S.P. Wood

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.

Targeted pharmacological depletion of serum amyloid P component for treatment of human amyloidosis.

The normal plasma protein serum amyloid P component (SAP) binds to fibrils in all types of amyloid deposits, and contributes to the pathogenesis of amyloidosis. In order to intervene in this process we have developed a drug, R-1-[6-[R-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid, that is a competitive inhibitor of SAP binding to amyloid fibrils. This palindromic compound also crosslinks and dimerizes SAP molecules, leading to their very rapid clearance by the liver, and thus produces a marked depletion of circulating human SAP. This mechanism of drug action potently removes SAP from human amyloid deposits in the tissues and may provide a new therapeutic approach to both systemic amyloidosis and diseases associated with local amyloid, including Alzheimers disease and type 2 diabetes.

X-ray analyses of aspartic proteinases:the three-dimensional structure at 2.1 Å resolution of endothiapepsin

The molecular structure of endothiapepsin (EC 3.4.23.6), the aspartic proteinase from Endothia parasitica, has been refined to a crystallographic R-factor of 0.178 at 2.1 A resolution. The positions of 2389 protein non-hydrogen atoms have been determined and the present model contains 333 solvent molecules. The structure is bilobal, consisting of two predominantly beta-sheet domains that are related by an approximate 2-fold axis. Of approximately 170 residues, 65 are topologically equivalent when one lobe is superimposed on the other. Twenty beta-strands are arranged as five beta-sheets and are connected by regions involving 29 turns and four helices. A central sheet involves three antiparallel strands from each lobe organized around the dyad axis. Each lobe contains a further local dyad that passes through two sheets arranged as a sandwich and relates two equivalent motifs of four antiparallel strands (a, b, c, d) followed by a helix or an irregular helical region. Sheets 1N and 1C, each contain two interpenetrating psi structures contributed by strands c,d,d and c,d,d, which are related by the intralobe dyad. A further sheet, 2N or 2C, is formed from two extended beta-hairpins from strands b,c and b,c that fold above the sheets 1N and 1C, respectively, and are hydrogen-bonded around the local intralobe dyad. Asp32 and Asp215 are related by the interlobe dyad and form an intricate hydrogen-bonded network with the neighbouring residues and comprise the most symmetrical part of the structure. The side-chains of the active site aspartate residues are held coplanar and the nearby main chain makes a firemans grip hydrogen-bonding network. Residues 74 to 83 from strands aN and bN in the N-terminal lobe form a beta-hairpin loop with high thermal parameters. This flap projects over the active site cleft and shields the active site from the solvent region. Shells of water molecules are found on the surface of the protein molecule and large solvent channels are observed within the crystal. There are only three regions of intermolecular contacts and the crystal packing is stabilized by many solvent molecules forming a network of hydrogen bonds. The three-dimensional structure of endothiapepsin is found to be similar to two other fungal aspartic proteinases, penicillopepsin and rhizopuspepsin. Even though sequence identities of endothiapepsin with rhizopuspepsin and penicillopepsin are only 41% and 51%, respectively, a superposition of the three-dimensional structures of these three enzymes shows that 237 residues (72%) are within a root-mean-square distance of 1.0 A.

Molecular Dissection of Alzheimer'S Disease Neuropathology by Depletion of Serum Amyloid P Component.

New therapeutic approaches in Alzheimers disease are urgently needed. The normal plasma protein, serum amyloid P component (SAP), is always present in cerebrospinal fluid (CSF) and in the pathognomonic lesions of Alzheimers disease, cerebrovascular and intracerebral Aβ amyloid plaques and neurofibrillary tangles, as a result of its binding to amyloid fibrils and to paired helical filaments, respectively. SAP itself may also be directly neurocytotoxic. Here, in this unique study in Alzheimers disease of the bis(d-proline) compound, (R)-1-[6-[(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC), we observed depletion of circulating SAP and also remarkable, almost complete, disappearance of SAP from the CSF. We demonstrate that SAP depletion in vivo is caused by CPHPC cross-linking pairs of SAP molecules in solution to form complexes that are immediately cleared from the plasma. We have also solved the structure of SAP complexed with phosphothreonine, its likely ligand on hyperphosphorylated τ protein. These results support further clinical study of SAP depletion in Alzheimers disease and potentially other neurodegenerative diseases.

Trapping of palindromic ligands within native transthyretin prevents amyloid formation.

Transthyretin (TTR) amyloidosis is a fatal disease for which new therapeutic approaches are urgently needed. We have designed two palindromic ligands, 2,2-(4,4-(heptane-1,7-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (mds84) and 2,2-(4,4-(undecane-1,11-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (4ajm15), that are rapidly bound by native wild-type TTR in whole serum and even more avidly by amyloidogenic TTR variants. One to one stoichiometry, demonstrable in solution and by MS, was confirmed by X-ray crystallographic analysis showing simultaneous occupation of both T4 binding sites in each tetrameric TTR molecule by the pair of ligand head groups. Ligand binding by native TTR was irreversible under physiological conditions, and it stabilized the tetrameric assembly and inhibited amyloidogenic aggregation more potently than other known ligands. These superstabilizers are orally bioavailable and exhibit low inhibitory activity against cyclooxygenase (COX). They offer a promising platform for development of drugs to treat and prevent TTR amyloidosis.

Structure of human porphobilinogen deaminase at 2.8 Å: the molecular basis of acute intermittent porphyria

Mutations in the human PBGD (porphobilinogen deaminase) gene cause the inherited defect AIP (acute intermittent porphyria). In the present study we report the structure of the human uPBGD (ubiquitous PBGD) mutant, R167Q, that has been determined by X-ray crystallography and refined to 2.8 A (1 A=0.1 nm) resolution (Rfactor=0.26, Rfree=0.29). The protein crystallized in space group P2(1)2(1)2 with two molecules in the asymmetric unit (a=81.0 A, b=104.4 A and c=109.7 A). Phases were obtained by molecular replacement using the Escherichia coli PBGD structure as a search model. The human enzyme is composed of three domains each of approx. 110 amino acids and possesses a dipyrromethane cofactor at the active site, which is located between domains 1 and 2. An ordered sulfate ion is hydrogen-bonded to Arg26 and Ser28 at the proposed substrate-binding site in domain 1. An insert of 29 amino acid residues, present only in mammalian PBGD enzymes, has been modelled into domain 3 where it extends helix alpha2(3) and forms a beta-hairpin structure that contributes to a continuous hydrogen-bonding network spanning domains 1 and 3. The structural and functional implications of the R167Q mutation and other mutations that result in AIP are discussed.

Proteolytic cleavage of Ser52Pro variant transthyretin triggers its amyloid fibrillogenesis

Significance Transthyretin, a normal circulating plasma protein, is inherently amyloidogenic. It forms abnormal, insoluble, extracellular amyloid fibrils in the elderly, sometimes causing structural and functional damage leading to disease, senile amyloidosis. More than 100 different point mutations in the transthyretin gene cause earlier adult-onset, autosomal-dominant, fatal, hereditary amyloidosis. The transthyretin variant Ser52Pro is responsible for the most aggressive known clinical phenotype. Here we identify the crucial pathogenic role of specific proteolytic cleavage at residue 48 in triggering fibril formation by this variant. Genuine amyloid fibril formation in vitro is much more extensive than previously reported for wild-type transthyretin or any other transthyretin variant. Characterization of the fibrillogenic effect of this cleavage powerfully informs drug design and targeting for transthyretin amyloidosis. The Ser52Pro variant of transthyretin (TTR) produces aggressive, highly penetrant, autosomal-dominant systemic amyloidosis in persons heterozygous for the causative mutation. Together with a minor quantity of full-length wild-type and variant TTR, the main component of the ex vivo fibrils was the residue 49-127 fragment of the TTR variant, the portion of the TTR sequence that previously has been reported to be the principal constituent of type A, cardiac amyloid fibrils formed from wild-type TTR and other TTR variants [Bergstrom J, et al. (2005) J Pathol 206(2):224–232]. This specific truncation of Ser52Pro TTR was generated readily in vitro by limited proteolysis. In physiological conditions and under agitation the residue 49-127 proteolytic fragment rapidly and completely self-aggregates into typical amyloid fibrils. The remarkable susceptibility to such cleavage is likely caused by localized destabilization of the β-turn linking strands C and D caused by loss of the wild-type hydrogen-bonding network between the side chains of residues Ser52, Glu54, Ser50, and a water molecule, as revealed by the high-resolution crystallographic structure of Ser52Pro TTR. We thus provide a structural basis for the recently hypothesized, crucial pathogenic role of proteolytic cleavage in TTR amyloid fibrillogenesis. Binding of the natural ligands thyroxine or retinol-binding protein (RBP) by Ser52Pro variant TTR stabilizes the native tetrameric assembly, but neither protected the variant from proteolysis. However, binding of RBP, but not thyroxine, inhibited subsequent fibrillogenesis.

Fragment screening using capillary electrophoresis (CEfrag) for hit identification of heat shock protein 90 ATPase inhibitors

CEfrag is a new fragment screening technology based on affinity capillary electrophoresis (ACE). Here we report on the development of a mobility shift competition assay using full-length human heat shock protein 90α (Hsp90α), radicicol as the competitor probe ligand, and successful screening of the Selcia fragment library. The CEfrag assay was able to detect weaker affinity (IC50 >500 µM) fragments than were detected by a fluorescence polarization competition assay using FITC-labeled geldanamycin. The binding site of selected fragments was determined by co-crystallization with recombinant Hsp90α N-terminal domain and X-ray analysis. The results of this study confirm that CEfrag is a sensitive microscale technique enabling detection of fragments binding to the biological target in near-physiological solution.

Structural basis of ligand specificity in the human pentraxins, C-reactive protein and serum amyloid P component.

The normal physiological roles of the phylogenetically conserved human plasma proteins C‐reactive protein (CRP) and serum amyloid P component (SAP) are not known. Novel drugs targeting their ligand specificities are in clinical development as both proteins have significant pathophysiological effects, SAP in promoting amyloidosis and CRP in exacerbating ischemic injury. Both proteins bind to phosphoethanolamine and we show here that, under physiological conditions, phosphoethanolamine is bound with higher affinity by human SAP than by human CRP. An explanation is provided by X‐ray crystal structures that show SAP residue Tyr74 allowing additional hydrophobic protein–ligand interactions compared with the equivalent Thr76 of CRP. Docking simulations show many more low energy positions for phosphoethanolamine bound by CRP than by SAP and are consistent with the crystallographic and functional binding results. These fundamental observations on structure–activity relationships will aid the design of improved pentraxin targeting drugs. Copyright

Crystallographic Studies of Reduced Bond Inhibitors Complexed with an Aspartic Proteinase

To aid in the design of an effective inhibitor to human renin, it is essential to have a detailed knowledge of how this aspartic proteinase interacts with its substrate, angiotensinogen. Human renin shows a stringent specificity toward the Leu–Val bond in its natural substrate. The minimal length for an effective substrate has been characterised as an octapeptide sequence derived from the amino terminal portion of angiotensinogen (residues 6 — 13): His–Pro–Phe–His–Leu–Val–Ile–His (Leu–Val is the scissile bond). This suggests that renin has a fairly extensive active site cleft, as has been observed in homologous enzymes whose three-dimensional structures have been solved using x-ray diffraction methods. The homologous fungal aspartic proteinase, endothiapepsin, has been cocrystallised with human renin inhibitors of the type His–Pro–Phe–His–Leu–R–Val–Ile–His, where R indicates a reduced carbonyl analogue of the scissile peptide bond. The three-dimensional crystallographic structures of two complexes of endothiapepsin with an inhibitor have been solved. The details of inhibitor binding at the active site cleft of endothiapepsin are described. These data allow a rational approach to the design of novel renin inhibitors, through studies of these inhibitors in a three-dimensional model of human renin constructed in our laboratory.