosis. There is thus an important unmet medical need for new cardioprotective and neuroprotective treatments. We have previously shown that human C-reactive protein (CRP), the classical acute-phase protein that binds to ligands exposed in damaged tissue and then activates complement, increases myocardial and cerebral infarct size in rats subjected to coronary or cerebral artery ligation, respectively. Rat CRP does not activate rat complement, whereas human CRP activates both rat and human complement. Administration of human CRP to rats is thus an excellent model for the actions of endogenous human CRP. Here we report the design, synthesis and efficacy of 1,6-bis(phosphocholine)-hexane as a specific small-molecule inhibitor of CRP. Five molecules of this palindromic compound are bound by two pentameric CRP molecules, crosslinking and occluding the ligand-binding B-face of CRP and blocking its functions. Administration of 1,6-bis(phosphocholine)-hexane to rats undergoing acute myocardial infarction abrogated the increase in infarct size and cardiac dysfunction produced by injection of human CRP. Therapeutic inhibition of CRP is thus a promising new approach to cardioprotection in acute myocardial infarction, and may also provide neuroprotection in stroke. Potential wider applications include other inflammatory, infective and tissue-damaging conditions characterized by increased CRP production, in which binding of CRP to exposed ligands in damaged cells may lead to complement-mediated exacerbation of tissue injury.

The physiological structure of human C-reactive protein and its complex with phosphocholine.

BACKGROUND Human C-reactive protein (CRP) is the classical acute phase reactant, the circulating concentration of which rises rapidly and extensively in a cytokine-mediated response to tissue injury, infection and inflammation. Serum CRP values are routinely measured, empirically, to detect and monitor many human diseases. However, CRP is likely to have important host defence, scavenging and metabolic functions through its capacity for calcium-dependent binding to exogenous and autologous molecules containing phosphocholine (PC) and then activating the classical complement pathway. CRP may also have pathogenic effects and the recent discovery of a prognostic association between increased CRP production and coronary atherothrombotic events is of particular interest. RESULTS The X-ray structures of fully calcified C-reactive protein, in the presence and absence of bound PC, reveal that although the subunit beta-sheet jellyroll fold is very similar to that of the homologous pentameric protein serum amyloid P component, each subunit is tipped towards the fivefold axis. PC is bound in a shallow surface pocket on each subunit, interacting with the two protein-bound calcium ions via the phosphate group and with Glu81 via the choline moiety. There is also an unexpected hydrophobic pocket adjacent to the ligand. CONCLUSIONS The structure shows how large ligands containing PC may be bound by CRP via a phosphate oxygen that projects away from the surface of the protein. Multipoint attachment of one planar face of the CRP molecule to a PC-bearing surface would leave available, on the opposite exposed face, the recognition sites for C1q, which have been identified by mutagenesis. This would enable CRP to target physiologically and/or pathologically significant complement activation. The hydrophobic pocket adjacent to bound PC invites the design of inhibitors of CRP binding that may have therapeutic relevance to the possible role of CRP in atherothrombotic events.

High-resolution structure of myo-inositol monophosphatase, the putative target of lithium therapy

Inositol monophosphatase is a key enzyme of the phosphatidylinositol signalling pathway and the putative target of the mood-stabilizing drug lithium. The crystal structure of bovine inositol monophosphatase has been determined at 1.4 A resolution in complex with the physiological magnesium ion ligands. Three magnesium ions are octahedrally coordinated at the active site of each of the two subunits of the inositol monophosphatase dimer and a detailed three-metal mechanism is proposed. Ligands to the three metals include the side chains of Glu70, Asp90, Asp93 and Asp220, the backbone carbonyl group of Ile92 and several solvent molecules, including the proposed nucleophilic water molecule (W1) ligated by both Mg-1 and Mg-3. Modelling of the phosphate moiety of inositol monophosphate to superpose the axial phosphate O atoms onto three active-site water molecules orientates the phosphoester bond for in-line attack by the nucleophilic water which is activated by Thr95. Modelling of the pentacoordinate transition state suggests that the 6-OH group of the inositol moiety stabilizes the developing negative charge by hydrogen bonding to a phosphate O atom. Modelling of the post-reaction complex suggests a role for a second water molecule (W2) ligated by Mg-2 and Asp220 in protonating the departing inositolate. This second water molecule is absent in related structures in which lithium is bound at site 2, providing a rationale for enzyme inhibition by this simple monovalent cation. The higher resolution structural information on the active site of inositol monophosphatase will facilitate the design of substrate-based inhibitors and aid in the development of better therapeutic agents for bipolar disorder (manic depression).

The structures of crystalline complexes of human serum amyloid P component with its carbohydrate ligand, the cyclic pyruvate acetal of galactose.

Two monoclinic (P2(1)) crystal forms of human serum amyloid P component (SAP) in complex with the 4,6-pyruvate acetal of beta-D-galactose (MObetaDG) were prepared. Structure analysis by molecular replacement and refinement at 2.2A resolution revealed that crystal form 1 (a=95.76A, b=70.53A, c=103.41A, beta=96.80 degrees) contained a pentamer in the asymmetric unit with a structure very similar to that of the published search model. The mode of ligand co-ordination was also similar except that four of the five subunits showed bound ligand with an additional H-bond between O1 of the galactose and the side-chain of Lys79. One sub-unit showed no bound ligand and a vacant calcium site close to a crystal contact. The 2.6A resolution structure of crystal form 2 (a=118.60A, b=109.10A, c=120.80A and beta=95.16 degrees ) showed ten sub-units in the asymmetric unit, all with two bound calcium ions and ligand. The most extensive protein-protein interactions between pentamers describe an AB face-to-face interaction involving 15 ion pairs that sandwiches five molecules of bound MObetaDG at the interface.

Interaction of Serum Amyloid P Component with Hexanoyl Bis(D-Proline) (Cphpc)

Serum amyloid P component is a pentameric plasma glycoprotein that recognizes and binds to amyloid fibres in a calcium-dependent fashion and is likely to contribute to their deposition and persistence in vivo. Five molecules of the drug CPHPC avidly cross-link pairs of protein pentamers and the decameric complex is rapidly cleared in vivo. Crystal structures of the protein in complex with a bivalent drug and cadmium ions, which improve crystal quality, allow the definition of the preferred bound drug isomers.

Characterisation of the SUMO-Like Domains of Schizosaccharomyces pombe Rad60

The S. pombe Rad60 protein is required for the repair of DNA double strand breaks, recovery from replication arrest, and is essential for cell viability. It has two SUMO-like domains (SLDs) at its C-terminus, an SXS motif and three sequences that have been proposed to be SUMO-binding motifs (SBMs). SMB1 is located in the middle of the protein, SBM2 is in SLD1 and SBM3 is at the C-terminus of SLD2. We have probed the functions of the two SUMO-like domains, SLD1 and SLD2, and the putative SBMs. SLD1 is essential for viability, while SLD2 is not. rad60-SLD2Δ cells are sensitive to DNA damaging agents and hydroxyurea. Neither ubiquitin nor SUMO can replace SLD1 or SLD2. Cells in which either SBM1 or SBM2 has been mutated are viable and are wild type for response to MMS and HU. In contrast mutation of SBM3 results in significant sensitivity to MMS and HU. These results indicate that the lethality resulting from deletion of SLD1 is not due to loss of SBM2, but that mutation of SBM3 produces a more severe phenotype than does deletion of SLD2. Using chemical denaturation studies, FPLC and dynamic light scattering we show this is likely due to the destabilisation of SLD2. Thus we propose that the region corresponding to the putative SBM3 forms part of the hydrophobic core of SLD2 and is not a SUMO-interacting motif. Over-expression of Hus5, which is the SUMO conjugating enzyme and known to interact with Rad60, does not rescue rad60-SLD2Δ, implying that as well as having a role in the sumoylation process as previously described [1], Rad60 has a Hus5-independent function.